Volume 4, Issue 1 (2017) Tropical Plant Research

ISSN (E): 2349 1183
ISSN (P): 2349 9265

4(1): 0106, 2017
DOI: 10.22271/tpr.2017.v4.i1.001
Research article
Seed germination inhibitory effect of Caryota urens L.

seed pericarp on rice and associated weeds
S. I. Fonseka1, S. Adikari1, L. R. Jayasekera1*, P. Ranasinghe2 and G. A. S. Premakumara2
1
Department of Botany, University of Kelaniya, Sri Lanka
2
Industrial Technology Institute, Colombo, Sri Lanka
*Corresponding Author: ranjith@kln.ac.lk [Accepted: 05 January 2017]
Abstract: Previous studies have shown that the Caryota urens seed pericarp possesses botanicals
capable of inhibiting seed germination. Extracts made from C. urens pericarp were tested at
different concentrations to find out its inhibitory activity. Three rice varieties (Bg 305, Bg 358, Bg
368) and the rice weeds (Echinochloa crus-galli, Ischaemum rugosum and Ipomoea aquatica)
were tested. Percentage germination was measured for 7 days at 2 day intervals. The methanol
extract of dried seed pericarp significantly reduced the seed germination, indicating that the
concentration of the inhibitory substance/s in C. urens pericarp is higher in methanol extracts than
the water extracts. Dried seed pericarp showed the highest inhibitory effect on the seed
germination. The germination of all rice cultivars and weed species tested were completely
inhibited by 100 and 200 mg.ml-1 concentrations, suggesting that the C. urens seed pericarp has a
seed germination inhibitory effect on all weeds tested. In general, the germination inhibitory
effects were maximal at high concentrations than at lower concentrations. Potential for using C.
urens pericarp for weed control is highlighted.
Keywords: Caryota urens - Seed pericarp - Germination - Rice - Weeds.
[Cite as: Fonseka SI, Adikari S, Jayasekera LR, Ranasinghe P & Premakumara GAS (2017) Seed germination
inhibitory effect of Caryota urens L. seed pericarp on rice and associated weeds. Tropical Plant Research 4(1):
16]
INTRODUCTION
The ever increasing demand for food with the exponential increase in human population demands maximal
achievements in crop production. Development of effective and environmental friendly weed control measures
is one area of importance in this respect. Weeds and weed control have become a major cost factor determining
the economic profitability of crop production worldwide. Weeds, being the major biotic stress for most crops
including rice, compete with crops for light, nutrients and moisture, resulting in significant decrease of yield and
quality of crop harvest. In the light of the above, studies on locally available natural sources that have a potential
to control weeds become relevant to develop appropriate, environmental friendly control measures that suit the
agricultural background and economy of Sri Lanka.
Allelopathy is defined as a mechanism by which plant, directly or indirectly affects, inhibits or stimulates
growth of other plants by the production of chemical compounds or allelochemicals released to the environment
(Ridenour & Callaway 2001). The use of allelochemicals by allelopathic plants/plant parts for weed
management has received attention in recent times (Weston 1996) in view of their environmental friendly nature
as opposed to synthetic chemicals. Hence the use of natural substances from plants is considered as a low input
and sustainable approach to integrated weed management, a practice that helps reduce the increasing incidences
of herbicide resistance in weeds as well (Mayer & Mayber 1989, Materechera & Hae 2008).
Studies have shown the inhibitory effects of certain plants not only on weeds but also on growth and yields
of crop species. The allelopathic and herbicidal effectiveness of different plant species have shown to depend on
the plant part (Oudhia 2003). Therefore investigations are required to explore plants and respective plant parts
with effective allelopathic activity, especially in the control of agricultural weeds (Materechera & Hae 2008).
The inhibitory effects of extracts obtained from different seed pericarps and plant parts on seed germination of
www.tropicalplantresearch.com 1
Received: 11 October 2016 Published online: 31 January 2017
https://doi.org/10.22271/tpr.2017.v4.i1.001
Fonseka et al. (2017) 4(1): 0106
various other crops and weeds have been studied by Humaid & Warrag (1999), Kivi et al. (2010).
The study reported here is based on the seed germination inhibitory effect of Caryota urens (Family:
Arecaceae) seed pericarp. In C. urens the inflorescence is about 3 m in length and emerges at each leaf node
from top to bottom. The plant produces pendent clusters of white, unisexual flowers resulting in about 35000 to
40000 seeds per inflorescence. Mesocarp is fleshy, filled with abundant irritant, needlelike crystals. When these
fruits fall on the ground, it takes a long time to germinate. Therefore, it can be assumed that the seed pericarp of
C. urens may contain seed germination inhibitory substances (Wijesinghe 1992). No study is known to have
been conducted related to the seed germination inhibitory effects of C. urens seed pericarp on rice and
associated weeds.
As no information is available on the pattern of C. urens seed germination, Ranasinghe et al. (2008)
conducted an investigation to study the C. urens seed germination pattern and to develop a method for induction
of rapid germination. In their study, it was found that the complete removal of fleshy pericarps of ripe C. urens
fruits is the most effective way to achieve a higher rate of germination. The prevention of C. urens seed
germination may be due to the presence of inhibitory substances in their pericarps which may contain relatively
high concentrations of growth inhibitors that can suppress germination of the embryo (Taiz & Zeiger 2010).
This research was designed to study the inhibitory effect of C. urens seed pericarp on rice and on some
associated weeds. Echinochloa crus-galli (L.) P.Beauv. is a grass weed that can germinate and grow for
extended periods of time in an anaerobic environment (Kennedy et al. 1983) which is ecologically similar to
rice. Being a highly competitive weed with rice, it can reduce rice yield up to 100% (Nyarko & Datta 1991).
Ischaemum rugosum Salisb. is an annual grass weed that grows up to 120 cm in height. This aggressive, highly
competitive weed is propagated by seeds. Five plants per square meter reduced 15% rice yield, while 80 plants
per m2 reduced 82% rice yield in a study conducted by Nyarko & Datta (1991). Ipomoea aquatica Forssk. is a
fast growing, perennial broad leaf vine propagated by seeds and stem cuttings, and can cause up to 30% rice
yield loss (Nyarko & Datta 1991). The three lowland rice cultivars, Bg 305, Bg 358 and Bg 360 tested have
been developed by the Rice Research and Development Institute in Bathalagoda (Bg) in Sri Lanka (Personal
communication, 18 September 2013).
MATERIALS AND METHODS

Selection of seeds
Three rice cultivars (Bg 305, Bg 358 and Bg 368) and the rice weeds (Echinochloa crus-galli (L.) P.Beauv.,
Ischaemum rugosum Salisb. and Ipomoea aquatica Forssk.) were selected for the experiment.
The inhibitory effects of the aqueous extract of Caryota urens seed pericarp on seed germination
Ripe fruits of Caryota urens were crushed and the pericarp was separated, homogenized and filtered. The
extract was freeze dried. Lipolyzed seed pericarp extract (WESP) was used in germination inhibition
experiments. A concentration series (0.5, 5, 50 and 500 mg.ml-1) was made with distilled water. 2000 l of this
concentration series was added to the Petri dishes containing 20 seeds of test species on filter papers separately.
Three replicates were used for each concentration. Distilled water was used in control experiment. During the
experimental period, seeds were treated with distilled water. Number of germinated seeds and lengths of roots,
shoots of germinated seedlings were recorded. Results obtained on percentage germination, root length and
shoot length of each treatment were statistically analyzed.
The inhibitory effect of the methanol extract of Caryota urens seed pericarp on seed germination
Seed pericarps were oven dried for one week at 50 C. Dried pericarps were immersed in methanol for a
week and filtered through a muslin cloth. Methanol in filtrate was evaporated by rotator evaporation and the
crude was freeze dried to remove excess water. The crude obtained from dried seed pericarps (DSPE) was
stored in a freezer at -12C. Seed germination inhibitory effect was observed using this DSPE crude. In the
bioassay on filter papers, a concentration series (0.5, 5, 50 and 500 mg.ml-1) from the DSPE was applied
separately into Petri dishes containing 20 seeds of test species. 2000 l of the extract was added per Petri dish.
The dishes were kept wet by adding distilled water during the experimental period. Three replicates were used
for each treatment. Distilled water was used for the control experiment. Germination of seeds, the length of
shoots and roots of germinated seedlings were recorded. Results of each treatment were statistically analyzed.
Bioassay on soil
In the bioassay on soil, a higher concentration series (25, 50, 100 and 200 mg.ml-1) was applied separately into
Fonseka et al. (2017) 4(1): 0106
the Petri dishes with paddy soil containing 20 seeds of test species. 5000 l of the extract was added per Petri
dish. The soil was kept wet by adding distilled water, and the number of germinated seeds in each Petri dish was
recorded daily. Three replicates were used for each treatment and distilled water was used for the control
experiment. Percentage inhibition, shoot and root length of seedlings of each treatment were statistically
analyzed.
RESULTS
Germination inhibitory effect of water-extracted seed pericarps on Echinochloa crus-galli seeds
Reference to table 1, E. crus-galli seeds in the control experiment were started to germinate on the 2nd day,
and reached a percentage germination of 84.6% after six days, whereas the germinability of seeds treated with
500 mg/ml of WESP was significantly lower (p<0.05) compared to the control. However, the other
concentrations of WESP (50, 5 and 0.5 mg.ml-1) did not show a significant difference (p>0.05) in germination.
In comparison with the control and other concentrations, a significantly (p<0.05) shorter root length (4.82.3
mm) after 6th day was observed at 500 mg.ml-1 WESP treatment. The lowest shoot growth (p<0.05) was
observed with the 500 mg.ml-1 WESP treated E. crus-galli seeds (5.92.0 mm), whereas the shoot length of the
control was 27.62.0 mm after six days.
Table 1. Effect of water extract of Caryota urens pericarp on germination of Echinochloa crus-galli.
Mean Root length Mean Shoot
Concentration (mg.ml-1) Germination (%) SE
(mm) SE length (mm) SE
500 47.13.3c 4.82.3c 5.92.0c
50 73.83.2ab 18.12.3a 16.32.0b
5 77.33.4a 21.42.2a 22.12.2a
0.5 79.63.3a 30.32.3a 26.92.0a
Control 84.63.3a 26.22.3a 27.62.0a
Note: Values are means of three replicates standard error with twenty seeds each. Values in a column with the same
superscript are not significantly different (p>0.05).
Germination inhibitory effect of methanol-extracted seed pericarps on Echinochloa crus-galli seeds
The seeds of E. crus-galli in the control began to germinate on the 2nd day resulting in a percentage
germination of 80.5% after six days, whereas the germinability of seeds treated with 500 mg.ml-1 and 50 mg.ml-1
of DSPE were significantly lower (p<0.05) compared with the control (Table 2). The other concentrations of
DSPE (5 and 0.5 mg.ml-1), showed a significantly lower (p<0.05) germination rate (Table 2). A significant
(p<0.05) shorter root lengthening was also observed after the 6th day in 500 and 50 mg.ml-1 DSPE treatments.
Even the other two treatments, 5 mg.ml-1 and 0.5 mg.ml-1 reduced the root growth (Table 2). In the case of shoot
development, a significantly lower shoot growth was observed in all 500, 50 and 5 mg.ml-1 DSPE treatment,
except in the 0.5 mg.ml-1 treatment where no significant (p>0.05) effect was found in the shoot elongation
(Table 2).
Table 2. Effect of methanol extract of Caryota urens pericarp on germination of Echinochloa crus-galli.
Mean Root length Mean Shoot
Treatment (mg.ml-1) Germination (%) SE length (mm) SE
(mm) SE
500 19.03.7c 0.92.9c 0.72.1c
50 28.33.5c 3.22.7c 2.82.1c
5 66.63.4b 18.02.8b 16.62.2b
0.5 68.33.7b 31.82.9a 27.22.1a
Control 80.53.3a 43.12.8a 32.92.0a
Note: Values are means of three replicates standard error with twenty seeds each. Values in a column with the same
Effect of DSPE extract on rice seeds and other associated weeds on filter papers
Reference to Table 3, 0.5 and 5 mg.ml-1 treatments showed a significant inhibition in germination of Bg 305,
Bg 358 and Bg 360 rice seeds, while 50 and 500 mg.ml-1 treatments completely inhibited the seed germination
of Bg 305, Bg 358 and Bg 360. Compared to the control 0.5 mg.ml-1, 5, 50 and 500 mg.ml-1 treatments inhibited
the seed germination of I. rugosum. Among 0.5, 5 and 50 mg.ml-1 treatments, the highest inhibitory activity was
observed with the 50 mg.ml-1 concentration, whereas 100% inhibition was observed in 500 mg.ml-1 treatment. A
Fonseka et al. (2017) 4(1): 0106
significant inhibition in germination was observed in I. aquatica seeds compared to its control. Both 50 and 500
mg.ml-1 treatments completely inhibited the germination of I. aquatica (Table 3).
Table 3. Effect of the DSPE on the three rice cultivars and the three rice weeds.
Concentration Percentage inhibition
(mg.ml-1) Bg 305 Bg 358 Bg 360 I. rugosum I. aquatica
Control 63.31.7a 7.51.1a 22.51.1a 45.82.4a 49.23.7a
0.5 74.22.4b 12.51.1b 53.31.7 b
68.33.3b 78.34.0b
5 76.71.7b 12.51.1b 40.81.5c 66.73.8b 79.25.2b
50 100c 100c 100 d
95.02.2c 100bc
500 100c 100c 100 d
100c 100bc
Note: Values are means of six replicates SE, each with twenty seeds. Values in a column with the same
superscripts are not significantly different (p>0.05).
Effect of DSPE extract on rice seeds and associated weeds on soil
The 25 mg.ml-1 treatment did not inhibit the germination of Bg 305 rice seeds significantly (Table 4). But the
50 mg.ml-1 treatment showed a significant inhibition of germination, while the 100 and 200 mg.ml-1 treatments
completely inhibited the Bg 305 seed germination. In the case of Bg 358, no significant difference was observed
in seed germination treated with 25 and 50 mg.ml-1 concentrations, whereas the germination inhibition was
statistically significant in the 100 mg.ml-1 treatment. It was observed that the 200 mg.ml-1 treatment completely
inhibited the seed germination of Bg 358. The effect of the three concentrations on the germination of Bg 360
seeds was somewhat different from the other two rice cultivars tested. While showing a no significant effect on
seed germination by 25 mg.ml-1, the 50 and 100 mg.ml-1 concentrations inhibited the germination of Bg 360
seeds significantly, and the 100% germination inhibition was observed with the 200 mg.ml-1 concentration.
Table 4. Effect of the DSPE on the three rice cultivars and the three rice weeds on soil.
Concentration Percentage of inhibition
(mg.ml-1) Bg 305 Bg 358 Bg 360 I. rugosum I. aquatica
Control 36.71.7a 5.01.3a 35.82.0a 40.81.5a 27.53.4a
25 45.82.4a 14.21.5a 35.82.0 a
60.09.1b 35.81.5a
50 82.51.7b 22.52.1a 81.71.1b 91.71.1c 52.51.1b
100 100c 83.34.0b 95.81.5 c
95.81.5d 97.51.1c
200 100c 100d 100 d
100e 100d
Note: Values are means of six replicates SE, each with twenty seeds. Values in a column with the same
E. crus-galli seeds in the control started to germinate on the 2nd day. 25, 50, 100 and 200 mg.ml-1 treated
seeds inhibited the germination with a significant difference (p<0.05) compared with the control. The inhibitory
effect of 100 mg.ml-1 treatment was higher than that of the 25 and 50 mg.ml-1 treatments. The 200 mg.ml-1
treatment was able to inhibit the seed germination of E. crus-galli completely. Compared with the control, all
four treatments inhibited the seed germination of I. rugosum with a significant difference. The highest inhibition
was observed in the 100 mg.ml-1 treated I. rugosum seeds, whereas the 200 mg.ml-1 treatment completely
inhibited the seed germination. There was no significant difference in inhibition between the control and the 25
mg.ml-1 treated I. aquatica seeds, whereas the effect of germination inhibition by the 50, 100 and 200 mg.ml-1
treatments was statistically significant. The highest inhibition was observed in 100 mg.ml-1, while the 200
mg.ml-1 treatment completely inhibited the seed germination of I. aquatica seeds.
DISCUSSION AND CONCLUSION

The inhibitory effect of C. urens seed pericarp was tested against the three rice varieties, Bg 305, Bg 358 and
Bg 360, and three rice weeds, Echinochloa crus-galli, Ischaemum rugosum Salisb. and Ipomoea aquatica
Forssk. The seeds with the viability above 80% only were selected for germination experiments. Most of the
previous studies on seed germination inhibitory effects of plant extracts have used water extracts (Junttila 1997,
Chung & Miller 1995) because of the fact that most of the plant substances are water soluble. Therefore in this
study too, the water was used to extract the contents of the C. urens seed pericarp followed by freeze drying.
The methanol extracts have also been used in some studies. Using lettuce and red beet seeds, Junttila (1997)
studied about the germination inhibitors in methanol extracts of red beet fruits (Beta vulgaris cv. rubra. The
Fonseka et al. (2017) 4(1): 0106
filter paper method, one of the easiest ways to observe the seed germination in the laboratory has been used to
observe seed germination (Gressel & Holm 2006).
The methanol extract of C. urens pericarp significantly reduced the seed germination of E. crus-galli than
the water extracts, indicating that the concentration of the inhibitory substance/s in C. urens pericarp was higher
in methanol extracts. Thus, the inhibitory action of methanol extracts was increased even at low concentrations.
In the light of these observations, further experiments using the methanol extracts were conducted to understand
the effect of germination inhibition of the rice and the weed seeds tested.
The germination of all rice cultivars and weed species tested were completely inhibited by 100 and 200
mg.ml-1 concentrations in this experiment. To test the inhibitory effect of C. urens on dicotyledons, Ipomoea
aquatica was used. A complete inhibition of I. aquatica seed germination was also observed at 200 mg/ml,
suggesting that the C. urens seed pericarp has a seed germination inhibitory effect on all weeds tested
irrespective of monocotyledons or dicoteledons. In general, the germination inhibitory effects were maximal at
high concentrations than at lower concentrations. In this study, it was revealed by the tetrazolium test that the
viability of all the weed and rice had not been affected by the DSPE. According to the results obtained, there is
an inhibitory effect on the germination of both rice and associated weeds, but the rice seeds resume germination
before the weed seeds. Therefore there is a possibility to use the extract of C. urens dried seed pericarp to
control the rice weeds tested.
ACKNOWLEDGEMENTS
Thanks are due to the lab staff of the Department of Botany, University of Kelaniya and the Industrial
Technology Institute, Colombo 07 for their assistance. Facilities and materials were provided by both
institutions. Seeds of the three rice cultivars were provided by the Rice Research and Development Institute,
Bathalagoda.
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Journal 87: 920925.
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inhibition by Abutilon theophrasti. Planta 57: 212224.
Humaid AI & Warrag MOA (1999) Effect of mesquite (Prosopis juliflora) pericarps aqueous extracts on seed
germination and plumule and radicle elongation of bermuda grass (Cynodon dactylon). Journal of
Agricultural Sciences 11(2): 149156.
Junttila O, Jorunn E & Moritz T (1997) Long day induced bud break in Salix pentandra is associated with
transiently elevated levels of GA1 and gradual increase in Indole-3-acetic acid. Plant and Cell Physiology
38: 536540.
Kennedy RA, Rumpho ME & Vanderzee D (1983) Germination of Echinochloa crusgalli (Barnyard grass)
seeds under anaerobic conditions. Plant Physiology 72: 787794.
Kivi MP, Tobeh A, Shahverdikandi MA & Somarin SJ (2010) Inhibitory impact of some crop plants extracts on
germination and growth of wheat. American Eurasian Journal of Agricultural & Environmental Sciences 9
(1): 4751.
Materechera SA & Hae ME (2008) Potential of aqueous extracts from parts of the pepper tree (Schinus molle L.)
to affect emergence and seedling development of wheat (Triticum sativa L.) and weeds in a manure amended
soil. The Open Agriculture Journal 2: 99104.
Mayer AM & Mayber AP (1989) The Germination of Seeds, Fourth edition. Pergamon Press, New York, pp.
206.
Nyarko K & Datta SK (1991) Handbook for weed control, First edition. International Rice Research Institute,
Manila, Philippines, pp. 739.
Oudhia P (2003) Traditional medicinal knowledge about useful herb Doobi (Cynodon dactylon) in Chhattisgarh,
India, (20012003). Available from http://www.botanical.com/site/Column_poudhia/111_doobi. (accessed 8
May 2013).
Ranasinghe P, Wickramasinghe WGU, Premakumara GAS & Weerasinghe PA (2008) Germination pattern of
Kithul (Caryota urens) seeds and a method for induction of rapid germination. Proceedings of the 28th
Annual Sessions of the Institute of Biology, Sri Lanka.
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Ridenour WM & Callaway RM (2001) The relative importance of allelopathy in interference: The effects of an
invasive weed on a native bunch grass. Oecologia 126: 444450.
SAS/STAT 9.2 Users Guide (2008) Available from: http://support.sas.com/documentation /cdl/en/statugglm,
(accessed 10 Aug 2012).
Taiz L & Zeiger E (2010) Plant Physiology, Fifth edition. Sinauer Associates, Inc., Publishers, Sunderland,
USA, pp. 686687.
Weston LA (1996) Utilization of allelopathy for weed management in agro ecosystems: Allelopathy in cropping
systems. Agron Journal 88(6): 860866.
Wijesinghe R (1992) Seed characteristics and pattern of germination of Kitul palm (Caryota urens L.).
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ISSN (E): 2349 1183
ISSN (P): 2349 9265
4(1): 0712, 2017
DOI: 10.22271/tpr.2017.v4.i1.002
Research article
Citrus varieties of Kokrajhar District, BTAD, Assam: Its

propagation and cultivation prospect
Mohesh Gogoi1* and Miniswrang Basumatary2
1
Department of Botany, Biotech Hub, Science College, Kokrajhar, BTAD, Assam
2
Biotech Hub, Science College, Kokrajhar, BTAD, Assam
*Corresponding Author: mohesh.gogoi@rediffmail.com [Accepted: 08 January 2017]
Abstract: Citrus spp. grows in different localities of Kokrajhar district (BTAD), Assam and this
place is a natural home to many varieties of the said fruit. Even though a favorable agro-climatic
condition for citrus growth prevails in the region the production is not sufficient. Using
inappropriate rootstock as well as the seedling is cultivation bottleneck that has been learned from
the orchardists. The studies revealed that right type of seedling produced by using standardized
nursery technique ensure healthy growth and optimum production. This paper deals with
distribution of the different citrus varieties that grow in wild, semi-wild and cultivated forms in
Kokrajhar district and usefulness of different vegetative propagation technique along with seed
germination.
Keywords: Citrus - Propagation - Cutting - Layering - Orchard.
[Cite as: Gogoi M & Basumatary M (2017) Citrus varieties of Kokrajhar District, BTAD, Assam: Its
propagation and cultivation prospect. Tropical Plant Research 4(1): 712]
INTRODUCTION
Citrus (Citrus L.) is one of the worlds most important fruit crops, distributed in the tropical and subtropical
regions of the world (Randhwa & Srivastava 1986). They are grouped under the family Rutaceae and subfamily
Aurantioideae (Singh 1981, Gogoi et al. 2003). Swingle (1943) have divided Citrus into two subgenera, Citrus
(formerly Eucitrus) and Papeda. In this regard, Bhattacharya & Dutta (1956) have postulated that the species
belonging to the subgenus Citrus are economically more important than those of the subgenus Papeda. Citrus is
the third most important fruit crop in India (Ghosh 1999). NE region of the country alone produces an annual
production of 7,91,000.36 metric tons (Govt. of Assam report 201213) which are considered to be a major
citrus growing zone (Singh 1999). As per Govt. of Assam report 201213 the state alone could ably produce 3,
23,000.39 metric tons which account for 40% of the total annual production of Citrus in the region. Except for
few upper Assam districts, the border areas of Nagaland and Arunachal Pradesh and a large part of BTAD
provide ample scope for citrus cultivation. BTAD comprises four districts viz. Kokrajhar, Chirang, Baksa and
Udalguri with a total area of 8, 970 km2 out of which 2, 562 km2 is delineated as forest area (28.6%), while non-
forest area is 6, 408 km2 (Gogoi & Bhansali 2015). Among the four districts of BTAD, Kokrajhar district
provides a suitable agro-climatic condition for large-scale citrus cultivation which is bounded by Bhutan in
North, Dhubri district in South, Bongaigaon and Chirang district in East and West Bengal in West. As per BTC
report (Dept. of Forest), the total geographical area of Kokrajhar district is 3,98,635 ha out of which total
151680 ha is cultivable land, and the gross crop and net crop area is 168561 ha and 108916 ha respectively.
Santana (Citrus reticulata) varieties are extensively cultivated all over the district where the total production
was 173 metric ton in 201011 and 365 metric ton in 201112. On the other hand lemon (C. limon) and lime (C.
aurantifolia) varieties has high market demand that encourages the orchardists to grow citrus in their backyard
and kitchen garden as well. It has been reported by district Agriculture & Horticulture office, Kokrajhar that the
total citrus production was 238 metric ton in 201011 and 365 metric ton in 201112 but the production was
reported to be too less to fulfill the market demand. To understand the distribution pattern, availability, and
problems related to citrus cultivation, an extensive study was carried out with the financial support of UGC in
Gogoi & Basumatary (2017) 4(1): 0712
the form of the minor research project. Various propagation methods have been employed for improvement of
the quality of seedling by developing nursery.
Figure 1. Map of Kokrajhar district showing the localities from where the Citrus varieties have been collected.
A thorough survey was conducted in between 201314 in different areas of Kokrajhar district (Fig. 1) for
Citrus species in two different seasons covering its complete phenophase (Gogoi 2014). Detailed observation of
plant species was made scoring the data on habitat, morphology of the whole plant, phenology, ecology and
associated plants. All the taxonomic details and other information have been recorded in a descriptive blank as
per the model of H. J. Webber (Webber 1943) and UPOV TF/83/3. The geographical, environmental and soil
parameters of the places were noted and later a map was drawn showing the citrus growing localities of the
district. A general identification of the collected species was done with the help of standard taxonomic literature.
The careful collection was made for herbarium specimen following the methodology of Lawrence (1951) and
Jain & Rao (1977). Thereafter, comparison was made with authentic specimens deposited at BSI herbarium,
Shillong and BSI herbarium, Calcutta.
For sapling preparation and propagation fresh cuttings about 510 cm lengths (Rajput & Haribabu 1985) of
Citrus limon Brum f., C. medica L. and C. aurantifolia Swin. by applying growth promoting hormone were later
planted during MarchMay, 2013. One year old branches of above mentioned three Citrus species were planted
in the nursery bed. The tubs were regularly irrigated with an adequate amount of water. The data were taken at
fortnightly interval up to 90 days. Simultaneously mature fruits of each plant were collected for histological
observation.
Further to examine the quality of propagules a series of nursery experiment was conducted within the shade
house for their performance by using the method of Dutta (1964), Mukharjee & Majumder (1983), Williamson &
Jackson (1994), Kalita et al. (2002) and Singh (2012). The rooting behavior was also studied by means of
multiple nodes cutting and layering method followed by Chaudhury & Chakwawar (1980), Singh et al. (2004).
RESULTS AND DISCUSSION

After an extensive survey and detailed morphological observation, a total of 8 species and 5 varieties
including both wild and cultivated forms have been identified (Table 1). Three varieties of Citrus limon (Assam
lebu/China lebu, Elachi lebu/Cardamom lemon, Pati lebu/Jora nemu), two varieties of Citrus aurantifolia
(Abhayapuri lime/oval-fruited and Karimganj lime/round-fruited) and each variety of Citrus jambhiri, Citrus
reticulata, Citrus nobilis, Citrus grandis, Citrus medica, Citrus sinensis were recorded.
Table 1. Identified Citrus species of Kokrajhar district.

Sl. No. Species identified Varieties identified
1 Citrus limon Brum f. Assam lebu/China lebu, Elachi lebu/Cardamom lemon, Pati
lebu/Jora nemu
2 Citrus aurantifolia (Christm.) Swin. Abhayapuri lime (Oval fruited) and Karimganj lime
(Round-fruited)
3 Citrus jambhiri Lush. A round fruited variety locally called gulnemu
4 Citrus reticulata Blanco A common mandarin variety
5 Citrus nobilis Lour Semi-domesticated variety locally called jeneru tenga
6 Citrus grandis L. Osbeek Juice vesicle pink coloured variety locally called jambura
7 Citrus medica L. Wild growing variety with mamilate fruit apex
8 Citrus sinensis L. Osbeek A hybrid variety locally called mithamuri
Figure 2. Photograph showing different Citrus species found in Kokrajhar district: A, Citrus limon Burm f. var. Assam
lebu/China lebu (Assam lemon); B, Citrus limon Burm f. var. Elachi lebu (Cardamom lemon); C, Citrus limon Burm f. var.
Pati lebu/Jora nemu; D, Citrus aurantifolia (Christm.) Swin. var. Oval fruited; E, Citrus aurantifolia (Christm.) Swin. var.
Round-fruited; F, Citrus jambhiri Lush.; G, Citrus reticulata Blanco (Santara/ Sumthira/ Kamala); H, Citrus nobilis Lour
(Mitha tulia/ Jamir tenga/ Jeneru tenga); I, Citrus grandis L. Osbeek var. Jambura /Robab tenga; J, Citrus sinensis L.
Osbeek (Mithamuri); K, Hybrid variety (Mithamuri); L, Citrus medica Linn. var. Nare/Narang/Jora tenga
The survey on the distribution pattern of both wild and domesticated Citrus species in the different locality
of the district (Fig. 1) showed that their distribution is more or less similar both in rural and semi-urban areas. A
variety of Citrus species were found to be distributed in different places of Kokrajhar district in wild, semi-wild
and cultivated forms. In addition, a distinct morphological variation of the species with healthy growth of semi-
wild and wild forms is observed which indicates their adaptability to the local agro-climatic conditions. Among
them, citron (C. medica) an indigenous species of Assam (Bhattacharya & Dutta 1951) was found in tropical
forests of the district. Roxburgh (1832) encountered citron in Garo hills of Assam, and Hooker (1875) and
further revised by BSI, Dehradun in (1990) and reported it in Sikkim, Garo hills, Khasi hills and also in the
Western Ghats and Satpura range. Another species lemon (C. limon) has shown diverse forms of varieties as
well as healthy growth habits in different localities. The rough lemon (C. jambhiri) was also found widely
distributed in the region which was earlier reported as indigenous species to north eastern Himalayan Zone
(Bhattacharya & Dutta 1951). The king orange (C. nobilis) was found to grow in semi-domesticated form.
Similarly, both mandarin orange (C. reticulata) and sweet orange (C. sinensis) are two other popular Citrus
species that grow in the district and generally cultivated in orchards commercially. Moreover, sweet pummelo
(C. grandis) is also commonly cultivated in orchards and grow in the backyard.
Citrus is considered to be one of the most important fruit crops since prehistoric time because of its
consumer demand and wide uses in different kinds of processed product (Singh 1999, Ghosh 1999). It is the
most widely studied genus (Webber 1943, Swingle 1943, Aiyappa & Srivastava 1965, Hodgson 1967, Tanaka
1977, Bhattacharya & Dutta 1951 & 1956, Singh 2001) on different aspects of classification and cultivar
development. The morphological character of fruit is considered as a basic tool for identification as well as
classification and the important characters can be derived from the observation of both live and herbarium
specimens. During the study, a large array of variation of the fruits has been observed among the eight species
which has genetic as well as taxonomic potential. The species bear special kind of fruit i.e. hesperidium of
variable shape and size such as spherical, globose, ovate, ovate-oblong etc. Close similarities have been
observed in between C. reticulata Blanco and C. nobilis Tan. in regard to globose shaped fruit and thin rind
(Fig. 2). However, the size of the fruit of C. reticulata Blanco (size 4.55.9 cm. ht., 6.06.8 cm. diam.) is larger
than C. nobilis Tan. (size 3.06.4 cm. ht., 3.26.5 cm. diam.). The species, C. grandis (L.) Osbeck has large fruit
with spherical shape (size 13.019.5 cm. ht., 14.018.5 cm. diam.) and with a thick and spongy rind as well as
mesocarp while C. sinensis Osbeck. has a medium-large fruit (size 6.08.5 cm. ht., and 4.56.0 cm. diam.) of
spherical-oblong shape are distinct from other species. However, it has been observed that C. aurantifolia
(Christm.) Swin. is medium large (size 4.35.1 cm. ht., 4.25.1 cm. diam.), round shaped fruit, (7.39.1 cm. ht.,
4.25.1 cm. diam.) oval shaped fruit with light yellow colour and the mamillate apex. C. limon Burm f. can be
easily identified on the basis of the oval shaped fruit (size 1014 cm. ht., 58 cm. diam.) with necked base,
mamillate apex and lemon yellow colour. Similarly, C. medica L. has oblong shaped fruit, large to medium-
large (size 911 cm. ht., 6.07.3 cm. diam. to 5.26.0 cm. long, 3.54.4 cm. diam.), large mamillate apex and
yellow colour. In addition, oval shaped fruited C. sinensis (6.08.5 cm. ht., 4.56.0 cm. diam.) can be separated
from C. jambhiri (6.07.5cm. ht., 6.57.5 cm. diam.) easily.
While conducting an experiment on the propagation of selected Citrus varieties, it has been observed that the
rate of seed germination in all hormone treatment irrespective of concentration was found to be superior over
distilled water treatment. The treatment GA3-800 ppm (gibberellic acid) resulted in the highest rate of
germination (90%). The lowest germination percentage was recorded (40%) in control plot. On the other hand
cutting of C. limon Brum f. and C. medica L. has easily sprouted within 15 days after planting, but C.
aurantifolia Swin. grew slowly. After three months of observation, it was found that the success rate in case of
two species i.e. C. limon and C. aurantifolia was 80% and 30% respectively. Moreover, data recorded on air
layering practice (Fig. 3) from the field has revealed that it the mostly used technique to propagate C.
aurantifolia Swin and C. limon Brum f. with the highest percentage of survival rate up to 90% with maximum
production.
According to horticulturist vegetative propagation methods is the greatest single step which can be taken up
for the improvement of tropical fruit culture. The methods commonly used in the tropics are cutting, layering,
budding, inarching, marcottage and tissue culture. The new technique of mist propagation for improving the
rooting of cuttings has been very widely applied in NE regions although the use of special rooting composts is
gaining importance (Mukherjee & Majumder 1983). Another method, air-layers of varieties to be multiplied and
rooted shoots are separated and can be planted directly in the field or in the nursery.
Figure 3. Photograph showing nursery preparation and different propagation techniques: A, Nursery beds prepared for
planting of Citrus spp.; B, Two months old Citrus reticulata seedling; C, Planted multiple node cutting; D, Sapling prepared
through air layering; E, Sprouted sapling of C. limon, C. medica and C. aurantifolia in the nursery.
CONCLUSION
Citrus species and varieties grow naturally in different localities all around the Kokrajhar district. The
studied species possess distinct morphological characters which have determined their systematic position as
separate species and the large array of variation of citrus fruits shows its genetic potential. Rare varieties like C.
nobilis (Jeneru tenga) and C. limon (Elachi lebu) are required to be conserved immediately by adopting adequate
strategies. Regarding its propagation, it can be hypothesized that Citrus is a seed propagated plant and
vegetative propagation can be done by means of cutting, layering, and tissue culture. Pre-sowing seed treatment
with hormone can improve the rate of germination and growth of the seedling. Air layering is the most common
method used to propagate C. aurantifolia Swin and C. limon Brum f. by orchardists of Kokrajhar district. Citrus
production in Kokrajhar district is not encouraging even though suitable agro-climatic conditions prevail for
large scale cultivation. Therefore, it is required to cultivate with the right variety of seedling that can be
developed by using proper nursery technique.
ACKNOWLEDGEMENTS
Authors are thankful to UGC-NERO, Guwahati for financial support in the form of Minor Research Project
sanctioned in 2013 and DBT sponsored Biotech Hub, Science College, Kokrajhar for providing lab facility.
Thanks are due to the Principal, Science College, Kokrajhar for his constant support. The help received from
forest guard, laboratory assistant and orchardist of Kokrajhar district are also highly acknowledged.
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ISSN (E): 2349 1183
ISSN (P): 2349 9265
4(1): 1319, 2017
DOI: 10.22271/tpr.2017.v4.i1.003
Research article
In Vitro antioxidant, antibacterial and phytochemical screening of

Cochlospermum religiosum (L.) Alston - A potent medicinal plant
Pooja Ponnamma, G. Manasa, M. S. Sudarshana, M. Murali and C. Mahendra*
University of Mysore, Department of Studies in Botany, Manasagangotri,
Mysore-570006, Karnataka, India
*Corresponding Author: mahendra.c149@gmail.com [Accepted: 12 January 2017]
Abstract: The work is undertaken to evaluate the preliminary phytochemicals, antibacterial and
antioxidants activity of Cochlospermum religiosum leaf extracts with three solvents via
chloroform, ethyl acetate and methanol based on polarity index. The antibacterial activity was
assessed against five bacterial pathogens like Escherichia coli, Bacillus subtilis, Bacillus cereus,
Staphylococcus aureus and Pseudomonas aeruginosa by well diffusion assay. Among the tested
pathogens, the maximum zone of inhibition was observed against E. coli (26 mm) followed by P.
aeruginosa (23 mm) in ethyl acetate extracts compare to other solvent extracts. Phytochemical
analysis also revealed the presence of various pharmaceutically active secondary metabolites like
alkaloids, phenolic, flavonoids, saponins, carbohydrates, proteins, glycosides, sterols, etc.
Antioxidant activity was determined by DPPH scavenging, total phenolic and phospho-
molybdenum method. In DPPH assay, ethyl acetate extract was found to be the most effective.
Similarly, total phenols and phospho-molybdenum assay the methanol extracts was found to
contained good sources of antioxidants. The outcomes of the present study specified the plant
possess various potentially active secondary metabolites which help for the developing
pharmaceuticals, especially antioxidant and antimicrobial drugs.
Keywords: Cochlospermum religiosum, Phytochemical, Antibacterial, Antioxidants, DPPH.
[Cite as: Ponnamma P, Manasa G, Sudarshana MS, Murali M & Mahendra C (2017) In Vitro antioxidant,
antibacterial and phytochemical screening of Cochlospermum religiosum (L.) Alston - A potent medicinal plant.
Tropical Plant Research 4(1): 1319]
INTRODUCTION
Nature has been a source of medicinal agents for thousands of years and by using the natural resources
impressive number of the drugs have been isolated. Most of these isolations commonly based on their uses in
traditional medicine (Cragg & Newman 2001). More than 65% of the global population uses medicinal plants as
a primary health care modality. The medicinal plants are useful for healing and as well for curing human
diseases because of the presence of phytochemical constituents (Fabricant & Farnsworth 2001). Phytochemicals
are naturally occurring compounds in medicinal plants which have defense mechanism and protect various
diseases (Motaleb 2011). In recent years natural antibiotics have been used for several infectious diseases,
regarding to this, the work on new antimicrobial agents from plants are even more essential especially in the
countries like India where infectious diseases of bacteria are not only rapid but are also more resistant to
common antibiotics (Shah et al. 2014). Most of the medicinal plants holding active secondary metabolites with
high antioxidant property which are playing important role in the prevention of various diseases (Lobo et al.
2010). Natural antioxidants from plant sources are potent and safe due to their harmless nature.
Cochlospermum religiosum (L.) Alston is a small sparsely branched tree belonging to family Bixaceae. It is
commonly called yellow silk cotton tree, butter cup tree, and torch wood tree because of flowers are large,
bright golden yellow and seeds are covered with silky hairs (Sasikala et al. 2013). The name of the tree
Religiosum derived from the fact that the flowers are used for temple offerings. The plant can easily identify
by its characteristic feature of deeply furrowed bark, lobed leaves and bright golden yellow bisexual flowers.
Received: 22 September 2016 Published online: 31 January 2017
Ponnamma et al. (2017) 4(1): 1319
The tree especially known for its gum- yielding character, the gum is orange in color which exudes from the
bark. The gum is medicinally used for the treatment of stomachic, sedative, gonorrhea, syphilis and asthma
(Pandhure et al. 2013). Hence the attempt has been made in this study to examine various phytochemical
constituents, and their antibacterial and antioxidant efficacy of various solvent extracts of C. religiosum.
MATERIALS AND METHODOLOGY

Plant material collection
The fresh leaves of Cochlospermum religiosum (L.) Alston was collected from Chamundi Hills, Mysuru and
identified with the help of Flora of Presidency of Madras (Gamble 1935) healthy leaves were separated then
washed with running tap water, and then cut into small pieces and shade dried at room temperature until it is
complete gets dried.
Preparation of plant extract
Dried leaf powder was extracted in three different solvents like chloroform, ethyl acetate and methanol 30 g
of plant powder extracted in 300 ml of solvents for 24 hrs in a rotary shaker at room temperature. The
supernatant was collected and evaporated to get extracts and same procedure was followed to remaining
solvents and obtained crude extracts was stored at 4oC in air tight glass bottles for further studies (Maizura et al.
2011)
Phytochemical Analysis
The dried powdered plant material was extracted sequentially with solvents, viz., Chloroform, Ethyl acetate
and methanol based on polarity index. The obtained solvent extracts were subjected to qualitative phytochemical
screening to detect the presence of various phytoconstituents like Carbohydrates, Alkaloids, Glycosides, Sterols,
Flavonoids, Saponins, Triterpenes, Resins and Proteins followed by the standard procedure given by Harborne
(1972).
Antibacterial activity
i. Test micro-organisms:
Bacterial cultures were procured from Microbial Type Culture Collection and Gene bank (IMTECH,
Chandigarh, India). The antibacterial activity was carried out against both Gram positive and Gram negative
bacteria viz. Gram-positive (Staphylococcus aureus MTCC - 7443 and Bacillus subtilis (MTCC -121) Gram-
negative (Escherichia MTCC -7410 and Pseudomonas aeruginosa MTCC -1688).
ii. Agar-well Diffusion Method:
The antibacterial activity was determined by agar well diffusion method. Nutrient agar plates were swabbed
with 24 hrs. old culture of selected bacteria. 10 mm wells were made in each NA plates using sterile cork borer.
Standard and Stock solution of leaf extract was prepared at the concentration of 100 mg.ml -1 of each extracts.
About different concentrations of plant extracts 25, 50 and 75 l were added to the wells by using micro pipettes
and allow to diffusing at room temperature for 2 hours. The plates were incubated at 37 C for 24 hrs. The
diameter of inhibition zone was measured in millimeter (Antarasen et al. 2012).
Antioxidant assay
i. DPPH Free radical scavenging activity assay:
DPPH free radical scavenging activity was performed as described by Abdulwahab et al. (2011) and
Mahendra et al. (2016). Briefly 1ml of leaf extracts and the standard solution of Gallic acid from 8 different
concentrations of the absolute methanol were added to 4 ml of DPPH reagent in 8 test tubes. DPPH and absolute
methanol were used for reagent blank. All reagents were mixed and incubated for 30 minutes at room
temperature, protected from light. The absorbance was measured at 517 nm by using a spectrophotometer.
Experiments were done in triplicates. The percentages of the DPPH free radical scavenging activity were
calculated as follows:
( ) ( )
( )
ii. Estimation of total phenolic content:
Total phenolics in the leaf extract of plant were determined using the Folin - Ciocalteu reagent method
(Chlopicka et al. 2012). A stock solution of leaf extracts was prepared to the concentration of 1 mg.ml-1. To 1
mL of each extract, 5 mg.ml-1 of FC Reagent were added. The mixture of solution was vortexed and incubated
Ponnamma et al. (2017) 4(1): 1319
in the dark chamber for 3 min, respectively. After that 5 mg.ml-1 of sodium carbonate (75 g.l-1) solution was
added to the mixture and mixed thoroughly. The mixture was again incubated in the dark for 1 hr. The
absorbance was read at 765 nm. Blank consisted of 5 mg.ml-1 FC reagent, 1 mL ethanol, and 4 mL sodium
carbonate solution.
iii. Phospho - molybdate assay:
The total antioxidant capacity of the extracts was determined by phospho - molybdate method by using
Gallic acid as standard. An aliquot of 0.1 ml of the sample solution was mixed with 1 ml of reagent solution.
The tubes were incubated in a water bath at 95C for 90 minutes. After the samples had cooled to room
temperature, the absorbance of a mixture of the sample was measured at 765 nm against a blank. A typical blank
contains 1 ml of reagent solution and the appropriate volume of the solvent was incubated under the same
conditions.
RESULTS
Phytochemical Screening
The preliminary phytochemical screening of C. religiosum leaf crude extracts of various solvents showed the
presence of secondary metabolites like alkaloids, steroids, flavonoids, terpenoids, glycosides, carbohydrates,
tannins, saponins and phenols and the results were tabulated in the (Table 1). Among the tested extract, ethyl
acetate was showed maximum phytochemicals followed by methanol extracts and sterols, triterpenes and
flavonoids were absent in both ethyl acetate and chloroform extracts and present in methanol extracts.
Table 1. Preliminary Phytochemical screening of Cochlospermum religiosum.
Phytochemical Tests Chloroform Methanol Ethyl acetate
Sterols 1. Salkowski Test - + -
2. Liebermann-Burchard Test - + -
Triterpenes Salkowski Test - + -
Liebermann-Burchard Test - + -
Saponins Foam test - + -
Alkaloids Mayers Test - - +
Dragendroffs Test + - +
Wagner sTest + - +
Hagers Test + - +
Tannins FeCl3 Test + - -
GelatinTest + - -
Flavonoids Shinado Test - + -
FeCl3 Test - + -
Lead acetate test: - + -
Carbohydrates Molischs - + -
Fehlings Test + + -
Benedicts Test - + -
Phenols FC reagent test + + +
Proteins Biuret Test - - -
Ninhydrin Test - - -
Glycosides Keller killiani test + - -
Note: += Present -= Absent
Antibacterial activity
The results of antibacterial activity studies was performed by agar well diffusion assay by using crude
chloroform, ethyl acetate, and methanol solvent extracts of leaf parts of C. religiosum analyzed against 5
different gram positive and gram negative human pathogenic bacteria with compared standard antibiotic
streptomycin. It was found that the maximum zone of inhibition was observed in ethyl acetate extract which was
most effective against E. coli (26 0.27) and P. aeruginosa (23 0.35) followed by B. cereus (18 0.43),
Bacillus subtilis (17 0.24) and Staphylococcus aureus (14 0.10) compare to chloroform and methanol
extracts. It was also noticed that the bactericidal properties of the treated crude extracts was concentration
dependent which gives higher zone of inhibition with increasing the concentration and the results are presented
in (Table 2, Fig. 1).
Ponnamma et al. (2017) 4(1): 1319
Figure 1. Ethyl acetate leaf extract of Cochlospermum religiosum against different strains of bacteria: A, Bacillus cereus; B,
Bacillus subtilis; C, Pseudomonas aeruginosa; D, Staphylococcus aureus; E, Escherichia coli (Scale: a, 25 l; b, 50 l; c,
75l; n, Negative, s, Streptomycin).
Table 2. Antibacterial activity of leaf extracts of C. religiosum.

Plant extract
Pathogens Zone of Inhibition measured in mm Negative Standard
used CHL EA METH control 10 mcg
25 mcg 50 mcg 75 mcg 25 mcg 50 mcg 75 mcg 25 mcg 50 mcg 75 mcg
B. subtilis 000.00 000.00 000.00 120.14 130.10 170.24 000.00 000.00 070.24 000.00 240.25
S. aureus 000.00 000.00 070.83 100.38 110.08 140.10 000.00 000.00 100.38 000.00 220.29
P. aeruginosa 000.00 000.00 000.00 080.32 130.28 230.35 000.00 000.00 000.00 000.00 260.34
E. coli 000.00 000.00 060.71 120.19 140.14 260.27 000.00 060.87 090.47 000.00 300.42
B. cereus 000.00 000.00 080.32 110.23 130.20 180.43 000.00 000.00 070.54 000.00 240.21
Note: Values are means of three independent replicates standard error. CHL: Chloroform extracts, EA: Ethyl acetate extracts, METH:
Methanol extracts, Negative control: Respective solvents and Standard: Streptomycin (for comparative study, the maximum activity
given extracts standard values are mentioned in the table).
DPPH Free radical scavenging activity
Figure 2. Antioxidant activity of Cochlospermum religiosum leaf extracts by DPPH Scavenging method.
Ponnamma et al. (2017) 4(1): 1319
Medicinal plants known to possess natural antioxidants which preventing the oxidative stress. The free
radical scavenging activity of different crude solvent extracts of C. religiousum was studied and the results
revealed that both chloroform and ethyl acetate extract showed excellent antioxidant activity when compared to
methanol extracts and there was complete discoloration of purple colored solution into yellow color solution in
chloroform and ethyl acetate, extract of leaf. The degree of discoloration by plant sample was recorded and IC50
was calculated and the results are graphically represented (Fig. 2).
Total phenolic content
The antioxidant activity of the most of the green and herbaceous plants crude extracts is often allied with
phenolic substances hence, they constitute together will make major group of compounds is known as primary
antioxidants which are easily react with active oxygen free radicals. Total phenolic content was measured
according to Folin - Ciocalteu method with all the three extracts. The results showed that all the extracts of leaf
samples used for the present investigation contain considerable amount of polyphenols. The methanol extract
was found to contain the highest amount of phenols (35.4%) when calculated with the standard Gallic acid curve
(Fig. 3).
Figure 3. Antioxidant activity of Cochlospermum religiosum leaf extracts by Total phenolic method (Gallic acid curve).
Total antioxidant capacity by Phospho-molybdenum method
Total antioxidant capacity of leaf extract of C. religiosum was evaluated by phospho - molybdenum
method and was expressed as Gallic acid as a standard per gram of plant extract. Total antioxidant capacity
leaf extract of C. religiosum was calculated using the standard curve of Gallic acid (y = 0.0428x+ 0.2391).
Among all the three extract of plant, the methanol was found to possess the highest total antioxidant capacity
of 4.5% and antioxidant capacity of Gallic acid has been used as a reference standard from which plant
extracts which possess potential antioxidant activity are comparable with standard graph (Fig. 4) plotted
against tested samples.
Figure 4. Antioxidant activity of Cochlospermum religiosum leaf extracts by Phospho-molybdenum method (Gallic acid
curve).
Ponnamma et al. (2017) 4(1): 1319
DISCUSSION
It was understood from the present study that the extracts of C. religiosum contain many phytochemicals as
revealed by biochemical tests. Most of the plants which possess vast number of secondary metabolites some
times which act as defense mechanism for external invaders such kind of phytochemical constituents produced
by them self they played major role in defense system. In this study, the phytochemical analysis of the
methanol extract of the C. religiosum showed the presence of more number of secondary metabolites like
terpenoids, saponins, phenolics, alkaloids, glycosides comparable with that of other solvent extracts. Likewise,
Cooper et al. (2006) was also reported presence of more group of phytochemical diversity which gives synergic
effects in many biological applications.
Further, each extracts were subjected to explore for their antibacterial activity against some gram positive
and gram negative bacterial strains with various range of concentrations. The outcome of the antibacterial assay
revealed that there is an increasing the concentration of the extracts will leads to inhibit the growth of bacteria
and the same was compared with standard antibiotic drug. In concurrence to this, Shakeri et al. (2012) in
Anabasis aphylla also reported increased zone of inhibition with increasing concentration of extracts. The
antioxidant activity of the extracts and is measured by the ability to scavenge DPPH free radicals, is observed in
figure 2. The percentage inhibition of free radical by C. relgiosum extracts significantly reduced color of the
DPPH reagent from purple to yellow. The highest antioxidant activity of was observed by the Ethyl acetate
followed by Chloroform extracts. In corroborated with findings of Marzouk et al. (2006) stated that Tecoma
stans has a good antioxidant activity in various extracts of aerial parts showed significant antioxidant activity as
measured by DPPH as a scavenging reagent. Likewise, Torane et al. (2011) also reported, the antioxidant
activity of Tecoma stans ethyl acetate extracts is higher than methanol and acetone extract. Based on antioxidant
activities, the total phenolic content and phospho- molybdenum method was also conducted and the results are
moderate for C. relgiosum. Most of the previous researches have also been reported, majority of the plant
compounds responsible for antioxidant activity are due to the presence of polyphenols, similar way, the verdicts
of our result shows the phenolic content is slightly higher than some other medicinal plants. The antioxidant
activity of phenolic compounds is due to their redox property which plays an important role in absorbing and
neutralizing free radicals, quenching singlet and triplet oxygen and a metal chelation potential. The results of the
present study are agreement with that of Abdelwahab et al. (2009).
CONCLUSION
The present study summarizes that C. religiosum is a good source of various metabolites like steroids,
flavonoids, triterpenoids, glycosides, tannins, saponins, resins, alkaloids and carbohydrates. Ethyl acetate leaf
extract of the plant showed effective antibacterial activity against all the tested bacterial pathogens. The
methanol extract of leaf showed excellent radical scavenging activity which was significantly comparable to
free radical scavenging activity of ascorbic acid. The finding of this study suggests that the leaves of this plant
could be a potential source of natural antioxidant. Further investigation on the isolation and characterization is,
however, to be required.
ACKNOWLEDGEMENTS
The authors are thankful to the University of Mysore, Mysore for providing laboratory facility through
Institution of Excellence (IOE) and DST-FIST in the botany department.
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ISSN (E): 2349 1183
ISSN (P): 2349 9265
4(1): 2030, 2017
DOI: 10.22271/tpr.2017.v4.i1.004
Research article
Taxonomical and phytosociological studies on Chithalikavu- A

sacred grove, Thrissur district, Kerala
Deepa M. R.1*, P. S. Udayan1 and Anilkumar K. A.2
1
P.G. and Research Department of Botany, Sree Krishna College, Guruvayur, Ariyannur,
Thrissur-680102, Kerala, India
2
Spices Board, Ministry of Commerce and Industry, Government of India, West Sikkim-737111, India
*Corresponding Author: deepakrishna56@gmail.com [Accepted: 15 January 2017]
Abstract: Groves contains natural forest and also rich in biodiversity. They are protected by local
communities because of their deities linked with these forest patches. The present study was
conducted in Chithalikavu, a sacred grove of Thrissur district to know the plant diversity and their
structural parameters. Floristic composition of Chithalikavu sacred grove revealed that the
occurrence of 57 species of angiosperms belonged to 54 genus and 35 families. Among them
29.82% trees, 24.56% shrubs, 15.79% herbs and 29.83% climbers. The parameters like frequency,
relative frequency, density, relative density and importance value index were estimated by using
standard procedures. Strychnos nux-vomica was recorded as the most dominant species in the
community as it constituted highest IVI. Other dominant species of the community were Ficus
benghalensis and Terminalia paniculata. The highest basal area was reported in Ficus
benghalensis.
Keywords: Chithalikavu - Sacred grove - Kerala - Phytosociology - Quadrets - Taboos.
[Cite as: Deepa MR, Udayan PS & Anilkumar KA (2017) Taxonomical and phytosociological studies on
Chithalikavu- A sacred grove, Thrissur district, Kerala. Tropical Plant Research 4(1): 2030]
INTRODUCTION
Sacred groves are small patches of native forests and act as abodes of gods, protected by local communities,
exist all over the world. These are known as Kavu or Sarpakavu in Kerala. According to Malhotra et al.
(2001) groves are those area dedicated by local communities to their ancestral spirits or deities. These have
immense value from genetic diversity as well as ecological point of view and rich in flora. They are the
repository of several medicinal and economically important plants. Attached with socio-cultural and religious
sentiments these exist as undisturbed islands. But today these are adversely affected by human activities.
These sacred groves are protected usually through taboos and sanctions with cultural and ecological
implications. There are more than 2000 sacred groves occur in Kerala (Pushpangadan et al. 1998). These acts as
treasure houses of plants and animals and can satisfy scientific, cultural and aesthetic needs of mankind. In
Kerala groves are mainly dedicated to Serpant gods and Folk deities. Many threatened species existed in the
sacred groves of Kerala (Nair & Mohanan 1981) and it preserving unique species of plants, insects and animals
(Venkatachalam et al. 2005). Therefore it acts as a tool for biodiversity conservation (Gaikwad et al. 2004).

Study Area
The study was conducted in Chithalikavu, a sacred grove of Talappilly taluk, Thrissur district, Kerala state,
lies in107321 N and 764273 E with an area of ca.1.73 ha. The management of these kavu is under the
control of Chithali family members. The main deities are Nagam (Snake God) and Bhagavathy (Godes).
Protection of this kavu is mainly due to the presence of deities. This area receives both Southwest and Northeast
monsoons during JuneAugust and OctoberDecember respectively. In Chithalikavu compound wall or fencing
were absent, therefore external disturbance like grazing, stem cutting, illegal medicinal plants collection are
present and one side of grove soil had less humus cover.
Received: 21 September 2016 Published online: 31 January 2017
Deepa et al. (2017) 4(1): 2030
Figure 1. Map showing the study area: Thrissur district of Kerala, India. [Adopted from Deepa et al. (2016)]
Plant collection
Floristic composition of this grove was recorded during field visits and plant collection conducted over
different seasons between April 2013 and September 2015. All the Angiosperms including trees, shrubs, herbs
and climbers were considered for the study. Important field observation such as habit, habitat, local names and
medicinal uses available were noted in the field book. Plant materials of proper size with relevant parts were
collected from the field and sealed in polyethylene covers after treating with formaldehyde. Herbaria were
prepared, processed and labeled by following standard herbarium methods given by Jain & Rao (1977). Each
species in fresh condition were critically studied with the help of floras (Gamble 19151936, Sasidharan &
Sivarajan 1996), Monographs, publications, etc and provisional determination was made. The identity of the
taxon was confirmed with type materials deposited at E, K, CAL, MH, KFRI and protologue. The voucher
specimens are deposited at Sree Krishna College, Guruvayur, Thrissur, Kerala.
Ecological data collection and analysis
Phytosociological study by quadrate method was also conducted for describing the Structural features of the
grove. Size and number of the quadrate was determined by the species area curve method (Shailaja & Sudha
2001). Two quadrates of 20 m 20 m size were randomly established in the study site for the studies of tree
species (Bajpai et al. 2015). Each quadrate was then systematically surveyed by identifying and all trees with
girth at breast height (gbh) greater than and equal to 30 cm were recorded for analysis. Tree girth measurements
were made as per Poffenberger et al. (1992).
The numerical value obtained were analyzed to find out frequency, relative frequency, density, relative
density, relative basal area and importance value index were estimated by using standard formula (Curtis &
McIntosh 1950, Krebs 1989, Phillips 1959).
RESULTS
The present taxonomic study in Chithalikavu resulted in the collection and identification of 57 species of
angiosperms belonged to 54 genus and 35 families. Among them 29.82 % trees, 24.56 % shrubs, 15.79 % herbs
and 29.82 % climbers. During the study an endemic plant to Peninsular India (Canthium rheedei DC.), and
under two near threatened species like Piper longum L. and Tinospora sinensis (Lour.) Merr. (Ravikumar &
Ved 2000) were collected. Almost all the plants found in the grove have medicinal properties.
During this study Euphorbiaceae was recorded as the dominant family with seven species of six genera,
followed by Fabaceae (five species of five genera) while Amaranthaceae, Meliaceae, Poaceae, Oxalidaceae,
Bombacaceae, Caricaceae, Combretaceae, Convolvulaceae, Cucurbitaceae, Dioscoreaceae, Hypoxidaceae,
Leeaceae, Liliaceae, Loganiaceae, Oleaceae, Periplocaceae, Piperaceae, Poaceae, Ranunculaceae are the
Deepa et al. (2017) 4(1): 2030
families represented by only one species. Phyllanthus, Sida and Tinospora were the largest genus with 2 species
while 51 genera were denoted by only one species in each. Because of their medicinal values Aerva lanata (L.)
Juss. ex Schult., Azadirachta indica A. Juss., Canthium rheedei DC., Cassia fistula L., Ficus benghalensis L.,
Gloriosa superba L., Glycosmis pentaphylla (Retz.) DC., Naravelia zeylanica (L.) DC., Piper longum L.,
Strychnos nux-vomica L., Terminalia paniculata Roth. and Tinospora cordifolia (WIlld.) Miers. are the some
important plants of the study area (Fig. 2). Botanical name of each plant with their family, local name, habit
phenology (flowering and fruiting) and medicinal uses have been provided in table 1.
Figure 2. Some important plants in Chithalikavu: A, Aerva lanata (L.) Juss. ex Schult.; B, Azadirachta indica A. Juss.; C,
Canthium rheedei DC.; D, Cassia fistula L.; E, Ficus benghalensis L.; F, Gloriosa superba L.; G, Glycosmis pentaphylla
(Retz.) DC.; H, Naravelia zeylanica (L.) DC.; I, Piper longum L.; J, Strychnos nux-vomica L.; K, Terminalia paniculata
Roth.; L, Tinospora cordifolia (WIlld.) Miers.
Only eight tree species were observed in the study area. Among them Strychnos nux-vomica L. was the
dominating species because of having the maximum value of Importance Value Index (IVI) Beside Strychnos
nux-vomica (IVI = 1.085), Ficus benghalensis L. (0.634), Terminalia paniculata Roth (0.338) and Schleichera
oleosa (Lour.) Oken (0.309) were the other important tree species from Chithalikavu. Maximum density was
showed by Strychnos nux-vomica as 325 ha-1 followed by Caryota urens L. (37.5 ha-1) and Terminalia
paniculata Roth (25 ha-1) (Table 2). The highest relative basal area was recorded in Ficus benghalensis L.
(0.515).
Deepa et al. (2017) 4(1): 2030
Table 1. Species recorded from sacred groves with their family, local name, habit, phenology (flowering and fruiting) and medicinal uses.
(H- Herb, S- Shrub, T- Tree, C- Climber)
S. Col.
Botanical name Family Local name Habit Phenology Medicinal uses
No. No.
1 198 Aerva lanata (L.) Juss. ex Amaranthaceae Cherula H September Used in urinary obstructions,
Schult. April bladder stones and haemorrhages
associated with pregnancy.
2 061 Albizia saman (Jacq.) F. Mimosaceae Mazhamaram T March Used in hot baths for stomach
Muell. May cancer and remedy for colds,
diarrhea, headache, intestinal
ailments, stomachache and sore
throat.
3 014 Amorphophallus Araceae Kattuchena H May Used in dysentery, piles, cough,
paeoniifolius (Dennst) June applied externally to treat
Nicolson rheumatism, opthalmia, cure
diarrhea, rheumatic swellings,
elephantiasis and vomiting.
4 114 Azadirachta indica A. Meliaceae Ariyavepu T February Used in skin diseases, ulcers,
Juss. September eczema, rheumatism, intestinal
worms, impurity of blood, eye
diseases, diabetes, small pox,
chiken pox, ringworm, scabies etc.
5 144 Bambusa bambos (L.) Poaceae Mula S July Haemorrhoid, diarrhoea, wounds,
Voss February skin diseases, fever, cough,
shortness of breath, vomiting,
cardiac diseases and skin diseases
6 146 Biophytum reinwardtii Oxalidaceae Mukkutti H July It is used in strangury, urninary
(Zucc.) Klotzsch December calculi, hyperdipsia, wounds,
asthma, stomachalgia, snakebite
and insomnia.
7 070 Bombax ceiba L. Bombacaceae Poola T January Used in calculous affections and
April ulceration of bladder and kidneys,
dysentery, pulmonary tuberculosis,
influenza, menorrhagia, fever,
burning sensation and skin
eruptions.
8 073 Canthium rheedei DC. Rubiaceae Edalimaram S March Usedfor whitish ulcers on the
June surface of a mucous membrane,
better for obstructions of the liver,
purifies, blood and cheers up the
patient.
9 058 Capsicum frutescens L. Solanaceae Kantharimul- H Throughout Used as carminative and
aku the year rubefacient.
10 274 Cardiospermum Sapindaceae Uzhinja C July Whole plant is used for hair growth,
helicacabum L. February rheumatism, glandular swellings,
constipation, nervous disorders,
piles, chronic bronchitis, fever,
hydrocele, sprains and cardiopathy.
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11 148 Carica papaya L. Caricaceae Pappaya S Throughout Fruits, seed, leaf and latex used in
the year hemiplegia, rheumatoid arthritis,
anorexia, indigestion, sprue, colic,
stomachalgia, dyspepsia, intestinal
worms, inflammations, piles,
cardiac diseases, oedema, fever,
ringworm, skin diseases and
leprosy.
12 006 Caryota urens L. Arecaceae Aanapana T January The leaf bud, seed and toddy are
April used for diarrhoea, migraine and
scorpion-sting poisoning.
13 149 Cassia fistula L. Caesalpiniaceae Konna T February Remedy for skin diseases, leprosy,
September fever, promotes digestion,
leucoderma, eczema, diabetes,
cardiac diseases, jaundice, polyuria,
and urticaria.
14 150 Cayratia pedata (Lam.) Vitaceae Veluthasori- C June Leaf decoction is used to check
A. Juss. ex Gagnep. valli July uterine reflexes. Roots made into a
paste and slightly heated are
applied on cracked heels.
15 115 Chromolaena odorata Asteraceae Communist- S November Leaf juice is applied externally on
(L.) King & Robins. pacha May cuts and wounds to stop bleeding.
16 016 Cissus latifolia Lam. Vitaceae Chunnambu- C June Used for the treatment of burning
valli September fever, cough, purifies blood, cure
the ulcer of lungs.
17 306 Cleistanthus collinus Euphorbiaceae Odugu T December Leaves, roots and fruits act as
(Roxb.) Benth. ex Hook. f. November gastrointestinal irritant. Fruits used
for treating cancer.
18 054 Curculigo orchioides Hypoxidaceae Nilappana H June Tuberous roots used in skin
Gaertn. December troubles, demulcent, diuretic, tonic.
Useful in leucorrhoea, urinary
diseases, piles, jaundice, asthma,
diarrhoea, gonorrhoea, itch and skin
diseases.
19 053 Delonix regia (Boj. ex Caesalpiniaceae Poomaram T February Leaves are used for diseases of
Hook) Rafin. July vata, constipation, inflammations,
arthritis, hemiplegia and
dysmenorrhoea.
20 055 Dioscorea bulbifera L. Dioscoreaceae Kattukachil C September Tubers used for ulcers, piles,
October leprosy, worm infestation, cardiac
diseases, polyuria, urinary calculi,
aphrodisiac, rejuvenator, dysentery
and syphilis.
21 052 Diploclisia glaucescens Menispermaceae Vattavalli C March Leaf powder with milk given in
(Blume) Diels August biliousness, gonorrhoea and
syphilis.
Deepa et al. (2017) 4(1): 2030
22 266 Ficus benghalensis L. Moraceae Peraal T May Bark is used in skin diseases, cures
August dysentery, diarrhoea, leucorrhoea,
nervous disorders and reduces
blood sugar in diabetes.
23 049 Gliricidia sepium (Jacq.) Fabaceae Seemakonna T March Used for headache, cold and cough.
Kunth ex Walp. May
24 050 Gloriosa superba L. Liliaceae Menthonni C July Tubers used for swelling, piles,
December oedema, leprosy, cronic ulcers,
colic pain in the bladder, itching,
antidote against cobra poison; easy
and quick expulsion of the placenta
after delivery.
25 229 Glycosmis pentaphylla Rutaceae Panal S September Leaf juice used in fever and liver
(Retz.) DC. April complaints and as a vermifuge.
Leaves considered good antidote
for inflammations, fever,
helminthiasis, cough, bronchitis,
rheumatism, jaundice, anaemia,
hepatopathy and skin diseases.
26 123 Grewia nervosa (Lour.) Tiliaceae Kotta S August The plant is used for indigestion,
Panigrahi April eczema and itch, typhoid fever,
dysentery and syphilitic ulceration
of the mouth.
27 203 Hemidesmus indicus (L.) Periplocaceae Nannari C August Roots used for cooling and blood
R.Br. December purifying action, dyspepsia,
dysentery, cough, bronchitis,
uterine haemorrhage, wounds,
leprosy, blood diseases, anaemia,
jaundice, fever, thirst, vomiting,
rheumatism and skin diseases.
28 253 Jasminum sambac (L.) Oleaceae Mulla C Throughout Roots, leaves and flowers used in
Ait. the year ophthalmopathy, pruritus,
cephalalgia, otopathy, skin diseases,
haemorrhage, wounds, ulcers, fever,
itching, headache, vomiting,
hiccough and galactorrhea.
29 048 Leea indica (Burm.f.) Leeaceae Chorianthali S March Roots used in diarrhoea, dysentery,
Merr. August hyperdipsia, ulcer and skin
diseases.
30 095 Macaranga peltata Euphorbiaceae Vatta T January Decoction of leaves and bark used
(Roxb.) Muell.-Arg. February as vulnerary. Gum used for
venereal sores.
31 211 Mallotus philippensis Euphorbiaceae Sindooram T October Used against tapeworms, abdominal
(Lam.) Muell.-Arg. March disorders, haemopathy, calculus,
flatulence, leprosy, skin diseases
and ringworm.
Deepa et al. (2017) 4(1): 2030
32 206 Merremia vitifolia Convolvulaceae Manjavayar- C November Whole plant used for strangury and
(Burm. f.) Hall. f. avalli February urethral discharges. Root eaten by
tribals as a stomachic.
33 189 Morinda pubescens J.E. Rubiaceae Manjapavitta T March It is used for eczema, fever, ulcers,
Smith June glandular swellings and digestive
disorders especially in children.
34 025 Mukia maderaspatana Cucurbitaceae Kasappuch- C Throughout Whole plant used for burning
(L.) Roem. edi the year sensation, flatulence, constipation,
ulcers, cough, neuralgia, odontalgia
and vertigo. Fruits used for dysuria,
piles, polyuria, tuberculosis and
alleviating pitta.
35 085 Murraya koenigii (L.) Rutaceae Kariveppu S March Juice of roots used for relief from
Spreng. July renal pains. Leaves improves voice,
stimulates digestion and destroys
concocted poisons in the system;
skin diseases, worm troubles and
neurosis.
36 190 Naravelia zeylanica Ranunculaceae Vathamkodi C October Whole plant is used in
(L.) DC. April helminthiasis, dermatopathy,
leprosy, rheumatalgia, odontalgia,
cephalalgia, inflammations, wounds
and ulcers.
37 282 Pedilanthus tithymaloides Euphorbiaceae Zigzag plant S April Wound healing property.
(L.) Poir. August
38 045 Phyllanthus amarus Euphorbiaceae Keezharnalli H July The plant is used for flu, dropsy,
Schum. & Thonn. October diabetes, jaundice, asthma,
bronchial infections, diseases of the
liver, stomach, genito-urinary
system, liver and kidney. The plant
is reported to show antiviral activity
against hepatitis B-virus and related
hepadna virus.
39 160 Phyllanthus reticulatus Euphorbiaceae Neeroli S August Bark used in rheumatism, dysentery
Poir. in Lam. December and venereal diseases. Leaves used
for burning sensation, gastropathy,
sores, burns, skin eruptions and
obesity. Fruits are used in dressing
syphilitic sores.
40 307 Physalis angulata L. Solanaceae Njottanjod- H July Roots boiled in water suppresses
ian December diabetes, heal sores of mouth.
Whole plant used for burning
sensation, ulcers, gastropathy,
cough and bronchitis.
41 088 Piper longum L. Piperaceae Thippali C August Roots and fruits are used for
January improve intellect memory power
and regain health by dispelling
diseases. It also cures cough,
asthma, indigestion, worm troubles,
anaemia and chronic fever.
Deepa et al. (2017) 4(1): 2030
42 009 Pothos scandens L. Araceae Paruvakodi C October Whole plant is used in skin
November diseases, boils, swellings, wounds,
ulcers, dropsy, menorrhagia,
vomiting, flatulence, strangury and
burning sensation.
43 042 Putranjiva roxburghii Euphorbiaceae Poothilanji T March Leaves and seeds are used for
Wall. August burning sensation, thrist,
stomatopathy, opthalmopathy,
constipation, elephantiasis and
habitual abortion.
44 181 Schleichera oleosa Sapindaceae Poovam T March Bark useful in curing ulcers,
(Lour.) Oken June malaria and inflammations. Seed
oil used in leprosy, dermatopathy,
boils, ulcers, blood disorders,
intermittent fever, snakebite and
burns.
45 030 Senna tora (L.) Roxb. Caesalpiniaceae Thakara H August Leaves and seeds used in ringworm,
December leprosy, skin diseases, constipation,
abdominal disorders, obesity,
flatulence helminthiasis and
constipation.
46 108 Sida acuta Burm. f. Malvaceae Kurumthotti S August Used for uropathy, arthritis,
October leucorrhoea, gonorrhoea, diarrhoea
and to promote strength.
47 031 Sida cordifolia L. Malvaceae Anakurumt- S Throughout Roots given in urinary troubles,
hotti the year stranguary and haematuria, used in
combination with asafoetida in
hemiplegia, sciatica and facial
paralysis (with rock salt). Powdered
roots are given with milk in
leucorrhoea and frequent
micturition. Leaves demulcent and
febrifuge; also used in dysentery.
Roots are used for rheumatism,
headache, neurological disorders,
tuberculosis and ophthalmia.
48 090 Streblus asper Lour. Moraceae Paruka T January Bark, roots and seeds are used for
October sinusitis, inflammations,
elephantiasis, haemorrhages, cough,
bronchitis, foul ulcers, diarrhoea,
dysentery, fever, swellings,
neuralgia and haemorrhages.
49 039 Strychnos nux-vomica L. Loganiaceae Kanjiram C March Seeds are useful in intermittent
December fevers, dyspepsia, chronic
dysentery, paralytic and neuralgic
affections, insomnia, chronic
rheumatism, colic, impotence, heart
disease, spermatorrhoea and skin
diseases.
Deepa et al. (2017) 4(1): 2030
50 308 Terminalia paniculata Combretaceae Maruthu T August Bark is a cardio tonic and diuretic.
Roth February
51 165 Tiliacora acuminata Menispermaceae Vallikanjiram C April Roots are used as an antidote to
(Poir.) Miers ex Hook. f. December snake poison.
& Thomas.
52 222 Tinospora cordifolia Menispermaceae Chitamruthu C January Stems used in fever, jaundice,
(Willd.) Miers. June thirst, burning sensation, diabetes,
piles, skin ailments, respiratory
disorders, neurological disorders
and rheumatism.
53 037 Tinospora sinensis Menispermaceae Katamruthu C February Stems used for treatment of piles
(Lour.) Merr. June and ulcerated wounds, liver
complaints, chronic rheumatism
and also as muscle relaxant.
54 134 Triumfetta rhomboidea Tiliaceae Oorpam S August Roots used in dysentery, intestinal
Jacq. January ulcers and their hot infusion hasten
parturition. Bark and leaves used in
diarrhoea. Leaves and flowers used
in leprosy.
55 138 Urena lobata L. Malvaceae Uthiram S August Decoction of stem and roots are
December used in flatulent colic. Flowers are
used for cough and sore throat.
56 035 Vernonia cinerea (L.) Asteraceae Puvankuru- H Throughout Useful combination with quinine
Less. nal the year against malaria. Roots used for
fever, dysuria, leucorrhoea,
excessive bleeding, chronic skin
diseases, bladder stones, piles,
worms and haematological
disorders. The plant juice is good
for eyes.
57 184 Zanthoxylum rhetsa Rutaceae Mullilam T March Bark and fruits used in dyspepsia,
(Roxb.) DC. November asthma, bronchitis, heart diseases,
toothache, diseases of eye and ear,
worm infestation, leprosy, diseases
of head and rheumatism. Seeds are
used in cholera. Thorns are used in
treating pimples.
DISCUSSION
As the study area (Chithalikavu) is near to an all-weather road, passing through its Southern side and there is
no compound wall around the sacred grove; various anthropological disturbances are common and these affect
the normal growth of natural flora. Floral diversity studies show that regional diversity is well represented in
grove system, larger groves often have the relic species of the region and there are frequent changes in floral
composition due to various external influences (Khumbongmayum et al. 2006, Rao et al. 1990). Regeneration of
tree species in this area was very poor because of zoo-anthropogenic activities, which also causes the soil
erosion and finally decreasing the soil fertility.
Invasive species Chromolaena odorata (L.) King & Robins. and Carica papaya L. are present inside this
grove. Overgrowth of these plants is considered as a threat to the other vegetation of the sacred groves.
Deepa et al. (2017) 4(1): 2030
Chromolaena odorata is most widely spreading and encroaching species of the similar areas (Cronk & Fuller
1995, Richardson & Rejmnek 2011, Mandal & Joshi 2014a).
Table 2. Phytosocioloical analysis of major tree species in Chithalikavu.
S.N. Name of species D (No./ha) Ab F RD RF RBA IVI
1 Caryota urens L. 37.5 1.5 1.0 0.083 0.182 0.004 0.269
2 Delonix regia (Boj. ex Hook) Rafin. 12.5 1.0 0.5 0.028 0.091 0.003 0.121
3 Ficus benghalensis L. 12.5 1.0 0.5 0.028 0.091 0.515 0.634
4 Putranjiva roxburghii Wall. 12.5 1.0 0.5 0.028 0.091 0.001 0.119
5 Schleichera oleosa (Lour.) Oken 12.5 1.0 0.5 0.028 0.091 0.190 0.309
6 Strychnosnux-vomica L. 325.0 13.0 1.0 0.722 0.182 0.181 1.085
7 Terminalia paniculata Roth 25 1.0 1.0 0.056 0.182 0.101 0.338
8 Zanthoxylum rhetsa (Roxb.) DC. 12.5 1.0 0.5 0.028 0.091 0.007 0.125
Note: D- Density, Ab- Abundance, F- Frequency, RD- Relative density, RF- Relative frequency, RBA- Relative basal
area, IVI- Importance value index.
CONCLUSION
Groves are important for conservation of biological diversity. Its values and functions can be maintained
through effective conservation and management. Almost all species of flora is medicinal inside the grove and
used in various systems of medicine. This study shows due to diverse threats the floristic diversity reaches near
to its minimum level. Therefore increase in demand to conservation to maintain and increase biodiversity. Local
people must become aware of direct benefits of groves and they can learn more about the functions. The above
facts focus the aspects of conservation of groves, facing great danger of existence due to loss of sanctity values.
ACKNOWLEDGEMENTS
Authors are grateful to Sri. D. Jayaprasad, Principal and Dr. G. Jayakrishnan, Department of Botany, Sree
Krishna College, Guruvayur for providing valuable suggestions for the work. Authors acknowledge the family
members of these sacred groves for granting permission to conduct the study and providing information about
the groves.
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monotypic genus Indopiptadenia: a near threatened tree from the TeraiBhabar Region of Central Himalaya.
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Cronk QCB & Fuller JI (1995) Plant invaders: the threat to natural ecosystems. Chapman and hall, London.
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selected sacred groves in Thrissur district, Kerala. Tropical Plant Research 3(1): 230242.
Dregne HE (1987) Soil erosion: cause and effect. Land use policy 4(4): 412418.
Gaikwad SS, Paralikar SN, Chavan V & Krishnan S (2004) Digitizing Indian sacred groves An information
model for web interfaced multimedia database. In: Vinya G, Sane H and Ranade SS (eds) Focus on Sacred
Groves and Ethnobotany. Prisam Publications, Mumbai, India, pp.123128.
Gamble JS & Fischer CEC (19151936) The Flora of the Presidency of Madras. Parts 111 (parts 1-7 by
Gamble and 8-11 by Fischer), Vols. 13. Adlard & Sons Ltd., London.
Jain SK & Rao RR (1977) A Handbook of Field and Herbarium Methods. Today and Tomorrow, New Delhi.
Khumbongmayum AD, Khan ML & Tripathi RS (2006) Biodiversity conservation in sacred groves of Manipur,
northeast India: population structure and regeneration status of woody species. Biodiversity and
Conservation 15: 24392456.
Malhotra KC, Gokhale Y & Chatterjee S (2001) Cultural and Ecological Dimensions of Sacred Groves in India.
Indian National Science Academy, New Delhi and Indira Gandhi Rashtriya Manav Sangrahalaya, Bhopal.
Mandal G & Joshi SP (2014a) Quantitative vegetation dynamics and invasion success of Lantana camara from
the tropical forests of doon valley. International Journal of conservation science 5(4): 511526.
Nair NC & Mohanan CN (1981) On the rediscovery of four threatened species from the sacred groves of Kerala.
Journal of Economic and Taxonomic Botany 2: 233235.
Parthasrathy N & Karthikeyan R (1997) Biodiversity and population density of woody species in a tropical
evergreen forest in Courtallum reserve forest, Western Ghats India. Tropical ecology 38: 297306.
Deepa et al. (2017) 4(1): 2030
Pascal JP & Pelissier R (1996) Structure and floristic composition of a tropical evergreen forest in southwest
India. Journal of Tropical Ecology 12: 191214.
Poffenberger M, Mc Gran B, Ravindranath NH & Gadgil M (1992) Field methods Manual volume 1. Diagnostic
tool for supporting joint forest Management System. Prepared for the joint forest Management Support
programme.
Pushpangadan P, Krishnan PN & Rajendraprasad M (1998) The life form spectrum of sacred groves - a
functional tool to analyze the vegetation. Tropical Ecology 39(2): 211.
Rao P, Barik SK, Pandey HN & Tripathi RS (1990) Community composition and tree population structure in a
sub-tropical broad-leaved forest along a disturbance gradient. Vegetation 88: 151162.
Ravikumar K, Ved DK Assisted by Vijaya Sankar R & Udayan PS (2000) 100 Red-Listed Medicinal Plants of
Conservation Concern in Southern India. Foundation for Revitalisation of Local Health Traditions (FRLHT),
50, MSH Lay out, 2nd stage, 3rd main, Anand Nagar, Banglore, India.
Richardson DM & Rejmnek M (2011) Trees and shrubs as invasive alien species - a global review. Diversity
and Distribution 17: 788809.
Sasidharan N & Sivarajan VV (1996) Flowering Plants of Thrissur Forest (Western Ghats, Kerala, India).
Scientific Publishers, Jodhpur.
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Publishing Co. Pvt. Ltd. New Delhi.
Venkatachalam, Kalaiselvi ST & Ratha Krishnan P (2005) Sacred Groves - Conservation is Imperative.
Institute of Forest Genetics and Tree Breeding, PB 1061, R.S.Puram, Coimbatore 641002, India, pp. 9396.
ISSN (E): 2349 1183
ISSN (P): 2349 9265
4(1): 3136, 2017
DOI: 10.22271/tpr.2017.v4.i1.005
Research article
Optimization of extraction parameters for dye from

Pterocarpus santalinus L.f. (red sanders) wood
using response surface methodology
Surendra S. Bisht1*, Pradeep S. Hiremath2, P. L. Muralidhara2,
Mamata Ravindra1 and V. S. Shetteppanavar1
1
Institute of Wood Science and Technology, Bengaluru, Karnataka, India
2
Rashtreeya Vidyalaya College of Engineering, Bengaluru, Karnataka, India
*Corresponding Author: ssbchem@gmail.com [Accepted: 27 January 2017]
Abstract: The present study deals with the extraction of dye from Red sanders (Pterocarpus
santalinus) wood by chloroform and ethanol solvents using response surface methodology (RSM)
technique. Through RSM using three-level three-factor Box-Behnken Design, the optimal
conditions such as extraction temperature, time, and feed ratio were identified to be affecting
extraction efficiency. The best predicted yield of dye with chloroform solvent extraction was
17.9% at 35C, 105 minutes, feed ratio 200 mL.g-1 while experimental yield was 17%. Similarly
for ethanol solvent extraction 24.36% was the predicted yield at 65C, 105 minutes and feed ratio
of 200 mL.g-1 while experimental yield was found to be 23%.
Keywords: Pterocarpus santalinus - Response surface methodology - Dye - Extraction.
[Cite as: Bisht SS, Hiremath PS, Muralidhara PL, Ravindra M & Shetteppanavar VS (2017) Optimization of
extraction parameters for dye from Pterocarpus santalinus L.f. (red sanders) wood using response surface
methodology. Tropical Plant Research 4(1): 3136]
INTRODUCTION
Natural dyes such as indigo, chlorophyll, heme and red sanders are considered as sustainable and eco-
friendly (Strych & Trauner 2013). Red sanders (Pterocarpus santalinus) wood is renowned for its characteristic
timber of exquisite colour, beauty and yielding a red natural dye which belong to the molecular class of
condensed bioflavonoid (Arnone et al. 1975, 1977a,b). It also plays an important role in Ayurveda medicine for
treating digestive tract problems and coughs. Extraction of dye is influenced by various process parameters such
as solvent composition, pH, temperature, extraction time and solid to liquid ratio (Borges et al. 2011, Wijngaard
& Brunton 2010). Therefore it is very much important to optimize the extraction parameters, so that high yield
of dye can be achieved.
Response Surface Methodology (RSM) provides superb statistical tools for design and analysis of
experiments aimed at process optimization. Design of Experiments (DOE) deals many RSM designs with
options depend on the number of design variables or factors, which can range from one to ten offering Box-
Behnken designs (BBD) for three to seven factors require only three levels, coded as -1, 0, and +1 creating
designs with desirable statistical properties (Bafna 2012). In view of these points the present study deals with
the evaluation of RSM technique for the extraction of dye from Red sanders wood.

Red sanders wood was powdered by mechanical pulveriser. Chemically inert borosilicate glassware was
used during the experiments. Laboratory grade chemicals were purchased from HiMediaLaboratories Pvt. Ltd.,
while distilled water was used during all set of experiments. Instrument Buchi Rota vapour was used for
vaporisation of excess amount of solvents from samples. A Box-Behnken model of three factors was adopted in
this study for modelling using MiniTab17.
Bisht et al. (2017) 4(1): 3136
Optimization of extraction parameters using Response Surface Methodology (RSM)

10g of the wood powder was mixed with various volumes of chloroform and ethanol solvents to give a solid
to liquid ratio ranging from 1:50 to 1:200 (g.mL-1).The flask containing sample powder along with solvent was
incubated in thermostatic water bath at various temperatures (3565 oC) and various time intervals (30200
min). Observe the change in colour within solvent and filtering it into filtrate. Extract containing coloured
filtrate is subjected to evaporation in rota vapour at 50C and dye extract remained in the round bottom flask
was weighed. Design summary of extraction is given in table 1 and 2.
Table 1. Design summary of RSM for chloroform Table 2. Design summary of RSM for ethanol extraction
extraction method. method.
Factors Units Low High Factors Units Low High
Temperature C 35 55 Temperature C 35 65
Time min 30 180 Time min 30 180
Feed Ratio mL.g-1 50 200 Feed Ratio mL.g-1 50 200
Statistical screening and optimization by design of experiments
A Box-Behnken model of three factors was adopted in this study for modelling using MiniTab17. This
method is preferred as design model, since relatively few combinations of the variables are adequate to estimate
potentially complex response function. In total 15 experiments are needed in each sample of individual solvent
to calculate its 10 coefficients of the second order polynomial equation which was, fitted on the experimental
data (Table 3 & 4).
Percentage recovery of dye was taken as response of the system while the three process parameters i.e.,
temperature, extraction time and solid to liquid ratio were taken as input independent variables with respect to
solvent. The system was stated by the following equation:
Y=X0+X1A+X2B+X3C+X11A2+X22B2+X33C2+X12AB+X13AC+X23BC
Where, X0is the intercept; X1, X2, and X3 are linear coefficients; X11, X22, and X33 are squared coefficients;
X12, X13, and X23 are interaction coefficients and the experimental variables are temperature (A), Extraction time
(B) and Feed ratio (C), Dof- Degrees of freedom. The model adequacies were checked in terms of the values of
2and analysis of variance (ANOVA) was employed to determine the significance of the models.
RESULTS AND DISCUSSIONS

Box-Behnken analysis
BBD was used for three process variables i.e., extraction temperature, time, and feed ratio at three levels.
The design points fall within a safe operational limit, within the nominal high and low levels. Design
arrangements and responses of experimental and predicted values of chloroform and ethanol solvents were
shown in table 3 and 4 respectively.
Table 3. BBD responses for extraction by chloroform.
Experiment Time Temperature Feed ratio Experimental Predicted
S.N.
number (Minutes) (C) (mL.g-1) Yield (%) Yield (%)
1 1 105 45 125 12 12.34
2 2 30 45 200 13 14.35
3 3 105 45 125 11 12.34
4 4 180 45 50 16 14.65
5 5 30 45 50 05 4.13
6 6 105 35 50 06 7.27
7 7 180 35 125 15 15.14
8 8 105 45 125 14 12.34
9 9 105 35 200 17 17.99
10 10 105 55 200 16 14.73
11 11 30 35 125 08 9.04
12 12 105 55 50 11 12.02
13 13 30 55 125 09 8.86
14 14 180 55 125 15 15.39
15 15 180 45 200 17 17.87
Bisht et al. (2017) 4(1): 3136
Table 4. BBD responses for extraction by ethanol.

Experiment Time Temperature Feed ratio Experimental Predicted
S.N.
number (Minutes) (C) (mL.g-1) Yield (%) Yield (%)
1 1 105 50 125 22 20.09
2 2 30 65 125 18 16.83
3 3 180 35 125 13 14.33
4 4 30 35 125 09 9.83
5 5 105 50 125 21 20.09
6 6 105 65 50 19 19.81
7 7 180 50 50 14 14.07
8 8 30 50 50 15 15.56
9 9 30 50 200 10 10.11
10 10 180 65 125 21 20.35
11 11 180 50 200 20 19.62
12 12 105 35 200 14 13.36
13 13 105 35 50 19 17.80
14 14 105 50 125 22 20.09
15 15 105 65 200 23 24.36
Statistical analysis
Multiple regression analysis of the data yielded, the following equation for the recovery of natural dye using
chloroform and ethanol recovery of batch solvent extraction method in terms of coded factors:
For chloroform
Y1 = -224 + 1.161*A + 5.56*B+1.606*C - 0.00074*A2-0.0167*B2 + 0.00148*C2- 0.0033 A*B-
0.00311A*C- 0.0267B*C
For ethanol
Y2 = 89 + 1.40*A + 1.6*B - 1.12*C - 0.00778*A2- 0.017*B2 - 0.00156*C2 - 0.0022 A*B
+ 0.00489 A*C+ 0.0200 B*C
Where, Y1 and Y2 are chloroform and ethanol responses variables respectively. The student t-distribution
and the corresponding p-values along with the f-values of chloroform and ethanol responses are listed in table 5
and 6 respectively.
Table 5. ANOVA for response surface quadratic model of chloroform recovery.
Squares of Squares of
Source DoF F-value P-value
sum mean
Model 9 22231.7 2470.19 8.01 0.017
X1 1 9800 9800 31.78 0.002
X2 1 112.5 112.5 0.36 0.572
X3 1 9112.5 9112.5 29.55 0.003
X 12 1 64.1 64.1 0.21 0.668
X 22 1 10.3 10.26 0.03 0.862
X 32 1 256.4 256.41 0.83 0.404
X1*X2 1 25 25 0.08 0.787
X1*X3 1 1225 1225 3.97 0.103
X2*X3 1 1600 1600 5.19 0.072
Error 5 1541.7 308.33
Lack-of-fit 3 1075 358.33 1.54 0.418
Pure Error 2 466.7 233.33
Total 14 23773.3
In ANOVA for response surface quadratic model of chloroform recovery of batch solvent extraction method,
the Model F-value of 8.01 implies the model is significant and there is only a 1.7% chance that a "Model F-
Bisht et al. (2017) 4(1): 3136
Value" this large could occur due to noise. Values of "Prob < F" >0.05 indicate model terms are significant. In
this case feed ratio is significant model terms. Values greater than 0.1 indicate the model terms are not
significant. The "Lack of Fit F-value" of 1.54 implies it is not significant relative to the pure error. The values
for the coefficient of determination 2=0.9352 and Adjusted 2 =0.8184 represents the proportion of variation in
the yield or response in the model.
Table 6. ANOVA for response surface quadratic model of ethanol recovery.
Squares of Squares of
Source DoF F-value P-value
sum mean
Model 9 27566.7 3063 15.84 0.004
X1 1 3200 3200 16.55 0.01
X2 1 8450 8450 43.71 0.001
X3 1 7950 7950 22.64 0.017
X 12 1 10016 10016 51.81 0.001
X 22 1 539.1 539.1 2.79 0.156
X 32 1 1077.6 1077.6 5.57 0.065
X1*X2 1 25 25 0.13 0.734
X1*X3 1 3025 3025 15.65 0.011
X2*X3 1 2025 2025 10.47 0.023
Error 5 966.7 193.3
Lack-of-fit 3 900 300 9 0.102
Pure Error 2 66.7 33.3
Total 14 28533.3
In ANOVA for response surface quadratic model of ethanol recovery of batch solvent extraction method, the
model F-value of 1.18 implies the model is significant. There is only a 4.5% chance that a "model F-value" this
large could occur due to noise. Values of "Prob<F" >0.05 indicate model terms are significant. In this case
extraction temperature, time, feed ratio, squared temperature coefficient and temperature-solid to liquid
interaction coefficients are significant model terms. The "Lack of Fit F-value" of 9.0 implies it is not significant
relative to the pure error. The values for the coefficient of determination 2=0.9661 and adjusted 2=0.9051
represents the proportion of variation in the yield or response in the model.
Plot response surface design
To visualize the relationship between response and experimental levels of the independent variables for the
natural dye extraction, three dimensional surface and contour plots were constructed according to the quadratic
polynomial model equation. The variation of chloroform and ethanol recovery with extraction time and
temperature are graphically presented below in figures 1 to 4. As the extraction time of both solvents increases
and temperature were decreased, the natural dye recovery increased significantly with curvature contour lines.
Figure 1. Chloroform recovery surface and contour plots of experimental vs. time and temperature.
Bisht et al. (2017) 4(1): 3136
Figure 2. Chloroform recovery surface and contour plots of experimental vs. feed ratio and temperature.
Figure 3. Ethanol recovery surface and contour plots of experimental vs. time and temperature.
Figure 4. Ethanol recovery surface and contour plots of experimental vs. feed ratio and temperature.
CONCLUSION
Response surface methodology was used to optimize the batch solvent extraction of natural dye from
Pterocarpus santalinus wood. The optimum process parameters and the multiple regression analysis for
Bisht et al. (2017) 4(1): 3136
predicting responses were obtained by applying Box-Behnken design. Under optimum condition ethanol
extraction method showed the highest natural dye yield of 23% at 65C, 105 minutes and feed ratio of 200
mL.g-1 compared to chloroform extraction method, which had a maximum yield of 17% at 35C, 105 minutes
and feed ratio of 200 mL.g-1. In conclusion we found RSM using Box-Behnken design as a useful tool for
optimization of parameter for high yield dye from red sanders wood.
ACKNOWLEDGEMENTS
Director, Institute of Wood Science and Technology, Bangalore is acknowledged for providing the
infrastructure during this work. Pradeep is thankful to Dr. M. A. Lourdu Antony Raj, Professor and Head,
Department of Chemical Engineering, R. V. College of Engineering, Bengaluru for providing all possible help
and advice during the project.
REFERENCES
Strych S & Trauner D (2013) Biomimetic Synthesis of Santalin A,B and Santarubin A,B, the Major Colorants of
Red Sandalwood. Angewandte Chemie International Edition 52: 95099512.
Arnone A, Camarda L, Merlin L & Nasini G (1975) Structures of the red sandalwood pigments santalins A and
B. Journal of the Chemical Society, Perkin Transactions 1: 186194.
Arnone A, Camarda L, Merlin L & Nasini G (1977a) 13 C nuclear magnetic resonance spectral analysis of
santalin and santarubin permethyl ethers. Journal of the Chemical Society, Perkin Transactions 1: 2118
2122.
Arnone A, Camarda L, Merlini L, Nasini G, Taylor DAH (1977b) Colouring matters of the West African red
woods Pterocarpus osun and P. soyauxii. Structures of santarubins A and B. Journal of the Chemical
Society, Perkin Transactions 1: 21162118.
Borges GDSC, Vieira FGK, Copetti C, Gonzaga LV & Fett R (2011) Optimization of the extraction of flavanols
and anthocyanins from the fruit pulp of Euterpe edulis using the response surface methodology. Food
Research International 44(3): 708715.
Wijngaard HH & Brunton N (2010) The optimisation of solidliquid extraction of antioxidants from apple
pomace by response surface methodology. Journal of Food Engineering 96(1): 134140.
Bafna PG (2012) Optimization of Process Parameters for Extraction of Kokum (Garcinia Indica) Fruit Pulp
using Response Surface Methodology (RSM). International Journal of Scientific & Engineering Research
3(8): 12931299.
ISSN (E): 2349 1183
ISSN (P): 2349 9265
4(1): 3748, 2017
DOI: 10.22271/tpr.2017.v4.i1.006
Research article
Systematic wood anatomy of the tribes Euphorbieae and

Hippomanieae (Euphorbiodeae, Euphorbiaceae) from India
Prem Prakash Jangid1*, Soumana Datta2, Sandeep Yadav3 and Sangeeta Gupta1
1
Wood Anatomy Discipline, Botany Division, Forest Research Institute, Dehradun, India
2
Department of Botany, University of Rajasthan, Jaipur, Rajasthan, India
3
Advance Research Centre for Bamboo and Rattan, Aizawl, Mizoram, India
*Corresponding Author: ppjangid2010@gmail.com [Accepted: 10 February 2017]
Abstract: The present paper deals with detailed wood microstructure of 9 species belonging to six
genera covering tribe Hippomaneae and Euphorbieae of the subfamily Euphorbiodeae
(Euphorbiaceae). The study mainly focused on the systematic implication of wood anatomy to the
subfamily, also ecological and evolutionary aspects were evaluated. Wood microstructure of the
tribe Hippomaneae was found to be homogeneous considerably while Euphorbieae was
heterogeneous to certain extent reflecting upon unnatural classification of the tribe. All the genera
showed high vulnerability and mesomorphy reflecting adaptation to mesic environment. An
identification key has been developed based on constant wood anatomical properties.
Keywords: Euphorbieae - Euphorbiaceae - Hippomaneae - Systematic - Wood anatomy.
[Cite as: Jangid PP, Datta S, Yadav S & Gupta S (2017) Systematic wood anatomy of the tribes Euphorbieae
and Hippomanieae (Euphorbiodeae, Euphorbiaceae) from India. Tropical Plant Research 4(1): 3748]
INTRODUCTION
The subfamily Euphorbiodeae is one of the uniovulate subfamily of the spurge family Euphorbiaceae s.l.,
containing most of the species with white latex and is characterized by non-articulated laticifers and the flowers
without a disc or petals (Webster 1994, Radcliffe-Smith 2001). Webster (1994) classified the subfamily into five
tribes consisting of two large tribes, in addition to several more isolated genera that have been assigned to
smaller tribes. One of these two large tribes is the Euphorbieae, distributed in tropical, subtropical and temperate
regions around the world and characterized by its synapomorphic pseudanthial inflorescence (termed a
cyathium) composed of gland-bearing involucres of several united bracts and their associated flowers and
bracteoles. The tribe includes mainly the large genus Euphorbia L., as well as four other smaller genera in three
subtribes (Esser 2012). Webster (1994) included 11 genera in three subtribes (Anthosteminae (Baill.) G. L.
Webster, Neoguillauminiinae Croizat, and Euphorbiinae) in the tribe Euphorbieae. In India only one subtribe
(Euphorbiinae) occur with 3 genera ( pedilanthus, Euphorbia and synadenium) and 84 species (Balakrishnan &
Chakrabarty 2007). The second large tribe of Euphorbioideae is the Hippomaneae A.Juss. ex Bartl., pantropical
(with only few extratropical species) distributed in tropical America, Africa, asia and Australia. The
Hippomaneae do not include any single, large genera, but rather a larger number of smaller genera, several of
them monotypic. Floral bud of the tribe is unique and quite unlike any other euphorboid type (Esser 1997).
Esser (1994), Esser et al. (1998) and Esser (2001) proposed the generic circumscription of the tribe.
Balakrishnan & Chakrabarty (2007) even considered the tribe of such importance that they proposed a separate
subfamily (Hippomanioideae Chakrab. & N.P.Balakr.) for the tribe. According to Esser (2012) the tribe
comprise 33 genera and ca. 300 species of mostly woody plants (with few herbs and succulents. In Webster
(1994) circumscription the tribe hippomaneae comprises 19 genera in three subtribes (Carumbiinae Mll.Arg.,
Mabeinae Pax & K.Hoffm., and Hippomaninae) and 24 genera in two subtribes (Carumbiinae and
Hippomaninae) in circumscription of Radcliffe-smith (2001). This tribe is represented by only subtribe
(Hippomaninae) with 6 genera viz. microstachys, Balakata, Shirakoipsis, Triadica, Excoecaria and Falconeria.
Received: 14 May 2016 Published online: 28 February 2017
Jangid et al. (2017) 4(1): 3748
Gross wood microstructure of the subfamily Euphorbiodeae has been studied by Raturi et al. (2001);
Mennega (2005) studied wood structure of sub-tribe Flueggeinae and Securineginae; Bamber (1974) reported
thin to thick walled libriform fibres (Type I). Metcalfe & Chalk (1950) general microstructure of some genera.
Pearson & Brown (1932) studied wood microstructure of Excoecaria agallocha. Gamble (1922) studied gross
wood structure. The tribe Hippomaneae and Euphorbieae are the most controversial from systematic point of
view in the subfamily Euphorbiodeae. Thus the present study was performed to understand the systematic
implication of wood microstructure in Hippomaneae and Euphorbieae tribes.
MATERIAL AND METHODS

The present study is based on examination of 24 wood specimens of 9 species belonging to 6 genera of
tribes Hippomaneae and Euphorbieae of the subfamily Euphorbiodeae (Euphorbiaceae), available in Xylarium
of Forest Research Institute, Dehradun (Table 1). For microscopic examination 1520 m thick cross, radial and
tangential sections were cut on Reichert microtome. The sections were stained with Heidenhains haematoxylin
and safranin and permanent slides were prepared by following standard laboratory procedures. All samples were
macerated by following Schultzs method (30% nitric acid and a pinch of potassium chlorate). IAWA
terminology (IAWA 1989) has been followed for writing the anatomical descriptions. The two ecological
indices, vulnerability and mesomorphy, given by Carlquist (1988) of the genera under study was evaluated as
per following formulae:
Vulnerability = Mean vessel diameter / Mean vessel frequency
Mesomorphy = Vulnerability X Mean vessel element length
Table 1. Details of wood samples studied of the subfamily Euphorbiodeae.
1. Balakata baccata (Roxb.) Esser (Sapium baccatum Roxb.); DDw 1962, Chittagong, B.D.; DDw 6538,
Rangoon, Myanmar; DDw 8207, Buxa Div, W.B.; DDw 8238, Kameng , Arp.
2. Euphorbia neriifolia L.; DDw 7174, Dornal Forest.
3. Euphorbia royleana Boiss.; DDw 3075, Sabathu, Pune.
4. Euphorbia tirucalli L.; DDw 3509, Khirda, Orissa
5. Euphorbia tortilis Rottler ex Ainslie; DDw 5694, Coimbture, T.N.
6. Excoecaria agallocha L.; 2477, Andamans; DDw 6703, Bombay; DDw 7298, Sunderbans, W.B; DDw
7301, Sunderbans, W.B; DDw 7304, Sunderbans, W.B; DDw 7361, Khutna, W.B; DDw 7470, Sunderbans,
W.B.
7. Falconeria insignis Royle (Sapium insigne (Royle) Benth. & Hook.f.; DDw 615, Kullu, H.P.; DDw 5862,
Dehradun, U.A.; DDw 5863, Dehradun, U.A.; DDw 6471, Rangoon, Myanmar.
8. Shirakoipsis indica (Willd.) Esser (Sapium indicum Willd.); DDw 6507, Sunderbans, W.B.
9. Triadica sebifera (L.) Small (Sapium sebiferum (L.) Roxb.); DDw 3114, Dehradun, U.A.; DDw 7423,
Dehradun, U.A.; DDw 7424, Dehradun, U.A.; DDw 7487, Dehradun, U.A.
RESULTS
Wood Microstructure Descriptions
Balakata Esser
Containing two species from NE India to Vietnam and throughout SE Asia, up to parts of New Guinea, of
these only one found in India (Balakrishnan & Chakrabarty 2007) studied here.
Species studied: Balakata baccata (Roxb.) Esser (= Sapium baccatum Roxb.)
Physical features: Wood white to yellowish white, turning to greyish yellow on ageing, soft and light to very
light, straight grained and medium coarse textured, specific gravity ranging between 0.320.48 air dry
Microscopic features: Vessels frequency 7 (414) per mm2, mostly solitary vessels with some radial multiples of
23, rarely clusters present. Vessel outline rounded to oval, average tangential diameter 144 (83206) m,
mean vessel element length 610 (481749) m (Tables 2 & 3). Intervessel pits alternate, rounded, distinctly
bordered with 7.8 (5.513.7) m diameters. Perforation plate simple. Tyloses common. Vessel ray pits
simple, much reduced bordered, rounded to elliptical with 10.7 (8.213.7) m diameters. Fibres non-septate,
thin walled with distinctly bordered pits on radial walls only (Fig. 1G). Average fibre length 1089 (856
Table 2. Diagnostic qualitative wood anatomical properties of species studied of tribe Euphorbieae and Hippomanieae
Jangid et al. (2017) 4(1): 3748
39
Species Axial Rays Vessels
Prismatic/Rhomboidal
Parench Fibres
Crystals
yma Cellular Vessel Ray Pits
Composition
Distinctly bordered pits on both radial and

Chambered
Chambered
Silica bodies in axial parenchyma, rays

Non-
Mixed all type cells throughout body
Thick/ Thin To Thick/ Thin Walled

Simple rounded, reduced bordered
Two distinct type in same ray cell

Body cells procumbent with single
Homocellular (all cells upright/
row of upright cells at margins
vessel outline rounded/angular
Fibre trachied/ libriform fibre

Diffuse, short radial multiples
Laticifers ( Narrow/ broad )
IVP rounded/ scalariform

Perforation Plate Simple
Upright/square ray cells
Diffuse in aggregate
Septate/ non-septate
square/procumbent)
VRP similar to IVP

Axial parenchyma
Axial parenchyma
Procumbent cells
Tyloses common
tangential walls
Perforated cells
Seriation
Diffuse
Absent
E. nerifolia A - 1 U/S - - R+ + - - - - - + A + S + - - - LF - - TN
E. royleana F F 1 - + - R+ + - - - - - + R + S + - - - LF - - TN
E. tirucalli F F 1 - + + R+ + - - - - - + R + R - - + - LF - + TT
E. tortilis F F 1 - + - A,R+ + - - - - - + R + S + - - - - + - TN
E. agallocha F F 1 - - + - R- - A A A - - + R + R + - - - - + - TN
www.tropicalplantresearch.com
B. buccata F F 1 U/S - + R- + - - - - A + R + R - + - R FT - - TN
,
F. insignis F F 1-2 P - - - R- - - - - A - +
R R + R - - + - - + - TN
S .indica F F 1-2 - - + - R- + - - - - R + R + R - + - - FT - - TN
T. sebifera F F 1-2 P - - + R- - - - - A - + R + R - + - R - + - TN
Abbreviations :- A = Abundant, F = Frequent, O = Occasional, R = Rare, R+ = Broad laticifers in rays, R- = Narrow laticifers in rays, AR+ =
Broad laticifers in both axial parenchyma and rays, U/S = Upright and Square cells, P = Procumbent, S = Sclariform, R = Rounded, LF = Libriform,
FT = Fibre tracheids,
Jangid et al. (2017) 4(1): 3748
40
Table 3. Diagnostic quantitative wood anatomical properties of species studied of the tribe Euphorbieae and Hippomanieae
Mean Tangential Dia. of
Multiseriate Ray Height

Multiseriate Ray Width
Uniseriate Ray Height

Inter Vessel Pit Size
Fibre Length (m)
Fibre Double Wall

Mean V. Element
Vessel Frequency
V. Lumina (m)
Ray Frequency
Length (m)
(Rays.Mm-1)
Thickness
(V.Mm-2)
Species
(m)
(m)
(m)
(m)
Avg Avg Avg Avg Avg Avg Avg Avg Avg Avg
(MaxMin) (MaxMin) (MaxMin) (MaxMin) (MaxMin) (MaxMin) (MaxMin) (MaxMin) (MaxMin)
E. nerifolia 26 53 487 11 991 8 25.3 615 589 18
(16.544) (3969) (321749) (814) (8031284) (2227.5) (2141391) (375803) (1620)
E. royleana 38 81 519 14 907 6 42.3 1022 958 14
(16.555) (6996) (428642) (1018) (7491177) (3355) (2682140) (5351284) (1215)
E. tirucalli 4.3 66 551 15 886 8 17 628 530 23
(2.86.8) (5583) (375642) (1120) (7491070) (13.722) (1601498) (2681070) (2225)
39 62 412 7 873 9 22 460 375 16
E. tortilis (16.555) (4283) (214642) (48) (7491070) (19.227.5) (161803) (246695) (1518)
E. agallocha 3.0 68.7 667 12 994 6 22.9 432 476 12
(2.84.1) (39110) (375963) (525) (5351391) (1155) (107856) (161535) (818)
www.tropicalplantresearch.com
B. buccata 7.8 144 610 7 1089 6.8 16.3 637 - 18
(5.513.7) (83206) (481749) (414) (8561284) (1122) (1611284) (1322)
F. insignis 6.9 156 639 5 1040 6.6 19.9 615 14
(5.58.2) (82.5303) (375856) (39) (7491284) (16.530.2) (1611177) - (1018)
S .indica 5.2 151 594 6 908 8.1 39.3 332 223 14
(4.16.9) (124171) (482856) (410) (5351284) (27.555) (107589) (107535) (1216)
T. sebifera 3.68 111 559 7 1235 8.1 20.7 590 398 16
(2.85.5) (55179) (268910) (313) (6421819) (16.541.2) (1611605) (61749) (1020)
Jangid et al. (2017) 4(1): 3748
Figure 1. AC, T.S. showing diffuse to diffuse-in-aggregates axial parenchyma in Balakata buccata (A), Shirakiopsis indica
(B) and Excoecaria agallocha (C); DF, T.L.S. showing exclusively uniseriate rays and intervessel pits in B. baccata (D), S.
indica (E) and E. agallocha (F); G, Maceration showing thin walled fibres and intrusive cavity in B. buccata. H, R.L.S.
showing homocellular rays containing silica bodies in S. indica; I, laticifers in rays in E. agallocha.
1284) m, average fibre diameter 26.3 (19.233.0) m, double wall thickness 6.8 m. Parenchyma
frequent, diffuse to diffuse in aggregates and scanty paratracheal (Fig. 1A). Silica bodies present. Rays 17.7
(1322) per mm, exclusively uniseriate (Fig. 1E). Average uniseriate ray height 637 (1611284) m with 2
45 cells. Average ray width 16.3 (1122) m (Table 2 & 3). Rays homocellular, composed of mostly
upright/square cells only. Frequently silica bodies present.
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Euphorbia L.
Cosmopolitan, mainly in tropical, subtropical and warm temperate regions around the world, ca. 2000
species. 82 species belonging to 6 subgenera found in India (Balakrishnan & Chakrabarty 2007). In present
study four species described of subgenus Euphorbia, out of 17 species in India.
Species studied: Euphorbia neriifolia L., Euphorbia royleana Boiss., Euphorbia tortilis Rottler ex Ainslie,
Euphorbia tirucalli L.
Physical features: The wood is yellowish brown, soft and fine textured, specific gravity ranging between 0.21
0.52 air dry.
Microscopic features: Vessel frequency 12 (420) per mm2, radial multiples of 23 cells common with many
solitary vessels and rarely clusters. Vessel outline rounded, average tangential diameter 65.5 (3996) m,
mean vessel element length 492 (214749). Perforation plate simple. Inter vessel pits scalariform and similar
vessel ray pits present in E. nerifolia, E. tortilis and E. royleana (Fig. 2I). E. tirucalli containing alternate,
rounded to elliptical distinct bordered intervessel pits with 4.3 (2.86.8) m diameters and simple, reduced
bordered, rounded vessel ray pits. Fibres non-septate, thin walled, libriform fibres present in E. nerifolia, E.
tirucalli and E. royleana (Fig. 2H). E. tortilis contains fibres with distinctly bordered pits on both radial and
tangential walls. 914 (749.01284) m long, diameter 32 (2538) m. Double wall thickness 7.7 m.
Parenchyma frequent, diffuse to diffuse in aggregate and scanty paratracheal (Fig. 2AC). Rays 18 (1225)
/mm, exclusively uniseriate (Fig. 2DF). Some rays contain radial laticifers in all species. Laticifers large in
size and surrounded by cluster of secretory cells (Fig. 2E). Average uniseriate ray height 681 (1602140) m
with 225 cells. Average multiseriate ray width 27 (13.755.0) m, ray height 613 (2681284) m with 6
16 cells (Tables 2 & 3). Rays heterocellular composed of procumbent and square/upright cells mixed
throughout body (Fig. 2G) except E. neriifolia containing homocellular rays made up of upright/square cells
only. Perforated ray cells present in E. tirucalli. Both Radial and axial laticifers found in E. tortilis only.
Excoecaria L.
A Paleotropical genus of about 40 species, most in tropical Asia but a few taxa in Africa, Madagascar, and
Tropical Australia. 6 species (including cultivated one) represented in India (Balakrishnan & Chakrabarty 2007),
of which one species studied here.
Species studied: Excoecaria agallocha L.
Physical features: Wood white or greyish yellow to pale yellow, soft, light, straight grained and medium coarse
textured, specific gravity ranging between 0.350.52 air dry.
Microscopic features: Vessels frequency 12 (525) per mm2, radial multiples of 23 common with some solitary
vessels, few long multiples and clusters present. Vessel outline rounded to oval, average tangential diameter
68.7 (39110) m. Mean vessel element length 667 (375963) m (Tables 2 & 3). Intervessel pits alternate,
polygonal, distinctly bordered with 2.9 (2.84.1) m diameters. Perforation plate simple. Tyloses common.
Vessel-ray pits similar to intervessel pits. Fibres non-septate, thin walled with distinctly bordered pits on
both radial and tangential walls. Average fibre length 994.4 (5351391) m, average fibre diameter 28.2
(19.241.2) m, double wall thickness 6 m. Parenchyma frequent, diffuse to diffuse in aggregates
sometime forming interrupted fine lines and scanty paratracheal (Fig. 1C). Frequently non-chambered
prismatic/rhomboidal crystal present. Rays 12.2 (818) per mm, exclusively uniseriate (Fig. 1E). Average
uniseriate ray height 432 (107856) m with 228 cells and width 22.9 (1155) m. Rays heterocellular,
composed of procumbent body cells with single row of upright and square cells at marginal ends (Fig. 2I).
Frequently non-chambered prismatic/rhomboidal crystal present. Some rays containing radial laticifers.
Laticifers small in size, not surrounded by secretory cells (Fig. 2I).
Falconeria Royle
Monotypic genus distributed in India and Sri Lanka to Vietnam and China (Balakrishnan & Chakrabarty
2007).
Species studied: Falconeria insignis Royle (= Sapium insigne (Royle) Benth. & Hook.f.)
light, straight grained and medium coarse textured, specific gravity ranging between 0.390.50 air dry.
Microscopic features: Vessels frequency 5 (39) per mm2, mostly solitary vessels with some radial multiples of
23, rarely clusters present. Vessel outline rounded to oval, average tangential diameter 156 (82.5303) m,
mean vessel element length 639 (375856) m. Intervessel pits alternate, rounded, distinctly bordered with
Jangid et al. (2017) 4(1): 3748
Figure 2. AC, T.S. showing diffuse to diffuse-in-aggregates axial parenchyma in Euphorbia royleana (A), E. tirucalli (B)
and E. nerifolia (C); DF, T.L.S. showing exclusively uniseriate rays and radial laticifers in E. royleana (D), E. tirucalli (E)
and E. nerifolia (F); G, R.L.S. showing heterocellular rays made up of procumbent and upright/square cells mixed
throughout body in E. royleana; H, Maceration showing fibres and vessel elements in E. tirucalli; I, scalariform intervessel
pits in E. nerifolia.
6.9 (5.58.2) m diameters. Perforation plates simple. Tyloses common. Vessel ray pits two distinct types
(rounded to elliptical and horizontal), much reduced bordered in same ray cells. Fibres non-septate, thin
walled with distinctly bordered pits on radial walls only. Average fibre length 1040 (7491284) m, average
fibre diameter 31 (22.041.2) m, double wall thickness 6.6 m. Parenchyma frequent, diffuse to diffuse in
aggregates and scanty paratracheal. Abundant chambered crystal present in series up-to 10. Rays 14 (1018)
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per mm, exclusively uniseriate. Average uniseriate ray height 614.5 (1611177) m with 423 cells and
width 19.9 (16.530.2) m (Tables 2 & 3). Rays homocellular, composed of procumbent cells only. Rarely
chambered/ non-chambered, prismatic/rhomboidal crystal present.
Shirakiopsis Esser
Containing 6 species distributed in tropical Africa and Asia, from India to Combodica and throughout
Malaysia. Only type species represented in India (Balakrishnan & Chakrabarty 2007) studied here.
Species studied: Shirakoipsis indica (Willd.) Esser (Sapium indicum Willd.)
Microscopic features: Vessels frequency 6 (410) per mm2, radial multiples of 23 common with many solitary
vessels, rarely clusters present. Vessel outline rounded to oval, average tangential diameter 151 (124171)
m. Mean vessel element length 594 (482856) m. Intervessel pits alternate, rounded, distinctly bordered
with 5.2 (4.16.9) m diameters (Tables 2 & 3). Perforation plate simple. Tyloses absent. Vessel ray pits
simple, reduced bordered, rounded to elliptical 6.9 (5.58.2) m. Fibres non-septate, thin walled with
distinctly bordered pits on radial walls. Average fibre length 908 (5351284) m, average fibre diameter
33.8 (24.746.7) m, double wall thickness 8.1 m. Parenchyma frequent, diffuse to diffuse in aggregates
and scanty paratracheal (Fig. 1B). Silica bodies present. Rays 14 (1216) per mm, mostly uniseriate with
few locally biseriate (Fig. 1E). Average uniseriate ray height 332 (107589) m with 312 cells. Average
biseriate ray width 39.3 (27.555.0) m and height 223 (107535) m with 315 cells. Rays heterocellular,
composed of procumbent body cells with single row of upright and square cells at marginal ends. Abundant
silica bodies present (Fig. 1H). Crystal absent.
Triadica Lour.
About 4 species distributed in Asia, NE. India to China, Indo-China and West Malaysia. Two species
represented in India (Balakrishnan & Chakrabarty 2007), of which type species is studied here.
Species studied: Triadica sebifera (L.) Small (= Sapium sebiferum (L.) Roxb.)
Physical features: Wood white to yellowish white, turning to greyish-yellow on ageing, soft and light to very
Microscopic features: Vessels frequency 7 (313) per mm2, radial multiples of 23 common with some solitary
vessels, rarely clusters present. Vessel outline rounded to oval, average tangential diameter 111 (55179)
m, mean vessel element length 558.8 (268910) m (Tables 2 & 3). Intervessel pits alternate, rounded,
distinctly bordered with 3.7 (2.85.5) m diameters. Perforation plate simple. Tyloses absent. Vessel ray pits
distinctly bordered similar to intervessel pits with 4.6 (2.86.9) m. Fibres non-septate, thin walled with
distinctly bordered pits on both radial and tangential walls. Average fibre length 1235 (6421819) m,
average fibre diameter 22 (28.549.2) m, double wall thickness 8.1 m. Parenchyma frequent, diffuse to
diffuse in aggregates and scanty paratracheal. Abundant chambered crystal present in series up-to 10. Rays
16 (1020) per mm, mostly uniseriate with few locally biseriate. Average uniseriate ray height 590.7 (161
1605) m with 448 cells. Average biseriate ray width 20.7 (16.541.2) m and height 398.5 (61749) m
with 325 cells. Rays homocellular, composed of procumbent cells only. Crystal absent. Perforated ray cells
present.
Identification key to Indian species of the subfamily Euphorbiodeae

1. Radial laticifers broad and surrounded by subsidiary cells, frequently crystals present in rays and axial
parenchyma.... 2
-- Radial laticifers narrow and naked...5
2. Fibre non-septate, intervessel pits scalariform, vessel ray pits similar to intervessel pits...3
-- Fibre septate, intervessel pits rounded to elliptical, vessel ray pits two distinct type in same ray
cell..................Euphorbia tirucalli
3. Distinctly bordered pits present on both radial and tangential walls of fibres, both radial and axial laticifers
present..... Euphorbia tortilis
-- Libriform fibres, exclusively radial laticifers present.......4
4. Rays made up of upright/square cells only.......Euphorbia neriifolia
-- Rays made up of mixed upright/square and procumbent cells throughout of body...Euphorbia royleana
Jangid et al. (2017) 4(1): 3748
5. Vessel ray pits similar to intervessel pits.....6

-- Vessel ray pits simple, rounded with reduced border...7
6. Homocellular rays made up of procumbent cells only...Triadica sebifera
-- Heterocellular rays made up of procumbent body cells and single row of upright/square cells at marginal
ends...Excoecaria agallocha
7. Fibre trachied present, crystals absent, silica bodies present in axial parenchyma and ray cells.8
-- Distinctly bordered pits present on both radial and tangential walls of fibres, chambered crystal present in
axial parenchyma, silica bodies absent......Falconeria insignis
8. Homocellular rays made up of upright/square cells only, tyloses present.Balakata buccata
-- Heterocellular rays composed of procumbent body cells and single row of upright/square cells at marginal
ends, tyloses absent....Shirakiopsis indica

Webster (1994) and Radcliffe-Smith (2001) classified the subfamily into five tribes. Euphorbioideae consist
of two large tribes, in addition to several more isolated genera that have been assigned to smaller tribes. Webster
(1994) included 11 genera in three subtribes (Anthosteminae (Baill.) Webster, Neoguillauminiinae Croizat, and
Euphorbiinae) in the tribe Euphorbieae. In India only one subtribe (Euphorbiinae) occurs with 3 genera
(Pedilanthus, Euphorbia and Synadenium) and 84 species (Balakrishnan & Chakrabarty 2007). According to
Esser (2012) the tribe comprise 33 genera and ca. 300 species of mostly woody plants (with few herbs and
succulents. In Webster (1994) circumscription the tribe Hippomaneae comprises 19 genera in three subtribes
(Carumbiinae Mll.Arg., Mabeinae Pax & K.Hoffm., and Hippomaninae) while 24 genera in two subtribes
(Carumbiinae and Hippomaninae) in circumscription of Radcliffe-Smith (2001). This tribe is represented by
only subtribe (Hippomaninae) with 6 genera viz. Microstachys, Balakata, Shirakoipsis, Triadica, Excoecaria
and Falconeria.
Tribe Hippomaneae
We studied five genera viz. Excoecaria, Triadica, Shirakoipsis, Balakata and Falconeria, out of six genera
of the tribe Hippomaneae represented in India. Webster (1994) included the genera Triadica, Shirakoipsis,
Balakata and Falconeria in the genus Sapium Jacq. Sapium has been considered a genus of about 100 species in
circumscription of Pax & Hoffmann 1912 and Webster 1994. Further morphological phylogeny of
Hippomaneae by Kruijt (1996) and Esser (2001) supporting the, rearrangement of the previously unnatural
Sapium into several distinct genera and proposed a new overall generic classification of the Hippomaneae based
on a worldwide morphological survey of members of all genera and sections of the tribe. A number of species
recently been excluded from Sapium by Kruijt (1996) and Esser (1999, 2002), so that the actual number of
currently accepted species is around 20 only (Esser 2012).
We studied one species from each of five genera of tribe Hippomaneae. Excoecaria showed some difference
in vessel diameter, vessel ray pitting and crystal deposition from other four genera studied. The wood
microstructure of four genera viz. Balakata, Shirakoipsis, Falconeria and Triadica found uniform to certain
extent. Quantitative wood anatomical properties were nearly constant in all species studied of these four genera.
A little variation was noted in only qualitative features like fibre wall pitting ray cellular composition and
crystal/silica deposition in parenchyma and rays. Balakata buccata have exclusively uniseriate rays while other
three species F. insignis, T. sebifera and S. indica have predominantly uniseriate rays with few locally bi-seriate
rays. B. buccata and S. indica contains silica bodies and lack of crystals, whereas F. insignis and T. sebifera
contain chambered crystals in axial parenchyma and lack of silica bodies.
The generic distinctiveness between Sapium (Mennega 2005) and these four genera were noted in presence
of crystals in ray cells and absence of silica bodies in Sapium unlike the Balakata, Triadica, Falconeria and
Shirakoipsis. Present study supports the segregation of the genera viz. Balakata, Shirakoipsis, Triadica and
Falconeria from the genus Sapium by Esser (1999, 2002) and Kruijt (1996).
Tribe Euphorbieae
According to circumscription of Webster (1994), the subtribe contains seven genera: Chamaesyce Gray,
Cubanthus (Boiss.) Millsp., Endadenium Leach, Euphorbia, Monadenium Pax, Pedilanthus Necker ex Poit., and
Synadenium Boiss. Most authors treat these as infrageneric taxa of Euphorbia. We studied only genus
Jangid et al. (2017) 4(1): 3748
Euphorbia out of three genera (Pedilanthus, Euphorbia and Synadenium) of subtribe Euphorbiinae represented
in India.
The integrity of the genus Euphorbia has long been questioned, and many 20th century workers (Carter 1988,
1992, Gilbert 1987, Balakrishnan & Chakrabarty 2007) favouring subdivision of this vast genus into two or
more genus. There are 9 to 11 generally recognized subgenera. Seven sub-genera occurs in India (Balakrishnan
& Chakrabarty 2007), of these our species falls in Euphorbia subg. Euphorbia.
There was great deal of variation in qualitative wood anatomy of species studied of genus Euphorbia viz. E.
nerifolia, E. royleana, E. tirucalli and E. tortilis. E. tirucalli has distinct wood microstructure from other three
species, differential wood character found are, thin to thick walled septate fibres, small alternate intervessel pits,
vessel ray pits simple, rounded to elliptical unlike rest of species. Similarily, E. tortilis differ from E. tirucalli,
E. nerifolia and E. royleana in features such as presence of distinctly bordered pits on both radial and tangential
walls of fibres and presence of axial laticifers. On other hand E. royleana and E. nerifolia have similar wood
microstructure to certain extent.
In Webster (1994) infra-generic classifications of the Euphorbia, he included the four species studied here in
Euphorbia subg. Euphorbia. According to present study these species should not be maintained in same
infrageneric group (Euphorbia subg. Euphorbia) due to considerable wood anatomical dissimilarities among
species. Euphorbia subg. Euphorbia further can be classified in three different groups at section or subgenus
level.
Group I: axial and radial laticifers present, scalariform intervessel pits and non-septate fibres with distinctly
bordered pits on both radial and tangential walls. Species falls in this group is E. tortilis.
Group II: exclusively radial laticifers present, scalariform intervessel pits and non-septate libriform fibres.
Species falls in this group are E. royleana and E. nerifolia.
Group III: exclusively radial laticifers present, rounded to elliptical intervessel pits and septate libriform fibres.
Species falls in this group is E. tirucalli.
Figure 3. Graph showing vulnerability and mesomorphy values and their relationship within subfamily Euphorbiodeae.
Ecological consideration
Vulnerability and Mesomorphy are two indices proposed by Carlquist (1977) as ecological indicators for
wood anatomy trends. According to Carlquist (2001), the values of vulnerability index lower than one (<1)
reflect adaptation to drier areas, while values greater than three (>3) are found in plants living in areas with high
water availability. For mesomorphy index, values less than 100 indicate plants which grow in dry environments
(xeric) or in regions with a dry period for several months in the year, while higher mesomorphy index values
(>200) are found in species which grow in mesic environments. Plants adapted to water stress environments
have higher vessel grouping.
In the present study, all the genera showed relatively higher values of vulnerability and mesomorphy indices
(Fig. 3). Euphorbia showed lowest values of vulnerability (6) and mesomorphy (2849) whereas Falconeria
showed highest values of vulnerability (31) and mesomorphy (19937). Addition to this, presence of high vessel
groupings (Carlquist 1966) and relatively narrower vessels with higher frequencies (Baas et al. 1983, Carlquist
Jangid et al. (2017) 4(1): 3748
& Hoekman 1985) and thicker fibre walls (Alves & Angyalossy 2002, Barajas-Morales 1985, Luchi et al. 2005)
also represent adaptation to xeric environment. Higher values of mesomorphy and vulnerability indices within
the subfamily showed its adaptation to mesic environment which is favoured by presence of very thin fibres,
banded parenchyma and wide vessels with low frequencies which are more efficient in water conduction than
narrow ones (Baas et al. 1983, Zimmermann 1983).
Figure 4. Graph showing F/V and L/D values and their relationship within subfamily Euphorbiodeae.
Evolutionary consideration
According to the major trends of specialization in secondary xylem of dicotyledons (Yatsenko-Khmelevskyi
1954) and Carlquist (2001), wood anatomical features interpreted as primitive characters are, diffuse to diffuse
in aggregate parenchyma, long thick walled non-septate fibres, bordered pits on both radial and tangential walls
and small diameter of vessels, long vessel element, scalariform perforation plates, vessel ray pits similar to
intervessel pitting and absence of helical sculpture on fibre walls. According to Kribs (1935) relatively narrow
rays, presence of heterocellular, both uniseriate and multiseriate rays together are primitive characters.
According to Mennega (1987) the combination of characters considered most advanced includes vessel with
simple perforation plates, medium sized vessel element, small intervessel and vessel-ray pitting, axial
parenchyma absent/rare or scanty paratracheal and thin walled, septate libriform fibres.
In the species studied of the subfamily Euphorbiodeae, almost all the wood anatomical features are evident
of advancement such as presence of simple perforation plates, exclusively uniseriate rays, alternate intervessel
pits, relatively wider and shorter vessel elements and thin walled libriform fibres except Excoecaria and
Euphorbia. Excoecaria contains some primitive features also such as minute intervessel pits and vessel ray
pitting similar to intervessel pits and fibres with distinctly bordered pits on both radial and tangential walls,
similarly Euphorbia contains scalariform intervessel pits and vessel ray pitting similar to intervessel pits.
Length, width ratio of vessels (L/D), fibre and vessel element length ratios (F/V) normally serve as indices
of phylogenetic advancement (Bailey 1957). High L/D and concomitantly low F/V values are widely regarded
as primitive (Rury 1985). In the present study lowest F/V (1.5) and highest L/D (9.7) values were found for
Excoecaria. On other hand highest F/V (2.2) and lowest L/D (5) values were found for Triadica (Fig. 4).
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ISSN (E): 2349 1183
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4(1): 4954, 2017
DOI: 10.22271/tpr.2017.v4.i1.007
Research article
Seed dormancy testing and germination frequency determination

of Psoralea corylifolia L., an endangered medicinal plant
Poonam Arya* and Ragini Gothalwal
Department of Biotechnology, Barkatullah University, Bhopal-462026, Madhya Pradesh
*Corresponding Author: ms.poonam_arya@rediffmail.com [Accepted: 13 February 2017]
Abstract: Seed dormancy is one of important factor which prevent the cultivation of medicinally
important plant Psoralea corylifolia. Mechanical dormancy is present in the seed due to hard seed
coat. P. corylifolia is a slow growing species due to low germination percentage (57%). Long
gestation period and delicate field handling discourages the commercial cultivation of the plant so
different treatments were used to break dormancy in seeds. Out of five different methods used
98% concentrated H2SO4 for about one hour was found to be best method for seed dormancy
breakage. Statistical analysis of data was performed by one way ANOVA using Sigma State
Software version 4.
Keywords: Psoralea corylifolia - ANOVA - Dormancy - Gestation period - Dormancy.
[Cite as: Arya P & Gothalwal R (2017) Seed dormancy testing and germination frequency determination of
Psoralea corylifolia L., an endangered medicinal plant. Tropical Plant Research 4(1): 4954]
INTRODUCTION
Psoralea corylifolia L. (Indian bread root) is an endangered medicinally important plant (Fig. 1) distributed
in tropical and subtropical region of world belonging to Fabaceae family (Jain 1994). In India it is seen along
the road sides and waste place of the tropical regions. Especially in the semi-arid region of Rajasthan and
Eastern district of Punjab, Bihar and Karnataka (Agrawal et al. 2013). The Hindi name of the plant is Babchi
and bakuchi (Oudhiya 2001). It is an erect herb of height 30180 cm that grows in winter season high. Leaves
are broadly elliptical in shape, Flower shows indense axillary long-peduneled heads pods small 3.54.5 mm
2.03.0 mm, seeds are compressed mucronate dark chocolate to almost black one, smooth, adhering to the
pericarp (Uikey et al. 2010). The plant is harvested by drug industry when it sets in to flowering. P. corylifolia
is propagated by seed germination.
Scientific classification
Kingdom: Plantae
Division: Magnoliophyta
Class: Magnoliopsida
Order: Fabales
Family: Fabaceae
Genus: Psoralea
Species: Psoralea corylifolia L.
Figure 1. Plant of Psoralea corylifolia L. (Inset: Seeds)
Active constituent of Psoralea corylifolia

The major active constituents reported to be present in P. corylifolia seeds are corylifols a-c
(prenylflavonoid) (Yin et al. 2004). Dried ripe fruit contains Psoralen, isopsoralen and neobavaislflavones
Received: 30 July 2016 Published online: 28 February 2017
Arya & Gothalwal (2017) 4(1): 4954
(Rajput et al. 2008). Daidzein (4-7 dihydroxyisoflavonoid) and genistein (4, 5, 7 trihydroxy isoflavonoid) are
present in natural plant as well as in vitro culture (Shinde et al. 2009).
Medicinal Uses
P. corylifolia has aromatic, antihelminthic, antibacterial and antifungal properties. It is used as a diuretic,
diaphoretic, laxative and stimulant. The powdered seeds are applied externally to cure skin problems. It is
valued as Chinese herbal medicine as a tonic remedy and is used to improve vitiligo. The roots are used for
treating dental caries. Plant yields a useful medicinal oleoresin which treats kidney disorders, impotence and
lumbago. The drug psoralen obtained from seed of P.corylifolia used for the treatment of vitiligo and
leucoderma (Vaidya 2006).
Dormancy
Germination is prevented by dormancy mechanism during unsuitable ecological conditions, when the
probability of seedling survival is low (Black et al. 2006). P. corylifolia has mechanical dormancy because its
seed coats or other covering are too hard which prevent the embryo to expand during germination (Baskin &
Baskin 1999). In, environment condition the seed coats of physically dormant seeds become water permeable
over time through repeated heating and cooling over many months to years in the soil. Chemical or pigment that
is present around the covering of embryo may be leached out of the tissue by specific chemical method (Bewley
& Black 1994). P. corylifolia is a slow growing species due to low germination percentage (57%). Long
gestation period and delicate field handling discourages the commercial cultivation of the plant (Pandey et al.
2013).
The objective of this paper was to determine the effective method for breaking dormancy of Psoralea
corylifolia with the purpose of increasing cultivation of medicinally important plant.

Seeds were collected form MFP-PARC located at coordinate 23.2086 N, 77.4731 E Madhya Pradesh. State
Minor Forest Produce Trading & Development Co-operative Federation under the brand name "Vindhya
Herbals" Located in Van Parisar, Barkheda Pathani, Near BHEL, Bhopal, Madhya Pradesh.
Methodology for seed dormancy
Five different treatments were performed to break seed dormancy.
a) Control
b) 98% Concentrated H2SO4 treatment for 1 hour and Hot water treatment 100C for 20 min (Siva et al.
2014).
c) 1 N NaOH treatment for 1 hour and Scarification by sand paper number 100 (Baes et al. 2002).
d) Cold water treatment Overnight incubation at 4C (Fariman et al. 2011).
Seeds were washed with running tap water for 5 min to remove surface dust than washed with Tween 20 for
about 1 min and then seeds were given different treatment then under aseptic conditions seeds were washed
with double distilled water for about three times. 70% alcohol treatment was given to the seeds for about 1 min
again washed with double distilled water three times than 0.1% HgCl2 treatment was given for about 2 min
again then they were used with double distilled water for about three times. With the help of sterile forceps
seeds were transferred in petridishes having layers of blotting paper soaked with distilled water. Each
petridishes has 50 seeds shoot length was recorded at regular intervals and final result was recorded after 28
days.
According to International Seed Testing Association: ISTA (2004), the germination test was performed in
total 400 seeds in 4 groups each having 100 seeds. Present experiment was carried out in 8 petridishes each
having 50 seeds (850=400).
Methodology for seed germination
Germination percent and percent of germination speed were calculated according to Krishnaswamy & Seshu
(1990).
1. ( )
Arya & Gothalwal (2017) 4(1): 4954
2. ( )
3. Germination energy = Percentage of seed germinated at 72 hrs (Bam et al. 2006).
4. Vigor Index = Number of germinated seeds/day of first count + + Number of germinated seeds/days at
final count.
5. Fresh and dry weight of seed ling, seedling were dried in oven 70C overnight.
RESULT AND DISCUSSION

Five different treatment performed for seed dormancy shows that 98% concentrated H2SO4 has maximum
seedling length, germination frequency, germination energy, germination speed, germination vigor index, fresh
weight (g), dry weight (g) as compared to control.
Figure 2. Seedling germination after 28 days: A, Control; B, Effect of 1 N NaOH treatment for 1 hour on seed germination;
C, Effect of Scarification by sand paper number 100 P on seed germination; D, Effect of Hot water treatment 100C for 20
min on seed germination; E, Effect of Cold water treatment overnight incubation at 4C on seed germination; F, Effect of
98% Concentrated H2SO4 treatment for 1 hour on seed germination.
Figure 3. Shoot length determination after 28 days: A, Control; B, Effect of 1 N NaOH treatment for 1 hour on seed
germination; C, Effect of Scarification by sand paper number 100 P on seed germination; D, Effect of Hot water treatment
100C for 20 min on seed germination; E, Effect of Cold water treatment overnight incubation at 4C on seed germination;
F, Effect of 98% Concentrated H2SO4 treatment for 1 hour on seed germination.
Seed dormancy is a unique feature of family Fabaceae; in P. corylifolia mechanical dormancy is present. To
overcome the dormancy and to increase the germination frequency five different treatments were used which
breaks the dormancy. Sterile 400 seeds treated with different treatment for different time intervals were
inoculated in petridishes having presoaked filter paper and they were incubated for 28 days at 28C (Figs. 2A
F). Subsequently the shoot length was calculated after 28 days (Figs. 3AF).
Arya & Gothalwal (2017) 4(1): 4954
Figure 4. Effect of different treatment on dry weight of seedling after 28 days of germination.
Table 1. Effect of different treatment on germination parameter.

S. Treatment Number Average Grm. Grm. Grm. Grm. Vigor Fresh weight Dry weight
No. of Seeds Seedling frequency Energy Speed Index Average (g) Average (g) Average
(400) length (cm) (%) (%) (%) value value value
1 Control 400 7.852.64 1.75 0.75 0.18 0.3114.8 0.350.1 0.110.06
2 1N NaOH 400 4.781.99* 2.25 0.75 25 0.440.00 a 0.070.0a 0.020.00*
3 Scarification 400 6.52.58* 2.25 1 22.9 0.410.22 a 0.260.1a 0.040.02*
Sand Paper (100P)
4 Hot water (100C) 400 6.802.40* 2.5 1.5 29.1 0.580.38 a 1.430.5a 0.030.03*
5 Cold water (4C) 400 7.353.60* 9.25 0.75 6.11 0.580.38 a 1.440.55a 0.080.03*
6 H2SO4 400 8.452.84* 13.75 4.5 33.9 1.991.4* 2.451.7* 0.130.09*
Note: Grm. = Germination; * = Significance indicates significant value compared with control at (P = <0.001); a = indicates non
significant value with control at (P =<0.001).
Different treatments have been used for seed germination parameters study and breaking seed dormancy in
Psoralea corylifolia medicinal plant and they were compared with the control (Table 1). In the present study
sulfuric acid treatment for one hour was effective in all seed germination parameter after 28 days (Fig. 4).
Mechanical injury to the seed coat or chemical treatment has been used for breaking the seed dormancy of
Arya & Gothalwal (2017) 4(1): 4954
certain cultivated medicinal plants Gloriosa superb, Echinacea purpurea, Belladonna (Gupta & Shah 1971,
Supari et al. 1993, Mittar et al. 1993, Kumar & Sharma 2012).
The present study was first which compare different treatments for seed dormancy breakage in Psoralea
corylifolia. The plant is listed as an endangered species mainly due to the destruction of its natural habitats.
Sulfuric acid and hot water pre-treatment have been reported that to improve the seed germination and seedlings
growth of Cassia fistula (Soliman & Abbas 2013). Primary exogenous dormancy due to physical factors present
outside the embryo is present in most of the Fabaceae plants but in P. corylifolia mechanical dormancy is
reported. Seed coats are too hard to allow the embryo to expand during germination. In Coronilla varia physical
dormancy is present due to the low moisture level of seed, embryo gets quiescent. The outer macrosclereid and
mucilaginous cell layer becomes impermeable to water. Or a hardened endocarp is three reasons that make seed
coats impermeable to water. Such seed coats develop during the last stages of seed development. Whereas in P.
corylifolia, acid breaks the seed layers and impermeable seed coat with helps in germination. Acid treatment
was effective for breaking strong seed dormancy and impermeable seed coat which is the major cause low
germination frequency in P. corylifolia. It is an effective, reliable and reproducible method to get high frequency
seed germination in P. corylifolia, when compared to any other treatments, seeds Damaging and breaking
possibilities are very low at this acid treatment. Thus, this method can be adopted as a good alternative than the
other treatments for higher percentage of seed germination since; the aseptic seedlings were used as explants in
large number of in vitro studies.
ACKNOWLEDGEMENTS
I would like to thanks UGC for RGNF Fellowship and Department of Biotechnology, Barkatullah University
Bhopal for providing me lab facilities and chemicals for my research work.
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ISSN (E): 2349 1183
ISSN (P): 2349 9265
4(1): 5561, 2017
DOI: 10.22271/tpr.2017.v4.i1.008
Research article
Effect of Mancozeb on Mustard (Brassica juncea L.):

An In-vitro study
Monika* and Mohd. Kashif Kidwai
Department of Energy and Environment Sciences, Chaudhary Devi Lal University, Sirsa, Haryana, India
*Corresponding Author: monikakaswan88@gmail.com [Accepted: 15 February 2017]
Abstract: Pesticides are extensively used all over the globe for the control of pest population
responsible for reduction in yield and among them fungicides are the specifically used to control
fungal plant pathogens. An In-vitro plant study was conducted to assess the effect of Mancozeb on
early growth parameters on two Mustard (Brassica juncea) varieties i.e. RH- 30 and Laxmi.
Mancozeb (75% WP) is a widely applied contact fungicide for control of various diseases but
several toxicity issues are associated with Mancozeb. Mustard is one of the most important oil
yielding crops and is majorly used for various purposes such as medicinal uses, condiment and
industrial uses. In the experiment three different doses of Mancozeb (75% WP) i.e. 1 mg.kg-1 Half
Recommended Dose (RD), 2 mg.kg-1 Recommended Dose (RD) and 4 mg.kg-1 Double
Recommended Dose (2RD) applied on RH-30 and Laxmi varieties. Results revealed that, RD and
2RD reduced the germination up to 3060 % in variety RH-30 whereas in variety Laxmi reduced
germination percentage is 1755 % in comparison of control. The overall length of plants of
variety Laxmi was found to be increased in comparison of control but in plants of variety RH-30
the results was just opposite. Overall results indicate that excess amount of Mancozeb induced
stress in both mustard varieties which resulted in low germination percentage. Whereas after seven
days, Laxmi resulted in better growth than RH-30 plants. Also, Seeding vigour was recorded
higher in Laxmi than RH-30.
Keywords: Pesticides - Fungicides - Seeding Vigour - Mustard.
[Cite as: Monika & Kidwai MK (2017) Effect of Mancozeb on Mustard (Brassica juncea L.): An In-vitro study.
INTRODUCTION
Mustard (Brassica juncea L.) is one of the most important oil crops of the world after soybean and
groundnut (FAO 2012) and in India it is popularly cultivated as major oil seed crop (Meena et al. 2010). It is
predominantly cultivated in the states of Rajasthan, Uttar Pradesh, Haryana, Madhya Pradesh and Gujarat which
contribute 81.5% in terms of area and 87.5% in term of production and majorly used for various purposes such
as medicinal uses, condiment, and industrial uses. Mustard is rich in phytonutrients, minerals, vitamins and
antioxidants as well as constituents like sinalbin and sinigrin and serves as a functional food (Kumar & Andy
2012). According to Ayurveda, Mustard leaf is considered as a vegetable, while the seeds are used as a
condiment and constitute the source of mustard oil. Mustard seed is considered as rich source of oil and protein.
The seed has oil content as high as 4648 % while seed meal has 43.6% protein (use of meal) and low in
glucosinolate content. Also, seed residue used as an ingredient for cattle feed and poultry feed in India (Mandal
et al. 2002, Manohar et al. 2009).
Some anti-nutritional factors present in rapeseed-mustard crops are known to be associated with some plant
defence system and other important biological functions (Kumar et al. 2014).
Application of pesticide is strategy for effective management of pests and diseases and the productivity of
crops depends on their effective control (Pretty & Bharucha 2015). Pesticides are extensively used all over the
globe to control different pest population and among them fungicides are specially used for control fungal plant
pathogens (Mancini & Romanazzi 2013). In the past few, non-target environmental impact and Excessive use of
Received: 28 October 2016 Published online: 28 February 2017
Monika & Kidwai (2017) 4(1): 5561
fungicides have caused environmental pollution and development of fungicide resistance in plant pathogens
which led to the search for alternative methods (Hollomon 2015). In recent years, crop suffers from many
diseases out of them Alternaria blight [Alternaria brassicae (Berk.) Sacc. & A. brassicicola (Schw.)] is one of
the serious and widely occurred diseases all over the country (Meena et al. 2011, Chaurasia & Bhajan 2015).
Alternaria blight is reported to causes blight of leaf, pod, stem and seed abnormalities (Chattopadhyay et al.
2005). It is reported that Alternaria blight reduce upto 70% yield of mustard in India (Rathi et al. 2015).
Table 1. Physical and Chemical Properties of Mancozeb as described by (Harvest 2016).
Chemical Name Manganese ethylenebis (dithocarbamate) (polymeric) complex with zinc salt
Empirical Formula (C4H6MnN2S4)x(Zn)y
Molecular Weight 541
Structural Formula
Flashing point 138C (150C Dec.)

-1
Solubility 620 mg.l in water, practically insoluble in most organic solvents
Vapour pressure Negligible
Acute oral LD50 5 g.kg-1 (rat)
Mancozeb formulation 75% WP
Mancozeb (75% WP) is a contact fungicide mainly used for control various diseases on wide variety of
crops (Jankowski 2007, Mathivanan & Prabhavati 2007). Mancozeb [[1, 2-ethanediylbis-[carbamodithioato]]
(2- )] manganese, mixture with [[1, 2-etha-nediylbis-[carbamodithioato]]-(2- )] zinc is a fungicide of the
carbamate pesticide family. It is marketed by the trade names Dithane M45, Indofill, Manzeb, Nemispot,
Manzane etc. It is applied on various crops including oil bearing plants and field crops against a wide spectrum
of fungal diseases (Nirwan et al. 2016). The fungicide Mancozeb 75% WP [Indofil M-45] was used in the
present study.
Table 2. Diseases controlled by fungicide Mancozeb on Mustard (Brassica juncea L.).
Diseases Causal organism References
Sclerotinia rot Sclerotinia sclerotiorum (Lib.) de Bary Biswas et al. (2007),
Vishwanath & Kolte (1997)
White rust Albugo cruciferarum (Gray) Kuntze Bhatia & Gangopadhyay (2008)
Alternaria blight Alternaria brassicae (Berk.) Sacc. Meena et al. (2011)
and A. brassicicola (Schw) Wiltshire
Powdery mildew Erysiphe cruciferarum Opiz ex L. Junell Biswas et al. (2007),
Vishwanath & Kolte (1997)
Downy mildew complex Hyaloperonospora parasitica (Pers.) Biswas et al. (2007),
Constant. Vishwanath & Kolte (1997)
Mancozeb is applied on various crops against a wide spectrum of fungal diseases (Hayes & Paiford 1995,
Goswami & Ghosh 2012). Mancozeb has power to inhibit spore formation in pathogenic fungi thereby causing
its death (Elsamen et al. 2015). Mancozeb is reported to be associated with several health hazards when applied
in very high doses. The resulting symptoms are convulsions, slurred speech, confusion, slowed heartbeat etc.
Mancozeb is reported to be an established carcinogen (FAO/WHO 1993, Vettorazzi et al. 1995, Liu et al. 2016).
Mancozeb is reported to cause teratogenic, carcinogenic and goitrogenic effects on mammals. Ethylene thio urea
(ETU), carbon disulphide (CS2), thio urea etc are metabolites produced in degradation of Mancozeb reported to
be responsible for inducing thyroid toxicity and neurotoxicity (Kackar et al. 1997, Domico et al. 2007, Miller et
al. 2009). ETU is reported to cause ground water pollution due to its high solubility in water (Belpoggi et al.
2002, Srivastava & Singh 2013). The fungicide Mancozeb has a considerable deleterious impact on soil
microflora, nitrification, ammonification, soil microbial biomass, carbon mineralization and soil enzymes which
may result in harmful effects on nutrient uptake and plant growth. These findings suggest that the use of
Mancozeb to control plant diseases in apple orchard soil requires simultaneous application of large quantities of
nitrogen based fertilizers (Dias 2012).
Monika & Kidwai (2017) 4(1): 5561
There is very little information available with regard to the effect of fungicide on oil crops generally and on
Mustard crop particularly. Therefore the present study was undertaken with a view to understand the effect of
commonly used fungicide Mancozeb on initial plant growth and chlorophyll content of Mustard (Brassica
juncea L.).

The present study was conducted at the laboratory of Environment Toxicology and Microbiology,
Department of Energy and Environmental Science of Chaudhary Devi Lal University, Sirsa. Soil sample was
collected from the fertile field of village Umedpura of District Sirsa. In this field, wheat, cotton and mustard are
mainly cultivated crops. The soil was processed in Laboratory, excess moisture was removed by drying the soil
in shade and sieved properly to make it usable for pot trial study. The pots were prepared by 100 gm soil in each
pot, and this study was triplicated for each treatment (Kidwai 2007). Fungicide Mancozeb 75% WP [Indofil M-
45] was procured from local market of Sirsa. The three different doses of fungicide i.e. half recommended dose,
recommended dose and double recommended dose were used in this study.
Table 3. Different doses of Mancozeb 75% WP with their application rate used for seed treatment.
Doses Application rate (per kg seed)
Half recommended dose (RD) 1 mg.kg-1
Recommended dose (RD) 2 mg.kg-1
Double recommended dose (2RD) 4 mg.kg-1
Seed of two popular mustard varieties i.e. RH-30 and Laxmi (procured from Krishi Vigyan Kendra, Sirsa)
were surface sterilized with 0.1% HgCl2 (mercuric chloride) for 5 min then washed thoroughly with distilled
water (Hirve & Bafna 2013) and allowed to germinate in pots. Three sets in each dose were maintained along
with the control for comparative study. On the seventh day, growth parameters and chlorophyll content were
evaluated. Germination percentage was estimated as described by Rangwala et al. (2013),
Root and shoot length of seedlings were recorded using the standard centimetre scale (Kabir et al. 2008,
Rangwala et al. 2013). Seedling Vigour index was calculated applying formula suggested by Aery (2010),
( )
Three plants of each treatment were weighed in order to determine the fresh weight (Kabir et al. 2008,
Rangwala et al. 2013). Spectrophotometric estimation of the Chlorophyll a, Chlorophyll b and total
Chlorophyll of mustard crop was done by following the method as described by Aery (2010).
Chlorophyll a (mg.g-1) = 12.7A663-2.69A645V /1000W
Chlorophyll b (mg.g-1) = 22.9 A645-4.68A663 V /1000W
Total Chlorophyll (mg.g-1) = 27.8 A662 V /1000W
Where, V =Vol. of the extract in ml, A 645=Optical density measured at 645 nm, A 663=Optical density measured
at 663 nm and A 662=Optical density measured at 662 nm.
Table 4. Codes for the Treatment used in the study.

Treatments (T) Code
Control RH-30 TR1
Control Laxmi TL1
Half Recommended Dose of Mancozeb ( RH-30) TR2
Half Recommended Dose of Mancozeb (Laxmi) TL2
Recommended Dose of Mancozeb (RH-30 TR3
Recommended Dose of Mancozeb ( Laxmi) TL3
Double Recommended Dose of Mancozeb ( RH-30) TR4
Double Recommended Dose of Mancozeb (Laxmi) TL4
Monika & Kidwai (2017) 4(1): 5561

Table 5. Variation in early growth parameter and chlorophyll content in Mustard varieties.
T Germination Fresh Total Seedling Chl 'a' Chl 'b' Chl 'total'
% weight length Vigour (mg.g-1) (mg.g-1) (mg.g-1)
(gm) (cm) Index
TR1 800.2 0.82.01 6.8.005 544.057 0.502.0005 0.248.0005 0.742.0005
TR2 70.25 0.81.005 6.4.01 448.15 0.463 .001 0.228.001 0.643.002
(13) (1) (6) (18) (8) (8) (13)
TR3 60.05 0.71.01 6.1.05 3661 0.428 .001 0.201.002 0.599.003
(25) (13) (10) (33) (15) (19) (19)
TR4 50 2 0.43.22 3.1.2 1554 0.201.001 0.083.001 0.219.11
(38) (48) (54) (72) (60) (67) (70)
TL1 902 0.89.03 8.60.4 7741 0.691.01 0.535.005 0.751.001
TL2 80.05 0.88.03 8.5.15 6808 0.68.01 0.531.001 0.747.002
(0.11) (1) (1) (12) (2) (1) (1)
TL3 70.5 0.85.01 7.1.1 4974 0.677.002 0.527.01 0.698 .01
(22) (4) (17) (36) (2) (1) (7)
TL4 601 0.61.01 4.2 .1 2521 0.311.001 0.209.05 0.354.1
(33) (31) (51) (67) (55) (61) (53)
Note: * All the values are mean of triplicate value, figure in parenthesis indicates percent decrease in comparison of control.
The fungicide Mancozeb has highly influenced all the studied growth parameters of mustard seedlings.
Germination percentage of Mancozeb fungicide treated seed was decreased with the increase in concentration of
fungicide Mancozeb. Other studies also reported that more fungicide produces negative interference and abiotic
stress in germination of seeds and decreased it drastically in the treated sets (Horii et al. 2007, Cataneo et al.
2010, Marini et al. 2011, Parween et al. 2016). The results of our present study were conflict to Benicio (2015)
who reported that fungicides doses not produces interference in germination of seeds.
The results of present study demonstrated that shoot and root length was decreased with increasing
concentration of fungicide. Petit et al. (2012), Anitha & Savitha (2013) and Shakir et al. (2016) were also
reported that growth of plants was reduced after increasing the dose of Mancozeb fungicides. Results were in
agreement with the work done by Windham & Windham (2004), Bensoltane et al. (2006) and Mohammed &
Alrajh (2014) who have reported that Mancozeb is a systemic fungicides which are based on sterol biosynthesis
inhibitor are closely related to plant growth regulators the use of which at higher than labeled rates shorten the
internodes which may lead to slow-shoot and root growth.
Seedling vigour index was decreased at almost all the concentration of fungicide as compared to control in
the present study. But the results of our present study were contrary to Morales et al. (2012) who observed
higher vigour of rice seeds treated with Carboxin and Thiram fungicides.
In the study, results of mustard seedlings treated with Mancozeb indicate decrease in fresh weight with
increasing concentration of fungicide. These results were in parallel with the findings of Avinash & Hoshmani
(2012) that fresh weight of leaves of sorghum seeds treated with Carbendazim decrease with increase in
concentration of fungicide.
Chlorophyll content were also reduced in the study as increased in concentration of Mancozeb fungicide
and the results of our study were similar to the effect of tricyclazole on Maize seeds (Avinash 2012). According
to Anitha & Savitha (2013), Petit et al. (2012) and Shakir et al. (2016) chlorophyll content were also reduced
after increasing the dose of Mancozeb fungicides.
Majid et al. (2014), Singh & Kaur (2016) and Parween et al. (2016) reported oxidative stress caused by
Mancozeb that affect overall plant growth as well as biochemical parameters especially chlorophyll content. It
was also reported that higher dose of Mancozeb have negative effect on plant growth and activity of mycorrhizal
symbiosis (Saleh 2006, Chaurasia 2014). According to Kackar et al. (1997) and Mirkovic et al. (2015) chronic
oral feeding of Mancozeb has produced dose dependent toxicity and death of animal by structural and functional
changes like significant increase in thyroid, body weight ratio and histopathological changes. Easton et al.
(2001) reported that by the effect of Mancozeb fungicide novel proteins being induced and stress were
increased. Some new technology like Nano and PEG applied for Mancozeb claimed much eco-friendly
(Majumder et al. 2016).
Monika & Kidwai (2017) 4(1): 5561
CONCLUSION
From the present study it was concluded that the recommended dose of Mancozeb favours growth of
seedlings but concentration higher than recommended dose can be unfavourable for proper growth of plants and
chlorophyll content. Further field trial is in process to study the effect of Mancozeb involving other biochemical
parameters. Explore the most promising opportunities to increase benefits and reduce health and environmental
risk of fungicide use. We should educate and aware the farming community about using the proper quantity of
fungicide and adopt eco-friendly practices for sustainable agriculture which is direly needed in the larger interest
of planet earth.
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ISSN (E): 2349 1183
ISSN (P): 2349 9265
4(1): 6270, 2017
DOI: 10.22271/tpr.2017.v4.i1.009
Research article
Morphology and phylogeny reveal two new records of boletoid

mushrooms for the Indian mycobiota
Dyutiparna Chakraborty1, Kamal C. Semwal2, Sinchan Adhikari3,
Sobhan K. Mukherjee3 and Kanad Das1*
1
Cryptogamic Unit, Botanical Survey of India, P.O. Botanic Garden, Howrah-711103, India
2
Department of Biology, College of Sciences, Eritrea Institute of Technology, Mai Nafhi, Asmara, Eritrea
3
Department of Botany, University of Kalyani, Kalyani-741235, Nadia, West Bengal, India
*Corresponding Author: daskanadbsi@gmail.com [Accepted: 16 February 2017]
Abstract: A detailed macro- and micromorphological studies coupled with the LSU-based
phylogenetic inference of Aureoboletus nephrosporus, a tubulose member of the family
Boletaceae is presented. Similarly, another tubulose bolete, Strobilomyces mirandus which was
collected both from Eastern and Western Himalayas of India is also reported here with
morphological details and ITS-based phylogeny. Both are the new records for this country.
Keywords: Boletales - India - Macrofungi - New records - Phylogeny - Sikkim.
[Cite as: Chakraborty D, Semwal KC, Adhikari S, Mukherjee SK & Das K (2017) Morphology and phylogeny
reveal two new records of boletoid mushrooms for the Indian mycobiota. Tropical Plant Research 4(1): 6270]
INTRODUCTION
The members of the family Boletaceae are mostly ectomycorrhizal in tropical to subalpine regions and thus
are well represented in Sikkim of the Eastern Himalaya (Lakhanpal 1996, Das 2009, 2012, 2013, Chakraborty &
Das 2015, Das & Chakraborty 2014, Das & Dentinger 2015, Das et al. 2012, 2013, 2014, 2015) because of the
abundance of suitable host trees like Abies Mill., Picea Mill., Tsuga Carrire, Lithocarpus Blume, Castanopsis
(D. Don) Spach, Quercus L., etc. and favourable microclimates. A hitherto poorly recorded (from India)
ectomycorrhizal and tubuloid bolete genus Aureoboletus Pouzar is separated from the rest of the tubulose
members of Boletaceae by its glutinous pileus, brightly yellow colored hymenophore which is unchanging on
bruising, ixotrichodemis pileipellis (rarely trichodermis) and smooth basidiospores. In a recent macrofungal
foray in 2016 to South and East districts of Sikkim, a number of boletoid mushrooms were collected by authors
(DC & KD) along with other mushroom members and after thorough morphological examination and molecular
phylogenetic studies of those mushrooms, one appeared as a recently established species: Aureoboletus
nephrosporus G. Wu & Zhu L. Yang which is reported so far from China (Wu et al. 2016). It is described here
for the first time from India with a detailed macro- and micromorphology along with an LSU-based phylogeny.
The representatives of another genus Strobilomyces Berk. (Boletaceae) are characterised mainly by greyish
or blackish pileus, the presence of squamules or scales on pilear surface, ornamented basidiospores (Smith &
Thiers 1971, Singer 1986). They are fairly common in India and are distributed from subtropical to subalpine
zones (Bilgrami et al. 1991, Lakhanpal 1996, Kour 2013, Das et al. 2014). During last couple of forays to
Eastern and Western Himalayas of India from 2007 to 2016 authors (DC, KCS & KD) came across repeatedly
an interesting member of this boletoid genus. Thorough studies (morphology and ITS-based phylogeny) of the
collected materials revealed a species of this genus with unusual morphology i.e. Strobilomyses mirandus
Corner. A detailed macro- and micromorphology along with the ITS-based phylogeny of this species are also
presented here for the first time from the country.

Morphology
Macrofungal forays were undertaken by three of us (DC, KCS & KD) to different parts of Eastern (South
and East districts of Sikkim) and Western Himalaya during the rainy season (JulyAugust) in 2016.
Received: 08 November 2016 Published online: 28 February 2017
Chakraborty et al. (2017) 4(1): 6270
Macromorphological characters were observed in the field and or basecamp from the fresh and dissected young
to mature basidiomata. Samples were duly dried with respective field-drier. Images of these basidiomata were
captured with the help of Canon Power Shot SX 220 HS and Nikon Coolpix P510. Micromorphological
characters were observed with the help of a compound microscope (Nikon Eclipse Ni-U) from the dry samples
mounted in a mixture of 5% KOH, 1% Phloxin and 1% Congo red or in distilled water. Color codes and terms
mentioned here are mostly after Methuen Handbook of Color (Kornerup & Wanscher 1978).
Micromorphological drawings were prepared with a drawing tube (attached to the Nikon Eclipse Ni or Olympus
CX 41) at 1000. Basidium length excludes sterigmata. Basidiospore measurements were recorded in profile
view from 20 basidiospores mounted from a spore print. Spore measurements and length/width ratios (Q) are
given here as minimummeanmaximum. Methods for SEM follow Das et al. (2015). Herbarium codes follow
Thiers (continuously updated).
DNA extraction, polymerase chain reaction (PCR) and sequencing
Genomic DNA (for a molecular phylogeny) was extracted from 100 mg of dried basidiome of each of the
species with the help of InstaGeneTM Matrix Genomic DNA isolation kit (Biorad, USA) following the
manufacturers instructions. The nrITS gene region was amplified with primer pairs ITS5 and ITS4 (White et al.
1990). Similarly, the nrLSU gene region was amplified with primer pairs LR0R and LR7
(http://www.biology.duke.edu/fungi/mycolab/primers.htm). PCR was performed in volumes of 25 L
containing 2.5 L of 10x assay buffer (100 mM TrisCl; pH 8.3, 500 mM KCl, 15 mM MgCl2), 200 M dNTP
mix (Bangalore Genei, Bangalore, India), 10 picomoles of primer, 1.0 unit of Taq DNA polymerase (Bangalore
Genei), and 30 ng of template DNA. Then PCR-amplification was done with a thermal cycler (Eppendorf,
Germany) programmed for 2 min at 94C, followed by 35 cycles of 45 sec at 94C, 1 min at 55C, 1 min at
72C and a final stage of 10 min at 72C for ITS region. The PCR condition for nLSU was as follows: 5 min at
95C, followed by 30 cycles of 1 min at 95C, 30s at 52C (for D1D2 region), 2 min at 72C and a final 7 min
extension step at 72C. The PCR products were purified using the QIAquick PCR Purification Kit (QIAGEN,
Germany). Both strands of the PCR fragment were sequenced on the 3730xl DNA Analyzer (Applied
Biosystems, USA) using the amplifying primers. The DNA sequence of the reverse strand was edited with
Sequence Navigator version 1.0.1 (Applied Biosystems). The final consensus sequences were deposited at
GenBank to procure the accession numbers (KY412776 for LSU of Aureoboletus nephrosporus and KY412777
for ITS of Strobilomyces mirandus).
Phylogenetic analysis
Phylogenetic analyses based on ITS and LSU sequences data were carried out to establish the phylogenetic
placement of our isolated taxa. Reference sequences and outgroups were selected from the relevant literature
and GenBank. Alignment were performed using CLUSTAL W (http://www.ebi.ac.uk/clustalw/) and
phylogenetic analyses were conducted in MEGA 6.0 (Tamura et al. 2013). No manual editing was done within
the alignment. The evolutionary history was inferred by using the Maximum Likelihood method based on the
Kimura 2-parameter model (Kimura 1980). One thousand bootstrap replicates were analysed to obtain nodal
support values. The European material of Boletus edulis was chosen as out group taxon in our ITS-based
analysis whereas, Phylloporus rhodoxanthus (North American sample) and Xerocomus subtomentosus
(European sample) were chosen as outgroups in our LSU-based analysis.
RESULTS
Phylogeny
Our LSU-based phylogenetic analysis (Fig. 1) with 22 LSU sequences (including the present species)
resolved genus Aureoboletus with full support. The sequence (GenBank accession no. KY412776) derived from
Indian collection of Aureoboletus nephrosporus G. Wu & Zhu L. Yang is clustered with the sequences derived
from its Chinese counterpart (represented by GenBank accession numbers KT990516 and KT990517) showing
the conspecificity (100% identity in BLAST search) with strong support (BS value). Similarly, our ITS based
phylogeny (Fig. 2) clearly supports the existence of two distinct clades (Clade A and Clade B) in the genus
Strobilomyces which are also reported by the earlier workers like, Gelardi et al. (2012) and Antonin et al.
(2015). Clade A includes Strobilomyces confusus Singer, Strobilomyces seminudus Hongo, Strobilomyces
verruculosus Hirot. Sato where basidiospores are never with reticulation. Distinguishingly, Clade B represented
by Strobilomyces strobilaceous (Scop.: Fr) Berk., Strobilomyces sp. (from India), S. echinocephalus Gelardi &
Figure 1. Phylogram of DC 16-29 (Aureoboletus nephrosporus G. Wu & Zhu L. Yang, in bold and red font) inferred from
Maximum Likelihood analysis of nrLSU sequences using MEGA 6.0.
Figure 2. Phylogram of DC 16-27 (Strobilomyces mirandus Corner, in bold and red font) inferred from Maximum
Likelihood analysis of nrITS sequences using MEGA 6.0.
Vizzini., S. pteroreticulosporus Antonn & Vizzini, S. mirandus where basidiospores are typically reticulate
(Gelardi et al. 2012, Antonin et al. 2015). The sequence (GenBank accession no. KY412777) from our present
Indian material of Strobilomyces (DC 16-27) showing conspecificity (99% identity in BLAST search) is
clustered with the sequence derived from S. mirandus from Japan with strong support (bootstrap value 99).
Taxonomy
Aureoboletus nephrosporus G. Wu & Zhu L. Yang, Fungal Diversity, DOI 10.1007/s13225-016-0375-8
(Figs. 3 & 4)
Pileus 4570 mm diam, subhemispherical to convex, sometimes with a broad umbo when young; surface
mat, subvelvety, reddish brown (8D6) or brownish red to greyish red (10D65) when dry, turning dark brown
(8F8) with KOH and greenish grey (26E2) with FeSO4; margin with sterile flap of tissue. Pore surface yellow
(2A8), becoming dingy with time, unchanging when bruised; pores 0.7 per mm in mature basidiomata, rounded
to angular, compound. Tubes up to 50 mm long, subdecurrent to decurrent, light yellow (2A5). Stipe 90170
715 mm, central, cylindrical, tapering at base; surface covered with striations; light yellow (5A6) to yellow
ochre (5C7), olive (3E3) or greenish black when bruised. Context pale yellow (3A3) at pileus, turning golden
yellow (5B7) with KOH; pastel yellow (3A4) or darker at stipe, turning reddish brown (8D8) with KOH.
Figure 3. Aureoboletus nephrosporus G. Wu & Zhu L. Yang: A & B, Young and mature fresh basidiomata in field; C, Pore
surface; D, Pileipellis; E & F, Pleurocystidia; G, Caulocystidia; H, Basidiospores. Bars. [Scale: DE, 50 m; FH, 10 m]
Basidiospores 8.59.5(10.8) 5.66.2(6.7) m, (Q = 1.411.53(1.71)), ovoid to ellipsoid or nephroid,
inequilateral, smooth under a light microscope. Basidia 3755 1013 m, 4-spored, clavate. Pleurocystidia
5070 913 m, emergent up to 60 m, fusoid to ventricose, mostly with thick covering, rarely thin walled.
Subhymenial layer up to 25 m thick, hyphal. Tube edge fertile with basidia and cystidia. Hymenophoral trama
mostly parallel to subparallel or sometimes interwoven. Pileipellis a trichodermis, up to 200 m thick,
composed of erect hyphae of slightly inflated cells; terminal cells 2344 7.210 m, cylindrical to
subcylindrical, sometimes subfusoid. Stipitipellis up to 160 m thick, composed of hyphae and cystidia in
several clusters; basidia not observed; caulocystidia 4055 820 m, broadly ventricose to lanceolate, clavate
to subclavate or cylindrical with fusoid apex.
Figure 4. Aureoboletus nephrosporus G. Wu & Zhu L. Yang: A, Basidiospore; B, Pleurocystidia without covering of
refractive substance; C, Pleurocystidia with covering of refractive substance; D, Basidia; E, Pileipellis; F, Caulocystidia.
[Scale: AF, 10 m]
Distribution: China and India (Sikkim).
Specimen examined: India, Sikkim, South district, Maenum Wild life Sanctuary (Maenum top 2), 2315 m,
N271918.7" E882207.9", under Quercus sp., 21st August 2016, D. Chakraborty & K. Das, DC 16-29 (CAL).
Note: The combination of macro- and micromorphological characters like fleshy tubulose basidiomata with
reddish, dry or mat pilear surface, bright yellow pore surface, unchanging (on bruising) context color and
smooth nephroid basidiospores place the Indian collection under the genus Aureoboletus. Further, A.
nephrosporus is micromorphologically distinct from rest of the species of this genus by the presence of nephroid
basidiospores and surface of pleurocystidia being covered with a thick layer of a strongly refractive substance
which is often dissolved in KOH (Wu et al. 2016). Morphology of the Indian material [except the stipe (which is
longer) and pleurocystidia (which is smaller)] is mostly in conformity with that of its counterpart from China.
Moreover, our LSU-based phylogeny strongly shows the conspecificity of our collection to Aureoboletus
nephrosporus (represented by KT990516 in Fig. 1), the Chinese counterpart both in morphology and phylogeny.
But, the Chinese material shows somewhat different altitudinal variation (collected from subtropical belt: 1700
m). Another Chinese species, Aureoboletus zangii X.F. Shi & P.G. Liu (represented by JQ734420 to JQ734422
in Fig. 1) is quite similar to A. nephrosporus, however, the former differs by its viscid pileus and stipe, fox red
or English red colored stipe-surface and narrower basidiospores (34 m) (Wu et al. 2016) from the latter.
Aureoboletus thibetanus (Pat.) Hongo & Nagas. (the only other species from this genus reported from India) can
easily separated from A. nephrosporus by presence of strongly reticulate and highly glutinous pileus surface
(Sharma et al. 2005).
Strobilomyces mirandus Corner, Boletus in Malaysia: 61 (1972). (Figs. 5, 6)
Pileus 3775 mm. diam.; convex, vivid yellow to sunflower yellow (3A84A7); surface densely squamulose
with flat to bluntly pyramidal or wart-like squamules, which are more dense towards centre, brown (7EF4) to
blackish brown, surface brownish red (89C8) with KOH; margin wavy with cottony veiler remnant, vivid
yellow (3A8). Pore surface covered by partial veil when young, smoky white; depressed near stipe, brownish
initially then blackish on bruising; pore 2/mm, simple, angular. Tube 1216 mm long, adnatesinuate, chalky to
smoky white, then greyish brown (7D3). Stipe 5590 1728 mm, central, concolorous with pileus, with the
cotton-like annular region; surface with reticulations on the upper portion of the annular region, striations with
pit like openings throughout the rest; basal mycelium greyish magenta (13B3). Context solid in pileus and stipe;
context in pileus chalky white but immediately turning greyish orange (7C4) and then brownish grey (7E2) or
brownish black on exposure, turning reddish orange to brownish orange (7BC8) with KOH, greenish grey to
dull green (26B226D3) with FeSO4 in pileus, stipe context chalky white turning dark brown to brownish black
with an intermediate orange brown to red brown coloration when exposed. Spore print blackish brown. Taste
indistinct. Odour indistinct.
Basidiospores 7.28.8(10.5) 66.9(7.7) m, (Q = 1.091.26(1.45)), mostly subglobose or broadly
ellipsoid, ornamented, forming complete reticulation. Basidia 3848 1016 m, four-spored, clavate.
Pleurocystidia 6090 1520 m, fusoid to ventricose or ventricose rostrate, brown pigmented. Hymenophoral
trama divergent. Pileipellis a trichodermis, 200400 m thick, composed of erect to suberect hyphae of slightly
inflated to elongated cells, with minute incrustations; terminal cells 2275 8112 m, cylindrical to
subcylindrical. Stipitipellis hyphal, same as pileal hyphae; cystidia in several clusters; basidia not observed;
caulocystidia 3253 0113 m, broadly ventricose to lanceolate, clavate to fusoid.
Distribution: Malaysia, Japan, China and India (Sikkim).
Specimens examined: India, Sikkim, South district, Rabangla, 1985 m, N271514.8 E882303.7, under
Lithocarpus sp., 20th August 2016, D. Chakraborty & K. Das, DC 16-27; ibid., South district, Kewzing, 1888 m,
N271746.5 E882126.6, under Lithocarpus sp., 21st August 2016, D. Chakraborty & K. Das DC 16-028
(CAL); Uttarakhand, Rudraprayag, Kund, 1160 m, under Cinnamomum tamala and Quercus glauca, 13th
August 2007, K.C. Semwal, KCS 1102; 12th August 2016, K.C. Semwal, KCS 2556.
Note: Unlike other (Asian or extralimital) species of Strobilomyces, present one i.e. S. mirandus has entirely
distinct combination of macromorphological features like golden yellow to yellowish orange or yellow colored
pileus (distinct from any known species of this genus) with blackish brown squamules, pileus margin with
yellow veilar remnants, smoky white pore surface (turning brownish after bruising), yellow stipe with cottony
annular region. Morphological features of Indian collection is on conformity to its counterparts reported from
other Asian countries like Malaysia, Japan or China (Corner 1972, Sato et al. 2005, Ge &Yang 2005) and the
combination of our ITS-based phylogeny and morphological studies further warrants its wider range of
distribution in different countries of Asia. Moreover, the occurrence of S. mirandus strengthens the
representation of this genus in India with eight species (Strobilomyces strobilaceus (Scop.) Berk., S. nigricans
Berk., S. polypyramis Hook. f., S. velutipes Cooke & Massee, S. annulatus Corner, S. mollis Corner, S. mirandus
Corner and Strobilomyces sp. (unpublished and represented by Strobilomyces sp. in our ITS-based tree: Fig.
2).
Figure 5. Strobilomyces mirandus Corner: AC, Young and mature fresh basidiomata in field and in basecamp; D, Pore
surface; E, Pileipellis; FG, Pleurocystidia; H, Basidiospores; I, SEM of basidiospores. [Scale: E, 50 m; FI, 10 m]
In the western Himalaya, S. mirandus was collected from the present locality (Kund Forest) very first time in
2007. That time the forest was so fruitful with abundant suitable hosts, leaf litter and humus and about 25
mushroom species had been encountered in a small area in a day on August 13, 2007, but in the year of 2016
when the forest area has been visited again on August, 12, it was observed that the forest trees were so less with
very less leaf litter and humus. Only 3 wild mushrooms species has been encountered. So from August 2007 to
August 2016 several factors dramatically altered the scenario of the concerned forest. It has been assumed that
the several anthropogenic activities (construction of hydropower dam and subsequent shifting of the respective
national highway) which developed in recent years are the cause of the declination of the mushroom diversity in
this area. Moreover, the host trees are likely to be cut shortly in order to shift the national highway. Therefore,
we are afraid that there will be the declination of S. mirandus from Western Himalaya of India once all these on-
going construction activities will be over.
Figure 6. Strobilomyces mirandus Corner: A, Basidiospore; B, Basidia; C, Pleurocystidia; D, Caulocystidia; E, Pileipellis.

[Scale: AE, 10 m]
ACKNOWLEDGEMENTS
The authors are grateful to the Director, Botanical Survey of India (Kolkata) and the Scientist-in-Charge,
Botanical Survey of India (Gangtok) for providing facilities. DC & KD are thankful to the entire forest
department of Sikkim for allowing us (KD & DC) to undertake the macrofungal surveys to the restricted areas
of South district of Sikkim. Assistance (to KD & DC) rendered by Subhash Pradhan (BSI, Gangtok) in the field
is duly acknowledged.
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ISSN (E): 2349 1183
ISSN (P): 2349 9265
4(1): 7176, 2017
DOI: 10.22271/tpr.2017.v4.i1.010
Research article
Callus induction and organogenesis from Tinospora formanii

Udayan and Pradeep: A rare endemic plant
Sheema Dharmapal P.1*, M. Najla1, E. S. Swetha1, P. S. Udayan1 and K. K. Elyas2
1
Department of Botany, Sree Krishna College, Guruvayur, Ariyannur P.O., Thrissur-680102, Kerala, India
2
Department of Biotechnology, University of Calicut, Calicut University P.O., Malapuram-673635, Kerala, India
*Corresponding Author: sheemamohandaas@gmail.com [Accepted: 16 February 2017]
Abstract: Tinospora is a genus within the family Menispermaceae renowned for its medicinal
properties. The present study was undertaken to develop an effective protocol for optimum callus
induction and organogenesis in Tinospora formanii, a rare endemic plant reported from Western
Ghats of Kerala. Data of number of days required for callus induction, callus induction percentage,
callus morphology, callus fresh weight and number of days required for shoot regeneration were
recorded. Nodal segments from one year old plants were used as explants for callus induction and
shoot regeneration. After proper treatment of explants with surface sterilants, 70% (v/v) ethyl
alcohol and 0.1% (w/v) mercuric chloride (HgCl2), it was transferred to Murashige and Skoogs
(MS) medium supplemented with different concentration of 6-Benzylaminopurine (0.5, 1.0, 2.0,
3.0 and 5.0 and 8.0 mg.L-1) with 1-Naphthaleneacetic acid (0.2 mg.L-1). All these hormonal
combinations gave callus. Maximum callus induction (96%) was observed on medium containing
1.0 mg.L-1 6- Benzylaminopurine (BAP) with 1-Naphthaleneacetic acid (NAA) 0.2 mg.L-1.
Number of days taken for callus induction in this medium was 50.28. Organogenic creamy brown
friable callus developed. The fresh weight of the callus was 4.750.08 gm after 60 days of culture.
Small green shoot buds developed from this callus after 65 days of culture which developed into
shoots of about 2.53.0 cm length.
Keywords: Callus - Nodal explants - Menispermaceae - Tinospora formanii.
[Cite as: Sheema DP, Najla M, Swetha ES, Udayan PS & Elyas KK (2017) Callus induction and organogenesis
from Tinospora formanii Udayan and Pradeep: A rare endemic plant. Tropical Plant Research 4(1): 7176]
INTRODUCTION
Plant based drugs are being increasingly preferred in medical science. The medicinal properties of plants are
attributed to the presence of biologically active compounds like terpenes, alkaloids, polyphenols, flavonoids etc.
Several phytochemical surveys have been carried on for detecting diverse groups of naturally occurring
phytochemicals. Annual growth rate between 515% for trade of plant based drugs and raw materials is
indicative of growing demand for herbal drugs. It is therefore, necessary to select, characterize, multiply and
conserve genetically superior planting material of important medicinal plants for assured uniformity and desired
quality. The biotechnological tools can play an important role in selecting, multiplying and conserving the
critical genotypes of species. Further, biotechnological tools and techniques offer scope for multiplication and
genetic enhancement of desirable genotypes and in vitro plant cell culture systems have potential for
commercial exploitation of secondary metabolites from the plant (Manisha et al. 2012).
The genus Tinospora belonging to the family Menispermaceae has about 32 species distributed in Tropical
Africa, Madagascar, Asia to Australia and Pacific Islands (Kubitzki et al. 1993, Mabberley 2008). In India, the
genus is represented by 4 species. Among them, 2 species Tinospora cordifolia (Willd.) Miers. ex Hook. f. &
Thoms. and T. sinensis (Lour.) Merr. are known to occur in South India. The other two species T. crispa (L.)
Miers. ex Hook. f. & Thoms. and T. glabra (Burm. f.) Merr. are reported from Northeast India and Andaman
Islands (Pramanik & Gangopadhyay 1993).
Tinospora is a genus within Menispermaceae reputed for its medicinal properties (Pathak et al. 1995).
Species of the Tinospora genus have prominent roles in the traditional medicinal practices of Australia, Africa
Sheema et al. (2017) 4(1): 7176
and Asia. The great majority of compounds isolated from Tinospora species have been furanoid diterpenes of
the clerodane type, and their glycoside derivatives (Hungerford et al. 1998).
Tinospora formanii Udayan & Pradeep, a new species of the family Menispermaceae has been reported from
the Western Ghats of Thrissur district, Kerala, South India (Udayan & Pradeep 2009). Tinospora formanii is a
woody dioecious climber growing at an altitude of 500650 m and is endemic to Kerala. Tinospora formani is
allied to T. smilacina Benth. with smooth, shining, papery bark peeling off into scales and prominent leaf-
scars. Leaves are alternate, ovate to elliptic-lanceolate, coriaceous, and glabrous with reticulation more
prominent on lower surface. Female inflorescence is greenish-yellow, glabrous, stout compound elongated
pseudoracemes with six petals. Drupes are globose and red when mature.
Tinospora formanii is a rare, endemic plant. Therefore, the present experiment has been designed to develop
an efficient protocol for callus culture and organogenesis of T. formanii from nodal explants under in vitro
conditions.

Nodal explants collected from one year old vegetatively propagated disease free plants were cultured on MS
media for callus initiation. The explants were first washed in running tap water for 30 minutes and then soaked in
distilled water for 30 minutes. The explants were then treated with Tween 20 emulsifier solution (0. 01% v/v) for five
minutes. After distilled water wash for 23 times, the explants were taken to the laminar air flow chamber for further
sterilization. The nodal explants were initially sterilized in ethyl alcohol (70 %) for 30 seconds followed by 0.1 %
mercuric chloride for five minutes. The treated explants were then washed four times with sterile distilled water to
make them free from sterilants.
Finally using sterile forceps, explants were transferred to sterile Petri dishes and the nodes were cut into
vertical sections of 1.01.5 cm length with sterile scalpel and inoculated into the media. The nodes were then
cultured on semi solid MS medium (Murashige & Skoog 1962) supplemented with 3% (w/v) sucrose and 0.8%
(w/v) agar. The pH of the media was adjusted and maintained to 5.8 by using 0.1 N NaOH or 0.1 N HCl after
the addition of growth regulators. The medium was autoclaved at 121C, 15 psi pressure for 15 min. All these
cultures were incubated in growth room at 25 2 C under 16 hour photoperiod at a relative humidity of 5060
percent with a light intensity of 3000 lux provided by cool white fluorescent lamps.
Single nodal explants inoculated in each test tube having MS medium supplemented with different
combinations of auxins, BAP (0.5, 1.0, 2.0, 3.0, 5.0 and 8.0 mg.L-1) with NAA (0.2 mg.L-1) was used to find out
a suitable concentration for callus induction and proliferation. MS without growth regulators served as control.
Subcultures were done every 20 days interval into fresh medium. Observations like percentage of callus
induction, number of days required for callus initiation, colour and texture of callus were recorded. To find out
the growth of callus on different concentrations of above mentioned growth regulators, the callus were harvested
after 60 days of initiation and measured in terms of fresh and dry weights.
The experiments were carried out in completely randomized block design and repeated three times each with
12 replications for each of the treatment. The observations were tabulated and statistical analysis was carried
out, as per Sukhatme & Amble (1985) and the results were interpreted.
RESULTS
The effect of auxins on callus initiation was studied by culturing nodal explants on MS medium
supplemented with BAP and NAA in combinations. The results are summarized in table 1. It is observed that
nodal segments showed highest percentage of survival when treated for 5 min with 0.1% HgCl2. Cultures raised
on basal medium (without any growth regulators) served as control. The results showed that explants failed to
produce callus on hormone free medium tested for callus induction. From the results, it is clear that the
concentration of 1.0 mg.L-1 BAP with NAA 0.2 mg.L-1 was optimum for callus induction. 96% of explants
responded at this concentration and the callus was initiated within 5 days of inoculation (Fig. 1). After 23 days
of inoculation, cultured explants were enlarged and became swollen, however, they remained green in colour.
Then the explants were gradually covered by thin layer of cream friable callus. Callus continued to proliferate
into a large mass. The maximum fresh weight observed at this concentration of phytohormones was 4.750.08
gm after 60 days of culture (Table 2). Large creamy brown friable organogenic callus developed. After 65 days
of culture, small green buds developed from this callus. This green bud developed into 2.53.0 cm long shoots
within 14 days (Fig. 1).
Sheema et al. (2017) 4(1): 7176
Table 1. Effect of MS media supplemented with BAP and NAA on callus induction from nodal explants of Tinospora
formanii Udayan & Pradeep.
MS + Plant growth regulators % of explants showing Number of days for
Treatments
(mg.L-1 ) callus formation callus induction
T0 MS basal (control) 0.00 0.00
T1 BAP 0.5 + NAA 0.2 51.0 0.92d 18.0 1.53b
T2 BAP 1.0 + NAA 0.2 96.0 0.95a 5.0 0.28a
T3 BAP 2.0 + NAA 0.2 82.6 0.69b 19.3 0.57bc
T4 BAP 3.0 + NAA 0.2 75.3 1.66 c 21.6 1.52cd
T5 BAP 5.0 + NAA 0.2 26.6 3.33e 25.0 1.33e
T6 BAP 8.0 + NAA 0.2 0.00 0.00
Note: Values are means S.E. of three independent experiments, each consisted of 12 replicates per treatment. Treatment
means followed by same letter within column are not significantly different from each other at P= 0.05; comparison by
Duncans Multiple Range Test.
Figure 1. Callus development and organogenesis in MS supplemented with 1 mg.L-1 BAP and NAA 0.2 mg.L-1: A, Mother
plant; B, Nodal explant; C, Swelling at basal part of nodes; D, Callus initiation after 5 days of inoculation; E, Callus
proliferation; F, Development of green shoot but after 65 days of inoculation; G & H, Shoot development from callus.
Sheema et al. (2017) 4(1): 7176
Table 2. Fresh and dry weights of callus cultured from nodal explants of Tinospora formanii Udayan & Pradeep on MS
media supplemented with BAP and NAA.
MS + Plant growth
Type of callus Fresh weight (gm) Dry weight (gm)
regulators (mg.L -1)
c
BAP 0.5 + NAA 0.2 Soft yellow watery 0.87 0.01 0.34 0.33e
BAP 1.0 + NAA 0.2 Creamy brown friable 4.75 0.08a 2.45 0.13a
BAP 2.0 + NAA 0.2 Brown friable 2.36 0.10b 1.53 0.06b
BAP 3.0 + NAA 0.2 Creamy yellow compact 0.90 0.01c 0.74 0.02c
BAP 5.0 + NAA 0.2 Brown compact 0.35 0.02d 0.12 0.01d
Note: Values are means S.E. of three independent experiments, each consisted of 12 replicates per treatment.
Treatment means followed by same letter within column are not significantly different from each other at P = 0.05;
comparison by Duncans Multiple Range Test.
82.6 % response was obtained with 2.0 mg.L-1 BAP and NAA 0.2 mg.L-1 and the callus developed after 19.3
days of inoculation in culture. At lower concentration of 0.5 mg.L-1 BAP with NAA 0.2 mg.L-1, soft yellow
watery callus developed. 51 % of callus initiated after 18 days of inoculation in culture medium. At higher
concentrations of BAP with NAA 0.2 mg.L-1, compact callus was obtained. Creamy yellow compact callus
developed with 3.0 mg.L-1 BAP and NAA 0.2 mg.L-1 after 21.6 days whereas brown compact callus developed
with 5.0 mg.L-1 BAP and NAA 0.2 mg.L-1. At higher concentration of 5 mg.L-1 BAP with NAA 0.2 mg.L-1, it
was observed that the number of days taken for callus initiation was increased to 25 days and the percentage of
response was reduced to 26.6%. At a concentration of BAP 8 mg.L-1 with NAA 0.2 mg.L-1 there was no
callusing (Fig. 2).
Figure 2. Different types of callus formation: A, Soft yellow watery callus; B, Brown friable callus; C, Creamy brown
friable callus; D, Creamy yellow compact callus; E, Brown compact callus.
Sheema et al. (2017) 4(1): 7176
DISCUSSIONS
Frequency of callus induction is influenced by many factors: media composition, explant source, genotype
and environment. Callusing was obtained from nodal explants on Murashige - Skoog (MS) medium
supplemented with varying concentrations of BAP with NAA 0.2 mg.L-1. In vitro propagation in T. cordifolia
was reported from nodal segments through axillary shoot proliferation (Raghu et al. 2006, Gururaj et al. 2007).
Singh et al. (2009) cultured vegetative parts such as stem, leaf and nodal explants from an elite in vivo grown
mature plant of Tinospora cordifolia on MS medium supplemented with different hormonal concentrations and
reported callus induction and organogenesis. MS medium containing 1.0 mg.L-1 BAP combined with NAA 0.2
mg.L-1 was found to be optimum for callus induction in T. formanii. Similar findings have been reported in
Tectona grandis where MS medium supplemented with 0.5 mg.L-1 NAA and 1.5 mg.L-1 BAP produced compact
and fibrous callus after two weeks (Egodawatta et al. 2014). Hormonal regulation of auxin and cytokinin
balance is a key factor in the control of cell division in tissue culture. Media composition plays a key role in
morphogenesis as nutritional requirement for optimal growth of a tissue under in vitro conditions varies with
species (Bhojwani & Rajdan 1996). The colour and texture of callus formed varied with different concentration
of BAP. As reported by Roberts et al. (1984) different tissue types within the same plant or the same tissue at
various stages of development produce different responses. With higher concentration of BAP, number of days
taken for callus induction increased and the percentage of response decreased. Frequent sub culturing of callus
was done to fresh medium to prevent browning and to enhance the survival rate. Brown exudates promote dying
of cells by interfering with the metabolic activities of cells.
CONCLUSION
An efficient protocol for callus culture and indirect organogenesis of Tinospora formanii Udayan & Pradeep
was established in the present study. In vitro culture can be used to propagate and conserve such rare and
endemic plants. Callus culture can be employed to reduce the over exploitation of plants from their in situ
habitats and harvest secondary metabolites from rare medicinal plant. The medicinal properties of this plant are
to yet be scientifically explored in a systematic way. This protocol may open a new way to facilitate secondary
metabolites production and isolation of pharmaceuticals from callus without harvesting the whole plant.
ACKNOWLDGEMENTS
The authors express gratitude to UGC, Government of India for the financial support as research grant. The
authors are also thankful to Dr. G. Jayakrishnan, Head of the Department, Botany, Sree Krishna College,
Guruvayur for his valuable suggestions and support.
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ISSN (E): 2349 1183
ISSN (P): 2349 9265
4(1): 7780, 2017
DOI: 10.22271/tpr.2017.v4.i1.011
Short communication
Eleocharis atropurpurea (Retz.) J. Presl & C. Presl and Eleocharis

acutangula (Roxb.) Schult. (Cyperaceae): Two new distributional
records for Andaman and Nicobar Islands, India
Sanjay Mishra*, Vivek C. P., Gautam Anuj Ekka and Lal Ji Singh
Botanical Survey of India, Andaman and Nicobar Regional Centre, Port Blair-744102,
Andaman and Nicobar, India
*Corresponding Author: sanjayalld74@gmail.com [Accepted: 18 February 2017]
[Cite as: Mishra S, Vivek CP, Ekka GA & Singh LJ (2017) Eleocharis atropurpurea (Retz.) J. Presl & C. Presl
and Eleocharis acutangula (Roxb.) Schult. (Cyperaceae): Two new distributional records for Andaman and
Nicobar Islands, India. Tropical Plant Research 4(1): 7780]
Eleocharis R.Br. is a cosmopolitan genus of the family Cyperaceae which comprises about 200 species,
distributed in tropical, subtropical, and temperate regions of the world (Gonzalez-Elizondo & Peterson 1997,
Mabberley 2009). Prasad & Singh (2002) mentioned about 21 species of this genus from India. During field
expedition for the project of Flora of Kyd, James and Pitman islands, the authors came across with interesting
specimens of Eleocharis from water logged field in Shoal bay area of South Andaman. On Critical study,
perusal of literature and consultation of the herbarium at PBL, the specimens were identified as
Eleocharis atropurpurea (Retz.) J. Presl & C. Presl, and Eleocharis acutangula (Roxb.) Schult. A thorough
scrutiny of the literature (Rao 1986, Lakshminarasimhan & Rao 1996, Mathew 1998, Pandey & Diwakar 2008,
Murugan et al. 2016) revealed that Eleocharis atropurpurea (Retz.) J. Presl & C. Presl has so far not been
reported from the Andaman and Nicobar Islands. Kamble (2013) mistakenly determined one Eleocharis
specimen collected from Andaman (Mayur Y Kamble 30664) as Eleocharis acutangula (Roxb.) Schult.
However, investigation on the particular specimen housed at PBL revealed that the specimen is actually of
Eleocharis spiralis (Rottb.) Roem. & Schult. Hence, our collection of Eleocharis acutangula (Roxb.) Schult.
from Shoal bay forms the first record of the species from the Andaman and Nicobar Islands.
Eleocharis atropurpurea (Retz.) J. Presl & C. Presl in C. B. Presl, Reliq. Haenk. 1: 196. 1828; C.B. Clarke in
Hook. f. Fl. Brit. Ind. 6: 627. 1893; Bhattacharya, Bull. Bot. Surv. In. 6: 208. 1964; Panigrahi et al., Bull. Bot.
Surv. Ind. 11: 111. 1969; Rao & Verma, Cyp. N. E. Ind. 25. 1982; Karthik. et al., Fl. Ind. Enum. Monocot. 48.
1989; Cook, Aqu. Wet. Pl. Ind. 127. f. 121. 1996. Scirpus atropurpureus Retz. Obs. Bot. 5: 14. 1789. (Fig. 1)
Annual herbs. Rhizome absent; roots fiberous. Stems 525 cm 0.251.0 mm, green, glabrous, erect, tufted,
slender. Leaves absent; sheaths 2, ca. 1.5 cm long, obliquely truncate at apex, membranous, purplish below, pale
brownish above, glabrous. Inflorescence solitary with terminal spikelets. Bracts absent. Spikelets ca.5.0 2.5
mm, ovoid to oblong-ovoid, lanceolate, subterete, brownish, turning fuscous dark in fruit, acute, wider than
stem, many-flowered. Glumes ca. 1.5 1.0 mm, membranous, elliptic, oblong, loosely imbricating, muticous,
keeled; keels green, sides hyaline, nerveless with purplish bands on both sides of the green keel. Perianth-
bristles 7 in numbers, ca. 1.0 mm long, slender, white, translucent, antrorsely scabrid, shorter than a nut,
sometimes much reduced or absent. Stamens 2; anthers ca. 0.5 mm long, yellow, oblong, glabrous; filaments ca.
1.0 mm long, slender, glabrous, longer than anthers. Pistils ca. 1.5 mm long; ovaries ca. 0.3 mm long, ovate,
glabrous; styles ca. 0.5 mm long, bicleft, base discoid, glabrous; stigmas 2, ca. 0.7 mm long, glabrous,
acuminate. Nuts ca. 0.7 mm 0.5 mm, obovoid, biconvex, smooth, glossy, shining black on maturity, stipitate
at the base, truncate at the apex with grayish and flattened appendage, surface cells obscure, subquadrate,
persistent minute style base, persistent perianth bristle whitish, shorter than to slightly exceeding the nut.
Flowering & Fruiting: JulyNovember.
Habitat: Grassy areas on the edges of waterlogged soil, open wet places.
Mishra et al. (2017) 4(1): 7780
Figure 1. Eleocharis atropurpurea (Retz.) J.Presl & C.Presl: A, Habit; B, Cross section at distal end of culm below the
spikelet; C, Culms with spikelets; D, Glume, adaxial view; E, Glume, abaxial view; F, Nut.
Mishra et al. (2017) 4(1): 7780
Distribution: Pantropical and subtropical distribution, occurring throughout Africa, Middle East, Afghanistan,
China, Japan and Taiwan, Southeast Asia to Australia, North, Central and South America. India: Andaman and
Nicobar Islands, Jammu and Kashmir, Delhi, Himachal Pradesh, Punjab, Rajasthan, Uttar Pradesh, West
Bengal, Assam, Madhya Pradesh Gujarat, Maharashtra, Karnataka, Andhra Pradesh, Kerala, Tamil Nadu.
Specimens examined: India; Andaman and Nicobar Islands, South Andaman, Shoal Bay-12, near Government
Secondary School, 6 m (1150.964' N and 9243.81' E), 12.08.2016, Sanjay Mishra, C.P. Vivek & Gautam Anuj
Ekka 32692 (PBL).
Taxonomic Notes: Eleocharis atropurpurea (Retz.) J. Presl & C. Presl is easily confused with Eleocharis
geniculta (L.) Roem. & Schult., due to similar morphological appearance. But it can be distinguished from the
later by the presence of tightly imbricate glumes with a green keel and whitish perianth bristles during fruiting,
whereas, in E. geniculata glumes are loosely imbricate, green keel absent and perianth bristles are brownish
while fruiting.
Figure 2. Eleocharis acutangula (Roxb.) Schult.: A, Habit; B, Cross section at distal end of culm below the spikelet; C,
Culms with spikelets; D, Glume, adaxial view; E, Glume, abaxial view; F, Nut.
Eleocharis acutangula (Roxb.) Schult. in Roem. & Schult., Syst. Mant. 2: 91. 1824; Kern in van Steenis, Fl.
Males. 1, 7: 525. 1974; Rao & Verma, Cyp. N.E. Ind. 23.f.9-9a. 1982; Karthik et al., Fl. Ind. Enum. Monocot.
48. 1989; Cook, Aqu. Wet. Pl. Ind. 127.f. 121 a-d. 1996; Clarke in Hook.f. Fl. Brit. Ind. 6: 626. 1893. Scirpus
acutangulus Roxb. Fl. Ind. 1: 216. 1820. (Fig. 2)
Perennial herbs with ca. 1.0 cm long rhizome. Stems triquertrous, acute at apex, 3075 cm 35 mm.
Leaves reduced to few basal sheaths; sheaths 23, ca. 8.0 cm long, loose, purplish, acute, oblique at mouth, soon
disintegrating, membranous, tubular, truncate. Inflorescence solitary with terminal spikelets. Bracts absent.
Spikelets ca. 3.5 cm 2.5 mm, terete, cylindrical, slightly thicker than stem, acute, glaucous, green-
stramineous, 832 flowered; rachilla persistent. Glumes ca. 6.0 4.0 mm, coriaceous, tightly imbricating, ovate,
oblong, scarious margined, stramineus, red-brown dotted, several, striate, acute, obtuse at apex, spiral, caduceus,
mid-nerves visible, brown with many faint nerves on both sides. Perianth-bristles 6, very unequal, ca. 2.0 mm
long, slightly broader at apex, brown, smallest one half to two third or as long as the nut, longest one equaling or
Mishra et al. (2017) 4(1): 7780
exceeding the beak of nut, retrorsely scabrid, acute, base rounded. Stamens 3, rarely 2; anthers ca. 2.0 mm long,
deep brown, linear-lanceolate, ovate-oblong, muticous, glabrous, acuminate, base rounded, connective
appendage very minute; filaments ca. 4.0 mm long, slender, hyaline, longer than anther, attached with pistil to
the base. Pistils ca. 4.0 mm long; ovaries ca. 1.0 mm long, obovate, glabrous, base acuminate; styles ca. 2.0 mm
long, glabrous, dilated at base, with a constriction above the nut, slender; stigmas 3, ca. 1.0 mm long, acute,
brown, fimbriate. Nuts ca. 2.0 1.0 mm, broadly obovoid, biconvex, slightly compressed, constricted at apex
into a short but distinct neck, glossy, brownish-stramineus, surface cells transversely oblong in 1520 vertical
rows on each face.
Flowering & Fruiting: JulyFebruary.
Habitat: Shallow stagnant water in ditches and margin of ponds.
Distribution: Nepal, Myanmar, China, Sri Lanka, Malaysia, Cambodia, Laos, Philippines, Japan, Tropical
Africa, Madagascar, America and Australia, India: Andaman and Nicobar Islands, Punjab, Rajasthan, Uttar
Pradesh, Madhya Pradesh, West Bengal, Assam, Maharashtra, Karnataka, Andhra Pradesh, Kerala and Tamil
Nadu.
Specimens examined: India; Andaman and Nicobar Islands, South Andaman, Shoal bay-12, near Government
Secondary School, 6 m (11 50 00.97 N and 9244 00.81 E), 12.08.2016, Sanjay Mishra, C.P.Vivek &
Gautam Anuj Ekka 32693 (PBL).
Taxonomic Notes: Eleocharis acutangula (Roxb.) Schult. has some morphological similarity with Eleocharis
spiralis (Rottb.) Roem. & Schult., but can be easily distinguished from the later by its loosely imbricating
glumes which are broadly oblong or oblong ovate and obtuse to acute at apex. Whereas, glume of E. spiralis is
tightly imbricating which are broadly obovate and truncate at apex. Moreover, the stem of E. spiralis is
gradually narrowed towards the apex unlike in E. acutangula where the stem is more or less in same thickness
from base to apex.
ACKNOWLEDGEMENTS
The authors are thankful to Dr. Paramjit Singh, Director, Botanical Survey of India, Kolkata, for providing
facilities and constant support. The authors are also thankful to the Ministry of Environment, Forests and
Climate Change for providing necessary facilities and support through the Director BSI, Kolkata. Thanks are
also due to the Forest Department, Andaman & Nicobar Islands, for extending logistic support during the field
visit.
REFERENCES
Gonzalez-Elizondo MS & Peterson PM (1997) A classification of and key to the supraspecific taxa in
Eleocharis (Cyperaceae). Taxon 46: 433449.
Kamble MY (2013) Eleocharis acutangula (Roxb.) Schult.(Cyperaceae): A New Record for Andaman and
Nicobar Islands. Indian journal of Forestry 36(2): 253254.
Lakshminarasimhan P & Rao PSN (1996) Supplementary list of angiosperms recorded (19831993) from
Andaman and Nicobar Islands. Journal of Economic and Taxonomic Botany 20: 175185.
Mabberley DJ (2009) Mabberley's plant book, a portable dictionary of plants, their classification and uses, 3rd
Edition. Cambridge University Press.
Mathew SP (1998) A supplementary report on the flora and vegetation of Bay Islands, India. Journal of
Economic and Taxonomic Botany 22: 249272.
Murugan C, Prabhu S, Sathiyaseelan R & Pandey RP (2016) A Checklist of Plants of Andaman and Nicobar
Islands. Available from: http://bsienvis.nic.in/Database/Checklist-of-Andaman-Nicobar-Islands_24427.aspx
(accessed: 10 Aug. 2016).
Pandey RP & Diwakar PG (2008) An integrated checklist of plants in Andaman & Nicobar Islands, India.
Journal of Economic and Taxonomic Botany 32: 403500.
Prasad VP & Singh NP (2002) Sedges of Karnataka (India). Reprinted from Journal of Economic and
Taxonomic Botany, Additional Series No. 21. Sci. Publ., Jodhpur.
Rao MKV (1986) A preliminary report on the angiosperms of Andaman and Nicobar Islands. Journal of
Economic and Taxonomic Botany 8: 107184.
ISSN (E): 2349 1183
ISSN (P): 2349 9265
4(1): 8189, 2017
DOI: 10.22271/tpr.2017.v4.i1.012
Research article
Effects of different nitrogen forms on growth, phenolics, flavonoids

and antioxidant activity in amaranth species
Rozy Munene1*, Evans Changamu2, Nicholas Korir1 and Gweyi-Onyango Joseph1
1
Department of Agricultural Science and Technology, Kenyatta University, Nairobi, Kenya
2
Department of Chemistry, Kenyatta University, Nairobi, Kenya
*Corresponding Author: rozymunene@gmail.com [Accepted: 20 February 2017]
Abstract: Higher plants, accumulate large number of polyphenolic compounds which are believed
to act as defence compounds against different environmental constraints. Nitrogen (N) is a critical
element for plant growth, absorbed as (NH4+) and (NO3-), which affects plant growth and to some
extent contributes to secondary metabolites accumulation. Greenhouse experiment was carried out
to determine the effect of N forms on growth and phytochemical accumulation in Amaranthus
species. Two amaranth varieties; AB6 and AB7 constituted the main plot while three N forms;
ammonium, nitrate, ammonium nitrate and control (no N form) represented the subplot.
Destructive sampling was done and plant height was recorded. Folin-Ciocalteus and aluminium
trichloride methods were used to determine total phenolic content (TPC) and total flavonoid
content (TFC) respectively. DPPH (diphenylpicrylhydrazyl) radical scavenging activity assay was
used to obtain total antioxidant activity. Nitrogen forms significantly (p0.05) affected plant
height between two amaranth varieties. Under nitrate treatment, AB7 exhibited greater height
(40.2 cm) than AB6 (35.2 cm). Furthermore, N effect was more evident in AB6 variety, where by
compared to the control, NO3- as exclusive N source enhanced shoot length by 64% in AB6 and
51% in AB7 which was twice that of the NH4+ -N treated plants. Sole NH4+ and no N form
enhanced accumulation of both TFC and TPC, unlike nitrate and ammonium-nitrate mixture.
Compared to NH4+ treatment, NO3- reduced TFC by 17.4% in AB6- variety and 14.7% in AB7
variety and TPC accumulation by 23% AB6 and 20% AB7 varieties respectively.
Correspondingly, NH4+ - N form resulted to superior antioxidant DPPH scavenging activity
indicated by high scavenging activity and lower IC50 value (concentration which scavenged 50%
of the DPPH radicals). Plant height displayed a significant negative correlation with TFC and TPC
accumulation of r= 0.75 and r= 0.81 respectively. The results indicated that ammonium-induced
stress enhanced total flavonoids and phenolics accumulation; a salient phytochemical plasticity
observed during plant growth and survival trade-off in a vegetable amaranth.
Keywords: Nitrogen forms - Ammonium- Nitrate - Phenolics - Flavonoids - Amaranth.
[Cite as: Munene R, Changamu E, Korir N & Gweyi-Onyango J (2017) Effects of different nitrogen forms on
growth, phenolics, flavonoids and antioxidant activity in amaranth species. Tropical Plant Research 4(1): 81
89]
INTRODUCTION
African indigenous vegetables, including leafy amaranth occupy a very important place in the human diet in
African communities and they are believed to have some form of medicinal and therapeutic properties
(Mavengahama 2013). They are not only excellent host of numerous vitamins and mineral elements (Kwenin et
al. 2011, Habwe et al. 2009) but also reported to contain immense bioactive phytochemical compounds strongly
associated with the health and remedial benefits (Amabye 2015). Higher plants portray a considerable metabolic
plasticity by increased production and accumulation of myriad phytochemicals such as flavonoid and phenolic
compounds, enabling them survive the various biotic and abiotic stresses (Nakabayashi & Saito 2015, Altiok
2010). As the polyphenolic compounds effects positively on human health because of their antioxidative and
Munene et al. (2017) 4(1): 8189
protective properties, it achieved more interest in the last decade. Several studies have shown that
phytochemicals have antibacterial (Amabye 2015), anticancer (Mates et al. 2011); anti-aging attributes (Xiang
et al. 2011). Observational epidemiology studies have indicated that significant dietary intake of flavonoids and
phenolics are associated with a lower incidence of various cancers (Collins 2005).
Both the primary and secondary metabolism of higher plants is influenced by mineral nutrition (Caretto et al.
2015). Species growing in nutrient-poor habitats often have traits that lead to high nutrient retention and high
levels of secondary metabolites (Lillo et al. 2008). Deficiency in mineral elements such phosphorous and
potassium have been reported to up-regulate the amounts of polyphenols either as existing pools or by inducing
their de novo synthesis (Kovcik et al. 2007, Glynn et al. 2008). Nakabayashi et al. (2014) observed that
increased amount of flavonoids as a consequence of phosphorous and water limitation. Drought often causes
oxidative stress (Akula & Ravishankar 2011) and was reported to show increase in the amounts of flavonoids
and phenolic acids in Solanum species and Ligustrum vulgare (Okello 2015, Tattin et al. 2004). Phosphorous
stress was reported to elevate total phenolic and antioxidant activity in black nightshade (Ogembo 2015).
Nitrogen is a one of the most critical elements affecting plant growth and development (Zhou et al. 2011).
Unlike other elements nitrogen is metabolized by plants in two ionic forms NH4+ andNO3- (Olfati et al. 2012)
which not only affects plant growth but also nutritional quality of higher plants (Sun et al. 2014, Sabir et al.
2013). Nitrogen nutrition is of great importance as it influences both the primary and secondary metabolic
pathways thus secondary plant metabolites accumulation (Chen et al. 2011). Deficiency of crucial elements for
instance nitrogen has been found to enhance accumulation of phenolics compounds in the plant tissues (Ibrahim
et al. 2011). Previous studies (Argyropoulou et al. 2015, Salahas et al. 2011) have demonstrated that different
rates of N application can influence phytochemical buildup in plant tissues; however very little has been done on
effects of different N forms. Therefore the aim of the present study was to evaluate the effects of N forms on
growth and total flavonoids and phenolic concentration in leafy amaranth. The information is relevant in N
forms management for optimum derivation of these vital therapeutic components.

Experimental Design and Treatments
The experiments were laid out in a split plot arrangement on a Randomized Complete Block Design (RBCD)
replicated three times. The main plot comprised of two vegetable amaranth varieties i.e. Abukusa 6 (AB6) and
Abukusa 7 (AB7) while three N-forms (NO3-, NH4+ and NH4NO3) and control (no N form used) constituted the
sub-plots. Sole NH4+ and NH4NO3 were stabilized with padin as the nitrification inhibitor composed of a
mixture of dicyandiamide and 3, 4 methylpyrazole phosphate.
Agronomic practices
Amaranth seeds were obtained from Jomo Kenyatta University of Agriculture and Technology (JKUAT).
The seeds were directly sown 2 kg containers and about two grams of Triple superphosphate (TSP) was used as
basal fertilizer. Watering of the amaranth plants was done daily.
Harvesting and Preparation of plant samples
Harvesting of the plant samples was done by uprooting the whole plant. The shoots and roots were separated
and shoot height recorded in centimeter (cm). Plant shoots (stems and leaves) were oven dried at 6065 C for
72 hours until the weight was constant and dry weight recorded. The dried plant samples were groundusing a
grinder-MIKA to fine powder (0.2 mm) which was kept in zip lock polythene bags, appropriately labeled and
stored in the dark awaiting phytochemical analysis.
Extraction of plant material
About 20 g of the powdered plant material was weighed, placed in a flask, 100 ml methanol AR was added
and allowed to stand for 4872 hours. It was then filtered through Whatman filter paper No. 1 and distilled using
rotary evaporatorat 65C until methanol-free paste was obtained (Mibei et al. 2012). The resulting extracts were
properly labeled and preserved at 5C in airtight plastic bottles for further use.
Total flavonoids contents analysis
The AlCl3 method (Mervat et al. 2009) was used for the determination of total flavonoid content (TFC) of
the sample extracts. Portions of 1.5 ml of 1:10 g.ml-1 extracts were added to equal volumes of a solution of 2%
AlCl3 6H2O. The mixture was vigorously shaken and allowed to stand between 1015 min. and absorbance read
Munene et al. (2017) 4(1): 8189
with a spectrophotometer (Spectro SC labmed inc.) at 425 nm. Flavonoid contents were expressed as mg catchin
equivalent (mgCE.g-1) dry weight.
Total phenolic contents Analysis
Total phenolics content (TPC) was determined by Folin Ciocalteu reagent (Esmaeili et al. 2009, Nabavi et
al. 2008). Dilute solution of amaranthextracts (0.5 ml of 1:10 g.ml-1) or gallic acid (standard phenolic
compound) was mixed with Folin Ciocalteu reagent (5 ml, 1:10 diluted with distilled water) and aqueous 5%
Na2CO3 (4 ml). The mixture was allowed to stand for 15 minutes and absorbance determined at 765 nm with a
spectrophotometer (Spectro SC labmed Inc.). The standard curve was prepared by 0, 50, 100, 150, 200, and 250
mg.ml.l-1 solutions of gallic acid to determine the total phenolic concentrations. Total phenolic content values
are expressed in terms of gallic acid equivalent (mgGAE.g-1) of dry weight.
Anti-oxidant analysis
Diphenylpicrylhydrazyl (DPPH) was used to determine radical-scavenging capacity of samples according to
(Mibei et al. 2012). Different concentrations 0.05, 0.1, 0.5, 1.0, 2.0 and 5 mg.ml-1 of the extracts were prepared,
in methanol. One ml of the extract was placed in a test tube, 3ml of methanol added followed by 0.5 ml of 1 mM
DPPH in methanol. This was shaken vigorously and left to stand for about five minutes. Concentrations of
ascorbic acid were prepared at the same concentrations as the extract and used as standard. Blank solution was
prepared with same amount of DPPH and methanol. Absorbance of the solutions was obtained at 517 nm with a
spectrophotometer (Spectro SC labmed Inc.). Radical scavenging capacity of the samples determined using
formula:
Where, Ab = absorption of the blank sample; Aa = absorption of the extract.

Data analysis
Data was subjected to analysis of variance (ANOVA) at 95% confidence level using SAS- computer
software (SAS 2015, Version 9.0). Where significant, mean separation was obtained by LSD. Data was
presented inform of tables and graphs.
RESULTS
Plant height
Nitrogen forms significantly (P 0.05) affected shoot height in the greenhouse experiment (Fig. 1). Data
showed that ammonium depressed plant growth (height) for both varieties compared to nitrate as a sole N
source. Variety AB7 had greater height (40.2 cm) than AB6 (35.2 cm) under nitrate treatment. Compared to the
control, provision of nitrate as sole source of N enhanced shoot length by 64% in AB6 and 51% in AB7 which
was twice that of the ammonium treated amaranth plants.
Figure 1. Effects of nitrogen forms on amaranth plant height.
Munene et al. (2017) 4(1): 8189
This plant height was same for both accessions when no N was supplied but once ammonium was supplied,
AB7s performance was statistically superior as compared to control plants. Nitrate treatment led to 2 folds
increase in height when compared to control for AB7 variety while the increase was almost 4 folds for AB6.
The accession AB7 grows better than AB6 but what is significant is that responds better to N in nitrate and
ammonium-nitrate N source.
Total flavonoids and phenolics concentration
Nitrogen forms had a significant (P0.05) effect on the total shoot flavonoids and phenolics content. The
results revealed a stimulatory effect of sole ammonium on shoot TFC and TPC accumulation in amaranth plants.
Compared to nitrate, ammonium increased TFC by 17.4% in AB6 and 14.7% in AB7. Ammonium increased
TPC by 23% in AB6 and 20% in AB7 as opposed to when nitrate was added as N source. Amaranth plants not
treated with N form (control) had comparatively higher TFC to ammonium form while accumulation of TPC in
the plants treated with ammonium form was statistically at par with the control for AB6 variety (Table 1).
Table 1. Effects of nitrogen forms on total phenolic content (TPC) and total flavonoid content (TFC) accumulation.
Treatments Total flavonoids Total phenolics contents
Contents (mg.g-1 GAE) (mg.g-1 GAE)
AB6 Cntl 28.2b 72.6b
Am 25.2d 75.5b
AmNi 23.1e 61.4d
Ni 20.8f 58.1e
AB7 Cntl 30.6a 74.7b
Am 26.7c 79.9a
AmNi 23.0e 64.2d
Ni 22.8e 63.9d
P value 0.001 0.001
LSD 1.1 3.0
NxV * NS
Interesting, AB7 was still superior to AB6 variety (Fig. 1 and Table 1). Though the ammonium treatment
elicited greater accumulation of TFC and TPC, AB7 was superior. Naturally it would be expected that
production of these phytochemicals incur some energy costs and hence the variety accumulating less should
grow slowly (shorter).
Antioxidant DPPH Radical-Scavenging Activity
The IC50 (concentration which scavenged 50% of the DPPH radicals) values of the amaranth extracts ranged
from 0.06 mg.ml-1 to 1.80 mg.ml-1. Ammonium as sole N source had superior antioxidant DPPH scavenging
activity indicated by lower IC50 value for AB7 (0.06 mg.ml-1 and AB6 (0.3 mg.ml-1) compared to nitrate
treatment for AB7 (0.7 mg.ml-1) and AB6 (1.8 mg.ml-1) (Table 2); an indication of ammonium treated plants to
possess great antioxidative capacity as compared to nitrateand ammonium nitrate mixture which require a bit
bigger volumes to scavenge 50% of DPPH radicals.
Table 2. IC50 and maximum percentage inhibition values for the 2 amaranth extracts.
Treatments IC50 Max Inhibition Concentration
(mg.ml-1) (%) (mg.ml-1)
AB6 Cntl 0.40 82.0 2
Am 0.30 72.8 2
AmNi 1.00 60.9 5
Ni 1.80 65.0 5
AB7 Cntl 0.09 75.0 2
Am 0.06 86.1 1
AmNi 0.60 70.1 2
Ni 0.70 64.3 5
Ascorbic acid 0.07 89.5 5
In addition ammonium form extract revealed a remarkable maximum inhibition percentage 72.8% for AB6
and 86.1% for AB7 at a lower concentration of 2 mg.ml-1 unlike nitrate (65% for AB6 and 64.3% for AB7) and
ammonium nitrate mixture (60.9% for AB6 and 70.1%) which with maximum inhibition of 5 mg.ml -1 which
was 2.5 folds higher than that of ammonium. Control had equally superior antioxidant capacity as ammonium
Munene et al. (2017) 4(1): 8189
(Table 2). Maximum inhibition of ascorbic acid (89.5%) was higher than that of amaranth extract, however at a
higher concentration (5 mg.ml-1) than that of ammonium treated amaranths and the control and indication of
notable stimulatory antioxidative effect of ammonium form.
Relationshipbetween plant growth and phytochemical accumulation
Enhancement of TFC and TPC accumulation was observed under reduced plant shoot growth (height)
exhibited by negative correlation coefficient (r= 0.75 and r= 0.81) respectively (Table 3).
Table 3. Pearson correlations coefficient between measured plant growth parameters and phytochemical
accumulation and antioxidant DPPH scavenging activity.
1 2 3 4 5 6
Shoot fresh weight 1
Root fresh weight 0.75** 1
Shoot height 0.91* 0.86* 1
TFC -0.75** -0.66* -0.75* 1
TPC -0.85* -0.64* -0.81** 0.68** 1
Ant. activity -0.65* -0.60* -0.69* 0.63* 0.78** 1
Note: * and ** significant at P 0.05 or P 0.01 respectively. TFC- Total flavonoids compounds, TPC-Total
phenolics compounds, Anti= Antioxidant.
In this study polyphenolics (TFC and TPC) concentration in vegetable amaranth related negatively with
plant growth (shoot and root fresh weight) with correlation coefficient r = -0.75 and -0.85 respectively. As
expected antioxidative activity of the amaranth extracts had a significant positive relationship with TFC and
TPC which implies a higher possibility that DPPH antioxidative capacity is as a result of higher accumulation of
flavonoids and phenolics.
DISCUSSION
Plant height
Depression in shoot height agrees with previous work by Borgognone et al. (2013) who observed that plant
height was sharply decreased when NH4+ was the dominant N source. The restriction in shoot height for the
plants supplied with sole ammonium compared to sole nitrate and mixed form (ammonium nitrate) may
probably be related to decline in cell number and cell size, which is manifested by impaired root- to- shoot
translocation of cytokinins as indicated by (Walch-Liu et al. 2000) on tobacco. Working with tomatoes Rahayu
et al. (2005) reported superiority among nitrate treated plants as compared to ammonium treated plants which
were even inferior to ammonium-nitrate-N plants. Nitrate N was associated with induction of phytohormonal
cascade transduction from root to shoot, leading to expansive leaf growth.
This is in concurrence with findings of Lobit et al. (2006), who found that a 49% reduction in the shoot
length under dominant NH4+ nutrition. These results were also in agreement with the findings of Gweyi-
Onyango et al. (2009) who observed a higher relative growth rate under nitrate even with lower concentrations
as compared to ammonium.
Total flavonoids and phenolics
The current study revealed up-regulated build-up of TFC and TPC that when no N form (control) was
supplied to the amaranth plants (Table 1). This is in concurrent with results of other workers (Kovik & Bakor
2007, Ibrahim et al. 2011) which show that the accumulation of polyphenolic components in plant tissues is
often enhanced under conditions of restricted nitrogen nutrition. Salahas et al. (2011) observed that N-
deficiency stimulated biosynthesis of secondary plant metabolites, such as total phenolics and betacyanins in red
beet. Previous findings by Argyropoulou et al. (2015) further revealed that total phenolics concentration
significantly increased in N-starved plants, indicating that biosynthesis of secondary plant metabolites is
stimulated by nitrogen deficiency, and this is in agreement with the report of Scheible et al. (2004). Lower
levels of phenolic and flavonoid compounds in plants grown under high N supply have been reported for apple
trees (Leser & Treutter 2005). Ammonium-N source was superior in poly-phenolic accumulation compared to
other sources. Application of plants with sole ammonium source leads to acidification of rhizosphere (Sabir et
al. 2013), which associated with poor plant growth (Gweyi-Onyango et al. 2009). This in turn induces plant
defense mechanisms by increased poly-phenolic accumulation (Caldwell et al. 2003) as defense mechanism
against nutritional stress. This may be supported by carbon nutrient balance (CNB) hypothesis which anticipates
Munene et al. (2017) 4(1): 8189
that the accumulation of excess carbon in response to nutrient stress leads to the increased production of carbon-
based secondary metabolites (CBSM) and their precursors (Yongke et al. 2005). Ammonium nutrition
stimulates build-up of high levels of polyamines (Chen et al. 2011, Gill & Tuteja 2010), which act as precursors
of secondary metabolites biosynthesis (Smith 1990). Increased carbon skeletons for ammonium assimilation,
might have stimulated shikimic acid pathway activities which in turn could have enhanced the production of
plant secondary metabolites (Fan et al. 1998) under sole ammonium treatment. Reduction in sink size to some
extend reduces translocation of carbohydrates to other plant parts (Reddy et al. 1996.) and extra carbohydrates
might be channeled towards secondary metabolism (Tognetti &Johnson 1999).
DPPH Anti-oxidant Activity
Similar to the TFC and TPC accumulation plants supplied with NH4+-N exhibited superior scavenging
capacity unlike other (NO3- and NH4+/NO3- mixture). Maisarah et al. (2013) observed that phytochemicals such
as flavonoids and phenolics constitute a major group of compounds that act as primary antioxidants. Plants
exposed to high NH4+ concentration as N source accumulate low molecular osmolytes among them polyamines
(Claussen et al. 2006) which enhances the plants tolerance to stresses (Tassoni et al. 2008, Yamaguchi et al.
2007). Moreover polyamines to some extent are involved in detoxification of nitrogen stress acting as nitrogen
reservoirs, and free radical scavengers maintaining the integrity of membranes, consequently protecting cells
from nitrogen toxicity (Chen et al. 2011). Flavonoids have been reported to possess strong antioxidant activity,
indicated by their ability to chelate metals, scavenge singlet oxygen radicals and inhibit oxidation of low density
lipoprotein in vitro studies (Kandaswami & Middleton 1994). Anthocyanins; which are flavonoids (Kumar &
Pandey 2013) lower the accumulation of ROS in vivo under oxidative stress (Nakabayashi et al. 2014). Previous
work has been done to support total phenols as effective antioxidants or free radical scavengers (Ogembo 2015,
Okello 2015) in leafy vegetables.
Relationship between plant growth and TFC and TPC concentration
Pearson correlation indicated a strong negative relationship between plant height and TFC and TPC which is
in line with the findings of (Ogembo 2015, Sousa et al. 2008) who demonstrated higher levels of total phenolics
were accompanied by lower plant growth in Solanum and Brassica species. Phenolics and flavanoids (quarcetin)
concentrations increase was observed under inhibited plant growth in Amaranthus hypochondriacus (Onyango
et al. 2012). High level of poly-phenolic compounds may be associated with nutritional-induced stress under the
sole ammonium and no N form due to resource allocation trade-off between plant growth and defense for
survival, therefore suppressed plant growth. This is in parallel with results of Donaldson et al. (2006) who
reported that nutrient limitation decreased growth, leaf mass ratio, and photosynthesis but augmented leaf
condensed tannin concentrations. Negative relationship between growth and condensed tannins indicated an
additional indirect cost of allocation to secondary metabolites (Donaldson et al. 2006).
CONCLUSION
Amaranth plants responded differently to different N forms. Ammonium N form restricted plant growth
which correlated negatively with polyphenols (total flavonids and phenolics) accumulation. Sole NH4+ induced
stress enhanced total flavonoids, phenolics and natural antioxidants with effective antioxidative capacity; a
striking metabolic plasticity observed during plant growth and survival trade-off in vegetable amaranth. Variety
AB7 was superior to AB6 both in terms of growth and accumulation of TFC and TPC which are important for
health and hence this would be recommended to farmers as well as consumers.
ACKNOWLEDGEMENTS
We sincerely thank Kenyatta University- Vice Chancellors research Grant Programme without which this
work may not have been completed. We also want to sincerely thank Prof Christof Engels of Humboldt
University, Berlin for providing us with Padin and Prof. Abukutsa-Onyango of JKUAT for the provision of
amaranth seeds.
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ISSN (E): 2349 1183
ISSN (P): 2349 9265
4(1): 9094, 2017
DOI: 10.22271/tpr.2017.v4.i1.013
Research article
Nitrate reductase and peroxidase activity in growth and

productivity of Santalum album L.
Pramod Kumar1*, Randhir Kumar2 and S. A. Ansari3
1
Tropical Forest Research Institute, PO- R.F.R.C., Mandla Road, Jabalpur, Madhya Pradesh, India
2
I.P.S. College, Chhindwara, Madhya Pradesh, India
3
Institute of Forest Productivity, Ranchi, Jharkhand, India
*Corresponding Author: pramod_kt@rediffmail.com [Accepted: 22 February 2017]
Abstract: Santalum album is an important oil bearing species contains 46% oil rich in -santalol
and -santalol. Accessing genetic variation within the population, 30 sandal trees of different age
and girth were evaluated for their leaf nitrate reductase (NR) activity and peroxidase activity. A
significant variation in leaf NR activity and peroxidase activity in selected trees was observed. NR
activity ranges from 0.062 to 0.219 mol NO2- g.fr.wt-1.h-1 and peroxidase activity from 15.400 to
69.480 470 min-1mg-1protein. However, correlation of girth of the trees (GBH) with NR and
peroxidase activity was not significant thus, these three parameters are independently affecting the
growth and oil yielding capacity. The maximum phenotypic and genotypic coefficient of variance
was recorded for peroxidase activity (PCV 58.02, GCV 29.16) followed by NR activity (PCV
30.68, GCV 28.03). More PCV for both NR and peroxidase activity indicates that the variation is
influenced by the environment.
Keywords: Genotypic variation - Nitrate reductase activity - Peroxidase activity - Santalum
album.
[Cite as: Kumar P, Kumar R & Ansari SA (2017) Nitrate reductase and peroxidase activity in growth and
productivity of Santalum album L. Tropical Plant Research 4(1): 9094]
INTRODUCTION
Santalum album L. (Family- Santalaceae), a small evergreen tree, commonly known as East Indian
Sandalwood is a partial root parasite and is widely distributed throughout India, Australia and Pacific Islands.
Species is hemiparasitic, having photosynthetic capacity but water, mineral nutrients and organic substances are
acquired via the host plant (Nagaveni & Srimathi 1985, Radomiljac et al. 1998, Radomiljac et al. 1999). This
means that natural regeneration or artificial establishment is dependent on the presence of suitable host plant as
well as suitable environmental conditions. In addition, sandalwood is vulnerable by various abiotic and biotic
factors including illicit felling, poaching, change in land use and low natural regeneration (Srinivasan et al.
1992).
Indian sandalwood is highly valued for its fragrant heartwood containing oil which is used for centuries for
religious and customary purposes and contributing to the economy of the country by its use in cosmetics,
aromatherapy, scenting of soaps, perfumery and medicines. However, trees vary in their oil content depending
upon the site conditions where they grow. The heartwood formation generally begins after 8 years of plantation
and the best heartwood form in more than 20 years old trees having girth of above 50 cm (Sen-Sarma 1977).
Though, the heartwood formation governed by genetic factors (Srimathi & Kulkarni 1980) but the age of the
tree and colour of heartwood is indicative to the quality and content of sandal oil. Generally there is a decrease
of about 45% in oil content from root to tip and about 20% from core to the periphery. The root portion roughly
contains 36 %, stem 35 % and branches 13 % of the oil. Light coloured wood generally contain a higher
percentage of oil than the dark coloured ones (Shankaranarayana 1985). Identification of superior genotypes
with a higher quantity of heartwood was emphasized during the second All India Sandal Seminar held in 1981.
Data on heartwood yield for different girth size trees was recorded by Rai & Kulkarni (1986). The majority of
the oil is composed of sesquiterpene alcohols, dominated by and -santalol (Verghese et al. 1990, Jones et al.
Kumar et al. (2017) 4(1): 9094
2006). Imbalance in commercial production and harvesting resulted in a sharp decline in the natural supplies of
sandal (Brennan & Merlin 1991). International prices for sandalwood have therefore been consistently rising
over the past few decades (Page et al. 2012). Sandalwood plant material has both genuine demand in India and
abundant exporting potential (Arun Kumar et al. 2012). Present need is to expand its distribution range by
raising plantations and also protection of existing natural populations.
Nitrate reductase is a key enzyme in the assimilation of exogenous nitrate and thus, responsible for entry of
nitrogen in the biological system particularly in non-nitrogen fixing plants. Its activity gives a good estimate of
the nitrogen status of the plant and is very often correlated with growth and yield (Srivastava 1980). In higher
plants, cytosolic NAD(P)H-nitrate reductase (NR) is rapidly modulated by environmental conditions such as
light, CO2, or oxygen availability. Photosynthesis activates 6080 % NR activity in leaves whereas after
stomatal closure, leaf NR is inactivated down to 20 or 40% of its maximum activity (Kaiser et al. 1999).
Peroxidases (donor: H2O2-oxidoreductase) are a single polypeptide chain, iron containing enzymes participating
in many physiological processes viz. lignification, suberization, cross linking of cell wall proteins, stress
response, defense against pathogens, salt tolerance and senescence has often been used as a marker enzyme.
Peroxidases exists in several isozyme forms and the expression pattern of these enzymes is organ-specific and
developmentally regulated (Ghamsari et al. 2007).
Indian sandalwood exhibits very high heritable value (h2=0.92) for heartwood production (Ansari et al.
2008). However, information on genotypic variation for developing conservation strategies, genetic
improvement and sustainable use of the species is inadequate. Considering the economic importance of the
species, NR activity and peroxidase activity based assessment of genotypic variation may be used as a marker
for the conservation of superior genotypes.

Present work was conducted on 30 trees of different age and girth after randomization of trees on the basis of
a random number for experimental sampling purpose (Gomez & Gomez 1984). Leaf samples were taken from
the selected trees for the assay of nitrate reductase activity and peroxidase activity. Nitrate Reductase (EC:
1.6.6.1) activity was estimated following method of Jaworski (1971). 500 mg leaf pieces were incubated for 2
hrs in dark in a reaction mixture. Absorbance recorded at 540 nm using a UV-vis spectrophotometer (GBC,
USA). NRA was expressed as mol NO2- h-1.g-1 fresh leaf tissue and compared with a standard curve prepared
from a known concentration of KNO3.
Peroxidase activity (EC 1.11.1.7) was measured from acetone powder formed after homogenization of
100mg leaf samples in chilled acetone (Mahadevan & Sridhar 1986). Acetone powder was suspended in 1ml
0.02 M sodium phosphate buffer (pH 6.4) in an eppendorf tube and centrifuged for 10 minutes at 10000 rpm.
The supernatant was used for estimation of peroxidase activity following the method of Rama Rao et al. (1982).
Absorbance recorded at 470 nm for 3 min with an interval of 30 sec. The soluble protein content in the enzyme
extract was estimated following the method of Lowry et al. (1951) and the absorbance was measured at 750 nm.
A standard curve from known concentrations of bovine serum protein was prepared for quantifying protein
content. The peroxidase activity was computed in accordance with protein content and expressed as 470 protein
min-1.mg-1.
The data recorded for five replications each of nitrate reductase and peroxidase activity was subjected to
analysis of variance (ANOVA) by the standard statistical procedure (Gomez & Gomez 1984). The significance
of the data was ascertained by F-test and critical difference at p=0.05 computed for comparison of various
treatments (selected trees) means.
RESULTS
Data of GBH, NR activity and peroxidase activity are depicted in figure 1A, B, C. Statistical analysis (Table
1) reveals a significant genotypic variation in NR and peroxidase activity among selected trees. Variation in NR
activity ranges from 0.062 (Tree 17) to 0.219 (Tree 13) mol NO2- g.fr.wt.-1.h-1. Peroxidase activity varied
between 15.400 (Tree 30) to 69.480 470 min-1.mg-1 protein (Tree 6) in selected trees. The maximum phenotypic
and genotypic coefficient of variance was for peroxidase activity (PCV: 58.02, GCV: 29.16) followed by NR
activity (PCV: 30.68, GCV: 28.03). PCV for peroxidase activity was about of the double magnitude of GCV.
Simple linear regression analysis exhibits NR and peroxidase activity as an independent parameter for growth
and productivity in sandal (Fig. 2A, B).
Kumar et al. (2017) 4(1): 9094
Table 1. Analysis of variance (ANOVA) for NR Activity and Peroxidase activity.

Parameters Source df Sum of Mean F value Standard CD
squares square Error (P=0.05)
NR Activity Replication 04 0.0550 0.013743 11.6479 0.021724 0.043123
Genotype 29 0.21 0.007132 6.0445
Error 116 0.14 0.001179
Total 149 0.40
Peroxidase Replication 04 18361.55 4590.387 6.8195 16.408763 32.571396
Activity Genotype 29 26119.55 900.6740 1.3380
Error 116 78081.78 673.1188
Total 149 122562.88
Figure 1. A, GBH; B, Nitrate reductase activity; C, Peroxidase activity of selected trees.
Figure 2. Linear regression between: A, Girth at breast height (GBH) and NR activity; B, GBH and Peroxidase activity.

Roots of Santalum album depend upon other plants especially leguminous plants for nutrition and
establishment corresponds with its slow growth and late heartwood production. A high Km value (0.255 mM
KNO3) of nitrate reductase enzyme (Tiwari 2008 unpublished) is an indication of low NR activity in Santalum
album which is related with its hemiparasitic nature. Ananthapadmanabha et al. (1988) categorized host plants
of sandal in three groups by different physiological activities. Significant NR activity of sandal plants growing
Kumar et al. (2017) 4(1): 9094
with different host species was observed by Nagaveni & Vijayalakshmi (2003). Poorly grown sandal plants
showed low NR activity and hence directly proportional to the growth parameters of the plants. Among the
various metabolic processes, nitrate reductase activity was found directly correlated with the biomass production
(Pokhariyal et al. 1993).
The phenotypic variance was greater in magnitude compared with their corresponding genotypic variance
for both the parameters i.e. NR activity and peroxidase activity. These two parameters show a moderate level of
coefficient of variance. Further, fluctuation observed in peroxidase activity in different genotypes is much more
influenced by the environment than the genotype. But in the case of nitrate reductase, the fluctuation is almost
equally attributable to both environment and genotype. Parthasarthi et al. (1986) observed a negative correlation
between the specific peroxidase isozyme activity and oil percent in the mature sandal plants. Mor et al. (2008)
found peroxidase activity in the interaction between the host (Arabidopsis thaliana) and the parasite (Orobanche
aegyptica) and proposed that peroxidases could have a role in generating extracellular reactive oxygen species
(ROS) for a loosening of the cell wall of the host in order to facilitate penetration. Alternatively, the ROS could
act in facilitating the root elongation of the parasite.
Correlation analysis reveals non-significant correlation of NR activity and peroxidase activity with the GBH
of trees which is advocating the independent role of these three parameters in the growth and yield. It may be
because of their genes are located at a distance beyond the linkage, i.e. > 1 c Morgan on different loci on the
same chromosome or on different chromosomes. However, while working with 37 accessions of sandal, Arun
Kumar et al. (2011) recorded a weak significant positive correlation between tree diameter and heartwood
proportion. Since, the economic importance of sandalwood depends on the quantity and quality of heartwood
and its oil-bearing potential, NR activity and peroxidase activity may be used as an independent biochemical
marker for selection of high oil yielding trees of sandal for tree improvement. However, seasonal and diurnal
changes also affecting these enzyme activities and thus growth and productivity.
ACKNOWLEDGEMENTS
Authors are grateful to the Director, Tropical Forest Research Institute, Jabalpur for providing necessary
facilities for the study.
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ISSN (E): 2349 1183
ISSN (P): 2349 9265
4(1): 95102, 2017
DOI: 10.22271/tpr.2017.v4.i1.014
Research article
Host range, anatomy, biochemistry and impacts of Cuscuta

reflexa Roxb.: A case study from the Betla National Park,
Jharkhand, India
Preeti Kumari1, S. K. Tiwari2*and A. K. Choudhary1
1
Department of Botany, Ranchi University, Ranchi-834002, Chhattisgarh, India
2
BRSM College of Agricultural Engineering and Technology, IGAU, Raipur, Chhattisgarh, India
*Corresponding Author: shashikant.au@gmail.com [Accepted: 25 February 2017]
Abstract: The Cuscuta reflexa is a leaf less parasitic angiosperm belonging to the family
Convolvulaceae and it is directly attaches to the host plants through the haustoria. During this
investigation we find that the plant is a hemiand holoparasite, living on wood yielding, fruit yield
in great medicinally important plants species. In present study, systematic survey and
identification of the different host plants was undertaken. Surveys were conducted to find out the
host plants of Cuscuta reflexa from different localities of Betla National Park areas of Jharkhand,
India. In a survey 33 species, representing 30 genera belong to 23 families were recognized as host
plants for it. Host plants were also examined for anatomical and biochemical studies. Haustorium
penetration in host stem and size of the haustorium was found specific to the host. Each transverse
section of host stem showed haustorium reached up to the secondary xylem. Poly-phenol oxidase
activity and protein content were also studied in healthy and infected stem of Ziziphus mauritiana,
Cajanus cajan and Ficus glomerata by Cuscuta reflexa. It was interesting to note that the protein
content is markedly stimulated in all infected host plants. The maximum stimulation was recorded
in Ziziphus mauritiana while minimum in Artocarpus integrifolia. The impact of Cuscuta reflexa
on host growth, allometry and reproduction was also studied and found that it was major factor,
which lead to changes in competitive balances between host and non-host species and therefore
affect community structure, vegetation and population dynamics. Impacts on hosts may further
affect herbivores, pollinators and seed vectors, and behaviour & diversity of these is often closely
linked to the presence and abundance of parasitic plants.
Keywords: Host plants - Parasite - Wood yielding trees - Anatomy - Poly-phenol oxidase - Protein.
[Cite as: Kumari P, Tiwari SK & Choudhary AK (2017) Host range, anatomy, biochemistry and impacts of
Cuscuta reflexa Roxb.: A case study from the Betla National Park, Jharkhand, India. Tropical Plant Research
4(1): 95102]
INTRODUCTION
Cuscuta is an obligate angiosperm parasitic climber found commonly throughout India. It has about 100170
species which attach various trees, shrubs, herbs and affect commercially valuable plants. We know that the
plants water and other essential inorganic nutrients absorbed through conducting cells. The xylem connections
between host and parasite, while organic substances transported from phloem tissue of host plants to parasite.
Cuscuta ranges in severity based on its species and the species of host, the time of attach and whether any
viruses are also present in the host plants (Kumar et al. 2012). Cuscuta is also known as Amerbel and it is used
for various purposes viz. as a purgative, in the treatment of liver disorders, cough and itching, respiratory dieses
and treatment of falling of hairs. It is known that it contains phenolics and flavonoids compound. Due to
presence of such organic compounds it acts as Anti-inflammatory and anti-cancerous properties (Udavant et al.
2012). Fungi, nematodes, bacteria, and viruses are probably the first things that come to mind in the current list
of parasites available in nature. Above parasite are responsible for development, growth and production of
plants and crops i.e. it affects the economy of any country, but it may surprise to know that parasitic flowering
Received: 26 September 2016 Published online: 28 February 2017
Kumari et al. (2017) 4(1): 95102
plants are also important parasite. The purpose of this research work is to provide complete information about
relationships of these fascinating and unusual plants and also to focus upon those that negatively affect the plant
nutrients and production of crops. Most of the plants available in nature are autotrophs and produce their own
carbon sources through photosynthesis. Although some plants such as Indian pipe (Monotropa) lack chlorophyll
and appear to be parasitic, they are mycoheterotrophs (parasites of mycorrhizal fungi) and, hence, only
indirectly parasitize the trees on which the mycorrhizal fungi are found. A haustorium is a modified root that
forms a morphological and physiological link between the parasite and host (Kujit 1969). It is useful to make a
distinction between the terms "parasite" and "pathogen." Parasite is from the Greek para (beside) and sitos
(grain or food) which literally means "beside the food". If a plant also induces disease symptoms in a host, then
it is a pathogen as well as parasite. A general term that refers to both parasites and mycotrophs that derive
carbon from sources other than their own photosynthesis is heterotrophic, which simply means "different
feeding."Cuscuta, dodder (Convolvulaceae-Morning Glory Family) The species of Cuscuta, is commonly
known as dodder, and among the entire parasite it is the best known and common parasitic plants. The biology
and control of dodders was reviewed in (Dawson et al. 1994). Dodders have a broad host range, although
monocots are less preferred. The genus Cuscuta contains three subgenera. Members of the subgenus Monogyna
are robust vines that may attack and kill fruit trees. Many researchers have been reported about cuscuta but no
anybody has been studied and work carried out related to Cuscuta in BNP.
The present investigation is emphasizing on the host plants of Cuscuta reflexa Roxb. from different localities
of Betla National Park areas of Jharkhand, India. Anatomy of Cuscuta and its host plants has been studied. We
have also studied the biochemical attributes like enzyme poly-phenol oxidase and protein content from healthy
and infected host plants, as well as impacts of Cuscuta reflexa has been also reported.
MATERIALSAND METHODS
Study Area
Betla National Park (BNP) is located in the District of Plamu situated between latitude 2325 and 2355'
North and longitudes 8350' and 8436' East, in the state of Jharkhand, India. The total area of the project Tiger
circle is 1306 km2. Out of above areas 979.97 km2 area of the PTR has been declared as plamu wildlife
sanctuary and out of that an area of 226.32 km2 has been notified as a Betla National Park in 1996. It is located
in the western part of Chhootanagpur and covered by Plamu, Garhwa and Lathehar District. It is also part of the
Central India Landscape and extends into the Sanjay-Dubri Tiger reserve and Achanakmar-Kanha tiger
landscape through the Jashpur and Mahan forest of Chhattisgarh. In this areas mainly dry mixed forest, dry sal
forest, moist sal forest and moist mixed forest are presents. BNP is also becoming home to many unwanted
nonnative plants.
Data collection
The diversified host species of Cuscut reflexa Roxb. were studied in Betla National Park of Jharkhand, India.
Extensive field survey was undertaken during the year i.e. from 20152016 at different areas including forest
villages and out skirts, higher plant parasites occurring on the host species. These are photographed and
collected the host species and parasites for identification and confirmed with the help of existing literature.
The host plants of Cuscuta were collected from different localities of Betla National Park of Jharkhand,
India and identified by using recent standard books and current literature. The hosts were categorized in herbs,
shrubs, climbers, lianas, trees; angiosperms, gymnosperms, and their families; medicinal, insecticidal and
economically important plants. The transverse sections of Cuscuta host stem have been taken from highly
affected area. Then these sections have been stained with dilute safranin and dilute light green using double
staining technique. And now these ready slides have observed by using light microscope to study the anatomical
details in host stem and Cuscuta shoot association. The activity of an oxidative enzyme poly-phenol oxidase and
protein content were studied from healthy and infected host plants of Cuscuta using the following methods.
Poly-phenoloxidase analysis
The Poly-phenol oxidase (PPO) activity has been analyzed as per the procedure of Mahadevan & Sridhar
(1982). The reaction mixture consisted 1.5 ml of 0.1 M Sodium phosphate buffer (pH 6.5) and 200 l of the
enzyme extract. 0.01 ml Catechol has added to the reaction mixture to start the reaction. Poly-phenol oxidase
activity has expressed as change in absorbance at 412 nm per minute/g fresh weight of tissue.
Kumari et al. (2017) 4(1): 95102
Protein analysis
Fresh steam sample 100 mg has extracted in 0.1 M Sodium phosphate buffer (pH 7.0) at 04 oC. The
homogenate has centrifuged for 20 minutes at 12000 rpm. The protein content of the sample has been
determined by the method of Lowry et al. (1951). Protein (100 g) from different tubes has taken and mixed
with 10 l of sample buffer in eppendorf tube, boiled for 34 minutes and incubated at 4oC for 30 minutes. The
samples containing equal amount of protein have loaded into wells of 12% polyacrylamide gel. Electrophoresis
has carried out at constant voltage of 75 volts for two hours. The gels have stained with 0.2% coomassie brilliant
blue (R-250) solution and distained with acetic acid/water. The hierarchical cluster analysis has performed on
gel documentation system using NTSYS-pc software and the dendrogram has prepared using average linkage
between groups based on presence/absence of protein bands in different lanes of the gel.

Study the host plant of Cuscuta reflexa
In the present investigation, surveys have made during 2015 to 2016 to locate the host plants of Cuscuta in
the Plamu Tiger reserve areas of Betla National Park in Jharkhand, India. In a survey 33 species, representing 30
genera belong to 23 families have been recorded as a host plants of Cuscuta (Table 1). In above observed host
plants some are herbs, shrubs, climbers and trees and it is agricultural, horticultural, medicinal, weeds and
economically important plants. Their life span may be annual, biennial or ephemeral. The present results clearly
indicate that, dodder ranges in severity based on the species of host. The very common plants viz. Ziziphus
mauritiana, Cajanus cajan and Ficus glomerata have very favorable hosts of Cuscuta, (Fig. 1) and when other
suitable hosts were nearby Cuscuta shoots, Cuscuta spread from host plant to host plant often forming a dense
mat of intertwined stems. It grows on each and every type of plants. In shaded areas, twining and attachment
were greatly reduced. Nikam et al. (2014) reported Vitex negundo and Duranta plumier plat is a most favorable
host plant in Baramati area of Pune district of Maharashtra and Reddy et al. (1990) reported Vitex negundo
Linn., hedge plant as a new host for Cuscuta reflexa in Bidar, Karnataka. According to Jayasinghe et al. (2004),
Cuscuta is widely distributed inSri Lanka. They searched 161 host plant species including rice, belonging to 59
families and 139 genera. Patel et al. (2004) presented tabulated data of 48 host plants parasitized by Cuscuta
species in North Gujarat, India. From the different experimental studies Schoolmaster (2005) concluded that,
Impatiens capensis Meerb. (Balsaminaceae) was anecessary nurse host for the parasitic plant Cuscuta grovonii
in Schultes in Southeastern Michigan wetlands. One very interesting thing was revealed by Kelly (1992) i.e. in
greenhouse experiments C. europaea accept (coil) host of high nutritional status and grow away from (reject)
hosts of poor quality.
Table 1. List of host plants of Cuscuta reflexa Roxb. collected from Betla National Park, Plamu Tiger reserve areas in
Jharkhand.
S. Botanical names of Cuscuta host Vernacular names of Family Uses
No. plants Cuscuta host plants
1 Carissa spinarum L. Karonda Apocynaceae Medicinal
2 Eugenia heyneana Duthie kathjamun Myrtaceae Medicinal
3 Acacia catechu (L.f.) Willd. khair Mimosaceae Formation of katha
4 Acacia nilotica (L.) Delile Babul / Kikar Mimosaceae Tanin product
5 Achyranthes aspera L. chirchiri Amaranthaceae Medicinal
6 Adhatoda zeylanica Medik Adosa Acanthaceae Medicinal
7 Artocarpus integrifolia L.f. Jack fruit / Kathal Moraceae Vegetable
8 Aegle marmelos (L.) Corra Bel Rutaceae Fruit
9 Syzygium cumini (L.) Skeels Jammun Myrtaceae Fruit
10 Aerva lanata L. Gorakhbuti Amaranhaceae Medicinal
11 Spondias mangifera Wild. Amra / Amda Anacardiaceae Pickle
12 Ageratum conyzoides L. Jangli pudina Asteraceae Medicinal
13 Ziziphus xylopyrus (Retz.) Willd. kathber Rhamnaceae Medicinal
14 Azadirachta indica A.Juss. Neem Meliaceae Wood
15 Bombax ceiba L. semal Bombacaceae For cotton
16 Butea monosperma (Lam.) Taub. Palas Fabaceae Gum
17 Albizia lebbeck (L.) Benth. Sirish Mimosaecae Wood
18 Emblica officinalis Gaertn. Amla. Euphorbiaceae Medicinal
19 Lawsonia inermis L. Mehendi, Apiaceae Dye
Kumari et al. (2017) 4(1): 95102
20 Moringa oleifera Lam. Sahjan, Moringaceae Vegetable

21 Tamarindus indica L. Imali Caesalpiniaceae Medicine
22 Terminalia bellirica (Gaertn.) Roxb. Bahera Combretaceae Medicinal
23 Terminalia chebula Retz. Harra Combretaceae Medicinal
24 Madhuca longifolia L. Mahua Sapotaceae Medicinal drug
25 Vitex negundo L. Nirgundi Verbenaceae Medicinal
26 Cajanus cajan (L.) Millsp. Arhar Fabaceae Pulses
27 Ziziphus mauritiana Lam. Ber / plum Rhamnaceae Fruit
28 Shorea robusta Gaertn. Sal / Sakhua Dipterocarpaceae Wood
29 Annona squamosa L. Sitaphal Annonaceae Fruit
30 Pyrus communis Decne. Naspati Rosaceae Fruit
31 Dalbergia sissoo Roxb. Shisam Fabaceae Wood
32 Nerium oleander L. Kaner Apocynaceae Ornamental
33 Ficus glomerata Roxb. Gular Moraceae Medicinal
Figure 1. Plant affected by Cuscuta reflexa Roxb. in Betla National Park, Jharkhand: AB, Ziziphus mauritiana Lam.; CD,
Cajanus cajan Lam. EF, Ficus glomerata Roxb.
Anatomical study of host plant of Cuscuta reflexa
The light microscopic anatomical observations of Cuscuta reflexa Roxb and its host stem showed
tremendous diversity (Fig. 2). The present result clearly indicates that, Cuscuta houstorium is easily penetrate in
to the host plant steam and penetration depends on the size of both steam as well as Cuscuta. Each transverse
section of host stem shows Cuscuta haustorium reached up to the secondary xylem. Here one of the interesting
things has been observed that food material is available from phloem tissue instead of this Cuscuta inserted in to
Kumari et al. (2017) 4(1): 95102
the next of phloem tissue of host plants. And this insertion shows only limited growth. The another common
character was observed that the Cuscuta penetration has been affected the steam of cortex tissue and this tissue
elongated towards the Cuscuta there for stem of host plants became completely changed its structure. In Cuscuta
haustorium apical meristem and root caps are absent and its develops from cortical parenchyma cells of the
pericycle. In addition, during the formation of the haustoria, it is also clear that cell elongation dominates over
the cell division therefore the number of cells of the parasite endophytic system in the host is determined by the
number of Cuscuta cortical parenchyma cells undergoing transformation. Furthermore, the haustoria have
limited growth capacity. The anatomical studies of Cuscuta made by Ihl & Wiese (2000). Haustoria formation
was restricted to a sub apical region (area where the most intensive elongation of the steam) of C. reflexa stem.
According to Arnaud et al. (1998) while the Cuscuta easily attached itself to its hosts, the first difficulty was to
establish connection between xylem vessels and sieve-tubes. As per the studies of Dey & KandPati (1998),
transverse sections of the affected area of the stem of Digitaria ciliaris showed that the haustoria penetrate the
host by rupturing the bulliform cells or epidermal pores.
Figure 2. Anatomical structure of host plant stems infected by Cuscuta reflexa Roxb.: A, Ziziphus mauritiana
Lam.; B, Cajanus cajan (L.) Millsp. C, Ficus glomerata Roxb.
Kumari et al. (2017) 4(1): 95102
Study the Biochemistry of Cuscuta reflexa

Phenolic compounds are believed to impart resistance to disease in plants and poly-phenol oxidase
(Catecholase and Cresolase) enzyme has been reported to be responsible for in vivo synthesis and accumulation
of these compounds (Vaughan & Duke 1984). In many cases, a close correlation has been found between the
enhanced activity of poly-phenol oxidase andper-oxidase and the concentration of Phenolic substances on one
hand and between plant resistances on the other (Dickinson & JandLuca 1982). In the present investigation
poly-phenol oxidase activity studied in healthy and infected stem of Ziziphus mauritiana, Cajanus cajan, Ficus
glomerata, Emblica officinalis Gaertn and Artocarpus integrifolia (Table 2). The common trend of enzyme
activity is stimulatory in infected host plants. None of the infected host shows decreasing trend of polyphenol
oxidase activity. Present results clearly indicates the role of poly-phenol oxidase activity in plant diseases, so
here it may concluded that increasing activity of poly-phenol oxidase enzyme markedly involve in physiological
defense mechanism of host plants. The similar results are also proposed by many workers. The effect of poly-
phenol oxidase activity have been studied by Jite & Tressa (1999) and found that an increase in poly-phenol
oxidase activity in infected Jasminum plants with Uromyces hobsoni. Gawande et al. (2002) concluded that
enzymes polyphenol oxidase and peroxidase are responsible for resistance or susceptibility of host plants against
pathogen.
Table 2. Poly-phenol oxidase activity in healthy and infected host plant steam by Cuscuta reflexa Roxb. in Betla
National Park areas.
S. Poly-phenol oxidase activity (OD.min-1g-1 fresh wt)
Plant material (Stem material)
No. Healthy Infected
1 Ziziphus mauritiana Lam. 5.20 12.78
2 Cajanus cajan (L.) Millsp. 6.31 11.45
3 Ficus glomerata Roxb. 5.63 9.12
4 Emblica officinalis Gaertn. 6.63 7.78
5 Artocarpus integrifolia 9.34 9.92
Protein content studied in healthy and infected host plants steams of Ziziphus mauritiana, Cajanus cajan,
Ficus glomerata, Emblica officinalis and Artocarpus integrifolia by Cuscuta reflexa has recorded in table 3. It
is interesting to note that the protein content is markedly stimulated in all infected host plants. The maximum
stimulation occurs in Ziziphus mauritiana compared to another plants. Again increasing protein content proves
its role in plants defense mechanism.
Table 3. Protein content in healthy and infected host plant steam by Cuscuta reflexa Roxb. in Betla National Park areas.
S. Protein content (g per 100 gm dry tissue)
Plant material (Stem material)
No. Healthy Infected
1 Ziziphus mauritiana Lam. 60 190
2 Cajanus cajan (L.) Millsp. 85 205
3 Ficus glomerata Roxb. 72 139
4 Emblica officinalis Gaertn. 68 120
5 Artocarpus integrifolia L.f. 167 210
Impact of Cuscuta reflexa (Dodder plant)
Cuscuta is a parasite and it affects different plans, agricultural crops such as Ziziphus mauritiana & Cajanus
cajan and also affects the horticultural crops. Others dodder ranges in severity based on its species and the
species of the host, the time of attack. Parasitism has major impacts on host growth; allometry and reproduction.
Cuscuta also affect the behaviours, pollinators, seed vectors and diversity of host plants.
Ecological and economic impact of dodder species
Cuscuta also affect the environment as well as ecological balance in the nature. It affect the economy of
farmer because Cuscuta spread in to the field and affect the growth of agricultural crops therefore very low
production of crops. Cuscuta also affect the soil quality of the field. It decreases the arable surface, results in
quantitative and qualitative crop losses, represents a vector concerning the transmission of such diseases as
viroses and microplasmoses to the host plant, and its impact on the biodiversity determines the degradation of
the landscapes decorative aspect.
Impacts of parasitic plants on the plant community
Over one season, for instance, a single Cuscuta plant may form thousands of connections with many host
Kumari et al. (2017) 4(1): 95102
species and may cover an area greater than 100 m (Kelly 1990), resulting in considerable impacts on the plant
community despite its being perhaps less than 5% of vegetation biomass (Pennings & Callaway 1996) .
Impacts of the parasite on other tropic levels
It has been found that not only plants are affected by Cuscuta many other organisms such as birds and insect
herbivores, other parasites and mycorrhizal fungi can be affected, either directly or indirectly. Parasite also
affects the trophic levels because it is part of our biodiversity. Monitoring Cuscuta species and their spreading
tendencies, as well as their prevention and therapy generates positive and immediate economic and social
effects, by means of creating an integrated protection system of cultures. Moreover, it might also determine a
qualitative and quantitative increase of agricultural production, which benefits farmers on the short, medium and
long run. Furthermore, they will be mirrored in the quantity and quality of fodders, animal health and welfare of
farmers; since it is universally acknowledged that the decrease in risk of diseases, parasites and weeds within
agricultural ecosystems would influence public health and environmental protection in a very positive way.
Impacts of the parasite on the abiotic environment
The abiotic environment is also affected by parasitic plants and most significant types of ecosystems affected
by Cuscuta species are the pratologic ecosystems. However, there is a huge number of species which represent
host plants for dodders; this fact affecting the biodiversity of ecosystems at a process level, as well as with
regard to human society and animal health.
CONCLUSION
In present study systematic survey and identification of the host's plants has been conducted to find out the
host plants of Cuscuta reflexa Roxb. from different localities of Betla National Park areas of Jharkhand in India.
In a survey total 33 species, representing 30 genera belong to 23 families have been observed as host plants of
Cuscuta and Ziziphus mauritiana, Cajanus cajan and Ficus glomerata are most favorable host of Cuscuta. Host
plants have also examined for anatomical and biochemical studies. Cuscuta haustorium penetration in host stem
and size of the haustorium is specific to host and Cuscuta species. Each transverse section of host stem clearly
shows that Cuscuta haustorium has been reached up to the secondary xylem. But these haustoria insertion was
not up to the pith and shows limited specific growth. The another common character has observed that, the
Cuscuta haustorium penetration in the host stem has affected on the cortex tissue and this tissue shows markedly
elongation towards the Cuscuta stem and host stem structure has completely changed. The common trend of
enzyme activity is stimulatory in infected host plants. Here it may concluded that increasing activity of poly-
phenol oxidase enzyme markedly involve in physiological defense mechanism of host plants. Protein content is
markedly stimulated in all infected host plants. The maximum stimulation occurs in Ziziphus mauritiana
compared to another plants. Parasitism has major impacts on host growth, allometry and reproduction. Impacts
on hosts may further affect herbivores, pollinators and seed vectors, and the behavior and diversity of these is
often closely linked to the presence and abundance of parasitic plants.
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plantes cultives deux phanrogames parasites (Cuscuta reflexa etStriga hermonthica. Les Phanerogames
Parasites 192(1): 101119.
Dawson JH, Musselman LJ, Wolswinkel P & band Dorr I (1994) Biology and control of cuscuta. Reviews of
Weed Science 6: 265317.
Dey D & KandPati BR (1998) Record of parasitism of cuscuta reflexa Roxb. on digitaria cilliaris Pers. Journal
of Economic and Taxonomic Botany 22(1): 235236.
Dickinson C & JandLucas JA (1982) Plant Pathology and Plant Pathogen, Edition II, Vol. 6. Black-well
Scientific Publication.
Gawande VL, Patil JV, Naik RM & Kale AA (2002) Plant biochemical defense against powdery mildew
(Erysiphe polygoni DC) disease in mungbean (Vigna radiate (L.) Wilczek). Journal of Plant Biology 29(3):
337341.
Ihl B & Wiese K (2000) Studie an Cuscuta reflexa ROXB. VIII. Mechanische Haustorieninduktion an
nichtwindenden Achsen des Parasiten. Flora Jena 195(1): 18.
Kumari et al. (2017) 4(1): 95102
Jayasinghe C, Wijesundara DSA, Tennekoon KU & Marambe B (2004) Cuscuta species in the lowlands of Sri
Lanka, their host range and host parasite association. Tropical Agricultural Research 16: 223241.
Jite PK & Tressa J (1999) Biochemical changes in Jasminum grandiflorum infected by Uromyces hobsoni.
Indian Phytopathology 52(1): 7778.
Kelly CK (1990) Plant foraging: a marginal value model and coiling response in Cuscuta subinclusa. Ecology
71: 19161925.
Kelly CK (1992) Resource Choice in Cuscuta europaea. Proceedings of the National Academy of Sciences of the
United States of America 89(24): 1219412197.
Kujit J (1969) The Biology of parasitic Flowering plants. University of California press, Berkeley, CA, USA.
Kumar A, Rani S & Niketa SS (2012) Review on plant molecular biology, phytophysiology, phytochemistry
and ethonopharmacology of Cuscuta reflexa Roxb. A wonderful parasitic plant. International Research
Journal of Pharmacy 3(7): 3038.
Lowry OH, RosebroughN J, Farr AL & Randall RJ (1951) Protein measurement with the folin phenol reagent.
Journal of Biological Chemistry 193(1): 265275.
Mahadevan A & Sridhar R (1982) Methods in physiological plant pathology, 2nd Edition. Sivakami publication,
Indra Nagar, Madras.
Patel DM, Bhatt DC, Dodia SK & Parmar RP (2004) Selection of host plants by Cuscuta L. species in semi-arid
area of Visnagar, North Gujarat. Advances in Plant Sciences 17(2): 549552.
Pennings SC & Callaway RM (1996) Impact of a parasitic plant on the structure and dynamics of salt marsh
vegetation. Ecology 77: 14101419.
Reddy PP, Nalini AS & Prabhakar AS (1990) Vitex negundo Linn.:- A new host for Cuscuta reflexa Roxb.
Current Research 19(4): 5556.
Nikam SS, Pawar SB & Kanade MB (2014) Study of Cuscuta reflexa Roxb. with reference to host diversity,
anatomy and biochemistry. Central European Journal of Experimental Biology 3(2): 612.
Schoolmaster DR (2005) Impatiens capensis (Balsaminaceae) Meerb. is a necessary nurse host for the parasitic
plant Cuscuta gronovii (Convolvulaceae) Willd in Southeastern Michigan wetlands. American Midland
Naturalist 153(1): 3340.
Udavant PB, Satyanarayana SV & Upasani CD (2012) Preliminary screening of Cuscuta reflexa stems for Anti
inflammatory and cytotoxic activity. Asian Pacific Journal of Tropical Biomedicine S1: 303307.
Vaughan KC & Duke SO (1984) Function of polyphenol oxidase in higher plants. Physiologia Plantarum 60:
106112.
ISSN (E): 2349 1183
ISSN (P): 2349 9265
4(1): 103, 2017
DOI: 10.22271/tpr.2017.v4.i1.015
Letter to editor
Use of Aloe vera (L.) Burm.f. in the treatment of piles

Gammel Ahmed Baghaffar
Department of Chemistry, Faculty of Science, Hadramout University of Science and Technology,
Mukalla, Yemen
*Corresponding Author: djamil_12@yahoo.com [Accepted: 25 February 2017]
[Cite as: Baghaffar GA (2017) Use of Aloe vera (L.) Burm.f. in the treatment of piles. Tropical Plant Research
4(1): 103]
Aloe vera (L.) Burm.f. is widely cultivated throughout the globe for their various medicinal properties. The
native range of the species is doubtful, but it is assumed that the species may be native of Socotra (Yemen),
Somalia and Sudan (Treutlein et al. 2003). The plant has been used by the people of a number of civilisations
i.e. Greece, Egypt, India, Mexico, China and Japan for different purposes (Morton 1961, Crosswhite &
Crosswhite 1984, Marshall 1990, Mehra et al. 2014).
The use of Aloe extract orally in the treatment of inflammation (Reynoldsa & Dweckb 1999), diabetic
wounds (Chithra et al. 1998b), high cholesterol (Dixit & Joshi 1983) and other gastrointestinal difficulties
(Bland 1985) have already been described.
In the summer of 2004, I had vigorous piles. I used drugs under the guidance of physician for a month. After
not getting any significant benefit, my doctor decided to operate the piles. In the meanwhile, one of my friends
Mr. Abdunnaser Albataty, advised me an ethnic method for treating it. As per his instruction, I prepared a dilute
solution of Aloe (i.e. 1 g / 250 ml water). Now, filtrated the impurities to obtain pure solution. In the morning,
before breakfast, 30 ml of this purified solution with 35 drops sesame oil was taken. This was repeated for one
month. After one month continuous use of this method, I felt comfortable. I shared this method among the
people and also broadcast this method with the help of a radio programme. Number of people had been
medicated by this method. Thats why, here I am informing you this method of piles treatment.
DECLARATION
These are the authors own views, experience and observation. Journal is not responsible for any kind of
discomfort to anyone.
REFERENCES
Bland J (1985) Effect of orally consumed Aloe vera juice on gastrointestinal function in normal humans.
Preventive Medicine 14: 152154.
Chithra P, Sajithial G.B & Chandrakasan G (1998) Influence of Aloe vera on the healing of dermal wounds in
diabetic rats. Journal of Ethnopharmacology 59: 195201.
Dixit VP & Joshi S (1983) Effect of Aloe barbedensis and clofibrate on serum lipids in triton induced
hyperlipidaemia in Presbytis monkeys. Indian Journal of Medical Research 78: 417421.
Crosswhite FS & Crosswhite CD (1984) Aloe vera, plant symbolism and the threshing floor. Desert Plants 6:
4350.
Morton JF (1961) Folk uses and commercial exploitation of Aloe leaf pulp. Economic Botany 15: 311319.
Marshall JM (1990) Aloe vera gel: what is the evidence? Pharmaceutical Journal 244: 360362.
Mehra A, Bajpai O & Joshi H (2014) Diversity, utilization and sacred values of Ethno-medicinal plants of
Kumaun Himalaya. Tropical Plant Research 1(3): 8086.
Reynoldsa T & Dweckb AC (1999) Aloe vera leaf gel: a review update. Journal of Ethnopharmacology 68 (1
3): 337.
Treutlein J, Smith GF, van Wyk BE & Wink W (2003) Phylogenetic relationships in Asphodelaceae (Alooideae)
inferred from chloroplast DNA sequences (rbcl, matK) and from genomic finger-printing (ISSR)". Taxon
52(2): 193.
ISSN (E): 2349 1183
ISSN (P): 2349 9265
4(1): 104108, 2017
DOI: 10.22271/tpr.2017.v4.i1.016
Research article
Effect of organic fertilizers on the performance of seed potato

R. K. Sikder1, M. M. Rahman1, SM Washim Bari2 and H. Mehraj3,4*
1
Horticulture Development Division, BADC, Dhaka-1000, Bangladesh
2
Seed Testing Laboratory, BADC, Dhaka-1216, Bangladesh
3
The United Graduate School of Agricultural Sciences, Ehime University, Matsumaya-shi,
Ehime 790-8556, Japan
4
Lab of Vegetable and Floricultural Science, Faculty of Agriculture and Marine Science, Kochi University,
Monobe 200, Nankoku-shi, Kochi 783-8502, Japan
*Corresponding Author: hmehraj02@yahoo.com [Accepted: 03 March 2017]
Abstract: The experiment was conducted for the evaluation of the performance of seed potato to
organic fertilizers. Two potato varieties viz. Asterix (V1) and Diamant (V2) were subjected to
different inorganic fertilizers viz. Control (T1), Cowdung (T2), Annapurna organic fertilizer (T3),
75% Annapurna organic fertilizer + 25 % Vermicompost (T4) and Vermicompost (T5). Early 80%
emergence was found from T1 (V1: 26.3 days; V2: 23.3 days) while early tuberization from T2 for
V1 (36.0 days) and T1 for V2 (29.0 days). Both varieties performed differently to the organic
fertilizers used in the experiment. T4 was the best for V1 (14.3 kg per plot and 28.8 t.ha-1) while T3
was best for V2 (14.1 kg per plot and 28.3 t.ha-1). Asterix yielded more tuber than diamant variety.
The performances of both varieties were not varied significantly among the treatments for different
graded tuber except for 2855 mm graded tuber in asterix. From the study it is suggested to use of
75% Annapurna organic fertilizer + 25% Vermicompost for asterix variety with BARI
recommended inorganic fertilizers in order to get more yielding seed potato.
Keywords: Annapurna - Vermicompost - Asterix - Diamant - Yield.
[Cite as: Sikder RK, Rahman MM, Bari SMW & Mehraj H (2017) Effect of organic fertilizers on the
performance of seed potato. Tropical Plant Research 4(1): 104108]
INTRODUCTION
Potato (Solanum tuberosum) is the 3rd (just after rice and wheat) most consumed food crop in the world
(Champouret 2010, Verzaux 2010, Visser et al. 2009). The growth and yield of potato largely depends on the
soil and soil conditions can be improved throughout the use of different organic fertilizer. Approximately 4%
organic matter is essential of any agricultural soil while soil of our 60% cultivable land contains organic matter
below 1% (Ferdoushi et al. 2010). Deficiencies of soil organic matter reduced the crop yield which is in
important fact for Bangladeshs agriculture. Bangladeshi farmers generally used the inorganic fertilizers
judiciously to get high yield and this judicious application of the inorganic fertilizers destroy our agricultural
soil. Now it is important to concern about the soil health. Though single nutrient source may supply the
respective required nutrients for plant but integrated use of all sources is required for balanced plant nutrition
(Arora 2008). Vermicompost used as a fertilizer and soil conditioner (Munroe 2007, Rajesh et al. 2003)
responsible for the improvement of the physical properties of soil and supply vital plant nutrients (Smith et al.
2014). Tuber yield of potato is much more after using organic manures than the recommended dose of inorganic
fertilizers only (Boke 2014). Balance fertilization is required for most of the crops also for potato (Alam et al.
2007, Sharma et al. 2003) but tuber yield increases with the application of high amount of manures (Roy et al.
2001, Fageria et al. 1997, Johnston 1986). Lack of quality seed potato and high yielding varieties with poor
agricultural management was the key factor for low yield of potato (Amede et al. 2006, Mehdin et al. 2000).
The hypothesis tested in this study to improve the tuber yield using organic fertilizers. Organic fertilizers were
supplemented with adequate nitrogen in available form for plant (Atiyeh et al. 2000, Bayite-Kasule 2009). The
current study was done to evaluate the growth and yield performance of seed potato throughout the application
of organic fertilizers.
Received: 21 November 2016 Published online: 31 March 2017
Sikder et al. (2017) 4(1): 104108
MATERIALS AND METHOD

An experiment was conducted at Domar Foundation Seed Potato Production Farm, BADC, Nilphamari,
Bangladesh. Two potato varieties viz. Asterix (V1) and Diamant (V2) were assigned to different inorganic
fertilizers viz. Control (T1), Cowdung (T2), Annapurna organic fertilizer (T3), 75% Annapurna organic fertilizer
+ 25% Vermicompost (T4) and Vermicompost (T5) using three replication. In total 60 tubers (in three rows i.e.,
20 tubers/row) were planted on 2.0 m 2.5 m plot. The row to row distance was 60.96 cm. The tuber size
ranged from 2040 mm and tuber to tuber distance was 12.7 cm. Urea, TSP, MP, gypsum and zinc sulphate
were applied @ 220, 120, 220, 100 and 10 kg.ha-1 as basal dose (BARI 2011). Half of urea and entire dose of
the rest inorganic fertilizers were applied during final land preparation. Rest half of urea was applied at 30 days
after planting. Data were collected on different parameters and analyzed by MSTAT-C computer package
program. Means for all the treatments were calculated and the analysis of variance for each of the character was
performed by F (variance ratio) test. Data are presented as the mean standard error (SE). Difference between
treatments was evaluated by least significant difference (LSD) test at 1% level of significance (Gomez &
Gomez 1984).
RESULTS
Day to 80% emergence and tuberization: Days to 80% emergence was varied significantly among the
treatments in both varieties. Early 80% emergence was found from T1 (V1: 26.3 days; V2: 23.3 days) while late
80% emergence was found from T4 (V1: 29.7 days; V2: 26.0 days) (Table 1). Days to tuberization were
statistically identical among the treatments in both varieties. However, earliest tuberization was found in T 2 for
V1 (36.0 days) and in T1 for V2 (29.0 days) (Table 1). V2 showed early 80% emergence and tuberization than V1.
Plant height: Plant height was varied significantly among the treatments. The tallest plant was found from T4
(V1: 63.4 cm; V2: 63.3 cm) while the shortest plant was found from T1 (V1: 61.5 cm; V2: 56.7 cm) (Table 1).
Table 1. Effect of different fertilizer on days to 80% emergence, days to tuberization and plant height of two potato varieties.
Days to 80% emergence Days to tuberization Plant height (cm) at 60 DAP
Treatments
V1 V2 V1 V2 V1 V2
T1 26.3b 0.19 23.3c 0.38 36.3a 0.19 29.0a 0.07 61.5c 0.33 56.7d 1.58
T2 27.2b 0.19 24.0bc 0.33 36.0a 0.07 29.3a 0.19 62.3b 0.33 58.3c 1.58
b
T3 27.3 0.39 24.0bc 0.14 a
36.3 0.13 29.3a 0.19 62.8b 0.33 59.7b 0.19
T4 29.7a 0.19 26.0a 0.11 36.3a 0.17 29.7a 0.15 63.4a 1.00 63.3a 1.64
b
T5 27.4 0.39 25.3a 0.19 a
36.3 0.18 29.3a 0.18 62.1b 0.67 59.3b 0.69
LSD0.01 2.01 1.46 1.46 1.42 0.51 1.12
CV% 3.4 3.24 1.42 1.76 3.12 6.25
Note: Values are means of three replicates SE; Values in a column with having similar and dissimilar superscript letter(s) are
significantly similar and different (p>0.01) respectively; Control (T1), Cowdung (T2), Annapurna organic fertilizer (T3), 75%
Annapurna organic fertilizer + 25% Vermicompost (T4) and Vermicompost (T5).
Table 2. Effect of different fertilizer on tuber yield of two potato varietiesX.

Tuber yield
Treatments kg per plot t.ha-1
V1 V2 V1 V2
T1 10.7b 0.39 10.1b 0.34 23.5cd 0.48 21.0b 0.13
T2 11.3b 0.20 11.2b 0.35 22.2d 0.15 23.5b 0.66
T3 12.7ab 0.11 14.1a 0.37 26.0bc 0.20 28.3a 0.72
T4 14.3a 0.37 13.7a 0.05 28.8a 0.41 27.7a 0.13
T5 12.8ab 0.27 11.2b 0.29 26.2b 0.42 22.9b 0.21
LSD0.01 2.2 1.7 2.5 3.695
CV% 6.6 5.0 3.62 5.47
Note: Values are means of three replicates SE; values in a column with having similar and dissimilar superscript
letter(s) are significantly similar and different (p>0.01) respectively; Control (T1), Cowdung (T2), Annapurna organic
fertilizer (T3), 75% Annapurna organic fertilizer + 25% Vermicompost (T4) and Vermicompost (T5).
Yield: Yield of potato varieties varied significantly among the treatments. In case of V1, maximum yield was
found from T4 (14.3 kg per plot and 28.8 t.ha-1) while minimum from T1 (10.7 kg per plot and 22.2 t.ha-1)
whereas for the V2, maximum yield was found in T3 (14.1 kg per plot and 28.3 t.ha-1) and minimum was found
Sikder et al. (2017) 4(1): 104108
from T1 (10.1 kg per plot and 21.0 t.ha-1) (Table 2). V1 was found as more yielder variety than V2.
Grade wise tuber yield: Yield of different graded tuber was not varied significantly among the treatments in
both varieties (except V1: 2855 mm). Maximum yield was found in T4 at 2855 mm graded tuber (V1: 12.74 kg
per plot and V2: 12.31 kg per plot) (Table 3). In this case V1 also found as the better performer than V2.
Table 3. Effect of different fertilizer on grade wise tuber yield of two potato varietiesX.
Yield (kg per plot) according to different tuber grade
Treatments <28 mm 2855 mm >55 mm
V1 V2 V1 V2 V1 V2
T1 0.65a 0.07 0.81a 0.09 9.30d 0.42 9.02a 0.31 0.73a 0.06 0.49a 0.11
T2 0.75a 0.05 1.02a 0.06 9.92cd 0.25 10.12a 0.79 0.66a 0.11 0.61a 0.10
T3 0.91a 0.09 a
0.74 0.07 ab
11.86 0.45 12.31 0.53a a
0.57 0.02 0.69a 0.06
T4 0.78a 0.04 0.95a 0.06 12.74a 0.66 11.48a 0.41 0.41a 0.10 0.74a 0.09
T5 0.64a 0.13 a
0.57 0.14 bc
10.67 0.72 10.43 0.59a a
0.44 0.13 0.53a 0.13
LSD0.01 0.71 0.68 1.34 3.92 0.83 0.74
CV% 14.63 10.45 14.42 11.42 13.96 12.94
Note: Values are means of three replicates SE; values in a column with having similar and dissimilar superscript letter(s)
are significantly similar and different (p>0.01) respectively; Control (T1), Cowdung (T2), Annapurna organic fertilizer (T3),
75% Annapurna organic fertilizer + 25% Vermicompost (T4) and Vermicompost (T5).
DISCUSSION
The results showed seed potato performed differently on growth and yield to different organic fertilizers.
Nitrogen content increases in soil by the application of organic fertilizers may stimulate the faster plant growth
that lead to more yield (Nogales et al. 2005). The stimulation of the plant growth in organic fertilizers arises by
the presence of the phytohormones (Nogales et al. 2005, Smith et al. 2014). Our results showed that additional
application of organic fertilizers with inorganic fertilizers increases the total tuber yield also different graded
tuber. Integrated nutrient management by the application of both inorganic fertilizers and organic manures
increases the different grades tuber production (Kumar et al. 2008, 2011, Das et al. 2009) and total tuber yield
(Kumar et al. 2001, Raghav & Kamal 2008). Yield of tuber increases due to the availability of N, P and K
contents in soil through the application of organic manures (Kumar et al. 2008, Baishya 2009, Zaman et al.
2011). The maximum advantages from applications of additional organic fertilizers with recommended doses of
inorganic fertilizers might be found and i.e., to enhance uptake of fertilizer, to increased soil physical and
chemical properties. Besides, by providing macro and micronutrient organic manure improve crop production.
Potato yielded more tuber from manure application along with inorganic fertilizers (Johnston 1986, Nyiraneza &
Snapp 2007, Bereez et al. 2005, Alam et al. 2007, Gruhn et al. 2000, Daniel et al. 2008). In our study potato
tuber size <28 mm, 2855 mm, >55 mm were considered as undersized, marketable and oversized as similar to
Chilephake & Trautz (2014). In case of the 2855 mm grade tuber, all the treatments had significant effect in
asterix but it was not found any significant effect in diamant variety. Significant difference for grade wise tuber
yield was found among different genotypes (Bhardwaj et al. 2008) and different treatments (Banjare et al. 2014,
Chilephake & Trautz 2014) while non-significant difference was also found by Banjare et al. (2014).
CONCLUSION
Both the asterix and diamant variety were very popular to farmers in Bangladesh. It was found that asterix
was better than diamant variety considering tuber yield. The asterix variety showed best performance in T4 (75%
Annapurna organic fertilizer + 25% Vermicompost) with BARI recommended inorganic fertilizers among the
treatments used in the study. Annapurna organic fertilizer (T3) was found as the best treatment for diamant. It is
recommended to use BARI recommended inorganic fertilizers with T4 treatment for asterix and T3 for diamant.
But further research is suggested using combination of organic and inorganic fertilizers in different areas of
Bangladesh. From the results of the current study it can be concluded that use of the organic fertilizers with
BARI recommended inorganic fertilizers can improve the tuber yield of potato.
ACKNOWLEDGEMENT
Authors are highly grateful to Bangladesh Agriculture Development Corporation (BADC) for providing the
entire experimental facilities.
Sikder et al. (2017) 4(1): 104108
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ISSN (E): 2349 1183
ISSN (P): 2349 9265
4(1): 109114, 2017
DOI: 10.22271/tpr.2017.v4.i1.017
Research article
Colonization of stem borer damaged maize plants by aflatoxigenic

Aspergillus species in Zimbabwe
N. Nleya*, Y. O. Nyararai, M. Mawanza and F. N. Mlalazi
National University of Science and Technology, Department of Applied Biology and Biochemistry, Box AC
939, Ascot, Bulawayo, Zimbabwe
*Corresponding Author: ndangwa@gmail.com [Accepted: 05 March 2017]
Abstract: The worldwide contamination of food and feed with mycotoxins is a significant
problem. Aflatoxins are major mycotoxins of agro-economic importance produced by some
Aspergillus species. Aflatoxigenic Aspergillus species were isolated from insect larvae damaged
maize stalks and characterized. The fungi were isolated from the stems, ears and silks of the plant.
Aflatoxin production by the isolates was determined using the ammonium hydroxide method as
well as the ultra violet fluorescence method. The Lepidopteran species feeding on the crop was
identified as Busseola fusca. The Aspergillus species isolated were identified as Aspergillus flavus
and Aspergillus parasiticus. This was done using macro- and microscopic features. A total of 35
isolates were obtained of which only 17 were aflatoxigenic. From the aflatoxigenic species 88%
were A. parasiticus whilst 12% were A. flavus. Insects were however negative for aflatoxigenic
fungi implying that these insects do not act as direct vectors of aflatoxigenic fungi, but however
predispose the crop to fungal infection by damaging the physical barrier.
Keywords: Aflatoxins - Busseola fusca - Stalk borer - Vectors.
[Cite as: Nleya N, Nyararai YO, Mawanza M & Mlalazi FN (2017) Colonization of stem borer damaged maize
plants by aflatoxigenic Aspergillus species in Zimbabwe. Tropical Plant Research 4(1): 109114]
INTRODUCTION
Corn (Zea mays) is the most important cereal crop in the world after wheat and rice and belongs to the grass
family Gramineae (Karthikeyan et al. 2013). Cereal grains and their products are the main foods for human
consumption throughout the world. In Zimbabwe maize is of great importance as it the primary staple food and
occupies about half of the agricultural land (Sithole 1989). It is consumed in a wide variety of ways. Green
maize fresh on the cob is eaten roasted or boiled; grain is used in traditional dishes (such as umxhanxa and
inkobe) or ground to mealie-meal to make porridge and paste (sadza/ isitshwala). Maize is also an important
livestock feed both as silage and as crop residue.
The maize grain is vulnerable to contamination by mycotoxigenic fungi if damaged by insect pests. These
moulds include Aspergillus, Penicillium and Fusarium species. Aflatoxins are a group of mycotoxins produced
by some Aspergillus species namely, A. flavus, A. parasiticus and rarely A. nomius (Dvorackova 2000). These
moulds are able to colonize a wide range of crops both in the field as non-destructive pathogens and in storage.
Damage from insects feeding provides preferential sites for penetration by fungi, with some insects acting as
vectors of fungi (Samapundo 2006). The main insects implicated in maize production is the maize stem borer
complex which comprise of the maize stalk borer; Busseola fusca, the pink stalkborer; Sesamia calamistis, the
spotted stem borer; Chilo partellus and the sugarcane stem borer; Eldana saccharina (Capinera 2008). Stem
borer infestations range from 3070% in fields of poor resourced farmers and less than 30% in commercial
farms where insecticides are used for control (Sithole 1989).
Insect larvae that feed on developing corn kernels are implicated in the establishment of Aspergilli infection
and subsequent contamination of the seed with aflatoxins before harvest. These insects facilitate the entrance of
Aspergilli from outside the plant into the ears of the crop (Lawley et al. 2012). Insects infect kernels through the
following ways; (i) they transport primary inoculum to the ears, (ii) they move inoculum from the silks into the
ear, (iii) they disseminate inoculum within the ear and (iv) they wound intact tissue providing more infection
Nleya et al. (2017) 4(1): 109114
sites. Wounding may also allow kernels to dry down to moisture levels that support the growth of Aspergillus
and subsequent aflatoxin production (Giorni 2007, Samapundo 2006).
Aflatoxins are one of the major etiological factors in the development of hepatocellular carcinoma (IARC
2002). There are four naturally occurring aflatoxins aflatoxin (AF) B1, B2, G1 and G2. Mammals that ingest
AFB1 and AFB2 contaminated diets eliminate into milk amounts of the principal 4-hydroxylated metabolite
known as milk toxin or AFM1 and AFM2. These hydroxylated metabolites are potential contaminants in dairy
foods. As such, aflatoxins are not only of importance in humans alone but in animals as well (Samapundo 2006).
This results in corn, when consumed as a staple food, being a major dietary source of mycotoxins to both public
and animal health (Samapundo 2006). The aim of the study was to isolate and characterize aflatoxigenic
Aspergillus that are associated with insect larvae damaged maize stalks in order to assess the impact of damage
to aflatoxin contamination in maize.
METHODS AND MATERIALS

Sampling
A total of 20 maize plants damaged by the maize stalk borer were obtained from a small scale field in
Manningdale, Bulawayo in Zimbabwe. The maize plants were taken to the laboratory where they were sundried
to reduce the moisture content. Nodes of the stem showing insect damage were cut and the surface swabbed
using 70% ethanol. Using a sterile blade, each node was cut vertically so as to expose the damaged tissue.
Corn ears showing insect damage were removed from the stalk, and the leaves were removed to expose the
damaged tissue. Scrapings of the damaged grain were aseptically placed on malt extract agar and the plates
incubated at 25C for 5 days. From each of the 20 ears, 2 mm portions of the silks were excised and placed on
MEA and incubated at 25C for 5 days.
The insect larvae that were found feeding on the stems and ears were surface sterilized using 2% sodium
hypochlorite, followed by two washes in sterile water. This was done so as to remove any microorganism on the
surface of the insect, and to allow isolation of fungi present within the insect. Each insect was then placed on
MEA, and the plates incubated at 25C for 5 days. Insect pupae obtained from both stems and ears were placed
in jars, where they were allowed to complete their life cycles so as to enable easy identification of the species.
Figure 1. A, Maize plant showing signs of stalk borer attack; B, Maize stalk showing points through which the stalk borer
penetrated the plant; C, Pupae of the stalk borer inside the maize stalk; D, Ears showing damage by the stalk borer; E,
Busseola fusca larvae isolated from maize plants.
Nleya et al. (2017) 4(1): 109114
Identification of isolates
Identification of the fungal isolates was done using colony morphology and microscopy. Microscopy was
done using Motic digital microscope and the captured images were identified by comparing images from
(Baquaiao et al. 2013, Ellis et al. 2007, Watanabe 2002).
Detection of aflatoxins
The isolates ability to produce aflatoxins was carried out using the ammonium hydroxide method by Saito
& Machida (1999) after growing the isolates on yeast extract sucrose (YES) agar. To determine the type of
aflatoxin produced by positive isolates from the ammonium hydroxide test, the UV fluorescence determination
according to Davies et al. (1987) using coconut agar media was carried out.
RESULTS
Figure 2. The distribution of Aspergillus in the maize plants.

The insects that were isolated from the maize stalks belonged to one species i.e. Busseola fusca as shown in
figure 1. From the 20 maize plants that were sampled Aspergillus was isolated from 12 maize plants. Figure 2
shows the distribution of Aspergillus species in maize plants. Aspergillus was isolated from 63% of the plants
sampled whereas no Aspergillus was present in the remaining 37%.
Table 1. Macroscopic features used to identify the Aspergillus isolates.
Colony morphology (Macroscopic) Colony colour Identification
(i)
Yellowish green Aspergillus flavus
(ii)
Dark green Aspergillus parasiticus
Nleya et al. (2017) 4(1): 109114
Table 2. Microscopic features used in the identification of Aspergillus isolates.

Microscopic image Features Identification
(i)
Large globose sclerotia

Aspergillus flavus
Biseriate metualae
(ii)
Large globose sclerotia
Uniseriate metualae Aspergillus parasiticus
Septate conidiophore
A total of 35 isolates was obtained. Isolates were obtained from the ears, silks and stems. No Aspergillus was
isolated from the insects. The 35 isolates belonged to two species namely Aspergillus flavus and Aspergillus
parasiticus. The predominating species was A. parasiticus. Macroscopic and microscopic features were used in
the identification of the species as shown in table 1 and 2 respectively. The isolates ability to produce aflatoxins
was done using the ammonium hydroxide test were aflatoxin producers were identified by a change in colour as
shown in figure 3.
Figure 3. Photograph of an aflatoxigenic isolate before (A) and after (B) screening. Before screening the underside of the
plate was cream in colour, and after screening the plate had developed an orange-pinkish colour due to reaction of aflatoxin
biosynthetic intermediates and ammonium hydroxide.
DISCUSSION
Stalk tunnelling was evident in all the maize plants that had been attacked by the stalk borer (Fig. 1C) due to
the feeding of the insect larvae feeding on the plant stem tissue. The insect species was identified as Busseola
fusca (Fig. 1D) which is also known as the African maize stalk borer or the Fuller, and it is the most common
maize pest in Zimbabwe and Sub-Saharan Africa (Sithole 1989). The tunnelling action of B. fusca predisposes
the plant to penetration by fungi, some of which are aflatoxigenic (Samapundo 2006). Members of the
Aspergillus species section Flavi where isolated from the tunnel scrapings and these were A. flavus and A
parasiticus. These were identified by their characteristic conidial heads which occur in shades of yellow-green
Nleya et al. (2017) 4(1): 109114
to brown (Gupta 2012) as shown in table 1. Aspergillus species were isolated from 63% of the maize plants. A.
parasiticus and A. flavus are the major aflatoxigenic species in the world (Samapundo 2006). Both species
produce the most potent aflatoxin, AFB1. This ability to produce AFB1 in maize makes them a significant threat
to both humans and livestock who may consume this contaminated maize and maize stalks respectively. In
addition to AFB1, A. flavus and A. parasiticus produce AFB2. Together AFB1 and AFB2 when consumed by
mammals are metabolically biotransformed to AFM1 and AFM2 and are excreted in milk. This has a negative
impact on nursing animals which may suffer from aflatoxicosis (Gupta 2012). Also, in the commercial set up,
contamination of feed with A. flavus and A. parasiticus may result in contamination of the milk designated for
consumer use. If regulatory laws are thorough, such milk may not be released for consumer use, thus entailing
economic losses to dairies.
In order to determine the role of insects as vectors of fungi, insect larvae were surface sterilized and cultured
on MEA. No aflatoxigenic fungi or Aspergillus species was isolated from them. This means that insects may not
directly act as hosts of fungi. The stalk borer breaks open the physical barrier allowing the fungi to access the
plant tissue. According to Giorni (2007) wounding of the kernels and stems by insects also reduces the moisture
content and this favours the proliferation of A. flavus with subsequent aflatoxin production.
It was observed that the stems were damaged at several sites per stem node as compared to the damage per
ear (Figs. 1B &1D). This trend is greatly explained by the lifecycle of B fusca on maize plants. The female B.
fusca moth oviposit its eggs behind the vertical edges of leaf sheaths. After hatching the first instars migrate to
the whorl where they feed on young and tender leaves deep inside the whorl. From the third instar onwards,
larvae migrate to the lower parts of the plant where they penetrate into the stem. These larvae begin to feed on
the stem, going up the plant to the ear. Thus the ear is usually infested by the pests at much later stages
(Calatayad et al. 2014, Frrot et al. 2006). Results show that the stems had the highest prevalence of
aflatoxigenic fungi of 65%, followed by the silks (42%) and the ears had the least prevalence of 33% (Fig. 2).
The observed trend can be explained by the nutritional content, moisture content and environmental differences
between these plant tissues. Stems have a high nutritional content packed with sugars and complex
carbohydrates (Plessis 2003). This is favourable for both insect and fungal growth. Thus most fungi were able to
proliferate within the stems as compared to the ears and silks. The silks had a moderate aflatoxigenic fungi
prevalence of 42%. Silks are exposed to the atmosphere as such these toxigenic fungi isolated may not be a
result of insect activity, but a result of aflatoxigenic fungal spores flying in the atmosphere which adhere to silk
surfaces.
In Zimbabwe, maize is the staple food consumed by the vast majority of the population. In the rural areas,
households grow the crop themselves and use it to make the mealie-meal, which is consumed on a daily basis all
year round. These communities often do not make use of pesticides due to financial constraints. As a result,
damage of the crop by insects such as B. fusca as demonstrated in the study, allows growth of A. flavus and A.
parasiticus. This means that these communities are at a risk of aflatoxin poisoning. Furthermore, crop residues
that remain after a seasons harvest are given to cattle as feed during the dry season exposing the livestock to
aflatoxin poisoning as the dry stalks and cobs offer the desirable conditions for Aspergillus proliferation, as
demonstrated in the study. Therefore there is a need to develop strategies of controlling or eliminate stem borers
in the communal areas such as cultural control. Cultural control is an economical method of stem borer control
and has been adopted in West Africa. It includes methods such as removal and destruction of crop residues,
intercropping, crop rotation and manipulation of planting dates (Ogah & Ogbodo 2012). Biological methods can
also be used to control the maize stalk borer for example the exchange of species and strains of natural enemies
between regions, and the use of non-co-evolved natural enemies, as well as habitat management solutions,
namely the use of trap plants such as wild grasses on which larval mortality can be very high (Cherry et al.
1999).
ACKNOWLEDGEMENTS
The authors would like to thank the support provided by the National University of Science and Technology
and the Technical staff of the Department of Applied Biology and Biochemistry for their support.
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ISSN (E): 2349 1183
ISSN (P): 2349 9265
4(1): 115125, 2017
DOI: 10.22271/tpr.2017.v4.i1.018
Research article
Evaluation of land-use land-cover change with changing climatic

parameters of a watershed of Madhya Pradesh, India
Sandeep Soni
Remote Sensing and GIS Lab, Mahatma Gandhi Chitrakoot Gramoday Vishwavidyalaya Chitrakoot,
Satna, Madhya Pradesh, India
*Corresponding Author: sandeepsoni80@gmail.com [Accepted: 09 March 2017]
Abstract: In this present study an attempt has been made to determine trend of climatic
parameters (precipitation and temperature) as well as LULCC for the watershed located near the
Achanakmar-Amarkantak biosphere reserve of Central India. For analyzing trends of LULCC,
Remote Sensing technique is used and Landsat satellite data of five different years (1990, 2000,
2005, 2011, and 2013) are procured and eight LULC classes such as settlement, river, water
bodies, high dense vegetation, low dense vegetation, fallow land, open land and agriculture are
identified. The trend analysis carried out over the LULCC data which shows that the high dense
vegetation and agricultural lands are decreasing while settlement, fallow land and low dense
vegetation lands are increasing. Simultaneously, when both parametric and nonparametric methods
of trend analysis are applied over the annual precipitation and temperature (maximum, mean and
minimum) data for the period of 1981 to 2011, significant (p-value < 0.05) decreasing and
increasing trends are observed, respectively. Although, the present study does not include an
establishment of an empirical relationship of LULCC and climate change, result of this study is a
strong indicator of decreasing high dense vegetation having local impacts of decreasing rainfall
and vice-versa.
Keywords: Land-use land-cover - Remote Sensing - GIS - Precipitation & Temperature - Trend
Analysis.
[Cite as: Soni S (2017) Evaluation of land-use land-cover change with changing climatic parameters of a
watershed of Madhya Pradesh, India. Tropical Plant Research 4(1): 115125]
INTRODUCTION
The anthropogenic activities of post Industrial era, have caused significant emission enhancement of
greenhouse gases resulting the global warming. Subsequently, climate change has become the most important
environmental plight of the present time (IPCC 2007 a,b,c,d). However, along with the climate change issue of
present time, rapid land use and land cover change (LULCC) has also taken place throughout the world due to
urbanization, deforestation and population growth (Duncan et al. 1993, Parker & Alexander 2002, Delang 2002,
Duram et al. 2004, Fromard & Vega 2004). It is now presumed that LULCC and climate change are interlinked
with each other (Turner et al. 2007). Since the global and hemispheric climate change can have direct impacts
on temperature and precipitation (rainfall, snow etc.) distribution, enhancement of flood and avalanche
frequency along with changes in the agricultural productivity of a country or region, impact assessments of
climate change for a particular ecosystem or targeted land practices such as national parks, forests or river
basins are necessary.
Simultaneously, LULCC is an important parameter contributing to local and regional climate change (Chase
1999, Pielke et al. 2002). A recent study estimated that 40% of the global temperature rise is due to world-wide
change in land use (Kalne & Kai 2003, Munoz-Villers & Lopez-Blandco 2008). The LULCC of a region is so
pervasive that, when aggregated globally, the LULCC significantly affects the key aspects of Earth system
functioning (Muttitanon & Tripathi 2005). The LULCC is the primary source of soil and forest degradation
(Tolba & El-Kholy 1992) which alters the ecosystem services of a particular land type and affect the ability of
biological systems to support human needs (Sala et al. 2000, Vitousek et al. 1997). However, enough attention
Soni (2017) 4(1): 115125
was not provided to the LULCC during several climate studies which involve detection of climate controlling
factors (Reddy & Gebreselassie 2011). According to the third assessment report of Intergovernmental Panel on
Climate Change (IPCC), climate studies involving detection and attribution techniques of climate controlling
factors have not taken into account the anthropogenic forcing such as changes in the Land use and Land cover
(LULC) (IPCC 2001).
Several national and international research communities have studied the reason, trend and impacts of
climate change at global, hemispherical and regional scale (Chase 1999, Joeri 2011, Paeth 2009). Temperature
and precipitation are assumed to be one of the important climatic parameters that represent climate change on a
long-term basis. Therefore, several explorations on climatic trends have been conducted by analyzing
precipitation and temperature data at different periods of records throughout the world (Dessens & Bucher 1995,
Serra et al. 2001, Marengo 2004, Longabardi & Villani 2009). Most of these studies have shown that the trend
of temperature or precipitation distribution of a particular region is either decreasing or increasing. A recent
study by Karl et al. (1993) have shown that the monthly minimum temperature, from countries comprising 37%
of the global landmass, is increasing by 0.84oC compared to only 0.28oC increase in maximum temperature for
the period 19511990. The IPCC (2007e) report has also demonstrated that the global surface warming is
occurring at a rate of 0.740.18 C during 19062005.
The maximum and minimum temperature datasets are important because minimum temperature alone is
almost certainly not a good parameter to detect heat accumulation in the atmosphere associated with climate
changes (Pielke & Matsui 2005). In addition, minimum temperature is much more sensitive to land use change
than maximum temperature (Hale & Gallo 2008, Runnalls & Oke 2006, Walters et al. 2007).
In India, the climate change is expected to adversely affect forestry, agriculture, temperature, and rainfall
distribution. Climate change is also expected to change the monsoon onset (Lal et al. 1994, Panigrahy et al.
2009) and increase extreme events such as floods (Booij 2005), droughts (Loukas et al. 2008) and devastating
cyclonic storms (Knutson et al. 2010) which have direct consequences on the population and the economy of the
country (Fulekar & Kale 2010). Therefore, climate change studies are of paramount importance and efforts are
continuing to understand the trend of climatic regimes over the Indian subcontinent (Aggarwal et al. 2004, Mall
et al. 2006, Rupa-Kumar et al. 2006, Joshi & Rajeevan, 2006, Samui & Kamble 2009). A study by Kothawale &
Rupa-Kumar (2005) has indicated that the mean annual temperature of the Indian subcontinent has increased at
a rate of 0.05C per decade during 19012003 mostly due to the rise of maximum temperature (0.07C per
decade) rather than the increase of minimum temperature of 0.02C per decade.
However, the LULCC has an inter-relationship with the temperature and precipitation distribution of an area,
and depending on the feedback processes within land and atmosphere, impact of the climate change can be
assessed. A study by Douglas et al. (2006) on the changes in moisture and energy fluxes due to agricultural land
use and irrigation in the central Indian region has shown that the increasing agricultural land use has contributed
significantly in increasing the vapor flux of this region which could modulate the local to regional scale cloud
formation and subsequently, modulate the precipitation distribution. Therefore, it is important that we
understand the LULCC of a particular river basin area or a targeted land practice area, so that the inter-
relationships between the climate parameters and the LULCC can be explored (Turner et al. 1994, Knorr et al.
2011).
Therefore, the present study aims to explore the long-term land use land cover changes of a watershed with
particular focus on investigating changes in the meteorological parameters of the area and their relationships
with LULCC. The Chakrar watershed of Madhya Pradesh is chosen for this purpose. The Chakrar watershed is
an important sub-tributary of the Narmada basin and constitutes part of the Achanakmar-Amarkantak Biosphere
Reserve. The watershed has dominant Sal (Shorea robusta Gaertn.) forest and due to increasing human
population in the last few decades, it is hypothesized that the forest area within this watershed is degrading. An
in-depth analysis of land-use land-cover change of the watershed and subsequent analysis of meteorological
parameters over the study area is, therefore, envisaged to provide (i) whether the underlying hypothesis of
degrading forest cover over the watershed is true and (ii) enhanced understanding of the inter-relationships
between climatic and LULCC drivers.

Study Area
The study area is Chakrar Watershed of Dindori District, Madhya Pradesh, India (Fig. 1). The watershed has
Soni (2017) 4(1): 115125
rich Sal (Shorea robusta Geartn.) dominant forest with altitudinal range 700980 m. The watershed extends
from 22o31' 12.24" to 22o52' 44.93" N latitude and 81o14' 41.23" to 81o28' 29.42" E longitude. Total catchment
area of the Chakrar watershed is 415 km2. The area falls under sub-tropical monsoon climatic region. Average
annual precipitation of this watershed varies between 12001306 mm and annual temperature varies between
1843 C. Maximum amount of rainfall occurs during monsoon season i.e. June to September.
Figure 1. Location map of the study area.

Data Used
In order to investigate the LULCC of the study area, Landsat 5 TM (path 143, row 44) satellite data (Ioannis
& Meliadis 2011) are used for the year of 1990 and Landsat 7 TM (path 143, row 44) satellite data are used for
2000, 2005, 2011 and 2013. Satellite data was procured from USGS websites (www.glovis.usgs.gov). Yearly
continuous data of LULCC was unavailable from the Landsat products over the experimental area. Hence, only
five years of LULCC of the study area was investigated. However, the Meteorological time series data of annual
precipitation, mean temperature, maximum temperature and minimum temperature for 30 years (19812011) are
acquired from India Water Portal (www.indiawaterportal.org/met_data/) for the study area.
Land-Use Land-Cover Change Analysis
Satellite data were processed using ERDAS IMAGINE 9.2 software for geometric corrections (image to
image georectification) with the coordinate system UTM WGS 84, Zone 44 North to make these images
compatible (Lillesand & Kiefer 1994). The images of 1990, 2000, 2005, 2011 and 2013, resampled to 30m x
30m pixel size using the nearest neighbor resampling technique (Serra et al. 2003, Jensen 2005). Pixel based
supervised image classification with maximum likelihood classification algorithm was used to map the land-use
land-cover classes (Lillesand & Kiefer 1994, Shalaby & Ryutaro 2007). Eight LULC classes viz., Settlement,
River, Water bodies, High Dense Vegetation, Low Dense vegetation, Fallow land, Open land and Agriculture
were identified for image classification.
The pre and post field visit was done with a GPS receiver and using a set of questionnaire designed for the
purpose. GPS points were selected for ground truth validation and verification of location (latitude and
longitude) and elevation. The accuracy assessments were performed for classified images of 1990, 2000, 2005,
2011 and 2013. A minimum of about 40 random points were generated per class using stratified random
sampling approach for efficient accuracy assessment (Congalton & Green 2009). The corresponding reference
class for each LULC type was collected from different data sources, including data from field visits, topographic
maps, and raw images. Raw images were used for those visually visible classes, e.g., forests and water bodies
Soni (2017) 4(1): 115125
(Congalton & Green 2009). Topographic maps were utilized to collect reference samples for 1990 classified
images while field visits data were mainly used for the 2013 classified image. Reference points for the 2000,
2005 and 2011 classified image were collected through visual interpretation of the raw Landsat TM 2000, 2005
and 2011 image. This was supplemented by field visits and discussion with elders in the study landscape that
made it possible to establish reference points of different classes.
Trend Analysis
The trend is a significant change over time exhibited by a random variable, detectable by statistical non-
parametric and parametric procedures. According to nz & Bayazt (2003), parametric t-test has less power
than the non-parametric Man-Kendall test when the probability distribution is skewed. With due trend detection
and cross verification, both parametric and non-parametric statistical procedures are applied to the precipitation
and temperature time series.
Mann-Kendall Test
Mann-Kendall test is a non-parametric statistical test used to assess the significance of trends in climatic
time series data such as precipitation and temperature (Mavromatis & Stathis 2011). Non-parametric tests are
thought to be more suitable for non-normally distributed data which are encountered in climatic time series (Yue
et al. 2002). Man-Kendall test was suggested by Mann (1945) for randomness against time, which constitutes a
particular application of Kendalls test for correlation commonly known as the Mann-Kendall or the Kendall t
test (Kendall 1962). The test has been extensively used with environmental time series (Hipel & McLeod 1994,
McLeod et al. 1990). Let X1, X2,Xn represents data points over time, in the test null hypothesis H0 is
tested where the random variables are independent and identically distributed. The alternative hypothesis H A, is
that the data are not identical. Under H0, the Mann-Kendall test is calculated by using following equations
(Lazaro et al. 2001, nz & Bayazit 2003, Kahya & Kalayci 2004).
Where,
Under the hypothesis of independent and randomly distributed random variables, when n 8, the S statistics is
approximately normally distributed with the mean.
The variance statistic is given as
Where, p is the number of tied groups in the data and tj is the number of data values in the jth tied group. As a
consequence, the standard test statistic Z is computed as follows
The test statistic Z is used a measure of significance of trend and used to test the null hypothesis, H0. The test
statistic Z follows a standard normal distribution. A positive (negative) values of Z signifies an upward
(downward) trend.
Spearmans Rho
Spearmans Rho is a rank based test to determine the significance of correlation between two variables that
can be used to test for a correlation between time and the data series (Siegel & Castellan 1988). This test
evaluates the degree to which individuals or cases with high rankings on one variable were observed to have
similar ranking on another variable (Sprent 1989).
Soni (2017) 4(1): 115125
The test statistic s is the correlation coefficient, which is obtained in the same way as the usual sample
correlation coefficient, but using ranks:
Where,
and xi (time), yi (variable of interest), and refer to the ranks ( , , Sx and Sy have the same value in a
trend analysis).
For time series, the quantity is normally distributed mean of 0 and variance of 1.
Linear Regression
Linear regression is a parametric test which is one of the most common trend test and in its basic form
assumes that data is normally distributed. The test statistic for linear regression is the regression gradient. The
test is used to test for linear trend by the linear relationship between time and the variables of interest. The linear
regression gradient is calculated by
And the intercept is estimated as
The test statistic S is
Where,
The application of this test assumes that the errors (deviations from the trend) are independent and follow the
same normal distribution with 0 mean.

Accuracy assessment of the supervised classification of the satellite imagery was derived by using a
reference template from the margining data with 40 randomly selected samples on the latest imagery, from
which overall accuracy and Kappa statistics were derived. The Kappa statistics incorporated the diagonal
elements of the error matrices (Yuan et al. 2005). Satellite imageries of 1990, 2000, 2005, 2011 and 2013 were
classified (Fig. 2) and validated using error matrix and Kappa statistics. The overall accuracy was found to be 91
percent whereas overall Kappa statistics was 0.8898. The statistics shows that the result was overall good.
Land use land cover maps from Landsat imageries of 1990, 2000, 2005, 2011 and 2013 were produced and
trend analysis of LULCC were carried out. Regardless of proportion of changes in the size and type of land
cover, significant changing trends were observed between 1990 and 2013. The major land cover classes such as:
settlement, fallow land, and low dense vegetation show increasing trend in land cover areas (Fig. 3A,B,C)
having r2 of 0.90, 0.89 and 0.72, respectively. Settlement area was found to increase by 1.87%, whereas the
fallow land and low dense vegetation were found to increase by 8.99% and 4.78%, respectively. Significant
decreasing trends were observed for high dense vegetation and agriculture classes. High dense vegetation, which
was spread over 139.83 km2 area in 1990, degraded to only 95.85 km2 in 2013 (Table 1) and remaining
proportion of lands were found to transform to low dense vegetation and fallow land (Fig. 2AE). A significant
Soni (2017) 4(1): 115125
decrement of 10.60% is observed in high dense vegetation with r2 of 0.84 (Fig. 3AF). Agriculture land which
were spread over 53.38 km2 during 1990 reduced to 31.29 km2 in 2013 showing a decreasing trend with r2 =
0.54 and loss of 5.32%. A trend relationship between high dense vegetation with rainfall and agriculture with
rainfall shows a decreasing trend (Fig. 4A, B).
Table 1. Land use land cover changes in Chakrar Watershed during 19902013. [Reproduces from Soni et al. 2015]
Class 1990 2000 2005 2011 2013
Area Area Area Area Area Area Area Area Area Area
(km2) (%) (km2) (%) (km2) (%) (km2) (%) (km2) (%)
Settlement 4.91 1.18 6.41 1.54 10.96 2.64 12.29 2.96 12.69 3.06
River 2.67 0.64 2.71 0.65 2.73 0.66 2.73 0.66 2.72 0.66
Waterbodies 0.24 0.06 0.26 0.06 0.34 0.08 0.43 0.10 0.48 0.11
High Dense 139.83 33.69 132.15 31.84 131.73 31.74 96.25 23.19 95.85 23.10
Vegetation
Low Dense 54.54 13.14 58.46 14.09 54.92 13.23 75.55 18.20 74.37 17.92
Vegetation
Fallow Land 143.04 34.46 141.15 34.01 159.25 38.37 162.56 39.17 180.33 43.45
Open Land 16.40 3.95 11.83 2.85 17.74 4.28 16.63 4.01 17.29 4.17
Agriculture 53.39 12.86 62.07 14.95 37.35 9.00 48.60 11.71 31.29 7.54
Total 415.03 100.00 415.03 100.00 415.03 100.00 415.03 100.00 415.03 100.00
Figure 2. Land use land cover map of Chakrar Watershed during: A, 1990; B, 2000; C, 2005; D, 2011; E, 2013.
[Reproduces from Soni et al. 2015]
Soni (2017) 4(1): 115125
Figure 3. Graph of trend analysis of land use/cover classes: A, Settlement; B, High dense vegetation; C, Low dense
vegetation; D, Fallow land; E, Open land; F, Agriculture land. [Reproduces from Soni et al. 2015]
Figure 4. Trend relationship between: A, High dense vegetation and rainfall; B, Agriculture and rainfall with respective
years.
Soni (2017) 4(1): 115125
Since, it is hypothesized that a major proportion of this LULCC is associated with changing climatology of
the area; trends of two major climatic indicators (precipitation and temperature) were also computed. Along
with the non-parametric Mann-Kendall test for identifying trend of rainfall and temperature for 30 years over
the study area, parametric Linear Regression test for trend analysis and Spearmans Rho test for correlation
analysis with time were also carried out for the climatic indicators. In the Mann-Kendall test, Z statistics and S
score for annual rainfall revealed negative trend at p-value < 0.05 (Table 2A). For annual maximum
temperature, mean temperature and minimum temperature positive trend at p-value < 0.1 were observed (Table
2B,C,D). In Spearmans Rho test, correlation coefficient value for total annual rainfall was found to be negative
(-0.42) at a p-value < 0.05. However, for annual maximum temperature, mean temperature and minimum
temperature correlation coefficient values were found to be 0.16, 0.19 and 0.26, respectively, with p-values <
0.1. In order to compare the trend analysis results from the nonparametric methods with parametric method,
linear regression tests were also performed over the climatic parameters. Sign of slopes of the linear regression
tests for all the climatic parameters were found to be comparable with the Mann-Kendal method. However, the
magnitude of slopes was found to vary marginally within an error range of 10%.
Table 2. Trend analysis: A, Total annual rainfall; B, Minimum temperature; C, Mean temperature; D, Maximum temperature.
A. Total annual rainfall
Test statistic (Statistical table) (Resampling) Result
Z statistics a=0.1 a=0.05 a=0.01 a=0.1 a=0.05 a=0.01
Mann- Total S -2.278 1.645 1.96 2.576 1.615 1.819 2.583 Statistically significant trend
Kendall score = -135 (at a < 0.05). Decreasing trend.
Spearman's Rho = -2.279 1.645 1.96 2.576 1.645 1.997 2.474 Statistically significant trend
Rho -0.416 (at a < 0.05). Decreasing trend.
Linear Sigma = -2.345 1.699 2.045 2.756 1.687 1.968 2.708 Statistically significant trend
regression 4.439 (at a < 0.05). Decreasing trend.
B. Minimum temperature
Z statistic a=0.1 a=0.05 a=0.01 a=0.1 a=0.05 a=0.01
Mann- Total S 1.139 1.645 1.96 2.576 1.7 2.057 2.549 No statistically significant trend
Kendall score = 68 (at a = 0.10). Decreasing trend.
Spearman's Rho = 1.411 1.645 1.96 2.576 1.743 2.061 2.754 No statistically significant trend
Rho 0.258 (at a = 0.10). Decreasing trend.
Linear Sigma = 1.558 1.699 2.045 2.756 1.682 2 2.974 No statistically significant trend
regression 0.005 (at a = 0.10). Decreasing trend.
C. Mean temperature
Kendall score = 52 (at a = 0.10).
Rho 0.187 (at a = 0.10).
Linear Sigma = 1.247 1.699 2.045 2.756 1.754 2.091 2.99 No statistically significant trend
regression 0.005 (at a = 0.10).
D. Maximum temperature
Kendall score = 42 (at a = 0.10).
Rho 0.159 (at a = 0.10).
Linear Sigma = 0.966 1.699 2.045 2.756 1.756 2.096 2.779 No statistically significant trend
regression 0.006 (at a = 0.10).
CONCLUSION
The primary aim of this present study was to evaluate the changing land use and land cover and climatic
parameter of a watershed area near a biosphere reserve of the central India. It was hypothesized that the land use
and land cover change of an area is interlinked with the local climate. Therefore, trend analysis of LULCC and
major climatic parameters were carried out. It was observed that the high dense vegetation and agricultural land
are decreasing for the study area since 1990, while low dense vegetation, settlement and fallow land are
Soni (2017) 4(1): 115125
increasing. Simultaneously, trend analysis of the climatic variables for the period of 1980-2011 over the study
area are revealed that the annual rainfall trend is decreasing whereas, trend of annual maximum temperature,
mean temperature and minimum temperature is increasing. It should be noted that the present study does not
include establishment of a direct relationship between LULCC and climatic control, albeit, an indication of
decreasing high dense vegetation with decreasing rainfall is noted. Therefore, this present study lays the
foundation of a future land use land cover climate model scenario for testing sensitivity of both climate and
LULC to each other.
ACKNOWLEDGEMENTS
Author is thankful to the Vice Chancellor of MGCGV University M.P. India to provide Remote Sensing and
GIS laboratory.
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ISSN (E): 2349 1183
ISSN (P): 2349 9265
4(1): 126133, 2017
DOI: 10.22271/tpr.2017.v4.i1.019
Research article
Effects of genotype and agro-ecological conditions on storability

of soybean [Glycine max (L.) Merr.] seed
Grace J. Chirchir1*, Maina Mwangi1, Desterio O. Nyamongo2 and
Joseph P. Gweyi-Onyango1
1
Kenyatta University, School of Agriculture and Enterprise Development, Department of
Agricultural Science and Technology, Nairobi, Kenya
2
Genetic Resource Research Institute, Kenya Agriculture and Livestock Research Organization, Kenya
*Corresponding Author: gjchirchir@gmail.com [Accepted: 11 March 2017]
Abstract: Soybean seed storability in the tropics is especially important due to rapid deterioration
that varies with the environment, genotype and management practices. The objective of this study
was to investigate soybean seed quality during storage as influenced by genotype and agro-
ecological conditions in Meru South Sub-County, Kenya. Seeds of two commonly grown soybean
genotypes- Gazelle and TGx 1740-2F (SB19) harvested in February 2013 were used to investigate
seed storability. Seed samples (2.5 kg) were stored in gunny bags in a completely randomized
design with three replications, giving a total of 6 experimental units per site. The storage
treatments were set on farm under ambient conditions in two contrasting agro-ecologies: Upper
Midlands II (altitude- 1529 m above sea level; temp.- 18.220.6 C) at Kirege and Lower
Midlands IV (altitude- 1129 m above sea level; temp.- 21.023.5 C) at Igambatuntu. Changes in
seed quality were monitored at 0, 123 and 246 days of storage by subjecting them to germination,
electrical conductivity and accelerated aging tests. Data were analyzed using SAS 2009. Results
revealed that smaller-seeded soybean genotype TGX 1740-2F was a better storer seed than larger
seeded Gazelle as shown by a higher vigor at end 246 days (8 months) of storage. In addition, seed
storage environmental conditions significantly influenced the degree of seed deterioration.
Soybean seed stored at cooler Upper Midlands II had greater vigor than that stored at the warmer
Lower Midlands IV agro-ecology. This study concludes that soybean seed deteriorates in storage
and that genotype by environment interaction plays an important role in expression of seed
storability. It is recommended that soybean be stored in the cooler higher agro-ecologies of the
tropics for improved storability.
Keywords: Soybean - Seed storability - Genotype - Agro-ecology.
[Cite as: Chirchir GJ, Mwangi M, Nyamongo DO & Gweyi-Onyango JP (2017) Effects of genotype and agro-
ecological conditions on storability of soybean [Glycine max (L.) Merr.] seed. Tropical Plant Research 4(1):
126133]
INTRODUCTION
Availability of high quality seed that ensure adequate plant stand is necessary for the production and
expansion of soybean. Between harvest and the next planting season farm- saved seed is usually stored under
ambient tropical storage conditions. The preservation of seed viability and quality in storage is an important trait
both for food usage and for seed use (Bentsink et al. 2000). Generally, viability and quality of seeds gradually
deteriorate after harvest (Coolbear 1995, McDonald 1999), but the deterioration in long-term storage depends on
environment, biochemical, biological, and genetic factors. Loss of seed viability and vigor under high
temperature and RH conditions is a common phenomenon in many crop seeds (Baleevi-Tubi et al. 2010) but
it is well marked in soybean (Burris 1980, Tatipata 2009). Field seed storage conditions of high humidity and
temperature synergistically accelerate physiological deterioration and pathological damage of seed. In addition,
significant genotypic differences in soybean seed storability have been found by several researchers (Kurdikeri
et al. 1996, Shelar 2002, El-Abady et al. 2012, Wien & Kueneman 1981). Such differences have been attributed
Chirchir et al. (2017) 4(1): 126133
to biochemical characteristics of soybean genotypes which affect the degree of seed damage and the ability of
seed to resist the negative consequences of aging (Baleevi-Tubi et al. 2011). Genetic factors such as hard-
seediness, seed size, seed coat color, resistance to diseases and seed chemical composition influence the
expression of seed vigor (AOSA 2009). Soybean seed quality deterioration has been associated with large seed
size and permeable seed coats (Horlings et al. 1991). Soybean seed size was found to have a direct effect on
seed germination and vigor (Sung 1992). In addition, a strong negative correlation was found between
germination and seed weight among soybean genotypes (Singh et al. 1978). However, the physiological and
biochemical mechanisms by which this variability is expressed are still not fully understood, although it has
been found to be significantly influenced by genotype, environment, management practices and their
interactions (Bellaloui et al. 2011). Hence, in the present investigation, efforts have been made to study the
storability of soybean as influenced by genotype and contrasting storage agro-ecological conditions of Meru
South sub-county of Kenya.

Site description:
The storage experiment was conducted in Meru South Sub-county of Kenya, in two soybean growing areas,
representative of the environmental conditions soybean seed would be subjected to in a typical farmers storage
facility. Site 1 was at Kirege village located at 00 20.580S, 037 37.189 E, a higher altitude (1529 m above
sea level ) site within cooler humid agro-ecological zone - Upper Midlands II (UM2) with an annual mean
temperature of 18.2 to 20.6 C (Jaetzold et al. 2006). Site 2 was at Igambatuntu Village located at 00
0619.4N, 037 54 49.7E; altitude 1129 m above sea level, which represented the warmer semi-humid agro-
ecological zone - Lower Midlands IV (LM4) with annual mean temperature of 21.0 to 23.5 C (Jaetzold et al.
2006 ).
Experimental layout:
Farm saved seed of two commonly grown soybean genotypes Gazelle and TGx 1740-2F (SB19), harvested
in February 2013 were used in the experiment. The seed was obtained from two farmers from areas
representative of Upper Midlands III agro-ecology in Meru South and Maara Sub-counties in February 2013.
Seed samples (2.5 kg) from each seed lot was stored in three replicates in synthetic gunny bags (Biryani
Pakistani Rice bags, M/S.H.M Traders) and tied with a sisal twine giving a total of 6 experimental units per
site. The storage treatments were set in continuous non-climate controlled farmers in-house stores, in a
Completely Randomized Design (CRD) with three replications and stored for eight months from 14th March
2013 to 14th November 2013. The seeds were sampled after 0, 123 and 246 days (0, 4 and 8 months
respectively) of storage for quality tests at the Genetic Resource Research Institute Laboratories (S 01 12.955;
E 036 37.859, altitude 2100 m above sea level, AEZ LH3). The seed moisture, standard germination, electrical
conductivity and the accelerated aging tests (AOSA 2009) were performed to monitor any changes in seed
quality during storage.
1000 seed weight:
Weight of 1000 seeds was evaluated by randomly sampling seeds from each seed lot, counting and weighing
them. The values were then corrected at 13% moisture content.
Determination of seed moisture:
Seed moisture contents were determined using a Grain moisture meter (GMK-303RS, G-Won Hitech Co.
Ltd) which measures the electric properties of seed moisture either by conductivity or capacitance within the
range of 625% range. Four replicates per seed lot were sampled, placed inside the moisture meter, ground and
readings taken.
Germination tests:
Seeds of soybean [Glycine max (L.) Merr.] were treated for 40 seconds with sodium hypochlorite solution
(3.85% active ingredient) diluted with water in 1:2 ratio for 40 seconds and then surface washed with distilled
water three times, to retard saprophytic fungal growth. Seeds were then germinated by placing 50 seeds per
replication in four replicates in germination boxes using 500 ml plastic containers with lids, containing 1%
water agar under laboratory conditions (ISTA 2007). The germination boxes were arranged in a completely
randomized design (CRD) in a walk-in germination chamber with alternating 12 h fluorescent light at 30C and
Chirchir et al. (2017) 4(1): 126133
12 hours darkness at 20C. Counts of germinating seeds were made daily, starting on the first day of imbibitions
and terminated 11 days after sowing, when maximum germination was obtained. Seeds were identified as
germinated when 2mm of the radicals protruded (ISTA 2007). Normal seedlings were recorded for calculating
germination percentage (GP) at last count. Germination percent (GP) was calculated as follows:
Electrical Conductivity test:

Electrical conductivity tests were determined by weighing four replicate samples of 50 seeds per treatment
and placing them in 250 ml plastic cups containing 200 ml of distilled water. The seeds were gently stirred to
remove air bubbles. Any floating seeds were removed and the cups covered with aluminum foil. The seeds
were then left to soak in the water for 24 hours at room temperature 202 C. Conductivity of seed leachates
(electrical conductivity) was measured using a Jenway 4020 conductivity meter and CRT-CAA- 515B electrode
dip type cell (Fisons Scientific Equipment) The conductivity meter was standardized with 0.01N potassium
Chloride. The solution was prepared afresh by dissolving 0.7456 g KCl in 1,000 ml of distilled water at room
temperature. The cell constant (K) value at 1.000, and temperature coefficient per C of 2% rise at 25C was
used to take the readings. The electrical conductivity of a control sample of an equivalent quantity of distilled
water was also determined. Conductivity was expressed on a weight basis in micro Siemens per cm per gram
(s.cm-1.g-1) of seed (ISTA 2007).
Accelerated ageing test:
For each seed lot, seeds were first preconditioned by exposing them to humid environment, created by
placing seeds in open trays, above a water pan at room temperature for 48 hours in order to raise the seed
moisture content to between 10% and 14%. After allowing the seed moisture to equilibrate, 100 seed weight,
adjusted to 13% moisture content was taken. Accelerated aging tests were then conducted by placing four
replicates of 100 seeds per seed lot on a screen inside a 382810 cm accelerated aging boxes (Hoffman
Manufacturing Company, Albany, OR) containing 500 ml of distilled water. The boxes were tightly sealed and
placed in an ageing chamber maintained at 41C and 100 per cent relative humidity for 72 hours (Delouche
1965). At the end of ageing, germination tests were conducted in four replicates of 50 seeds per seed lot on 1%
water agar in a germination chamber with alternating 12 h darkness (20C) and 12h of fluorescent light (30C).
The ageing response was measured based on accelerated ageing germination percent of normal seedlings (ISTA
2007).
Statistical analysis:
Data were analyzed using PROC GLM (GLM Procedure) model of the Statistical Analysis Systems
software. Parameters were subjected to Analysis of Variance (ANOVA) and means separated using Least
Significance Difference (LSD) at p < 0.05 based on Tukeys Studentized Range (HSD) test.
RESULTS
The means of seed viability and vigor for tested genotypes of soybean - TGx1740- 2F (SB 19) and Gazelle
before and after storage in both storage agro-ecological conditions of Upper Midlands II (UM2) and Lower
Midlands IV (LM4) was investigated. Results revealed that seed quality traits during storage varied amongst
tested genotypes and storage environments, with statistically highly significant differences (p < 0.05).
Effects of genotype on storability of soybean seed
The genotypic differences in seed viability and vigor of soybean genotypes SB 19 (TGx1740-2F) and
Gazelle in storage under ambient conditions was significant (p < 0.05). There were significant differences in
1000-seed weight between the soybean genotypes (Table 1).
Table 1. Seed weight of soybean genotype Gazelle and TGx 1740-2F.
Genotype 1000 Seed weight Seed coat color
Gazelle 159.20a Cream
TGx1740 2F (SB19) 119.60b Cream
LSD0.05 7.52 -
Note: Values followed by the same letter(s) in each column are not significantly different (p<0.05)
Chirchir et al. (2017) 4(1): 126133
Genotype Gazelle (159.2 g) had a higher 1000 seed weight than TGx 1740-2F (119.6 g). Although both
soybean genotypes were of cream seed coat color, Gazelle was larger seeded than TGx 1740-2F. Further,
soybean genotypes seed viability and vigor during storage showed marked differences (Tables 2 & 3 and Fig. 1).
The seed moisture content fluctuated during storage but generally increased over the 246 DAS from a minimum
of 7.4% to a maximum of 8.9%. However genotypic differences in seed percentage moisture content varied with
site and storage duration.
Table 2. Genotypic differences in studied traits of soybean seed during storage at Upper Midlands II (UM2) Kirege
Meru South Sub-county.
Seed Moisture Electrical conductivity Final
Soybean Content (%) (s.cm-1.g-1) Germination (%)
Genotype 0 123 246 0 123 246 0 123 246
DAS DAS DAS DAS DAS DAS DAS DAS DAS
Gazelle 7.43b 9.36a 8.93a 42.98b 47.85a 44.96b 93.50b 97.67a 97.08a
TGx1740-2F 7.47a 9.35a 8.66b 46.53a 47.38a 47.37a 97.00a 97.42a 96.83a
LSD 2.87 0.07 0.07 2.119 2.736 1.86 2.567 1.271 1.59
r2 1.00 0.99 0.99 0.69 0.82 0.82 0.46 0.24 0.22
Note: Values followed by the same letter(s) in each column are not significantly different (p<0.05). DAS = Days after
storage, UM2-Upper Midland II Agro-ecology.
Table 3. Genotypic differences in studied traits of soybean seed during storage at Lower Midlands IV (LM4)
Igambatuntu, Meru South Sub-county.
Seed Moisture Electrical conductivity Final
Genotype 0 123 246 0 123 246 0 123 246
DAS DAS DAS DAS DAS DAS DAS DAS AS
Gazelle 7.43b 8.23a 7.61a 42.98b 48.55b 50.25a 93.50 b 97.0 a 93.25a
TGx 1740-2F 7.47a 8.08b 7.71a 46.53a 51.75a 50.89a 97.00 a 96.4a 92.75a
LSD0.05 2.871 0.05 0.10 2.12 1.92 2.29 2.56 2.13 2.48
r2 1.00 0.96 0.84 0.69 0.90 0.49 0.46 0.17 0.41
Note: Values followed by the same letter(s) in each column are not significantly different (p<0.05), DAS = Days after
storage, LM4=Lower Midlands IV Agro-ecology.
Throughout storage, both LM4 and UM2 agro-ecologies, soybean seed maintained a high germination
percent of above 92% (Table 2 & 3). Genotypic differences in germination % were only observed at the onset
of storage with TGx-1740-2F (97%) having higher viability than Gazelle (93.5%) but with no differences by
123 and 246 DAS.
Figure 1. Genotypic differences in accelerated ageing (72h, 41C) germination percent of soybean seed during storage at
Lower Midlands IV (LM4) and Upper Midlands II (UM2) after 0 and 123 days of storage.
Chirchir et al. (2017) 4(1): 126133
Seed vigor however reduced over time, with significant genotypic differences across sites and storage
durations as shown by the seed leachate conductance (Tables 2 & 3) and accelerated ageing germination % (Fig.
1). The electrical conductivity (EC) of seed membrane leachates generally increased during storage, signifying
deterioration of seed membranes with aging. By the end of storage (246 days) the EC values of the genotypes
were not significantly different at the warmer LM4 storage site. However, at the cooler UM2 storage site, seed
leachate conductance was higher for TGx 1740-2F (47.3 s.cm-1.g-1) than Gazelle (44.9 s.cm-1.g-1), probably
implying that membrane integrity varied with site and genotype. In addition, the accelerated ageing (AA)
germination percent prior to storage and after 123 days of storage (Fig. 1), revealed significantly higher vigor of
the small seeded genotype TGx-1740-2F than the larger seeded genotype Gazelle at both seed storage sites.
At onset of storage, TGx 1740-2F (83.7%) had a higher vigor than Gazelle (45.7%). After 123 days of
storage, the Accelerated Ageing germination %, hence seed vigor reduced with aging to 34.7% and 55.8% for
smaller seeded TGx 1740-2F which was still significantly higher than for the larger seeded genotype Gazelle
(24.5% and 13.1%) at the UM2 and LM4 storage agro-ecologies respectively.
Effect of seed storage agro-ecology on storability of soybean
A considerable variation in soybean seed germination and vigor across different seed storage agro-ecologies
of Meru South Sub-county are presented on tables 4 and 5. The results revealed that storage agro-ecology
significantly affected the seed germination and vigor of soybean (p < 0.05).
Table 4. Effects of seed storage agro-ecology on soybean seed germination and vigor in year 2013 in Meru South Sub-
County.
Seed Moisture Electrical Conductivity Final
storage
Agro-
genotype 0 123 246 0 123 246 0 123 246
ecology
DAS DAS DAS DAS DAS DAS DAS DAS DAS
UM2 7.43a 9.36a 8.93a 42.98a 47.84a 44.96b 93.50a 97.66a 97.08a
Gazelle LM4 7.43a 8.23b 7.61b 42.98a 48.55a 50.25a 93.50a 97.00a 93.25b
LSD0.05 2.87 0.02 0.02 2.11 2.07 1.95 2.56 2.04 2.12
TGx 1740- UM2 7.47a 9.35a 8.60a 46.53a 47.38b 47.37b 97.00a 97.41a 96.83a
2F (SB19) LM4 7.47a 8.08b 7.71b 46.53a 51.75a 50.89a 97.00a 96.41a 92.75b
LSD0.05 2.87 0.05 0.01 2.11 2.43 2.14 2.56 1.23 2.06
Note: Values followed by the same letter(s) in each column are not significantly different (p<0.05); Upper Midland II
(UM2) Kirege site and Lower Midland IV (LM4) Igambatuntu site.
Table 5. Effects of seed storage agro-ecology on soybean seed vigor by Accelerated ageing (72h, 41C) germination
percent in year 2013 in Meru South Sub-County.
Soybean TGX-1740-2F Soybean GAZELLE
Seed storage agro-ecology
0 DAS 123DAS 0 DAS 123DAS
UM2 83.66a 55.58a 45.66a 24.50a
LM4 83.66a 34.67b 45.66a 13.08b
LSD0.05 3.558 7.622 10.27 9.493
r2 0.48 0.85 0.69 0.64
Note: Values followed by the same letter(s) in each column are not significantly different (p<0.05); DAS = Days after .
storage, LM4=Lower Midlands IV Agro-ecology (Kirege site) and UM2=Upper Midland II agro-ecology (Igambatuntu site).
Soybean seed stored in cooler (1821C) Upper Midlands II- (UM2) had significantly higher seed moisture
than seed stored in warmer LM4 (2124C) by the end of 246 days of storage. However, despite the increased
moisture levels, seed stored better in the cooler agro-ecology of UM2 than the warmer LM4 as shown by the
higher seed germination and vigor. At the end of storage, germination percent was significantly higher at UM2
for Gazelle (97%) and TGx 1740-2F (96.8%) as compared to the warmer LM4 with Gazelle (93.2%) and TGx
1740-2F (92.7%). Also seed vigor was higher at cooler UM2 than at warmer LM4 agro-ecology as shown by the
reduced seed leachates of Gazelle (44.9 s.cm-1.g-1) and TGx 1740-2F (47.4 s.cm-1.g-1) than at LM4 with
Gazelle (50.3 s.cm-1.g-1) and TGx 1740-2F (50.9 s.cm-1.g-1). Similarly, seed vigor test by accelerated ageing
(Table 5) after 123 days of storage showed that soybean seeds were of lower vigor at the warmer LM4 agro-
ecology with TGx 1740-2F (34.67%) and Gazelle (13.08%) than at the cooler UM2 with TGx 1740 2F
Chirchir et al. (2017) 4(1): 126133
(55.58%) and Gazelle (24.5%). These observations suggest greater deterioration of seed membranes and seed
ageing in warmer LM4 than at the cooler UM2 agro-ecology.
DISCUSSION
The demand for soybean in Kenya is continuously growing but poor seed viability and vigor between
harvest and next planting season is a constraint towards its production. Genetic and environmentally based
variation on soybean seed storability was observed in the study.
Effects of genotype on storability of soybean seed
Differences in the initial level of deterioration or ageing of seed lots can be identified by accelerated ageing
(AA) test for Glycine max, which is an ISTA-validated vigor test (ISTA 2011, AOSA 2009). In addition,
Electrical conductivity (EC) test of seed leachates provides a quick decision about seed vigor. High EC of seed
is an indicator of seed membrane deterioration of lower quality seed. In this study, genotypic differences in
soybean seed storability were observed. In particular, larger seeded soybean genotype Gazelle produced higher
seed leachates and lower accelerated ageing germination % values suggesting greater deterioration of seed
membranes and enhanced ageing than in the smaller seeded TGx-1740-2F. In addition, since the initial vigor
before storage was still higher for the smaller seeded TGx 1740-2F than larger seeded Gazelle, resistance to
field weathering (Changrong et al. 2007) may have played a great role. Such genotypic differences in resistance
to field weathering and storability of seed have been found by various researchers. Soybean lines with small
seed size and/or black seed coats have been shown to exhibit greater resistance to field weathering than large
seeded types with lighter colored seed coats (Horlings et al. 1991). Sung (1992) showed that soybean seed size
had a direct effect on seed germination and vigor. Wien & Kueneman (1981) on the other hand found consistent
differences in storability among soybean lines, with some small-seeded lines maintaining more than 50%
germinability after 8 months of adverse ambient storage than large seeded ones; while Saranga et al. (1998)
found that seed vigor was negatively correlated with embryo mass and that large seeds germinated and emerged
later than small seeds. Similar results were obtained by Peksen et al. (2004) in pea (Pisum sativum L.); which
revealed that cultivars with low 100 seed weight had higher germination percentage than larger seed ones. In
soybean, Rastegar & Kandi (2011) reported that smaller seeded cultivars had better germination uniformity and
got food reserves to seedlings more and faster than for larger seeded ones. Hence the differences observed in the
current study on soybean seed viability and vigor highlights variations in resistance to field weathering and in
storability of soybean genotypes; since the smaller seeded genotype TGx 1740-2F resisted the negative
consequences of ageing and stored better than larger seeded Gazelle after 8 months of ambient storage in Meru
South Sub-county. These results demonstrate that seed deterioration and eventual seed storage life may be
dependent on genetic factors.
Effects of agro-ecological conditions on storability of soybean seed
During storage, temperature, moisture, and time are critical factors affecting physical, physiological and
biochemical changes that impact seed properties. These changes include loss of viability, grain color changes,
alterations in moisture content, decomposition of lipids, degradation of protein, and damage to plasma and
organelle membranes (Bailly et al. 1996, 1998, Kumar et al. 1999). Losses in seed quality occur during field
weathering, harvesting and storage, and are exacerbated if seeds are exposed to high temperatures and/or
humidity (Basra et al. 2000). After storage for 8 months, variations in seed storability due to storage agro-
ecological conditions were evident in the current study. The reduced seed storability in the warmer LM4 was
associated with increased electrolyte leakage and lower accelerated ageing germination %. Temperature changes
may have played a greater role than moisture in the stored seed because all moisture content values ranged from
7.4% to 9.4% which was within acceptable levels for seed storage hence were unlikely to adversely affect seed
longevity. However, the upto 6C higher temperature at warmer Lower Midlands IV agro-ecology may have
caused accelerated seed deterioration than in seed stored at the cooler Upper Midlands II. These results agree
with those reported elsewhere that unfavorable storage conditions (high air temperature and high humidity of
air) accelerate seed deterioration, leading to losses in seed germination and vigor of stored seed (Burris 1980,
Tewari & Gupta 1981, Harrington 1973, Chirchir et al. 2016). Warmer storage temperatures cause higher levels
of lipid peroxidation (Buchvarov & Gantcheff 1984) and enzymatic activity leading to increase in the
permeability of seed membranes (Ching & Schoolcraft 1968), increased electrolyte leakage at germination,
higher pathogen loads (Copeland & McDonald 1995) and eventual death of the seed while in storage. From the
Chirchir et al. (2017) 4(1): 126133
current study, soybean seed storage agro-ecology significantly influenced vigor of soybean with the cooler
higher Upper Midlands II agro-ecology of Kirege providing a better storage environment than the warmer
Lower Midlands IV agro-ecology of Igambatuntu of Meru South Sub-county.
CONCLUSION
The genotype by environment interactions played a key role in storability of soybean genotypes. The smaller
seeded soybean TGX 1740-2F was better storer seed than larger seeded Gazelle and seed should be stored for
shorter periods of up to 123 days (4 months) for better viability and vigor. In addition, storing soybean seed in
cooler higher elevations (UM2) of Kirege maintained seed of higher seed germination and vigor than storage in
the lower warmer elevations (LM4) of Igambatuntu of Meru South Sub-county.
ACKNOWLEDMENT
We acknowledge the National Commission for Science, Technology and Innovation for partially funding
this study and the Genetic Resources Research Institute for providing the seed testing laboratory facilities. We
acknowledge the two farmers from Meru South sub-county - Mrs. Leah Gicheru and Mrs. Lucy Moffat who
provided their stores for the soybean seed storage experiment. There was no conflict of interest related to this
work.
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ISSN (E): 2349 1183
ISSN (P): 2349 9265
4(1): 134144, 2017
DOI: 10.22271/tpr.2017.v4.i1.020
Research article
Comparative study of qualitative phytochemical and quantitative

GC-MS analysis of Olea dioica Roxb. from different forest types
of Western Ghats, Karnataka, India
Ashwathanarayana R.* and Raja Naika
Department of PG Studies and Research in Applied Botany, Jnanasahyadri, Kuvempu University,
Shankaraghatta-577451, Shimogga, Karnataka, India
*Corresponding Author: ashwinjamadagni497@gmail.com [Accepted: 15 March 2017]
Abstract: Olea dioica is a medicinal angiospemic tree of Western Ghats India. Roots of the plant
used for cancer and snake bite treatment in siddha and bark, fruit paste is used in the treatment of
rheumatism; decoction of the bark is used to wash old wounds and given in fever. Plant material
was collected from different forest types of Western Ghats, Karnataka were air dried subjected for
soxhlet extraction and these extracts were subjected for preliminary phytochemical and GC-MS
analysis using standard procedures. The results obtained was compared with the different forest
types of Western Ghats revealed that the Sagara forest area showed higher yield of crude extract,
with higher secondary metabolites compared to the Chakra and Kigga forest area. which may be
due to the influence of climatic factors like stress, temperature, rainfall, humidity, wind speed,
light intensity, the supply of water, minerals, and CO2 etc.
Keywords: Olea dioica - Western Ghats - Phytochemical analysis - GC-MS - Benzeneethanol - 4-
hydroxy.
[Cite as: Ashwathanarayana R & Naika R (2017) Comparative study of qualitative phytochemical and
quantitative GC-MS analysis of Olea dioica Roxb. from different forest types of Western Ghats, Karnataka,
India. Tropical Plant Research 4(1): 134144]
INTRODUCTION
Secondary metabolites play a major role in the survival of the plant in its environment. Secondary
metabolites synthesis triggered in response to predators and diseases, stress and also in attraction of pollinators
(Harborne 1978, Wink 1988).
A wide array of external stimuli are capable of triggering changes in the plant cell which leads to a cascade
of reactions resulting in the formation and accumulation of secondary metabolites which helps the plant to
overcome the stress. The biotic and abiotic elicitors trigger enhancement of the secondary metabolite
production. The stimuli are perceived by receptors, which result in the activation of the secondary messengers.
These then transmit the signals into the cell through the signal transduction pathways leading to gene expression
and biochemical changes and the synthesis of secondary metabolites (Sudha & Ravishankar 2002).
Many secondary metabolites like glycosides (Wang et al. 2010), alkaloids (Christiansen et al. 1997, Szabo et
al. 2003), Flavonoids (Larson 1988, Nogues et al. 1998), Phenols (Hernndez et al. 2006), Terpenoids (Nacif &
Mazzafera 2005), Chlorogenic acid is an important intermediate in lignin biosynthesis (Del-Moral 1972) etc.,
were observed whenever stress was induced in the plant.
Botanical classification of Olea dioica Roxb.
Kingdom: Plantae
Phylum: Tracheophyta
Class: Magnoliopsida
Order: Lamiales
Family: Oleaceae
Genus: Olea
Species: dioica Roxb.
Ashwathanarayana & Naika (2017) 4(1): 134144
Olea dioica Roxb. Is an important ethno-medicinal tree belonging to the family Oleaceae. The tree grows up
to 15 m tall. Bark of the tree is brownish, rough; blaze pale brown. Young branchlets are subquadrangular,
lenticellate, glabrous. Leaves are simple, opposite, decussate; petiole 0.61.3 cm long, canaliculate; lamina 7.5
17.5 cm long & 2.37.5 cm wide, elliptic to elliptic-oblong, apex gradually acuminate to subacute, base acute or
attenuate, margin distantly serrate (with strong teeth) or entire, coriaceous to subcoriaceous, glabrous; midrib
flat above, usually reddish when dry; secondary nerves 812 pairs; tertiary and higher order nerves obscure or
faintly impressed. Inflorescences are axillary & divaricate panicles; flowers polygamodioecious, cream-white;
pedicel 0.4 cm long. Fruit is drupe, ellipsoid, blue when ripe; one-seeded. Roots of the plant have medicinal
properties and are used for treatment of cancer and snake bite in siddha medicine. In Maharashtra, the tribes use
Olea dioica fruits for treatment of skin disease. Bark and fruit paste are used in rheumatism; decoction of the
bark is used to wash old wounds and given to counter fever (Pullaiah 2006). Ripe fruits are traditionally used by
the tribes in Kerala forest (Yesodharan & Sujana 2007). Olea dioica leaf ethanolic extract showed appreciable
antibacterial and antifungal activity (Ashwathanarayana & Naika 2015).
Despite of many work on this genus Olea dioica Roxb. a very important medicinal plants were unexplored
for many pharmacological activities which is traditionally used by the folklore and tribes. Therefore, the aim of
the study was to provide data on the expression of secondary metabolites in Olea dioica Roxb. collected from
different type of forest of Western Ghats, Karnataka with slightly different climatic conditions.

Study site
Figure 1. Location of sampling site.

The study site was three different areas with different forest types (Kigga- everngreen, Chakra- semi
evergreen, Sagara- moist deciduous) situated in Shimoga and Chikmagalur district within the Western Ghats,
Karnataka, India with altitude range 630840 m (Kigga- N 14 8'9.4992'' E 74 57'33.0480'', Chakra- N 13
25'5.1636'' E 75 10' 26.1804'', Sagara- N 13 48'28.3212'' E 74 58' 1.3980'') (Fig. 1).
Sample collection
The plant samples were collected from Kigga forest, Chakra forest, Sagara forest, Karnataka. The botanical
identification of the plant was done by Prof. K G Bhat, Udupi and the voucher specimen was conserved under
the reference number KU/AB/RN/AS/001.
Purification and extraction

The plant samples were shade dried for about 30 to 45 days and mechanically powdered. Powdered material
was subjected to soxhlet extraction with ethanol, air dried and kept in an air tight bottles.
Qualitative tests on plant material for secondary metabolites
Extracted plant samples were screened (Gartlan et al. 1980) for the presence of tannins, alkaloids, saponin,
glycosides, flavonoids, steroids/sterols and phenols using the methods described by Harborne (1998).
Alkaloids
Hagers test- test solution was treated with few drops of Hagers reagent (saturated picric acid solution). A
positive result for the alkaloids presence was shown by the formation of yellow precipitate.
Mayers test- test solution was treated with Mayers reagent cream colour precipitate is formed.
Saponins
Foam test- test solution was mixed with water and shaken well and observed for the formation of foam froth
which is stable for 15 minutes for a positive result.
Tannins
Ferric chloride test- Plant material was boiled with water for a few minutes, this was filtered and diluted with
more water. Bluish-black colour formation after addition of few drops of ferric chloride, is indicative of the
presence of tannins.
Gelatin test- test solution treated with gelatin solution gives white precipitate indicating the presence of tannins.
Flavonoids
Ferric chloride test- test solution when treated with few drops of ferric chloride solution would results in the
formation of blackish red colour indicating the presence of flavonoids.
Alkaline reagent test- test solution treated with sodium hydroxide solution shows increase in the intensity of
yellow colour which would become colourless by the addition of few drops of dilute Hydrochloric acid,
indicates the presence of flavonoids.
Lead acetate test- test solution treated with few drops of lead acetate (10%) results in the formation of yellow
precipitate.
Shinda test- test solution and add few fragments of Magnesium ribbon and add concentrated Hydrochloric acid
magenta red colour formed.
Steroids / Sterols
Liebermann burchard test- crude extract mixed with few drops of acetic anhydride, boiled and cooled.
Concentrated sulphuric acid was added from the sides of the test tube and then observed for the formation of a
brown ring at the junction of two layers. Green colouration of the upper layer and the formation of deep red
colour in the lower layer would indicates a positive test for steroids and sterols respectively.
Glycosides
Keller killiani test- Test solution was treated with some drops of glacial acetic acid and ferric chloride solution
then mixed. Concentrated sulphuric acid was added and observe for the formation of two layers. Lower reddish
brown layer and upper acetic acid layer which turns bluish green would indicates positive test for glycosides.
Bromine water test- test solution was dissolved in bromine water and observed for the formation of yellow
precipitate to show a positive result for the presence of glycosides.
Phenols
Ferric chloride test- test solution and add 0.5 ml of ferric chloride results in the formation of intensive colour.
Glacial acetic acid test- test solution and add few drops of 5% glacial acetic acid and 5% of sodium nitrate
result in the formation of muddy yellow or Niger brown or deep chocolate colour precipitate.
RESULTS
Comparison of physical parameters of Olea dioica collected from different places of Western Ghats,
Karnataka
Physical parameters are compared within the forest types, in that, Kigga forest situated in higher altitude
841m above the sea level compared to other to (Chakra- 640 m and Sagara- 613 m) and Kigga is highest rain
fall area (1905 mm) compared to other two (Chakra- 1544 mm and Sagara- 843 m), forest type of three
sampling site varies from evergreen to semi evergreen to moist deciduous or mixed of two or more forest type
observed. Kigga forest has least average annual temperature (2030 C) compared to other two (Chakra- 2334
C and Sagara- 2636 C). Wind speed of Sagara and Chakra forest (5 km.h-1) area is more compared to Kigga
(4 km.h-1). Wind direction of Kigga forest is east and north east side while wind direction of chakra forst is east
and south east and Sagara forest wind blow towards east direction. Average humidity of Kigga forest (95%) is
more compared to the rest of two forest types (Chakra- 80% and Sagara- 79%). Average precipitation also high
in case of Kigga forest (1200 mm) compared to the rest of two forest types (Chakra- 1180 mm and Sagara- 911
mm). All the data collected form metrological stations of Shimoga and Chikamagaluru (Table 1).
Table 1. Comparison of physical parameters of Olea dioica collected from different places of Western
Ghats, Karnataka (represented from IIRs 2013).
S.N. Parameters Kigga forest Chakra forest Sagara
1 Altitude (m) 841m 640m 613 m
2 Rainfall (mm) 1905 1544 843
3 Forest type Evergreen Evergreen Semi evergreen,
Semi evergreen Semi evergreen Moist deciduous
4 Average 2030 C 2334 C 2636 C
temperature (C)
5 Average humidity 95% 80% 79%
6 Average 1200 1180 911
precipitation (mm)
7 Wind speed 4 km.h-1 5 km.h-1 5 km.h-1
8 Wind direction E NE E SE E
9 Latitude longitude N 14 8' 9.4992'' N 13 25' 5.1636'' N 13 48' 28.3212''

E 74 57' 33.0480'' E 75 10' 26.1804'' E 74 58' 1.3980''
Compound yield of Olea dioica plant with respect to different climatic zones of Western Ghats, Karnataka.
For soxhlet extraction 750 grams of different plant parts like Matured leaves, Immature leaves, Flower,
immature fruit, mature fruit, Immature seed, Matured seed, Inner bark, Outer bark & Roots were used and run
using ethanol. The compound yield was compared between three forest types (Kigga, Cakra and Sagara) reveals
that the Sagara forest yields highest extract compared to chakra and Kigga. The moderate Olea dioica extract
yield is observed in chakra and least extract of Olea dioica was observed in Kigga forest (Table 2; Fig. 2).
Table 2. Extract yield of ethanolic extracts of Olea dioica collected from different places of Western Ghats, Karnataka.
S. Solvent Quantity of Quantity of yield of extract (gram)
Plant parts
N. used sample used Kigga forest Chakra forest Sagara forest
1 Matured leaves 14.32 15.33 17.31
2 Immature leaves 10.56 9.32 14.35
3 Flower 9.54 10.43 15.67
4 immature fruit 12.34 14.54 17.22
750 grams
Ethanol
5 mature fruit 15.34 13.43 16.98

6 Immature seed 11.22 12.65 15.35
7 Matured seed 14.32 15.97 19.65
8 Inner bark 19.67 20.87 20.45
9 Outer bark 20.32 23.44 29.67
10 Root 19.67 21.83 30.54
Distribution of secondary compounds in plant parts with respect to different climatic zones of Western Ghats,
Karnataka.
1. Kigga forest: Olea dioica plant parts collected form Kigga forest subjected for qualitative phytochemical
analysis which revealed the presence of many secondary metabolites. Alkaloids were present only in matured
leaves, inner bark, outer bark and root. Saponins were present in all the parts except immature leaves and
immature seed. Tannins were distributed in matured leaves, immature leaves, inner bark and roots. Flavonoids
were present in all the parts examined except immature leaves and immature seed. Steroids were present in
matured leaves, matured fruit and matured seed. Glycosides were present in matured leaves, matured seeds,
inner bark, outer bark and roots. Phenols were absent in immature leaves and immature seed (Table 3).
Figure 2. Extract yield of ethanolic extracts of Olea dioca collected from different places of Western Ghats, Karnataka.
Table 3. Presence of secondary metabolites in Olea dioica collected near Kigga, Chakra and Sagara forests (Qualitative assay).
Alkaloids Saponins Tannins Flavonoids Steroids/sterols Glycosides Phenols
Plant
S.
Chakra
Chakra
Chakra
Chakra
Chakra
Chakra
Chakra
Kigga
Sagara
Sagara
Sagara
Sagara
Sagara
Sagara
Sagara
parts
Kigga
Kigga
Kigga
Kigga
Kigga
Kigga
N.
used
1 Matured - - + + + + + + + + + + + + + + + + + + +
leaves
2 Immature + + + - - - + - + - - + - - - - - - - - +
leaves
3 Flower - - - + + + - - - + + + - + + - + + + + +
4 Immature - - + + + - - - - + - + - - - - + - + - +
fruit
5 mature - - + + + + - + + + + + + + + - - + + + +
fruit
6 Immature - - - - - - - - - - - + - - - - + + - - +
seed
7 Matured - - + + + + - - - + + + + + + + + + + + +
seed
8 Inner + + + + - + + + + + - + - - - + - - + - -
bark
9 Outer + + - + + - - - + + + + - + + + + + + + +
bark
10 Root + + + + + + + - - + + + - - - + + - + + +
Note: +, presence; -, absence.
2. Chakra forest: Olea dioica plant parts collected form Chakra forest has different metabolite confirmation,
alkaloids present in immature leaves, inner bark, outer bark and root parts. Saponins were present in all the parts
except immature leaves, inner bark and immature seeds. Tannins were distributed in matured leaves, matured
fruit, and inner bark. Flavonoids were present in parts like matured leaves, flower, matured fruit, matured seed,
outer bark and root except immature leaves, immature seeds and inner bark. Steroids were present in matured
leaves, flower, matured fruit, matured seed and outer bark. Glycosides were present in matured leaves, flower,
immature fruit, immature seed, mature seed, outer bark and roots. Phenols were absent in immature leaves,
immature fruit, immature seed and inner bark, but in all the plant parts phenols were present (Table 3).
3. Sagara forest: Olea dioica plant parts collected form Sagara forest subjected for qualitative phytochemical
analysis revealed the confirmation of different secondary metabolites compared to other study sites. alkaloids
present in all the plant parts except flower, immature seed and outer bark. Saponins were present in all the parts
except immature leaves, immature fruit, immature seeds and outer bark. Tannins were distributed in matured
leaves, immature leaves, mature fruit, inner bark and outer bark. Flavonoids were present in all parts. Steroids
were present in matured leaves, flower, matured fruit, matured seed, and outer bark. Glycosides were present in
matured leaves, flower, mature fruit, immature seed, mature seed and outer bark. Phenols were absent in inner
bark, but in all the plant parts phenols were present (Table 3).
Figure 3. GC-MS of crude ethanolic extract collected from Sagara forest showing percentage of different compounds.
Table 4. Presence of metabolites in GC-MS analysis of crude ethanolic extract collected from different sampling sites with their properties.
S. Chemical compound Average Properties of the compound Kigga Chakra Sagara
N. present Percentage forest (18 forest (33 forest (38
compounds compounds compounds
present ) present) present)
1 Glyceraldehyde 1.97 Parental compound of + + +
glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) enzyme
induced under environmental stress
2 3-Hydroxy-2(5H)- 0.23 anti-oxidants and anti-inflammatory - + +
furanone
3 Formamide, N- 0.71 Hematoxic to animals - + +
(cyanomethyl)-
4 Benzenemethanol 0.23 Bacteriostatic agent - - +
5 2,4-Hexanedione 0.29 Antimicrobial activity - + +
6 Pentanoic acid, 4-oxo- 0.37 Anti-inflammatory and medication - + +

for cancer; anti-bacterial
7 2-Furancarboxylic acid 0.16 Bactericide and fungicide - + +
8 Cyclopentane, 1-acetyl- 1.62 Unknown + + +

1,2-epoxy-
9 Cyclohexanone, 2,6- 0.26 Cytotoxic, Antimicrobial, - - +
dimethyl- Anticancer, Anti-malarial activity
10 Phenylethyl Alcohol 0.33 Antimicrobial - + +
11 2-acetyl-2-hydroxy- 0.41 Aecreational intoxicant, - + +
.gamma.-butyrolactone
12 4H-Pyran-4-one, 2,3- 2.22 Sedative, Cytotoxicity, Anticancer, + + +
dihydro-3,5-dihydroxy- Anti-inflammatory
6-methyl-
13 4H-Pyran-4-one, 3,5- 0.52 Antioxidant - + +
dihydroxy-2-methyl-
14 1,2-Benzenediol 0.17 Pesticides, Carcinogen, Cytotoxic - - +
15 2,3-DIHYDRO- 0.82 Entactogen agent - + +

BENZOFURAN
16 2-Furancarboxaldehyde, 3.81 Mutagenic + + +
5-(hydroxymethyl)-
17 1,2,3-Propanetriol, 1.25 Antifungal agent + + +
diacetate
18 2H-Pyran-5-carboxylic 1.08 Unknown + + +
acid, 2-oxo-, methyl
ester
19 2-Methoxy-4- 0.52 Flavoring agent - - +
vinylphenol
20 Borolo[1,2-a]borine, 1.01 Unknown + + +
octahydro-
21 Benzeneethanol, 4- 37.44 Antioxidant + + +
hydroxy-
22 Benzene, 1- 2.82 Unknown + + +
(bromomethyl)-3-nitro-
23 Tyramine, N-formyl- 1.28 Unknown - + +
24 1,3-Benzenediol, 4- 6.46 Antioxidant + + +
propyl-
25 2-Amino-3-(3,4- 16.47 psychoactive drug, cytotoxic to rats + + +
Dihydroxy-Phenyl)-
Propionic Acid (L-
Dopa)
26 Benzeneacetic acid, 4- 0.41 Cytotoxic - + +
hydroxy-3-methoxy-,
methyl ester
27 2-(2-Hydroxy-2- 0.38 unknown - + +
phenylethyl)-3,5,6-
trimethylpyrazine
28 2H-Benzocyclohepten-2- 0.32 unknown - + +
one, 3,4,4a,5,6,7,8,9-
octahydro-
29 3,7,11,15-Tetramethyl-2- 0.49 chemical deterrents against + + +
hexadecen-1-ol predation
30 2,4-Dimethoxytoluene 5.01 insect repellant activity + + +
31 Cyclohexene, 1,5,5- 0.6 unknown - - +

trimethyl-6-
acetylmethyl-
32 2,4-Hexadienedioic acid, 0.84 unknown + + +

3,4-diethyl-, dimethyl
ester, (E,Z)-
33 2H-Pyran, tetrahydro-4- 0.85 Flavor Ingredients, fragrance - + +
methyl-2-(2-methyl-1- chemical
propenyl)-
34 Benzaldehyde, 2,3- 4.63 Antioxidants, Flavor Ingredients + + +
dimethoxy-
35 Benzoic acid, 3-amino- 0.79 unknown + + +
6-(4-morpholyl)-
36 4-tert-Butoxystyrene 1.51 unknown + + +
37 Benzenamine, 3,4- 0.59 precursor for vitamin B2, with - + +
dimethyl- ( modest toxicity
38 Pyridinium, 2,6- 1.14 unknown + + +
dimethyl-1-
[(phenylsulfonyl)amino]-
, hydroxide
Note: +, presence; -, absence.
Figure 4. GC-MS of the Olea dioica crude extract: A, Kigga forest; B, Chakra forest; C, Sagara forest.
GC-MS analysis of ethanolic crude extract collected from different sampling sites
In GC-MS analysis of medicinal Olea dioica ethanolic extract revealed the presence 18 compounds form
Kigga forest, 33 compounds from chakra forest, 38 compounds from Sagara forest in that major compounds
present in all the sampling site was Benzeneethanol, 4-hydroxy - (37.44), 1,3-Benzenediol, 4-propyl - (6.46), 2-
Amino-3-(3,4-Dihydroxy-Phenyl)-Propionic Acid (L-Dopa) - (16.47), 2,4-Dimethoxytoluene - (5.01),
Benzaldehyde, 2,3-dimethoxy - (4.63) in average percentages. GC-MS analysis of ethanolic crude extract
revealed that it has Benzeneethanol, 4-hydroxy - (37.44%) has anti-oxidant and cytotoxic properties and 2-
Amino-3-(3,4-Dihydroxy-Phenyl)-Propionic Acid (16.47%) commonly called as L-Dopa which is used as
psychoactive drug and also cytotoxic properties (Table 4; Figs. 34).
DISCUSSION
Presence of secondary metabolites in Olea dioica collected near Kigga, Chakra and Sagara forest
(Qualitative assay)
Alkaloids were absent from semi ripened and ripe fruit, which may declines the seed dispersal chances
through animals (McKey 1974) in all the other parts alkaloids were present main defense compound and main
compound released during stress conditions.
Saponins were found in all plant parts examined. The lowest occurrence of saponins was found in tree twigs.
Most of the saponins observed were poisonous to fishes, pests etc. but its expression to the physical test is
unknown. Saponins are widespread in plant, studies also revealed that saponins and tannins should not co-occur
in plant parts but in our experiments plant parts like leaf, fruit, bark and root parts both tannin and saponins were
present which has to be investigated.
It is belief that condensed tannins have evolved mainly in defense against microbes and fungi (Azaizeh et al.
1990). In our study area there is varied forest type with or without human interference and for about 4 months of
rainy season also triggers favorable conditions for common pathogens as well as opportunistic to infect plants
so, the plants must produce metabolites like condensed tannins against these pathogens to survive and
propagate. Having powerful antioxidant activity and anticancer properties of hydrolysable tannins its plant
defense activity was not clearly known.
We observed tannins mainly in matured plant parts like inner bark, entire bark and root and matured leaf and
fruit parts because plant should protect these parts otherwise it will be dead in the growth stage and will in turn
affects the plant growth. In immature leaf, flower, immature fruit, matured fruit, immature seed and matured
seeds we observed nil tannins because the tannins will resist water, seeds must be germinate as soon as possible
so it must be less defense compounds and rich in other nutritive compounds which attracts the animals as well as
microbes, in other parts the absence of tannin is unknown. In some matured and immature leaf parts tannins
were observed which may hydrolysable tannins and in matured fruit we observed tannins which may be
condensed tannins not hydrolysable tannins (Mali & Borges 2003).
In entire bark, root parts we observe nil tannins, because they protected by high levels of fiber and lignin
therefore do not require protection from other compounds. But Inner bark shows positive results for tannin
reason is unknown. We observed tannins mainly in matured plant parts like inner bark, entire bark, matured
fruit, matured leaf and root parts. In immature leaf, flower, immature fruit, immature seed and matured seeds we
observed nil tannins. Inner bark and entire bark tannins were observed may help in regulating the growth of
phloem, xylem, cortex and epidermis. Tannins has main role in controlling the pest invading stem root etc.
Different climatic zones of Western Ghats will not affect the presence of tannins in different plant parts of Olea
dioica but in Kigga forest we observe less tannins form all the parts.
In our study flavonoids mainly present in all the parts except immature leaves and immature seeds.
Flavonoids mainly present in flowers because they are the important plant pigments for flower colouration and
pigmentation in petals intended to attract pollinator insects and animals. Flavonoids also present in matured
leaves, roots, matured seed, inner and outer bark because flavonoids has many tasks like UV filtration,
symbiotic nitrogen fixation, chemical messengers, physiological regulators, and cell cycle inhibitors. Some
flavonoids have inhibitory activity against pathogens that cause plant diseases, e.g. Fusarium oxysporum.
Flavonoids also have protective functions in drought stress.
Sterols and Steroids were helps plant to tolerance/resistance a wide range of biotic and abiotic stresses, like
drought, salinity, heat, cold, virus infection, and pathogen attack (Divi & Krishna 2009). In our study the
research plant steroids were mainly found in matured leaves, flower, matured seeds, matured fruit and inner bark
which may be localized in the this part against the physical stress and also pathogenic defense.
Glycosides mainly found in matured leaves, matured seed, inner bark, outer bark, roots. Glycosides appear to
be very much less as defense chemicals than alkaloids, saponins and phenolics.
Phenols were found in in all the parts except immature leaves and immature seeds. Phenols were main
defense secondary metabolites against almost all pathogens (phytoalexins) and major compound active during
the physical stress.
Climatic factors affecting on the bio synthesis of secondary metabolites
Altitude matters in case of plant to encounter stress. Sagara forest is nearer to coastal area compared to
Kigga and chakra has less altitude with high stress compared with higher altitude plants. Rainfall data was
evaluated showed that the Kigga forest was well rain fed area with evergreen type of forest compared to Chakra
and Sagara which has semi- evergreen and moist deciduous forest types. Sagara forest has least rain fall which
in turn triggers the stress tolerating machineries with the other stress factors like high temperature, less
humidity, less precipitation and high wind speed compared to Kigga and chakra forest. Due to stress plants
trigger the production of stress tolerating metabolites for its existance. GC-MS analysis was compared with the
three forest types showed that many secondary metabolite present in the extract has some role in tolerating
physical and biological stress.
GC-MS analysis
In GC-MS analysis of Olea dioica ethanolic extracts revealed the presence of 18 compounds collected form
Kigga forest, 33 compounds collected from chakra forest, 38 compounds collected from Sagara forest (Table 4;
Figs. 34). The major and the common compounds present in all the sampling site was Benzeneethanol, 4-
hydroxy - (37.44), 1,3-Benzenediol, 4-propyl - (6.46), 2-Amino-3-(3,4-Dihydroxy-Phenyl)-Propionic Acid (L-
Dopa) - (16.47), 2,4-Dimethoxytoluene - (5.01), Benzaldehyde, 2,3-dimethoxy - (4.63) in average percentages.
These compounds has many medicinal properties as showed in table 4 along with other unexplored metabolites
has some medicinal value yet to be discovered.
CONCLUSION
The results obtained was compared with the different zones of Western Ghats revealed that, the Sagara forest
area shows higher yield of crude extract, compared to the Chakra and Kigga forest area and preliminary
phytochemical analysis also revealed that the presence of secondary metabolites was higher in case of Sagara
forest area compared to the Chakra and Kigga forest area. Climatic factors like stress, temperature, rainfall,
humidity, wind speed, light intensity, the supply of water, etc. were the main reason which triggers the stress in
the Sagara forest to synthesize more secondary metabolites to tolerate it and to survive in that condition.
AKNOWLEDGEMENTS
Author thankful to Department of PG studies and Research in Botany, Kuvempu University, for providing
facility to conduct the experiment.
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ISSN (E): 2349 1183
ISSN (P): 2349 9265
4(1): 145152, 2017
DOI: 10.22271/tpr.2017.v4.i1.021
Review article
Quality protein maize (QPM): Genetic basis and breeding perspective

Swapan K. Tripathy*, Dinesh M. Ithape, Manasmita Maharana and A. M. Prusty
Department of Agricultural Biotechnology, College of Agriculture, OUAT, Bhubaneswar, Odisha, India

*Corresponding Author: swapankumartripathy@gmail.com [Accepted: 10 April 2017]
Abstract: Major fraction (60%) of seed storage protein in maize is zein which determines the
quality of food and feed. Zeins comprise four subfamilies e.g., , , and zeins. Among these,
- zeins are the major prolamin subunits in maize. -zeins are rich in glutamine, leucine and
proline but, deficient in essential amino acids like lysine and tryptophan causing malnutrition. The
opaque-2 (o2)-a natural recessive mutation in maize led to nearly double the lysine and tryptophan
content in endosperm due to a decrease in the synthesis of zein proteins and increase in the other
seed protein bound lysine and tryptophan. RNAi studies proved down regulation of 22kD zeins
than the 19kD component as the biochemical basis of QPM phenotype. However, the opaque-2
mutation made the endosperm chalky and soft resulting damaged kernel while harvesting, poor
germination, increased susceptibility to pest and diseases, inferior for food processing and in
general reduced yield. Later, combining opaque-2 allele with its desirable genetic modifiers made
it possible to breed QPM genotypes having hard kernel with high lysine and tryptophan content.
Since, opaque-2 is a recessive mutation and endosperm specific, and biochemical analysis of
lysine and tryptophan content is expensive; conventional backcross breeding alone is inefficient
for the nutritional enrichment of maize. However, use of opaque-2 gene specific markers provided
excellent opportunities for conversion of elite normal inbreds to homozygous o2/o2 forms through
marker assisted selection (MAS). In India, Vivek QPM-9: a hybrid of two QPM introgression lines
is being widely used for commercial cultivation.
Keywords: Quality protein maize (QPM) - Opaque-2 - Nutritional value - Introgression lines -
QPM hybrids.
[Cite as: Tripathy SK, Ithape DM, Maharana M & Prusty AM (2017) Quality protein maize (QPM): Genetic
basis and breeding perspective. Tropical Plant Research 4(1): 145152]
INTRODUCTION
Maize is the queen of cereal crops with highest grain yield potential. Endosperm is the store house of seed
storage proteins. Maize grains contain around 9% protein. The major fraction (60%) of seed protein in maize is
zeins (a prolamin group-alcohol soluble) (Leite et al. 1999) followed by glutelin (34%), while albumin and
globulin occur in traces (3% each). A balanced protein is required to assist body building process and therefore,
amino acid balance seems to be a determining factor for quality of any food and feed. Daily protein requirement
for average Indian adult is 52 gm as against the availability of merely 2630 gm in the daily diet. To alleviate
malnutrition, protein content can be increased to as high as 18% by increasing the prolamine (zein) fraction in
maize endosperm (Dudley & Lambert 1969), but unfortunately it consequently led to lysine and tryptophan
deficiency. Many researchers around the globe have tried to address the problem using quality protein maize
(QPM). In this pursuit, we focus on the genetic basis and prospects of quality protein maize for amino acid
amelioration and enhancing the productivity of maize through the development of heterotic hybrids using elite
QPM introgression lines.
GENETIC BASIS
Zein seed proteins are the products of multigene families (Lending & Larkins 1989) and located within
protein bodies on the rough endoplasmic reticulum (Larkins et al 1993). Each of the zein polypeptide is a
product of a differential structural gene (Zp). These Zp genes are simply inherited and are members of a large
Received: 07 December 2016 Published online: 30 April 2017
Tripathy et al. (2017) 4(1): 145152
group of genes (upto 150). Zein is particularly rich in glutamine (2126%), leucine (20%), proline (10%) and
alanine (10%), but deficient in important essential amino acids e.g., lysine and tryptophan leading to protein
malnutrition. - zeins are the major prolamin subunits in maize, although other minor groups (-15kD, -16 &
27kD and -10kD zeins) (Coleman & Larkins 1999, Leite et al. 1999) are also present in seeds. In normal
maize, - zeins consist of two major sub-classes e.g., 19kD and 22kD zeins. Polymorphism arises from the
presence of multigene families. and -zeins form the protein body core which is covered by peripheral and
- zeins (Lending et al. 1992, Esen & Stetler 1992).
The discovery of opaque-2 (o2) natural mutants by Purdue university researchers paved the way for
improving protein quality of maize endosperm protein (Mertz et al. 1964, Nelson et al. 1965). These mutants
alter amino acid profile and composition of maize endosperm protein and result in two-fold tryptophan increase
in the levels of lysine and tryptophan compared to normal maize. However, the pleiotropic effects of opaque-2
mutation made the endosperm unpleasant taste, chalky, lighter and soft (starch granules loosely packed)
resulting damaged kernel while harvesting, increased susceptibility to pest and diseases, inferior for food
processing and in general reduced cob weight and lower yield due to reduced dry matter accumulation. For
many years, this became the major hindrance in genetic improvement for higher productivity with enriched
nutritional quality. Subsequently, the opaque-2 mutation is reported to be associated with numerous modifiers
which together behave as polygenic trait for kernel virtuousness. Later, concerted effort of Surinder K. Vasal
and Evangelina Villegas (CIMMYT) made it possible to improve kernel hardness and grain yield by combining
the opaque-2 and its complex genetic modifier systems using modified backcrossing and recurrent selection.
This gave birth to the quality protein maize (QPM).
There are several mutants that alter the amino acid profile of maize endosperm protein. These include
opaque-2(o2), floury-2 (fl-2), Mucronate (Mc) and Defective endosperm B30 (DEB30). The opaque mutants are
recessive (o1, o2, o5, o9-11, o13, 016, o17), the floury mutations are semi-dominant (fl-1, fl-2 and fl-3) where
as Mucronate and Defective endosperm are dominant mutations. The opaque mutations affect the regulatory
network (Mertz et al. 1964, Nelson et al. 1965, Krivanek et al. 2007) whereas floury, Mucronate and defective
endosperm affects the amino acid profile of storage proteins (Gibbon & Larkin 2005). Similar to opaque 2;
opaque-16 mutant allele is also reported to increase lysine and tryptophan content in the endosperm. Recently, a
transposable element rbg is reported to induce differential expression of opaque-2 mutant gene in two opaque
2 NILs derived from the same inbred line (Chen et al. 2014).
The molecular evolution of the opaque-2 gene was investigated by Henry et al. (2005). Sequencing of most
of the coding regions and parts of non-coding sequences of the o2 gene in a set of cultivated and teosinte (wild
progenitor of cultivated maize) accessions revealed 5.4% polymorphic sites and 72 insertions/deletions, located
mostly in noncoding regions. Cultivated accessions retained about 70% of the diversity observed in teosintes.
Besides, the molecular diversity in the o2 transcriptional activator was reported to be quite high compared to
that of other transcription factors in maize (Henry et al. 2005).
In fact, opaque-2 (o2) is a natural recessive mutation in the transcriptional activator conditioning negative
expression of zein protein (down regulation). This led to nearly double the lysine and tryptophan content in
maize endosperm due to a decrease in the synthesis of zein proteins and increase in the other seed protein bound
lysine and tryptophan (Henry et al. 2005). The o2 gene has a large effect on lysine and protein content while
minor on oil content (Lou et al. 2005). It seems that developing maize seeds possess compensatory mechanisms
that sense protein content when zein synthesis is interrupted, leading to translation of other mRNAs instead of
zein mRNAs. Combining deletion mutagenesis with current methods in genome and transcriptome profiling can
make it possible to reveal alteration in amino acid composition (Holding 2014). RNA interference based down
regulation of 22kD RNAi lines is reported to cause opaque phenotype more profoundly as compared to 19kD
component. This is probably due to the greater interaction of 22 KDa components with and -zeins resulting
in a disruption in protein body formation which causes the opaque phenotype (Segal et al. 2003, Huang et al.
2004). Besides, Zhang et al. (2010) were able to trace the inheritance of opaque-16 mutant allele which also
linked to the elevated synthesis of lysine and tryptophan content. Transfer of this high lysine gene (o16)
into opaque -2 genetic background can further enhance the lysine content.
The details of proteome modulation that operates to alter amino acid composition are not clear. A microarray
analysis of 1400 maize genes revealed that the 60 genes up-regulated more than three times in the mutant are
related to stress responses, molecular chaperones and protein turn over (Prioul et al. 2008). Interestingly, among
Tripathy et al. (2017) 4(1): 145152
the 66 down-regulated genes, many genes are involved in carbon, carbohydrate metabolism and branched chain
amino acids (Hunter et al. 2002). Studies revealed that LKR/SDH (lysine-ketoglutarate reductase/saccharopine
dehydrogenase) -the first enzyme of lysine catabolism is strongly depressed in o2 mutants as compared to wild
type (Brochetto-Braga et al. 1992, Azevedo et al. 2003). During the process of evolution, segmental duplication
in the progenitor genome of maize (Teosinte) is reported to result in diverged copies of the regulatory opaque-2
gene (Xu & Messing 2008). Recently, a 15.26kb duplication segment (qy27) at the 27-kDa -zein locus is
reported to be a major modifier QTL which confers enhanced expression of this protein and leads to endosperm
hardness (Liu et al. 2016). They mapped the said major QTL within a narrow interval of 100kb between the
marker 0916-2 and Ch7-120.35 through fine mapping. Proteomic analysis using SDS-PAGE indicated that o2
introgression decreased the accumulation of various zein proteins except for 27-kDa -zein and also affected
other endosperm proteins related to amino acid biosynthesis, starch-protein balance, stress response and signal
transduction (Zhang et al. 2015, Zhou et al. 2016).
Pleiotropic effects of opaque-2 gene has been recognized. Expression of the o2 gene environment labile and
also depends on background polygenes which accounts for appreciable variation in amino acid composition and
opaque phenotypes (Lou et al. 2005). However, In addition to alteration in amino acid composition, it affects
starch organization making the kernel softer, opaque in appearance and unpleasant taste. A set of modifier genes
(QTLs) in the opaque -2 genetic background are known to impove hard and vitreous kernels (Bjarnason et al.
1976, Ortega & Bates 1983, Burnett & Larkins 1999). Inheritance of o2 modifiers is complex (Vasal et al.
1980). Two major QTLs associated with endosperm modification have been identified. Vasal et al. (1980)
combined the opaque-2 allele with QTLs for genetic modifiers and produced elite germplasm with the hard
kernel and much higher quantity of lysine and tryptophan. The modified opaque -2 mutants are reported to bring
about reduced levels of 22 KDa -zeins and 23 times higher levels of 27 KDa -zeins (Geetha et al. 1991) along
with increased hardness of the kernel (Moro et al. 1995). Besides, Krivanek et al. (2007) reported the
involvement of a series of amino acid modifier genes for improvement of lysine and tryptophan. The genetics of
amino acid modifiers is not well understood. Nevertheless, accumulation of favourable amino acid modifiers in
o2 genetic background was accomplished by recombination breeding, and subsequently, several QPM pools
were generated to provide genetic stocks for QPM breeding programmes (Sofi et al. 2009).
The opaque-2 gene was identified near to defective endosperm gene DEB 30 in the short arm of
Chromosome 7 (Holding & Larkins 2008, Holding et al. 2008, Sofi et al. 2009); while, following RFLP analysis
in an F2 population, two major QTLs associated with endosperm modification have been identified near the
centromere and telomere, respectively, on the long arm of the same chromosome (Lopes et al. 1995). A large
number of reports followed wherein varying degrees of endosperm modifications were observed (Paez et al.
1969). The mechanism underlying the change of grain structure from chalky to vitreous in modified opaque-2
mutants (mo2) is yet to be elucidated. But, breeders can undertake selection approach to accumulate QTLs to
regulate this tightly controlled program (Dudley & Lambert 2004). Vasal et al. (1980) combined the opaque-2
allele with QTLs for genetic modifiers and produced modified version of QPM germplasm with the hard kernel
and much higher quantity of lysine and tryptophan.
A number of researchers revealed polymorphic molecular profiles of QPM lines using RAPD (Nkongolo et
al. 2011, Hemavathy 2015), ISSR (Nkongolo et al. 2011, Lenka et al. 2015), SSR (Bantte & Prasanna 2003)
and SNP (Semagn et al. 2012) markers. Bantte & Prasanna (2003) identified a few SSR markers e.g., bnlg 105,
bnlg 125, bnlg 439, phi 037 and dupssr 34 with high polymorphic information content to differentiate QPM
lines. But, none of these proved efficient to detect polymorphism between QPM and Normal maize inbreds.
However, Identification of molecular markers that co-inherit with the opaque 2 phenotype is a crucial step for
their use in marker assisted breeding. CIMMYT designed o2-gene specific SSR primers viz., phi 057, phi 112
and umc 1066 which are located as internal repetitive elements within opaque-2 gene on the short arm of
chromosome 7(www.agron.missouri.edu). Among these phi 057 and Umc 1066 are reported to be co-dominant
while phi 112 is a dominant marker (Magulama & Sales 2009). Allelic polymorphism among QPM and normal
maize inbreds was surveyed by several workers (Babu et al. 2005, Jompuk et al. 2006, Magulama & Sales 2009,
Gupta et al. 2013) using these gene specific SSR primers and confirmed the codominant status of phi 057 and
umc 1066. Phi 057 is reported to reveal a very good polymorphism with QPM donors showing a 169bp band
and normal maize inbreds with a 159bp fragment (Magulama & Sales 2009). Bantte & Prasanna (2003) detected
30 unique SSR alleles to differentiate QPM inbreds. They also identified the SSR primer phi 057 to detect QPM
Tripathy et al. (2017) 4(1): 145152
inbreds carrying opaque 2 mutation. Kata et al. (1994) used RFLP technique using Hind III digestion of
genomic DNA and opaque-2 locus specific cDNA probe to detect o2/o2, o2/o2, and o2/o2 genotypes of
individual plants in breeding populations. Besides, Zhang et al. (2010) reported the use of molecular marker
umc1141 to trace the inheritance of the erstwhile mentioned opaque-16 mutant allele that led to the elevated
synthesis of lysine and tryptophan content.
BREEDING FOR QUALITY PROTEIN MAIZE (QPM)
Maize is a cross pollinated crop. The major breeding approach for increasing productivity is production of
hybrids using heterosis breeding. The success of this method depends on development and identification of
suitable inbred lines using an appropriate mating design; and selection of most promising heterotic normal
maize hybrid. Now, QPM hybrids have been developed and tested for varying climatic and growing conditions.
QPM varieties are grown on roughly 9 million acres worldwide following their spread from Mexico to
throughout Latin America and to Africa, Europe, and Asia (Kataki & Babu 2003). For the production of QPM
hybrid, the ultimate aim is to combine the advantage of heterosis along with amelioration of amino acid
composition using QPM donors. To achieve this, conversion of both parental non-QPM inbreds to QPM status
is the first step to develop a heterotic QPM hybrid. A number of reliable QPM donors (composite K; Ver 181-
Ant gp venezula-1, Thai composite, PD 9MS6 and composite-1) are now available at CIMMYT. These QPM
donor stocks have been developed through intra-population selection for genetic modifiers in opaque-2
backgrounds and are currently being used for large scale conversion of non-QPM inbreds to their QPM version.
Subsequently, combining ability studies in QPM germplasm have been conducted and published (Vasal et al.
1993a, 1993b) so as to assist QPM hybrid development. Vivek et al. (2008) reported tryptophan content more
than 0.60% and lysine content more than 2.6% in a set of CIMMYT QPM inbreds. Besides, Ortega & Villegas
(1988) reported on an average 0.8% tryptophan and 3.1% lysine content among a set of QPM inbreds as against
0.4% and 1.6% respectively in normal maize lines. The inbred lines e.g., CML 176 and CML 186 are reported
to be potential QPM donors (Manna et al. 2005, Danson et al. 2006) for introgression of o2 allele to non-QPM
maize.
The opaque-2 allele is recessive (Vasal et al. 1993a, b) in nature, but the endosperm modifiers follow
polygenic inheritance. At CIMMYT, a conservative approach is adapted to develop modified opaque 2 (mo2)
genotypes to strike a balance between proteins levels and grain quality and competitive yield levels. However,
molecular marker based screening for QPM status coupled with phenotypic selection to improve endosperm
characteristics seems to be an appropriate strategy for the development of QPM introgression lines. The
erstwhile mentioned gene-specific co-dominant markers phi 057 (Manna et al. 2005, Danson et al. 2006,
Magulama & Sales 2009) and umc 1066 (Singh & Ram 2014, Gupta et al. 2009, Gupta et al. 2013) are
worthwhile to trace the opaque-2(o2) allele in conversion programme. Distinct polymorphism revealed by both
the primers can discriminate the QPM donors from respective non-QPM recurrent parents and also between
homozygous (o2o2) and heterozygous (o2o2) opaque-2 back cross progeny. This paves the way for rejection of
non target BC progenies (dominant homozygous) resulting short cutting the breeding cycle (eliminates the need
to grow F2) and substantial savings of labour and material resources for amino acid estimation (Tanksley et al.
1989, Frisch et al.1999, Gupta et al. 2013). This made it possible to bred a QPM hybrid in less than half the
time required in conventional breeding. Besides, foreground selection for opaque-2 combined with phenotypic
selection for recipient parent at early back cross generations can bring about rapid recovery of recurrent parent
genotype. The introgression lines developed using marker assisted back cross breeding may serve as important
breeding material for the development of QPM hybrids.
Two Brazian QPM varieties e.g., BR 451 and BR 473 developed by the Breeding Program of the National
Maize and Sorghum Research Center (CNPMS - EMBRAPA) were released in 1988 and 1994 respectively for
commercial cultivation. The former has been successfully used as a substitute for wheat due to its white color;
while the yellow colour kernel of the later resembles normal maize resulting consumers acceptance as a
substitute of normal maize. A mixture of wheat flour with that of BR 451 in a suitable proportion was reported
ideal for industrial production of bread, cookies, and pasta (Peixoto et al. 1989). Besides, a double cross QPM
hybrid was released in Brazil during 1997. Thereafter, a QPM hybrid Zhongdan 9407 was soon released in
China. India has also released Shakti-1 in 1998 and is deeply involved in testing hybrids from CIMMYT. In
India, Vivek QPM-9: a hybrid of two QPM introgression lines was released in 2008 (Gupta et al. 2009, Gupta et
al. 2013) for commercial cultivation. CML 180 and CML 170 were selected as QPM donors for introgression of
Tripathy et al. (2017) 4(1): 145152
opaque-2 allele into the parental normal maize inbreds CM 212 and CM 145 through marker assisted backcross
breeding. The said QPM hybrid, showed 41% increase in tryptophan and 30% increase in lysine over the
original hybrid. However, the grain yield of the improved hybrid was on par with the original hybrid. Soon
after, many countries participated in QPM network. The Republic of South Africa had earlier released hybrids
HL-1, HL-2, and has recently released HL8 which has hard endosperm, good yield potential, and tolerance to
diseases. In the similar line of work, marker-assisted backcross breeding (MABB) has been also used to develop
maize genotypes for reduced anti-nutritional factors (Naidoo et al. 2012). Muthusamy et al. (2014) developed -
Carotene rich maize hybrids through marker-assisted introgression of -carotene hydroxylase allele. Chander et
al. (2008) identified a major locus (y1) for carotenoid content using gene targeted molecular marker (Y1ssr).
CONCLUSION
Quality protein maize has a far-reaching impact on nutritional security with the discovery of opaque-2
mutation. Such a natural recessive mutation led to selective down-regulation of specific zein genes resulting
alteration in amino acid composition and opaque phenotype of endosperm. Modified marker assisted back cross
breeding made it possible to develop QPM versions of normal maize inbreds with desirable endosperm
characteristics and seed yield. These QPM introgression lines may be combined to develop QPM hybrids.
ACKNOWLEDGEMENT
We sincerely acknowledge and thank all researchers for their valuable contributions included in the text as
references.
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4(1): 153160, 2017
DOI: 10.22271/tpr.2017.v4.i1.022
Research article
Pharmacognostical studies on Saraca asoca (Roxb.) Willd. flower

Anupam Bisht1*, Saba Irshad2, A. K. S. Rawat3 and Harinath Dwivedi1
1
School of Pharmacy, Babu Banarasi Das University, Faizabad Road, Lucknow-226028, India
2
Phytoinformatics Department, CSIR- Unit for research & development of information products,
Pune-411008, India
3
Pharmacognosy and Ethnopharmacology Division, CSIR-National Botanical Research Institute,
Lucknow-226001, India
*Corresponding Author: anupam_sajwan@yahoo.com [Accepted: 12 April 2017]
Abstract: Saraca asoca is a medium sized handsome evergreen tree of Caesalpiniaceae family. It
is distributed in the central and eastern Himalayas and Kashi, Garo and Lushai hills. It is used in
folk medicine for the treatment of many diseases. Bark is useful in menorrhagia, leaves have blood
purifying properties and seeds are used for treating bone fractures and vesical calculi. Flowers of
S. asoca are considered to be uterine tonic and used in dysentery and dried flowers are used in
diabetes. The aim of present study was to investigate pharmacognostical and phytochemical profile
of S. asoca flower. Powder microscopy of flower showed covering trichomes, spiral xylem
vessels, oval-spherical pollen grains, oil gland, prismatic crystals and minute starch grains. -
sitosterol, a phytosterol was identified at Rf 0.80 and quantified to 0.06 % in the flower.
Phytochemically the plant was found to contain flavonoids, glycosides, saponins, phenolics and
tannins.
Keywords: Microscopy - HPTLC - Standardization - Phytochemical profile.
[Cite as: Bisht A, Irshad S, Rawat AKS, Dwivedi H (2017) Pharmacognostical studies on Saraca asoca (Roxb.)
Willd. flower. Tropical Plant Research 4(1): 153160]
INTRODUCTION
Saraca asoca (Roxb.) Willd. = Saraca indica L. of Caesalpiniaceae family is medium sized handsome
evergreen tree up to 9 m. in height. It is found throughout India in evergreen forests up to an altitude of 750 m.
in the central and eastern Himalayas and Kashi, Garo and Lushai hills. The branches of the tree are glabrous and
drooping. The leaves are 3060 cm. in length, in 23 pairs of lanceolate leaflets. The flowers are fragrant,
orange-yellow in colour and found in dense corymbs (Anonymous 2005). Ashoka bark is bitter, acrid,
refrigerant, astringent to the bowels, cures dyspepsia, enlargement of the abdomen, piles and ulcers (Kirtikar &
Basu 1975). Bark is useful in menorrhagia due to uterine fibroids, in internal bleeding hemorrhoids and
hemorrhagic dysentery (Nadkarni 1982). Leaves have blood purifying properties and their juice mixed with
cumin seeds is used for stomach-ache. Flowers of S. asoca are considered to be uterine tonic and used in
dysentery, scabies, syphilis, burning sensation and in hemorrhoids. Dried flowers are used in diabetes. Seeds are
used for treating bone fractures and vesical calculi (Warrier et al. 1997). Pharmacologically the plant have been
reported to possess, antibacterial (Acharyya et al. 2009), analgesic (Verma et al. 2010, Mohod et al. 2014),
anticancer (Kaur & Misra 1980, Ghosh et al. 1999, Cibin et al. 2012), antiulcer (Maruthappan et al. 2010),
oxytocic (Satyavati et al. 1970), anti-inflammatory (Shelar et al. 2010), anthelmintic (Sarojini et al. 2011) and
larvicidal activity (Mathew et al. 2009). Reported phytoconstituents from S. asoca bark are catechin,
epicatechin, procyanidin B2, leucocyanidin and epiafzelechin-(48)-epicatechin. Leaves contain quercetin, -
sitosterol, gallic acid and ellagic acid. Flowers contained oleic, palmitic, linoleic acid, quercetin, gallic acid and
ellagic acid. Seeds have been reported to contain various fatty acids such as oleic, linoleic, palmitic and stearic
acid (Chatterjee & Pakrashi 2006). The most important and widely used part of the ashoka plant is its bark,
flowers are another important ingredients in many ashoka herbal formulations. Literature survey on this plant
revealed pharmacognostic and pharmacological details on stem bark but on the flowers there is minimal
Bisht et al. (2017) 4(1): 153160
information on pharmacological and pharmacognostical work.

Therefore, the objective of present study is to investigate macroscopical, microscopical characters,
physicochemical parameters, preliminary phytochemical screening, fluorescence analysis and HPTLC
fingerprinting of the Saraca asoca flowers and standardize the raw drug on the basis of pharmacognostical and
phytochemical studies.

Plant material
The flowers of Saraca asoca (Roxb.) Willd. were collected from the Central Institute of Medicinal &
Aromatic Plants, Lucknow in the month of April 2015 and was identified by Botanical survey of India (BSD)
Northern Regional Centre, Dehradun as S. asoca Accession number 115720. A herbarium of this plant was
deposited in the herbarium of Botanical Survey of India, Northern Regional Centre, Dehradun (BSD). The
flowers were dried in shade, powdered and stored in air tight container for further studies.
Macroscopical studies
Macroscopical studies helps in the evaluation of raw drug. The fresh flowers of S. asoca were washed
thoroughly under tap water to remove adhering dirt. The flowers were morphologically evaluated for external
characters such as colour, size, shape, odour, taste and texture etc.
Microscopic studies
Plant drugs are generally used in powder form. In the powdered form generally histological structures
destroyed hence powder drug study is essential. For powder drug study, the dried flowers were grinded in
pestlemortar then passed through 60 mesh sieve. The fine powder was mounted in chloral hydrate and glycerin
solution (1:1) and slides were prepared (Jackson & Snowdon 2000). Slides were observed under microscope at
15x 10x magnification. The cell structures were observed under microscope and drawn by the help of Camera
lucida. Photomicrographs were taken with Leica USA (Leica 2000 ATC model) Digi 3 Photomicrographic unit
at 10x 40x magnification.
Physico-chemical studies
Physico-chemical constants like ash values, extractive values, moisture content and foaming index were
determined as per prescribed methods (Anonymous 1996).
Fluorescence analysis
Fluorescence analysis was done according to the prescribed method (Mukherjee 2002). Powder sample was
treated with different chemical reagents and observed under different UV range and visible light for various
colour reactions.
Preliminary phytochemical screening
Dried, coarsely powdered flowers of Saraca asoca was extracted successively with petroleum ether (60
80C), chloroform, acetone, methanol and water using soxhlet apparatus. Extracts thus obtained were
concentrated under reduce pressure to get different extracts. Further these extracts were screened for the
presence of various phytoconstituents (Khandelwal 2004).
HPTLC studies
For HPTLC fingerprinting, methanolic extract was prepared by extracting 2 g of the powdered flowers of
Saraca asoca with 10 ml methanol by maceration at room temperature. The procedure was repeated thrice with
methanol (10 ml) at room temperature. All the extracts were combined, filtered through Whatmann No.1 filter
paper. The combined filtrate were concentrated under vacuum in a rotary evaporator at 40C. 10 mg of this
dried extract was redissolved in 1 ml of methanol. Stock solution of standard marker -sitosterol was prepared
by dissolving 1 mg -sitosterol in 10 ml methanol to get 0.1 mg.ml-1 solution of standard marker.
Chromatography was performed on Merck HPTLC precoated Silica gel G60 F254 (10x10) TLC plate of
uniform thickness of 0.2 mm. 15l of sample and 10 l of standard were applicated with Camag Linomet V
sample applicator on Plate. The plate was developed to a distance of 8 cm from the lower edge of the plate in
solvent system Toluene: Ethyl acetate: Formic acid (7:3:1). The plate was visualized under UV 254 nm, UV 366
nm and under visible light after dipping in anisaldehyde-sulphuric acid reagent followed by drying and heating
at 110C for 5 minutes in order to develop the chromatogram. The plate was documented in visible light after
Bisht et al. (2017) 4(1): 153160
derivatization (Wagner & Bladt 1996).
RESULTS
Macroscopic features
Flowers of Saraca asoca orange or orange- yellow in colour, aromatic, astringent in taste, found in dense
axillary corymb inflorescence. Hermaphrodite flowers, 2.53.5 cm in length. Ovate bracts and 2 sub acute
bracteoles, appearing like a calyx; petaloid 4 tubular calyx, imbricate, yellowish orange- red in colour; corolla
absent; stamens 67, exerted, long filiform filament, versatile anther; minute capitate stigma, style curved into
ring and ovary pubescent (Fig. 1).
Figure 1. Different parts of Saraca asoca: A, Tree habit; B, A flowering twig; C, A flower; D, Gynoecium; E, Stamens.
Microscopic studies
The powder was brown in colour with aromatic odour, astringent in taste. Under microscopic observation
powder showed uniseriate, small covering trichomes present on the outer epidermis of calyx. Pollen grains were
large, oval to spherical in shape with smooth exine. Small brown ovoid oil gland, stomata, prismatic crystals of
calcium oxalate were present. Spiral xylem vessels observed and epidermal cells of calyx were rectangular in
surface view. Fragments of fibrous layer of anthers were composed of small cells. The occasional fragments of
the walls of ovary were composed of small polygonal cells. Minute starch grains were scattered into the powder
(Figs. 23).
Physico-chemical studies
Various physico-chemical parameters viz. total ash, acid insoluble ash, water soluble ash, water soluble
extractive, alcohol soluble extractive, moisture content and foaming index were determined according to Indian
Pharmacopoeia (Anonymous 1996) and results are given in (Table 1). Water soluble extractive value of S. asoca
is found to be higher in comparison to alcohol soluble soluble extractive value which denotes the possibilities of
presence of higher amount of polar water soluble constituents in the flower of S. asoca. Ash values are
important quantitative standard and it denotes the inorganic components and impurities present in drugs. Total
Bisht et al. (2017) 4(1): 153160
ash value and acid insoluble ash of S. asoca were found low which indicate low content of carbonates,
phosphates, silicates and silica and low contamination with silicon material or sand. Moisture content
determination is important for maintaining the pharmacopoeial standards and it indicates the stability of the
drug. Foaming index is found less than 100 and it denotes the foaming ability of drugs.
Figure 2. Powder microscopy of flower of Saraca asoca (at 10x 40x magnification): A, Epidermal cells in surface view; B,
Epidemal cells of calyx form a disk; C, Outer epidermis of bract with trichomes; D, Stomata; E, Starch grains; F, Covering
trichome ; G, Upper epidermis of calyx showing palisade cells; H, Outer epidermis near the base of bracteoles in surface
view showing calcium oxalate crystals; I, Prismatic crystal of calcium oxalate; J, Part of gynoecium showing stigma and
style; K, Parenchymatous cells; L, Xylem vessels with spiral thickening; M, Oil gland; N, Part of ovary; O, Endothecium
of anther; P, Pollen grains.
Figure 3. Microscopic examination of powdered flower of Saraca asoca with camera lucida (at 15x 10x magnification): A,
Epidermal cells in tangential view; B, Oil gland; C, Prismatic crystals of calcium oxalate; D & E, Covering
trichome; F, Pollen grain; G, Stomata; H, Spiral xylem vessels; I, Epidermal cells of calyx in surface view; J,
Trichomes with epidermal cell.
Bisht et al. (2017) 4(1): 153160
Table 1. Quantitative standards for the flower of Saraca asoca.

Parameters MeanSD
Ash Values
(i) Total ash 4.73 0.20
(ii) Acidinsoluble ash 0.23 0.03
(iii) Watersoluble ash 2.5 0.25
Extractive Values
(i) Alcohol soluble 7.0 1.50
(ii) Water soluble 24.33 0.76
Moisture content 68.136.34

Foaming index Less than 100
Fluorescence analysis
Many herbs fluorescence when cut surface or powder is exposed to UV light and this can help in their
identification. For a few drugs (Rhapontic, Indian and Chinese rhubarb) it is used for identification when they
are in powdered form. Many phytochemicals present in plant materials shown fluorescence when examined
under UV light. For each compound the colour of fluorescence is specific. Powdered drug was treated with
different reagents and was examined under UV light (254 & 366 nm). The S. asoca powder treated with
different reagents shown no fluorescence when examined under UV light (254 & 366 nm) but the distinctive
colour changes in treated powder observed under UV light examination and the results are given in (Table 2).
Table 2. Florescence analysis of the powdered flower of Saraca asoca.
Treatment Day light Short UV light (254 nm) Long UVlight (365 nm)
Powder as such Chocolate brown Dark brown Dark brown
Powder + 1N NaOH Rust brown Black Black
Powder+CH 3 COOH Chocolate brown Dark chocolate Dark chocolate
Powder + 1N HCl Light brown Black Black
Powder + Cocn. NH 3 Brownish-orange Rusty brown Blackish-brown
Powder+50% H 2 S0 4 Brown Brown Dark brown
Powder+Conc.HNO 3 Brown Brownish-green Blackish-green
Table 3. Successive soxhlet extractive values of Saraca asoca flower.

Extract Colour Nature Value (MeanSD)
Petroleum ether Yellow Waxy 1.570.14
Choloroform Light green Waxy 1.130.09
Acetone Yellowish-brown Sticky 1.700.16
Methanol Rusty brown Viscous 9.420.90
Water Blackish-brown Gummy 9.110.95
Table 4. Preliminary detection of phytoconstituents in the flower of Saraca asoca

Extracts
Phytoconstituents
Petroleum ether Chloroform Acetone Alcohol Water
Sterols + + - - -
Carbohydrates - - + + +
Glycosides - - + + +
Phenolic & Tannins - - - + +
Flavonoids - - + + +
Saponins - - - - +
Alkaloids - - - - -
Note: + = Present; - = Absent.
Preliminary phytochemical studies
The percentage successive soxhlet extractive values of the raw drug with different solvents (solvents used
according to increasing polarity order) were determined with reference to the air dried drug. The extractive
value of different flower extracts of S. asoca along with colour and nature of extract is given in (Table 3). The
results showed that the % yield of extract increases as solvent polarity increased and maximum yield was
obtained in methanol solvent. The Phytochemical analysis of different solvent extracts of S. asoca flower
Bisht et al. (2017) 4(1): 153160
Figure 4. TLC Profile of methanolic extract of Saraca asoca: A, Visualization under UV 254 nm; B, Under UV 366 nm; C,
Under visible light after derivatization. 1, Saraca asoca 2. - sitosterol.
Figure 5. HPTLC Densitogram: A, Area under curve of standard beta sitosterol at 600 nm; B, HPTLC chromatogram of
Saraca asoca showing beta sitosterol at 600 nm.
Bisht et al. (2017) 4(1): 153160
showed the presence of carbohydrates in acetone, alcohol and water extracts. Phenolic compounds and tannins
were present in alcohol and water extracts, flavonoids in acetone, alcohol and water extracts, saponins in water
extracts, glycosides in acetone, alcohol and water extract, sterol in petroleum ether and chloroform extracts only.
These extracts exhibit negative tests for alkaloids. Results are shown in (Table 4).
HPTLC finger print profile
Quantitative HPTLC analysis of -sitosterol in ethanolic extract of S. asoca has been performed. A
densitogram and banding pattern obtained from extract shows -sitosterol 0.06 %. Rf value of -sitostetrol was
recorded 0.80 (Fig. 45).
DISCUSSION
The purpose of standardization of raw drugs is to ensure its therapeutic efficacy. Standardization ensures the
use of correct herbs and determines its quality. S. asoca flower was standardized on the basis of macroscopic,
microscopic studies, physico-chemical standards, fluorescence analysis and HPTLC. Microscopic studies give
detailed information about the raw drugs by identifying the known histological characters of the drugs. For the
powdered drug identification is based on presence or absence of different cytomorphological characters viz.
parenchyma, collenchyma, fibres, stones cells, vessels, trichomes, secretory cells, epidermal cells and the cell
inclusion characteristics viz. starch grains, pollen grains aleurone grains, calcium-oxalate crystals. Moisture
content determination is important for denoting the stability of raw drug as excess water in raw drugs favours
spoilage of drugs by molds and bacteria and make possible the enzymatic destruction of active principles. Most
drugs may be stored safely if the moisture content is reduced to 6 per cent or less (Mukherjee 2002). Ash values
denote the inorganic materials phosphates, metallic salts and silica present in raw drugs and it is important
parameter for determination of quality, purity and detection of adulteration in raw drugs. Extractive values
indicate the nature of constituents present in a raw drug. Such extractive values provide an indication of the
extent of polar, medium polar and non-polar components present in the plant material. The preliminary
phytochemical screening gives the general idea regarding the nature of chemical constituents present in raw
drugs and it is useful in finding the quality of the drug (Kokate 1999). Many medicinal plants contain saponins
and their aqueous decoction on shaking produces persistent foam. Foaming index measures the foaming ability
of an aqueous decoction of plant materials (Anonymous 1998). Fluorescence analysis is an important
standardization parameter. Few drugs viz. ergot powder, berberis, gentian, aconitine gives specific fluorescence
when exposed to UV light. A nonfluorescent compound may produce fluorescence if they are mixed with
fluorescent impurities (Ansari 2006, Nawagish et al. 2007). Various physico-chemical parameters, viz. moisture
content, ash values, extractive values, foaming index, HPTLC, fluorescence analysis and phytochemical
screening of various extracts were performed to substantiate standardization data on the flower of Saraca asoca.
These parameters help in the evaluation of purity of drugs. The present study concludes that the complete
pharmacognostic parameters of the flower of S. asoca would be useful in identifying the flowers for safe
applications in drug manufacturing.
ACKNOWLEDGEMENTS
Authors duly acknowledge Dr. Sayyada Khatoon, Scientist, Pharmacognosy and Ethnopharmacology
Division, N.B.R.I, Lucknow for their kind help.
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ISSN (E): 2349 1183
ISSN (P): 2349 9265
4(1): 161167, 2017
DOI: 10.22271/tpr.2017.v4.i1.023
Research article
Impact of dust accumulation on three roadside plants and their

adaptive responses at National Highway 37, Assam, India
Bitopan Sarma1,2, Sanjay Kumar Chanda2 and Mantu Bhuyan2*
1
Academy of Scientific and Innovative Research, CSIR-North East Institute of Science and Technology,
Jorhat, Assam, India
2
Biological Science and Technology Division, CSIR-North East Institute of Science and Technology,
Jorhat, Assam, India
*Corresponding Author: mantubhuyan@gmail.com [Accepted: 13 April 2017]
Abstract: Roadside plants are consistently exposed to dust. Deposition of dust on roadside plant
and its impact on leaf epidermal traits as well as leaf pigment concentration, the percentage of
carbon and nitrogen has been studied of three roadside plants i.e. Cassia alata, C. tora and C.
sofera at National Highway - 37, Assam. Significant variation in terms of dust deposition with
species specific result observed during the study. Declination of leaf pigment concentration, leaf
area and nitrogen percentage along with increased leaf thickness indicate the dust pollution impact.
Above findings may be helpful to find out some representative species for developing some model
to cope with automobile generated dust pollution in future.
Keywords: Dust pollution - Roadside plant - Chlorophyll - Leaf epidermal traits - Cassia spp.
[Cite as: Sarma B, Chanda SK & Bhuyan M (2017) Impact of dust accumulation on three roadside plants and
their adaptive responses at National Highway 37, Assam, India. Tropical Plant Research 4(1): 161167]
INTRODUCTION
Dust is an important abiotic factor and has a key influence upon the organism. Sever pressure of dust may be
responsible for altering bio-chemical as well as morphological setup of an organism which further initiated
adaptive evolution to cope with the changing environment. Based on the source of generation as well as the
structure of road, the role of dust may be variable (Farmer 1993, Anthony 2001). In an urban area, road dust
contains a mainly huge amount of different metals as well as a small amount of clay and minerals too (Beckett
et al. 2000). Vehicles are the prime source of dust generation for roadside plants. Poor road infrastructure, the
frequency of running vehicles in terms wheel determine the rate of dust generation. Therefore, adaptive
evolution of roadside plant species is more visible in poor road infrastructure with more running vehicles. The
response of the plant to dust accumulation may vary according to different species, as dust deposition fluctuates
with plant species due to leaf orientation, leaf surface geometry, phyllotaxy, epidermal and cuticular features,
leaf pubescence, height and canopy of roadside plants (Davison & Blakemore 1976, Chaphekar et al. 1980,
Farmer 1993, Chaturvedi et al. 2013) With the accumulation of dust, the roadside plant may exhibit adaptive
response by changing morphological and physiological attributes.
A number of registered motor vehicles in India have reached from 0.3 million to 159.5 million from 1951 to
2012 (Report: Road Transport Year Book 201112, Government of India, Ministry of Road Transport and
Highways Transport Research Wing, www.morth.nic.in). In Assam, vehicles are increased from 53, 4885 in
200001 to 1,98,4880 in 201314 indicating 73.05% boost of the growth of motor vehicle in the state. But the
roads are limited and poor in condition (Source: Office of the Commissioner of Transport, Assam and
Directorate of Economics and Statistics, Assam). National Highway - 37 (NH-37) is the backbone of road
transport in Assam and the road starts from Panchatantra of Goalpara district of Assam to Roing of Arunachal
Pradesh with a length of 740 km and considered one of the busiest road connecting all the major cities of the
south of the Brahmaputra river.
In the present study, we emphasised the impact of dust deposition in terms of epidermal traits of leaf,
chlorophyll contents, carbon and nitrogen percentage upon roadside plant and their adaptive responses to cope
Sarma et al. (2017) 4(1): 161167
with the vehicular dust deposition. Three common shrub species of the genus Cassia i.e. Cassia alata L., C. tora
L. and C. sofera L. selected to study the impact of vehicular dust and their adaptive response.

CSIR-North East Institute of Science and Technology (CSIR-NEIST) is situated towards south to NH-37. A
site on the NH-37 in front of CSIR-NEIST was selected to study vehicular load/ hour on NH-37 and the road
was monitored continuously in a different time interval of the day up to one month. Total numbers of vehicles
including two, three and >three wheelers (four wheelers and above) vehicles crossed from both sides has been
counted and the average value was calculated per hour.
Based on the frequency of occurrence on the roadside of NH-37, 3 months old three plant species of the
genus Cassia i.e. Cassia alata L. Cassia tora L. and Cassia sofera L. have been selected to study the
accumulation of dust and its impact on the plants. Leaves were collected randomly of from plants with equal
height from each species growing along with the roadside of NH-37, after the seasonal rainfall of Assam in
April, 2015. Leaves from the three roadside plant species were collected in zipper pouches separately, in
triplicate and brought to the laboratory for estimation of dust deposition. Each leaf was washed with distilled
water using a spray bottle and suspended dust along with the water was collected carefully on pre-weighed
Whatmans filter paper (pore size 110 mm). The filter papers were dried in an oven at 4050 C for 2 hours to
remove the water and weighed later to calculate the dust deposition in mg/cm of the leaf. Washed leaves were
blotted dry and then traced on graph paper to measure the total leaf area in mm (Chaphekar et al. 1980, Vora &
Bhatnagar 1986). Prominent secondary veins, as well as midrib of the leaf was avoided to reduce sample
variation during measuring leaf area. Number of stomata and epidermal cell present per m of each of the
collected leaf from all the three plant species were calculated to estimate the Stomatal Index of the leaves.
Mature leaf was fixed in FAA solution (acetic acid: alcohol: formalin: water = 2:5:1:12) for 24 hours followed
by washing the leaf in 70% ethanol and then each leaf samples were cut out into circular form for easy peeling.
Cut leaves were boiled in 5% aqueous solution of KOH for 5-10 minute and adaxial and abaxial surface was
peeled and peeled section was stained with 1% safranin followed by temporary mounting in glycerin to observe
the number of stomata as well as epidermal cell under the microscope (Leica DM-3000 LED, Made in
Germany) (Satao et al. 1993). At least three microscopic observations at 20X were considered for counting.
Stomatal index (I) was calculated by the following formula (Meidner & Mansfield 1968),
Where, (S) = Numbers of stomata, (E) = Numbers of epidermal cells present in a unit area2
Leaf pigments i.e. chlorophyll a, b and total chlorophyll concentration was counted with the method
described by (Arnon 1949) and the pigment concentration was expressed in mg.g-1 tissue. Carbon and nitrogen
percentage in leaf were determined by the Perkin Elemental CHN analyser. Microsoft Excel 2010 version is
used for analysis. Data were entered and arranged accordingly to calculate Standard Deviation (SD) and
Standard Error (SE) from different replicated samples (Federrer 1947). One way ANOVA is using with Sigma
Plot statistical software v13 (Jandel Scientific, San Rafael, CA). All groups were compared using the Student-
Newman-Keuls post hoc analysis method. The p value less than 0.05 was considered to be statistically
significant.
RESULTS
National Highway - 37 is one the one of the busiest roads of communication, connecting maximum numbers
of cities of Assam. The road bears the vehicular load of an average of 580 numbers of vehicles i.e. two, three
and >three wheelers (four wheelers and above) per hour. More than three wheelers vehicles were more frequent
followed by two and three wheeler vehicles on the NH-37 (Fig. 1A). Irrespective of plant species, deposition of
dust on leaf of roadside plant was found to increase significantly with time. Amount of dust deposited in the leaf
of C. tora was maximum followed by leaf of C. sofera and C. alata during 1st and 2nd week (Fig. 1B). However,
during 3rd and 4th week dust deposition was found higher in C. sofera followed by C. tora and C. alata.
Leaf chlorophyll is an important index to measure the impact of dust on the roadside plant species. Leaf pigment
concentration i.e. chlorophyll was found to decreased considerably in the roadside plant species compare to
control. It is evident from table 1 that roadside plant produces less chlorophyll a, chlorophyll b and total
Sarma et al. (2017) 4(1): 161167
Figure 1. A, Average numbers of different vehicles per hour at NH-37; B, Dust deposition (mg/cm2) on the leaf of Cassia
tora, C. alata and C. sofera.
Table 1. Chlorophyll (chlorophyll a, b and total) contents of leaves of three different Cassia species in different sites.
Species Site Chlorophyll a (mg.g-1) Chlorophyll b (mg.g-1) Total
Cassia sofera
Control 1.87a0.026* 0.89A0.012* 2.47aa0.023*
NH-37 1.57b0.012 0.87A0.022 2.21bb0.025
Reduction (%) 16.04 2.24 10.52
1.88c0.041 1.05B0.032 2.62cc0.030

Cassia tora
Control
NH-37 1.43d0.020 0.86C0.008 2.12dd0.032
Reduction (%) 23.93 18.09 19.08
Cassia alata
Control 1.94e0.027 0.92D0.010 2.91ee0.011

NH-37 1.55f0.031 0.73E0.009 2.31ff0.013
Reduction (%) 20.1 20.62 20.61
Note: *Values are mean SE (n = 10). Each rows having different letters are significant at p<0.05 level.
chlorophyll than the control. The percentage of reduction of chlorophyll content was found highest in C. alata
plant species. Reduction of Specific Leaf Area (SLA), Stomatal index (adaxial and abaxial) and Leaf Dry Matter
Content (LDMC) were observed in all the three plant species growing along the roadside of NH-37 compare to
control (vehicle free zone) (Fig. 2AD). All those parameters were different among all the three species even in
control. However irrespective of plant species, the above mentioned leaf parameters were recorded lower in the
leaf of roadside plant species. Maximum percentage of reduction of SLA was observed in the leaves of C. sofera
(Fig. 2A), but highest percentage reduction of the stomatal index for both abaxial and adaxial surface of the
leaves was recorded in C. tora plant. Similarly, C. tora also exhibited maximum percent reduction of LDMC
(Fig. 2BD).
On the other hand, leaf thickness of each plant species from roadside was found to increase in compare to
control (Fig. 2E). Leaf thickness of C. tora was significantly higher than rest of the two plant species, but
highest percentage increase was recorded in C. sofera. Nitrogen content of plant is a limiting resource for plant
growth, reproduction and defence. Roadside plant exhibited less nitrogen with highest reduction in C. sofera
followed by C. alata and C. tora (Fig. 3A). However, dust exposure roadside plant presenting higher
accumulation of carbon content in all studied plant and highest percentage of increase was recorded in C. sofera
plant followed by C. alata and C. tora (Fig. 3B). It is evident from (Fig. 3AB) that highest reduction of
nitrogen of the plant is associated with the highest increase of carbon (C. sofera) and lowest nitrogen of the
plant associated with lowest carbon (C. tora).
Sarma et al. (2017) 4(1): 161167
Figure 2. A, Specific leaf area (SLA); B, Stomatal index (Adaxial); C, Stomatal index (Abaxial); D, Leaf dry matter content
(LDMC); E, Leaf thickness of leaf of the control and roadside plant of Cassia tora, C. alata and C. sofera.
Figure 3. A, Nitrogen content (%); B, Carbon content (%) of the leaf of the control and roadside plant of Cassia tora, C.
alata and C. sofera.
Sarma et al. (2017) 4(1): 161167
DISCUSSION
Rolling vehicles put force to the road surface and pulverizes the roadbed materials in the form of dust; eject
it in shearing force and turbulent vehicle force (Khan et al. 2015). The pulverization of roadbed materials will
be more in the poor road with more vehicles. NH-37 in Assam is poor in quality and >three wheelers (four
wheelers and above) are dominant on the road. In poor road, more numbers wheels can faster dust generation.
Roadside plants i.e. Cassia sofera, C. tora and C. alata of NH-37 are consistently exposed to dust. The dust
particles settled on the leaf surfaces every day, but deposition was not uniform in all the plant species.
Settlement of dust particle on the leaf surface mainly depends on the phylotaxy of leaf, smoothness of the leaf
surface, shape of leaf, petiole length as well as its position in the plant (Davison & Blakemore 1976). On the
basis of leaf structure and texture, local air movement influence to dislodge deposited dust from the leaf.
However, during the study period, local air movement was found to be insignificant. Therefore, variation of dust
deposition was observed in leaves of different plant species. Dust deposition was also found to increase with
time. Dust generated from day to day traffic activity will be accumulated on the leaf up to a limit. Hence,
irrespective of plant species, dust deposition was found to increase in different time intervals. It was also
observed that during the first two weeks, dust deposition was found highest on the leaf of C. tora followed by C.
sofera and C. alata. But after 2nd week the pattern of dust deposition changed and highest accumulation was
recorded in C. sofera then to C. tora and C. alata. Leaf structure and texture determine the limit of dust
deposition. According to plant species, dust load per day may very i.e. initial rate of dust deposition may
increase in some plant species and deposition rate decrease with time. Here the size of leaf and overall height of
the plant species may also contribute. Leaf petiole and surface characteristics may be responsible for the higher
rate of dust deposition in C. sofera during 3rd and 4th week. In addition to the leaf surface characteristics, the
position of leaves on the plant and their orientation also crucial in terms of dust deposition.
Structural properties of the leaf are influenced by deposition of dust, which may lead to lower photosynthesis
activity (Pourkhabbaz et al. 2010). All the studied roadside plant species showed reduction of chlorophyll
contents (chlorophyll a, b and total chlorophyll) in compare to vehicle free site (control). Dust particles
physically obstruct sunlight as well as block stomatal pore of leaf and thus dust deposition hampered
photosynthetic activities of plant (Nicholson et al. 1989, Keller & Lamprecht 1995, Pandey & Kumar 1996,
Hope et al. 1999, Manno et al. 2006). Shading effect of dust as well as alkaline condition caused by dissolution
of dust particles in cell sap degrade leaf pigment concentration has been reported earlier (Prajapati & Tripathi
2008a). Degradation of chlorophyll contents in the leaf is a common phenomenon from dust deposition on leaf
(Prajapati & Tripathi 2008b). A plenty of studies (Satao et al. 1993, Prusty et al. 2005, Gostin 2009) reviewed
declination of chlorophyll concentration due to deposition of dust in a number of the annual non-leguminous
crop.
SLA which is a supposed to be an indicator of plant morphology was found to decrease in roadside plant
species in compare to control. Among the roadside plant species, C. sofera exhibited maximum reduction in
SLA. Reduction in SLA specifies poor physiological as well as the biochemical status of the plant. Strong
negative correlation has been reported earlier in between dust load and SLA (Raupach et al. 2001, Chaturvedi et
al. 2013). Continuous exposure to dust in leaf surface leading to the formation of dense dust layers, which
reduce light capturing capacity of plants (Prajapati & Tripathi 2008a, Pourkhabbaz et al. 2010) and finally
hamper plant photosynthetic activity. Light capturing capacity of leaf via lowering photosynthetic rates, clogged
stomata and increased foliage temperature are the cause behind dust deposition reported earlier (Anthony 2001).
Stomatal index has been proven to be an indicator of environmental stress (Gostin 2009). Decreased rate of
photosynthesis and alteration of stomatal conductance are responsible for reduction of the stomatal index as well
as dry matter content of leaf (Khan et al. 2015). Reduction of the stomatal index (abaxial and adaxial) of the
roadside plant may be due to shading effect of dust layers, which may block the stomata and reduce the
photosynthesis rate of roadside plants. The Same impact also reflected on reduced leaf dry matter content.
Reduction percentage of the stomatal index and leaf dry matter were found higher in C. tora plant compared to
other two roadside plant species. It was already evident that leaf of C. tora accommodated more dust in first two
weeks, hence initial response of reduction of stomatal index and leaf dry matter may be higher than the other
two plant species. Leaf thickness was found to increase in all the roadside plants; however, maximum leaf
thickness was observed in C. sofera than other two plant species. It is also evident that plant under the
environmental stress, produces thicker leaves as an adaptive response (Satao et al. 1993, Hope et al. 1999,
Gostin 2009) and to cope with the stress of vehicular dust, roadside plants may produce thicker leaves.
Sarma et al. (2017) 4(1): 161167
Roadside plants indicate a reduction in the percentage of nitrogen in compare to control. Actually, dust
deposition alters the photosynthesis capacity and hampered the overall growth and development of the plant
(Thomson et al. 1984). Therefore, those plants are unable to uptake required nitrogen under such condition.
Accumulation of carbon was more resulting higher carbon percentage in plant tissue. It may be a part of plant
defence mechanisms because under stress condition plant produces more carbon based secondary metabolites.
Finally, present study suggested that alteration of leaf chemistry may be the result of heavy dust load which
lowered the nitrogen concentration in plant tissue and greater carbon uptake as a part of the adaptive response.
CONCLUSION
Present work provides basic information about the variation in dust accumulation of three roadside plant
species i.e. Cassia sofera then to C. tora and C. alata. Variation of dust loads positively correlated with the
alteration of biochemical and epidermal constituents of the leaf. Significant variation in terms of dust deposition
with species specific effect observed during the study. In future, to monitor dust fall at roadside areas these
representative plant species can be used as an indicator.
ACKNOWLEDGEMENTS
This work was funded by Department of Science and Technology (DST), Government of India for INSPIRE
fellowship and Council of Scientific and Industrial Research (CSIR), Govt. of India under Project No. BSC 111.
Authors are also indebted to Director, CSIR-North East Institute of Science and Technology, Jorhat, Assam,
India for giving necessary infrastructure and support.
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ISSN (E): 2349 1183
ISSN (P): 2349 9265
4(1): 168175, 2017
DOI: 10.22271/tpr.2017.v4.i1.024
Review article
A mini-review on the pharmacognosy and phytochemistry of a

tropical medicinal plant: Annona senegalensis Pers. (Annonaceae)
Koto-te-Nyiwa Ngbolua1, 2, 3*, Emmanuel Lengbiye Moke1, John Likolo Baya2,4,
Ruphin Djolu Djoza2, Colette Masengo Ashande2 and Pius T. Mpiana5
1
Dpartement de Biologie, Facult des Sciences, Universit de Kinshasa, B.P. 190 Kinshasa XI,
Rpublique Dmocratique du Congo
2
Dpartement des Sciences de lEnvironnement, Facult des Sciences, Universit de Gbadolite, B.P. 111
Gbadolite, Province du Nord Ubangi, Rpublique Dmocratique du Congo
3
Institut Suprieur Pdagogique dAbumombazi, Province du Nord Ubangi, Rpublique
Dmocratique du Congo
4
Universit de Lisala, Province de la Mongala, Rpublique Dmocratique du Congo
5
Dpartement de Chimie, Facult des Sciences, Universit de Kinshasa, B.P. 190 Kinshasa XI,
Rpublique Dmocratique du Congo
*Corresponding Author: jpngbolua@unikin.ac.cd [Accepted: 15 April 2017]
Abstract: The aim of this mini-review was to collect data obtained from various studies carried
out by different authors concerning the phytochemistry and pharmacognosy of Annona
senegalensis (Annonaceae). This review has been compiled using references from major databases
such as PubMed, PubMed Central, Science Direct and Google scholars. An extensive survey of
literature revealed that A. senegalensis is a good source of health promoting secondary metabolites
such as terpenoic acids (kaurenoic acid) among others that could have many wonderful
applications (like antisickling properties).Traditionally, the plant is used as stimulant, pain reliever
etc. whereas the plant possess beneficial effects such as anti-oxidant, antimicrobial, Antidiarrheal,
anti-inflamatory, anti-parasitic, anticonvulsant, antimalarial, anti-trypanosomal, antisnake venom
and Antinociceptive and many other medicinal properties. The results of the present review of
literature makes A. senegalensis an interesting candidate for advanced anti-sickle cell anemia
investigations such as erythroid differentiation and fetal hemoglobin induction effects of this plant
using K562 cell lines as model system.
Keywords: Traditional Medicine - Annona senegalensis - Phytochemicals - Scientific validation -
Biodiversity valorization.
[Cite as: Ngbolua KN, Moke EL, Baya JL, Djoza R, Ashande CM & Mpiana PT (2017) A mini-review on the
pharmacognosy and phytochemistry of a tropical medicinal plant: Annona senegalensis Pers. (Annonaceae).
INTRODUCTION
As per the records of World Health Organization, it is assumed that more than 60% of global population is
using the traditional medicine system to overcome several health related issues (Farnsworth 1994, Cotton 1997).
This percentage goes up in the developing countries where, rural and tribal population is higher because of its
low cost (Mehra et al. 2014, Bajpai et al. 2016). Recent findings revealed that over 80% of the African
population relies on medicinal plant species for their primary healthcare (Ngbolua et al. 2011a, b).
In the case of Sickle cell disease (SCD) treatment based plants, Bianchi et al. (2009) reported that
phytochemicals (like resveratrol) are able to ameliorate the state of SCD patients by shifting from monthly
blood transfusion dependency to a stable transfusion-free condition. The correlation between in vitro effects of
such fetal hemoglobin (HbF) inducers and in vivo treatment is well established. The complementary and
alternative medicine constitutes therefore a solution for the management of this hemoglobinopathy. Thus, the
Received: 09 January 2017 Published online: 30 April 2017
Ngbolua et al. (2017) 4(1): 168175
search and the development of antisickling herbal drugs are a priority agenda in Africa, where SCD is endemic.
In such region, herbal medicines are widely used to relieve the symptoms of SCD. In our efforts to search for
novel and bioactive antisickling hits from indigenous plants, we recently investigated plant species indigenous
to Democratic Republic of the Congo which are prescribed by traditional healers or sold in the markets for the
management SCD (Ngbolua et al. 2014a, Ngbolua et al. 2016). Annona senegalensis Pers. is one of the plants
traditionally used for this purpose. The aim of the present review is to document research results on the
pharmacognosy and phytochemistry of this promising medicine. These findings are highlighted in this mini
review. Such informations should pave the way for future directional research on this plant species as valuable
source of new hits against Sikle cell anemia (epigenetic modulator agents) and associated pathogenic bacteria
(antibacterial agents).
BOTANY AND DESCRIPTION
Annona senegalensis Pers. is a tropical plant species also known as wild custard apple or wild soursop. It
is a shrub (26 m) or small tree (11 m) under some suitable ecological conditions. The bark is smooth to
roughish, silver grey or grey-brown. The leaves of this medicinal plant are alternate, simple, oblong, ovate or
elliptic, green to bluish green, mostly lacks hairs on upper surface, with brownish hairs on lower surface.
Flowers are up to 3 cm in diameter on stalks 2 cm long, solitary or in groups of 24, arising above the leaf axils.
The fruits are formed from many fused carpels, fleshy, lumpy, egg shaped, 2.55.0 by 2.54.0 cm, ovoid or
globose, unripe fruit green, turning yellow to orange on ripening. Wild fruit trees of this species are found in
semi-arid to sub-humid regions of Africa, it is native to tropical east and northeast, west and west- central, and
southern Africa, as well as southern subtropical Africa, and islands in the western Indian Ocean. The species
occur along river banks, fallow land, swamp, forests and at the coast. It commonly grows as a single plant in the
understorey of savannah woodlands (Orwa et al. 2009).
ETHNOBOTANY
Annona senegalensis Pers. (Annonaceae) is a multipurpose plant with a high traditional and medicinal uses
for the maintenance of free health life. Traditionally the plant is used as stimulant, pain reliever etc. Several uses
of the plant species is reported for example anti-oxidant, antimicrobial, antidiarrheal, antiinflammatory,
antiparasitic, anticonvulsant, antimalarial, antitrypanosonal, anti-snake venom and antinociceptive properties
and many other biomedical properties of pharmaceutical relevance. These properties of the plant possess is due
to its important phytochemical constituents like triterpenes, anthocyanes, glucids, coumarins, flavonoids and
alkaloids etc. (Samuel et al. 2016).
As per the traditional medicine practices, all the plant parts of A. senegalensis are useful in several diseases.
The leaves have been used in treating yellow fever, tuberculosis, and small pox (Ajaiyeoba et al. 2006,
Mustapha et al. 2013). The stem bark has been used in snakebite and hernia treatment (Dambatta & Aliyu
2011). The root is used in conditions such as difficulty in swallowing, gastritis, snake bites, male sexual
impotence, erectile dysfunction, tuberculosis, and as antidote for necrotizing toxins; the root bark is effective in
infectious diseases (Ofukwu et al. 2008, Jiofack et al. 2009, Noumi & Safiatou 2015). Juice from the tree is
used in the treatment of chicken pox (Faleyimu & Akinyemi 2010). Many of the plant parts are used as antidotes
for venomous bites and in the management of diabetes (Ogoli et al. 2011, Ahombo et al. 2012). In Guinea, A.
senegalensis has been employed in the treatment of malaria (Traore et al. 2013). Among the Igede people of
Benue State in North Central Nigeria, the plant is used in combination with Ageratum conyzoides for diarrhoea
and in combination with Nauclea latifolia for dysentery (Igoli et al. 2005).
PHARMACOGNOSY
Antidrepanocytary (antisickling) activity
Annona senegalensis Pers.from Democratic Republic of the Congo was reported to possess
antidrepanocytary (anti-sickle cell anaemia) activity (Mpiana et al. 2007, Mpiana et al. 2012).
Antidiarrhoeal activity
The methanol stem-bark extract of Annona senegalensis was investigated using both in vivo and in vitro
models by oral application of effective dose (5000 mg.kg-1). In order to investigate intestinal transit time, the
plant extract was given by oral route to mice fed with charcoal as meal. The extract decreased intestinal transit
time by decreasing the spontaneous contractions of the intestine, thus the findings provided a scientific basis for
Ngbolua et al. (2017) 4(1): 168175
the use of Annona senegalensis stem bark extract in the treatment of diarrhoea. Therefore, A. senegalensis is a
potent phytomedicine for diarrhoea (Suleiman et al. 2008).
Antimalarial and filarial mosquito vectors activity
The methanol extract of Annona senegalensis possess antimalarial activity against Plasmodium berghei and
the extract showed better antimalarial activity than compared to the standard reference drug Chloroquine
disphosphate which had a 96.2% chemosuppression activity (Ajaiyeoba et al. 2006). In DRC antimalarial and
cytotoxic activities of tested plant extract moderate in vitro (i.e. 10< IC50 < 50 g/mL: A. senegalensis IC50 = 32,
526, 97 g.ml-1) and weak in vivo (i.e. %I < 33, 002: %I=16, 932, 00). The ethanolic crude extract from the
leaves of A. senegalensis displayed also cytotoxic effect towards P-388 cells (IC50 < 10 g.ml-1, therapeutic
index= 0, 27). The observed cytotoxic effect of the leaves could be due to presence of aporphine alkaloid
(Ngbolua et al. 2014b). These results are not consistent with previously reported research work. Indeed
(Ajaiyeoba et al. 2006) reported that A. senegalensis harvested in Nigeria had intrinsic antimalarial property that
was dose- dependent. They found that, at dose of 100 mg.kg-1 body weight of mice, methanolic extract produced
significant chemosuppression of parasitemia (> 57%) when administered orally. It had the highest activity at
800 mg.kg-1 weight of mice (91.1%). Their extract exhibited low cytotoxicity against A2780 ovarian cancer
cells (with an IC50 of 28, 8 g.ml-1).The antimalarial effectiveness of A. senegalensis from DRC has not
formerly demonstrated in vivo, it could be a question of a plant which used by traditional healers to alleviate or
prevent a wide range of malaria symptoms because of its anti-inflammatory, immunostimulant, antipyretic or
vasorelaxant effects or a plant species which potentiates other plants and thus its effectiveness would depend on
associations of the plants (Rasoanaivo et al. 2004, Ngbolua et al. 2014b).
Spermatogenic activity
Oladele et al. (2014) tested the aqueous leaf extract of A. senegalensis at different doses of 200, 300, and
500 mg.kg-1 body weight for its spermatogenic effect. Results showed the weight of the testes and epididymis
increased significantly for the 300 and 500 mg.kg-1 doses. The sperm concentration for the 200, 300 and 500
mg.kg-1 doses also significantly increased and the sperm motility at 300 and 500 mg.kg-1 also increased
significantly. Decrease in abnormal sperm morphology was not significant for any of the doses. However,
another study revealed that aqueous leaf extract of A. senegalensis may possess the potential to adversely affect
testicular function in rat (Nwonuma et al. 2015).
Antimicrobial activity
In a study of methanol-methylene chloride extract of the root bark of A. senegalensis, the ethyl acetate
fraction on further fractionation gave two active subfractions, a lipophilic oily and another fraction (AS2) which
on purification precipitated white crystalline compound, later characterized to be kaurenoic acid. MICs of the
ethyl acetate fraction, the lipophilic oily fraction and kaurenoic acid against Bacillus subtilis were 180, 60, and
30 g.ml-1 respectively. AS2 exhibited activity against Staphylococcus aureus with an MIC of 150 g.ml-1,
while the lipophilic oily fraction was active against Pseudomonas aeruginosa with an MIC of 40 g.ml-1.
However, the extract and kaurenoic acid exhibited no effects against Candida albicans and Aspergillus niger
(Jada et al. 2015).
Ethanol extract of the plant had in vitro antimicrobial activity against some oral pathogens (More et al.
2008). An extract of recipe containing six plants including A. senegalensis had significant antibacterial activity
with (MIC) of 62.5 g.ml-1 against Staphylococcus aureus and 250 g.ml-1 against Candida albicans (Aiyeloja
& Bello 2006).
Antioxidant activity
It is concluded that antioxidant activity and drug detoxification activity of Annona senegalensis leave in
carbon tetrachloride-induced hepatocellular damage in rats using 2, 2-diphenyl-1-picrylhydrazyl (DPPH)
radical, superoxide ion, hydrogen peroxide (H2O2), 2, 2'-azinobis-(3-ethylbenzthiazoline-6-sulfonate) (ABTS)
and ferric ion models decreased significantly. The responsible chemical constituent of antioxidant activity may
be due to the presence of flavonoids in the extracts (Ajboye et al. 2010).
Anti-inflammatory activity
Anti-inflammatory activities of the leaves extract of A. senegalensis were determined in rats in inflammatory
models. The extract induced a significant decrease in the number of inflammatory cells. This effect is probably
due to higher concentrations of tannins and phenolic compounds in the extract of plant (Yeo et al. 2011).
Ngbolua et al. (2017) 4(1): 168175
Haemostatic activity
A. senegalensis is sold by herbalists in South Benin for treatment of bleeding. Dandjesso et al. (2012), in a
bid to find scientific evidence for this use, performed in vitro haemostatic tests on hydro alcoholic extracts of the
leaves. Results confirmed its anticoagulant properties, as indicated by a 39% reduction of plasma re-
calcification time. A. senegalensis was also shown to have astringent property, so it could act on primary
haemostasis through vasoconstriction.
Anticonvulsant activity
Anticonvulsant activities of the root bark extract on pilocarpine-induced seizures in animal model was
evaluated. The results proved the efficacy of A. senegalensis in the treatment of epilepsy and convulsions
(Konate et al. 2012). Kaurenoic acid (KA) was identified as the anticonvulsant principle in the root bark extract
of A. senegalensis. The anticonvulsant effect of KA is most likely being mediated through central inhibitory
mechanisms.
In vivo trypanocidal activity
The aqueous extract of A. senegalensis possesses trypanocidal activity against Trypanosoma brucei in
infected mice (Ogbadoyi et al. 2007).
Anti-snake venom activity
(Adzu et al. 2005), tested the power of the methanol extract of the root bark of the Annona senegalensis was
tested on brine shrimp (Artemia saline Leach) against cobra (Naja nigricotlis nigricolis Wetch) venom in rats.
They further reported that the reduction in the induced hyperthermia directly detoxified the snake venom used
by 1633%. However, the extract doesnt restore the liver functions.
Antinociceptive activity
The methanolic extract of Annona senegalensis was reported to display antinociceptive property in a various
test models. The analgesic effect of the methanolic this plant extract might be through peripheral mechanisms.
This pharmacological property justifies the use of the plant species in traditional medicine to treat rheumatic
pain (Adzu et al. 2003).
Anthelmintic activity
Alawa et al. (2003) investigated the efficacy of the extract of Annona senegalensis against Haemonchus
contortus eggs and was shown a significant reduction in the egg hatch and larval recovery as the concentration
increases.
Hypnotic activity
Effect of extract and fractions of A. senegalensis leaves on pentobarbitone-induced sleeping time was
assessed. The extract and fractions significantly (p<0.05) shortened the sleep onset time (sleep latency) and
prolonged sleeping time in a dose-related manner. A bioactive fraction significantly (P<0.05) prolonged sleep
time but increased sleep latency in a dose-related manner (Okoli et al. 2010). Extracts of the root bark also
potentiated the central nervous system depressant effect of phenobarbitone in a dose dependent fashion
(Otimenyin & Omeri 2014).
PHYTOCHEMISTRY
Phytochemical screening of Annona senegalensis revealed the presence of various secondary metabolites
including tannins (Jada et al. 2014), flavonoid (Jada et al. 2015), saponins (Afolabi & Afolabi 2013), alkaloids
(You et al. 1995), glycosides, steroids (Ijaiya et al. 2014), volatile oil (Ngamo et al. 2007), anthocyanins
(Mpiana et al. 2012), triterpenes and coumarins. GC/MS study of stem bark of A. senegalensis showed the
presence of 1, 2 benzenediol, butylated hydroxytoluene (BHT), Phenol, 2, 6 bis (1, 1-dimethylethyl-4methyl,
methylcarbamate, n hexadecanoic acid, hexadecane, 13- hexyloxacyclotridec-10-en-2one, oleic acid,
tetracosane, 9- octylheptadecane, heneicosane, 12-mehtyl-E, E-2, 13- octadecadien-1-ol, octadecanoic acid, 9,
17-octadecandienal, pentadecane, tetratriacontane and squalene (Awa et al. 2012). Biochemist in Ahmadu Bello
University, Zaria, Nigeria has been reported that a hydroxylated phenol which is 2-benzenediol or catechol is
toxic to microorganisms (Awa et al. 2012).
The diterpenoid, kaur-16-en-19-oic acid or kaurenoic acid was reported as phytochemical constituent
responsible for the antibacterial effects of root bark (Okoye et al. 2012). A. senegalensis has also been found to
contain various minerals such as Ca, K, Mg, Zn, Fe, Cu, Mn, Pb, Cr as well as ascorbic acid and amino acids,
making it an important source of nutrients (Yisa et al. 2010).
Ngbolua et al. (2017) 4(1): 168175
The GC and GCMS analyses showed that p-cymene (36.0%), -phellandrene (25.0%), -pinene (8.3%), Z-
sabinol (6.9%) and limonene (4.8%) are the major compounds A. senegalensis stem bark essential oil
(Khallouki et al. 2002). In another study, the leaf essential oil of A. senegalensis had oxygenated monoterpenes
(65.0%) as the major compounds and contains also citronellal (30.0%), citronellol (14.8%), geranial (17.2%),
thymol (8.1%), caryophyllene (7.8%) and carvacrol (6.92%) (Ameen et al. 2011). The GC and GC/MS
analyses of the essential oil of A. senegalensis from Burkina Faso displayed the presence of germacrene D
(19.2%), -caryophyllene (19.1%), - cadinene (11.1%) and -humulene (9.7%) as major components (Nbia
et al. 2002). In Brazzaville (Congo), essential oils were found in all parts of A. senegalensis including leaves,
stem bark, root bark, epicarp and mesocarp (Nkounkou et al. 2010).
In Nigeria, nineteen monoterpenes and sesquiterpenes were identified in the essential oils of the leaves and
fruits of A. senegalensis. The major constituents were car-3-ene in the fruit oil and linalool in the leaf volatile oil
(Ekundayo & Oguntimein 1986).
Two new cyclopeptides, cyclosenegalin A, cyclo (Pro1-Gly2-Leu3-Ser4-Ala5-Val6-Thr7-) (1) and
cyclosenegalin B, cyclo (Pro1-Gly2-Tyr3-Val4-Tyr5-Pro6-Pro7-Val8-) (2), were isolated and structurally
characterized from the methanol extract of the seeds of Annona senegalensis Pers., along with the known cyclic
peptide, glabrin A. The structures of the isolated compounds were characterized on the basis of the MS/MS
fragmentation, using a Q-TOF mass spectrometer equipped with an ESI source, chemical degradation and
extensive 2D-NMR (Alassane et al. 2002). Bioactive-guided fractionation of the methanol-methylene chloride
root bark extract (MME) of A. senegalensis using pentylenetetrazole (PTZ)-induced seizures in mice, afforded
ethyl-acetate fraction (EF) with anticonvulsant activity. The chromatographic fractionation of the EF yielded
eight sub-fractions (F1F8) which were submitted to anticonvulsant screening assay. The white crystals from the
sub-fraction F2 were purified to afford A. senegalensis crystals, AS2. The AS2, which exhibited potent
anticonvulsant effects, was characterized by 1D and 2D NMR spectroscopy, mass spectroscopy and X-ray
crystallography (Okoye et al. 2013). Chromatographic fractionation of the methanol-methylene chloride root
bark extract of A. senegalensis afforded a potent antibacterial ethyl acetate soluble fraction (EF) which gave
after additional column chromatography, two active sub-fractions F1 and F2. F1 yielded a lipophilic liquid
component while F2 on purification, precipitated a white crystalline compound that was characterized by proton
NMR and X-ray crystallography as kaur-16-en-19-oic acid. F1 was analyzed using GC-MS to obtain 6 major
constituents: 1- dodecanol, kaur-16-en-18-oic acid, 1-Naphthalenemethanol, 6, 6-dimethyl-bicyclo [3.1.1] hept-
2-ene-2-ethanol,3,3-dimethyl-2-(3-methylbuta-1,3-dienyl) cyclohexan-1-methanol and 3-hydroxyandrostan-17-
carboxylic acid (Okoye et al. 2012).
CONCLUSION
The pharmacological relevance and diversity of compounds reviewed in this manuscript demonstrate that
there is much to be discovered in this medicinal plant. As an anti-sickling plant candidate, there is therefore an
urgent need to evaluate this plant species for it biological activity and modes of action of derived organic acids
extracts which may shelter some epigenetic modulators drugs for the management of SCD in the future.
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ISSN (E): 2349 1183
ISSN (P): 2349 9265
4(1): 176179, 2017
DOI: 10.22271/tpr.2017.v4.i1.025
Research article
Effect of fungal inoculants on growth and establishment of

Gmelina arborea Roxb. in transplantation conditions
Manas Ranjan Panigrahi, Soumya Ranjan Nayak and Nibha Gupta*
Plant Pathology & Microbiology Division, Regional Plant Resource Centre, Bhubaneswar-51015, Odisha, India
*Corresponding Author: nguc2003@yahoo.co.in [Accepted: 16 April 2017]
Abstract: To evaluate the effect of fungal inoculants, a pot experiment was carried out on 30 days
old transplanted seedlings of Gmelina arborea under nursery condition. Transplants were
inoculated with seven day old liquid culture of Aspergillus ellipticus, Fusarium incarnatum,
Aspergillus aculeatus, Aspergillus candidus, Penicillium species, Alternaria alternata, Penicillium
species, Fusarium javanicum, Penicillium cyclopium, Aspergillus flavus separately. Growth of
plants were measured in terms of plant height, leaf no, leaf size, biomass, NAR, RGR, LAR after
120 days. Data recorded on growth performance of Gmelina arborea plant exhibited the positive
impact of fungal inoculation especially in case of Fusarium incarnatum, Penicillium species,
Penicillium cyclopium and Aspergillus flavus over un-inoculated control. Successful establishment
after transplantation and improvement in growth over control has been demonstrated well when
plants were treated with Aspergillus flavus.
Keywords: Gmelina arborea - Phosphate solublization - Transplantation - Growth.
[Cite as: Ngbolua KN, Moke EL, Baya JL, Djoza R, Ashande CM & Mpiana PT (2017) A mini-review on the
pharmacognosy and phytochemistry of a tropical medicinal plant: Annona senegalensis Pers. (Annonaceae).
INTRODUCTION
Filamentous fungi are widely used as production of organic acid and are capable of solubilising the insoluble
form of phosphate, make them available to the plant root system indirectly (Manoharachary et al. 2005, Richa et
al. 2007, Pandey et al. 2008, Yadav et al. 2011). It is important to note that sufficient and balanced quantities of
nutrients needed for optimal growth of plants. Most of the nutritional contents in soil are present in bonded form
and released only through biological and/or chemical activity. In such circumstances supplementation of
chemical fertilizers is the only alternative to enrich the soil. But organic fertilizers are not the long term nutrient
supplier (Pascual et al. 1997, Patil et al. 2002, Mehrvarz & Chaichi 2008, Reis et al. 2008). In such cases,
bioinoculation of mineral solubilisers have shown a vital role in growth and development of plants especially
forest tree species (Pascual et al. 1997, Sahgal et al. 2004, Thatoi et al. 2005). The development of proper soil
mixture for use in the nursery may be the cost effective process to attain better growth and production of tree
species (Dash et al. 2013). Many such uses of biofertilizer for the production of quality planting material of
forest trees are well reported (Barua et al. 2010, Dhar & Mridha 2012). Gmelina arborea Roxb. is a large,
tropical, evergreen perennial tree, an important medicinal plant and preferred in afforestation programs because
of its unique timber (Orwa et al. 2009). It is reported that in poor soil, plantation of the tree may suffer from
nutrient disorder (Sturmann et al. 1994). The importance of fungal inoculation in Gmelina arborea under heavy
metal stress condition has also been reported (Barua et al. 2010). Under nutrient depletion condition, the
inoculation of mineral solubilising fungi play a vital role in growth and establishment of forest tree seedlings. In
view, an experiment under nursery condition has been carried out along with fungal inoculation in transplanted
seedlings of G. arborea and growth performance was evaluated.

The pot experiment was carried out in poly bags (size: 1216 cm containing 5.5 kg red laterite soil) received
average 352C temperature & 6080 % relative humidity. The soil contains a high amount of iron, manganese
Panigrahi et al. (2017) 4(1): 176179
and copper. Textural class of the soil was loamy sand the soil pH was 5.61. Average nitrogen (N), average
phosphate (P2O5) and average potassium (K2O) of the soil was 232.0 kg.Ha-1, 25.2 kg.Ha-1 and 99.12 kg.Ha-1
respectively. The soil was fumigated with 1% formalin (25 ml per pot) for 48 hrs prior to the experiment.
The seeds were collected from native plant and were treated with organic manure water for 72 hrs to depulp.
After drying at room temperature, seeds were sown in experimental poly bags 1 inch below the upper surface.
The seedlings were transplanted after 30 days from DAS (days of sowing) to other polypots of the similar
capacity and filled with the same type of soil. Seven days after the transplantation, 25 ml of 7 days old fungal
culture (prepared in Czapek dox medium pH 4.5) of 10 different fungi were added separately in 20 replications
to the polybags contain Gmelina arborea Roxb. seedlings. Fungal inoculations were repeated thrice in one
months interval of the experiment duration of 120 days. Observations after 4 months from DAS (date of
sowing) were recorded for shoot height, a number of leaves, leaflets, branches, the fresh and dry biomass of
leaves and stem (Leopold & Kriedemann 1975). The data were evaluated for RGR (Relative growth rate), NAR
(Net Assimilation Rate) and LAR (Leaf area ratio) and quality index (Dickson et al. 1960, Basak et al. 2004,
Tewari et al. 2006).
RESULT AND DISCUSSION

Fungal inoculation exhibited the improved shoot height as compare to uninoculated control. Penicillium
species and Aspergillus flavus both fungi had shown enhancement in shoot and root as well (Table 1). Treated
and transplanted plants had developed 34 branches under fungal inoculation of Fusarium javanicum,
Penicillium cyclopium and Fusarium incarnatum. The highest no of leaves were found in plants treated with
Fusarium incarnatum followed by Aspergillus aculeatus, Penicillium species and Fusarium javanicum. Only
these fungal inoculations i.e. Penicillium species, Penicillium cyclopium, Aspergillus flavus have shown the
highest no of leaf area as compared to uninoculated control.
Table 1. Growth performance of Gmelina arborea at transplantation stage and inoculated condition.
Growth Parameters
Shoot Root No. of No. of Leaf Area Initial Biomass (g) Dry Biomass (g)
Treatments
Height (cm) Length Branch Leaves (mm2) Shoot Root Shoot Root
(cm)
Control 58.84.1 22.23.2 21.0 35.18.6 8296.4800.5 32.17.7 17.75.6 15.34.3 9.12.2
Aspergillus ellipticus 52.08.6 25.23.9 20.5 30.513.6 6124.01067.4 20.33.9 19.93.3 13.43.5 11.33.4
Fusarium incarnatum 63.08.1 27.22.9 31.4 55.011.2 7425.0828.9 50.95.2 39.413.3 22.77.3 17.06.7
Aspergillus aculeatus 57.26.7 22.23.9 21.4 46.87.1 8107.0882.6 31.817.2 26.79.7 17.63.2 11.93.4
Aspergillus candidus 61.37.7 26.36.6 21.7 41.313.0 5727.480.7 43.813.1 25.28.6 20.05.0 12.73.8
Penicillium species 70.714.3 27.54.2 21.6 52.38.6 11995.81321.9 50.76.9 49.711.0 21.65.5 20.26.5
Alternaria alternata 46.510.8 29.84.7 11.3 32.211.9 4255.870.8 22.05.1 17.24.4 10.93.2 7.01.5
Penicillium species 54.76.2 31.22.9 20.6 42.511.5 4469.4185.7 33.210.1 32.29.3 15.05.0 12.44.6
Fusarium javanicum 52.21.1 33.23.1 41.5 48.84.8 4714.896.7 37.18.0 30.33.8 19.32.4 15.22.3
Penicillium cyclopium 63.03.7 25.32.8 31.0 42.210.1 15982.6699.0 43.84.5 33.97.9 19.33.7 13.91.7
Aspergillus flavus 75.712.5 28.72.4 20.8 36.27.6 14228.0923.0 59.16.0 38.06.2 23.84.3 16.91.4
Note: RGR= Relative Growth Rate; QI= Quality Index; NAR= Net Assimilation Rate; RSR= Root Shoot Ratio; LAR= Leaf Area Ratio.
In general, biomass in seedlings was higher under inoculated conditions as compared to control. Seedlings
exhibited maximum biomass production when inoculated with fungal strains Fusarium incarnatum, Penicillium
species, Penicillium cyclopium, Aspergillus flavus. Mean Biomass (fresh and Dry) measured after four months
indicated the maximum increment in growth of plants inoculated with this fungi. It is apparent that, in the term
of biomass, the seedlings inoculated with different fungi showed a higher production and superiority over
control. Fusarium incarnatum, Penicillium species, Penicillium cyclopium, Aspergillus flavus inoculated plants
attained maximum biomass with respect to control. Except for Aspergillus ellipticus, almost all other inoculants
resulted increased in biomass in comparison to control. Relative growth rate (RGR) was also changed due to the
enhancement in dry biomass, stem height and leaf area in transplanted conditions (Table 2). However,
transplanted seedlings of Fusarium incarnatum, Penicillium species, and Aspergillus flavus showed higher RGR
as well as Net assimilation rate. Growth analysis revealed that NAR (net assimilation rate), and LAR (leaf area
ratio) accounted for the differences in RGR (relative growth rate) in the treatments. Data recorded for the quality
index of Gmelina arborea Roxb. grown with selected fungal isolates in non transplanted and transplanted
conditions showed better quality and comparatively more growth than the un-inoculated control. Application of
Panigrahi et al. (2017) 4(1): 176179
selected fungal cultures resulted in an increase of biomass as compared to control, which leads to the successful
establishment of Gmelina arborea.
Table 2. Physiological growth performance of Gmelina arborea under transplanted and inoculated condition.
Wet RGR Dry RGR NAR LAR
Treatments QI RSR
(d-1) (d-1) (g .m-2.d-1) (m2 .g-1)
Control 0.161 0.107 0.30 0.657 0.402 0.377
Aspergillus ellipticus 0.030 0.085 0.06 0.609 0.465 0.485
Fusarium incarnatum 0.370 0.189 0.37 0.455 0.617 0.432
Aspergillus aculeatus 0.158 0.133 0.35 0.583 0.504 0.388
Aspergillus candidus 0.291 0.160 0.04 0.436 0.520 0.429
Penicillium species 0.368 0.177 1.15 0.640 0.587 0.392
Alternaria alternata 0.049 0.058 0.07 0.587 0.371 0.641
Penicillium species 0.174 0.104 0.10 0.480 0.491 0.571
Fusarium javanicum 0.217 0.151 0.11 0.409 0.644 0.636
Penicillium cyclopium 0.291 0.151 1.59 0.860 0.514 0.402
Aspergillus flavus 0.461 0.202 1.77 0.667 0.530 0.379
Note: RGR= Relative Growth Rate; QI= Quality Index; NAR= Net Assimilation Rate; RSR= Root Shoot Ratio;
LAR= Leaf Area Ratio.
Application of liquid inoculants to seedlings was better than seed inoculation. It is recommended that
seedlings raised in the nursery should be inoculated with liquid inoculants immediately or soon after
germination (Odee et al. 2002). Organic and inorganic fertilizers are not only supply limited amount of nutrients
to the plants but also needed in huge quantity. As Gmelina arborea is a forest grown plant, regular organic
manure supplement is not possible. So it is an ideal choice to develop microbial consortia which help the plant
root system to grow and absorb nutrients. It also improves the physiochemical and biological properties of soil.
It has been reported that Gmelina arborea has weak establishment property but once it establishes in an
environment, it grows quickly (Lamb 1968). In this regard, the supplementation of fungal cultures having
phosphate solubilising potential to the transplanted seedlings may be the better option to overcome the
transplantation and establishment problems. This process can also be applied to another forestry plant, those
who has weak establishment property.
ACKNOWLEDGMENT
We are thankful for the financial support obtained from Forest and Environment Department, Govt. of
Odisha under State Plan Project 201516.
REFERENCES
Barua A, Gupta SD, Mridha AU, Bhuiyan MK, Menon S, Mohan V & Krishnakumar N (2010) Effect of
arbuscular mycorrhizal fungi on growth of Gmelina arborea in arsenic contaminated soil. Journal of
Forestry Research 21(4): 423432.
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Dash S, Mohapatra AK & Gupta N (2013) Growth response of Dalbergia sissoo Roxb. to mineral solubilizing
bacteria and fungi in nursery conditions. Tropical Ecology 54(1): 09115.
Dhar PP & Mridha MAU (2012) Arbuscular mycorrhiza associations in different forest tree species of Hazaikhil
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nurseries. Forestry Chronicle 36: 1013.
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Oxford, UK.
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Mehrvarz S & Chaichi MR (2008) Effect of phosphate solubilizing microorganisms and phosphorus chemical
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Odee DW, Indieka SA & Lesueur D (2002) Evaluation of inoculation procedures for Calliandra calothyrsus
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Pandey A, Das N, Kumar B, Rinu K & Trivedi P (2008) Phosphate solubilization by Penicillium spp. Isolated
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Pascual JA, Garcia C, Hernandez T& Ayuso M (1997) Changes in the microbial activity of an arid soil amended
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Patil MG, Sayyed RZ, Chaudhari AB & Chincholkar SB (2002) Phosphate solubilizing microbes: a potential
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Richa G, Khosla B & Reddy MS (2007) Improvement of Maize plant growth by phosphate solubilizing fungi in
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Sahgal M, Sharma A, Johri BN, Prakash A (2004) Selection of growth promontory Rhizobia for Dalbergia
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Sturmann M, Bergmann, Zech W (1994) Mineral nutrition, soil factors and growth rates of Gmelina arborea
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Tewari P, Saxena AK & Rao OP (2006) Effect of sodicity and salinity on seedling growth of two early
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ISSN (E): 2349 1183
ISSN (P): 2349 9265
4(1): 180182, 2017
DOI: 10.22271/tpr.2017.v4.i1.026
Technical Report
Micronutrient management for mango and guava orchards:

farmers perspective
Tarun Adak*, Kailash Kumar and Vinod Kumar Singh
Division of Crop Production, ICAR-Central Institute for Subtropical Horticulture,
Rehmankhera, Lucknow-226101, Uttar Pradesh, India
*Corresponding Author: tarunadak@gmail.com [Accepted: 18 April 2017]
[Cite as: Adak T, Kumar K & Singh VK (2017) Micronutrient management for mango and guava orchards:
farmers perspective. Tropical Plant Research 4(1): 180182]
Indian soils are generally deficient in micronutrients. However, the degree of deficiency is higher in case of
Zinc (Zn) and Boron (B). The average level of Zn deficiency in Indian soils is estimated to be around 50% and
is projected to increase to 63% by 2025 (Singh 2001). The micronutrient deficiency is significantly related to the
food and nutritional security of human being. It was estimated that around 26% of Indias population is at risk of
zinc deficiency. The problem of micronutrient deficiency in ubiquitous, with deficiency of micronutrients had
leads to health risk across the globe (Alloway 2007, Black et al. 2008). The problem of micronutrient deficiency
is not the only man-made problem but also a number of soil related issues are associated with it.
Figure 1. Symptoms of micronutrient deficiency: A, Zink deficiency in mango; B, Boron deficiency in guava (cracking of
fruit); C, Boron deficiency in mango cracked mango (cracking of fruit).
Adak et al. (2017) 4(1): 180182
Table 1. Soil reaction and micronutrient distribution in 20 mango orchards of Malihabad region, Lucknow, Uttar Pradesh.
Sampling Sites pH Zn (mg.kg-1) Cu (mg.kg-1) Mn (mg.kg-1) Fe (mg.kg-1)
1 6.167.51 0.381.64 1.043.12 5.2614.22 6.4817.68
2 7.197.86 0.581.14 1.262.74 4.9612.34 6.5210.48
3 6.978.40 0.361.54 0.782.78 1.2812.88 2.6817.22
4 6.517.34 0.360.98 2.463.34 7.5613.92 11.1418.12
5 6.026.87 0.280.84 1.543.02 6.7813.62 7.6418.48
6 6.687.99 0.342.28 1.243.34 2.8212.86 6.5617.26
7 6.667.25 0.320.68 1.683.26 5.5614.48 6.4415.94
8 7.248.41 0.321.26 0.842.36 1.629.74 2.7612.22
9 7.398.04 0.321.18 0.922.16 3.488.78 4.3611.52
10 7.678.12 0.320.82 1.162.48 2.768.98 4.5411.04
11 7.157.66 0.462.52 0.782.96 3.6610.22 7.8018.92
12 7.227.77 0.642.86 2.363.42 3.147.82 11.7219.46
13 7.107.71 0.361.74 0.983.36 2.727.36 7.3216.28
14 7.018.10 0.421.36 1.683.62 3.6810.96 6.0810.26
15 6.767.76 0.541.96 1.863.42 3.547.22 3.2414.32
16 7.008.27 0.382.24 1.963.32 2.584.92 7.3413.64
17 6.987.54 0.361.22 2.283.40 1.128.42 2.8614.26
18 6.818.39 0.422.06 1.282.90 0.925.26 2.6214.02
19 7.198.66 0.442.54 1.563.36 1.226.18 2.7612.88
20 6.918.12 0.342.08 1.382.76 1.366.26 1.989.12
The problem of micronutrient management and deficiencies if present at all, needs to be addressed seriously,
particularly in the case of fruit crops. Orchard farming needs special attention in micronutrient management as
fruit quality and productivity is directly related to it. Sometimes plant shows hidden hunger symptoms also and
may not show any deficiency symptoms in leaf tissues even if soils showed deficient in a particular nutrient. It
has been estimated that widespread micronutrients deficiencies existed in mango orchards of Uttar Pradesh. The
orchards of Moradabad, Rampur, Bareilly, Shahjahanpur, Hardoi, Lakhimpurkhiri and Sitapur districts of Uttar
Pradesh showed wide variations in micronutrient contents and deficiencies across management practices
adopted by mango growers. The percentage deficiency of Zn ranged between 5.9 to 75.0 percent in soil and 33.3
to 100 percent in leaf tissue analysis (Kumar et al. 2015). Spatial distribution indicated that orchards of
Shahjahanpur districts are deficient in Zn (75 per cent) followed by orchards of Hardoi (50 per cent) and
Bareilly (36.6 per cent) districts respectively. Cent per cent orchards of Bareilly, Shahjahanpur and Hardoi were
deficient in both Cu and Mn while the soils of the orchards of Rampur and Lakhimpurkhiri districts were
deficient in Mn (Kumar et al. 2015). Available Fe was in optimum range in most of the orchards in all the
districts however, only few orchards of Rampur and Bareilly showed Fe deficiency. In another study, 250 soil
samples were collected from 20 villages of mango orchards of Malihabad, famous for its Dashehari mango
production in Uttar Pradesh and widespread variations in micronutrient content were revealed (Table 1). Mango
orchards deficient in Zn and Mn content were also recorded. Factors like coarse texture of soils, low organic
matter content, microbial activity and non-application of micronutrients in the orchards under subtropical
environment may be responsible for this. Zn deficiency symptoms were widely seen in mango orchards in India
and particularly in Uttar Pradesh (Fig. 1A) and to correct the deficiency, spraying of 0.5% ZnSO4 is advocated.
Figure 2. Mango fruit loss by black tip due to SO2 emission from brick kline.
Adak et al. (2017) 4(1): 180182
Boron is another crucial micronutrient limiting crop production (Shukla et al. 2014). Boron plays an
important role in reproductive as well increasing quality fruit production. Mango crop varies in their
requirements. This variation may be due to source of irrigation water, climate and season, rootstock, age and
developmental stages of plants. Of course, B is present in soils in the form of mineral tourmaline which is
extremely resistant to wreathing, thus relatively immobile in plants resulting in deficiency symptoms. The
symptoms are showing first in the growing part (young levels) and cracking of fruits finally (Fig. 1B, C). To
correct B deficiency, application of 100g borax per plant is recommended during the month of September-
October. Foliar application of boron, useful for optimum improvement in fruit quality of mango with spray of
0.5% boric acid solution at peanut and marble size of fruits. Sometimes, B was also observed in high pH soils
causing toxicity of B. It has been noted that Zn concentration in the lower level, enhances boron accumulation.
Mango orchards situated near the brick Kline many times affected by black tip due to SO2 emission. It forms
an acid that burns the tissue of mango fruit and deteriorate the quality. The intensity of the affected fruit quality
depends on the height of the brick Kline and wind direction. Farmers often came across yield and profit loss
(Fig. 2). To counter this man-made problem, setting up of brick Kline should not be permitted nearby clusters of
mango orchards.
REFERENCES
Alloway BJ (2008) Zinc in Soils and Crop Nutrition, 2nd edition. IZA and IFA Brussels, Belgium and Paris,
France, 135 p.
Black RE, Allen AH, Bhutta ZA, Caulfield LE, Onis M, Ezzati M, Mathers C & Rivera J (2008) Maternal and
child undernutrition: global and regional exposures and health consequences. The Lancet 371: 243260.
Kumar K, Adak T & Singh VK (2015) Status and distribution of micronutrients in mango orchards under
subtropical region of Uttar Pradesh, India. Journal of Agricultural Physics 15(2): 127139.
Shukla AK, Tiwari PK & Prakash C (2014) Micronutrients Deficiencies visvis Food and Nutritional Security
of India. Indian Journal of Fertilisers 10 (12): 94112.
Singh MV (2001) Evaluation of micronutrient status of difference agroecological zones of India. Fertiliser News
46(2): 2542.
ISSN (E): 2349 1183
ISSN (P): 2349 9265
4(1): 183191, 2017
DOI: 10.22271/tpr.2017.v4.i1.027
Research article
Isolation and characterization of nitrogen fixing bacteria that

nodulate alien invasive plant species
Prosopis juliflora (Swart) DC. in Marigat, Kenya
John O. Otieno1,2*, David W. Odee1, Stephen F. Omondi1,
Charles Oduor1 and Oliver Kiplagat2
1
Kenya Forestry Research Institute, P.O. Box 20412-00200, Nairobi, Kenya
2
School of Agriculture and Biotechnology, University of Eldoret, P.O. Box 1125-30100, Eldoret, Kenya
*Corresponding Author: jochiengo@gmail.com [Accepted: 22 April 2017]
Abstract: A total of 150 bacterial strains were isolated from the root nodules of Prosopis juliflora
growing in soils collected from Marigat area of Kenya. Soil samples from representative colonized
zones of Tortilis, Grass and Prosopis were used in trapping the microsymbionts. A physiological
plate screening allowed the selection of 60 strains which were characterized based on
morphological, cultural and biochemical characteristics. Tolerance to salinity, acid and alkaline pH
and resistance to antibiotics were studied as phenotypic markers. Morphological characteristics
allowed the description of a wide physiological diversity among tested isolates. Establishing
mutualistic interactions in novel environments is important for the successful establishment of
some non-native plant species. The associations may have negative impact on the interaction
networks of the native species whereby non-native species becoming dominant. Our study
suggests that P. juliflora may have led to the diversity of N-fixing microsymbionts observed in the
study area. The study provides basis for further research on the phylogeny of rhozobial strains
nodulating P. juliflora, as well as their use as inoculants to improve growth and nitrogen fixation
in arid lands of Kenya. The data obtained in this study can be used for strain improvement and
cross-inoculation experiments with different species when searching for well adapted and
compatible partners.
Keywords: Prosopis juliflora - Rhizobia - Microsymbionts - Diversity.
[Cite as: Otieno JO, Odee DW, Omondi SF, Oduor C & Kiplagat O (2017) Isolation and characterization of
nitrogen fixing bacteria that nodulate alien invasive plant species Prosopis juliflora (Swart) DC. in Marigat,
Kenya. Tropical Plant Research 4(1): 183191]
INTRODUCTION
Prosopis juliflora (Sw.) DC is an evergreen shrub but sometimes grows into a small tree in the family
Fabaceae, sub-family Mimosaidea (Polhill et al. 1981). The species is native to South America, Central America
and the Caribbean (Pasiecznik et al. 2001). In the United States, it is well known as mesquite. It is fast growing,
nitrogen-fixing and tolerant to arid conditions and saline soils (El-Keblawy & Al-Rawai 2007). It is fast
growing, nitrogen-fixing and tolerant to arid conditions and saline soils. The family leguminosae comprises
approximately 650 genera, 1800 species and is the largest family of flowering plants (Polhill et al. 1981). Its
species have worldwide distribution, are adverse, survive in a wide climatic condition and have a multiplicity of
use. The leguminosae is divided into three sub families; Caesalpinoidae, Mimosoidae and Papilionoidea. The
greatest occurrence of nodulation, 97% is in the Papilionaidae compared to 90% in the Mimosoidae and only
23% in the Caesalpinidae (Faria et al. 1989).
The current taxonomy has revealed a wide diversity of microsymbionts that can form N2-fixing symbioses
with legume roots in a manner that is similar to rhizobia at the genus, species and intra species level.
Traditionally, rhizobia were exclusively members of the Rhizobiaceae family in the Alphaproteobacteria class
of bacteria, which includes the genera Allorhizobium, Azorhizobium, Bradyrhizobium Mesorhizobium,
Rhizobium and Sinorhizobium (Sprent 2008). The term rhizobia have been used for all the bacteria that can
Otieno et al. (2017) 4(1): 183191
produce nodules and fix atmospheric nitrogen in legumes (Brewin 2004, Cheng 2008). Rhizobia are bacteria
capable of entering their legume hosts through root hairs, sites of lateral root emergence, or directly through root
epidermis (Sprent et al. 2013), where they can induce the development of nodules where biological nitrogen
fixation takes place (Gage 2004). The formation of root nodules involves complex molecular signaling pathways
between legumes and rhizobia (Stacey 2007). Within root nodules, organic forms of reduced atmospheric
nitrogen produced by the bacteria are utilized by the host plant and ultimately enter the earths food webs. In
exchange, bacterial symbionts acquire carbohydrates from legumes. Surprisingly, rhizobia are not monophyletic
and represent a diverse array of bacteria found in both the Alphaproteobacteria (alpha rhizobia, e.g. genera
Rhizobium and Bradyrhizobium) and Betaproteobacteria (beta rhizobia, e.g. genera Burkholderia and
Cupriavidus) classes (Gyaneshwar et al. 2011). This symbiosis plays a very important role in agriculture as it
can relieve the requirements for nitrogenous fertilizers during the growth of leguminous crops. However,
relatively little information is available regarding microsymbionts species associated with P. juliflora an
emerging alien invasive plant species in Kenya.
Invasive species are now considered as the second most important cause of the reduction in biodiversity in
the world, after habitat loss. Loss of species biodiversity of the soil biota may be an acceptable consequence of
the development and maintenance of agricultural systems. Habitat destruction at best erodes genetic diversity of
the legume host and at worst leads to extinction, the subsequent fate of the rhizobial flora, through time, would
be similar. A rich diversity of rhizobial species has been found in the tropics (Dreyfus et al. 1988, Odee et al.
1997). The need for conservation of both legume and host is therefore, paramount. There are numerous
instances of woody legume introductions becoming weedy resulting (in some cases) in the need for expensive
control programs. In particular, P. juliflora and Leucaena leucocephala have become noxious weeds in exotic
environments (Fagg & Stewart 1994). Of the 45 species of Prosopis described by Allen & Allen (1981), 12
showed nodules, the efficiency of which varies among the species and among varieties within a species.
Rhizobium strain isolated from P. juliflora nodualted peanut plants and was classified as belonging to the
cowpea group (Subba Rao et al. 1982); the same occurred for rhizobium isolated from 5 species of the genera
Acacia and Albizzia (Basak & Goyal 1975). P. juliflora nodules have apical meristem, with indeterminate
growth, thus with standing harsher stress condition provoked by temperature, drought and salinity, than species
with globose nodules (Felker & Clark 1980). Invasive trees in the Australian legume genus Acacia Mill. sensu
stricto (Leguminosae subfam. Mimosoideae, formerly Acacia subgen. Phyllodineae DC; Maslin (2008), have
received much research attention because of their invasion success and severe impacts on native ecosystems
globally (Richardson et al. 2011). Acacias are known to form successful rhizobial interactions in their
introduced ranges (Birnbaum et al. 2012, Wandrag et al. 2013) and have, in some instances, been co-introduced
with their rhizobia (Crisstomo et al. 2013, Ndlovu et al. 2013).
The unique partnership between legumes and rhizobia has been suggested as a major contributing factor to
the success of some legumes as prominent invasive species in many parts of the world (Parker 2001) and that
the ability to find compatible rhizobia in introduced regions plays an important role in establishment success
of legumes (Rodrguez-Echeverra et al. 2011). Variation in colonizing ability may be related to opportunities
for symbionts acquisition from legume taxa that are indigenous to the invaded habitat (Richardson et al. 2000,
Parker 2001). However, due to the variously restricted host ranges of rhizobia (Parker et al. 2004) only a subset
of native legume taxa are likely to be potential sources of symbionts for any particular invasive legume. Most
invaders will therefore encounter high spatial variability in symbionts availability owing to the heterogeneous
distribution of native legumes across the landscape.
Under the right conditions, P. juliflora can produce a variety of valuable goods and services: construction
materials, charcoal, soil conservation and rehabilitation of degraded and saline soils. Concern about
deforestation, desertification and fuel wood shortages in the late 1970s and early 1980s prompted a wave of
projects that introduced P. juliflora and other hardy tree species to new environments across the world. P.
juliflora has survived where other tree species have failed and in many cases become a major nuisance. In 2004
it was rated one of the worlds top 100 least wanted species (IUCN 2009). P. juliflora has invaded and continues
to invade, millions of hectares of rangeland in South Africa, East Africa, Australia and coastal Asia (Pasiecznik
et al. 2001). The tree species plays a leading role in the afforestation of arid lands. Their capability of growing
on degraded land under arid conditions has made them especially suitable for this purpose. In general,
mutualisms between invasive plants and rhizobia have been shown to increase plant biomass and to improve
establishment success (Weir et al. 2004).
Otieno et al. (2017) 4(1): 183191

Host Plant and Soil sampling
Soil samples in this study were collected from three zones within the experimental site. In each zone, soil
samples were taken from six random sampling points, at a depth of 1530 cm. The soil samples were pooled
into a single sample and transferred to the laboratory for analysis and trapping experiment in the green house at
KEFRI Muguga as described by Odee et al. (1997). Each soil was collected with aseptic precautions to avoid
cross-contamination between soils from different zones. The soils were analyzed using the methods described in
Anderson & Ingram (1989). The pH was measured in calcium chloride CaCl2) as follows: 10 g of soil was
suspended in 20 cm-3 of 10 mol.m-3 Cacl2 solution and measured using a Corning 240 pH meter.
Seeds of P. juliflora were obtained from Kenya Forestry seed Center Muguga. The seeds of uniform size
were selected and nipped using a blade and surface sterilized by immersion in 90% alcohol for 30s, followed by
3% sodium hypochlorite (NaOCl) solution for 3 min and rinsing several times in sterile water. Seeds were then
germinated on 1% water agar plates and incubated at 26oC (Vincent 1970). The experiment was established in a
greenhouse with temperature ranging from 19oC to 30oC, using complete randomized blocks design (CRBD)
with 10 replications, in PVC pots. Four seeds were planted which were later thinned out to two per pot. The tree
plants were then harvested after 90 days.
Nodulation Assessment and Rhizobial Isolation
The plants were cut at the level of the growth media to separate the shoot and the root. Shoot height was
recorded in centimeters using a meter ruler. Nodules were detached from the root, counted and fresh weight also
recorded at the same time. The different plant parts were then put in brown paper bags and then oven dried at
60oC for 2 days. The dry weights in mg of the plants were then taken separately using an electronic weighing
balance. Ten nodules were picked per pot giving a total of 100 nodules per trap host per zone. Fifty of those
nodules were selected at randomly for isolation and the rest were kept in McCartney bottles containing silica
gel. Fresh nodules were transferred into 95% ethanol for 510 s and then in 1% NaOCl for 6 min. Each nodule
was then successively rinsed in sterile distilled water (at least 6 changes) to remove traces of the sterilant, each
time sterilizing the forceps by dipping it into 95% alcohol followed by flaming with Bunsen burner. The surface
sterilized nodule was then placed in a drop of sterile petri-dish and then crushed with a blunt tipped forceps. A
loopful of the crushed nodule was then picked and streaked across the surface of yeast extract mannitol agar
(YMA) plates. The composition of YMA was according to Vincent (1970). The streaked plates were incubated
at 28oC until colonies appeared. Different types of isolates were re-isolated on diagnostic YMA media before
being transferred to YMA. The isolates were then stored at -70oC for further work.
Intrinsic antibiotic resistance (IAR)
Stock solution of the following antibiotic were prepared; Ampicillin (Sigma) 20 g.ml-1, Streptomycin
(Sigma) 200 g.ml-1, Kanamycin (Sigma) 100 g.ml-1, Tetracycline (Sigma) 50 gml-1 Rifamycin (Sigma) 50
g.ml-1, Nalicidixic acid (Sigma) 20 g.ml-1. The solutions were filter-sterilized using 0.22 micro ml Millipore
filters and stored in one use aliquots at -20oC at each agar plate preparation, an aliquot of each antibiotic was
thawed at room temperature and added aseptically to freshly prepared sterile molten YEMA (Yeast extract and
Agar from Difco Labaoratories) at 50oC to 60oC according to Vincent (1970) to give the desired final
concentration. Rhizobial isolates were grown in yeast Manitol broth to late exponential phase and diluted
accordingly before being used to inoculate the agar plates with a Denley multipoint inoculator. There were two
replicates for each antibiotic concentration combination. Growth on antibiotic plates was compared with control
(antibiotic free) plates and scored as follows, (+) good growth same as control, (+/-) growth less or weak than
control and () no growth.
Salt and pH tolerance level determination
The level of tolerance to sodium chloride (NaCl) was determined on YMA plates containing the following
levels of NaCl concentration in mol.m-3; 0, 2, 4, 6, 9, 10. Rhizobia isolates were grown and inoculated as for
IAR. Growth was compared with YMA plates with the normal concentration and scored as follows, (+) good
growth same as control, (+/-) growth less or weak than control and () no growth. The same was done for pH
tolerance level determination.
Statistical analysis
The data obtained from the ten replicates were subjected to statistical analysis by using Genstat version 12.0
Otieno et al. (2017) 4(1): 183191
computer program. Morphological characterization data was converted into an absence/presence binary matrix
(0, 1) using the method described by (Rohlf 1993).

Soil analysis
The soil pH of the zones varied from 7.48 Tortilis zone, 7.56 Prosopis zone and 8.32 Grass zone in water. In
CaCl2 soil pH varied from 6.85 Tortilis zone, 7.08 Prosopis zone and 7.41 Grass zone in. Electrical
conductivity ranged from 0.043 mS.cm-1 Tortilis zone, 0.053 mS.cm-1 Prosopis zone and 0.064 mS.cm-1 Grass
zone. Available ammonia varied from 1.78 ppm Grass zone, 2.37 ppm Prosopis zone and 2.11 ppm Tortilis
zone. Available nitrates varied from 1.12 ppm Grass zone, 2.21 ppm Prosopis zone and 1.18 ppm Acacia zone.
Height, fresh, nodulation and dry weight determination
Tortilis zone showed significance difference in terms of plants height compared to the other zones. Prosopis
zone was the best in terms of nodulation. The other two zones did not show significance difference in terms of
nodulation. In terms of nodule fresh weight, Prosopis zone had higher significant difference compared to the
other two zones which did not show significance difference. Dry shoot matter was highest in Prosopis zone and
Acacia zone but there was no significance difference. Grass zone was poor in terms of shoot dry matter
compared to the other two zones.
Isolation and Identification of Microsymbionts
A total of 150 microsymbionts were successfully isolated from the root nodules of the host plants. The
isolates were differentiated by their growth rate into either fast-growing (3 days) or slow-growing 5 days. Most
of the fast growing bacteria were observed as acid producers, while the slow growers were alkaline producers
based on the changes of pH in YEMA incorporated with BTB. Most of the strains failed to absorb the red colour
from Congo red.
Physiological characteristic
Table 1. Differentiating physiological traits of the rhizobial isolates recovered from Prosopis juliflora using soil
collected from grass zone (GZPJ).
pH tolerance NaCl tolerance (%) Intrinsic antibiotic resistance (IAR)
Isolate
4 6 8 9 10 0 1 2 3 4 5 AMP STR KAN RIF TET NAL
GZPJ-1 - + - - - + - - - - + - - - - - -
GZPJ-2 + + + + + + + + - + + + - - - + -
GZPJ-3 + + + + + + + - + + - + - - - - -
GZPJ-4 - - - - - - - - - - + - - - - - -
GZPJ-5 + + + + + + - - - + + - - - - + -
GZPJ-6 + + + + + + + + + + - - - - + - -
GZPJ-7 - + + + + + + - + + + + - - - - -
GZPJ-8 + + + + + + + + + + + + + - - + -
GZPJ-9 - + + + + + + - - + + + - - - - -
GZPJ-10 + + + + + + + + + + + + + - - + -
GZPJ-11 + + + + + + + + + + + + - - + + -
GZPJ-12 + + + + + + + + + + + + - - + + -
GZPJ-13 + + + + + + + + + + + - - - - + -
GZPJ-14 + + + + + + + + + + + - - - - + -
GZPJ-15 + + + + + + + + + + + - - - - + -
GZPJ-16 + + + + + + + - + + + - - - - + -
GZPJ-17 + + + + + + + - - + - - - - - + -
GZPJ-18 + + + + + + + - - + - + - - - + -
GZPJ-19 + + + + + + + + + + + + + - - + -
GZPJ-20 - + + + + + + - - + - + - - - - -
Note: +, -, strains were positive and negative, respectively.
As shown in tables (13), the isolates tested showed a wide diversity in their pH tolerance. From 90% to
100% of the isolates grew in lightly acid and neutral pH. A few of the isolates were unable to withstand either
very low or very high pH across the zones. Prosopis zone had over 60% isolates susceptible to pH4. Over 70%
of the isolates grew well in pH 6 showing a neutral and base-tolerant tendency. This tendency might be related
to the basic pH that characterizes most of the origin soil from which the isolates were recovered. As shown in
Otieno et al. (2017) 4(1): 183191
(Table 2), the isolates exhibited a wide diversity in their salt tolerance. The salt inhibitory concentration varied
among strains. There was tolerance to low salt levels but the percentage of tolerant strains decreased rapidly and
only a few of the strains showed moderate growth at pH 5. All the isolates performed well across the zones in
collected from tortilis zone (TZPJ).
Isolate
TZPJ-1 - + + + + + + + + + + + - - + + -
TZPJ-2 + + + + + + + + + + + + - - + + -
TZPJ-3 - - - - - - - - - - - - - - - - -
TZPJ-4 - + + + + + + + + + + - - - - + -
TZPJ-5 + + + + + + + + + + + + - - - + -
TZPJ-6 - + + - + + - - - - - - - - - - -
TZPJ-7 - + + + + + + + + + + + + + + + +
TZPJ-8 + + + + + + + + + + + + - - + + -
TZPJ-9 - + + + + + + - - + + + - - - + -
TZPJ-10 - + + + + + + - - + + - - - - + -
TZPJ-11 + + + + + + + + + + + + - - - + -
TZPJ-12 + + + + + + + - - - + - - - - + -
TZPJ-13 + + + + + + + + + + + + - - + + -
TZPJ-14 + + + + + + + + + + + - - - - + -
TZPJ-15 - + + + + + + - + + + - - - - + -
TZPJ-16 - + + + + + + - - + + - - - - - -
TZPJ-17 - + + + + + - - - - - - - - - - -
TZPJ-18 - + + - - - - - - - - - - - - - -
TZPJ-19 - + + + + + + + + + + - - - - + -
TZPJ-20 - + + + + + - - - - - - - - - - -
Note: +,-, strains were positive and negative, respectively.
collected from prosopis zone (PZPJ).
Isolate
PZPJ-1 + + + + - + + + + + + + + + + + +
PZPJ-2 + + + + + + + + + + + + + + + + +
PZPJ-3 + + + + + + + + + + + + + + + + +
PZPJ-4 + - + + - - + + + + + + + + + + +
PZPJ-5 + + + + + + + + + + + - - - - - -
PZPJ-6 + + + + + + - - - - - - - - - - -
PZPJ-7 + + + + + + + - - + + - - - - - -
PZPJ-8 - + + + + + + - - + - - + - - + -
PZPJ-9 - + + + + + + + + + + - - - + - -
PZPJ-10 - - + + - - - - - - - - - - - - -
PZPJ-11 - + + + + + + + + + + + - + - - +
PZPJ-12 - + + + + + + - - - - - - - - - -
PZPJ-13 - + + + + + + - - + + + - - - - -
PZPJ-14 - + + + + + + - - - - - - - - + -
PZPJ-15 + + + + + + + + + + + + - - + + -
PZPJ-16 - + + + - - + - - - - - - - - + -
PZPJ-17 + + + + + + + + - + + + - + - - -
PZPJ-18 + + + + + + + - - - - - - - - + -
PZPJ-19 - + + + + + + - - + + + - - - + -
PZPJ-20 + + + + + + + + + + + + + + + + -
Note: +,-, strains were positive and negative, respectively
terms of tolerance to salt. This result confirms a selection pressure for tolerance to salinity. The evaluation of
intrinsic resistance to antibiotics (IAR) showed that most of the isolates (67%) exhibited high resistance to
tetracycline (50 g.ml-1) and (50%) to ampicillin (20 g.ml-1) table 3. In the presence of nalidixic acid (20
g.ml-1) kanamycin (100 g.ml-1), rifamycin (50 g.ml-1) and streptomycin (200 g.ml-1), only 10% to 40% of
Otieno et al. (2017) 4(1): 183191
the isolates were resistant. All isolates from Grass zone were susceptible to nalidixic acid (20 g.ml-1) and
kanamycin (100 g.ml-1) except a few which were isolated from Prosopis zone and Tortilis zone (Table 3). In
terms of zones Prosopis had the highest number (38%) of strains which showed resistance to most of the
antibiotics followed by Tortilis zone (23%) and Grass zone was (18%). Cluster analysis grouped the 60 strains
by genomic similarity and resulted in the dendrogram as shown in (Figs. 13). The dendrogram are further
divided into sub-clusters of 6 in the Prosopis zone, 5 in Tortilis zone and 5 in grass zone and some isolates
without any grouping thus indicating that these isolates are probably unique and can be identified based on
16SrRNA gene sequencing technique.
Pj GZ -1
Pj GZ -4
Pj GZ -2
Pj GZ -8
Pj GZ -10
Pj GZ -19
Pj GZ -11
Pj GZ -12
Pj GZ -6
Pj GZ -13
Pj GZ -14
Pj GZ -15
Pj GZ -16
Pj GZ -3
Pj GZ -7
Pj GZ -9
Pj GZ -20
Pj GZ -5
Pj GZ -17
Pj GZ -18
1. 0 0. 9 0. 8 0. 7 0. 6 0. 5 0. 4
Figure 1. Dendrogram showing phenotypic relatedness based on Intrinsic Antibiotic resistance (IAR), pH and salt tolerance
among 20 rhizobial isolates from Grass zone (PjGZ). [Cluster analysis was performed using the Unweighted Paired Group
with Arithmetic Average (UPGMA)]
5
Pj TZ -1
Pj TZ -2
Pj TZ -8
Pj TZ -13
Pj TZ -5
Pj TZ -11
Pj TZ -4
Pj TZ -19
Pj TZ -15
Pj TZ -14
Pj TZ -9
Pj TZ -10
Pj TZ -16
Pj TZ -12
Pj TZ -7
Pj TZ -3
Pj TZ -18
Pj TZ -6
Pj TZ -17
Pj TZ -20
1. 0 0. 9 0. 8 0. 7 0. 6 0. 5
among 20 rhizobial isolates from Tortilis zone (PjTZ). [Cluster analysis was performed using the Unweighted Paired Group
Otieno et al. (2017) 4(1): 183191
Pj PZ-1
Pj PZ-2
Pj PZ-3
Pj PZ-20
Pj PZ-4
Pj PZ-5
Pj PZ-9
Pj PZ-15
Pj PZ-11
Pj PZ-17
Pj PZ-6
Pj PZ-12
Pj PZ-14
Pj PZ-18
Pj PZ-8
Pj PZ-7
Pj PZ-13
Pj PZ-19
Pj PZ-10
Pj PZ-16
1. 0 0. 9 0. 8 0. 7 0. 6 0. 5
among 20 rhizobial isolates from Prosopis zone (PjPZ). [Cluster analysis was performed using the Unweighted Paired Group
CONCLUSION
Our findings indicate that, like many other plant-mutualism interactions, native legume rhizobium
interactions are impacted by the presence of invasive species in communities. This study, therefore, provides the
basis for further research on the phylogeny of rhizobium strains nodulating Prosopis juliflora as well as their use
as inoculants to improve growth and nitrogen fixation in arid lands. Since no inoculation studies have been
carried out to experimentally measure the extent to which invasive legumes may be symbionts limited in
different sites within their natural range. Continued efforts should be made to understand the complex
association between the invasive plant species and the symbiotic partners and ultimately to employ efficient
strains in the sustainable agricultural practice.
ACKNOWLEDGEMENTS
The authors wish to thank the Director, Kenya Forestry Research Institute, for providing the infrastructure
facilities and support. This work was supported by funds from EU Marie Curie fellowsip Contract MIIFR-CT-
2006-980056. Technical assistance of James Were of KEFRI Biotechnology laboratory in conducting the
experiments is duly acknowledged.
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