Volume 4, Issue 3 (2017) Tropical Plant Research

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

4(3): 363–375, 2017
DOI: 10.22271/tpr.2017.v4.i3.048
Research article
Phytoecological study of Nzundu massif forest of Imbongo city,

Kwilu Province, Democratic Republic of the Congo
Y. B. da-Musa Masens1*, Koto-te-Nyiwa Ngbolua1,2, Mandung Masens3,
Tembeni M. Tambu1 and Ngiala Bongo Gédéon1
1
Faculty of Science, University of Kinshasa, Kinshasa XI, Kinshasa, Democratic Republic of the Congo
2
University of Gbadolite, Gbadolite, Province of Nord-Ubangi, Democratic Republic of the Congo
3
Faculty of Science, University of Kisangani, Kisangani, Democratic Republic of the Congo
*Corresponding Author: gedeonbongo@gmail.com [Accepted: 15 September 2017]
Abstract: Formerly, the Nzundu forest massif covered almost the two third of Kwenge and
Imbongo, and a part of Kipuka districts. Nowadays, it is represented by a reduced forest fragment
with a surface of about 50 to 70 hectares. The flora inventory was performed on the forest trees at
dbh ≥ 10 cm, measured at 1.30 m of high at the breast height trunk, allowed to identify 134
different kinds of plants divided into 109 genera and 31 families. Fabaceae, Malvaceae,
Rubiaceae, Euphorbiaceae, Moraceae, Meliaceae and Sapotaceae are the most represented
families. The value of the basal area got in this forest massif is high or 49.89 m 2.ha-1. As to the
ecological spectra and phytogeographical distribution, mesophanerophytes, sarcochores,
mesophyles and the Congolian-Guinea element are the most numerous. The density of the forest
trees listed in this forest massif is of 422 trunks.ha -1. Brachystegia laurentii is the species having
the highest number of feet or 11.8%. The values of Shannon and of Equitability indices calculated
are of 4.7 and 0.9 respectively.
Keywords: Forest ecology - Life forms - Diaspores - Foliar types - Phytogeography.
[Cite as: Masens da-Musa YB, Ngbolua K-t-N, Masens M, Tambu TM & Gédéon NB (2017) Phytoecological
study of Nzundu massif forest of Imbongo city, Kwilu Province, Democratic Republic of the Congo. Tropical
Plant Research 4(3): 363–375]
INTRODUCTION
The Nzundu forest massif is about ±8 km southeast to Kwilu Bridge on the Kikwit-Mukulu road
(Batshamba). The massif in question is derived from a dense forest of Guinean mesophilic semi-deciduous and
peri-Guinean mesophilic type predominantly in Brachystegia laurentii Louis ex Hoyle. Formerly, this forest
covered the low slopes of the left slope overlooking Kwilu River. In the past, it extended between Kwenge
sector and Kimbinga Makoloninga village in Imbongo district (Nicolaï 1963).
Currently, this large forest has been reduced to a forest massif, now subjected to an intense human activity
(as it was the case with the large forest from which it was derived) because of its proximity not only to Kikwit
city with a population estimated at more than 400,000 inhabitants and a majority of small farmers but also to
surrounding villages such as Giguidji, Mbundi, Kianga, Kakoy and Mbamba. Its maintenance is only due to its
erection as a private farm. Due to the anthropic pressure experienced by this massif, its surface is daily more and
more reduced; hence, the need of undertaking a phytoecological inventory. In order to preserve, protect and
better manage it, a thorough study of its various components (flora, specific diversity, structure, etc.) is
necessary. This concern integrated into the process (REDD +).
That is the reason why this phytoecological study was undertaken. Different parameters considered in this
survey were: structure, basal area, density, dominance, frequency as well as ecological and phytogeographic
spectra. This survey is significant especially for ecologists at this time where climate change is a global concern.
In fact, this massif forest with its florisitic cortege plays somewhat a role in the regulation of temperature which
exceeds more than 2°C according to many specialists. Data used to write this paper are based on observations
www.tropicalplantresearch.com 363
Received: 13 June 2017 Published online: 30 September 2017
https://doi.org/10.22271/tpr.2017.v4.i3.048
Masens et al. (2017) 4(3): 363–375
made between 2010 and 2012 in a series of studies carried out in various forested areas located in the interland
of Kikwit city following a degradation to which they are currently subjected.
MATERIAL AND METHODS

Study area
The Nzundu forest massif is located between Giguidzi-Mbundi and Kianga villages in Imbongo district. It is
at least 8 km far from Kwilu Bridge and 3 km upstream of Mwebe bridge (Fig. 1). Physically, this forest massif
which already undergoes aggressions from villagers and the population of the semi-rural of Kikwit city is
dominated by Brachystegia laurentii Louis ex Hoyle along with Pterocarpus mildbraedii Harms.
Figure 1. Map showing the location of study area.

Data sampling and analysis
A 1 Km long transect (divided into two sections of 10 m large and 500 m long) was drawn along which, dbh
≥ 10 cm, of trees were measured on both stretches. All species with dbh ≥ 10 cm were inventoried within each
delimited portion. The identification of collected specimens was performed using the flora of Central Africa
(consisting of 10 volumes and numerous fascicules) and in comparison with concerned herbarium specimens at
the Herbarium of the Department of Biology (Faculty of Sciences, University of Kinshasa).
Arborescent tree density was evaluated for all trees having a diameter of 1.30 m at breast height >10 cm,
present in various surveys. Basal area (BA), which is the sum of the sections of trunks at 1.30 cm above the
ground or 30 cm above tree spur, expressed in m2/ha, was calculated for all trees having a breast height diameter
(dbh) >10 cm, according to the following formula:
(1)
The total basal area of phytocenosis and/or any forest is obtained from the sum of areas of all trees of which
dbh is greater than a given value (Shaumba et al. 2016). In the current study, all trees of dbh >10 cm were
considered.
The circumference of trees was directly measured by considering the cylindrical or conical drums. Using the
following mathematical formula, different values of dbh was deduced,
C = π x d (2),
Masens et al. (2017) 4(3): 363–375
Henceforth,
(3)
Where, C = circumference and d: diameter
Relative dominance (expressed in percentage) was calculated using the formula:
(4)
Where, BA= basal area.
We used the following formula to calculate the relative Frequency:
(5)
Where, b = number of feet / ha for each species; bt = total number of individuals inventoried.
Concerning the relative importance (h) of various species, the following formula was used:
(6)
where, e = relative density; f = relative dominance and g = relative frequency.
As for the diversity of flora, we used the Shannon diversity index for its interpretation:
ISH = -Σpi ln pi (7)
This formula was given by PAST software where: pi, effective species, i and n total number of species.
Equitability (EQ) was established according to the following mathematical relationship:
(8)
+
Where, H = regularity and Hmax = maximum diversity.
Concerning diameter classes, the diameters of all species at dbh ˃ 10 cm, present in the various surveys,
were measured using a tape measure and grouped into different classes. Different eco-sociological groups were
distinguished on the basis of works of numerous authors (Masens 1997, Shaumba et al. 2017). The types of
foliar dimensions were defined from the Raunkiaer classification (Habari 2009, Belesi 2009). For the types of
phytogeographical distribution, we focused on the major chorological subdivisions of Africa (White 1983,
1992). As for the life forms (LF), we used the Raunkiaer classification adapted to the tropical regions (Habari
2009, Belesi 2009). As far as the types of dissemination of diaspores are concerned, we were inspired by the
works of Masens (1997).
RESULTS
Phytodiversity measure
The areal richness of Nzundu forest massif of dbh ≥10 cm species measured at 1.30 m breast height is of 134
woody plant species. Of these, five species have a dbh >70 cm; these are Brachystegia laurentii Louis ex Hoyle
(1.045 m), Baillonella toxisperma Stone (0.757 m), Bosqueiopsis gilletii De Wild. & Th. Dur. (0.757 m), Khaya
anthotheca (Welw.) C. DC. (0.717 m) and Antiaris toxicaria Lesch. (0.713 m). Out of these five species, two
belong to Moraceae family (Table 1). These 134 species are grouped in 31 families and 109 genera. The
following families are best supplied with species: Fabaceae (30 species), Malvaceae (12 species), Rubiaceae
(11 species), Euphorbiaceae (9 species), Moraceae (8 species), Sapotaceae, Annonaceae and Clusiaceae with 6
species each. These families alone account for 66.4% of all the species of the studied flora. As for the 23 other
families, they contain 33.5% of the total species inventoried in this forest massif. Of these, 16 families are
monospecific, representing 11.9% out of the total.
Table 1. Floristic inventory of Nzundu massif forest (Values of different structural parameters).
Plant species Family A B C D E F G H
Allophylus africanus P. Beauv. Sapindaceae 1 2 0.138 0.030 1.48 0.06 0.64 1.75
Amphimas pterocapoides Harms Fabaceae 2 4 0.320 0.321 2.96 0.63 1.28 1.62
Angylocalyx pynaertii De Wild. Fabaceae 2 4 0.302 0.287 2.96 0.06 1.28 1.43
Anisophyllea polynera Floret Anisophylleaceae 1 2 0.243 0.093 1.48 0.19 0.64 1.88
Anonidium mannii (Oliv.) Engl. & Diels Annonaceae 2 4 0.245 0.188 2.96 0.37 1.28 1.54
Masens et al. (2017) 4(3): 363–375
Anthonotha fragrans (Bak.f) Exell et Hill. Fabaceae 1 2 0.460 0.333 1.48 0.65 0.64 0.92
Anthrocaryon klaineana Pierre Annonaceae 1 2 0.426 0.285 1.48 0.56 0.64 0.89
Antiaris toxicaria Lesch. Moraceae 1 2 0.713 0.798 1.48 1.56 0.64 1.23
Antidesma sp. Euphorbiaceae 1 2 0.239 0.089 1.48 0.18 0.64 0.78
Antidesma vogelianum Müll.-Arg. Euphorbiaceae 1 2 0.484 0.367 1.48 0.72 0.64 0.95
Aphanocalyx margininervatus J.Leonard Fabaceae 2 4 0.566 1.007 2.96 1.97 1.28 2.07
Autranella congolensis (De Wild.) A. Chev. Sapotaceae 2 4 0.607 1.156 2.96 2.27 1.28 2.17
Baillonella toxisperma Pierre Sapotaceae 2 4 0.757 1.803 2.96 3.53 1.28 2.59
Berlinia grandiflora (Vahl.) Mikh. et Dalz. Fabaceae 1 2 0.426 0.285 1.48 0.56 0.64 0.89
Blighia unijugata Bak. Sapindaceae 1 2 0.350 0.192 1.48 0.38 0.64 0.83
Blighia welwitschii (Hiern) Radlk. Sapindaceae 2 4 0.413 0.537 2.96 1.05 1.28 1.77
Bosqueiopsis gilletii De Wild. & Th. Dur. Moraceae 2 4 0.757 1.801 2.96 3.53 1.28 2.59
Brachystegia laurentii Louis ex Hoyle Fabaceae 8 16 1.045 13.739 11.85 26.90 5.13 14.63
Canarium schweinfurthii Engl. Burceraceae 2 4 0.426 0.577 2.96 1.13 1.63 1.91
Carapa grandiflora Sprague Meliaceae 1 2 0.630 0.624 1.48 1.22 0.64 1.11
Carapa procera DC. var. procera DC. Meliaceae 1 2 0.331 0.172 1.48 0.34 0.64 0.82
Celtis adolfi-friderici Engl. Ulmaceae 1 2 0.552 0.479 1.48 0.94 0.64 1.02
Celtis durandii Engl. Ulmaceae 2 4 0.502 0.794 2.96 1.55 1.28 1.93
Celtis mildbraedii Engl. Ulmaceae 2 4 0.328 0.337 2.96 0.66 1.28 1.64
Celtis zenkeri Engl. Ulmaceae 2 4 0.300 0.284 2.96 0.56 1.28 1.60
Chrysophyllum lacourtianum De Wild. Sapotaceae 2 4 0.405 0.517 2.96 1.01 1.28 1.75
Coelocaryon botryoides Verm. Myristicaceae 1 2 0.178 0.049 1.48 0.10 0.64 0.74
Cola chlamydantha K.Schum. Malvaceae 1 2 0.280 0.123 1.48 0.24 0.64 0.79
Cola diversifolia De Wild. Malvaceae 1 2 0.111 0.019 1.48 0.04 0.64 0.72
Cola gigantea A. Chev. Malvaceae 1 2 0.292 0.133 1.48 0.26 0.64 0.79
Cola lateritia K. Schum. Malvaceae 2 4 0.128 0.051 2.96 0.10 1.28 1.14
Cola urceolata K. Schum. Malvaceae 1 2 0.283 0.125 1.48 0.25 0.64 0.79
Copaifera mildbraedii Harms Malvaceae 1 2 0.175 0.048 1.48 0.09 0.64 0.55
Corynanthe pachycera K. Schum. Rubiaceae 2 4 0.227 0.162 2.96 0.32 1.28 1.52
Crudia gabonensis Pierre ex Harms Fabaceae 1 2 0.280 0.123 1.48 0.24 0.64 0.79
Cynometra hankei Harms Fabaceae 2 4 0.248 0.193 2.96 0.38 1.28 1.54
Daniellia pynaertii De Wild. Fabaceae 1 2 0.452 0.321 1.48 0.63 0.64 0.92
Desplatsia chrysochlamys (Mildbr. & Bur,) Mildbr. Malvaceae 2 4 0.201 0.127 2.96 0.25 1.28 1.50
Detarium macrocarpum Harms Fabaceae 2 4 0.581 1.599 2.96 3.13 1.28 2.59
Dialium corbisieri Harms Fabaceae 1 2 0.414 0.269 1.48 0.53 0.64 0.88
Dialium pachyphyllum Harms Fabaceae 1 2 0.286 0.128 1.48 0.25 0.64 0.74
Dialium pentandrum Louis ex Stey. Fabaceae 1 2 0.343 0.185 1.48 0.36 0.64 0.83
Dialium tessmannii Harms Fabaceae 3 6 0.289 0.394 4.44 0.77 1.92 2.38
Dichostemma glauscens Pierre Euphorbiaceae 1 2 0.239 0.089 1.48 0.18 0.64 0.77
Diospyros canaliculata De Wild. Ebenaceae 1 2 0.132 0.027 1.48 0.05 0.64 0.73
Duboscia viridiflora (K. Schum.) Mildbr. Malvaceae 3 6 0.217 0.223 4.44 0.44 1.92 2.27
Enantia chloranta Oliv. Annonaceae 1 2 0.229 0.082 1.48 0.16 0.64 0.76
Entandrophragma angolense (Welw.) C. DC. Meliaceae 3 6 0.299 0.422 4.44 0.83 1.92 2.40
Entandrophragma utile Sprague Meliaceae 2 4 0.518 0.844 2.96 1.65 1.28 1.97
Masens et al. (2017) 4(3): 363–375
Erismadelphus exsul Mildb. Vochysiaceae 2 4 0.237 0.177 2.96 0.35 1.28 1.53
Erythrophleum suaveolens (Guill. & Perrot) Brenan Fabaceae 1 2 0.595 0.556 1.48 1.09 0.64 1.07
Fernandoaa dolfi-frederici (Gilg & Mildbr.) Heine Bignionaceae 1 2 0.304 0.145 1.48 0.29 0.64 0.80
Ficus lutea Vahl Moraceae 1 2 0.213 0.074 1.48 0.14 0.64 0.76
Ficus sur Welw. ex Ficalho Moraceae 1 2 0.232 0.084 1.48 0.17 0.64 0.76
Ficus thonningii Blume Moraceae 1 2 0.201 0.063 1.48 0.12 0.64 0.75
Filaeopsis discophora Harms Fabaceae 1 2 0.356 0.199 1.48 0.39 0.64 0.84
Funtumia elastica (Preuss) Stapf Apocynaceae 1 2 0.213 0.071 1.48 0.14 0.64 0.75
Gaertnera parvipaniculata Petit Rubiaceae 1 2 0.201 0.063 1.48 0.12 0.64 0.75
Ganophyllum giganteum (A. Chev.) Hauman Sapindaceae 2 4 0.426 0.571 2.96 1.12 1.28 1.79
Garcinia epunctata Stapf Clusiaceae 1 2 0.264 0.109 1.48 0.21 0.64 0.78
Garcinia kola Heckel Clusiaceae 2 4 0.491 0.758 2.96 1.49 1.28 1.91
Garcinia punctata Stapf Clusiaceae 2 4 0.283 0.252 2.96 0.49 1.28 1.58
Gilbertiodendron dewevrei (De Wild.) J. Léonard Fabaceae 2 4 0.283 0.252 2.96 0.49 1.28 1.58
Greenwayadendron suaveolens (Engl. & Diels) Verdc. Annonaceae 1 2 0.439 0.303 1.48 0.59 0.64 0.91
Grewia oligoneura Sprague Malvaceae 1 2 0.178 0.049 1.48 0.10 0.64 0.74
Guarea cedrata (A. Chev.) Pellerg. Meliaceae 1 2 0.277 0.120 1.48 0.24 0.64 0.79
Guibourtia tessmannii (Harms) J. Léonard Fabaceae 1 2 0.286 0.128 1.48 0.25 0.64 0.79
Hannoa klaineana Pierre Simaroubaceae 2 4 0.366 0.421 2.96 0.82 1.28 1.69
Homalium africanum Mast. Santalaceae 1 2 0.416 0.271 1.48 0.53 0.64 0.88
Hua gabonii De Wild. Huaceae 1 2 0.264 0.109 1.48 0.21 0.64 0.78
Hugonia platysepala WelW. ex Oliv. Hugoniaceae 1 2 0.165 0.043 1.48 0.08 0.64 0.74
Khaya anthotheca (Welw.) C. DC. Meliaceae 1 2 0.717 0.807 1.48 1.58 0.64 1.23
Lannea welwitschii (Hiern) Engl. Santalaceae 2 4 0.173 0.094 2.96 0.18 1.28 1.48
Lovoa trichiloides Harms Meliaceae 2 4 0.283 0.252 2.48 0.49 1.28 1.09
Maesobotrya floribunda Benth. Euphorbiaceae 1 2 0.136 0.029 1.48 0.06 0.64 0.73
Mammea africana Sabine Clusiaceae 1 2 0.254 0.102 1.48 0.20 0.64 0.77
Manilkara fouilloyana Aubr. Ex Pellegr. Sapotaceae 1 2 0.214 0.072 1.48 0.14 0.64 0.75
Manilkara koechlinii Aubr. Sapotaceae 1 2 0.242 0.091 1.48 0.18 0.64 0.77
Maranthes chrysophylla (Oliv.) France ex F. While Sapotaceae 2 4 0.120 0.045 2.96 0.09 1.28 1.44
Massularia acuminata (G. Don) Hoyle Rubiaceae 1 2 0.238 0.089 1.48 0.17 0.64 0.77
Memecylon leucocarpum Gilg. Melastomataceae 1 2 0.144 0.032 1.48 0.06 0.64 0.73
Micrococca mercurialis (L.) Benth. Euphorbiaceae 2 4 0.114 0.041 2.96 0.08 1.28 1.44
Milicia excelsa (Welw.) Berg. Moraceae 2 4 0.343 0.370 2.96 0.72 1.28 1.66
Millettia sapinii De Wild. Fabaceae 1 2 0.242 0.091 1.48 0.18 0.64 0.77
Monodora myristica (Gaertn.) Dunal Annonaceae 1 2 0.241 0.091 1.48 0.18 0.64 0.77
Monopetalanthus pteridophyllus Harms Fabaceae 1 2 0.515 0.417 1.48 0.82 0.64 0.98
Nesogordonia kabingensis (K. Schum.) Cap. Malvaceae 1 2 0.394 0.244 1.48 0.48 0.64 0.87
Nesogordonia papaverifera (A. Chev.) Capur. Malvaceae 2 4 0.281 0.247 2.96 0.49 1.28 1.58
Omphalocarpum procerum P. Beauv. Sapotaceae 1 2 0.278 0.121 1.48 0.24 0.64 0.79
Ongokea gore (Hua) Pierre Olacaceae 2 4 0.146 0.067 2.96 0.13 1.28 1.46
Oxyanthus speciosus DC. Rubiaceae 1 2 0.222 0.078 1.48 0.15 0.64 0.76
Panda oleosa Pierre Pandaceae 1 2 0.312 0.152 1.48 0.30 0.64 0.81
Paramacrolobium coeruleum (Taub.) J. Léonard Fabaceae 1 2 0.496 0.387 1.48 0.76 0.64 0.96
Masens et al. (2017) 4(3): 363–375
Parinari excelsa Sabine Chrysobalanaceae 3 6 0.232 0.254 4.44 0.50 1.92 2.29
Parkia filicoidea Welw. ex Oliv. Fabaceae 1 2 0.213 0.071 1.48 0.14 0.64 0.75
Pausinystalia macrocera (K. Schum.) Pierre ex Dup. Rubiaceae 1 2 0.280 0.123 1.48 0.24 0.64 0.79
Pavetta sp. Rubiaceae 1 2 0.144 0.032 1.48 0.06 0.64 0.73
Penianthus preussii Miers Menispermaceae 1 2 0.159 0.039 1.48 0.08 0.64 0.73
Pentacletra macrophylla Benth. Fabaceae 1 2 0.204 0.065 1.48 0.13 0.64 0.75
Pentadesma butyracea Sabine Clusiaceae 2 4 0.248 0.193 2.96 0.38 1.28 1.54
Pentadesma excelliana Staner Clusiaceae 2 4 0.343 0.369 2.96 0.72 1.28 1.66
Petersianthus macrocarpus (Beauv.) Liben Lecytidaceae 3 6 0.284 0.380 4.44 0.75 1.92 2.37
Piptadeniastrum africanum (Hook.) Brenan Fabaceae 3 6 0.456 0.979 4.44 1.92 1.92 2.76
Plagiostyles africana (Müll.-Arg.) Prain. Euphorbiaceae 2 4 0.206 0.134 2.96 0.26 1.28 1.50
Prioria balsaminfera (Vermoesen) Breteler Fabaceae 4 8 0.326 0.670 5.93 1.31 2.56 3.27
Psydrax arnoldiana (De Wild.) Brid. Rubiaceae 2 4 0.243 0.186 2.96 0.36 1.28 1.54
Pterocarpus mildbraedii Harms Fabaceae 5 10 0.180 0.255 7.41 0.50 3.21 3.70
Pterocarpus soyauxii Taub. Fabaceae 1 2 0.267 0.112 1.48 0.22 0.64 0.78
Pterocarpus tinctorius Welw. Fabaceae 2 4 0.487 0.745 2.96 1.46 1.28 1.90
Pycnanthus angolensis (Welw.) Ekell. Myristicaceae 3 6 0.192 0.173 4.44 0.34 1.92 2.24
Quassia africana (Baill.) Baill. Simaroubaceae 2 4 0.130 0.053 2.96 0.10 1.28 1.45
Ricinerodendron heudelotii (Baill.) Pierre ex Heckel Euphorbiaceae 1 2 0.175 0.048 1.48 0.09 0.64 0.74
Sacoglottis gabonensis (Baill.) Urb. Humiriaceae 2 4 0.426 0.571 2.96 1.12 1.28 1.79
Santiria trimera (Oliv.) Aubr. Burceraceae 1 2 0.114 0.020 1.48 0.04 0.64 0.72
Sarchocephalus diderichii (De Wild.) Rubiaceae 1 2 0.191 0.057 1.48 0.11 0.64 0.74
Sarchocephalus pobenguini (Hua ex Pob.) Merril. Rubiaceae 1 2 0.199 0.062 1.48 0.12 0.64 0.75
Schrebera arborea A. Chev. Oleaceae 1 2 0.235 0.087 1.48 0.17 0.64 0.76
Schumanniophyton magnificum (K. Schum.) Harms Rubiaceae 2 4 0.246 0.190 2.96 0.37 1.28 1.54
Scorodophoeus zenkeri Harms Fabaceae 2 4 0.430 0.583 2.96 1.14 1.28 1.80
Scyphocephalium mannii (Benth. & Hook. f.) Warb. Myristicaceae 1 2 0.200 0.062 1.48 0.12 0.64 0.75
Spondianthus preussi Engl. Euphorbiaceae 1 2 0.197 0.061 1.48 0.12 0.64 0.75
Staudtia kamerunensis Warb. Myristicaceae 4 8 0.204 0.026 5.93 0.51 2.56 3.00
Sterculia tragacantha Lindl. Malvaceae 1 2 0.477 0.358 1.48 0.70 0.64 0.94
Strombosia grandiflora Hook.f. Olacaceae 2 4 0.191 0.057 2.96 0.11 1.28 1.45
Strombosia majuscula Hook. f. Olacaceae 2 4 0.334 0.350 2.96 0.69 1.28 1.64
Synsepalum stipulatum De Wild. Sapotaceae 3 4 0.235 0.173 2.96 0.34 1.92 1.74
Tetrapleura tetraptera (Schum. & Thon.) Taub. Fabaceae 1 2 0.191 0.057 1.48 0.11 0.64 0.74
Treculia africana Decne Moraceae 1 2 0.181 0.051 1.48 0.10 0.64 0.74
Trichalisia crepiniana De Wild. & Th. Dur. Rubiaceae 1 2 0.181 0.051 1.48 0.10 0.64 0.74
Trichilia welwitschii C. DC. Meliaceae 2 4 0.178 0.099 2.96 0.20 1.28 1.48
Trilepisium madagascariense DC. Moraceae 2 4 0.175 0.096 2.96 0.19 1.28 1.48
Uapaca sp. Euphorbiaceae 1 2 0.211 0.070 1.48 0.14 0.64 0.75
Xylopia chrysophylla Louis ex Bout. Annonaceae 1 2 0.121 0.022 1.48 0.04 0.64 0.72
Zanthoxylum leprieuri Guill. & Perr. Rutaceae 1 2 0.168 0.044 1.48 0.09 0.64 0.74
Note: A = total number of trunks in transect for each species; B = number of trunks/hectares for each species, or a × 2; C = average dbh
(m); D = basal area (m2); E = relative density (%); F = relative dominance (%); G = relative frequency (%); H = relative importance (%).
Masens et al. (2017) 4(3): 363–375
Basal area
The basal area values of trunk sections at dbh ≥10 cm, calculated over a total surface of 1 ha are 49.89 m2.
The most important basal area values were obtained from the following woody species: Brachystegia laurentii
Louis ex Hoyle (13.739 m2), Baillonella toxisperma Pierre (1.803 m2), Bosqueiopsis gilletii De Wild. & Th.
(1.801 m2), Detarium macrocarpum Harms (1.599 m2), Autranella congolensis (De Wild.) A. Chev. (1.156 m2)
and Aphanocalyx margininervatus J.Leonard (1.007 m2). The lowest basal area values were observed in five
plant species amongst which are Cola diversifolia De Wild., Staudtia kamerunensis Warb. and Xylopia
chrysophylla Louis ex Bout 0.019, 0.026 and 0.022 m2.ha-1 (Table 1). Altogether, the basal area of the upper
structural unit is of 26.37 m2.ha-1 while that of the low and medium structural units is of 23.46 m2.ha-1.
The density of taxa
The density of woody species in the Nzundu forest massif is of 422 feet per ha. This set is distributed in 366
stems.ha-1 for species with dbh ≤49.9 cm and 56 trees for species with dbh ≥50 cm (including 5 to dbh >70 cm).
The highest relative density values were obtained from the following species: Brachystegia laurentii Louis ex
Hoyle (11.85%), Pterocarpus mildbraedii Harms (7.41%) and Prioria balsaminfera (Vermoesen) Breteler
(5.93%). Seven other species have moderately high relative density values.
Emergent trees at dbh >70 cm, the most numerous are Brachystegia laurentii Louis ex Hoyle (16 trunk.ha-1),
Pterocarpus mildbraedii Harms (10 trunk.ha-1), Prioria balsaminfera (Vermoesen) Breteler and Staudtia
kamerunensis Warb. having 8 trunk.ha-1 each. The following families have a large number of stems/ha:
Fabaceae (110 trunk.ha-1), Malvaceae (34 trunk.ha-1), Rubiaceae and Sapotaceae each with 28 trunk.ha-1,
Meliaceae Clusiaceae and Euphorbiaceae with 20 trunk.ha-1 each, Myristicaceae (18 trunk.ha-1), Annonaceae
and Ulmaceae with 14 trunk.ha-1 each (Table 1).
Relative dominance
The highest values obtained with respect to relative dominance for dbh ≥10 cm species measured at 1.30 m
were observed in four species, Brachystegia laurentii Louis ex Hoyle (26.90%), Baillonella toxisperma Stone
(3.53%), Bosqueiopsis gilletii De Wild. & Th. (3.53%), Detarium macrocarpum Harms (3.13%) and Autranella
congolensis (De Wild.) A. Chev. (2.27%) (Table 1).
Relative frequency
As for the relative frequency calculated for species with dbh ≥10 cm, the most important values obtained are
for the following species: Brachystegia laurentii Louis ex Hoyle (5.13%), Prioria balsaminfera (Vermoesen)
Breteler (2.56%), Pterocarpus mildbraedii Harms (3.21%) and Staudtia kamerunensis Warb. (2.56%) (Table 1).
Relative importance
The highest values of relative importance, calculated for dbh ≥10 cm, were observed in four species
(Brachystegia laurentii Louis ex Hoyle - 14.63%, Pterocarpus mildbraedii Harms - 3.70%, Prioria
balsaminfera (Vermoesen) Breteler - 3.27% and Staudtia kamerunensis Warb. – 3.00%) and the lowest in one
species (Copaifera mildbraedii Harms with 0.55%).
Distribution of the frequencies of different classes of diameters and the trend of the basal area
Figure 2. Shows the trunk distribution by diameter classes and the basal area values obtained for each class.
Masens et al. (2017) 4(3): 363–375
The considered diameter classes (grouped from table 1), collect the values in dbh groups of 20 cm intervals,
i.e. 10–29.9 cm; 30–49.9 cm; 50–69.9 cm; >70 cm. Fig. 2 shows that the curve of the species obtained has a
hyperbolic appearance with a decrease in the number of trunks for the high dbh classes. The curve representing
the trend of the basal area as a function of the diameter classes has a curvilinear shape. It is observed that the
highest values of this basal area were obtained in the emergent and the smallest in the dbh range between 10 and
29.9 cm.
Ecological spectra
Table 2. Ecological spectra, phytogeographical types and phytosociological status of listed species.
Plant species Family LF PhgD DTD TFD PhSt
Allophylus africanus P. Beauv. Sapindaceae MsPh At Sar Me H
Amphimas pterocapoides Harms Fabaceae MgPh GC Bal Me SP
Angylocalyx pynaertii De Wild. Fabaceae MsPh GC Bal Me SP
Anisophyllea polynera Floret Rhizophoraceae MsPh CG Sar Me SP
Anonidium mannii (Oliv.) Engl. & Diels Annonaceae MsPh CG Sar Me SP
Anthonotha fragrans (Bak.f) Exell et Hill. Fabaceae MsPh CG Bal Me SP
Anthrocaryon klaineana Pierre Anacardiaceae MgPh CG Sar Me SP
Antiaris toxicaria Lesch. Moraceae MgPh GC Sar Me MT
Antidesma sp. Euphorbiaceae MsPh - Sar Me SP
Antidesma vogelianum Müll.-Arg. Euphorbiaceae MsPh At Sar Me SP
Aphanocalyx margininervatus J.Leonard Fabaceae MsPh CG Bal Me SP
Autranella congolensis (De Wild.) A. Chev. Sapotaceae MsPh At Sar Me SP
Baillonella toxisperma Pierre Meliaceae MgPh CG Sar Me SP
Berlinia grandiflora (Vahl.) Mikh. et Dalz. Fabaceae MsPh GC Bal Me SP
Blighia unijugata Bak. Sapindaceae MsPh At Sar Me SP
Blighia welwitschii (Hiern) Radlk. Sapindaceae MgPh GC Sar Me SP
Bosqueiopsis gilletii De Wild. & Th. Dur. Moraceae MsPh GC Sar Me SP
Brachystegia laurentii Louis ex Hoyle Fabaceae MgPh CG Bal Me SP
Canarium schweinfurthii Engl. Burseraceae MgPh GC Sar Me SP
Carapa grandiflora Sprague Meliaceae MgPh GC Sar Me SP
Carapa procera DC. var. procera DC. Meliaceae MsPh FC Sar Me SP
Celtis adolfi-friderici Engl. Ulmaceae MsPh GC Sar Mi SP
Celtis durandii Engl. Ulmaceae MsPh GC Sar Mi SP
Celtis mildbraedii Engl. Ulmaceae MgPh GC Sar Mi SP
Celtis zenkeri Engl. Ulmaceae MsPh CG Sar Mi SP
Chrysophyllum lacourtianum De Wild. Sapotaceae MgPh CG Sar Me SP
Coelocaryon botryoides Verm. Myristicaceae MsPh FC Sar Me SP
Cola chlamydantha K.Schum. Malvaceae MsPh CG Sar Me SP
Cola diversifolia De Wild. Malvaceae MsPh FC Sar Me SP
Cola gigantea A. Chev. Malvaceae MsPh CG Pté Me SP
Cola lateritia K. Schum. Malvaceae MsPh CG Sar Me SP
Cola urceolata K. Schum. Malvaceae MsPh CG Sar Me SP
Copaifera mildbraedii Harms Fabaceae MgPh CG Bal Me SP
Corynanthe pachycera K. Schum. Rubiaceae MsPh GC Sar Me SP
Crudia gabonensis Pierre ex Harms Fabaceae MsPh CG Bal Me SP
Cynometra hankei Harms Fabaceae MgPh GC Bal Me SP
Daniellia pynaertii De Wild. Fabaceae MgPh GC Bal Me SP
Desplatsia chrysochlamys (Mildbr. & Bur,) Mildbr. Malvaceae MsPh GC Sar Me MT
Detarium macrocarpum Harms Fabaceae MgPh CG Sar Me SP
Dialium corbisieri Harms Fabaceae MsPh FC Sar Me SP
Masens et al. (2017) 4(3): 363–375
Dialium pachyphyllum Harms Fabaceae MgPh CG Sar Me SP

Dialium pentandrum Louis ex Stey. Fabaceae MsPh FC Sar Me SP
Dialium tessmannii Harms Fabaceae MsPh CG Sar Me SP
Dichostemma glauscens Pierre Euphorbiaceae MsPh CG Sar Me SP
Diospyros canaliculata De Wild. Ebenaceae McPh At Sar Me SP
Duboscia viridiflora (K. Schum.) Mildbr. Malvaceae MsPh CG Sar Me SP
Enantia chloranta Oliv. Annonaceae MsPh GC Sar Me SP
Entandrophragma angolense (Welw.) C. DC. Meliaceae MgPh GC Pté Me SP
Entandrophragma utile Sprague Meliaceae MgPh GC Pté Me SP
Erismadelphus exsul Mildb. Vochysiaceae MgPh CG Sar Me SP
Erythrophleum suaveolens (Guill. & Perrot) Brenan Fabaceae MgPh GC Bal Me SP
Fernandoaa dolfi-frederici (Gilg & Mildbr.) Heine Bignoniaceae MsPh CG Pté Me SP
Ficus lutea Vahl Moraceae MsPh At Sar Me MT
Ficus sur Welw. ex Ficalho Moraceae MsPh At Sar Me MT
Ficus thonningii Blume Moraceae MsPh At Sar Me MT
Filaeopsis discophora Harms Fabaceae MgPh GC Bal Me MT
Funtumia elastica (Preuss) Stapf Apocynaceae MsPh GC Scl Me MT
Gaertnera parvipaniculata Petit Rubiaceae MsPh CG Sar Me SP
Ganophyllum giganteum (A. Chev.) Hauman Sapindaceae MgPh CG Sar Me SP
Garcinia epunctata Stapf Clusiaceae MgPh GC Sar Me SP
Garcinia kola Heckel Clusiaceae MgPh GC Sar Me SP
Garcinia punctata Stapf Clusiaceae MgPh GC Sar Me SP
Gilbertiodendron dewevrei (De Wild.) J. Léonard Fabaceae MgPh GC Bal Me SP
Greenwayadendron suaveolens 'Engl. & Diels) Verdc. Annonaceae MsPh GC Sar Me SP
Grewia oligoneura Sprague Malvaceae MsPh CG Sar Me SP
Guarea cedrata (A. Chev.) Pellerg. Meliaceae MgPh GC Sar Me SP
Guibourtia tessmannii (Harms) J. Léonard Fabaceae MgPh CG Bal Me SP
Hannoa klaineana Pierre Simaroubaceae MgPh GC Sar Me SP
Homalium africanum Mast. Flacoutiaceae MsPh GC Sar Me MT
Hua gabonii De Wild. Huaceae MsPh FC Sar Me MT
Hugonia platysepala WelW. ex Oliv. Menispermaceae MsPh GC Sar Me MT
Khaya anthotheca (Welw.) C. DC. Meliaceae MgPh GC Bal Me SP
Lannea welwitschii (Hiern) Engl. Santalaceae MsPh GC Sar Me H
Lovoa trichiloides Harms Meliaceae MgPh GC Scl Me SP
Maesobotrya floribunda Benth. Euphorbiaceae McPh FC Sar Me SP
Mammea africana Sabine Clusiaceae MgPh GC Sar Me SP
Manilkara fouilloyana Aubr. Ex Pellegr. Sapotaceae MgPh CG Sar Me SP
Manilkara koechlinii Aubr. Sapotaceae MgPh FC Sar Me SP
Maranthes chrysophylla (Oliv.) France ex F. While Sapotaceae MsPh CG Sar Me SP
Massularia acuminata (G. Don) Hoyle Rubiaceae McPh GC Sar Me SP
Memecylon leucocarpum Gilg. Melastomataceae McPh FC Sar Mi SP
Micrococca mercurialis (L.) Benth. Euphorbiaceae McPh GC Sar Me MT
Milicia excelsa (Welw.) Berg. Moraceae MgPh GC Sar Me MT
Millettia sapinii De Wild. Fabaceae McPh FC Bal Me SP
Monodora myristica (Gaertn.) Dunal Annonaceae MsPh CG Sar Me SP
Monopetalanthus pteridophyllus Harms Fabaceae MgPh FC Bal Me SP
Nesogordonia kabingensis (K. Schum.) Cap. Malvaceae MsPh GC Pté Me SP
Nesogordonia papaverifera (A. Chev.) Capur. Malvaceae MsPh GC Pté Me SP
Omphalocarpum procerum P. Beauv. Sapotaceae MgPh GC Sar Me SP
Masens et al. (2017) 4(3): 363–375
Ongokea gore (Hua) Pierre Olacaceae MgPh GC Sar Me SP

Oxyanthus speciosus DC. Rubiaceae McPh At Sar Me SP
Panda oleosa Pierre Pandaceae MgPh GC Sar Me SP
Paramacrolobium coeruleum (Taub.) J. Léonard Fabaceae MsPh At Bal Me MT
Parinari excelsa Sabine Chrysobalanaceae MgPh GC Sar Me SP
Parkia filicoidea Welw. ex Oliv. Fabaceae MsPh At Sar Me SP
Pausinystalia macrocera (K. Schum.) Pierre ex Dup. Rubiaceae MsPh GC Sar Me SP
Pavetta sp. Rubiaceae McPh - Sar Me SP
Penianthus preussii Miers Menispermaceae NnPh GC Sar Me MT
Pentacletra macrophylla Benth. Fabaceae MsPh GC Bal Me MT
Pentadesma butyracea Sabine Clusiaceae MsPh GC Sar Me SP
Pentadesma excelliana Staner Clusiaceae MsPh GC Sar Me SP
Petersianthus macrocarpus (Beauv.) Liben Lecytidaceae MgPh GC Pté Me MT
Piptadeniastrum africanum (Hook.) Brenan Fabaceae MgPh GC Bal Me SP
Plagiostyles africana (Müll.-Arg.) Prain. Euphorbiaceae MsPh CG Sar Me SP
Prioria balsaminfera (Vermoesen) Breteler Fabaceae MgPh CG Bal Me SP
Psydrax arnoldiana (De Wild.) Brid. Rubiaceae MgPh CG Sar Me SP
Pterocarpus mildbraedii Harms Fabaceae MgPh GC Pté Me SP
Pterocarpus soyauxii Taub. Fabaceae MgPh GC Pté Me SP
Pterocarpus tinctorius Welw. Fabaceae MgPh At Pté Me SP
Pycnanthus angolensis (Welw.) Ekell. Myristicaceae MsPh GC Pté Me MT
Quassia africana (Baill.) Baill. Simaroubaceae McPh GC Sar Me SP
Ricinerodendron heudelotii (Baill.) Pierre ex Heckel Euphorbiaceae MgPh GC Sar Me MT
Sacoglottis gabonensis (Baill.) Urb. Humiriaceae MsPh GC Sar Me SP
Santiria trimera (Oliv.) Aubr. Burseraceae MsPh GC Sar Me SP
Sarchocephalus diderichii (De Wild.) Rubiaceae MgPh GC Sar Me SP
Sarchocephalus pobenguini (Hua ex Pob.) Merril. Rubiaceae MsPh GC Sar Me H
Schrebera arborea A. Chev. Oleaceae MsPh GC Pté Me SP
Schumanniophyton magnificum (K. Schum.) Harms Rubiaceae MsPh GC Sar Ma SP
Scorodophoeus zenkeri Harms Fabaceae MgPh CG Bal Me SP
Scyphocephalium mannii (Benth. & Hook. f.) Warb. Myristicaceae MsPh GC Sar Me SP
Spondianthus preussi Engl. Anacardiaceae MsPh CG Pté Me SP
Staudtia kamerunensis Warb. Myristicaceae MsPh CG Sar Me SP
Sterculia tragacantha Lindl. Myristicaceae MsPh CG Pté Me SP
Strombosia grandiflora Hook.f. Olacaceae MsPh GC Sar Mi SP
Strombosia majuscula Hook. f. Olacaceae MsPh GC Sar Me SP
Synsepalum stipulatum De Wild. Sapotaceae MsPh GC Sar Me SP
Tetrapleura tetraptera (Schum. & Thon.) Taub. Fabaceae MsPh GC Bal Me MT
Treculia africana Decne Moraceae MsPh At Sar Me MT
Trichalisia crepiniana De Wild. & Th. Dur. Rubiaceae MsPh CG Sar Me SP
Trichilia welwitschii C. DC. Meliaceae MsPh CG Scl Me SP
Trilepisium madagascariense DC. Moraceae MsPh GC Sar Me MT
Uapaca sp. Euphorbiaceae MsPh - Sar Me H
Xylopia chrysophylla Louis ex Bout. Annonaceae MsPh CG Sar Me SP
Zanthoxylum leprieuri Guill. & Perr. Rutaceae MsPh GC Sar Me SP
Note: LF = Life forms (McPh = Microphanerophytes; MgPh = megaphanerophytes; MsPh = mesophanerophytes; NnPh =
nanophanerophytes); PhgD = Phytogeographical distributions (At = Afro-tropical; CG = Congolian-Guineo; FC = foreign-Congolian;
GC = Guineo-Congolian); DTD = Dissemination types of diaspores (Bal = Ballochores; Pté = Pterochores; Sar = sarcochors; Scl =
Sclerochores); TFD = Types of foliar dimensions (Me = Mesophyl; Mi = Microphyl) and PhSt = Phytosociological status (H =
Halleeteae; MT = Musango-Terminalietea; SP = Strombosio-Parinarietea).
Masens et al. (2017) 4(3): 363–375
The analysis of life forms shows the predominance of mesophanerophytes (MsPh) species (56%); followed
by megaphanerophytes (MgPh) species (36.6%) but Microphanerophytes (McPh) and nanophanerophytes
(NnPh) are poorly represented as 6.7% and 0.7% respectively. With regard to the dissemination of diaspores,
sarcochors (Sar) form the most abundant group (70.1%); then come next H and far behind this group,
Ballochores (Bal) and Pterochores (Pte) with 17.2 and 10.4% respectively of the total. Sclerochores (Scl) are
very poorly represented (2.2%). As for the types of the foliar dimensions, the mesophyl (Me) species
predominate and constitute ~94% of the total of the inventoried plants. On the other hand, microphyl (Mi)
species are very poorly represented (~5%) (Table 2). The Shannon index (ISH) calculated for all the 134 species
listed is of 4.77 and the Equitability is of 0.97 (Table 3).
Table 3. Biodiversity index values.
Indices Values
Regularity (H+) 4.77
Maximum diversity (Hmax) 4.90
Equitability (J) 0.97
Types of phytogeographical distribution
The Guinean base element (GC, CG and FC) is the most abundant; it accounts 90.1% of the total of species
listed. In this basic element, Guineo-Congolian (GC) species are the most numerous (51.9%) followed by CG
(29.8%) and FC (8.4%). Afro-tropical (At) species are relatively well represented (9.9%). This shows the sign of
degradation of this forest massif. Three taxa could not be determined to the species (Table 2).
Eco-sociological groups
Three eco-sociological groups have been identified. Among them, the species of Strombosio-Parinarietea
(SP) are the most abundant. They make up 81.3% of all the species listed in the Nzundu forest bulb. The species
belonging to Musango-Terminalietea (MT) are relatively well represented (15.7%). Their significant presence
already demonstrates sufficiently the state of degradation that currently characterizes this forest. Those of
Halleeteae (H) are very weakly represented (3%) (Table 2).
DISCUSSION
The Nzundu forest massif is part of the Guinean and Peri-guinian semi-deciduous tropical rainforests. It
differs from that of Kamaba (Masens 2015) by the high frequency of Brachystegia laurentii and the total
absence of certain noble species like Millettia laurentii However, these two forest massifs share a number of
plant species: Celtis mildbraedii, Piptadeniastrum africanum, Prioria balsamifera, Pterocarpus mildbraedii etc.
Unlike the Kamaba forest which gathered 155 different plant species while the Nzundu forest massif has only
134 species. The 134 species are divided into 31 families and 109 genera. Fabaceae (25 genera), Rubiaceae (10
genera), Euphorbiaceae (8 genera), Sapotaceae (7 genera), Annonaceae, Malvaceae, Meliaceae and Moraceae
with 6 genera each. The most supplied genera in species are: Cola (5 species), Celtis and Dialium with each 4
species and 4 genera each have 3 species. These include Ficus, Garcinia, Pentadesma and Pterocarpus.
Compared to what was observed in some forests studied in the tropics; these results are in the same order of
magnitude as those obtained in these various plant formations (Bosanza et al. 2017). Species of Kamaba
phytocenosis have a high proportion of trees with dbh measured at 1.30 m at breast height >30 cm. Thus, it
presents a curve with concavity facing downwards and the shape of this curve would undoubtedly explain a
rather remarkable presence of heliophilous species (Rollet 1969), while those of the Nzundu forest massif show
a large proportion of large trees at dbh ≥50 cm. Their distribution follows a curve of which concavity is oriented
upwards. According to the same authors, this demonstrates the presence of a large number of shade species.
Therefore, it is possible to paraphrase some authors (Lomba et al. 2017) that the dbh of Nzundu phytocenosis
trees, measured at 1.30 m, increases with the evolution of vegetation. Among the families listed in this forest
massif, we have Fabaceae, Malvaceae, Rubiaceae, Euphorbiaceae, Meliaceae, Moraceae, Sapotaceae,
Annonaceae and Clusiaceae which are the most supplied in plant species (Table 2). Apart from Myristicaceae
family, these results are similar to those obtained by Masens (2015) in the Kamaba forest massif in the lower
Kasaï vegetation in the Democratic Republic of the Congo (Belesi 2009). According to Kambale et al. (2017),
the basal area is a parameter commonly used to distinguish plant formations from the mainland. The basal area
values of the trunks measured at 1.30 m in Nzundu phytocenosis was significantly higher (49.89 m2.ha-1) than in
Masens et al. (2017) 4(3): 363–375
the Kamaba forest (20.0 m2.ha-1). This very significant difference could be explained by the high proportion of
trees with large trunks characterizing the Nzundu forest.
The basal area value obtained in this phytocenosis ranges from 23 to 50 m2.ha-1. It is clear that the basal area
values obtained in different equatorial and subequatorial forests are included in this interval (Mosango 1990).
Moreover, this value is relatively less than the tropical forests of Panama (59.6 m2.ha-1) as observed by Golley et
al. (1969), India (59.6 m2.ha-1) as observed by Bajpai et al. (2012) and slightly higher than the one obtained by
Mosango (1990) in the evergreen rainforest (Kongolo island/Kisangani).
For biological spectra, chorological types and phytogeographic status, mesophanerophytic and
megaphanerophytic species predominate (92.4%). The sarcochorous and mesophyl species are the most
abundant, with 70.1 and 94.8% respectively; the Guinean base element being the most predominant, accounting
for ~90% of all inventoried species. All these results are in the same order of magnitude as those obtained in
various tropical and intertropical forests as described in previous studies (Lejoly 1995, Belesi 2009, Masens
2015, Shaumba et al. 2017, Lomba et al. 2017).Concerning the relative frequency, Brachystegia laurentii
reaches 5.1% followed by three other species: Pterocarpus mildbraedii (3.2%), Prioria balsamifera and
Staudtia kamerunensis with 2.5% each.
As for relative dominance, Brachystegia laurentii is the most important (26.9%). Baillonella toxisperma and
Bosqueiopsis gilletii had a relative dominance of more than 3%. It is again the species Brachystegia laurentii
which reaches a value of the highest relative importance (14.6%), followed by three other species: Pterocarpus
mildbraedii, Prioria balsamifera and Staudtia kamerunensis gave values above 3% (Table 1). These results go
along with those obtained in the Ngoto forest (Lejoly 1995). It is clear that Brachystegia laurentii is the only
species that has exhibited large proportions of the values of different parameters considered in this study,
contrary to what was observed in the Kamaba forest massif (Masens 2015).
Finally, the high values of Shannon and Equitability indices can mean either a high specific richness due to a
high presence of rare species (species having only one individual) or diversity due to a regular distribution of
individuals between species or a high number of individuals in the observed distribution. The value of the
calculated Shannon index is 4.77. This value approximates the maximum diversity (4.90), which means that
diversity is important. That of Equitability is 0.97, which reflects the regularity in terms of the distribution of
individuals within the species. These values are in the same order of magnitude as those found by Sokpon
(1995) in the Strombosia glauscens and Triplochiton scleroxylon and Dialium guineense and Triplochiton
scleroxylum forests in Benin.
CONCLUSION
The study on the floristic inventory and some ecological parameters carried out in Nzundu forest massif
showed that this phytocenosis presents a moderately appreciable specific richness (134 species). The regressive
evolution observed within this phytocenosis is triggered with extreme rapidity by the slaughter and the reckless
clearing of trees more or less isolated and/or grouped and this by severe depredations, which are currently
inflicted on them. Even if these degradable practices had always existed in the past, they had never, according to
villagers surveyed, reached such a degree of acuity due to the high concentration of the population around this
forest massif. Considering woody species, species with dbh ≤ 49.9 cm showed a high specific richness
compared to trees with dbh ≥ 50 cm. The diameter distribution of species in this forest ecosystem is of the
hyperbolic type and having high basal area (49.89 m2.ha-1), located at the extreme top of the range of 23–50
m2.ha-1 established for Equatorial forests. This basal area was 23.46 m2.ha-1 for trees at dbh ≤ 49.9 cm and 26.37
m2.ha-1 for emerging species at dbh ≤ 50 cm. In view of this basal area value, we can ascertain that Nzundu
phytocenosis was in the recent past a mature and reworked forest. Currently this forest is under unprecedented
pressure because of its proximity to the urban-rural city of Kikwit. The population of this city and its
surroundings, composed of 2/3 of the farmers and charcoal burners, has largely destroyed this forest leaving
only a few hundred hectares of m2.
The density of species measured at 1.30 m was of 422 trunks.ha -1, divided into 366 trunks.ha-1 for trees at
dbh ≤ 49.9 cm and 56 trunks.ha-1 for dbh ≥ 50 cm. Fabaceae, Malvaceae, Rubiaceae and Euphorbiaceae were
the syntaxa accounting for the largest number of species and in addition to these families, Sapotaceae,
Meliaceae, Moraceae and Clusiaceae, collected a large number of trunks.ha-1. The calculated Shannon and
Equitability indices for this phytocenosis oscillated around 4.77 for the first and 0.97 for the second.
Masens et al. (2017) 4(3): 363–375
ACKNOWLEDGEMENT
The authors are thankful to the Head of Faculty of Science, University of Kinshasa, Democratic Republic of
the Congo for his valuable support.
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ISSN (E): 2349 – 1183
ISSN (P): 2349 – 9265
4(3): 376–382, 2017
DOI: 10.22271/tpr.2017.v4.i3.049
Research article
Phytochemical screening and antibacterial activities of

Tectona grandis L. f. (Teak) leaves on microorganisms isolated
from decayed food samples
O. T. Ogunmefun*, E. A. Ekundayo, F. C. Akharaiyi and D. Ewhenodere
Department of Biological Sciences, Afe Babalola University, Ado-Ekiti, Nigeria
*Corresponding Author: yinkatayo_08@yahoo.com [Accepted: 19 September 2017]
Abstract: Bacteria were isolated from decayed food samples (tomatoes, cooked beans and rice)
collected from Afe Babalola University, Ado Ekiti (ABUAD) cafeteria and characterized. Some of
these isolated microorganisms could pose serious harm to humans including animals and they are
normally treated with commercial antibiotics. However, the majority of bacteria are resistant to
many antibiotics therefore, the use of plant extracts with therapeutic potential against resistant
bacteria is necessary. In this investigation, eight bacteria were isolated from decayed food samples.
The bacterial isolates were identified as Bacillus cereus and B. siamensis from rice sample;
Klebsiella oxytoxa, Salimicrobium halophilium and Norcardia brasiliensis from beans sample;
Bacillus subtilis, Enterobacter taylore, and Brevibacillus agri from tomatoes. The leaf samples of
Tectona grandis were screened qualitatively and quantitatively for the phytochemicals while the
crude methanol and chloroform extracts were used as antimicrobial agents against the isolated
microorganisms. Alkaloids, carotenoids and tannins were present in large amount. The bacterial
isolates were more susceptible to commercial antibiotics than that of methanol extracts of T.
grandis. The methanol extracts of T. grandis have a higher antimicrobial activity than the
chloroform extracts.
Keywords: Chloroform extracts - Food samples - Methanol extracts - Microorganisms - Tectona
grandis.
[Cite as: Ogunmefun OT, Ekundayo EA, Akharaiyi FC & Ewhenodere D (2017) Phytochemical screening and
antibacterial activities of Tectona grandis L. f. (Teak) leaves on microorganisms isolated from decayed food
samples. Tropical Plant Research 4(3): 376–382]
INTRODUCTION
Food decay is any undesirable change in food that makes the food to lose its aesthetic value and becomes
unacceptable to humans for consumption. Food is also said to be decayed when the original nutritional value,
texture and flavor are altered through microbial activities. Various factors could be responsible for the
deterioration of various foods (Istifanus et al. 2014). Consumption of this decayed or contaminated food results
in food borne disease such as diarrhoea, gastroenteritis, respiratory infections and meningitis, if ingested (Mead
et al. 1999, Humphrey et al. 2007, Barth et al. 2009). Also, toxins from these microorganisms can be found in
contaminated food products and are pathogenic leading to disease (Edwin et al. 2013). Although, bacteria are
the main and important source of food spoilage, but other microbes such as fungi, viruses and protozoa have
also been implicated (Kumar et al. 2011). The most common pathogens causing serious infection in food
include Staphylococcus aureus, Pseudomonas sp., Streptococcus sp., Proteus sp., Clostridium sp. and
Coliforms (Lawrence et al. 1999). High pH (4.9‒6.5), water and nutrient contents enhance microbial growth
such as bacteria and fungi, which degrade the nutrients through enzymes production (Trias et al. 2008, Matthew
2011, Ogunbanwo et al. 2014). These also heighten spoilage susceptibility thereby reducing the nutritional and
market values (Bello et al. 2016). Most of the pathogenic microorganisms causing diseases have developed
resistance to modern antibiotics (Sharma et al. 2012). Bacteria generally have the genetic ability to transmit
diseases and acquire resistance to drugs used as therapeutic agents (Cohen et al. 1992). Therefore, there is
needed to look for new antimicrobials of plant origin.
Received: 23 May 2017 Published online: 30 September 2017
Ogunmefun et al. (2017) 4(3): 376–382
Medicinal plants are gaining popularity in usage due to a large number of people in search of health
remedies with little or no side effect which is the problem of most chemically synthesized drugs (Prance 1991,
Bajpai et al. 2016). Considerable attention is presently given to the use of eco-friendly and bio-friendly products
from plant origin for the prevention and cure of human and animal health challenges (diseases) (Gijtenbeek et
al. 1999, Johnson & William 2002). Plants based antimicrobials represent a vast untapped source of medicines
and further exploration of plant antimicrobials needs to occur. Antimicrobials of plant origin have enormous
therapeutic potentials (Evans & Turnbull 2004). The majority of bacteria are resistant to many antibiotics
therefore, the use of plant extracts against resistant bacteria leads to new choice for the treatment of infectious
diseases (Purushotham et al. 2010).
Tectona grandis L. f. (Teak) is a tropical tree species distributed naturally in countries including India,
Myanmar, Thailand, Myanmar, Nigeria and other tropical countries including Indonesia (Mahesh et al. 2016).
Teak is one of the world's premier hardwood tree species, highly famous for its quality, profile and durability of
timber (Sumthong et al. 2008). The generic name of T. grandis comes from „Tekka‟, which is the Malabar name
while the specific name, „grandis‟ is a Latin word for „large‟ or „great‟. Tectona grandis is a large, deciduous
tree reaching over 30 m in height in favourable conditions (Orwa et al. 2009). “Fruit is a drupe with 4 chambers;
round, hard and woody, enclosed in an inflated, bladder-like covering; pale green at first, then brown at
maturity” (Orwa et al. 2009). It occurs naturally in various types of tropical deciduous forests. In seasonal
climates, T. grandis is deciduous while trees grown in non-seasonal climates are semi-deciduous. It is often a
dominant member of a mixed deciduous forest, where its main associates are Xylia spp., Afzelia xylocarpa
(Kurz) Craib, Terminalia spp. and Lagerstroemia spp. Tectona grandis generally occurs scattered but can form
almost pure stands under favourable conditions. Young plants show a remarkable capability to recover after fire
(Orwa et al. 2009).
Diverse anti-microbial and substances that can serve as insect control agents can be produced by higher
tropical plants (Nakamura et al. 1991, Downum et al. 1993, Lis-Balchin et al. 1996). Substances such as
flavonoids, alkaloids, terpenoids are the secondary metabolites produced by the plants as a chemical defense
against pests and diseases attacks. It is estimated only 10% of these tropical plants have been investigated for
their pesticidal activity. Teak is one of these plants which produce secondary metabolic products containing
phenolic compounds. Astiti et al. (1998) found that the water extract of teak leaf obviously inhibited the growth
of Monilia species, the causative agent of wood decay. The sporulation of Alternaria cajani and
Helminthosporium sp. was inhibited by the T. grandis leaf methanol crude extract at concentration of 5 mg.ml-1
(Shalini & Srivastava 2008). The frontal leaves of the plant are widely used in folklore for the treatment of
various kinds of wounds, especially burn wounds. They are also useful to treat haemostatic, depurative, anti-
inflammatory and vulnerary and also useful in inflammation, leprosy, skin disease, pruritus, stomatitis, indolent
ulcer, haemorrhages and haemopstysis (Purushotham et al. 2010). The presence of high amounts of tannins in
the T. grandis leaves may be responsible for the pro-healing action of the extract and its antioxidant property is
attributed to it (Mrityunjoy et al. 2007). All these studies clearly showed the antibacterial potential of T. grandis,
thus the present study was designed to find out the effect of its leaves on the microorganisms isolated from
decayed food samples.
MATERIALS AND METHODS

Collection of food samples
Food samples (fresh tomatoes, cooked beans, cooked rice and fresh pepper) were collected from Afe
Babalola University, Ado-Ekiti (ABUAD) cafeteria/kitchen environment. These samples were allowed to decay
in ABUAD Microbiology Department laboratory.
Collection of plant sample
The leaves of Tectona grandis L. f. were obtained from ABUAD surroundings and were identified by Dr.
(Mrs.) O. T. Ogunmefun, a botanist in the Department of Biological Sciences of Afe Babalola University Ado -
Ekiti, Ekiti State, Nigeria.
Preparation of extracts
The collected plant materials were air dried at room temperature, ground with blender after which dry mass
of 887.2 g of the leaf sample was obtained (Purushotham & Sankar 2013). A 350 g each of powdered leaf
sample was soaked in 700 ml of chloroform and methanol separately using cold extraction method for 72 hours.
Ogunmefun et al. (2017) 4(3): 376–382
The extracts were separated from the solvents using a rotary evaporator. The extracts were concentrated on
water bath at a low temperature of 40°C (Ajaiyeoba et al. 2001, Ogbole et al. 2013).
Phytochemical screening of Tectona grandis
The powdered leaves of T. grandis were subjected to both qualitative and quantitative phytochemical tests
for plant secondary metabolites namely alkaloids, anthraquinones, tannins, saponins, cardiac glycosides,
flavonoids, carotenoids and phenols according to the method described by Harborne (1998) and Trease & Evans
(2004).
Isolation, characterization and identification of microbial species from the decayed food samples
Five-fold serial dilution was carried out on the food samples. An aliquot (1 ml) of the fifth dilution was pour
plated on nutrient agar and then incubated at 37°C for 24 hours. Colonies of bacterial isolates were selected
from each plate respectively and purified by sub-culturing on nutrient agar plates. Bacterial isolates were
preserved in slants at 4°C for temporary storage and were then identified both morphologically and
biochemically (Prescott et al. 2002).
Determination of the antibacterial activities of Tectona grandis using agar well diffusion technique
A sterile swab stick was placed into the broth bacterial culture corresponding to MacFarland standard of a
specific organism and then spread on already prepared sterile nutrient agar plate. This was repeated for all the
isolates in duplicates. The plates were allowed to dry for approximately 5 minutes. The wells were then filled
with dilutions of 50 mg.ml-1, 100 mg.ml-1, 150 mg.ml-1, 200 mg.ml-1, 250 mg.ml-1, 300 mg.ml-1, 350 mg.ml-1
and 400 mg.ml-1 with T. grandis extract reconstituted with 10% Dimethyl sulphoxide (DMSO) and sterilized by
0.45 µm membrane filter. The plates were incubated 37°C for 24 hours. The diameters of the inhibitory zones
were measured in millimeters. The DMSO was used as a negative control while streptomycin was used as the
positive control.
RESULTS
In this study, seven bacterial species were obtained from all the decayed food samples based on their
morphological and biochemical characteristics. Five of the isolates were Gram positive while the remaining two
isolates were Gram negative. The isolates were identified as Bacillus cereus, B. subtilis, B. siamensis,
Brevibacillus agri, Salimicrobium halophilum, Klebsiella oxytoxa, Norcadia brasiliensis and Enterobacter
taylore. The frequency of occurrence of the isolate is presented in table 1.
Table 1. Frequency of occurrence of bacterial isolates from decayed food samples.
Food samples
Bacterial isolates
Beans Rice Tomatoes
Bacillus cereus - + -
Bacillus siamensis - + +
Bacillus subtilis - - +
Klebsiella oxytoxa + - +
Salimicrobium halophilum + - -
Enterobacter taylore - - +
Norcadia brasiliensis + - -
Brevibacillus agri - - +
Note: +, Present; -, Absent.
Saponins, alkaloids, tannins among others were present while cardiac glycoside was not observed in the
qualitative screening. However, the quantitative analysis showed that 20 mg per 100 g of cardiac glycoside was
present. Alkaloids were most abundant followed by tannins and carotenoids respectively. The detail results of
the qualitative and quantitative phytochemicals of T. grandis are presented in tables 2 and 3.
The leaf extracts obtained with chloroform and methanol were used as antibacterial agents against the
bacterial isolates. The results showed that the extracts exhibited weak antibacterial activities on the isolates
(Table 4). The antibacterial activities of streptomycin used as positive control ranged from 11.00–30.00 mm
respectively and were without a doubt effective against food pathogens than the extract. All the bacterial isolates
were resistant to DMSO used as negative control (Fig. 1). At concentrations of 50, 100, 150 and 200 mg.ml-1,
the extracts were not effective but at concentrations of 250, 300, 350 and 400 mg.ml -1 there were clear zones of
inhibition (Table 4).
Ogunmefun et al. (2017) 4(3): 376–382
Table 2. Qualitative phytochemical analysis of Tectona grandis leaves.

Parameter Observation
Alkaloids +++
Flavonoids ++
Tannins +++
Saponins ++
Cardiac Glycosides -
Total Phenolics ++
Anthraquinones +
Carotenoids ++
Note: +++, Most abundant; ++, Abundant; +, Present; -, Absent.
Table 3. Quantitative phytochemical analysis of Tectona grandis leaves.

Phytochemical constituents Amount of Phytochemical constituents
Alkaloids (mg per 100 g) 1778.30
Flavonoids (mg per 100 g) 666.60
Tannins (mg per 100 g) 1583.00
Saponins (mg per 100 g) 406.60
Cardiac Glycosides (mg per 100 g) 20.00
Total Phenolics (GAE per g) 69.50
Carotenoids (μg per 100 g) 1268.33
Figure 1. Comparative antibacterial activities of methanol extract of Tectona grandis and Streptomycin at 37oC on nutrient
agar after 24 hours of incubation. [R, Rice; B, Beans; T, Tomatoes]
DISCUSSIONS
Different spoilage bacteria including fungi grow well at room temperature. A large number of
microorganisms and their waste products cause the objectionable changes in odor, taste and texture in food
samples (Kumar et al. 2011). The presence of these bacterial isolates may not be unconnected with the high
moisture contents of the food substances which aided the spoilage as opined by Akharaiyi & Adeyanju (2016).
Bacillus cereus has been associated with the production of toxins, diarrheal and emetic in food, which causes
food poisoning. It is found in dust, soil and raw food and can survive under normal cooking conditions as
produces heat resistant spore (Rajkowski & Bennett 2003). Bello et al. (2016) observed the prominence of B.
subtilis in spoilage of tomato fruits. Ghosh (2009) also suggested that the prevalence of microbial contamination
could be aggravated by poor sanitation including cross-contamination. Biological contaminant of bacterial
origin represents a major cause of foodborne illnesses (Edema et al. 2005). The presence of Bacillus subtilis has
been correlated with food poisoning. Klebsiella spp. and Enterobacter spp. call for concern as these organisms
Ogunmefun et al. (2017) 4(3): 376–382
are frequently associated with poor sanitary practices and could indicate the danger of possible food borne
infection (Oranusi et al. 2007, Eni et al. 2010). Klebsiella spp. has been isolated in food such as fruits and
vegetables, meat, milk and dairy products, salads, and drinking water, coming from the general environments of
soil, dust, air and water and from social environments.
Table 4. Antibacterial activity of methanol extract of Tectona grandis leaves.
Bacterial Concentration of crude extract (mg.ml -1)
isolates 50 100 150 200 250 300 350 400
P3B3 R R R R 10.00 10.00 11.00 11.50
P2B1 R R 13.00 13.00 15.00 15.00 18.00 18.00
P3B3 R R R 2.00 4.00 6.00 11.00 12.00
P2B2 R R R R R R 14.00 14.00
P1B1 R R R R R R R R
RIB1 R R R 16.00 11.00 15.00 15.00 15.50
RB1 R R R R R R R R
R1B2 R R R R R R R R
R1B1a R R R R R R R R
T1B2 R R 10.50 13.00 14.50 1.50 14.00 15.00
T3B1 R R R 12.00 12.50 13.50 15.00 18.50
T2B1 1.00 2.00 2.00 4.00 4.00 5.00 6.00 7.00
T1B1 R R 3.00 4.00 4.00 6.00 6.00 8.00
T3B2 R R 2.00 3.00 5.00 5.00 6.00 10.00
Note: PB, Isolates obtained from beans; RB, Isolates obtained from rice; TB, Isolates obtained from
tomatoes; B, Bacteria; R, Resistance.
Higher tropical plants can produce diverse antimicrobial and anti-insect substances (Nakamura et al. 1991,
Downum et al. 1993, Lis-Balchin et al. 1996). Substances such as flavonoids, alkaloids and terpenoids are
secondary metabolites produced by plants as a chemical defence against pests and diseases‟ attacks. Tannins are
astringent in nature and are used for treating intestinal disorders such as diarrhea and dysentery (Dharmananda
2003); they are referred to as natural antibiotics from plants.
The water extract of T. grandis leaf conspicuously inhibited the growth of Monilia sp., which is a causal
organism attributing in wood decay (Astiti 1998). The methanol crude extract of T. grandis leaves at 5 mg.ml-1
concentration inhibited the sporulation of Alternaria cajani and Helminthosporium sp. by 86.6 and 90.0%
respectively (Shalini & Srivastava 2008).
CONCLUSION
The methanol extract of Tectona grandis leaf significantly inhibited bacterial growth. Chloroform extract
was least effective on bacterial isolates as compared to methanol extracts. Teak extracts can be considered as
one of the alternatives to chemical food preservatives and for controlling food decay caused by these isolates. A
further study is needed to isolate and identify the active compounds that are responsible for the antimicrobial
potency of T. grandis.
ACKNOWLEDGEMENT
All the authors are thankful to the Department of Biological Sciences, Afe Babalola University, Ado-Ekiti,
Nigeria for providing necessary support. There is no conflict of interest among authors.
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ISSN (E): 2349 – 1183
ISSN (P): 2349 – 9265
4(3): 383–390, 2017
DOI: 10.22271/tpr.2017.v4.i3.050
Research article
New distributional records in lichen family Graphidaceae for

Andhra Pradesh, India
Satish Mohabe1, Anjali Devi B.1, A. Madhusudhana Reddy1*,
Gangadhar Pandhava1 and Sanjeeva Nayaka2
1
Department of Botany, Yogi Vemana University, Vemanapuram, Kadapa-516003, Andhra Pradesh, India
2
Lichenology Laboratory, CSIR-National Botanical Research Institute, Rana Pratap Marg, Lucknow-226001,
Uttar Pradesh, India
*Corresponding Author: grassced@yahoo.com [Accepted: 22 September 2017]
Abstract: The present paper reports 15 lichen taxa viz. Chapsa leprocarpoides, Diploschistes
scruposus, Fissurina capsulata, F. cingalina, F. comparimuralis, F. egena, Glyphis cicatricosa,
Graphis cincta, G. crebra, G. duplicata, G. furcata, G. handelii, G. leptocarpa, G. lineola and
Thecaria austroindica of family Graphidaceae for the first time from Andhra Pradesh. Out of
which, genera Chapsa, Fissurina and Thecaria are recorded for the first time from the state. The
brief description and updated distribution are provided for all the taxa studied.
Keywords: Taxonomy - Graphidaceae - Thelotremoid - New addition - Rayalaseema forest.
[Cite as: Mohabe S, Anjali DB, Reddy AM, Pandhava G & Nayaka S (2017) New distributional records in
lichen family Graphidaceae for Andhra Pradesh, India. Tropical Plant Research 4(3): 383–390]
INTRODUCTION
The lichen family Graphidaceae is well known for its tropical and subtropical distribution in the world. The
family was monographed by Staiger (2002) which has been recently reclassified on the basis of molecular
phylogeny (Plata et al. 2012, Lücking et al. 2017). At present Graphidaceae is the largest family comprising of
2,161 species in the world and 406 species in India (Singh & Sinha 2010). In the last five years, several taxa
under the family have been described from the various regions of India (Chitale et al. 2011, Sharma et al. 2012,
Joshi et al. 2012, Singh & Singh 2014, Mohabe et al. 2015, 2016a). Some of the studies especially focused to
know the diversity of the family within the different states of the country (Gupta & Sinha 2012, Singh & Singh
2015). In recent lichenological investigations on Rayalaseema region of Andhra Pradesh, Anantapur, Chittoor
and YSR districts additions have been made in lichen diversity of the region (Anjali et al. 2013, Mohabe et al.
2016b, 2017). So far seven species viz. Diorygma junghuhnii (Mont. & Bosch) Kalb & al., D. kurnoolensis
Mohabe, Nayaka & Reddy, Diploschistes scruposus (Schreb.) Norman, D. actinostomus (Pers. ex Ach.) Zahlbr.,
Graphis neeladriensis Mohabe, Anjali & Nayaka, G. plumierae Vain. and G. subalbostriata Lücking are known
from the state under Graphidaceae. The present investigation on Graphidaceous lichens reports new
distributional records as well as addition to the diversity of Graphidaceae within Rayalaseema forests of Andhra
Pradesh.

A total of 54 samples of Graphidaceous lichens were segregated from Herbarium Department of Botany,
Yogi Vemana University, Kadapa, Andhra Pradesh. The lichens were mostly observed on tree barks, which
were collected from Tirumala hills, Talakona mango orchards and Shikaram between altitudes ranges of 500 to
1000 m in Chittoor and Kurnool district respectively. These collections were made by the authors during 2012–
2014. The morphological features of lichen thallus and lirellae (ascomata) were observed under Leica S8AP0
stereo-zoom microscope. Spot test for colour reaction were carried out by 10% aqueous solution of potassium
hydroxide (K), Steiner’s stable para-phenylenediamine solution (PD) and calcium hypochlorite solution (C).
Light microscope of Leica DM500 was used for anatomical investigation of lirellae. All the measurements of
anatomical structures were taken in water and K while Lugol’s iodine solution was used especially for
Received: 18 June 2017 Published online: 30 September 2017
Mohabe et al. (2017) 4(3): 383–390
hymenium, ascus and ascospores observation. The lichen substances were identified with Thin Layer
Chromatography in solvent system ‘A’ following White & James (1985) and Orange et al. (2001). The other
literatures were followed for identification includes Makhija & Adawadkar (2007), Lücking et al. (2009)
Sharma et al. (2012), Joshi et al. (2012), Lücking et al. (2017) was consulted for nomenclature and
classification.
RESULTS
Taxonomic details and Indian distribution of endemic lichens of Graphidaceae in Andhra Pradesh, India
Figure 1. Endemic lichens of Graphidaceae in Andhra Pradesh, India: (A–D), Fissurina capsulata Makhija & Adaw. [A,
Habit (Scale: 0.5 mm); B, V.s. of lirellae (Scale: 50 µm); C–D, Ascospores within ascus in lectophenol cotton blue (Scale:
25 µm)]; (E–H), Thecaria austroindica (D. D. Awasthi & Upreti) K. P. Singh & G.P Sinha [E, Habit (Scale: 0.5 mm); F,
V.s. of apothecia showing completely carbonized exciple (Scale: 100 µm); G, Ascus with 8-spored ascospores (Scale: 25
µm), H, Brown muriform ascospores (Scale: 25 µm)].
1. Fissurina capsulata Makhija & Adaw., Lichenologist 39(2): 171. 2007. (Figs. 1A–D)
This corticolous species is characterized by yellowish brown, thin, ecorticated thallus; simple to
branched, 0.5–1.0 mm long ascomata, margin with swollen tissue; yellowish brown exciple; clear hyaline
hymenium; 8-spored ascus; hyaline, transversely 3–5-septate, 11–16 × 4–5 µm, halonate ascospores and
lacking lichen substances.
Distribution in India: The species is endemic to India and it was earlier reported from Tamil Nadu (Singh
& Sinha 2010).
Specimen examined: Andhra Pradesh, Chittoor district, Talakona, on bark of Mangifera indica, 539 m,
16.03.2013, ADB & SM 3638 (YVUH); Kurnool district, Shikaram, 7 km before Srisailam, on bark, 745 m,
12.01.2013, SM & KSR 3001 (YVUH).
2. Thecaria austroindica (D. D. Awasthi & Upreti) K. P. Singh & G.P Sinha, Indian Lichens: An Annotated
Checklist, 242, 2010. (Figs. 1E–H)
This corticolous species is characterized by epiphloedal, buff yellow to brownish, evanescent, smooth to
cracked thallus; rounded to elongate or oval, scattered, emergent, 0.2–0.5 mm apothecia; margin thin,
concolorous with thallus; entire, slightly irregular towards outside, divergent labia covered with thalline
Mohabe et al. (2017) 4(3): 383–390
layer; brown black, pruinose, dusty, open, concave disc; completely carbonized exciple; hyaline, inspersed
hymenium; 2–8 spored ascus; brown, muriform, transversely 4–8 septate, longitudinally 1–3 septate, I+ red
brown ascospores; lacking lichen substances.
Distribution in India: The species is endemic to India and it was earlier reported from Arunachal
Pradesh, Assam, Karnataka, Manipur and Odhisa (Singh & Sinha 2010, Devi et al. 2015).
Specimens examined: Andhra Pradesh, Chittoor district, Tirumala hills, on bark, 917 m, 06.02.2013, ADB
& SM 3251 (YVUH); Tirumala hills, Dharmagiri, on bark, 937m, 07.02.2013, SM & ADB 3435 (YVUH);
Shilathoranam, on bark of roadside tree, 973 m, 07.02.2013, ADB & SM 3331 3251, 3332, 3298, 3318, 3340
(YVUH); on bark, 958 m, 05.07.2014, SM & ADB 4059, 4051 (YVUH); Neeladri range, viewpoint, on bark,
06.07.2014, SM & ADB 4160, 4168 (YVUH); Starting of Tirumala hills, upside, on bark, 917 m, 06.02.2013,
ADB & SM 3242 (YVUH).
Taxonomic details and Indian distribution of new lichen records of Graphidaceae for Eastern Ghats
3. Fissurina egena (Nyl.) Nyl. Staiger, Biblioth. Lichenol. 85: 136. 2002. (Figs. 2A–C)
This corticolous species is characterized by greenish brown to olive brown, smooth to cracked,
sometimes verruculose thallus; 0.5–1.5 mm long, immersed, simple to furcate, straight to flexuous,
irregularly branched, ascomata surrounded by white roof like structure; epruinose, slit like disc; entire, non-
carbonized, convergent exciple; hyaline, clear, I− hymenium; indistinct, hyaline hypothecium; immature
ascus and ascospores; simple paraphyses and lacking lichens substances.
Distribution in India: It was earlier known from West-Bengal (Jagadeesh et al. 2006).
Specimen examined: Andhra Pradesh, Kurnool district, Shikaram, 7 km before Srisailam, on bark, 745
m, 12.01.2013, SM & KSR 3014 (YVUH).
4. Graphis crebra Vain. Hedwigia 38: 256. 1899. (Figs. 2D–G)
This corticolous species is characterized by smooth to cracked, whitish grey, 3–5 cm, corticated thallus;
white medulla, with large crystals at the base of exciple; indistinct to brownish prothallus; lirellate ascomata
erumpent, short to elongate, 0.5–2.0 mm long, 0.1–0.5 mm wide, branched, with lateral thalline margin;
entire, black, epruinose labia, exposed disc with white pruina; laterally carbonized 14–25 µm thick exciple;
pale brown, 12–17 µm thick epihymenium; hyaline, inspersed, 85–173 wide, 68–95 µm high, I− hymenium;
hyaline, 17–35 µm high hypothecium; hyaline, transversely 4–9-septate, 16–33 × 4–10 μm ascospores and
contains norstictic acid.
Distribution in India: It was earlier reported from West-Bengal (Jagadeesh et al. 2012).
Specimens examined: Andhra Pradesh, Chittoor district, Talakona, on bark of Mangifera indica, 539 m,
16.03.2013, ADB & SM 3645, 3749 (YVUH); Tirumala hills, Neeladri range, on bark, 06.07.2014, SM &
ADB 4074 (YVUH).
5. Graphis furcata Fée, Essai Crypt. Ecorc.: 40. 1824. (Figs. 2H–K)
This corticolous species is characterized by whitish grey, rough to cracked, ecorticated thallus with black
prothallus; short to elongate, erumpent, 0.1–0.2 mm long, 0.2–0.3 mm wide lirellate ascomata, simple to
partially branched with acute ends, flexuous lirellae laterally covered by the thallus; concealed disc; entire,
often thinly white-pruinose labia; laterally carbonized exciple; clear, hyaline hymenium; 8-spored ascus;
hyaline, ellipsoid to fusiform, transversely 5–11 septate, 22–38 × 8–9 µm ascospores and thallus lacking
lichen substances.
Distribution in India: It was earlier reported from Andaman & Nicobar Islands and Meghalaya (Singh &
Sinha 2010, Singh & Singh 2015).
Specimens examined: Andhra Pradesh, Chittoor district, Tirumala hills, Dharmagiri, on bark, 937 m,
07.02.2013, SM & ADB 3427, 3442 (YVUH); Shilathoranam, on bark, 937 m, 07.02.2013, SM & ADB 3315
(YVUH).
6. Graphis leptocarpa Fée , Essai Crypt. Exot. 36: 1825. (Figs. 2L–O)
This crustose corticolous species is characterized by whitish grey, rough to cracked, 2 cm across thallus
with indistinct prothallus; lirellate ascomata erumpent, short and sparsely branched 0.5–1.0 mm long, 0.01–
0.05 mm wide, with acute ends, with lateral thalline margin, slit like disc; entire, epruinose labia; laterally
carbonized, 19–48 µm thick exciple; greyish brown, 18–28 µm epihymenium; hyaline, inspersed, 73–106
µm high, I− hymenium; hyaline, 29–56 µm hypothecium; hyaline, 53–66 × 12–19 µm, 8-spored ascus;
Mohabe et al. (2017) 4(3): 383–390
Figure 2. New records for Eastern Ghats: (A–C), Fissurina egena (Nyl.) Nyl. [A, Habit (Scale: 0.5 mm); B, Clear
hymenium (Scale: 50 µm); C, Ascus (Scale: 50 µm)]; (D–G), Graphis crebra Vain. [D, Habit (Scale: 0.5 mm); E, Inspersed
hymenium with lateral carbonization (Scale: 50 µm); F, Ascospores in K (Scale: 10 µm); G, Ascopores in I (Scale: 10 µm)];
(H–K), Graphis furcata Fée [H, Habit (Scale: 0.5 mm); I, Clear hymenium with entire labia and lateral thalline margin
(Scale: 50 µm); J, Ascus with ascospores and pyraphyses (Scale: 50 µm.); K, Ascospore (Scale: 25 µm)]; (L–O), Graphis
leptocarpa Fée [L, Habit (Scale: 0.5 mm); M, Entire labia with lateral thalline margin (Scale: 50 µm); N, Ascospores within
ascus in K (Scale: 25 µm); O, ascopores in K (Scale: 25 µm)].
Mohabe et al. (2017) 4(3): 383–390
colourless, transversely 7–11-septate, 27–32 × 6–7 µm ascospores; paraphyses 1.6–3.8 µm thick, contains
stictic and constictic acid.
Distribution in India: It was earlier reported from Assam, Jammu & Kashmir (Daimari et al. 2014, Goni
& Sharma 2015).
Specimen examined: Andhra Pradesh, Chittoor district, Talakona, before jungle thrills, on bark of
Mangifera indica, 539 m, 16.03.2013, ADB & SM 3743 (YVUH).
Taxonomic details and Indian distribution new lichen records of Graphidaceae for Andhra Pradesh
Figure 3. New records for Andhra Pradesh: A, Chapsa leprocarpoides (Hale) Cáceres & Lücking (Scales: 0.5 mm); B,
Diploschistes scruposus (Schreb.) Norman (Scales: 0.5 mm); C, Fissurina cingalina (Nyl.) Staiger (Scale: 0.1 mm); D,
Fissurina comparimuralis Staiger (Scale: 0.1 mm); E, Glyphis cicatricosa Ach. (Scales: 0.5 mm); F, Graphis cincta (Pers.)
Aptroot (Scales: 0.5 mm); G, Graphis duplicata Ach. (Scales: 0.2 mm); H, Graphis handelii Zahlbr. (Scales: 0.5 mm); I,
Graphis lineola Ach. (Scales: 0.5 mm).
7. Chapsa leprocarpoides (Hale) Cáceres & Lücking, Libri Bot. 22: 52, 2007. (Fig. 3A)
This species is characterized by corticolous habitat, pale olive to fawn coloured, ecorticate thallus;
immersed to semi-immersed, rounded to angular, flesh coloured, 0.05–0.2 mm apothecia; pruinose disc and
low uneven to lobed margins; a free, pale to brownish or hyaline proper exciple; 2–4 spored, 40–60×20–30
µm ascus; hyaline muriform, oval-ellipsoidal, 20–25 × 10–15 µm ascospores and lacking lichen substances.
Distribution in India: It was earlier reported from Karnataka (Joshi et al. 2012).
Specimen examined: Andhra Pradesh, Chittoor district, Japali Anjaneya Swamy Temple, on bark, 746.5
m, 13.06.2012, SN & AMR 1819 (YVUH).
8. Diploschistes scruposus (Schreb.) Norman, Nyt Mag. Naturvidensk. 7: 232, 1853. (Fig. 3B)
This is a saxicolous species characterized by greenish grey to brown, rimose-areolate to cracked, upto 3
cm across, epruinose thallus; thin to thick, 0.5–1.5 mm areolae, plane to subconvex at ostiolar region;
Mohabe et al. (2017) 4(3): 383–390
urceolate, subimmersed to sessile, 0.5–1.0 mm, slightly pruinose ascomata; dark brown, concave disc;
hyaline hymenium; cylindricoclavate asci; brown, muriform, ellipsoid, 26–38 × 11–19 µm, transversely 4–7,
vertically 1–3 septate ascospores with longitudinal septa and contains diploschistesic, lecanoric and
orsellinic acid.
Distribution in India: It was earlier reported from Himachal Pradesh, Jammu & Kashmir, Madhya
Pradesh, Maharashtra, Meghalaya, Sikkim, Tamil Nadu, Uttarakhand, West Bengal (Singh & Sinha 2010).
Specimen examined: Andhra Pradesh, Chittoor district, Tirumala hills, starting point, top of the hill, on
rock, 917 m, 06.02.2013, ADB & SM 3176 (YVUH).
9. Fissurina cingalina (Nyl.) Staiger, Biblioth. Lichenol. 85: 128, 2002. (Fig. 3C)
This corticolous species is characterized by greenish grey to greenish brown, smooth to cracked, glossy
thallus delimited by black hypothallus; 1.0–2.5 mm long, branched, immersed, terminally acute lirelline
ascomata; epruinose, slit like disc; entire, non-carbonized, convergent exciple; hyaline, clear, I− hymenium;
indistinct, hyaline hypothecium; 8-spored ascus; hyaline, muriform, transversely 6–9, vertically 2–3 septate,
25–30 × 9–16 µm, halonate ascospores; simple paraphyses and lacking lichens substances.
Distribution in India: It was earlier reported from Kerala, Maharashtra, Manipur and Tamil Nadu (Singh
& Sinha 2010, Singh & Singh 2015).
Specimens examined: Andhra Pradesh, Chittoor district, Talakona, on bark of Mangifera indica, 539 m,
16.03.2013, ADB & SM 3699, 3709 (YVUH); Shilathoranam, on roadside tree bark, 958 m, 05.07.2014,
ADB & SM 4030 (YVUH).
10. Fissurina comparimuralis Staiger. Biblioth. Lichenol. 85: 134, 2002. (Fig. 3D)
This corticolous species is characterized by yellowish to dark brown, slightly verruculose to cracked,
slightly glossy thallus; delimited by thin black hypothallus; 0.3–1.0 mm long, simple to irregularly
branched, curved, immersed, terminally acute lirelline, ascomata with slightly raised thalline margin;
immersed, slit-like disc; entire, non-carbonized, orange, convergent exciple; hyaline, clear, I− hymenium;
indistinct hypothecium; simple paraphyses; 8-spored asci; hyaline, submuriform, transversely 5–6 and
longitudinally 1–2-septate, 21–24 ×5–8 µm, halonate, I+ weak blue ascospores and lacking lichen
substances.
Distribution in India: It was earlier known from Kerala (Sharma et al. 2012).
Specimen examined: Andhra Pradesh, Chittoor district, Talakona, on bark of Mangifera indica, 539 m,
16.03.2013, ADB & SM 3638 (YVUH); Kurnool district, Shikaram, 7 km before Srisailam, on bark, 745 m,
12.01.2013, SM & KSR 3013 (YVUH).
11. Glyphis cicatricosa Ach. Syn. Meth. Lich.: 107. 1814. (Fig. 3E)
This corticolous species is characterized by greenish to yellowish grey or yellowish brown, smooth
thallus; irregular lirellate apothecia embedded in carbonaceous stroma; dark brown, concave disc; hyaline,
clear, 65–125 µm high hymenium, simple paraphyses; 8-spored ascus; transversely 5–10 septate, halonate,
I+ blue, 25–65 x 8–12 µm ascospores with rounded lumina, lacking lichen substances.
Distribution in India: It was earlier reported from Andaman & Nicobar Islands, Goa, Karnataka, Kerala,
Sikkim, Tamil Nadu, West Bengal, Manipur, Meghalaya and Nagaland (Singh & Sinha 2010, Singh &
Singh 2015).
Specimens examined: Andhra Pradesh, Chittoor district, Tirumala hills, Dharmagiri, on bark, 937 m,
07.02.2013, SM & ADB 3440 (YVUH); on road side tree bark, 958 m, 05.07.2014, ADB & SM 4035, 4045,
4029, 4033 (YVUH); Shilathoranam, on bark, 937 m, 07.02.2013, SM & ADB 3318, 3332 (YVUH); Srivari
padalu, on bark, 1076 m, 07.02.2013, ADB & SM 3526, 3535 (YVUH).
12. Graphis cincta (Pers.) Aptroot in A.W. Archer, Fl. Australia 57: 651. 2009. (Fig. 3F)
This corticolous species is characterized by thin, smooth, whitish grey thallus; lirellate, 0.1–0.3 mm
wide, 0.2–1.2 mm long ascomata; epruinose, slit like disc; epruinose, entire labia, sometimes broad at apex;
laterally carbonised, 19–44 μm thick exciple; distinct, brown to dark brown, 15–20 μm thick epihymenium;
inspersed with oil globules, 56–79 μm high hymenium; hyaline, 19–22 μm high hypothecium; hyaline,
clavate, 59–79 × 5–10 μm, 8-spored ascus; hyaline, transversely 4–8-septate, 20–25 × 4–8 μm, I+ blue
ascospores and contains norstictic acid.
Mohabe et al. (2017) 4(3): 383–390
Distribution in India: It was earlier reported from Arunachal Pradesh, Goa, Karnataka, Kerala,
Maharashtra, Manipur, Meghalaya, Nagaland and Tamil Nadu (Singh & Sinha 2010, Singh & Singh 2015).
Specimens examined: Andhra Pradesh, Chittoor district, Tirumala hills, Dharmagiri, on the way, on bark,
937 m, 07.02.2013, ADB & SM 3422 (YVUH); Shilathoranam, on bark of road side tree, 958 m, 05.07.2014,
SM & ADB 4029 (YVUH).
13. Graphis duplicata Ach. Syn. Meth. Lich.: 81. 1814. (Fig. 3G)
This corticolous species is characterized by greyish white, smooth to rough, corticated thallus with brown
to black prothallus; lirellate ascomata, erumpent to prominent, 0.2–1.0 mm long, 0.05–0.1 mm wide, with
indistinct to basal thalline margin, elongate and irregularly branched, striate labia; laterally carbonized
exciple; clear hymenium; hyaline, transversely 7–11-septate, 13–42 × 5–8 µm ascospores and lacking lichen
substances.
Distribution in India: It was earlier reported from Arunachal Pradesh, Karnataka, Kerala, Maharashtra,
Manipur, Meghalaya, Nagaland, Tamil Nadu and Uttarakhand (Singh & Sinha 2010, Singh & Singh 2015).
Specimens examined: Andhra Pradesh, Chittoor district, Thamballapalli, near Shiva temple, on bark, 900
m, 05.01.2013, SM & AMR 2833, 2834 (YVUH).
14. Graphis handelii Zahlbr. in Hand.-Mazz., Symb. Sin. 3: 39 & 40. 1930. (Fig. 3H)
This corticolous species is characterized by greyish white, smooth to cracked or areolate, corticated
thallus; lirellate apothecia erumpent, short to elongate and sparsely to irregularly branched, with lateral
thalline margin; epruinose, exposed disc; entire labia; laterally carbonized exciple; hyaline, inspersed
hymenium; clavate, 8-spored ascus, transversely 5–11-septate, 16–46 × 5–10 µm ascospores and contains
norstictic acid.
Distribution in India: It was earlier reported from Manipur, Meghalaya and West Bengal (Singh & Sinha
2010, Singh & Singh 2015).
Specimen examined: Andhra Pradesh, Chittoor district, Tirumala hills, Kousthubham Cottage, 859 m,
06.02.2013, ADB & SM 3159 (YVUH).
15. Graphis lineola Ach. Lichenogr. Universalis: 264. 1810. (Fig. 3I)
This corticolous species is characterized by greenish grey to yellowish green, rough to cracked, lirellate
apothecia erumpent, with lateral to complete thalline margin; concealed disc; entire, epruinose labia;
laterally carbonized exciple; inspersed hymenium; 8-spored ascus; hyaline, transversely 5–11-septate, 22–42
× 6–9 µm ascospores and lacking lichen substances.
Distribution in India: It was earlier reported from Arunachal Pradesh, Jammu & Kashmir, Karnataka,
Manipur, Tamil Nadu, Uttarakhand, Uttar Pradesh and West Bengal–hills (Singh & Sinha 2010, Goni &
Sharma 2015, Devi et al. 2015, Gupta & Sinha 2016).
Specimen examined: Andhra Pradesh, Chittoor district, Tirumula hills, Kousthubham cottage, on bark,
859 m, 06.02.2013, ADB & SM 3172, 3162 (YVUH).
CONCLUSION
At present Graphidaceae is the third largest family in Andhra Pradesh with 22 species under 7 genera which
is followed by families Physciaceae and Parmeliaceae (Mohabe 2016a). Among these Fissurina capsulata
Makhija & Adaw. and Thecaria austroindica (D. D. Awasthi & Upreti) K. P. Singh & G.P Sinha recorded as
endemic to India. The study also recorded four species viz. Fissurina egena (Nyl.) Nyl., Graphis crebra Vain.,
G. furcata Fée and G. leptocarpa Fée are found first time from southern part (Eastern Ghats) of India. Further,
the present study on lichen diversity of Andhra Pradesh would represent 167 species, while Chittoor district
alone has 92 species. The available information on Graphidaceous lichens will helpful as base line records to
lichen flora of Andhra Pradesh and can be used for biomonitoring studies in future.
ACKNOWLEDGEMENTS
The first author is very grateful to University Grant Commission, New Delhi (F./PDFSS-2015-17-MAD-
12168) and Department of Science & Technology, New Delhi and CSIR, New Delhi (Letter No. 60(0100)/11/
EMR - II, 2012 for financial assistance; the Director and Dr. D.K. Upreti, Chief Scientist, CSIR-National
Botanical Research Institute, Lucknow for providing infrastructure facilities of Lichenology Laboratory for
lichens identification; Mr. K. Suresh Raju for helping in lichen collection and to the Forest official of Andhra
Mohabe et al. (2017) 4(3): 383–390
Pradesh to permit for sample collections.
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4(3): 391–395, 2017
DOI: 10.22271/tpr.2017.v4.i3.051
Research article
Testing of genetic homogeneity of elite eucalyptus clones

using DNA marker
Naseer Mohammad*, Ankur Dahayat, Yogesh Pardhi,
Yogeshwar Mishra and Fatima Shirin
Genetics and Plant Propagation Division, Tropical Forest Research Institute, Jabalpur, India
*Corresponding Author: naseer35518@gmail.com [Accepted: 20 October 2017]
Abstract: Under ‘Hariyali Prasar Yojna’ Chhattisgarh State Forest Department has procured and
planted elite eucalyptus clones on large scale in different forest circles/divisions of the state during
the monsoon of 2015–16 and 2016–17. To assess the genetic homogeneity of the supplied clones,
genetic fidelity testing of the procured clones was carried out using ISSR marker. The
monomorphic pattern of ISSR profiles observed for the ramets of the respective clones in
comparison with their mother plant confirmed the genetic purity. This also demonstrates the
application of molecular marker technology for quality control in social forestry plantation.
Keywords: Eucalyptus - Clonal fidelity - ISSR - Molecular marker.
[Cite as: Mohammad N, Dahayat A, Pardhi Y, Mishra Y & Shirin F (2017) Testing of genetic homogeneity of
elite eucalyptus clones using DNA marker. Tropical Plant Research 4(3): 391–395]
INTRODUCTION
Due to short rotation age, faster growth, multipurpose utility, fire hardiness, good coppicing vigour,
capability to over top weeds, browse resistance, wider adaptability and ease of mass propagation, eucalyptus
became one of the world’s leading industrial plantation species (Palanna 2017, Sumathi & Yasodha 2014). Tipu
Sultan, the ruler of Mysore introduced eucalyptus in India and planted in his palace garden on Nandi hills near
Bangalore around 1790 (ShyamSunder 1984). Afterwards, it was introduced in Niilgiri hills, Tamil Nadu, in
1843 and by 1856; regular plantations of E. globulus were raised for firewood (Wilson 1973). Wider
adaptability of the eucalyptus to degraded and wastelands made it one of the prime species for social forestry
plantations. Presently, India is one of the largest eucalypt growing countries in the tropics with an estimated area
of over 20 million hectares (Varghese et al. 2009). Thus eucalyptus has helped to reduce pressure on natural
forests by meeting requirements of people and industries.
In order to meet the increasing demand for wood and wood industry mass multiplication is being carried out
clonally. Cloning of mature trees is generally preferred over seedling because it fixes genetic gain, interim or
permanent within breeding program, whereas, through seedlings it is often not possible to determine whether
these seedlings have the desired qualities as reflected by its mother plant (Nanda et al. 2004, Venkataramanan et
al. 2015). Vegetative propagation technique is the handiest way to multiply eucalyptus quickly in industrial
forestry.
To realise the advantages of clonal-propagation technique, it is very much necessary to maintain the genetic
purity of the regenerants. Several strategies have been adopted for genetic purity testing. In past, morphological
descriptions, physiological traits, cytological studies, isozymes (Gupta & Varshney 1999, Devarumath et al.
2002, Agnihotri et al. 2009, Singh et al. 2012a, 2012b) and many other techniques have been deployed to assess
the genetic purity of clonally mass propagated plants. However, expression of the morphological and
physiological traits may changes in response to the prevalent environmental conditions (Singh et al. 2013). Due
to cytological aberrations, there are always possibility of phenotypically homogeneous looking plants may
behave differently during flowering/fruiting and later stages, making conclusion about genetic purity invalid.
Both hybridization and PCR based DNA markers have become useful tools for confirming the genetic
uniformity of clonally propagated plants and screening out the off-types. The inherent characteristics of DNA
markers such as abundantness and insensitivity to environmental conditions makes them more useful than
morphological and physiological traits in establishing the identity of particular tree/clone or testing the genetic
Received: 05 August 2017 Published online: 31 October 2017
Mohammad et al. (2017) 4(3): 391–395
purity or tracing its genetic relationship. Earlier, random amplified polymorphic DNA (RAPD), inter simple
sequence repeats (ISSR), amplified fragment length polymorphism (AFLP), restriction fragment length
polymorphism (RFLP), sequence characterized amplified region (SCAR), DNA amplification fingerprinting
(DAF), Arbitrarily primed polymerase chain reaction (AP-PCR) have been successfully employed for fidelity
testing in various plant species (Rani & Raina 2000, Archana et al. 2013, Singh et al. 2013, Mohammad et al.
2016). Among these, ISSR is a very simple, quick, cost-effective, highly discriminative and reliable method that
combines most of the advantages of SSRs and AFLP with the universality of RAPD (Reddy et al. 2002). They
are more useful and reproducible than isozymes, RAPD and less cumbersome and cost effective for routine
application than RFLP (Fang et al. 1997, Reddampalli et al. 2007).
Table 1. Details of the eucalyptus clones procured & planted under ‘Hariyali Prasar Yojna’ by Chhattisgarh State
Forest Department.
Sl. Name of forest circles / divisions Clonal detail
No. selected for Eucalyptus plantation
1 Durg Clone no. 03, Clone no. 07, Clone no. 316 & Clone no. 413
2 Bastar Clone no. 06, Clone no. 07 & Clone no. 316
3 Bilaspur Clone no. 07, Clone no. 316 & Clone no. 413
4 Kanker Clone no. 07, Clone no. 288, Clone no. 316, Clone no. 413
& Clone no. 526
5 Raipur Clone no. 07, Clone no. 288, Clone no. 316, Clone no. 413
6 Manendragarh Clone no. 413
7 Koriya Clone no. 413
8 Surguja Clone no. 413
9 Gariyaband Clone no. 316, Clone no. 413
10 Surajpur Clone no. 413
In the year 2015–16 and 2016–17, Chhattisgarh State Forest Department under the ‘Hariyali Prasar Yojna’
procured more than two cores of plantlets of different eucalyptus clones for plantation in different forest
divisions/circles of the state (Table 1). We have assessed the clonal purity of the plantlets forwarded by the CG
forest department to the institute. Therefore, present study was carried out with a definite aim to ascertain the
genetic homogeneity of the plantlets of different elite eucalyptus clones procured and planted under the scheme
using inter simple sequence repeats (ISSR) markers.

Plant material and DNA extraction
Figure 1. Samples of the eucalyptus clone number 7, 413 and 316 received from Balodabazor forest division (Raipur
Circle), Chhattisgarh for clonal fideliy testing.
Mohammad et al. (2017) 4(3): 391–395
Three to five random samples (ramets) of the each clonal lot along with mother leaf samples were provided
by the Chhattisgarh State Forest Department (Fig. 1). These ramets along with mother leaf samples were
subjected to genomic DNA extraction following extraction method described by Deshmukh et al. (2007). Total
DNA was quantified and its quality was verified by UV spectrophotometer (Cintra 404, Australia) and each
sample was diluted to 40 ng per 3 ul with TE buffer and stored at 4°C. No further purification of DNA before
amplification was found necessary.
PCR Amplification
Five ISSR primers (Tables 2) that were successfully used in our earlier study in Litsea glutinosa (Patel 2015)
were screened on eucalyptus samples. Primer UBC-854 which produces well resolved and consistently
reproducible fragments was selected for further testing.
Table 2. Details of the ISSR primers screened with eucalyptus clonal samples.
Sl. No. Primer Code Primer sequence (5’–3’) Tm GC (%)
1 UBC-821 GTGTGTGTGTGTGTGTT 50.3°C 47
2 UBC-853 TCTCTCTCTCTCTCTCAT 47.6°C 50
3 UBC-854 TCTCTCTCTCTCTCTCGG 51.5°C 44.4
4 UBC-859 TGTGTGTGTGTGTGTGGC 56.1°C 55.5
5 UBC-880 GGAGAGGAGAGGAGA 47.9°C 60
ISSR amplifications were performed in a volume of 10 ul containing 40 ng of genomic DNA, 1X Taq
polymerase buffer, 0.1 mM of each dNTPs, 2.5 mM MgCl 2, 1U Taq polymerase and 0.8 uM of ISSR primer.
The amplification reaction consisted of an initial denaturation step at 94°C for 3 min, followed by 35 cycles of
30 seconds at 94°C (denaturation), 30 seconds at a 50°C annealing temperatures and 1 min at 72°C (extension)
followed by a final extension step at 72°C for 10 min. DNA amplification fragments were separated in 2.0%
agarose gel (SeaKemR LE Agarose) using 0.5X TBE buffer and stained with ethidium bromide. Gels were
visualized using a gel documentation system (Alfa Innotech, USA). The size of the amplification products was
estimated from GeneRulerTM 100-bp DNA ladder (Genetix, Biotech Asia Pvt. Ltd).
RESULTS AND DISCUSSION

Clonal propagation is serving as an important tool for increasing the competitiveness of the forestry based
plantation industry (Sivarajan et al. 2014). However, for use of clonal propagation as continuous source of
planting material for commercial utilization on large scale, periodic monitoring of the genetic purity is of utmost
importance.
Due to continuous cycles of clonal propagation, there are possibilities of somaclonal variations, mutagenic
changes, cytoplasm effects and even admixtures when propagation is on large scale. Due to these, desired
characters may even lost that formed the basis of selection of the elite genotype, thereby resulting in
considerable economic losses. True-to-type clonal fidelity is important for realising the advantages of clonal
propagation. Therefore testing of genetic fidelity becomes very much essential especially in forest trees having
long rotation cycles (Lakshmanan et al. 2007).
Figure 2. Amplification profile of the eucalyptus clones 7, 413 and 316 received from Balodabazor Forest Division (Raipur
Circle), Chhattisgarh using ISSR primer. Where, M- mother plant, 1–3: ramets of the respective clone.
Mohammad et al. (2017) 4(3): 391–395
Many approaches ranging from morpho-metric-physiologic to biochemical features were tried to assess the
genetic fidelity. However, these traits are found not reliable as it got affected by the environment and expression
is stage dependent. DNA-based molecular markers have emerged as a powerful technique for this purpose and
therefore are being used in many crops and trees (Cuesta et al. 2010, Negi & Saxena 2011, Pandey et al. 2012,
Singh et al. 2013, Mohammad et al. 2016).
The amplification profiles of the clonally propagated plantlets of eucalyptus and their mother plant generated
using the ISSR marker UBC-854 is shown in figure 2. Both, ramets of the clones and the mother plant of
respective clones showed an identical banding pattern. These provide the representative example of
monomorphic bands obtained with ISSR primers and has clearly shown the absence of genetic
impurity/admixtures among the tested ramets of the respective eucalyptus clones. The scoring data of well-
resolved bands were subjected to calculation of similarity matrix based on Jaccard’s similarity coefficient. The
pair-wise value of the ramets and the mother plant of respective clones was 1, indicating 100% similarity. This
confirmed the true-to-type nature of the clonal lot supplied for testing. This also establishes the usefulness of
ISSR marker system in ascertaining the genetic purity of clones. Earlier also, ISSR markers were successfully
employed in bamboo species (Agnihotri et al. 2009, Singh et al. 2013), date palm (Kumar et al. 2010), apple
(Gupta et al. 2009), jalamdasa (Chandrika & Rai 2009), acacia (Nanda et al. 2004), stevia (Lata et al. 2013),
albizia (Mohammad et al. 2016) and by many others.
CONCLUSION
From the monomorphic pattern of ISSR amplification, we may conclude that ramets of the respective
eucalyptus clones supplied for testing is genetically homogeneous and corresponds to mother sample provided
for testing.
ACKNOWLEDGEMENTS
The authors are grateful to Dr. U. Prakasham, Director, Tropical Forest Research Institute, Jabalpur for
providing necessary facilities. Authors are also thankful to the. Chhattisgarh State Forest Department for
providing the clonal material for testing.
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ISSN (E): 2349 – 1183
ISSN (P): 2349 – 9265
4(3): 396–400, 2017
DOI: 10.22271/tpr.2017.v4.i3.052
Research article
Studies on the reproductive biology of

Wiesnerella denudata (Mitt.) Steph. - a rare hepatic
Anu Sharma* and Madhu Bhagat
Department of Botany, University of Jammu, Jammu-180006, Jummu & Kashmir, India
*Corresponding Author: anu4botany@gmail.com [Accepted: 22 October 2017]
Abstract: Wiesnerella denudata is a rare monotypic thalloid hepatic of the family Wiesnerellaceae
(Order: Marchantiales). During the course of present investigation, ten districts of Jammu region in
Jammu& Kashmir were explored, but the only single population was collected from Dhera Ki
Gali, Poonch. As it is a rare hepatic, the present communication deals with its reproductive
biology. It was interesting to note that the species does not undergo propagation by any vegetative
means. Further, the thalli with only female receptacles were recorded. However, well-developed
sporogonium differentiated into the foot, seta and capsule were observed along with a very high
spore-elater ratio. The status of the species in the Jammu region of Jammu and Kashmir (North-
West Himalayas) with special emphasis on its unique reproductive biology and the consequences
of its dependence on only a single mode of propagation (sexual) have been discussed.
Keywords: Hepatic - Monotypic - Rare - Spore-elater ratio - Mycorrhiza.
[Cite as: Sharma A & Bhagat M (2017) Studies on the reproductive biology of Wiesnerella denudata (Mitt.)
Steph. - a rare hepatic. Tropical Plant Research 4(3): 396–400]
INTRODUCTION
Wiesnerella denudata (Wiesnerellaceae; Marchantiales) is distributed in Eastern and Western Himalayas,
Indonesia, China, Korea, Hawaii and Borneo (Chang & Wu 2006, Ludwiczuk & Asakawa 2010). In India, it has
been reported from Jammu & Kashmir (Tanwir & Langer 2007), Himachal Pradesh (Kaur et al. 2010),
Nagaland (Eshuo 2012), Sikkim (Singh et al. 2008), West Bengal (Hattori 1966) and Uttarakhand (Kanwal
1977). From Nainital, it was recorded from Dhobighat, growing on the black muddy stream associated with
several other liverworts (Conocephalum conicum (L.) Dum., Dumortiera hirsuta (Sw.) Nees, Marchantia
paleacea Bertol. and Pellia endivaefolia (Dicks.) Dum. (Kanwal 1977). However, it could not be collected from
Dhobighat nor anywhere else in Nainital (Pant 1983). According to her, indiscriminate and ruthless collection of
this liverwort resulted in its disappearance and she listed it in the threatened category along with other nine
hepatic taxa [Conocephalum conicum (L.) Dum., Dumortiera hirsuta (Sw.) Nees, Reboulia hemispherica (L.)
Raddi, Sewardiella tuberifera Kashyap, Wiesnerella denudata (Mitt.) Stephani, Cryptomitrium himalayensis
Kash., Fossombronia himalayensis Kashyap, Athalamia pinguis Falc. and Stephensoniella brevipedunculata
Kash.]. Other workers like Udar & Srivastava (1983), Pant et al. (1994) and Srivastava (1998) enlisted it in the
rare category in Kumaon and Eastern-Western Himalaya.
Several studies have been conducted on the chemical constituents of Wiesnerella denudata (Asakawa et al.
1980, Kumar et al. 2007, Kaur et al. 2010, Huang et al. 2012) but studies on its other aspects have received less
attention. During the present investigation, a single population of the genus was collected from Poonch district
at an elevation of 2350 m inhabiting rock surface. Ecological data collected for this accession has been tabulated
in table 1. Present paper deals with the morphology, anatomy and reproductive biology of Wiesnerella denudata
thus collected.

Frequent visits were made in different districts (Doda, Jammu, Kishtwar, Kathua, Poonch, Rajouri, Ramban,
Reasi, Samba, Udhampur) of Jammu division ranging in altitude from 305–2350 m. Only one accession
inhabiting rock surface was collected from Dherakigali (Poonch). In order to record various stages of the life
Received: 26 June 2017 Published online: 31 October 2017
Sharma & Bhagat (2017) 4(3): 396–400
cycle, periodic visits were made at the site of collection of Wiesnerella denudata in Poonch district. Data on the
date of collection, altitude, temperature and relative humidity of the place of the collection was recorded.
Observations on habitat, morphology, number of receptacles/thallus, their shape, size and position of antheridia,
archegonia, sporophytes/receptacle per thallus and number of spores and elaters/capsule were made. Spore elater
ratio per capsule was determined by applying the formula:
Total number of spores per capsule / Total number of elaters per capsule.
Repeated the same procedure for five capsules and took out the mean value.

Phenology
In the month of August with frequent showers, thalli started rejuvenating from the dried patches. The thalli
remained in vegetative phase until the end of September. In the second week of October, sessile and small
female receptacles started appearing. Archegonia with long necks developed between mid-November and the
end of December. Male receptacles could not be collected due to heavy snowfall in the site of the collection
during January to March. Fertilization might have occurred during mid-December to January and sporophytes
started developing by the end of January. The mature sporophytes containing spores and elaters were seen in the
first week of April. Temperature range has been tabulated in table 1.
Table 1. Details of asexual and sexual reproduction.
Site of Altitude Date of Temperature Humidity Asexual Male Archegonia
pH
collection (m) collection (ºC) (%) reproduction Receptacle Y M CD
Dherakigali 2350 7.5 15.10.2012 20–29 46–68 Not observed Not + - -
(DKG) 25.12.2012 4.7–16 39–53 collected + - -
(Epilithic) 05.04.2013 25–29 78–90 - + +
23.02.2014 5.8–7.2 78–94 + - -
Note: Y = young; M = mature; CD = capsules dehisced; + = Present; - = Absent.
Morphology
Thalli of W. denudata are dichotomously branched, 2.0–3.5 cm long and 1–2 cm broad, nearly flat above,
margin wavy, lobes oblong to quadrate, notched at the apex and occur in large extended yellowish green or light
green patches of irregularly overlapping individuals as shown in figure 1A. Dorsal surface with distinct areolae;
ventral surface green with inconspicuous scales along the midrib region (Fig. 1B); air pores large, slightly
convex bounded by 2–3 rings of cells with 5–8 cells per ring, spherical to elliptical in surface view (Fig. 1C);
Scales in two rows, lunate with a single filiform appendage. Rhizoids of two types, smooth walled and
tuberculated, the former being more in frequency as shown in figure 1D.
Anatomy
Thallus differentiated into dorsal narrow photosynthetic and ventral wider storage zone. The air pore lacks
the papillae. Photosynthetic zone comprises of unistratose air chambers, chlorophyllous; air chambers small,
distant, filled with rudimentary filaments (Fig. 1E).
Reproduction
It reproduces only by sexual means. So far, no vegetative/asexual means of reproduction have been reported
nor seen in the population under study (Table 1).
Plant monoecious or dioecious; Tanwir (2005), reported male plants from the area and described their
position on the mid-dorsal groove of the thallus, behind the stalk of female receptacle or at notch, sessile to
subsessile, cushion-like surrounded by short, hyaline papillose scales; female receptacle in the present study
were 1–3 (1.8±0.45) per thallus, terminal arising from deep narrow notchat the apex, stalked; stalk 3–11
(6.9±1.79) mm with two rhizoidal furrows, receptacle convex, 4–6 (5.1±0.50) lobed; diameter of disc 2–5
(3.8±0.53) mm, involucres under the lobe (Fig. 2A; Table 1). Female receptacles had long necks before
fertilization (Fig. 2B) and after fertilization neck started degenerating and venter broadened. Sporogonium
differentiated into the foot, seta and capsule (Fig. 2C). The diameter of the capsule was 1510.6–1643.4 µm
(1565.38±29.29). Spores having diameter 36.56–49.80 µm (34.69±3.73) were yellowish brown to dark brown,
reticulate, winged; wings broad, lobed, wavy (Fig. 2D); elaters yellowish brown, narrow, and bispiralled (Fig.
2E); 265.6–398.4 µm (335.32±22.94) long and 4–8 µm (7.28±0.77). Spore and elater output were 1302–3435
(2744.42±492.6) and 107–319 (251±45.52) per capsule, respectively. Though Tanwir (2005), collected male
Sharma & Bhagat (2017) 4(3): 396–400
receptacles from this area, during the present study, it was not possible due to heavy snowfall in the area from
January to March.
Figures 1. A, Patch of Wiesnerella denudata growing on epilithic habitat; B, Thalli in dorsal and ventral view; C, Whole
mount of epidermal peeling showing air pore; D, Whole mount of smooth walled and tuberculated rhizoids showing hyphal
strands and a vesicle; E, V.S. of thallus showing upper photosynthetic and lower storage zone (Inset- Notice raised air pore).
Huang et al. (2012) and Chang & Wu (2006) have reported the genus to be dioecious. According to
Lindberg (2000), a higher proportion of dioecious species as compared to monoecious ones fail to produce
sporophytes. Low spore output may be a possible cause of rarity for dioecious hepatics but in the present case,
no rarity has been observed in spore production. Moreover, the spore elater ratio has been recorded to be higher
compared to the normal ratio found in Marchantialean members (4:1) (Schuster 1996). Thus, high spore output
indicates its successful sexual reproduction and survival rate. Furthermore, a smaller number or absence of
asexual propagules coincides with having a large number of spores (Soderstorm & During 2000). In present
work also, the authors observed the absence of any asexual structures. It means that asexual reproduction has no
role to play in the propagation of the species. But what about its rare nature? It is a serious question to ponder
about. Since the population is producing sporophytes, it is obvious that sexual reproduction is taking place
successfully. Though the authors were not able to collect male plants because of high snowfall in the area from
January to March, it is quite obvious that both the sexes were present in the population. The serious situation is
that if the plant is not producing any vegetative structures for propagation, what would happen if in near future
Sharma & Bhagat (2017) 4(3): 396–400
the habitat destruction ruins the limited population. According to Miles & Longton (1990), spores act as a more
efficient means of long-distance dispersal than vegetative propagation. In the present case, the populations are
not distributed to the wide range; it means that spore dispersal is occurring within a narrow patch. This may lead
to less genetic diversity/low variability within the population.
Thus the possible causes of its rarity in Jammu & Kashmir may be:
(1) lack of vegetative reproduction,
(2) low dispersal range of spores and thus low genetic variability,
(3) low spore viability in spite of high spore output.
Figures 2. A, Fully mature female receptacles on the thallus; B, V.S. of female receptacle passing through archegonia
bearing long necks; C, V.S. of female receptacle showing post fertilization stage with mature sporophyte bearing foot, seta
and capsule; D, Whole mount of spores and elaters (Inset- Spores at high magnification); E, Whole mount of elater showing
long and bispiralled bands.
CONCLUSION
The genus is rare, and if in near future the restricted populations are not able to withstand the adverse
environmental threats, the species may most likely fall into the category of threatened hepatics. So, the final
conclusion may be drawn only after studying more and more populations with an emphasis on its genetic
structure along with their spore viability. Further studies and comparison of spore elater ratios of different
populations are needed to arrive at any definite conclusion.
Sharma & Bhagat (2017) 4(3): 396–400
ACKNOWLEDGEMENTS
The authors wish to thank the Head, Dept. of Botany, University of Jammu, for providing the necessary
laboratory facilities. Special thanks to Dr. S. K. Singh (Scientist C, BSI, Eastern Regional Centre) for
identifying the plants and to Meteorology Department, J & K for providing the temperature and relative
humidity data. The authors declare that they have no conflict of interest.
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metapopulation dynamics. Journal of Bryology. 27: 261–268.
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Tanwir M (2005) Studies on the diversity of hepatic flora of district Poonch (North-West Himalaya). M. Phil.
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ISSN (E): 2349 – 1183
ISSN (P): 2349 – 9265
4(3): 401–404, 2017
DOI: 10.22271/tpr.2017.v4.i3.053
Research article
Phytochemistry and total phenolic content of methanol extract of

Pometia pinata J.R. Forst. & G. Forst. fruit flesh
from Papua, Indonesia
Candra Irawan1, Hanafi2, Lilis Sulistiawaty1* and Henny Rochaeni1
1
Departement of Analytical Chemistry Polytechnic of AKA Bogor, Bogor-16158, Indonesia
2
Departement of Food Industrial Quality Assurance Polytechnic of AKA Bogor, Bogor-16158, Indonesia
*Corresponding Author: lilis.anira@gmail.com [Accepted: 25 October 2017]
Abstract: The Pometia pinnata fruit flesh from Papua, Indonesia is widely used in traditional
medicine has been extracted. The extraction has used the solvent of methanol, ethyl acetate, and n-
hexane. The yield of extraction in methanol, ethyl acetate, and n-hexane were 21.65%, 2.92% and
0.91%. Qualitative analysis of phytochemical constituents in methanol were tannins, phenolic and
steroid, more complete contents of secondary metabolite, compared to those of n-hexane and ethyl
acetate extracts. Quantitative analysis for total phenolic compound was 393.38±0.28 mg gallic acid
equivalent (GAE.g-1).
Keywords: Phytochemistry - Total phenolic content - Pometia Pinata.
[Cite as: Irawan C, Hanafi, Sulistiawaty L & Rochaeni H (2017) Phytochemistry and total phenolic content of
methanol extract of Pometia pinata J.R. Forst. & G. Forst. fruit flesh from Papua, Indonesia. Tropical Plant
Research 4(3): 401–404]
INTRODUCTION
The Pometia pinnata J.R. Forst. & G. Forst. fruit flesh from Papua, Indonesia is widely used in traditional
medicine has phytochemical compound. Biologically active, phytochemicals are naturally occurring in the
plants, which provide health benefits for humans (Hasler et al. 1999, Trimedona et al. 2015). They protect
plants from disease and damage and contribute to plant’s color, aroma and flavor. Secondary constituents are the
remaining plant chemicals such as alkaloids, terpenes, flavonoids, lignans, plant steroids, curcumines, saponins,
phenolics, flavonoids and glucosides (Alapati & Sulthana 2015).
Pometia pinnata J.R. Forst. & G. Forst. a member of Sapindaceae family, is widely distributed in Asia
Pacific included Papua, Indonesia (Trimedona et al. 2015). The flesh of Pometia pinnata are used in traditional
medicine are hypertensi, abdominal ailments including stomach complaints, diarrhea, dysentery, obstetric and
gynaecological complaints. The compunds of the flesh of Pometia pinnata was different from the other organ
of Pometia pinnata, so phytoscreening in flesh of Pometia pinnata were investigated.
Polyphenolic nature enables them to scavenge injurious free radicals such as super oxide and hydroxyl
radicals (Dewick et al. 2002). The flesh of Pometia pinnata can be regarded as promising plant species for
natural plant sources of antioxidants with high potential value for drug preparation (John et al. 2014). The aims
research to knowed composition of the secodary metabolites and total phenolic compound of n-hexane, ethyl
acetate and methanol extract from Pometia pinnata.

Protocols and instruments
Pometia pinnata were harvested from local market in Pontianak, West Kalimantan, Indonesia The fruit flesh
of Pometia pinnata was drained in room temperature, and then were powdered. In addition all chemicals used
were of analytical grade, are Folin-Ciocalteu reagent, galic acid, sodium carbonate solution, Dragendorff’s
reagent, Mayer’s reagent, methanol, ethyl acetate, n-hexane, concentrated sulfuric acid, concentrated HCl, ferric
chloride hexahydrate (FeCl3.6H2O), DMSO, acetic acid anhydride, acetic acid glacial, and chloroform were
purchased from Merck.
Received: 25 July 2017 Published online: 31 October 2017
Irawan et al. (2017) 4(3): 401–404
Sample extraction
Sample preparation was conducted by maceration process using several organic solvents used Irawan
method (Irawan et al. 2017). 135 g of powdered fruit flesh of Pometia pinnata were immersed in 100 mL of n-
hexane for 3 days, and then filtered. Filtrate was then evaporated until dry sample was obtained, and this step
resulted in raw extract of n-hexane. The residue from first immersion was entirely immersed back in 100 mL
ethyl acetate for 3 days to obtain raw extract of ethyl acetate. The solution was then filtered and evaporated, and
the residue from this step was immersed in 100 mL methanol for 3 days, resulted in raw methanolic extract. The
maceration process was repeated several times to obtain clear extract containing all expected chemical species.
Phytochemical assay
The reason of solvent choosing based on caracteristic of polarity metabolite seconder compound. The assay
included several test for alkaloid, tannin, saponin, reducing sugar, flavonoid, glucoside, phenolic,
glycosidesteroid, and sterol - triterpenoid according to the method describe by Tiwari et al. (2011).
Total phenolic content
The total phenolic content of the extract was determined by the Folin–Ciocalteu method (Tiwari et al. 2011),
200 μL of crude extract (1 mg.mL-1) were made up to 3 mL with distilled water, mixed thoroughly with 0.5 mL
of Folin–Ciocalteu reagent for 3 min, followed by the addition of 2 mL of 20% (w/v) sodium carbonate. The
mixture was allowed to stand for 60 min in the dark condition, and absorbance was measured at 650 nm. Total
phenolic content was then calculated from the calibration curve, and the results were expressed as mg of gallic
acid equivalent of g dry weight.
RESULT AND DISCUSSION

Sample extraction
The results showed that different extracting agent resulted in different percentage of yield. From 135 g dry
flesh of fruit of Pometia pinnata, it yielded 1.23 g (0.91%) of yellow solution of raw n-hexane extract, 3.94 g
(2.92%) of brownish yellow solution of raw ethyl acetate extract and 29.24 g (21.65%) of blackish yellow of
raw methanolic extract. The results showed that the methanolic extract contains the largest yield compared to
the other types of extracts. The percentage of yield of extract indicated the extracting capacity of extracting
agent. The highest yield of methanolic extract indicated that methanol has the highest extracting capacity for
secondary metabolite in the flesh of fruit of Pometia pinnata. On the other side, the lowest yield for n-hexane
related to the fact that n-hexane has the lowest extracting capacity. Azmir et al. (2013) stated that the
efficiencies of extraction methods mostly depend on the understanding the nature of plant matrix and chemistry
of bioactive compounds. The possible explanation for this phenomenon was the fact that the secondary
metabolites contained in the methanolic extract were polar or semipolar thus needed the extracting agent which
has the similar polarity. This explanation must be supported by further phytochemical assay.
The physical appearance of the extract solution also provided supporting information that different kinds of
the secondary metabolites were extracted from different solvent. Phytochemical assay of n-hexane, ethyl
acetate, and methanol raw extract revealed that methanolic extracts had phenolic and tannin compound (Table
1). Methanolic extract showed the most complete contents of secondary metabolite, compared to those of n-
hexane and ethyl acetate extracts.
Table 1. Phytochemical assay of flesh of fruit of Pometia pinnata J.R. Forst. & G. Forst.
n-hexane ethyl acetate methanolic
No Parameter
extract extract extract
1 Alkaloid + - -
2 Flavonoid - - -
3 Tannin - - +
4 Phenolic - - +
5 Saponin - + -
6 Sterol Triterpenoid + - +
Total phenolic contents

Total phenolic content of the methanolic flesh of fruit of Pometia pinnata, calculated from the calibration
curve R2 = 0.9978 (Fig. 1.), was 393.38±0.28 mg gallic acid equivalent (GAE.g-1) (Table 2). Phenolic
compounds have redox properties, which allow them to act as antioxidants. The phenolics are composed of one
Irawan et al. (2017) 4(3): 401–404
or more aromatic rings bearing one or more hydroxyl groups and are therefore potentially able to quench free
radicals by forming stabilized phenoxyl radicals (Soobrattee et al. 2005). Phenolic compound could be content
of terpenoid compund, flavonoid or the other phenols. The total phenolic concentration could be used as a basis
for rapid screening of antioxidant activity. The antioxidant activity of which depends on the presence of free OH
groups, especially 3-OH.
Figure 1. Calibration curve of Gallic acid
Table 2. Total phenolics content of methanolic extract of fruit flesh of Pometia pinnata.
No. Total phenolics contenta
1 39.31
2 39.85
3 40.26
Average 39.81±0.48
Note: a = mg gallic acid equivalent (GAE.g-1).
CONCLUSION
This present study proved that the methanol extract of fruit flesh of Pometia pinnata was the most complete
compund compeared n-hexane and ethylacetate extract. The methanol extract of fruit flesh of Pometia pinnata
presence of total phenolics contained 393.38±0.28 mg GAE.g-1. Our result suggested that fruit flesh of Pometia
pinnata is a potential source of antioxidant agents and can be used as a natural antioxidant and preservative in
food and non-food systems. However, further phtyochemical analysis is required to isolate the elements of the
plant that show a broad spectrum of pharmacological activity.
ACKNOWLEDGEMENTS
This work was financially supported by Polytechnic of AKA Bogor, Indonesia. The authors are also grateful
to the institution for providing several apparatus and instrumentation. There is no conflict of interest among
authors.
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International Journal of Advanced Research 3(10): 1391–1398.
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(2013) Techniques for extraction of bioactive compounds from plant materials : A Review. Journal of Food
Engineering 117: 426–436.
Dewick PM (2002) Medicinal natural products: A Biosynthetic Approach. John Wiley & Sons England, pp. 76–
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Nutrition 129: 756S–757S.
Irawan C, Foliatini & Hanafi (2017) GC-MS Composition of Leaf Extract of Piper cf.arcuatum Blume and Their
Antioxidant Activity and Toxicity Studies. Journal of Pharmacognosy and Phytochemistry 6(4): 461–468.
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John B, Sulaiman CT, George S & Reddy VRK (2014) Total Phenolics And Flavonoids In Selected Medicinal
Plants From Kerala Biju. International Journal of Pharmacy and Pharmaceutical Sciences 6(1): 406–408.
Soobrattee MA, Neergheen VS, Luximon Ramma A, Aruoma OI & Bahorun OT (2005) Phenolics as potential
antioxidant therapeutic agents: mechanism and actions. Mutation Research/Fundamental and Molecular
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Tiwari P, Kumar B, Kaur M, Kaur G & Kaur H (2011) Phytochemical Screening and Extraction : A Review.
Internationale Pharmaceutica Sciencia 1(1): 103–104.
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pinnata Forst & Forst. Journal of Chemical and Pharmaceutical Research 7(11): 225–227.
ISSN (E): 2349 – 1183
ISSN (P): 2349 – 9265
4(3): 405–418, 2017
DOI: 10.22271/tpr.2017.v4.i3.054
Research article
Pseudomonas putida and Bacillus amyloliquefaciens alleviates the

adverse effect of pesticides and poise soil enzymes activities in
chickpea (Cicer arietinum L.) rhizosphere
Manoj Kumar1,2, Mohd Aslam Yusuf2, Puneet Singh Chauhan1,
Manisha Nigam3 and Manoj Kumar1*
1
Division of Plant Microbe Interactions, CSIR-National Botanical Research Institute, Rana Pratap Marg,
Lucknow-226001, Uttar Pradesh, India
2
Department of Bioengineering, Integral University, Lucknow-226026, Uttar Pradesh, India
3
Department of Biochemistry, Hemvati Nandan Bahuguna, Garhwal University, Srinagar,
Garhwal-246174, Uttarakhand, India
*Corresponding Author: manojyd1234@gmail.com [Accepted: 17 November 2017]
Abstract: Pesticide application for disease management is a major action for crop protection from
last seven decades. The repeated application of pesticide is the most important cause of the
reduction in microbial population. Soil microorganisms play an important role in efficient
acquisition and transportation of nutrients to plant. Pesticides leached in soil disturb the activities
of soil enzymes, such as β-glucosidase, dehydrogenase, phosphatases, protease and urease secreted
by these microorganisms. This drastically reduces nutrient availability to the plants and soil
fertility. In vitro experimental studies revealed that our PGPR (Pseudomonas putida and Bacillus
amyloliquefaciens) have the ability to tolerate pesticides at concentrations such as Carbendazim
(0.512%), Imidacloprid (3.27%) and Glyphosate (3.27%). We have observed an increase in PGP
activities like IAA production, exopolysachchride production, biofilm synthesis, phosphate
solubilization and siderophore production on the addition of pesticides at concentrations below
there threshold values, on the contrary reduction in activities was noticed above these values. Soil
enzymes activities from chickpea rhizosphere without PGPR inoculation showed variability on the
application of pesticides whereas activities were found normal or increased with PGPR inoculation
and pesticides application. Thus PGPR remains panacea for soils by managing adverse effects of
pesticide application. Hence our results concluded that P. putida and B. amyloliquefaciens have
the ability to reduce the negative impact of three pesticides and poise soil enzymes activities.
Hence our PGPR acts as efficient biofertilizers to improve soil fertility and soil health.
Keywords: Carbendazim - Chickpea - Glyphosate - Imidacloprid - PGPR - Soil enzymes.
[Cite as: Kumar M, Yusuf MA, Chauhan PS, Nigam M & Kumar M (2017) Pseudomonas putida and Bacillus
amyloliquefaciens alleviates the adverse effect of pesticides and poise soil enzymes activities in chickpea (Cicer
arietinum L.) rhizosphere. Tropical Plant Research 4(3): 405–418]
INTRODUCTION
Chickpea (Cicer arietinum L.) is the third most important legume crop. India produces nearly 75% of
world’s chickpea production. Often it is grown after cereal crops to interrupt onset of disease, weed control and
improve soil nitrogen content. Seed is the main edible part of the plant and is a rich source of carbohydrate
(48.2–67.6%), protein (12.4–31.5%) and fat (6%) especially for the vegetarian people (Rao 2010). Chickpea can
fix atmospheric nitrogen through its symbiotic association with Rhizobium species thus helping in enhancing the
soil quality. Pests involving insects, fungi and weeds are the major biotic factors which reduce crop yield by
affecting the root-shoot growth. The key pest that limits pulses improvement in India comprises pod borers,
foliar diseases and weeds. Pesticides uses remain minimal for pulses in India (Reddy & Reddy 2010). In a
survey of pesticide use on pulse crop in four regions revealed that use of fungicides is 4–12%, herbicides 0–24%
Received: 02 August 2017 Published online: 30 November 2017
Kumar et al. (2017) 4(3): 405–418
and insecticides 16–50% (IIPR 2010–11). To increase pulses production for the ever-increasing population
application of chemical pesticides ensured high crop productivity.
Pesticides are substances or mixtures of substances proposed for destroying, preventing, mitigating or
repelling pests (Grube et al. 2011). Pesticide application to the crop exposes soil and its microflora to its adverse
effects. These includes pesticide interaction with soil enzymes (Gianfreda & Rao 2008), its binding with the
active site of the soil enzymes affecting their catalytic activities (Tabatabai 1994) and/or pesticides utilized as a
nutrient source by soil microbes resulting in biodiversity changes. A number of pesticides stimulate the growth
of microorganisms, but other pesticides inhibit or have no effects on microorganisms when used at normal rates.
We have included three pesticides i.e. Carbendazim, Imidacloprid and Glyphosate in our study. Glyphosate (N-
(phosphonomethyl) glycine) a herbicide was commercialized in 1974. It has been demonstrated that the
glyphosate can impact the rhizosphere community (Sheng et al. 2012) and rhizosphere processes (Ahemad &
Kibret 2013). Among herbicides glyphosate [N-(phosphonomethyl) glycine] is the non-selective systemic
herbicide most commonly used in agricultural practices on a global scale (Mijangos et al. 2009). Once
glyphosate is absorbed by foliage, it is transported throughout the whole plant via the phloem to the growing
tissues such as the shoot and root meristems. This mechanism results in glyphosate being exuded into
rhizosphere soil (Neumann et al. 2006). Glyphosate [N-(phosphonomethyl) glycine] inhibits the enzymatic
activity of 5-enolpyruvoyl-shikimate-3-phosphate synthase (EPSPS) in the shikimate pathway, thus preventing
the synthesis of aromatic amino acids needed for plant growth and survival (Duke 2005). Carbendazim (methyl
benzImizol-2-yl carbamate) is a systemic fungicide. It acts by interfering with microtubule formation during
mitosis in fungal cells. The repeated use of pesticides into the crops may reach into the soil which disturbs local
metabolism or enzymatic activities (Engelen et al. 1998, Liu et al. 2008, Hussain et al. 2009). Negative impacts
of pesticides on soil enzymes such as dehydrogenase, oxidoreductases and hydrolase activities have been widely
reported in the literature (Perucci & Scarponi 1994, Malkomes & Dietze 1998, Monkiedje & Spiteller 2002,
Menon et al. 2005, Caceres et al. 2009). Imidacloprid (N-{1-[(6-Chloro-3-pyridyl) methyl]-4,5-dihydroImizol-
2-yl}nitramide) is a systemic insecticide which works as insect neurotoxin of neonicotinoids class and acts on
the central nervous system of insects. It is much low toxic for mammals also. Imidacloprid has become a
popular insecticide worldwide due to systemic mode of action and low toxicity to humans and its use in field
crops, vegetables and ornamentals (Ishaaya & Horowitz 1988, Matsuda et al. 2001, Nauen & Denholm 2005)
Imidacloprid is registered in approximately 120 countries and is used on over 140 different agricultural crops
(Buffin 2005) including chickpea.
Soil fertility is the result of the effective and cumulative role played by beneficial microbes in the soil.
Pesticides uses have an adverse effect on useful microorganisms in rhizoshpere. Vice versa microbes have the
ability to degrade pesticides and nullify influence of pesticides on microbial diversity in soil. The knowledge of
these processes and extent of its ability to minimise losses by pesticides are still not well studied. To prevent the
adverse effect of pesticides, rhizobial inoculants often used as bio-fertilizer and have the ability to maintains the
nutrients and degrade the pesticides when applied to soil/seeds of legumes (Lo 2010). Since plant growth
promoting rhizobacteria (PGPR) play positive role on plant health through a variety of mechanisms, including
mineralization of nutrients, suppression of disease, improving plant stress tolerance and production of
phytohormones (Berendsen et al. 2012, Figueiredo et al. 2011, Gupta et al. 2000). Soil enzyme activities are the
direct reflection of the soil community to metabolic requirements and available nutrients. The interaction
between soil components and pesticides influences the biochemical activity of bacteria. Telluric fungi
(Hernandez-Rodriguez et al. 2006, Ronhede et al. 2007) and bacteria (Dong et al. 2005) are able to degrade or
mineralize pesticides by enzymatic reactions. There is evidence that soil enzymes may provide valuable
information on the transformation of pesticides in soils (Gianfreda & Baollag 1994, Kalam et al. 2004, Gil-
Sotres et al. 2005, Hussain et al. 2009). The enzymes are active outside the cell where they catalyze reactions to
break down the structure of the nutrient source to make it more accessible, but pesticides affect the soil enzyme
and disturb the cycle. In India Maharashtra, Andhra Pradesh (including Telangana & Seemandhra), and Punjab
are top three states which contribute about 45% consumption of pesticides. Andhra Pradesh is the highest
pesticides consumer with 24% share. Soil enzymes are dehydrogenase, acid/alkaline phosphatase, β-
glucosidase, urease, protease. Decrease urease activity in soil due to the application of pesticides reduces urea
hydrolysis, which is generally beneficial, because it helps to maintain nitrogen availability to plants.
In the this paper we have reported that plant growth promoting traits of the bacteria such as biofilm
Kumar et al. (2017) 4(3): 405–418
formation, mineral phosphate solubilisation, siderophore production, IAA production and EPS formation
activity were estimated in presence of different concentrations of Carbendazim (fungicide), Imidacloprid
(insecticide) and Glyphosate (herbicide) with the help of standard protocols routinely used in our laboratory. In
our study, we have taken chickpea plant (BG-362) to evaluate the PGPR effect in presence of different
pesticides on plant growth parameters. For this, we have used the B. amyloliquefaciens (SN13) and P. putida
(RA) to see the effect of pesticides on them and Maximum Tolerance Level (MTL) was determined in vitro. In
vitro tests were performed to find the interaction between the PGPR and Pesticides, while in vivo tests were
performed to see the effects of pesticides on chickpea fortified with PGPR. We also did glasshouse experiments
to see the effect of pesticide sprays on soil enzymatic activities of chickpea rhizosphere treated with PGPR.

In vitro PGP traits evaluation of PGPR in presence of pesticides
Pseudomonas putida (RA) and Bacillus amyloliquefaciens (SN13) used in the study were procured from the
lab depository, Division of plant-microbe interactions, CSIR-NBRI. Pesticides used in this study were
Carbendazim (Car), Glyphosate (Gly) and Imidacloprid (Imi). Earlier we have performed in vitro PGP traits
evaluation of both the bacterium and their synergistic effect for drought amelioration in chickpea (Kumar et al.
2016).
Assessment of bacterial strains for pesticides tolerance
The bacterial strains were tested for their pesticide tolerance onto agar plate 4/5 dilution method (Gupta et al.
1994) using nutrient agar medium. The freshly prepared agar plates were spreaded with 48 hrs grown P. putida
and B. amyloliquefaciens cultures having 1 OD at 600 nm. These plates were spotted with 10 μl of different
concentrations of Imi (10 to 2.097 %), Gly (10 to 0.156 %) and Car (1 to 0.156 %) based on recommended
spraying concentration in field for disease management for chickpea crop in published literatures (Mishra et al.
2005, Andrabi et al. 2011, Yogeeswarudu & Venkata-Krishna 2014). Plates were incubated at 28°C for 48 hrs
and the highest concentration of Car, Imi and Gly supporting bacterial growth were defined as the Maximum
Tolerance Level (MTL) (Ahmed & Khan 2012).
Quantitative estimation of auxin, biofilm and exopolysachharide under pesticides treatments
Quantification of auxin production by bacterial strains was analysed using modified version (Brick et al.
1991) of method proposed by Gordon & Weber (1951) using Salkowski’s reagent. For IAA production assay 5
ml of auxin medium was supplemented with varying concentrations viz. 2.09%, 3.2%, 4.09%, 5.14% Imi,
2.62%, 2.09%, 3.27%, 4.09% Gly and 0.32%, 0.4%, 0.51%, .6% Car respectively in culture vials in triplicates.
Now a set of these culture vials were added with a single isolated colony of RA and a separate set with SN13.
Cultures tubes were incubated in a rotatory shaker at 180 rpm at 28ºC. After 5 days, 5 ml culture from each
treatment was centrifuged at 10,000 rpm for 15 min. 2 ml of supernatant was taken in fresh test tubes and 100 μl
of 10 mM of Orthophosphoric acid followed by 4 ml of Salkowski’s reagent was added. The mixture was
incubated at 28˚C in dark for one hour. The absorbance of the samples resulting orange colour was measured at
530 nm in UV spectrophotometer (Thermo Fisher Scientific, USA). Reagent mixture without culture was taken
as blank.
Biofilm formation assay was performed by inoculating bacterial strains in NB media. Take 5 ml of 48 hrs
grown culture at 28ºC in test tubes supplemented with above-mentioned pesticides concentrations in triplicate.
Now, 250 µl of this culture was transferred in a microtiter plate (Tarsons Pvt. Ltd., India) with the help of
pipette and overnight incubated at 30°C for 24 hrs. Discard the culture and gently tapped on filter paper. Stain
with 250 µl of 0.1% crystal violet then incubate at room temperature for 30 min. Crystal violet was removed by
washing with distilled water. Finally absorbed crystal violet extracted with 250 µl of 95% ethyl alcohol for 1
hour. O.D was taken at 595 nm (Khan et al. 2012).
Exopolysachharide production assay was performed according to Dubois et al. (1956). Bacterial strains were
grown in 50 ml Luria Bertani broth with 5% sucrose supplemented with different concentration of pesticides
and incubated for 5 days at 28°C on a shaker at 100 rpm. Take 10 ml of this 5 days grown culture in 50 ml
falcon tubes and add an equal volume of ethanol (absolute). Samples were overnight incubated at 4°C.
Centrifuge the tubes at 10,000 rpm for 10 mins at 4°C, dry the pellet in hot air oven. Add 0.5 ml saline, 0.5 ml
phenol (5%) and 1 ml H2SO4 (36%). Mix it and place it in a dark for 30–60 min. Absorbance was measured at
490 nm.
Kumar et al. (2017) 4(3): 405–418
Qualitative estimation of phosphate solubilization and siderophore production under pesticides treatments
Phosphate solubilisation assay was performed using NBRIP medium (Nautiyal, 1999). A single colony of
SN13 and RA was inoculated in each test tube containing 5 ml specially formulated medium NBRIP
supplemented with varying concentration of three pesticides in triplicates. Inoculated tubes were incubated for 7
days at 28°C in a rotatory shaker at 180 rpm and daily observed for change in phosphate solubilizing activity
visually. Tricalcium phosphate (TCP) solubilizing activity by SN13 and RA in the presence of three pesticides
was observed for color change. Siderophore production was tested by CAS (Chrome azurol S) assay (Meyer &
Abdallah 1978). RA and SN13 culture were inoculated in NB medium supplemented with different pesticides.
CAS agar media plate was spot inoculated with 10 μl of the overnight grown bacterial culture of RA and SN13
containing pesticides. These plates were incubated at 28°C for 5 days. Siderophore production was detected by
observing the yellow-orange halo zone.
Seed bacterization, sowing, growth parameter and pesticide spray
Chickpea seeds cultivar viz. BG362 was used for this experiment. Seeds bacterization was performed
according to Nautiyal (1997). The overnight grown culture of RA and SN13 were used separately for seeds
bacterization. Plastic pots (24 cm × 12 cm × 12cm) were filled with sterile soil composition (50% soil, 25%
vermiculate, 25% coccopit, and 10% sand). Total 30 seeds for each treatment RA, SN13 and control were taken.
Five seeds per pot were sown at 5 cm depth in 1.2 kg soil. Pots were kept in the glass house at temperature
24±2°C and 16: 8 light and dark conditions. The plants were irrigated with sterilized water. Plant growth
parameters were recorded 30 days after sowing. RA and SN13 inoculated plants along with control were
harvested in triplicates before pesticides spray for growth parameters like shoot, root length, fresh weight and
dry weight. These plants were dried in hot air oven at 65°C for 5 days. After this shoot and root dry weight were
recorded. Pesticides spray on chickpea plants was performed at different concentrations of Car, Imi and Gly
choosen from MTL results (Ahmed and Khan 2012). For pesticides spray 1% Car (50% WP), 10% Gly (41%
WP) and 10% Imi (17.8% S.L.) were sprayed on chickpea plants. The first spray was done after 30 days from
sowing of chickpea seeds and second sprays after 25 days from the first spray.
Soil enzymes assay
Soil enzymes assay were performed after first and second spray of different pesticides from chickpea
rhizosphere soil. To determine the positive or negative effect of pesticides on soil enzymes activity like
dehydrogenase, acid/alkaline phosphate, urease activity, β-glucosidase activity and protease activity were
analysed. This experiment was performed using three biological and three technical replicates. The data were
statistically analyzed using SPSS 16.0 software (Statistical Package for the Social Sciences 16.0, SPSS Inc.,
USA, 1999) for DMRT (Duncan's Multiple Range Test).
The activity of Dehydrogenase enzyme was analysed according to Alef & Nannipieri (1995). Weigh 5 gm
soil in 50ml tubes, containing 5 ml of 0.8% Triphenyl tetrazolium chlororide (TTC) solution. Tubes will be
tightly sealed and incubated at 28˚C for 24 hrs in the dark on a shaker at 120 rpm. A control contains 5 ml Tris-
HCl buffer (without TTC) will set up. After incubation, 40 ml acetone was added in each tube (intermediately
shaken) and again incubated at RT for 2 hrs in the dark with manual shaking after every 10–15 min. Later, 3 ml
from each tube was transferred falcon tubes, centrifuged and the absorbance of the supernatant was recorded at
the 540 nm. Soil phosphatase activities were analysed according to Tabatabai & Bremner (1969) using p-
nitrophenyl phosphate solution as the substrate. One gm of soil taken in 50 ml flask and add 0.25 ml toluene.
Take 4 ml Modified Universal Buffere (MUB) (pH 6.5 for acid phosphates and pH 11 for the alkaline
phosphates) and add 1 ml p-nitrophenyl phosphate (15 mM). Mix solution properly and incubate at 37˚C for 1
hr. After incubation, add 1 ml of CaCl2 (0.5 M) and 4ml of NaOH (0.5 M). For control, add 1 ml of PNPP
solution after the additions of CaCl2 (0.5 M) and 4 ml of NaOH (0.5 M) immediately before filtration of the soil
suspension. Mix the content properly and filter the soil suspension through whattman filter paper. Measure the
absorbance at 400 nm.
β-glucosidase activity was examined by taking one gm soil in 50 ml Erlenmeyer flask, add 0.25ml of
toluene, 4 ml of MUB solution and 1ml p-nitrophenyl-β-D glucoside (PNG) solution, stopper the flask and
mixed properly and incubate at 37˚c for 1hr. After the incubation, add 1 ml of CaCl2 solution, 4 ml tris-buffer
(pH 12) swirl the flask and immediately take a filter through the Whatman filter paper (Eivazi & Tabatabai
1988). Measure the colour intensity at 400 nm (if intensity comes high then we dilute the filtrate with tris-
buffer). For blank preparation, add substrate PNG before adding CaCl 2 and tris-buffer. The activity of urease
Kumar et al. (2017) 4(3): 405–418
enzyme was assayed according to (Kandeler & Gerber 1988). Five gm of moist soil was taken in Erlenmeyer
flask and 2.5 ml urea solution was added. Stopper the flask and incubate it, add 50 ml of KCl solution (74.6 g
KCl and 10 ml of 1M HCl to make 1 litre solution) and shake the flask for 3 min. After the filtering the resulting
suspension, filtrates were analyzed for the ammonium content. Take a 1ml filtrate in 50 ml flask, then add 9 ml
DW, 5 ml of Na- salicylate/NaOH solution and 2 ml of the sodium dichloro isocyanurate solution and allow
standing at RT for 30 min prior to measure O.D at 690 nm. For blank take 10 ml DW and add 2.5 ml urea
solution, 5 ml of Na- salicylate/NaOH solution, and 2ml of sodium dichloro isocyanurate solution at the end of
incubation and immediately before KCl addition. Protease activity was analysed by taking one gm of moist soil
in a centrifuge tube, add 5 ml tris-buffer and 5 ml of Na-Caseinate solution stopper the tubes and mix properly,
incubated at 50˚C for 2 hrs on the shaker bath. After incubation adds 5 ml 15% TCA solution and mix properly.
Centrifuge the soil suspension at 10,000–12,000 rpm for 10 min. Take a 5 ml of the clear supernatant into the
fresh tubes and add 7.5 ml alkaline reagent, incubate it at RT for 15 min. Finally add, 5 ml of folin’s reagent.
Filter the mixture through the filter paper and measure the absorbance at 700 nm. To prepare a control, add 5 ml
of Na-Caseinate solution, incubate and finally adding the TCA solution (Ladd & Butler 1972).
Colony forming unit (CFU) assay
CFU assay of rhisophere soil was performed on 24, 48 and 72 hrs after the first and second pesticides spray.
CFU was done by taking the rhizosphere soil samples of all treatments along with control to determine the effect
of pesticides on viable microbial colonies. One gram soil sample of each treatment was taken in falcon and used
to prepare a suspension of 10 ml using 0.85% NaCl saline, then vortex it and allow rotating at 28˚C on a rotator
shaker at 180 rpm for one hour and performed spotting on NA plates. The numbers of colonies in different
dilutions were determined by average CFU/ml values populations in triplicates per treatment and observe the
effect of pesticides on colonies. CFU measurement was performed in biological triplicates and their technical
triplicates.
Functional microbial diversity analysis using Biolog
Functional diversity of the soil microbial community was characterized using community level physiological
profiles generated by carbon source utilisation pattern on Biolog EcoPlate (BIOLOG, Hayward, CA, USA).
Microbial diversity analysis was performed for diversity indices like Shannon diversity, evenness, Simpson
diversity and McIntosh diversity and evenness. Rhizosphere soil samples were used for biolog assay. One gram
soil was dissolved in 10 ml of 0.85% of NaCl saline and incubated at 28ºC in rotator shaker for one hour at 200
rpm. From this, 125 microlitre suspension cultures was added to each well on Biolog Eco plate. Samples were
loaded in triplicate and incubated at 28ºC in the incubator. Absorbance was taken at 590 nm after 24 hrs interval
till 10 days.
RESULTS
Maximum tolerance level (MTL) of pesticides by bacterial strains
The highest concentration of Car (0.512%), Imi (3.27%) and Gly (3.27%) that supported the growth of RA
and SN13 is referred as MTL (Table 1). These MTL values results were observed after 24 and 48 hrs from
inoculation of pesticides on PGPR grown plates in vitro. There was similar size of hallo zone formation after 24
and 48 hrs of pesticide inoculation in case of both PGPR.
PGP traits evaluation of bacteria in presence of pesticides
Quantitative estimation of auxin, EPS and biofilm production by RA and SN13 in presence of different
concentrations of pesticides has been performed. Auxin production by RA with 0.4% Car (52.17 µg.ml -1),
3.27% Imi (56.09 µg.ml-1) and 3.27% Gly (52.69 µg.ml-1) were found maximum whereas SN13 produced
maximum auxin at 0.4% Car (26.39 µg.ml-1), 3.27% Imi (29.40 µg.ml-1) and 2.62% Gly (26.95 µg.ml-1) (Table
2). EPS production was measured in presence of pesticides showed its highest production by RA at 0.51% Car
(306.80 µg.ml-1), 3.2% Imi (330.20 µg.ml-1) and at 3.27% Gly (287.52 µg.ml-1) concentration in vitro. Similarly,
EPS production by SN13 was maximum at 0.4% Car (306.80 µg.ml-1), 3.2% Imi (359.67 µg.ml-1) and at 3.27%
Gly (309.62 µg.ml-1). Biofilm production for both the bacteria was found highest at 0.4% Car, 3.2% Imi and
3.27% Gly concentrations. On further increasing concentrations of these pesticides, decrease in auxin, EPS and
biofilm production was observed by RA and SN13.
Qualitative estimation of phosphate solubilisation and siderophore production by RA and SN13 in presence
of pesticides was performed and found promising results. Phosphate solubilisation (Fig. 1A) and siderophore
Kumar et al. (2017) 4(3): 405–418
production (Fig. 1B) was found the maximum in RA and SN13 samples containing Imi compared to control
sample. Car treated RA and Gly treated SN13 produced lesser siderophore activity than other treatments.
Figure 1. Qualitative estimation of in vitro PGP traits in presence of pesticides: A, Phosphate solubilisation by Pseudomonas
putida (RA) and Bacillus amyloliquefaciens (SN13), Blank (without inoculation of PGPR), control (bacteria inoculated
sample), Car (bacteria inoculated with carbendazim), Imi (bacteria inoculated with Imidacloprid), Gly (bacteria inoculated
with glyphosate); B, Siderophore production by RA and SN13. [Control (bacteria inoculated sample), Car (bacteria
inoculated with carbendazim), Imi (bacteria inoculated with Imidacloprid), Gly (bacteria inoculated with glyphosate)]
Table 1. Maximum tolerance level (MTL) values of three pesticides applied to 24 and 48 hrs grown Pseudomonas
putida and Bacillus amyloliquefaciens.
S. No. Pesticides Concentration (%) At 24 hrs At 48 hrs
RA SN13 RA SN13
1 Carbendazim (0.2–1%) 1 - - - -
0.8 - - - -
0.64 - - - -
0.512 - - - -
0.409 + + + +
0.327 + + + +
0.209 + + + +
0.262 + + + +
2 Glyphosate (2–10%) 10 - - - -
8 - - - -
6.4 - - - -
5.12 - - - -
4.096 - - - -
3.27 + + + +
2.62 + + + +
2.09 + + + +
3 Imidacloprid (2–10%) 10 - - - -
8 - - - -
6.4 - - - -
5.12 - - - -
4.096 - - - -
3.27 + + + +
2.621 + + + +
2.097 + + + +
Note: +, The tolerance of different pesticides by RA and SN13; -, The non-tolerance of different pesticides by RA and
SN13 in vitro
Chickpea plant growth parameters
Chickpea cultivar (BG-362) was evaluated for plant growth promotion to see the effect of PGPR. Results
(Table 3) clearly revealed that inoculation with RA and SN13 has significantly enhanced the plant biomass like
root and shoot length, the fresh and dry weight of root and shoot of chickpea plant as compared to un-inoculated
control.
Kumar et al. (2017) 4(3): 405–418
Kumar et al. (2017) 4(3): 405–418
Table 3. Plant growth parameters in chickpea treated with Pseudomonas putida (RA) and Bacillus amyloliquefaciens (SN13)
and under control condition measured 30 days after sowing.
Root length Shoot length Fresh weight Fresh weight Dry weight Dry weight R/S DW
Treatments
(cm) (cm) root (gm) shoot (gm) root (gm) shoot (gm) ratio
Control 27.67±2.33 19.50±0.29 0.63±0.16 1.64±0.18 0.08±0.01 0.25±0.02 0.31
SN13 49.33±2.03 24.33±0.33 0.67±0.01 2.08±0.04 0.09±0.00 0.28±0.00 0.32
RA 30.00±2.08 23.67±1.33 0.66±0.03 1.91±0.18 0.08±0.01 0.25±0.02 0.34
Soil enzymes activity under pesticide treatments
Soil enzymes assay using chickpea rhizosphere soil was performed after first and second spray of pesticides.
Dehydrogenase, alkaline/acid phosphatase, β-glucosidase, urease and protease activities were performed and
found that RA and SN13 poise these activities in soil. Soil enzymes activities were found higher in individually
RA and SN13 inoculated samples than control and other treatments. Dehydrogenase activity was inhibited in
Car and Gly treated samples whereas Imi didn’t affect this activity. Dehydrogenase activity was found induced
in samples inoculated with RA and SN13 in presence of these pesticides (Fig. 2A). Acid phosphatase activity
was enhanced in Car and Gly treated soil samples whereas activity was inhibited in Imi treated samples (Fig.
2B). RA and SN13 inoculated samples have higher acid phosphatase activity than other samples. RA and SN13
inoculated samples treated with Car, Gly and Imi resulted induced the activity of acid phosphatase. Alkaline
Figure 2. Soil enzymes activity in RA and SN13 inoculated soil samples after pesticides application: A, Dehydrogenase
activity; B, Acid phosphatase activity; C, Alkaline phosphatase activity; D, β-glucosidase activity; E, Urease activity; F,
Protease activity. [Control (without treatment), RA (samples inoculated with Pseudomonas putida), SN13 (samples
inoculated with Bacillus amyloliquefaciens), Car (samples sprayed with Carbendazim), RA+ Car (samples inoculated with P.
putida and sprayed with Carbendazim), Gly (samples sprayed with Glyphosate), RA+ Gly (samples inoculated with P.
putida and sprayed with Glyphosate), Imi (samples sprayed with Imidacloprid), RA+ Imi (samples inoculated with P. putida
and sprayed with Imidacloprid), SN13+Car (samples inoculated with B. amyloliquifaciens and sprayed with Carbendazim),
SN13+ Gly (samples inoculated with B. amyloliquifaciens and sprayed with Glyphosate), SN13+Imi (samples inoculated
with B. amyloliquifaciens and sprayed with Imidacloprid), bars represent the standard errors of the means (n = 3). Different
letters within column represents significant difference at (P = 0.05) by using DMRT]
Kumar et al. (2017) 4(3): 405–418
phosphatase activity was inhibited with Car and Imi but stimulated with Gly (Fig. 2C). RA inoculated sample
individually as well as RA with Gly treated sample stimulated highest alkaline phosphatase activity. β-
glucosidase activity was inhibited with all three pesticides. This activity was found higher in bacterized samples
than control (Fig. 2D). Imi treated sample showing the highly inhibited activity of β-glucosidase after first and
second spray. Sample inoculated with RA alone have highly stimulated this activity. Urease activity was found
inhibited by Gly and Imi whereas stimulated with Car (Fig. 2E). RA and SN13 inoculated samples treated with
Gly and Imi have enhanced urease activity than only Gly and Imi treated samples. Urease activity was highly
induced in RA inoculated soil treated with Car after first and second spray. Protease activity was found
stimulated in samples containing Car and Imi whereas highly reduced activity was found in Gly treated soil
samples (Fig. 2F). Surprisingly, most of the soil enzymes activities found reduced which we have performed
after second application of these three pesticides in comparison with the first application. These results proved
that repeated application of pesticides affects soil enzyme activities. Results indicated that RA and SN13 have
the ability to tolerate the pesticides and reduced its adverse effect in the soil at some point and play a beneficial
role in maintaining the soil fertility. Figure 2 showing the soil enzyme activities in presence of RA and SN13
inoculated with and without pesticides spray. Both the PGPR play an important role in maintaining the soil
enzyme activities and reduced the negative effect of pesticides in soil health.
Figure 3. Colony forming unit assay: A, CFU after 24 hrs from 1st and 2nd spray of pesticides; B, CFU assay after 48 hrs
from 1st and 2nd spray of pesticides; C, CFU after 72 hrs from 1st and 2nd spray of pesticides. [bars represent the standard
errors of the means (n = 3)]
CFU assay in pesticide treated soil
CFU of rhizosphere soil was measured after 24, 48 and 72 hrs from 1 st and 2nd spray of pesticides. The
numbers of viable colonies were higher in RA inoculated than control soil samples with and without pesticides
treatments. The decreased colonies were observed in Car, Imi and Gly treated soil samples after first and second
spray whereas colonies were increased in RA and SN13 inoculated soil samples (Fig. 3). Pesticides treated
Kumar et al. (2017) 4(3): 405–418
samples showed a reduction in CFU values after 72 hrs of 1st spray and after 48 hrs of 2nd spray. Samples with
PGPR inoculations and with pesticides sprays showed relative less reduction in CFU values. These results (Fig.
3) revealed that the RA and SN13 reduced the adverse effect of pesticides and survival of bacteria is increased.
Biolog assay in pesticide-treated soil
Average well color development (AWCD) was calculated which showed the microbial activity in each well
of the microplate. Microbial diversity indices like Shannon, McIntosh and Simpson functional diversity and
related evenness have been determined (Table 4). These results revealed the bacterial population in the
rhizosphere after pesticides spray. The Shannon and McIntosh diversity together with their evenness were
reduced in Gly treated samples in presence of both bacterial strains. But in case of Car and Imi samples diversity
indices were not changed with and without inoculation of RA and SN13.
Table 4. Functional diversities and evenness based on carbon source utilization pattern for chickpea rhizosphere treated
with Pseudomonas putida (RA) and Bacillus amyloliquefaciens (SN13) with and without pesticides treatment.
S.No. Sample ShD ShE ShipD McD McE
1 Control 3.34±0.02 0.99±0.01 0.99±0.00 0.98±0.00 0.99±0.00
2 RA 3.33±0.02 0.98±0.00 0.99±0.00 0.98±0.00 0.99±0.00
3 SN13 3.31±0.03 0.97±0.00 0.99±0.00 0.98±0.01 0.98±0.00
4 Car 3.30±0.02 0.96±0.00 0.98±0.00 0.98±0.00 0.97±0.00
5 Gly 3.21±0.01 0.94±0.01 0.97±0.00 0.95±0.00 0.96±0.00
6 Imi 3.25±0.01 0.95±0.00 0.96±0.00 0.98±0.00 0.98±0.00
7 RA+Car 3.31±0.01 0.97±0.00 0.99±0.00 0.98±0.00 0.98±0.00
8 RA+Gly 3.22±0.02 0.95±0.01 0.99±0.00 0.97±0.00 0.97±0.01
9 RA+Imi 3.27±0.01 0.96±0.01 0.99±0.00 0.97±0.00 0.98±0.00
10 SN13+Car 3.33±0.01 0.98±0.01 0.99±0.00 0.98±0.00 0.99±0.00
11 SN13+Gly 3.18±0.03 0.95±0.01 0.99±0.00 0.96±0.00 0.97±0.01
12 SN13+Imi 3.30±0.01 0.96±0.00 0.99±0.00 0.98±0.00 0.98±0.00
Note: ShD, Shannon diversity; ShE, Shannon evenness; SimpD, Simpson diversity; McD, McIntosh diversity; McE,
McIntosh evenness.
DISCUSSION
Pseudomonas putida and Bacillus amyloliquefaciens are well known for their PGP attributes. Production of
IAA, EPS and biofilm by these bacteria were maximum at threshold concentrations of pesticides but above that
production was reduced (Table 2). Pesticide uses may cause slight and temporary changes to microbial
populations of soil and their activities even if applied at normal rates (Johnsen et al. 2001). In case of repeated
application pesticides can interfere and disturb soil enzymatic activities and also affected local metabolism
resulting a reduction in soil fertility. Soil dehydrogenases are the enzymes belong to oxidoreductase enzyme
class and are its major representatives (Gu et al. 2009). Dehydrogenases are very important enzymes in the soil
environment which are an indicator of the overall microbial activity of soil (Gu et al. 2009, Salazar et al. 2011).
These occur intracellularly in all microbial cells (Yuan & Yue 2012). Dehydrogenase enzyme activity is
repeatedly performed for measurement of any disturbance due to pesticides, or direct measure of soil microbial
activity and trace elements or management practices to the soil. Our results revealed that dehydrogenase activity
was inhibited in Car and Gly treated samples whereas Imi didn’t affect this activity (Fig. 2). This enzyme
activity was higher in the sample inoculated with RA and SN13 alone without pesticides than control.
Phosphatases have five main groups of enzymes: phosphomonoesterases, phosphodiesterases,
phosphotriesterases, phosphoamidases and pyrophosphatases. Among these five enzymes,
phosphomonoesterases are most abundant in soils. This may be because of low substrate specificity of this
group of enzymes (De Cesare et al. 2000). Phosphomonoesterases are further classified into two groups which
are acid and alkaline phosphatase, on the basis of optimum pH for their activity. These two phosphatases are
mainly found in animals and microorganisms. It was found by researchers that alkaline phosphatase activity gets
inhibited when the fungicide is applied to soil (Rasool & Reshi 2010), while the activity of acid phosphatase
increased. Acid phosphatase activity was observed highly inhibited immediately after Car addition compared to
the control (Tortella et al. 2013). Researchers also found that other fungicides either had inhibited phosphatases
activity or no effect (Bello et al. 2008, Yan et al. 2011). On the application of herbicides the activity of acid and
alkaline phosphatase is either stimulated (imazethapyr) or unchanged (aurora 40 WG and rimsulfuron) (Perucci
et al. 2000, Baćmaga et al. 2012). Phosphatases activities were found to be inhibited by herbicide application
under different conditions of soil physicochemical properties and pesticides dose (Min et al. 2001, Tejada
Kumar et al. (2017) 4(3): 405–418
2009). The acid and alkaline phosphatase activities may respond differently to application of insecticides.
Sometimes the same insecticide may inhibit acid phosphatase and stimulate the activity of alkaline phosphatase,
and vice versa (Cycon et al. 2010, Defo et al. 2011, Jastrzebska 2011). Overall it was found in earlier studies
that pesticides play an inhibitory effect on the enzymatic activities involved in the phosphorus cycle. We found
enhanced acid phosphatase activity in samples treated with Car and Gly whereas this activity was inhibited in by
Imi. For bacterized samples treated with a recommended concentration of pesticides, an overall increase in acid
phosphatase activity was observed for both the PGPR. β-glucosidase is an enzyme which plays important role in
the decomposition/transformation of organic matter in the soil. Glucose is the final product which is an
important source of carbon energy for soil microorganisms (Deng & Tabatabai 1994). In our results, β-
glucosidase activity was found inhibited in samples treated with Car, Gly and Imi. This activity was found
higher in RA and SN13 inoculated soil samples than control.
Hydrolysis of urea into carbon dioxide and ammonia is catalyzed by urease. Researchers have proven that
herbicides and fungicides appear to have no effect (Cycon et al. 2010, Yan et al. 2011, Baćmaga et al. 2012) or
reduced effect on the activity of urease (Sukul 2006, Caceres et al. 2009, Tejada 2011). Application of
pesticides decreases the urease activity in the soil which is beneficial for plants by reducing the hydrolysis of
urea because it helps to maintain nitrogen availability to plant (Antonious 2003). On the other hand, the
fungicides validamycin and Car enhanced urease activity, respectively, up to 13–21% and to 70% (Qian et al.
2007, Yan et al. 2011). In our experiment, we found inhibited urease activity by Gly and Imi whereas stimulated
with Car. Protease enzyme plays an important role in nitrogen mineralization which regulates the amount of
available nitrogen to the plants. The protease activity was found to be higher after the application of endosulfan
and chlorpyrifos at lower and medium concentrations. Pesticides applications had stimulated the activity of
protease enzyme in comparison to control upto 21 days from incubation (Rasool & Reshi 2010). Protease
activity in soil decreased on applications of insecticides on concentration more than 25 ppm. Our results
revealed that protease activity was found stimulated in samples containing Car and Imi whereas reduced activity
was found in Gly treated soil samples. In overall experiments, it was common observations results for second
sprays were more drastic revealing that repeated use of chemicals leached to the soil and disturb the microbial
population and changed the soil enzyme activities. PGPR (RA and SN13) inoculation has reduced the adverse
effect by maintaining the microbial population and metabolic activities.
In biolog technique, the rate of colour development in wells provides information about the density and
metabolic activity of bacterial cells in an inoculum, while the diversity of colour development in wells about
microbial diversity in soil solution (Mondini & Insam 2003). We have concluded from the diversity study that
with the use of pesticides a slight reduction in microbial diversity and evenness was observed but the sample
inoculated with PGPR showed no such changes due to the survival of microbial population and poise of soil
enzymes.
CONCLUSIONS
Our experimental evidences concluded that repeated application of pesticides disturb soil microbial
dynamics. An increase in PGP trait activities like IAA production, exopolysachchride production, biofilm
synthesis, phosphate solubilization and siderophore production on the addition of pesticides at concentrations
below threshold values. Inoculation of P. putida and B. amyloliquefaciens maintain the equilibrium of microbial
population in soil and also balance soil enzymatic activities. Thus the application of pesticides (Car, Imi and
Gly) under PGPR (RA and SN13) inoculated soil would not reduce microbial population hence improve soil
fertility.
ACKNOWLEDGEMENT
This research was financially supported by New Initiative (as a Cross Flow Technology project) “Root
Biology and Its Correlation to Sustainable Plant Development and Soil Fertility” (Root SF, BSC0204) from the
Council of Scientific and Industrial Research (CSIR), New Delhi, India.
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ISSN (E): 2349 – 1183
ISSN (P): 2349 – 9265
4(3): 419–420, 2017
DOI: 10.22271/tpr.2017.v4.i3.055
Short communication
Rice brown spot Bipolaris oryzae (Breda de Haan) Shoemaker

in Paraguay
Lidia Quintana1*, Susana Gutiérez2, Manuela Arriola1,
Karina Morinigo1 and Aldo Ortiz1
1
Facultad de Ciencias Agropecuariasy Forestales-Universidad Nacional de Itapúa Encarnación, Paraguay
2
Universidad Nacional del Nordeste-Corrientes, Argentina
*Corresponding Author: lviedmaq@gmail.com [Accepted: 20 November 2017]
[Cite as: Quintana L, Gutiérez S, Arriola M, Morinigo K & Ortiz A (2017) Rice brown spot Bipolaris oryzae
(Breda de Haan) Shoemaker in Paraguay. Tropical Plant Research 4(3): 419–420]
Brown leaf spot is a serious disease in rice production worldwide, caused by Bipolaris oryzae (Breda de
Haan) Shoemaker, it is caused losses in stand due to seedling blight, in yield due to leaf and culm infection, and
in quality and yield by kernel infection. In the 2014/2015 season, disease survey was conducted in the different
rice-growing area of the country. Symptoms were observed on rice plants of IRGA 424 cultivar as leaf spot
throughout the growing season, mostly on leaf blade and leaf sheath. Small spots were dark brown to reddish
brown, circular to oval in shape, while older spots have a light, reddish-brown or gray center surrounded by a
dark to reddish-brown margin.
In the national bibliography no history published about this disease was found, thus the objective of this
study was to determine the etiology in this new disease in Paraguay. Naturally, diseased leaves of IRGA 424
cultivar infected in varied degrees with brown spot were collected from various districts of the country. Two
hundred leaf samples taken from each field with symptoms and signs of brown spot.
Affected tissues from the leaves were cut into small bits, washed thoroughly in running water to remove
dirts. These were dipped in 0.5% sodium hypochlorite (NaOCl) solution for 30–45 seconds and plated on 3
layers of moistened blotters in plastic petri dishes (ISTA 2003). The dishes were incubated at 25–30ºC, 12/12
hours light and darkness and examined under a stereomicroscope for the growth of B. oryzae after 7–10 days of
incubation. Subsequently, the fungus spores were isolated on PDA for the colonies observation.
Figure 1. A–B, Symptoms of brown spot on rice leaves; C, Conidia of Bipolaris oryzae (Breda de Haan) Shoemaker.
The brown spot was detected in green rice plants at reproductive stage in IRGA 424 cultivar grown in the
departments of Itapúa, Misiones and Caazapá. The mean disease incidence in leaves was 30–40 %. The disease
Quintana et al. (2017) 4(3): 419–420
has been described in all rice-producing countries in the world such, India, Pakistan, Bangladesh, Egypt, USA,
Philippines, Colombia, Brasil and Perú (Ou 1985, Webster & Gunnel 1992, Mew & Gonzales 2002). In Egypt,
the disease comes in the second rank after blast disease (El-Wahsh 1997). Gutiérrez et al. (2000) and Farias et
al. (2011) reported the identification of Bipolaris oryzae and Bipolaris spp. in seed lots of Northern of Argentina
and Rio Grande do Sul State, Brasil respectively.
The symptoms observed in this survey were similar with those described by Ou (1985), Webster & Gunnel
(1992), Mew & Gonzales (2002). Symptoms are visible as small and circular dark brown spot, sometimes purple
brown spots to ovalbrown spots with gray centers distributed on all leaf surface (Fig. 1A, B). Mycelia is gray to
dark greenish gray and conidiophore septate, solitary, or in smallgroups; straight or flexuous, simple; pale to
mid-brown; bearing conidia at the end and on sides. Microscopically conidia of Bipolaris oryzae club shaped to
cylindrical, generally curved, light brown to golden brown, with 6 to 13 transverse cell walls (Fig. 1C) and were
similar to those described by several authors (Ou 1985, Mew & Gonzales 2002).
The causal agent of rice brown spot was identified as Bipolaris oryzae (Breda de Haan) Shoemaker. This is
the first report of the disease in Paraguay.
ACKNOWLEDGEMENT
The authors are thankful to PROCIENCIA/CONACYT for their financial support.
REFERENCES
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ISSN (E): 2349 – 1183
ISSN (P): 2349 – 9265
4(3): 421–432, 2017
DOI: 10.22271/tpr.2017.v4.i3.056
Research article
New record of Cyanoprokaryotes from West Bengal

in Maldah district
Pratibha Gupta
Botanical Survey of India, Ministry of Environment Forests & Climate Change, Government of India,
AJCBIBG, CNH Building, Botanic Garden, Howrah-711103, India
*Corresponding Author: drpratibha2011@rediffmail.com [Accepted: 22 November 2017]
Abstract: Systematic survey and collection of Cyanoprokaryotes were carried out from different
water bodies of Maldah District, West Bengal. During survey samples were sampled from 55
different water bodies of this area comprising 05 sites in rivers, 32 bils, 07 dighis, 02 Jheels, 09
ponds for which surveyed all administrative blocks of Maldah District namely Ratua I, Ratua II,
Harishchandrapur I, Harishchandrapur II, Chanchal I, Chanchal II, Manikchak, Gazol, Habibpur,
Bamangola, Old Maldah, English Bazar and Kaliachak. During the study, altogether 22 genera and
105 species (comprising 93 species, 09 varieties and 03 forms) were identified from different types
of water bodies of Maldah District. Out of these 105, 27 species have been recorded from West
Bengal. These species have been described here along with nomenclature and distribution.
Keywords: Cyanoprokaryotes - New Record - Maldah District - West Bengal.
[Cite as: Gupta P (2017) New record of Cyanoprokaryotes from West Bengal in Maldah district. Tropical Plant
Research 4(3): 421–432]
INTRODUCTION
Maldah district is one of important district among 19 districts of West Bengal. The major river the Ganges
flows along south-western boundary of the district followed by another major rivers like Mahananda, Fulhar and
Kalindri. All above rivers originated basically from the Himalayan or sub-Himalayan region and flow Southerly
direction whereas some seasonal rivers like Tangoan, Punarbhaba, Pagla and Bhagirathi are also flowing
through district territory. Thus, in terms of duration and frequency of flood and its recurrences is concerned,
Maldah district is defined as a chronically flood-affected area. Swamps (bils) are extending along the right bank
of Mahananda from Kalindri. The practice of aquaculture, cultivation of rice and Makhana (Eurayle ferox
Salisb.) are other aspects on which socio-economic condition and livelihood of the district depend. To meet the
requirements, a huge number of big and small size „dighi‟ and „ponds‟ depending upon land area available to
individuals are made by them. Such water bodies often get eutrophicated by micro and macrophytes as a result of
contaminants from various non-point and point sources like runoff of agriculture fields in which huge quantity of
fertilizers and pesticides is used and civic as well as industrial pollutants respectively. Among the aquatic flora,
diversity and distribution of microscopic flora is much greater having both beneficial and harmful properties.
Therefore, exploration of all forms of different community is warranted which is not possible to be studied by
any individual. Thus, during the study, particularly concentrated on Cyanoprokaryota for proper exploration of
the unexplored area i.e. Maldah District of the State of West Bengal.
The ancient Cyanoprokaryota likely produced much of oxygen in the Earth's atmosphere, as they dominantly
metabolise and fix carbon in the form of sugars by using carbon dioxide. Increase in concentration of oxygen on
the earth crust, recorded during 2.4 billion years ago considered as an architect of earth's atmosphere as they are
“nature‟s first and foundational mother and father for causing photosynthesis”, entail to form pure ecological
niche on our planet and precisely stands as the founder of the aquatic food-chain. As per findings, they have
been distributed all over land and water system often in such an environment where there is no other vegetation
possibly due to their adaptive capability to extreme adverse environmental conditions with respect to different
environmental factors like temperature, pH, salinity, availability of nutrients, pollution load, etc (Sridhar et al.
2006, Bajpai et al. 2013). Their occurrence even in the wide range of ecologically stress conditions and extreme
Gupta (2017) 4(3): 421–432
habitats proves that they are very tolerant and thus placed in a unique group of micro-organisms. They occur in
a fresh-water ecosystem like bils, dighis, lakes, rivers, wetlands, waterfall, etc. and marine water system like -
salt marshes and pans, estuaries, brackish waters and ocean. Besides, it also occur on inter-tidal rocks, snow and
even in cold lakes underneath 5 m of ice pack as well as in thermal springs, soil, acid bogs and alkaline soils or
more, in sub-aerial habitats like tree trunks, moist walls and all other objects which remain moisten and get solar
light from any angle even in a short span of time.
Enormous work on quantitative and qualitative along with taxonomic studies, indicator of water pollution /
environmental assessments and R & D studies using selected species have been carried out in different parts of
India (Mohan et al. 2007, Bajpai et al. 2013, Malathi et al. 2014, Srivastava et al. 2014, Halder 2015, Jitendra &
Anand 2016, Patel et al. 2017). So far, out of 19 districts of West Bengal such studies have been carried out only
in some of the Districts and surrounding area comprising Bankura (Sinha & Mukherjee 1984, Mukherjee 1986),
Burdwan (Gupta & Sen 1978, Chatterjee & Choudhury 1980, Chatterjee & Chatterjee 1983), Hooghly (Gupta &
Sen 1987, Banerjee 1998, Sikdar & Keshri 2014), Howrah (Mukhopadhyay & Chatterjee 1981, Sabata & Nayer
1992, Sen & Gupta 1993), Kolkata (Biswas 1925, 1926, Santra 1987, Mitra & Gupta 1994, Banerjee 1997, Sen
2006), Midnapur (Pal & Santra 1985), Murshidabad (Pal & Santra 1982, 1984, Pal et al. 1986), 24-Paraganas
(Mukhopadhyay & Chatterjee 1981, Maity & Santra 1985, Singh et al. 2001, Naskar et al. 2008), Sunderban
(Pal et al. 1988, Banerjee & Santra 2001) and in continuation for further exploration in remaining Districts of
the West Bengal, Maldah district was considered first keeping in view to explore Cyanoprokaryotes forms
taxonomically.

Maldah district is situated in West Bengal of north-east India and lies between 24°41 20 and 25°32 08
North Latitude and 87°45 50 and 88°28 10 East Longitude, extends over 3733.17 km 2 with total population
32,90,468 as per Census, 2001 (BAES 2004) and English Bazar is the District Administrative Headquarter. The
district is bounded to its south by the district of Murshidabad across the river Ganga, by Rajshahi district of
Bangladesh and Dakshin Dinajpur district to its east and north-east, by Uttar Dinajpur district to its direct north
and by the Purnea of Bihar to its direct west and by Sahibganj of State of Jharkhand across the Ganga to the
south-west (Fig. 1).
Figure 1. Location of the study site.

During the survey all administrative blocks of Maldah district namely Ratua I, Ratua II, Harishchandrapur I,
Harishchandrapur II, Chanchal I, Chanchal II, Manikchak, Gazol, Habibpur, Bamangola, Old Maldah, English
Bazar and Kaliachak were visited and samples containing cyanophycean forms were sampled from 55 water
bodies comprising 5 sites in rivers like Kalindri (Bhaluka), Fulhar (Sankatala Ghat), Ganga (Mahadhap and
Gopalpur) and Mahananda (Alal Bridge); 32 bils (Amkhaki, Liltua, Barabilla, Singera, Ghogha, Chakla,
Bochamari, Ashi Dob, Makaiya, Hazartakia, Janipukur, Adhsoi, Manna, Dekul, Meetna, Singsar, Sanak, Singer,
Kendha, Kuchla, Bhatia, Chand, Tarkeshwar, Nawabganj, Balotuli, Jalsukha, Laxmipur, Chattara, Garhal,
Adhsoi, Pulintola and Madhaipur); 7 dighis (Raikhan, Thinnagar, Kalua, Paradhala, Sukan, Bara and Chota
Gupta (2017) 4(3): 421–432
Sagar); 2 Jheels (Bochahi and Mahadhap) and 9 ponds / Pukurs (Bhadobartola, Damua, Shivrampalli, Rohni,
Meenatulla, Jorkuppa, Kuppa, Samda and Salamidarwaza).
Samples were sampled randomly towing Phytoplankton net to a distance of 1.0–5.0 m depending upon the
depth of water bodies. The samples were preserved in 15.0 ml screw cap Borosil glass specimen vials to avoid
any chemical reaction. To take samples from another water bodies, Phytoplankton net was thoroughly washed
with clean water after collection of each sample. Samples were preserved by adding 2–3 drops of 4% Formalin
solution. Specimens were observed under Leica DM 2500 Microscope and photomicrograph of each specimen
was taken by DFC 500 digital camera with annotation using Leica QWin V 3.2 Image Processing and Analysis
Software and Leica Application Suit V4. Specimens were identified by consulting standard books, monograph
(Geitler 1932, Tiffany & Britton 1952, Desikachary 1959, Prescott 1982, Anand 1989, 1998, Komárek &
Anagnostidis 1998, 2005). The authority name of each species is cited in the text as described in „Authors of
Plant Names‟ (Brummitt & Powell 1992), title of the books in citation is cited in accordance with Stafleu &
Cowan (1976, 1979, 1981, 1983, 1985, 1986, 1988) and supplements as described by Stafleu & Mennege (1992,
1993, 1995, 1997, 1998, 2000), whereas Journals, Periodicals with Botanical content as described in “Botanico-
Periodicum-Huntianum”, BPH-2 (Bridson 2004a, b).
RESULTS
Details of each new record of Cyanoprokaryotes of West Bengal from different water bodies of Maldah
District in various blocks depicted in table 1 along with the geographical location.
Table 1. Cyanoprokaryotes new record from West Bengal in Maldah district.
S. Name of the Latitude, Name of the Name of the species
No. Block Longitude Water Bodies
1 Ratua I 25°12‟01.98"N, Bhadobartola Microcystis ichthyoblabe Kütz.
87°56‟56.26"E Pond Microcystis panniformis Komárek
2 Ratua II 25°08‟12.11"N, Barabilla Bil Pseudanabaena amphigranulata (Goor) Anagn.
88°00‟56.69"E Nostoc ellipsosporum var. violaceum C.B.Rao
Mahadhap Lake Pseudanabaena amphigranulata (Goor) Anagn.
Pseudanabaena limnetica (Lemmerm.) Komárek
Pseudanabaena redekei (Goor) B.A.Whitton
3 Harishchandr 25°24‟48.15"N, Damua Pond Oscillatoria crassa (C.B.Rao) Anagn.
apur I 87°53‟11.43"E Oscillatoria tenuis var. natans Gomont
Adhsoi Bil Dactylococcopsis raphidioides Hansg.
Hazartakiya Bil Oscillatoria perornata f. attenuata Skuja
Anabaena inaequalis Bornet & Flahault
4 Harishchandr 25°24‟22.46"N, Manna Bil Gloeocapsa nigrescens Nägeli
apur II 87°52‟00.23"E Aphanocapsa elachista var. conferta W.West &
G.S.West
5 Chanchal II 25°20‟08.65"N, Chakla Bil Komvophoron crassum (Vozžhenn.) Anagn. &
88°01‟09.92"E Komárek
6 Gazol 25°13‟10.64"N, Kuchla Bil Jaaginema geitleri (Frémy) Anagn. & Komárek
88°10‟13.19"E Raikhan Dighi Heteroleibleinia mesotricha (Skuja) Anagn. &
Komárek
7 Old Maldah 25°00‟31.46"N, Sukan Dighi Microcystis wesenbergii (Komárek) Komárek
88°09‟29.21"E Paradhala Dighi Synechocystis pevalekii Erceg.
Balutuli Bil Pseudanabaena limnetica (Lemmerm.) Komárek
Nawabganj Bil Anabaena vaginicola f. fertilissima B.N.Prasad
8 English Bazar 25°01‟08.41"N, Bara Sagar Dighi Microcystis novacekii (Komárek) Compère
88°14‟10.98"E Aphanocapsa rivularis (Carmich.) Rabenh.
Pseudanabaena dictyothalla (Skuja) Anagn.
Laxmipur Bil Cyanosarcina burmensis (Skuja) Kovácik
Jorkuppa Pond Pseudanabaena amphigranulata (Goor) Anagn.
Jalsukha Bil Oscillatoria angusta Koppe
Oscillatoria formosa f. loktakensis Brühl & Biswas
Jaaginema geminatum (Schwabe ex Gomont) Anagn.
& Komárek
Phormidium rimosum (Komárek) Anagn. & Komárek
Gupta (2017) 4(3): 421–432
Systematic Enumeration:
Systematic studies carried out on Cyanoprokaryotes from diverse water bodies of Maldah district, West
Bengal. Taxonomic enumeration of identified new record of Cyanoprokaryotes of West Bengal from Maldah
district is described here along with their details.
Chroococcales Wettst.
Chroococcaceae Nägeli
Cyanosarcina Kovácik
Cyanosarcina burmensis (Skuja) Kovácik, Arch. Hydrobiol. Suppl. 80 (Algol. Stud. 50-53): 176. 1988. (Fig. 2A)
Myxosarcina burmensis Skuja, Zur Süsswasseralgenflora Burmas 21, t. 1, f. 12. 1949.
Cells more or less angular with rounded corners, almost arranged in transverse and vertical series, pale blue-
green or olive-green, homogenous or finely granular; sheath very thin, mucilaginous, hyaline; colonies when
young 4 cells.
Dimension: Cells 2.0–3.5 μm in diameter.
Distribution: Bil (Laxmipur).
Dactylococcopsis Hansg.
Dactylococcopsis raphidioides Hansg., Notarisia 590, 1888; Desikachary, Cyanophyta 158, t. 29, f. 1-2. 1959.
(Fig. 2B)
Cells light blue-green, spindle shaped, sigmoid or lunulately bent, single to a few together; species highly
polymorphic.
Dimension: Cells 1.3–3.0 μm broad and 14.0–18.2 μm long.
Distribution: Bil (Adhsoi).
Microcystaceae Elenkin
Gloeocapsa Kütz.
Gloeocapsa nigrescens Nägeli, in Rabenh., Alg. Sachs. 63, 629, 1857; Desikachary, Cyanophyta 117, t. 24, f.
15, 17. 1959. (Fig. 2C)
Cells spherical, surrounded by individual envelops, not lamellated, hyaline; cells uniting laterally, contents
blue-green.
Distribution: Bil (Manna).
Microcystis Lemmerm.
Microcystis ichthyoblabe (G.Kunze) Kütz., Phycol. general. 921, 1843; Komárek & Anagn., Cyanoprokaryota
Part 1: Chroococcales 19(1): 226, f. 297. 1998. (Fig. 2D)
Granularia ichthyoblabe G.Kunze in E.Schmalz. Flora 6: 566. 1823.
Colony large, irregular, compact, without holes, mostly flattened, often form cell clusters or sub colonies in
common mucilage, later on disintegrated in to small groups of aggregated cells; margins of colonies irregular,
indistinct, diffuse, irregularly overlapping cells; cells spherical, densely homogeneously and evenly
accumulated.
Dimension: Cells 2.0–3.7 µm in diameter.
Distribution: Pond (Bhadobartola).
Microcystis novacekii (Komárek) Compère, Bull. Jard. Bot. Natl. Belg. 44: 19, 1974; Komárek & Anagn.,
Cyanoprokaryota Part 1: Chroococcales 19(1): 220, f. 302. 1998. (Fig. 2E)
Diplocystis novacekii Komárek, in Komárek & Ettl., Alg. Stud.: 63, t. 6, f. 1-4. 1958.
Colony almost spheroidal and slightly flattened, sometimes cells aggregated together; cells densely
agglomerated in the centre of the colony, few solitary cells in enveloping mucilage.
Distribution: Dighi (Bara Sagar).
Microcystis panniformis Komárek, Komárk.-Legn., C.L. Sant'Anna, M.T.P. Azevedo & Senna, Crypto. Algol. 23:
165, f. 14–28. 2002; Komárek & Anagn., Cyanoprokaryota Part1: Chroococcales 19(1): 226, f. 297. 1998. (Fig. 2F)
Gupta (2017) 4(3): 421–432
Colony flat, irregular with small holes; margins of the colonies smooth or irregular; cells regularly densely
and smoothly accumulated.
Distribution: Pond (Bhadobartola).
Microcystis wesenbergii (Komárek) Komárek ex Komárek, in Kondrateva Cvetenie vody, Naukova Dumka
Kiev 32, 1968; Komárek & Anagn., Cyanoprokaryota Part 1: Chroococcales 19(1): 232, f. 305. 1998.
(Figs. 2G–K)
Diplocystis wesenbergii Komárek, Komárek & Ettl., Alg. Stud.: 68, t. 7, f. 1-4. 1958.
Colony irregular, spheroidal to lobate or elongate with holes when old; mostly composed with connected
spheroidal sub-colonies; cells sparsely to densely accumulated often near the surface of sub-colonies.
Distribution: Dighi (Sukan).
Nostocales Borzì
Nostocaceae Eichler
Anabaena Bory ex Bornet & Flahault
Anabaena inaequalis Bornet & Flahault, Ann. Sci. Nat. Bot. ser. 7(7): 225, 231, 1886 (1888); N.D.Kamat,
Hydrobiologia 22: 277, 1963. (Fig. 2O)
Trichome almost straight or sometimes slightly twisted; cells short, barrel-shaped or truncate globose;
heterocysts globose; spore not found in the sample.
Dimension: Cells 4.0–5.3 μm broad; heterocysts 5.0–6.8 μm broad.
Distribution: Bil (Hazartakiya).
Anabaena vaginicola f. fertilissima B.N.Prasad, J. Indian Bot. Soc. 31: 361, f. 14-17. 1952; Desikachary,
Cyanophyta 401, t. 73, f. 3. 1959. (Fig. 2P)
Trichome single or in a common mucilaginous sheath, blue-green; cells quadratic barrel-shaped, constricted
at the cross-walls; heterocysts barrel-shaped slightly flattened; spore ellipsoidal, more than one or in the chain.
Dimension: Trichome 4.8–5.7 µm broad and cells 3.2–5.4 µm long; heterocysts 5.8–6.8 µm broad and 5.0–6.0
µm long; spores 5.6–8.2 µm broad and 6.8–9.0 µm long.
Distribution: Bil (Nawabganj).
Nostoc Vaucher ex Bornet & Flahault
Nostoc ellipsosporum var. violaceum C.B.Rao, Proc. Indian Acad. Sci., B. 6: 359, f. 4C, 1937; Desikachary,
Cyanophyta 383, t. 68, f. 4. 1959. („violacea‟). (Fig. 2Q)
Thallus gelatinous, irregularly expanded; filament flexuous, loosely entangled; trichome constricted at the
cross-walls; cells cylindrical or almost quadrate; heterocysts almost spherical or cylindrical with the rounded flat
end; spores ellipsoidal, almost spherical or cylindrical with the smooth outer wall.
Dimension: Trichome 3.2–3.6 µm broad and cells 2.5–7.9 µm long; heterocysts 4.0–6.2 µm broad and 4.8–7.8 µm
long; spores 5.0–7.0 µm broad and 5.6–12.0 µm long.
Distribution: Bil (Barabilla).
Oscillatoriales Schaffner
Gomontiellaceae Elenkin ex Geitler
Komvophoron Anagn. & Komárek
Komvophoron crassum (Vozžhenn.) Anagn. & Komárek, Arch. Hydrobiol., Suppl. (Algol. Stud. 50-53) 80:
373. 1988. (Fig. 2R)
Pseudanabaena crassa Vozžhenn., Bot. Mater. Bot. Inst. Akad. Nauk. SSSR, Spor. Rast. 9: 73, f. 1: 2. 1953.
Trichome curved sometimes more or less straight, short 8 to 80 celled, rarely more cells, distinctly
constricted at the cross-walls; cells cylindrical; apical cell almost rounded.
Dimension: Trichome 4.1–4.9 µm broad and cells 4.4–5.6 µm long.
Distribution: Bil (Chakla).
Oscillatoriaceae Kirchn.
Gupta (2017) 4(3): 421–432
Oscillatoria Vaucher ex Gomont

Oscillatoria angusta Koppe, Arch. Hydrobiol. Planktonk. 14: 641, 1923; Desikachary, Cyanophyta 227, 1959.
(Fig. 2S)
Trichome solitary, blue-green, not constricted at the cross-walls; cells cylindrical, longer than broad; cell
contents homogenous; apical cell almost cylindrical rounded.
Distribution: Bil (Jalsukha).
Figure 2. A, Cyanosarcina burmensis (Skuja) Kovácik; B, Dactylococcopsis raphidioides Hansg.; C, Gloeocapsa

nigrescens Nägeli; D, Microcystis ichthyoblabe Kütz.; E, M. novacekii (Komárek) Compère; F, M. panniformis Komárek;
G–K, M. wesenbergii (Komárek) Komárek; L, Aphanocapsa elachista var. conferta W.West & G.S.West; M, A. rivularis
(Carmich.) Rabenh.; N, Synechocystis pevalekii Erceg.; O, Anabaena inaequalis Bornet & Flahault; P, Anabaena vaginicola
f. fertilissima B.N.Prasad; Q, Nostoc ellipsosporum var. violaceum C.B.Rao; R, Komvophoron crassum (Vozžhenn.) Anagn.
& Komárek; S, Oscillatoria angusta Koppe.
Gupta (2017) 4(3): 421–432
Oscillatoria formosa f. loktakensis Brühl & Biswas, Mem. Asiat. Soc. Bengal 8: 264, t. 1, f. 5. 1926;
Desikachary, Cyanophyta 233, t. 39, f. 4. 1959. (Fig. 3A)
Trichome almost straight or slightly bent, flexible, slightly constricted at the cross-walls; end cell slightly
attenuated; septa prominent.
Dimension: Trichome 4.0–6.0 μm broad and cells 2.2–3.0 μm long.
Oscillatoria crassa (C.B.Rao) Anagn., Preslia 73: 372, 2001. (Fig. 3B)
Oscillatoria ornata var. crassa C.B.Rao, Proc. Indian Acad. Sci., B. 8: 165, f. 20. 1938.
Trichome straight and uniform in thickness, constricted at the cross-walls, granulated, dark blue-green in
colour; cells shorter than broad; end cell convex, not capitate; calyptra absent.
Distribution: Pond (Damua).
Oscillatoria perornata f. attenuata Skuja, Nova Acta Regiae Soc. Sci. Upsal. ser. 4(14): 47, t. 8, f. 10. 1949;
Desikachary, Cyanophyta 205, t. 41, f. 7. 1959. (Fig. 3C)
Trichome narrow, apices slightly attenuated and bent or curved, pale blue-green with or sometimes without
gas-vacuoles; cells highly granular including septa.
Dimension: Trichome 9.3–11.8 μm broad and cells 2.8–5.8 μm broad.
Distribution: Bil (Hazartakiya).
Oscillatoria tenuis var. natans Gomont, Ann. Sci. Nat. Bot. ser. 7(16): 221, f. 2-3. 1892; Prescott, Algae of the
Western Great Lakes Area 491, t. 110, f. 11. 1982; S.Sharma & M.L.Naik, Phykos 35: 140, 1996. (Fig. 3D)
Trichome straight, blue-green; cells broader than long; apical cell almost rounded.
Distribution: Pond (Damua).
Phormidium Kütz. ex Gomont
Phormidium rimosum (Komárek) Anagn. & Komárek, Arch. Hydrobiol., Suppl. (Algol. Stud. 50-53)80: 408,
1988; Komárek & Anagn., Cyanoprokaryota Part 2: Oscillatoriales 19(2): 440, f. 641. 2005. (Fig. 3E)
Lyngbya rimosa Komárek, Preslia 28: 374, f. 3 T - U & f. 4. 1956.
Thallus dark blue-green; filament short; sheath firm, attached to trichome, confluent within the colony;
trichome cylindrical, not constricted or sometimes very slightly constricted at the cross-walls; not attenuated
towards ends; cells usually broader than long, granulated; apical cell usually rounded; calyptra absent.
Synechococcales L.Hoffm., Komárek & J.Kastovsky
Heteroleibleiniaceae (Komárek & Anagn.) Komárek, J.Kastovsky, J.Mares & J.R.Johans.
Heteroleibleinia (Geitler) L.Hoffm.
Heteroleibleinia mesotricha (Skuja) Anagnostidis & Komárek, Arch. Hydrobiol., Suppl. 80: 434, 1988. (Fig. 3F)
Lyngbya mesotricha Skuja, Nova Acta Regiae Soc. Sci. Upsal. ser. 4, 14(15): 54, t. 9, f. 1-7. 1949.
Filament more or less curved, fixed to the substratum by the basal portion; sheath thin to moderately broad,
firm, colourless; trichome end not attenuated, not constricted at the cross-walls, marked with one or two large
granules on either side of cross-walls; cell contents pale blue-green, homogeneous; apical cell round or rounded
conical; calyptra absent.
Distribution: Dighi (Raikhan).
Pseudanabaenaceae Anagn. & Komárek
Jaaginema Anagn. & Komárek
Jaaginema geitleri (Frémy) Anagn. & Komárek, Arch. Hydrobiol. Suppl. 80 (Algol. Stud. 50-53): 395. 1988.
(Fig. 3G)
Oscillatoria geitleri Frémy, Arch. Bot. Mém. 3(2): 216, f. 185. 1930.
Gupta (2017) 4(3): 421–432
Trichome single, long, blue-green, not constricted at the cross-walls; end erect, not attenuated, subcapitate;
septa not granulated.
Distribution: Bil (Kuchla).
Jaaginema geminatum (Schwabe ex Gomont) Anagnostidis & Komárek, Arch. Hydrobiol. Suppl. 80 (Algol.
Stud. 50-53): 395. 1988. (Fig. 3H)
Oscillatoria geminata Schwabe ex Gomont, Ann. Sci. Nat. Bot. ser. 7(16): 222, t. 7, f. 6. 1892.
Trichome curved or sometimes straight, blue-green, constricted at the cross-walls; not attenuated at the end;
cells usually longer than broad; apical cell more or less rounded without calyptra.
Figure 3. A, Oscillatoria formosa f. loktakensis Brühl & Biswas; B, Oscillatoria crassa (C.B.Rao) Anagn.; C, O. perornata
f. attenuata Skuja; D, O. tenuis var. natans Gomont; E, Phormidium rimosum (Komárek) Anagn. & Komárek; F,
Heteroleibleinia mesotricha (Skuja) Anagnostidis & Komárek; G, Jaaginema geitleri (Frémy) Anagn. & Komárek; H, J.
geminatum (Schwabe ex Gomont) Anagnostidis & Komárek; I, Pseudanabaena amphigranulata (Goor) Anagn.; J. P.
dictyothalla (Skuja) Anagn.; K, P. limnetica (Lemmerm.) Komárek; L, P. redekei (Goor) B.A.Whitton.
Pseudanabaena Lauterborn
Pseudanabaena amphigranulata (Goor) Anagn., Preslia 73: 360. 2001. (Fig. 3I)
Oscillatoria amphigranulata Goor, Recueil. Trav. Bot. Néerl. 15: 255, t. 2, f. 2. 1918.
Gupta (2017) 4(3): 421–432
Trichome almost straight, constricted at the cross-walls with prominent granules; end not attenuated, not
capitates; end cell rounded, calyptra absent.
Distribution: Bil (Barabilla), Lake (Mahadhap) and Pond (Jorkuppa).
Pseudanabaena dictyothalla (Skuja) Anagn., Preslia 73: 360, 2001; Komárek & Anagn., Cyanoprokaryota Part
2: Oscillatoriales 19(2): 80, f. 52. 2005. (Fig. 3J)
Phormidium dictyothallum Skuja, Symb. Bot. Upsal. 9(3):51, t. 5, f. 8-11. 1948.
Trichome more or less straight or slightly bent; constricted at the cross-walls, not attenuated at the end; cells
almost cylindrical, blue-green with rounded ends; cell contents homogenous; apical cell rounded; calyptra
absent.
Pseudanabaena limnetica (Lemmerm.) Komárek, Sborn. Jihočesk. Muz. Ceských Budějovicich, Přir. Vedy 14:
162. 1974. (Fig. 3K)
Oscillatoria limnetica Lemmerm., Ber. Duetsch. Bot. Ges. 18: 310, 1900.
Trichome pale blue-green, straight sometimes slightly bent, distinctly constricted at the cross-walls, not
attenuated and capitates; end cell rounded; calyptra absent.
Distribution: Bil (Balutuli) and Lake (Mahadhap).
Pseudanabaena redekei (Goor) B.A.Whitton, Cyanobacteria (Cyanophyta) 110, 2011. (Fig. 3L)
Oscillatoria redekei Goor, Recueil. Trav. Bot. Néerl. 15: 258, t. 2, f. 3. 1918; Komárek & Anagn.,
Cyanoprokaryota Part 2: Oscillatoriales 19(2): 440, f. 641. 2005.
Trichome solitary, straight or slightly curved, pale blue-green or yellow-green, not or slightly constricted at
the cross-walls; not attenuated at the end, not capitate; cells usually longer than broad with two small or large
polar aerotopes at the septa; apical cell almost rounded; calyptra absent.
Distribution: Jheel (Mahadhap).
Merismopediaceae Elenkin
Aphanocapsa Nägeli
Aphanocapsa elachista var. conferta W.West & G.S.West, J. Linn. Soc., Bot. 40: 432, t. 19, f. 1. 1932;
Desikachary, Cyanophyta 133, t. 22, f. 10. 1959. (Fig. 2L)
Colony ellipsoidal or almost oval or spherical; mucilage colourless; cells more or less closely arranged.
Dimension: Cells 1.4–2.0 μm in diameter and colony 56.0–71.0 μm in diameter.
Distribution: Bil (Manna).
Aphanocapsa rivularis (Carmich.) Rabenh., Fl. eur. alg. 11: 49, 1865. Prescott, Algae of the Western Great
Lakes Area 454, t. 101, f. 17. 1982. (Fig. 2M)
Palmella rivularis Carmich., in Hook., Brit. Fl. 2(1): 397, 1833.
Cells globose, with bright blue-green granular contents, usually solitary or in pairs, scattered within the
colonial mucilage envelope.
Synechococcaceae Komárek & Anagno.
Synechocystis Sauv.
Synechocystis pevalekii Erceg., Acta Bot. Inst. Bot. Univ. Zagreb 1: 77, t. 1, f. 8. 1925; Desikachary,
Cyanophyta 145, t. 25, f. 11. 1959. (Fig. 2N)
Cells spherical, two together or sometimes single, generally without visible gelatinous matrix.
Distribution: Dighi (Paradhala).
Gupta (2017) 4(3): 421–432
DISCUSSION AND CONCLUSION

Altogether 27 species (including 03 variety and 03 forms) have been reported from 18 water bodies of
Maldah District, West Bengal. Out of 13 blocks of Maldah District Cynoprokaryotes were recorded from 03
Ponds, 10 Bils, 01 Lake and 04 Dighi of 08 blocks namely Ratua I, Ratua II, Harishchandrapur I,
Harishchandrapur II, Chanchal II, Gazol, Old Maldah and English Bazar. Blockwise maximum 09 new records
were observed from different water bodies of English Bazar viz. Aphanocapsa rivularis (Carmich.) Rabenh.,
Cyanosarcina burmensis (Skuja) Kovácik, Jaaginema geminatum (Schwabe ex Gomont) Anagn. & Komárek,
Microcystis novacekii (Komárek) Compère, Oscillatoria angusta Koppe, Oscillatoria formosa f. loktakensis
Brühl & Biswas, Phormidium rimosum (Komárek) Anagn. & Komárek, Pseudanabaena amphigranulata (Goor)
Anagn. and Pseudanabaena dictyothalla (Skuja) Anagn. and again maximum 04 species observed only from
Jalsukha Bil of English Bazar followed 02 species from each water bodies of block Gazol, Harishchandrapur I,
Harishchandrapur II, Old Maldah, Ratua I and Ratua II. However, single species observed from Chakla Bil of
block Chachal II i.e. Komvophoron crassum (Vozžhenn.) Anagn. & Komárek. It was observed that some of the
species have been observed in more than one water bodies like Pseudanabaena amphigranulata (Goor) Anagn.
from Barabilla Bil, Mahadhap Lake and Jorkuppa Pond and Pseudanabaena limnetica (Lemmerm.) Komáre from,
Balutuli Bil and Mahadhap Lake. From these studies, one can easily explain the ecological preferences of
various species of Cyanoprokaryota. These taxonomic studies may be used in experimental as well as ecological
studies. This study will fill the gap in our existing knowledge of the biodiversity of the Cyanoprokaryotes of this
area. A new distributional record recorded from West Bengal of Maldah District, which we expect to be utilized
by experimental and evolutionary researchers worldwide. Further Molecular studies in combination with
morphological studies will provide new insights into the real diversity of Cyanoprokaryota and their bio-
geographical distribution in this environment. However, many more studies are needed to unravel the enormous
diversity of Cyanoprokaryota and to better define their bio-geographical patterns.
ACKNOWLEDGEMENTS
The author is thankful to the Director, Botanical India, Ministry of Environment Forest & Climate Change,
Government of India, Kolkata for proving the necessary laboratory facilities for completion of this work.
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ISSN (E): 2349 – 1183
ISSN (P): 2349 – 9265
4(3): 433–440, 2017
DOI: 10.22271/tpr.2017.v4.i3.057
Research article
Isolation and characterization of hemagglutinins from

Clerodendrum phlomidis L.f.
Aaditi S. Khagar, Prerna S. Khagar, Shubhangi K. Pingle*, Rajani G. Tumane,
Aruna A. Jawade and Shraddha Jaiswal
National Institute of Miners’ Health, JNARDDC Campus, Wadi Nagpur-440023, Maharashtra, India
*Corresponding Author: pingle.shubhangi@gmail.com [Accepted: 23 November 2017]
Abstract: Lectins from the leaves, fruit, calyx and stem of Clerodendrum phlomidis plant were
isolated after screening of total 54 plants from central India. The crude extract of leaves, fruit,
calyx and stem of C. phlomidis plant was purified by ammonium sulfate precipitation, followed by
dialysis. The isolated lectins of C. phlomidis plant were able to agglutinate human as well as dog,
hen, goat mouse, mice and fish erythrocytes. The isolated lectins were found to be lactose specific.
The pH stability of C. phlomidis leaves lectins was between 4–10 pH, for fruit lectins it was 5–7
pH, and for calyx and stem lectins it was found stable in between 4 and 8 pH. The calyx and stem
lectins were active until 60ºC, fruit has shown activity upto 50ºC and in case of leaves, it was
found maximum 40ºC. Beyond their respective optimum temperature and pH range, all the lectins
were unable to agglutinate erythrocytes. Hemagglutination activity of C. phlomidis plant lectins
was also checked in the presence of different metal ions and it was found that it was partially
inhibited by aluminium metal ion.
Keywords: Clerodendrum phlomidis - Hemagglutinine - Lectins - Leguminosae.
[Cite as: Khagar AS, Khagar PS, Pingle SK, Tumane RG, Jawade AA & Jaiswal S (2017) Isolation and
characterization of hemagglutinins from Clerodendrum phlomidis L.f. Tropical Plant Research 4(3): 433–440]
INTRODUCTION
Lectins are the glycoproteinatious, non enzymatic, non immune origin structures having the ability to bind
specifically to carbohydrate moieties (Goldstein & Poretz 1986). The term lectin was first coined by Boyd and
Shapleigh in 1954, which literally means ‘to select’. Since they can bind to glycoconjugates present on the
surface of red blood cells of humans as well as some of the animals, and agglutinate the cells hence also termed
as hemagglutinins (Sharon & Lis 2004). The lectins are widely distributed among animals, microorganisms and
plants. Plants are the abundant source of lectins. Plant lectins or phytolectins are defined as plant proteins
possessing at least one non-catalytic domain, which binds reversibly to a specific mono- or oligosaccharides
(Peumans & Damme 1995). The plant family Leguminosae is considered as a rich source of
phytohemagglutinin. Plant lectins are involved in the symbiotic relationship between plant roots and symbiotic
microorganisms and fungi. Some of the lectins providesdefense against predatory animals and phytopathogens.
As compared to animal lectins, plant lectins are easily available to mankind and can utilize their properties in
various areas such as ABO blood grouping, cytotoxic, antimicrobial (Lam & Ng 2010) and antilarval activity
(Célia & Grossi-de-Sa’ 2002).
Clerodendrum phlomidis L.f. is a small herb commonly distributed in India, Sri Lanka, China and Australia,
belongs to family Verbenaceae. Clerodendrum phlomidis (CP) commonly known as Agnimatha is a part of
important Ayurvedic formulation known as Dasmoolarishta. It is commonly used as an herbal medicine in
Indian and China from ancient times. In the present study, lectins were isolated, purified and characterized from
various parts of CP such as leaves, fruits, calyx and stem (Sonawane et al. 2014).

Chemicals and reagents
The required chemicals such as Ammonium sulphate, Sodium chloride, Dialysis membrane-50, total protein
Khagar et al. (2017) 4(3): 433–440
kit (Bio-system reagents and chemicals), sugars (Arabinose, mammose, lactose, xylose, galactose, sorbitol,
maltose, sucrose, glucose, fructose, ribose) pH buffers (pH 2–12), Acrylamide, Bis-acrylamide, Coomassie
brilliant blue, urea, bromophenol blue, glycerol, TEMED and other chemicals were purchased from Himedia
and Serva chemicals.
Selection of plant material
Total fifty-four plants were screened for the presence of hemagglutination activity, among them
Clerodendrum phlomidis is abundant in central India. The plant is authenticated from Botany Department of
RTMNU Nagpur University, Nagpur. (Authentication No. CP: 10065). The hemagglutination activity of
extracts of study plant were screened against human blood group A, B and O collected from healthy donors and
also with some of the animal's blood collected from the veterinary hospital, Nagpur.
Plant extract preparation
The leaves, fruits, calyx and stems of Clerodendrum phlomidis were separated and washed thoroughly with
distilled water and soaked. The separated parts of both plants were homogenized with minimum amount of
saline with mortar & pestle at 4°C. Around 70 ml of extract was collected by filtering the homogenate with filter
paper, centrifuged at 1000 rpm for 10 min. The obtained supernatant was stored at 4°C for further use.
Isolation and characterization of lectins from selected plant
Ammonium sulphate (AS) precipitation of crude extracts: The ammonium sulphate purification relies on
decrease in interaction of proteins with water molecules as ammonium sulphate concentration increases from 60
to 90%. The lectins from dialysed extracts precipitate when protein precipitation was done by increasing the
concentration of ammonium sulphate by gradual stirring at 4°C. Ammonium sulphate was removed from
theextract by dialysing against normal saline at 4°C. The dialysis bag having micro pore size 2.4 nm to easily
diffuse small molecules from the pores while traps comparatively big size protein molecules inside the bag. The
ammonium sulphate dissolved extract was centrifuged at 1000 rpm for 1 hr. The precipitate was collected and
dissolved in minimum amount of saline followed by dialyses to remove excess amount of ammonium sulphate.
Protein estimation: Total protein was estimated by using Bio-system kit based on Biuret method, taking Bovin
Serum Protein as a standard and measured by semi- autoanalyzer (Gomall et al. 1949).
Heamaglutination activity of isolated CP lectins
Preparation of 2% erythrocytes: The human and animal blood samples were collected in heparinized vials and
washed 3–4 times with normal saline.One ml washed RBC’s were used to prepare 2% RBC’s in 50 ml normal
saline and stored at 4°C to test the hemagglutination activity against extracts (Olsen 1994).
Hemagglutination Assay: Using 2% RBC’s hemagglutination assay was determined against extracts in 96
microtiter well plate by serial dilution of extracts (Deshpande 2003). The activity was observed by button and
carpet pattern formation in the wells. The well-showing carpet was positive for hemagglutination and wells with
button pattern considered as inactive dilution for hemagglutination. Hemagglutination unit was calculated by
taking reciprocal of minimum dilution showing hemagglutination. The specific hemagglutination activity was
defined as a unit per mg protein. Specific activity was calculated in hemagglutination activity.
Hemagglutination Inhibition Assay: The effect of carbohydrate molecules on hemagglutination activity of
purified lectins was tested by incubating 100 μl extracts with 100 μl of 500 mM concentrations of different
sugars such as arabinose, maltose, lactose, xylose, galactose, sorbitol, glucose, fructose, ribose, mannose and
sucrose (Kurokawa et al. 1976) for 30 min. The inhibition was examined by adding 100 μl of 2% RBC’s to each
well. The minimum non-agglutinating dilution concentration was considered as threshold inhibitory
concentration.
Characterization of CP lectins
Optimization of pH for Hemagglutination activity of lectins: The optimum pH of lectins activity was examined
by treating extracts with different pH range 2–12. For the preparation of pH buffers, different compositions were
used. During assay 100 μl of the extract was incubated with pH buffers for 30 minutes at room temperature and
hemagglutination activity was determined against 2% RBC’s. Optimization of pH was determined by plotting
graph of pH range against percent agglutination (Suseelan et al. 1997).
Optimization of temperature for Hemagglutination activity of lectins: The 200 μl aliquots of extracts were
incubated at different temperature range 30–100°C for 30 minutes. Hemagglutination test was carried out
Khagar et al. (2017) 4(3): 433–440
against 2% RBC’s with different temperature ranges. The temperature for maximum stability was determined by
plotting graph of temperature range against percentage agglutination.
Effect of different metal ions: The metal ion requirement by purified lectins from extracts was determined by
treating extracts with different metal ions (Kawagishi et al. 1990). 100 μl of extracts were incubated with 400 µl
of 10 mM EDTA (pH 5) at 4°C for 20 hours. Then extracts were dialyzed against 25 mM sodium phosphate
buffer (pH 7). 50 μl of dialyzed samples was treated with 50 μl of 1 mM Mg+, Ca+, Hg+, Al+ ions and incubated
for 2 hr and hemagglutination assay was performed as above described standard protocol.
Native Polyacrylamide Gel Electrophoresis: Native PAGE was performed to determine the molecular weight of
lectins from leaves, fruit, calyx and stem of CP. 10% running gel and 5% stacking gel was used with a standard
marker protein (Serva Unstained SDS PAGE Protein Marker, 6.5–200kDa– catalogue no.39215.01). After
electrophoresis, the gel was kept in fixative for 30 min. and then stained with 0.2 % coomassie brilliant blue (R-
250) for overnight, Finally, the gel was destained in 10% acetic acid until the background gets cleared and then
analysed in densitometer (Bio Rad. PD Quest Software – 8.0.1)
RESULTS
Clerodendrum phlomidis L.f. was chosen on the basis of ample availability in surrounding campus and its
unknown medicinal use to local people. The various parts of Clerodendrum phlomidis (CP) plant such as leaves,
fruits, calyx; stem showed hemagglutination activity against human blood group as well as animal blood.
The method of ammonium sulphate partial purification (Ramteke & Patil 2005, Mothong 2009) of plant
lectins showed suitable saturation of 60–90% to precipitate almost total lectins from all parts of the plants.
Protein estimation
The protein content in extract of plants was found to be 10.3 mg.ml-1 in CP leaves, 10.6 mg.ml-1 in CP fruit,
10.0 mg.ml-1 in CP Calyx and CP Stem.
Heamaglutination activity of isolated CP lectins
Figure 1. Hemagglutinationtiter of partially purified extract of Clerodendrum phlomidis L.f.: A, Leaves; B, Calyx; C,
Fruits; D, Stem.
Hemagglutination Assay: Hemagglutination titer with serial dilution of all extracts against human blood group
was performed. In case of CP leaves A and O blood group showed carpet pattern till sixteen times dilution while
B blood group showed carpet pattern till eight times dilution. CP fruit had given carpet till sixty-four times with
A blood group and sixteen times in both B and O blood groups. Calyx from the same plant had given carpet
pattern up to four times dilutions in A and O while two times with B blood group. CP stem had given four times
dilution carpet with A blood group and upto eight times with B and O groups. The hemagglutination titer is
shown in figure 1. On the basis of titre, Hemagglutination Unit (HAU) and Specific Activity (SA) was
calculated which is presented in table 1.
Khagar et al. (2017) 4(3): 433–440
Table 1. Hemagglutination Unit (HAU) and Specific Activity (SA) calculations of partially purified lectins in
leaves, calyx, fruits and stem of Clerodendrum phlomidis L.f.
Dialyzed Estimated Protein HAU.ml-1 SA
extract (mg.ml-1) ‘A’ ‘B’ ‘O’ ‘A’ ‘B’ ‘O’
CP leaves 10.3 164.8 412 164.8 16 40 16
CP fruits 10.6 678.4 169.6 169.6 64 16 16
CP calyx 10.0 40 20 40 4 2 4
CP stem 10.0 40 80 80 4 8 8
Agglutination pattern of extracts with different blood group such as human and various animals is depicted
in table 2. All the extracts of CP showed strong hemagglutination activity against human blood group as well as
some of the animal blood such as dog, hen, goat, mouse, mice and fish. It indicates along with human red blood
cells, animal blood cells also present carbohydrate moieties which act as a ligand for plant lectins.
Table 2. Agglutination of human and animal erythrocytes by partially purified lectins from leaves, calyx, fruits
and stem of Clerodendrum phlomidis L.f.
Erythrocytes CP Leaves CP Fruit CP Calyx CP Stem
Human ‘A’ ++ + + +
Human ‘B’ ++ ++ ++ +
Human ‘O’ ++ ++ + +
Dog + + + +
Hen + + + +
Goat + + + +
Mouse + + + +
Mice + + + +
Fish + + + +
Hemagglutination Inhibition assay: Carbohydrate binding specificity or carbohydrate-affinity of lectin of CP
was examined in this inhibition experiment. Different sugars like D-Glucose, Sucrose, Lactose, Sorbitol, D-
Fructose, D-Maltose, D-Arabinose, D-Galactose, D-Xylose, D-Mannose and D-Ribose were used for
determination of inhibition of hemagglutination of lectin activity. Lactose was found to be the only inhibitor of
agglutination activity in CP plant extracts.
Further with lactose as the only inhibitor of hemagglutination activity, hemagglutination titer with various
dilutions of lactose was performed. The minimum concentration required to inhibit the agglutination of different
extracts is shown in table 3.
The values shown in the table 3 indicated that the leaves and fruits lectins activity can be inhibited by lactose
at lowest dilution of 125, at the same time activity inhibition for Calyx and Stem is 62.5 and 15.62 respectively.
Table 3. Inhibition of hemagglutination with different sugars.
Minimum concentration required to inhibit the hemagglutination (mM)
Sugars
CP Leaves CP Fruits CP Calyx CP Stem
D-Arabinose NI NI NI NI
D-Mannose NI NI NI NI
Lactose 125 125 62.5 15.62
D-Xylose NI NI NI NI
D-Galactose NI NI NI NI
Sorbitol NI NI NI NI
D-Maltose NI NI NI NI
Sucrose NI NI NI NI
D-Glucose NI NI NI NI
D-Fructose NI NI NI NI
D-Ribose NI NI NI NI
Note: NI, No Inhibition; CP, Clerodendrum phlomidis L.f..
Characterization of CP lectins
Optimization of pH for Hemagglutination activity of lectins: The varied pH stability of lectins and overlapping
of the ranges were observed. The leaves of CP plant showed a wide range of stability in 4–10 pH indicating
activity in acidic as well as basic pH. The calyx and stem of CP plant showed pH stability between 4–8 pH
range. The fruits lectins were stable in the range between 5–7 pH. Figure 2 depicted the graphical representation
of pH stability of lectins.
Khagar et al. (2017) 4(3): 433–440
Figure 2. The graphical representation of pH stability of lectins in Clerodendrum phlomidis L.f: A,

Leaves; B, Fruits; C, Calyx; D, Stem.
Optimization of Temperature for Hemagglutination activity of lectins: The results of the CP plant leaves showed
100% hemagglutination to the 40ºC temperature. Further increase in temperature resulted in the reduction of
activity of lectins. Fruits lectins were found to be active up to 50ºC. In case of CP calyx and stem, lectins were
actively showing hemagglutination until 60ºC and start to diminish from 70ºC onwards. The activity of lectins
in temperature variation is presented in figure 3.
Figure 3. The graphical representation of temprature stability of lectins in Clerodendrum phlomidis L.f: A, Leaves; B,
Fruits; C, Calyx; D, Stem.
Effect of Metal Ions on Hemagglutination
Table 4. Effect of metal ions (1mM) on agglutination of partially purified extracts.
Metal Ions CP Leaves CP Fruits CP Calyx CP Stem
Calcium - - - -
Magnesium ++ ++ ++ ++
Mercury ++ ++ ++ ++
Aluminium + + + +
Note: ++, Sign indicates hemagglutination; +, sign indicates slight hemagglutination; -, sign
indicates no hemagglutination and hemolysis.
The effect of metal ions on hemagglutination activity of plant lectins was observed in EDTA treated extracts
against metal salts and 2% RBC’s. The treatment with calcium salt hemolysed the RBC’s. Mercuric chloride
and magnesium chloride treatment was not effective to inhibit hemagglutination whereas Aluminium has shown
Khagar et al. (2017) 4(3): 433–440
light button pattern indicating partial inhibition of agglutination possed by Aluminium ions. Table 4 showed the
results of effects of metal ion (1mM) on agglutination of partially purified extracts.
Native Polyacrylamide Gel Electrophoresis
Native PAGE of the partially purified lectins from CP leaves, CP Fruit, CP Calyx and CP Stem gave protein
band with molecular weight CP leaves 67 kDa and 21 kDa, CP Fruit 70 and 21 kDa, CP Calyx and CP Stem 70,
50 and 45 kDa (Fig. 4).
Figure 4. Band pattern of lectins Marker (M), CM Leaves (L1), CM Fruit (L2), CM Calyx (L3), CM Stem (L4) on Native
PAGE.

The present study focused on isolation and characterisation of lectins form Clerodendrum phlomidis L.f.
plant. Most of the lectins those agglutinates erythrocytes are not specific towards blood groups, for example,
wheat germ agglutinins (Nagata & Burger 1974). With the same line obtaimed lectins of the CP plant are not
specific for blood groups, it can be infered that lectin specific glycoconjugates or glycorecepters are not present
on cell surface or vice varsa. It is known that, phytolectins have property to specifically bind glycoconjugates on
the cell surface and agglutinates RBCs of human as well as animals. Plant extract also agglutinates some of the
animal erythrocytes which is supported by study of Euphorbia tithymaloids by Jawade et al. (2016), Ahmed &
Cahtterjee (1987) and with Tridax procumbans by Ramteke & Patil (2005).
Many lectins are specific for their carbohydrate ligands with different specificity. In a report by
Chumkhunthod et al. (2006) Schizophyllum commune lectin has shown specificity towards N-acetyl-D-
galactosamine. To identify specific legands for lectins of the present sudy, hemagglutination inhibition assay
was performed. The results indicated that CP lectins is lactose specific. The effect of lactose dilution was
examined by serially diluting 500 mM lactose. The agglutination in case of leaves and fruits was inhibited at 4
times dilution while at 8 times and 16 times in case of calyx and stem respectively.
Optimization of pH is one of the important factor responsible for the structure of proteins. In the present
study, CP leaves lectins have shown a wide range of pH stability between 4–10. Calyx and stem lectins have pH
stability between 4–8 while fruit lectins are active within narrow range of pH 5–7. This indicates that leaves
lectins is compatible and active not only in highly acidic but also at highly basic pH. Almost similar results were
reported by Osukoya (2016) on the lectins of the fruit of cola nitida, kola nut.
The optimum temperature for maximum activity of CP calyx and stem lectins is shown upto 60°C and it
loses its activity at 70°C onwards. Whereas lectins from Vigna mungo (Suseelan et al. 1997) and
Erythrinaveluntina, Jackfruit (Artocarpus integrifolia) lectins (Ahmed & Cahtterjee 1987) were stable at 70°C.
Lectins from CP leaves and fruits are stable upto 50°C and 60°C respectively. From the above results we can
conclude that CP leaves lectins is tolerant for pH but not for temperature.
Khagar et al. (2017) 4(3): 433–440
In metal ion inhibition assay only aluminium metal ion has shown partial hemagglutination inhibition with
EDTA treated extracts. At the same time, Tridax procumbens calyx lectin inhibit hemagglutination activity by
Mercury (Ramteke & Patil 2005). The partially purified lectins from CP leaves, CP fruits, CP calyx and CP
stem showed multiple bands on Native PAGE with molecular wiegth 70 kDa, 67 kDa, 50 kDa,45 kDa and 21
kDa. The 70 kDa bands were observed in all the parts of the plant. It can be speculated that the MW of the
lectins is similar in all the parts of the plant.
Now a days, study on Lectins has been diverted towards their use in the development of blood grouping tool,
cancer therapeutics, antimicrobial and antiparasitic agents and many more diagnostic tools and curative drug
development areas. In the present work, we have screened CP lectins for the basic physical and chemical
characters. Further study on molecular charcterisation and activity of isolated lectins against the microbial
system, cancer cells, Diabetic cells and Immunogenicity can help us to introduce multiple uses of CP lectin in
future.
ACKNOWLEDGEMENT
Authors are thankful to Director, National Institute of Miner’s Health for their constant support and
encouragement. We are also thankful to the people who donate blood samples for our study.
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ISSN (E): 2349 – 1183
ISSN (P): 2349 – 9265
4(3): 441–448, 2017
DOI: 10.22271/tpr.2017.v4.i3.058
Research article
Pharmacological screening of Gymnanthemum coloratum (Willd.)

H. Rob. & B. Kahn (Compositae) and Terminalia ivorensis A.
Chev. (Combretaceae) from DR Congo: Spotlight on the
antisickling, antibacterial and anti-diabetic activities
Gédéon Ngiala Bongo1, Koto-te-Nyiwa Ngbolua1, 2, 3*, Colette Masengo Ashande2, Kevin
L. Karume1, Janvier Mukiza4, Dorothée D. Tshilanda5, Damien S.T. Tshibangu5,
Nadège K. Ngombe6, Théophile F. Mbemba1 and Pius T. Mpiana5
1
Département de Biologie, Faculté des Sciences, Université de Kinshasa, B.P. 190 Kinshasa XI, République
Démocratique du Congo
2
Département des Sciences de l’Environnement, Faculté des Sciences, Université de Gbadolite, B.P. 111 Gbadolite,
Province du Nord Ubangi, République Démocratique du Congo
3
Institut Supérieur Pédagogique d’Abumombazi, Province du Nord Ubangi, République Démocratique du Congo
4
Department of Biochemistry, Faculty of Medicine and Surgery, University of Gitwe, Nyanza, Republic of Rwanda
5
Département de Chimie, Faculté des Sciences, Université de Kinshasa, B.P. 190 Kinshasa XI, République
Démocratique du Congo
6
Faculté des Sciences Pharmaceutiques, Université de Kinshasa, Kinshasa XI, République Démocratique du Congo
*Corresponding Author: jpngbolua@unikin.ac.cd [Accepted: 25 November 2017]
Abstract: In Democratic Republic of the Congo (DRC), it was reported a rare association in a
patient, of two genetic diseases namely sickle cell anemia and diabetes which have a common
denominator that is to make patients susceptible to infections. They constitute a serious public
health problem in Africa. Given the difficult and limited management of these diseases, the use of
Traditional Medicine and medicinal plants can be an effective alternative. The leaves of both
Gymnanthemum coloratum and Terminalia ivorensis were collected in 2014 in Kinshasa city and
Gbadolite city (Nord Ubangi province) respectively and were selected through chemotaxonomic
approach. The bacterial strains used for assessing the antibacterial were Staphylococcus aureus
and Escherichia coli and mice for the antidiabetic activity. The phytochemical screening showed
the presence of total polyphenols, tannins, flavonoids, linked quinones, saponins,
leucoanthocyanins, alkaloids and anthocyanins. The organic extracts of G. coloratum and T.
ivorensis showed an antisickling activity. Only S. aureus was sensitive to the leaves of T. ivorensis
(MIC < 62.5 µg.mL-1) and G. coloratum (MIC ≤ 250 µg.mL-1) while no effect was observed on E.
coli. The mean values for glycemia in treated and untreated mice after 2 hours were 62±14.3
mg.dL-1 (Glibenclamide 20 mg.Kg-1) and 70.4±16.6 mg.dL-1 (ethyl acetate extract of T. ivorensis
500 mg.Kg-1). To our knowledge, it is for the first time that the antisickling activity of G.
coloratum and T. ivorensis is reported thus validating the chemotaxonomic approach used as a
criterion for selecting these two plants. It is also for the first time that anti-hyperglycaemic activity
of T. ivorensis is reported.
Keywords: Sickle cell anemia - Diabetes - Chemo-taxonomic approach.
[Cite as: Bongo GN, Ngbolua K-te-N, Ashande CM, Karume KL, Mukiza J, Tshilanda DD, Tshibangu DST,
Ngombe NK, Mbemba TF & Mpiana PT (2017) Pharmacological screening of Gymnanthemum coloratum
(Willd.) H. Rob. & B. Kahn (Compositae) and Terminalia ivorensis A. Chev. (Combretaceae) from DR Congo:
Spotlight on the antisickling, antibacterial and anti-diabetic activities. Tropical Plant Research 4(3): 441–448]
INTRODUCTION
In Democratic Republic of the Congo (DRC), it has been recently reported that a rare association in a patient,
Received: 22 July 2017 Published online: 30 November 2017
Bongo et al. (2017) 4(3): 441–448
of two genetic diseases, namely sickle cell disease and diabetes, diseases that constitute a serious public health
concern worldwide (Kamba et al. 2014). Sickle cell anemia is characterized by the presence of hemoglobin S in
the blood (Ngbolua 2012, Mpiana et al. 2013), and it is the first genetic disease in Africa by the number of
patients and it is clinically manifested by a vaso-occlusion and hemolytic anemia, resulting from the
polymerization of hemoglobin S molecules into tactoids (Ngbolua 2012).
Diabetes, on the other hand, is characterized by hyperglycaemia (sugar content > 1.2 g.dL-1 on an empty
stomach), hyper-ketone (keto acid > 3 mmol.L-1), an acid-base and hydro-electrolytic imbalance. At the
muscular level, there is a real competition between free fatty acids and glycose to be oxidized: free fatty acids
are oxidized in priority, leading to an increased production of acetyl CoA which in turn inhibits glycolysis
enzymes (Declerck 2002). Thus, the storage and use of glycose are decreased at the muscular level while there
is a stimulation of gluconeogenesis in the hepatic level and all of this contributes to increase the blood glucose
(Declerck 2003, De boeck et al. 2012).
Given the difficult and limited management of sickle-cell anemia and diabetes in DRC, the use of traditional
medicine and medicinal plants can be an effective alternative in the management of both diseases in emergency
situations. According to the World Health Organization (WHO), more than 80% of the population living in poor
areas in Africa uses medicinal plants to treat themselves (Kolling et al. 2010, Mangambu et al. 2012). Several
scientific works carried out in DRC and elsewhere highlight the antisickling and antidiabetic properties of
various plants (Katemo et al. 2012, Ngombe et al. 2013, Masunda et al. 2014, Ngbolua et al. 2014a, b, c, d, e,
Kasali et al. 2016).
In the present study Gymnanthemum coloratum and Terminalia ivorensis were used which were selected
using a chemo-taxonomic approach. In fact, some species of Vernonia and Terminalia genera are known for
their antisickling and antidiabetic properties (Mbodj 2003). Following this approach, it should therefore be
expected that T. ivorensis shows antisickling and antidiabetic activities while G. coloratum (Syn Vernonia
colorata) with antidiabetic properties is expected to show an antisickling activity. In addition, both hypotheses
(antisickling and antidiabetic activities) are validated, given that diabetes and sickle-cell anemia have a common
denominator which is to make patients susceptible to infections (Ngbolua 2012), henceforth both plants have to
show an in vitro antibacterial activity. The main objective of this study was to provide the scientific rationale of
what the ethnomedical use of these two plants would represent. And, the specific objectives were to perform a
phytochemical screening of aqueous extracts of these two selected plants, to subject the leaf powders to
fractional extraction using increasingly polar solvents (petroleum ether, ethyl acetate and methanol) and last to
assess the antisickling, antibacterial and anti-hyperglycemic activities of organic extracts. The significance of
this research is to value the use of medicinal plants in the treatment and management of sickle cell disease and
diabetes.

Plant materials
Plant materials used were leaves of Gymnanthemum coloratum and Terminalia ivorensis A. Chev. collected
at Makala commune in Kinshasa and Gbadolite in Nord Ubangi province respectively. G. coloratum was
identified in the Herbariun of Faculty of Sciences, University of Kinshasa while while T. ivorensis was
identified a botanist from “Centre de Surveillance de la Biodiversité”, University of Kisangani. Blood samples
used for assessing the antisickling activity of plant extracts were taken from a sickle cell adolescent patient at
the "Centre de Médecine Mixte et d'Anémie SS" of Kinshasa. Bacterial strains used were provided by the
Laboratory of Microbiology, Faculty of Pharmaceutical Sciences (University of Kinshasa) and mice were
provided by the National Institute of Biomedical Research (NIRB).
Methods adopted
A. Collection and conditioning of plant material
Plant samples were collected in 2014 in Kinshasa and Gbadolite, in the province of Nord-Ubangi
respectively. After collection, leaves were washed with tap water and dried under shade for one month, and then
crushed separately in a shredder (Moulinex brand). The powders obtained were sieved in order to obtain fine
powders.
B. Extraction with solvent of increasing polarity
Fifty grams of powder of each species were macerated in petroleum ether, ethyl acetate, and methanol (1:10,
Bongo et al. (2017) 4(3): 441–448
w/v) respectively for 48 hours. After filtration, various extracts were evaporated to dryness using a rotary
evaporator apparatus at 37°C.
C. Phytochemical Screening
The phytochemical screening was carried out according to the standard protocol as described by Bruneton
(1999), and it can be performed in aqueous or organic phases.
D. Biological experiments
a. Emmel test
The total SS blood previously diluted in saline solution (NaCl 0.9%) by reason of four to eight drops of the
saline solution. On a slide was placed a drop of diluted blood brought into contact with a drop of the drug along
with a drop of sodium meta-bisulfite (Na2S2O5 2%).
The mixture obtained constitutes the microscopic preparation, and it is then covered with a coverslip and is
super-cooled by paraffin put on the edges of the slide. The solutions of our extracts (petroleum ether, ethyl
acetate and methanol) are prepared by dissolving a few mg of these extracts in the saline solution. Different
preparations obtained were observed with the OLYMPUS optical microscope at 10X and 40 X magnifications
after 24 hours (Ngbolua et al. 2014a, b, c, d).
b. Antibacterial assay
The antibacterial activity was evaluated by the microdilution method in a liquid medium.
i. Preparation of the stock solution
In a sterile test tube, place 0.020 g of each extract diluted in 250 μL of DMSO then stir for 10 minutes and
add 5 mL of Mueller Hinton culture medium using a pipette and mix.
ii. Preparation of the bacterial suspension
Place 2 mL of the saline solution into two sterile test tubes. Using a sterile platinum loop, take two isolated
colonies of two strains to be tested namely Escherichia coli ATCC 27195 and Staphylococcus aureus ATCC
33591 and place each colony in the saline solution in both tubes. This bacterial suspension is diluted in the
appropriate culture medium at a rate of 9 mL.
iii. Dilution of extracts and inoculation of the microplate
A 96-well sterile polystyrene microplate (8 rows A-H. x 12 columns) with a round bottom was used. Then
each well was filled with 100 μL of culture medium as follows: A2 to A8, B2 to B8, C2 to C8, D2 to D8, E2 to E8
and F2 to F8 and then the 11th and 12th wells served as controls. Using a micropipette, place 200 μL of the stock
solution of extract 1 in A1 and B1 wells, 200 μL of the stock solution of extract 2 in C1 and D1 wells while 200
μL of the stock solution of extract 3 in E1 and F1 (i.e. 2 wells were used for each extract). Meanwhile, 100 μL of
each stock or control solution were taken and later the serial dilutions of 2 to 2 were performed.
Afterwards, 100 μL of A1B1, C1D1 and E1F1 wells were taken and transferred to A3B3, C3D3 and E3F3 wells,
then from the previous wells 100 μL were taken and were transferred to A4B4, C4D4, E4F4 wells. These solutions
were thoroughly mixed and the same procedure continued till we reached A 8B8, C8D8 and E8F8 wells. The last
100 μL taken from these wells were removed and thrown away (Ngbolua et al. 2014e, f).
iv. Determination of the minimum inhibitory concentration
The growth observed in different wells containing extracts or controls were compared to that in the bacterial
growth control well (well-having inoculum without extracts or antibiotics). For a test to be considered valid, an
acceptable growth has to be observed in the control wells. If the growth is insufficient in these wells, there is a
need to re-inoculate the microplate and after the addition of 5 μL of TCC 2% (2, 3, 5-
triphenyltetrazoliumchloride) to the control wells then the minimum inhibitory concentration can be read. The
principle of this method is based on the ability of living cells to reduce the tetrazolium salt to a red or formazan
precipitate. The minimum inhibitory concentration was then read from the first wells showing no bacterial
growth after 48 hours (Ngbolua et al. 2014e, f).
c. Oral glucose tolerance test
The study was carried out on an animal model consisting of 10 NMRI mice (male and female) subjected to
temporary hyperglycemia by gavage of a glucose solution (200 mg.mL -1). These 10 mice were divided into 3
groups as follows: a first group of two mice as a negative control (saline solution), a second group of three mice
as a positive control (Glibenclamid 10 mg.Kg-1) and the third group of five mice to be tested with the ethyl
acetate extract (500 mg.Kg-1). Blood glucose testing was performed using a contour TS blood glucose meter
from the tail (Williamson et al. 1996).
Bongo et al. (2017) 4(3): 441–448
d. Testing of normal blood sugar

Mice were given a 24-hour pre-feed and then administered the extracts. Blood glucose was measured at T 0,
T1, T2, T3, and T4.
e. Antihyperglycaemic activity
In our study, we caused a temporary hyperglycemia in mice by oral administration of glucose (diluted to
10% in distilled water) at a dose of 4 g.Kg-1 of body weight. The basic blood glucose of mice was first detected.
The basal glucose level of mice was determined after 24 hours of fasting (Baseline) and then glucose was
administered to the mice. After 30 minutes of glucose overload, the blood glucose was determined in order to
note the temporary hyperglycemia (transient hyperglycemia should reach the maximal value 30 min after
administration of glucose). And then three batches of mice according to sex, weight and especially temporary
hyperglycemia were formed. Different batches of mice were treated as follows: A control batch treated with a
saline solution at a dose of 0.9 %. A reference batch treated with Glibenclamid at 10 mg.Kg-1 of body weight
and a test batch treated with ethyl acetate extract at a dose of 500 mg.Kg -1 of body weight.

A. Phytochemical Screening
The result of the phytochemical screening is presented in the table below.
Table 1. Results of phytochemical screening of aqueous extracts of two plant leaves.
PLANT EXTRACTS
Compounds
Gymnanthemum coloratum Terminalia ivorensis
Total polyphenols +++ +++
Flavonoids +++ +++
Anthocyanins +++ -
Tannins +++ +++
Leucoanthocyanins +++ ++
Bound Quinones ++ +++
Alkaloids - +++
Saponins +++ +++
Note : -, Absence of the researched substance; +, Low concentration of the researched substance; ++, High
concentration of the researched substance; +++, The highest concentration of the researched substance.
From table 1 above, it is shown that G. coloratum leaves are rich in total polyphenols, tannins, flavonoids,
linked quinones, saponins, leucoanthocyanins and anthocyanins while the total absence of alkaloids was noted.
On the other hand, the leaves of T. ivorensis are rich in total polyphenol, tannins, flavonoids, linked quinones,
saponins, leucoanthocyanins and alkaloids; however they are devoided of anthocyanins.
B. Biological activities
a. Antisickling activity
Figure 1 shows the phenotype of untreated (1a) and treated (1b) sickle cells respectively.
Figure 1. A, Optical micrographic of untreated SS blood; B, red blood cells treated with petroleum extract of Terminalia
ivorensis (50 µg.mL-1) [NaCl 0.9% ; Na2S2O5 2%, X500].
Bongo et al. (2017) 4(3): 441–448
As it can be noticed in the above figures, the red blood cells are of sickle cell phenotype showing that, the
blood used was taken from a sickle cell patient. However, in the presence of petroleum extracts (b), sickle-cells
revert to the normal circular and biconcave form. These results are consistent with previous observations
(Ngbolua 2012, Mpiana et al. 2010).
The rate of normalization (Emmel test) of sickled cells in the presence of various extracts under conditions
of isotonic hypoxia (NaCl 0.9%, Na2S2O5 2%) is presented in table 2 below.
Table 2. Rate of normalization of sickled cells using Emmel test in presence of extracts.
RATE OF NORMALIZATION (RN)
Extracts
Gymnanthemum coloratum Terminalia ivorensis
Petroleum ether extract +++ +++
Ethyl acetate extract ++ +++
Methanol +++ +++
Note: -, Inactive; +, 10 < RN < 50 % (low activity); ++, 50 < RN < 70 % (higher activity); +++,
RN > 70 % (the highest activity) [Source: Mpiana et al. 2010]
As described in table 2, the presence of organic extracts (petroleum ether, ethyl acetate and methanol) in G.
coloratum and T. ivorensis demonstrate the normalization of the sickle-cells under conditions of hypoxia. This
normalization of SS erythrocytes under conditions of hypoxia constitutes partially scientific evidence that
justifies the integration of these two plants on the database of antisickling plants. The normalization of the SS
blood erythrocytes treated with the extracts of these plants results in the reappearance of the circular form of
sickled cells. These results are therefore are similar with those of previous works (Mpiana et al. 2007) as shown
in figures 1a and 1b. However, it has to be noted that the extract of the ethyl acetate of G. coloratum showed a
high antisickling activity with a normalization rate of between 10 and 50%.
b. Antibacterial Activity
Tables 3 and 4 present the results of the antibacterial test.
Table 3. Effects of Terminalia ivorensis extracts on bacterial growth in vitro (Micro-dilution method, dye: TCC 2%).
Concentration Escherichia coli ATCC 27195 Staphylococcus aureus ATCC 33591
(µg.mL-1) EEP EAE MeOH EEP EAE MeOH
4000 + - - - - -
2000 + + - - - -
1000 + + + - - -
500 + + + - - -
250 + + + - - -
125 + + + - - -
62.5 + + + - - -
MIC > 4000 > 2000 > 1000 < 62.5 < 62.5 < 62.5
Note: +, bacterial growth (appearance of red color, conversion of colorless TCC to red formaztan); -, Absence of visible
growth (the staining of the well is that of the extract); MIC, Minimum inhibitory concentration; ATCC, American Type
Culture Collection; EEP, Petroleum ether extract; EAE, Ethyl acetate extract; MeOH, Methanolic extract.
This table shows that only S. aureus is susceptible to T. ivorensis leaf extracts (MIC <62.5 μg.mL-1) while no
effect was observed on E. coli.
Table 4. Effects of Gymnanthemum coloratum extracts on bacterial growth in vitro (Micro-dilution method, dye: TCC 2%).
Concentration Escherichia coli ATCC 27195 Staphylococcus aureus ATCC 33591
(µg.mL-1) EEP EAE MeOH EEP EAE MeOH
4000 + - - - - -
2000 + - - - - -
1000 + - - - - -
500 + + - - - -
250 + + - - - -
125 + + - - - +
62.5 + + - - + +
MIC > 4000 1000 < 62.5 < 62.5 < 62.5 250
Note: +, bacterial growth (appearance of red color: conversion of colorless TCC to red); -, Absence of visible growth (the
staining of the well is that of the extract); MIC, Minimum inhibitory concentration; ATCC, American Type Culture
Collection; EEP, Petroleum ether extract; EAE, Ethyl acetate extract; MeOH, Methanolic extract.
Bongo et al. (2017) 4(3): 441–448
From this table, it is shown that only S. aureus is sensitive to G. coloratum extracts (MIC ≤ 250 μg.mL-1). In
fact, it is well known that extracts having a MIC less than 500 μg.mL-1 are considered active and it is the case
for these two plant extracts used in this study with respect to S. aureus (Molina-salinas et al. 2007). Contrary to
E. coli, it should be noted that S. aureus is a positive gram bacterium, and its wall is thick (having several layers
superimposed) while in E. coli the additional outer membrane would prevent the entrance of chemical
compounds in the bacterial cell (Maligan & Martinko 2007). The results obtained in this work indicated that the
extracts of the two plants used in this study may constitute a source of new drugs against S. aureus, knowing
that the sickle cell anemia patients are prone to repeated tissue infarction leading to a functional asplenia and are
therefore exposed to frequent infections due to immune deficiency and phagocytic cell abnormality to cytokines
(Bégué 2009). These two plants could be very useful in clinical trials.
C. Antidiabetic activity
The hyperglycemic test carried out with the ethyl acetate extract of T. ivorensis is presented in table 5 below.
Table 5. Antihyperglycemic test carried out with the ethyl acetate extract of Terminalia ivorensis.
Time (minutes)
Drug/Extract
0 30 60 90 120 % RG
NaCl 0.9% (Control) 96±14.1 106.5±14.8 122.5±17.6 108±1.41 98.5±3.5
Glibenclamid 68±10.5 111±19.8 185±15.08 67±10.9 62±14.3 37.05
(10 mg.kg-1)
EAE (500 mg.kg-1) 45.4±9.2 93.4±17.7 97.4±14.2 74.2±10.08 70.4±16.6 28.53
Following the above table, it is clearly displayed that the mean values of blood glucose after 120 minutes in
untreated and treated mice were 98.5±3.5 mg.dL-1 (NaCl 0.9%), 62±14.3 mg.dL-1 (Glibenclamid 10 mg.Kg-1)
and 62±14.3 mg.dL-1 (ethyl acetate extract of T. ivorensis 500 mg.Kg-1) respectively. These results show that T.
ivorensis has an antihyperglycaemic (hypoglycemic) activity. In fact, the reduction rate of glycaemia is of
28.53% versus 37.05% for Gilbenclamid. Up to date, numerous hypoglycemic plants have been identified by
Fezan et al. (2008). The results on animal models showed that plant extracts could act through various
mechanisms to lower blood glucose levels, thus reinforcing our results. To our knowledge, it is for the first time
that the antisickling activity of G. coloratum and T. ivorensis is reported, thus validating the chemo-taxonomic
approach used as a criterion for selecting these two plants while it is also for the first time that the anti-
hyperglycaemic activity of T. ivorensis is reported. Henceforth, this validates the hypothesis that an antidiabetic
plant would potentially possess an antisickling activity.
CONCLUSION AND SUGGESTIONS

The aim of this study was to assess the antisickling, antibacterial and anti-hyperglycemic potential of two
taxonomic Congolese plants of which Gymnanthemum coloratum and Terminalia ivorensis. Both plants contain
secondary metabolites capable of conferring the antisickling, antibacterial and anti-hyperglycaemic activities.
Only S. aureus was sensitive to T. ivorensis and G. coloratum extracts. The extract of ethyl acetate of T.
ivorensis is endowed with anti-hyperglycemic properties. Therefore, it would be necessary to carry out a
toxicological study on both plants in order to include them in the traditional antisickling pharmacopoeia and also
to consider carrying out an advanced phytochemical study on ethyl acetate extract of T. ivorensis in order to
isolate bioactive molecules.
ACKNOWLEDGEMENTS
The authors thank The World Academy of Sciences (TWAS) for Grant No. 15-156 RG/CHE/AF/AC_G–
FR3240287018 and the Switzerland embassy at Kinshasa (DRC) for providing financial assistance to RESUD
(Research for sustainable development/NGO).
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ISSN (E): 2349 – 1183
ISSN (P): 2349 – 9265
4(3): 449–455, 2017
DOI: 10.22271/tpr.2017.v4.i3.059
Research article
Interaction of a fly ash and root-knot nematode pathogens on

Pumpkin (Cucurbita moschata Duch. ex Lam.)
Gufran Ahmad, Abrar Ahmad Khan and Safiuddin Ansari*
Department of Botany, Aligarh Muslim University, Aligarh-202002, Uttar Pradesh, India
*Corresponding Author: safiuddin7ansari@gmail.com [Accepted: 26 November 2017]
Abstract: In the present investigation it was tried to find out the interactive effect of various level
of fly ash (abiotic) and root-knot nematode, Meloidogyne incognita (biotic) on plant growth, yield
and some biochemical contents in pumpkin (Cucurbita moschata). The experiments were
conducted in pots with different level of fly ash and soil mixture in the green house at Department
of Botany, Aligarh Muslim University, Aligarh, India. Seeds of pumpkin (F1 hybrid-Nutan) were
grown in pots filled with different level of fly ash (5, 10, 20, 30, 40 and 50%). At four leaves
stage, seedlings were inoculated with 2000 larvae (J2 stage) of M. incognita. Plants grown in soil
(uninoculated and inoculated stes) serve as control for comparing with different level of fly
ashamended soil. Plant growth and yield parameters were increased significantly in 10 to 30% fly
ash amended soils. However, at higher level of fly ash (40% and 50%), plant growth and yield
were reduced significantly. The suppression in plant growth and yield were maximum at 50% fly
ash level. Similarly the chlorophyll, carotenoids, carbohydrate and proline contents were also
increased at 10–30% levels, then increased in 40 and 50% of fly ash level compared to control
(uninoculated set). All the parameters; growth, yield, chlorophyll, carotenoids, carbohydrate and
proline content of plants were better for all the fly ash level as compared to nematode inoculated
sets.
Keywords: Carotenoids - Carbohydrate - Chlorophyll - Fly ash - Nematodes - Pumpkin.
[Cite as: Ahmad G, Khan AA & Ansari S (2017) Interaction of a fly ash and root-knot nematode pathogens on
Pumpkin (Cucurbita moschata Duch. ex Lam.). Tropical Plant Research 4(3): 449–455]
INTRODUCTION
Fly ash (FA) is the waste product of coal combustion process for generation of electricity and is accepted as
an environmental pollutant. Fly ash contains fine, powder-like particles that are spherical in shape, solid or
hollow, and mostly amorphous in nature. Depending on the quantity of unburned carbon in fly ash the color may
vary by gray to black. The surface area of FA may vary from 170 to 1000 m2.kg-1 while the specific gravity
ranges by 2.1 to 3.0. Basically four types of coal, each of which varies in terms of its chemical composition, ash
content, value of heating and geological origin and are anthracite, bituminous, sub-bituminous, and lignite.
Alumina, Silica, Iron oxide and Calcium, with variable amounts of carbon are the components of bituminous fly
ash. Higher concentrations of calcium and magnesium oxide and low percentages of iron oxide and silica and
carbon contents are characters of lignite and su-bituminous coal fly ashes.
Due to fly ash application, there is an increase in the availability of major nutrients in the soil was reported
by Ram et al. (2011). Except K, the increase in the availability major nutrients concluded by Dey et al. (2012).
There is an increase in organic carbon by the application of fly ash and farm-yard manure was reported by
Karmakar et al. (2009). As an amendment, fly ash is used in agriculture especially due to the presence of macro
and micro-nutrients (Wong & Wong 1986, Raghav & Khan 2002, Rizvi & Khan 2009). Fly ash has been found
beneficial for the growth of many plants (Mishra & Shukla 1986, Singh 1989, Pasha et al. 1990, Khan & Khan
1996, Raghav & Khan 2002, Rizvi & Khan 2009).
Root-knot nematodes (Meloidogyne spp.) are microscopic roundworms found in a broad range of habitats
and agro-ecosystems. Almost they complete their life cycle in the roots of the host plants; in spite of they can
Received: 27 May 2017 Published online: 30 November 2017
Ahmad et al. (2017) 4(3): 449–455
survive in the soil as eggs or as second-stage juveniles. The juveniles of first-stage develop within the eggs
while juveniles of second-stage hatch from the eggs and infect the roots of the various host plants. Giant cells
develop where they obtain nutrients from one permanent site in the root, called sedentary endoparasitism. For
the development of root-knot nematodes, the optimum soil temperature should be 25–28 °C. The test plant was
pumpkin is commonly applied to any plant in the taxonomically diverse Cucurbita genus and colour of fruit
varies yellow to orange. Pumpkin cultivars may belong to one of the several species: Cucurbita pepo L., C.
maxima Duch., C. moschata Duch. and C. mixta. Cv. This species, C. moschata has been considered to have
many functions for human health as antimicrobial and to preserve kidney function (Caili et al. 2006, El-Aziz &
El-Kalek 2011). Because of the high content of carbohydrates and fibre, this vegetable plant has been performed
as a food with an esteemed source of dietary fiber in human nutrition (Hussain et al. 2010) may decrease the
serum cholesterol level, the risk of coronary heart disease, and hypertension. Apart from, the seeds of Cucurbita
moschata have high amount of zinc, used in treatment of the early stages of prostate problem (Pandya & Rao
2010). Fly ash has shown its inhibitory effect on root-knot nematodes (Tarannum et al. 2001, Raghav & Khan
2002, Rizvi 2008, Rizvi & Khan 2009) however their combined effects on cucurbits so far.

The experiments were conducted in glass houses at the Department of Botany, Aligarh Muslim University,
Aligarh. The fresh fly ash was collected from Thermal Power Plant, Kasimpur for the experiment. The test
pathogen, Meloidogyne incognita Chitwood was maintained in pure form. Pumpkin (Cucurbita moschata) var.
NUTAN (F1 hybrid) was selected as a test plant for the experiments. Inoculums were prepared by incubating
the egg masses of pure M. incognita in distilled water. The freshly hatched second stage (J2) juveniles were
collected as water suspension and the number of J2 counted in ten 1ml samples from the suspension. The
average numbers of J2 were used to represent the number of second juveniles (J2) per ml of suspension. The soil
used in the experiment was collected from the unpolluted agricultural field up to 20 cm depth after scrapping the
surface of litters present. The collected soil was brought to the laboratory in gunny bags.
For this experiment, fly ash was mixed with autoclaved soil in different proportion to prepare 10, 20, 30, 40
and 50% levels. The clay pots of 12 inches height (20 cm diam.) were filled with 2 kg of each type of mixture.
Ten days old plants were inoculated with 2,000 juveniles. The treatments were given as below,
T1 = Control
T2 = Nematode (2000 J2 of Meloidogyne incognita)
T3 = 10% Fly ash + N
T4= 20% Fly ash + N
Fly ash effect on development of the nematode juvenile
Fly ash and autoclaved soil were mixed as above proportion. Pots containing only autoclaved soil served as
controls. A total of 140 pots (7 treatments × 4 weekly observations × 5 replicates) were prepared. A 15-day-old
seedling of pumpkin at the four leaves stage was kept on a glasshouse bench at 25–27 °C and each seedling was
inoculated with 2000 juveniles of M. incognita. Five seedlings were harvested from each treatment at different
intervals (1st, 2nd, 3rd and 4th week) and roots were cut and gently wash under tap water. After cutting, the roots
were taken into test tube with distilled water and boil under spirit lamp for 2 minutes. After boiling 2–3 drops of
acid fuschin add in each tube for staining and then roots were kept on slide. After this, different developmental
stages of the nematode were counted under a stereoscopic microscope.
Plant growth and yield
The plant growth (length, fresh weight and dry weight of root and shoot/plant) and yield (flower/plant;
fruit/plant) were taken after termination of experiments. Shoot length was taken from the point of emergence of
the root to the shoot apex. While root length was recorded from root emergence to longest root and both were
recorded in centimeter (cm). Fresh weight of roots and shoots were recorded in gram (g). After taking fresh
weight, roots and shoots were dried in a hot air oven at 80°C for 48 hrs and their dry weights were recorded.
Photosynthetic pigments
Photosynthetic pigments were estimated by Maclachlan & Zalik (1963) method. After 120 days of planting,
Ahmad et al. (2017) 4(3): 449–455
one g fresh leave was ground in 80 % acetone with the help of mortar and pestle. The suspension was filtered
through the Whatman filter paper No. 1 to the 100 ml volumetric flask and made to the known volume by
adding 80% acetone. Optical density (O.D.) was read at 645 nm and 663 nm for chlorophyll a and b and at 480
nm and 510 nm for carotenoids against 80% acetone as blank on spectrophotometer. The concentration of
chlorophyll a, chlorophyll b and total chlorophyll (a + b) and carotenoids present in the given extract were
calculated according to the formulae given below,
i) Chl a = 12.7(O.D. 663) – 2.69(O.D.645) × V/1000×W (mg/g)
ii) Chl b =22.9(O.D. 645) – 4.68(O.D.663) ×V/1000 ×W (mg/g)
iii) Total Chl (a+ b) = 20.2(O.D.645) – 8.02(O.D.663) ×V/1000×W (mg/g)
iv) Carotenoids = 7.6(O.D.480) – 1.49(O.D.510) / D×1000×W (mg/g)
Carbohydrate content
Carbohydrates are dehydrated with concentrated H2SO4 to form “Furfural”, which condenses with anthrone
to form a green color complex which can be measured by using colorimetrically at 620 nm (or) by using a red
filter. Anthrone reacts with dextrins, monosaccharides, disaccharides, polysaccharides, starch, gums and
glycosides. But they yields of color where is to form carbohydrate. Anthrone reagent: Dissolve 200 mg of
anthrone reagent in 100ml of concentrated H2SO4.
To take 0.2 to 1ml of working standard solution of five different test tube and add water to bring the volume
to 1ml in each test tube add 4ml of anthrone reagent and mix the contents as well and cover the test tube with
bath for 10 min then cool the test tube to the room temperature and measure the optical density in a
photoelectric colorimeter at 620 nm (or) by using a red filter. Simultaneously prepare a blank with 1ml of
distilled water and 4 ml of anthrone reagent. Construct a calibration curve on a graph paper, by plotting the
glucose concentration (10 to 100 mg) on x-axis and absorbance at 620 nm on the y-axis. Compute the
concentration of the sugar in the sample from the calibration curve. While calculating the sugar concentration in
the unknown sample, the dilution factor has to be taken into account. For the calculation of total carbohydrate
content following formula are required-
Amount of carbohydrate present in 100 mg of the sample = mg of glucose/Volume sample × 100
Proline estimation
Proline is very soluble and can be readily extracted by heating explants or aliquots of ground plant material
for 20 min in pure ethanol as well as in water. Proline can also be extracted together with total amino acids,
pigments, soluble sugars by heating plant material twice with 80% ethanol and once with 50% ethanol as
described by Cross et al. (2006), which results into a 70:30 ethanol: water mixture (v/v). Proline and total amino
acids may also be extracted using a cold extraction procedure by mixing 20–50 mg fresh weight aliquots with
0.4–1.0 ml of ethanol : water (40:60 v/v). The resulting mixture is left overnight a 4°C, and then centrifuged at
14000 g (5 min). The cold extraction procedure can be repeated on the pellet and supernatants pooled and used
for the analyses. The first extraction, however, already allows a recovery > 93% (Carillo et al. 2008).
Calculation
µ moles per g tissue = µg proline/ml x ml toluene/115.5 × 5/g sample
Where, 115.5 is the molecular weight of proline
RESULTS
The development of juveniles of meloidogyne incognita in the roots of pumpkin shows in table 1 was also
significantly suppressed by all fly ash and soil mixtures. The J2 developed to J3 /J4 stages at all levels of fly ash
amendment, but their number were less than in the control and decreased with the increase of the fly ash up to
40% soil mixture. At the end of the first week, neither premature nor mature females were found. During the
second week, J2 developed to older stages. However, while pre-mature females occurred in all roots only a few
mature females occurred in the control and at the 5–10% levels of fly ash and none at larger proportions of the
amendment. During the third week, the juveniles that had penetrated into the roots developed further. However,
numbers of pre-mature females were significantly suppressed by all proportions of fly ash while mature females
were significantly suppressed at 5–10% of this amendment and were still absent at larger proportions. After 4
weeks, all the J3 /J4 had developed further but pre-mature females were still significantly less than in the control
Ahmad et al. (2017) 4(3): 449–455
at all proportions of the amendment and a few mature females were observed only up to 20% of the amendment,
with none at all at greater proportions.
Table 1. Effect of different levels of fly ash on developmental stages of Meloidogyne incognita in roots of pumpkin (Cucurbita
moschata), after 1, 2, 3, and 4 weeks from inoculation of nematode juveniles.
Fly ash Developmental stages Developmental stages Developmental stages Developmental stages
level One week Tow week Three week Four week
(%) J2 J3/J4 P M J2 J3/J4 P♀ M♀ J2 J3/J4 P♀ M♀ J2 J3/J4 P♀ M♀
Control 155 405 - - 138 156 118 32 - 168 201 71 - - 98 140
5 166 378 - - 132 138 98 11 - 148 175 24 - - 82 46
10 201 310 - - 164 110 75 - - 142 171 16 - - 40 15
20 229 260 - - 175 98 53 - - 132 161 10 - 13 30 -
30 274 190 - - 188 79 36 - - 105 137 - 16 69 21 -
40 235 105 - - 134 95 17 - 108 160 89 - 21 102 14 -
50 187 75 - - 96 73 - - 88 139 28 - 27 130 09 -
LSD at 5% 14.2 23.7 2.1 5.3 7.0 9.1 8.1
The data given in table 2 shows that the growth (length of shoot and root, fresh and dry weight of shoot and
root) and yield (flowers/plant and fruits/plant) parameters of pumpkin were increased significantly in 5, 10, 20
and 30% levels of fly ash and M. incognita combinations compared to uninoculated (only soil) control set. The
maximum increment in all above parameters was observed at 30% level of fly ash and M. incognita
combination. But plant growth and yield parameters in higher levels (40% + M. incognita and 50% + M.
incognita combinations) were reduced significantly (P=0.05) compared to control set and maximum being at
50% FA and Mi level.
Table 2. Effect of different doses of fly ash and root-knot nematodes (Meloidogyne incognita) on growth and yield parameters of
pumpkin (Cucurbita moschata).
Concentration Length (cm) Fresh weight (gm) Dry weight (gm) Number/Plant
of fly ash Root Shoot Root Shoot Root Shoot Flower Fruits
C (Soil) 60±2.96 281±12.91 62.4±2.79 223.4±10.67 9.1±0.46 32.2±1.45 25±1.23 24±0.95
C (Nematode) 42±2.00 216±11.99 54.6±2.95 201.1±9.61 6.3±0.26 25.0±1.21 23±1.47 20±0.87
5% FA + N 52±2.25 232±9.75 59.6±2.11 214.4±9.87 7.1±0.20 29.2±1.54 21±0.88 20±0.87
10% FA + N 54±2.18 253±11.05 63.4±2.94 220.2±1.45 8.3±0.44 33.1±1.55 22±1.21 21±1.06
20% FA + N 57±3.00 265±11.68 68.2±1.93 231.1±10.15 10.2±0.36 35.2±1.30 24±1.39 22±1.18
30% FA + N 61±3.29 282±12.12 71.0±2.00 250.0±6.93 13.5±0.60 38.0±1.71 26±1.05 25±0.87
40% FA + N 50±3.00 224±8.85 54.6±2.33 220.4±10.13 7.2±0.30 29.3±1.47 21±1.00 17±0.85
50% FA + N 45±2.65 202±11.38 47.6±2.05 198.2±9.91 5.5±0.27 23.2±1.13 18±0.87 14±0.87
LSD-P≤0.05 6.5 20.8 4.4 19.2 1.32 2.1 2.3 2.0
Note: C, Control; Each value is a mean of five replicates, ± values shows the standard deviation with mean.
Table 3. Effect of different doses of fly ash and root-knot nematode (Meloidogyne incognita) on some biochemical parameters of
pumpkin (Cucurbita moschata).
Concentration Chlorophyll (mg.g-1 fresh leaf) Carotenoids Carbohydrate Proline
(%) of Fly ash a b Total (mg.g-1 fresh leaf) (µg fresh weight) (μmol.g-1 fresh weight)
Control 0.82±0.091 0.49±0.015 1.31±0.038 0.46±0.105 14.17±1.054 23.3±1.952
Nematodes 0.68±0.079 0.39±0.007 1.07±0.069 0.35±0.098 10.02±1.127 18.58±2.100
5%+N 0.81±0.088 0.46±0.009 1.27±0.096 0.44±0.130 11.23±0.954 19.02±2.032
10%+N 0.91±0.124 0.47±0.012 1.39±0.059 0.45±0.121 11.98±0.915 19.99±1.638
20%+N 0.95±0.048 0.52±0.010 1.47±0.046 0.46±0.136 12.05±1.014 21.22±1.947
30%+N 0.98±0.022 0.55±0.006 1.53±0.082 0.47±0.156 15.05±1.583 24.77±2.947
40%+N 0.81±0.188 0.48±0.007 1.30±0.103 0.43±0.182 11.99±1.890 22.32±2.629
50%+N 0.79±0.037 0.46±0.011 1.28±0.008 0.42±0.103 10.95±0.961 20.87±2.722
LSD-P≤0.05 0.016 0.008 0.009 0.007 0.83 1.34
Note: Each value is a mean of five replicates, ± values shows the standard deviation with mean.
Similar pattern of increase/decrease in photosynthetic pigments (chl. a, chl. b, chl. a+b and carotenoids),
carbohydrate and proline content of pumpkin were also observed in all fly ash + M. incognita treatments when
compared to control one shows in table 3. However, maximum increment was found at 30% fly ash + M.
incognita. When growth, yield and photosynthetic pigments, carbohydrate and proline content of pumpkin in
different fly ash and M. incognita combinations were compared to inoculated set (nematode alone), it was
Ahmad et al. (2017) 4(3): 449–455
observed that all parameters were increased significantly (P=0.05). And all above biochemical parameters
decreased significantly at 40% FA + Mi and 50% FA + Mi combinations and maximum at 50% level of FA and
nematode.

Several beneficial nutrients including S, B, Ca, Mg, Fe, Cu, Zn, Mn, and P, which are responsible for plant
growth, found in fly ash. It is known that plants take up nitrogen in the form of nitrate (NO3-) because nitrates
are more quickly available to plants as they move through the roots and as such lesser content of nitrate in 5%
and 10% fly ash containing fields may be due to more hydraulic absorption because of higher water holding
capacity in the fly ash amended soi. Fly ash decreases porosity and thus increases water holding capacity. This
would facilitate the absorption of nutrients as well as photosynthetic activity. Similar findings have been
reported by (Thetwar 2007). In the present study, soil amendment with fly ash was harmful to the nematode at
all levels. The alkaline nature of fly ash may have directly affected the juveniles, leading to less penetration into
the roots and subsequently delayed development. Edongali (1982) stated that juvenile penetration is affected by
the concentration of different elements, irrespective of the type of element in the soil solution. We found higher
chlorophyll a and b concentration in cucurbita moschata plant could be due to the micronutrients available in fly
ash than the control. Similar reports have been made by (Niyaz & Singh 2006, Hisamuddin & Singh 2007).
Pumpkin plant grows in soil with different doses of fly ash and did not show any visible injury and the lower at
30% level of fly ash was found beneficial to plant growth, yield, photosynthetic pigments, carbohydrate and
proline contents of pumpkin.
The soil application of fly ash ameliorated plant growth of pumpkin and suppressed the M. incognita in pots.
Improved plant growth with fly ash has been observed earlier (Elseewi et al. 1980, Mishra & Shukla 1986). Due
to the better health status of the plant, the yield, photosynthetic pigments, carbohydrate, proline contents of
pumpkin were also increased. The beneficial effects of fly ash were found from 10 to 30% levels in soil, and
optimum being at 30%. Similar beneficial effects on above parameters have also been observed on a number of
crops like cabbage, Capsicum, chickpea, collard greens, com, cucumber, Lactuca sativa, mustard green, radish,
soybean, sunflower, tomato, Vigna mungo, wheat etc. (Singh 1989, Menon et al. 1990, Khan & Khan 1996,
Rengifo et al. 1996, Sarangi & Mishra 1998, Sahu & Dwivedi 1999, Tarannum et al. 2001, Upadhyay & Khan
2002, Upadhyay 2004). However, the responses of various crops were different to different levels of fly ash
(10–50%). Higher level adversely affected the plant growth and other parameters of Pumpkin. The adverse
effects of fly ash at higher level of application are attributed to excess of micro-nutrients (Adriano et al. 1980)
and toxicity of compounds like dibenzofuron and dibenzo-p-dioxine as well as heavy metals found in fly ash
(Helder et al. 1982, Mishra & Shukla 1986, Wong & Wong 1986). Harmful effects of higher levels above 50%
have been observed on Brassica juncea, chickpea, cucumber, lentil, Linum usitatissimum, maize, potato,
soybean and tomato (Mishra & Shukla 1986, Pasha et al. 1990, Raghav 2006). On the other hand, the soil
application of fly ash noticed the effect of M. incognitai with respect to levels. This might be due to the excess
of salts, toxic compounds and heavy metals which caused nematicidal effects on M. incognita either directly or
within the host. Nematode might have lost its activities and later could not survive under the stress of fly ash.
Losing the activity and not reaching the mature stage of M. incognita is very important for the agriculture point
of view, because there will be no loss to the crop (Khan 2007, Iram 2010). Thus soil application of fly ash with
30% level is useful, as it suppresses the, M. incognita one hand, and improves the Pumpkin crop on the other
hand.
ACKNOWLEDGEMENTS
The authors wish to thank the Charirman, Department of Botany, Aligarh Muslim University, Aligarh for
providing the infrastructure facilities and support.
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ISSN (E): 2349 – 1183
ISSN (P): 2349 – 9265
4(3): 456–460, 2017
DOI: 10.22271/tpr.2017.v4.i3.060
Research article
Impact of three phosphate solubilizing species of Penicillium on

growth of Piper longum L. under inoculated condition
Hruda Ranjan Sahoo and Nibha Gupta*
Division of Plant Pathology and Microbiology, Regional Plant Resource Centre,
Bhubaneswar-751015, Odisha, India
*Corresponding Author: nguc2003@yahoo.co.in [Accepted: 02 December 2017]
Abstract: Efficient phosphate (P) solubilising strains of Penicillium such as Penicillium oxalicum,
Penicillium glabrum and Penicillium sp. isolated from endophytic sources was applied as
biofertiliser and it enhanced the productivity of Piper longum, a RET Medicinal plant which is one
of the endangered species of Odisha state. Periodical study of the growth performance of Piper
longum plants inoculated with different Penicillium sp. under pot culture conditions showed
outstanding impact of the fungal strains on plant growth. Maximum height was recorded in plants
treated with Penicillium oxalicum strains (76.75±22.9) cm after 120 days as compared to control
plants with (57.4±16.3) cm. In control plants, spike development was observed after 180 days.
Maturation of the fruits occurred in Penicillium glabrum and Penicillium sp. at 150 days and all
the spikes obtained were of the male type and maturation of female spikes occurred after 180 days.
The Relative Growth Rate (RGR) and Net Assimilation Rate (NAR) were higher in plants
inoculated with Penicillium sp. and recorded as 12.3 mg.g-1.day-1 and 4.49 g.cm-2.day-1
respectively.
Keywords: Piper longum - Pikovskaya’s medium - P solubilising fungi - Growth parameters.
[Cite as: Sahoo HR & Gupta N (2017) Impact of three phosphate solubilizing species of Penicillium on growth
of Piper longum L. under inoculated condition. Tropical Plant Research 4(3): 456–460]
INTRODUCTION
Phosphate is an essential irreplaceable element which is building block of all life forms. Globally, phosphate
needs are currently met from geochemical sedimentary rock formations which are available only in selected
areas of the world. These are finite resources that will last for half a century, depending upon the total resources
that exist or currently accessible sources of P in the world. Hence, phosphate rock deposits are non-renewable
and availability of high-grade P ores is finite. Since India suffers from lack of geological reserves of high-grade
P it is dependent on imports to meet up to 90% of its domestic P requirement and it also witnessed a steep
increase in the prices of P fertilizers in the recent years. Therefore, efficient usage of P to minimize wastage
/loss remains the only option. However, Phosphate represents an interesting case of resource management, a key
component in the pillar of information (DNA) and energy (ATP) molecules of the cell and is non-substitutable
for all living organisms. In Indian soils, inorganic P contributes 54–84% of total P whereas the share of organic
P varies from 16–46% in different states (Tomar 2000). However, since soils of more than 90% of the districts
belonged to low to medium fertility categories, P fertilization is necessary to produce optimum crop yields. On
the basis of accessibility and extractability, soil exists in 4 different pools such as Soil solution P, Surface
adsorbed P, strongly bonded or absorbed P and inaccessible or precipitated P (Syers et al. 2008). Soil P solution
concentration is affected by dissolution-precipitation (mineral equilibria), sorption-desorption, mineralization-
immobilization. Hence P in soils is present in two main insoluble forms- mineral forms such as apatite,
hydroxyapatite, oxyapatite and organic forms such as inositol phosphate (soil phytate), phosphomonoesters,
phosphodiesters, phosphotriesters etc.
Natural solubilisation of mineral phosphates is a characteristic exhibited by most of the Phosphate
Solubilising Microbes or PSM (Perez et al. 2007). In rhizosphere soil, these microbes play an ecophysiological
role by mobilizing the insoluble inorganic phosphates so that they can be absorbed by plant roots and thus PSM
Received: 08 August 2017 Published online: 31 December 2017
Sahoo & Gupta (2017) 4(3): 456–460
can be used for biofertilization to improve crop yields (Richardson 2001, Khan et al. 2007, Wafula et al. 2016,
Nayak et al. 2017). Soil inoculation with phosphate solubilising organisms improves solubilisation of fixed soil
P and applied phosphates so that P is available to the plant for growth and development. Plant growth and
development is also related to its access to minerals such as P (Santos et al. 2010, Ghosh et al. 2017).
Aspergillus and Penicillium among fungi are the most common P-solubilising fungi (Seshadri et al. 2004,
Wakelin et al. 2004, Sahoo & Gupta 2016, Nayak et al. 2017, Panigrahi et al. 2017). Different species of
Penicillium obtained from Agricultural soil, hill soil, mine soil and rhizosphere of plants were reported to have P
solubilising activity by various investigators (Singh & Reddy 2011, Morales at al. 2011, Mahamuni et al. 2012).
Various species of Penicillium with P solubilizing activity were tested under pot trial to evaluate their growth
promoting abilities with crops such as cereals (maize, corn, wheat); Vegetable crops (Lettuce); oilseed crops
(Groundnut); Forest trees (Dalbergia sisso) etc. (Kassim & Al-Zandinary 2011, Singh & Reddy 2011, Malviya
et al. 2011, Morales at al. 2011, Patil et al. 2012, Dash et al. 2013). Endophytic microbes associated with plant
also promote its growth and increases productivity due to their capacity of solubilising P mostly Ca and Fe
phosphates (Vitorino et al. 2012).
Piper longum L. is a slender aromatic climber having several medicinal properties due to the dry spikes of
female types which is economically important. This is the reason for its overexploitation and more than 100
metric tons/year of the dry weight of long pepper is being traded, as a result of which it is disappearing rapidly.
Therefore, large-scale cultivation should be promoted to enhance its population in the wild. However, several
problems such as lack of quality planting material, mortality in the field and poor growth are associated with
large-scale cultivation (Singh & Gogoi 2010). Field trial of P. longum with P solubilising fungi was not reported
earlier. The present study emphasizes on the application of efficient P solubilising strains of Penicillium such as
Penicillium oxalicum Currie & Thom, Penicillium glabrum (Wehmer) Westling and Penicillium sp. as
biofertiliser in order to enhance the productivity of P. longum which is one of the endangered species of Odisha
state.

A pot culture experiment was designed to test the effectiveness of P solubilising fungal strains on growth
and development of Piper longum L. under the greenhouse conditions. 3 Phosphate solubilising fungi belonging
to Penicillium genera were selected for the experimental trial. Polybags of 10" × 10" size containing red laterite
soil was used. All the polypots of 5 kg capacity were filled with sterile soil before transplanting the rooted stem
cuttings of Piper longum plant. This was done in 10 replications for each treatment. The experiment was done in
completely randomized block design with 3 treatments, F1, F8 and F11 inoculated with Penicillium sp.,
Penicillium oxalicum and Penicillium glabrum respectively in 10 replications.
The characteristic features of the soil used for the experiment was acidic with pH 6.06, electrical
conductivity 0.2 dSm-1, Organic carbon 0.9% with available N, P, K as 232, 25 and 99 kg.ha -1 respectively, Ca
and Na with 6.6 and 0.174 meq/100g respectively. However, the concentration of micronutrients such as Fe,
Mn, Cu and Zn was found to be 32, 11, 1 and 0.3 mg.kg-1 respectively. The pots were inoculated with fungal
cultures at regular intervals and were maintained in the greenhouse at an adequate temperature with daily water
supply to maintain the soil moisture level and without any application of fertilizer. The plant growth parameters
especially observation of morphological details was done. The parameters such as leaf number, branch number,
shoot height and fresh weight, root length and fresh weight, leaf area and fresh weight were recorded
periodically. The dry weight of plant biomass such as shoot, root and leaf samples were determined after drying
at 60ºC in the hot air oven for 48 hours. The observations recorded after 30, 60 and 90 days were compared with
the control setup. The RGR, NAR and LAR were also calculated for all the treatments. Fruiting was observed
after 120 days of fungal inoculation into the plants. The male and female spike development, their maturation
and spike length was also recorded after 150 and 180 days.

Periodical study of the growth performance of Piper longum L. plants inoculated with different Penicillium
sp. under pot culture conditions revealed that Penicillium oxalicum and Penicillium glabrum showed a higher
number of branches (11.25±2.5) and (11.25±4.03) respectively as compared to control plants (8.4±2.77) at the
end of 120 days as shown in table 1. However, no significant increase is observed in number of branches for
Penicillium sp. inoculated plant throughout the growth period. Leaf development in plants treated with different
Sahoo & Gupta (2017) 4(3): 456–460
strains of Penicillium showed a good increase in number at the end of 120 days. Penicillium oxalicum treated
plants showed (38.25±8.54) number of leaves as compared to other plants. All the strains showed higher number
of leaves from control plants. The plant height also increased after treatment with strains of Penicillium as
compared to control. Maximum height was recorded in plants treated with Penicillium oxalicum strains
(76.75±22.9) cm after 120 days. After fungal treatment at regular intervals, the plants showed a great increase in
their height. All the three strains improved the Growth of Piper longum plant. P. oxalicum also increased the
root length of plants and maximum of (33.75±2.47) cm is recorded after 90 days. The increment in root length
of these plants is almost the double of control plants. However, all the three strains showed an increase in root
length as compared with control. P. oxalicum also influenced the leaf development as higher leaf area
(386.5±17.7) cm2 is obtained for the same.
Table 1. Growth parameters recorded for Piper longum L. after regular interval inoculated with different species of Penicillium.
A. 30 days SHOOT ROOT LEAF
No. of No. of Fresh Dry Fresh Dry Fresh Dry
Height Length Area
Treatment branches leaves weight weight weight weight weight weight
Control 5.5± 7± 36.5± 2.133± 0.27± 9.25± 1.01± 0.138± 261.63± 3.615± 0.497±
0.71 1.41 12.02 0.89 0.115 0.35 0.13 0.018 23.16 0.61 0.038
F1 3.5± 4.5± 26.35± 1.11± 0.18± 20± 0.735± 0.124± 301.88± 1.94± 0.33±
2.12 2.12 17.89 0.37 0.094 12.73 0.53 0.082 273.12 1.14 0.19
F8 5.5± 6.5± 29.25± 1.187± 0.173± 19.7± 0.89± 0.151± 240.33± 2.81± 0.36±
0.71 0.71 12.37 0.56 0.082 0.71 0.28 0.046 21.8 0.5 0.08
F11 6± 7± 41.25± 1.695± 0.203± 23.5± 1.052± 0.142± 210.878± 3.113± 0.385±
0 0 7.42 0.8 0.045 2.12 0.142 0.034 37.13 0.99 0.11
B. 60 days SHOOT ROOT LEAF
Height Length Area
Control 5± 4± 43.45± 2.857± 0.343± 9± 1.15± 0.317± 253± 2.775± 0.44±
2.83 2.83 3.18 0.62 0.15 1.41 0.5 0.14 171 1.3 0.23
F1 4.5± 6± 32.2± 1.504± 0.228± 10.7± 0.678± 0.149± 173.88± 1.971± 0.313±
2.12 1.41 17.8 0.47 0.07 1.63 0.28 0.06 98.8 1.26 0.21
F8 8± 9.5± 55± 2.352± 0.417± 7.8± 0.482± 0.108± 353.63± 3.63± 0.683±
1.41 2.12 0.63 0.24 0.17 1.41 0.06 0.013 54.62 0.03 0.06
F11 6.5± 8± 53.25± 2.74± 0.344± 8.2± 0.769± 0.144± 273.38± 2.94± 0.6±
2.12 1.41 6.72 0.22 0.02 1.34 0.47 0.09 11.14 0.29 0.05
C. 90 days SHOOT ROOT LEAF
Height Length Area
Control 6.5± 8± 41.25± 2.672± 0.43± 17.5± 0.47± 0.101± 270.4± 3.17± 0.49±
3.54 4.24 5.3 0.54 0.04 3.53 0.27 0.059 214.4 2.32 0.32
F1 6± 8.5± 41.75± 2.26± 0.32± 22± 0.91± 0.20± 292.13± 2.71± 0.56±
2.83 4.95 18.74 1.66 0.27 2.82 0.68 0.16 257.2 2.04 0.53
F8 8.5± 12± 65.7± 3.11± 0.325± 33.75± 1.1± 0.21± 386.5± 4.77± 0.69±
0.71 2.82 8.7 0.35 0.07 2.47 0.31 0.06 17.7 0.11 0.008
F11 7± 11± 50.25± 2.41± 0.299± 30.75± 1.082± 0.286± 260.25± 4.07± 0.68±
2.83 4.24 23.69 1.49 0.16 0.35 0.013 0.056 29.34 2.84 0.33
D. 120 days
No. of No. of Shoot
Treatment leaves branches height
Control 27.63±9.43 8.4±2.77 57.4±16.3
F1 29.25±16.52 7.75±3.3 64.75±17
F8 38.25±8.54 11.25±2.5 76.75±22.9
F11 36.75±4.99 11.25±4.03 62±6.7
Observations recorded for plant biomass studies highlight that P. oxalicum showed higher fresh shoot weight
(3.11±0.35) g at 90 days as compared to plants inoculated with other strains and control plants whereas the dry
biomass of shoot is not much significant and less in treated plants than that of control plants. No effect of the
Sahoo & Gupta (2017) 4(3): 456–460
strains was observed on the biomass of the roots of the plants. As after 60 days, the fresh and dry biomass of
root was highest in control and recorded as (1.15±0.5) g and (0.317±0.14) g respectively. Higher fresh and dry
leaf biomass of (4.77±0.11) g and (0.69±0.008) g respectively is observed in P. oxalicum inoculated plants after
90 days. Hence, it is clear that among the 3 strains of Penicillium, Penicillium oxalicum can be used as efficient
P solubiliser for biofertilization studies as it enhanced the values of the parameters related to plant growth and
development.
Table 2. Fruit development recorded in Piper longum L.
Treatment 120 days 150 days 180 days
Control 0 0 0
F1 4 4 5
F8 6 7 8
F11 6 2 2
The fruit development was observed in plants treated with fungal strain but it is absent in control plants even
after 180 days as depicted in table 2. However, in control plants, spike development was observed after 180
days. A maximum number of fruits was recorded in P. oxalicum treated plants (6 in 120 days, 7 in 150 days and
8 in 180 days, respectively). However, maturation of the fruits occurred in Penicillium glabrum and Penicillium
sp. at 150 days and all the spikes obtained were of the male type. The maximum average length of the male
spike was found to be (6±1.3) cm which is highest in Penicillium sp. However, maturation of female spikes
occurred after 180 days. The maximum average length of the male spike was found to be (5.8±0.42) cm and the
female spike was (4.6±0) at 180 days as reflected in table 3.
Table 3. Harvesting of spikes of Piper longum L.
A. 150 days
Number of male Number of female Length of Male Length of female
Treatment spike harvested spike harvested spike (in cm) spike (in cm)
Control 0 0 0 0
F1 2 0 6±1.3 0
F8 0 0 0 0
F11 4 0 5.6±1.14 0
B. 180 days
Number of male Number of female Length of Male Length of female
Treatment spike harvested spike harvested spike (in cm) spike (in cm)
Control 0 0 0 0
F1 2 1 5.8±0.42 3.4±0
F8 2 1 4.9±0.71 4.6±0
F11 1 1 5.4±0 3.6±0
The Relative Growth Rate (RGR) and Net Assimilation Rate (NAR) were higher in plants inoculated with
Penicillium sp. and recorded as 12.3 mg.g-1.day-1 and 4.49 g.cm-2.day-1 respectively (Table 4). However, Leaf
Area Ratio (LAR) was highest in P. oxalicum treated plants and found to be 662 cm-2.g-1. Hence, all the
parameters recorded were mostly higher in treated plants as compare to the control plants.
Table 4. Relative growth rate (RGR), net assimilation rate (NAR) and leaf area ratio (LAR) recorded in Piper longum L.
Treatment RGR (mg.g-1.d-1) NAR (g.cm-2.d-1) LAR (cm2.g-1)
Control 3.25 0.68 616.25
F1 12.3 4.49 517.5
F8 6.42 0.9 662
F11 6.98 0.31 385.7
ACKNOWLEDGEMENT
The financial assistance obtained through INSPIRE programme, (No. DST/INSPIRE Fellowship/2013/506)
DST, Govt. of India is gratefully acknowledged.
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ISSN (E): 2349 – 1183
ISSN (P): 2349 – 9265
4(3): 461–467, 2017
DOI: 10.22271/tpr.2017.v4.i3.061
Research article
Response of dust accumulation on roadside plant species due to

open cast mining at Jhansi-Allahabad NH-76,
Uttar Pradesh, India
Priyanka Singh and Amit Pal*
Institute of Environment & Development Studies, Bundelkhand University, Jhansi-284128, India
*Corresponding Author: apu13@rediffmail.com [Accepted: 12 December 2017]
Abstract: Deposition of dust on roadside plant and its impact on leaf i.e. leaf pigment
concentration, the carotenoid and protein has been investigated on fifteen selected roadside plants
species namely Ailanthus excelsa, Azadirachta indica, Butea monosperma, Calotropis procera,
Cassia fistula, Datura metel, Ficus benghalensis, Ficus hispida, Ficus infectoria, Ficus religiosa,
Holoptelea integrifolia, Millettia pinnata, Nerium oleander, Phoenix dactylifera and Psidium
guajava respectively at Jhansi-Allahabad National Highway-76. The variation in terms of dust
deposition with species specific result observed during the entire study. Declination of leaf
pigment concentration, carotenoid content and protein content indicate the positive impact of dust
pollution. Above findings may be helpful to find out some species which is resistant or to cope
with open cast mining generated dust pollution in and around mining areas as well as
beautification of adjacent highways. The maximum deposition was found in Ficus hispida
followed by Calotropis procera, Butea monosperma, Ficus benghalensis, Ailanthus excelsa,
Azadirachta indica.
Keywords: Dust pollution - Chlorophyll - Carotenoid - Protein - Ficus spp.
[Cite as: Singh P & Pal A (2017) Response of dust accumulation on roadside plant species due to open cast
mining at Jhansi-Allahabad NH-76, Uttar Pradesh, India. Tropical Plant Research 4(3): 461–467]
INTRODUCTION
Dust is an important abiotic factor and has a key influence on the various plant species. The severe pressure
of dust may be responsible for the biochemical and morphological changes in the plant species which further
initiated adaptive evolution to merge 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). Vehicles are the prime
source of dust generation for roadside plants. Vehicular exhaust adds up huge amounts of soot particles, smoke,
poisonous gases (SO2, NO2, CO2, VOCs etc.), heavy metals and organic molecules on the roads all over the
world. All these air pollutants are known to produce adverse effects on the health of plants, animals and humans
(Kulshreshtha et al. 2009, Rezaei et al. 2010, Atkinson et al. 2012). Due to dryness of soil in the arid ecosystem,
the windblown dust is a common feature and plays a great role in increasing dust pollution in the environment
(Younis et al. 2013). Similarly, high-speed vehicles and agricultural activities also generate too much high dust
pollution in the air (Manins et al. 2001, Van Jaarsveld 2008). 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, Rai et al. 2010, Chaturvedi et
al. 2013). This accumulation mainly depends on vegetation type. Previous research has shown that at high
concentrations, many of the pollutants present in the exhaust gases can be damaging to plants (Wellburn 1990,
Ackerly & Bazzaz 1995, Pal et al. 2002, Grantz et al. 2003).
Basically, the outer surface of the plants like leaves plays a role in absorbance of these dust particles (Samal
& Santra 2002). Thus in these areas, the quality of air can be improved by planting more trees near road and
farm sides (Beckett et al. 2000, Raupach et al. 2001, Freer-Smith et al. 2005). Plants act as a sink in the
Received: 25 September 2017 Published online: 31 December 2017
Singh & Pal (2017) 4(3): 461–467
environment, so they are useful in reducing dust concentration and other particulate matters of air. Plants can act
as great candidates for reduction of air pollution at a particular place by absorption, deposition, accumulation
and detoxification of harmful pollutants (Prajapati & Tripathi 2008). Dust pollution causes a negative impact on
plants as it reduced photosynthesis and cause leaf fall with tissue death (Farooq et al. 2000, Shrivastava & Joshi
2002). Dust affects the synthesis of chlorophyll and resulted in leaf chlorosis (Seyyednejad et al. 2011).
Similarly, morphology and anatomy of leaves are also altered by dust (Gostin 2009, Sukumaran 2012). At the
same time, many plants are able to survive in high dust load due to the synthesis of carotenoids and proteins
which give non-enzymatic resistance to plants to numerous abiotic stresses (Prajapati & Tripathi 2008). The
present study emphasized the impact of dust deposition in terms of chlorophyll contents, carotenoids content
and protein content upon the roadside plant and their adaptive responses to cope with the vehicular dust
deposition. Fifteen different plant species have been selected to study the impact of vehicular as well as
opencast mining generated dust deposition on roadside plants.

Based on the frequency of occurrence on the roadside of NH-76 adjacent to opencast granite mining areas of
Bhagawantpura of Jhansi district of Uttar Pradesh, India (Fig. 1), matured fifteen plant species have been
selected to study the accumulation of dust and its impacts on the selected plant species growing along with the
roadside. Leaves were collected randomly of from plants with equal height from each roadside of NH-76 and as
well as from the Bundelkhand University campus (as control). Three replicates of the leaves of each species
have been analyzed. After removing leaves from branches they were packed in pre-weight polythene and
brought to the laboratory. Here the dust on the leaves was carefully cleaned by using the fine brush in polythene
and again taken the weight. The dust accumulation was calculated by using following formula (Prajapati and
Tripathi 2008),
W = (w2-w1)/a
Where, w is dust content (g/m), w1 is initial weight, w2 is final weight with dust and ‘a’ total area of the leaf
(m2).
Fresh weights of leaves were taken then oven dried at 70ºC for 72 hrs for dry weight measurements. The leaf
area (cm2) was recorded with a leaf area meter (CI-203, CID inc. USA). Leaf pigments i.e. chlorophyll,
carotenoid and protein concentration was counted with the method described by (Arnon 1949) and the pigment
concentration was expressed in the mg.g-1 tissue. Selected plant species are: Ailanthus excelsa Roxb.,
Azadirachta indica A.Juss., Butea monosperma (Lam.) Taub., Calotropis procera (Ait.) Dryand., Cassia fistula
L., Datura metel L., Ficus benghalensis L., Ficus infectoria Roxb., Ficus hispida L.f., Ficus religiosa L.,
Holoptelea integrifolia Planch., Millettia pinnata (L.) Panigrahi, Nerium oleander L., Phoenix dactylifera L. and
Psidium guajava L.
Figure 1. Map showing the location of study area.
RESULTS
In present study, the maximum dust deposition has been observed in leaf of Ficus hispida (18.44 mg.cm-2)
followed by Calotropis procera (16.46 mg.cm-2), Butea monosperma (10.25 mg.cm-2), Ficus benghalensis (9.11
Singh & Pal (2017) 4(3): 461–467
mg.cm-2), Ficus infectoria (5.30 mg.cm-2) respectively (Fig. 2). Lowest dust deposition has been found in
Nerium oleander i.e. 0.34 mg.cm-2.
Figure 2. Dust deposition on leaves of selected plant species.

Roadside plant species shows the reduction of chlorophyll contents in compare to dust free site (i.e.
Bundelkhand University campus). Dust particles physically obstruct sunlight as well as block stomatal pore of
leaf and thus dust deposition hampered photosynthetic activities of the plant. Effect of dust degrades leaf
pigment concentration has been reported earlier. In present study maximum chlorophyll content has been
observed in leaf of Ficus hispida in polluted site (13.632 mg.g-1) and in unpolluted site (19.227 mg.g-1); it was
followed by Calotropis procera (12.616 mg.g-1 and 16.778 mg.g-1), Butea monosperma (8.508 mg.g-1 and
15.122 mg.g-1), Ficus benghalensis (7.511 mg.g-1 and 14.641 mg.g-1), Ailanthus excelsa (7.184 mg.g-1 and
11.685 mg.g-1), Datura metel (7.715 mg.g-1 and 14.468 mg.g-1) respectively (Fig. 3). Lowest chlorophyll content
has been found in Nerium oleander and in Azadirachta indica.
Figure 3. Chlorophyll content in leaves of selected plant species.

The maximum carotenoid content has been observed in the leaf of Ficus hispida i.e. unpolluted (0.4772
mg.g-1) and in polluted (0.366 mg.g-1) followed by Calotropis procera (0.426 mg.g-1 and 0.3288 mg.g-1), Butea
monosperma (0.3584 mg.g-1 and 0.3252 mg.g-1), Ficus benghalensis (0.3244 mg.g-1 and 0.284 mg.g-1), Datura
metel (0.3144 mg.g-1 and 0.1684 mg.g-1), Ailanthus excelsa (0.2964 mg.g-1 and 0.1404 mg.g-1). The lowest
carotenoid content was recorded in Ficus religiosa and Nerium oleander (Fig. 4). The carotenoid content is less
Singh & Pal (2017) 4(3): 461–467
in roadside plant species as compared to control site due to dust particles settled on the leaf surface regularly
although deposition was not uniform in all the 15 plant species.
Figure 4. Carotenoid content in leaves of selected plant species.

In present investigation maximum protein content has been found in leaf of Ficus hispida (1.261 mg.g-1) in
unpolluted site and in polluted it was 0.504 mg.g-1 followed by Calotropis procera (0.744 mg.g-1 and 0.234
mg.g-1), Butea monosperma (0.344 mg.g-1 and 0.184 mg.g-1), Ficus benghalensis (0.292 mg.g-1 and 0.151 mg.g-
1
), Datura metel (0.244 mg.g-1 and 0.137 mg.g-1), Ailanthus excelsa (0.187 mg.g-1 and 0.116 mg.g-1). Lowest
protein content in unpolluted is Azadirachta indica (0.055 mg.g-1) and in polluted it was recorded in Holoptelea
integrifolia (0.045 mg.g-1). Overall the protein content is less in roadside plant species as compared to control
site i.e. unpolluted areas (Fig. 5).
Figure 5. Protein Content in leaves of selected plant species.

Singh & Pal (2017) 4(3): 461–467
DISCUSSION
The maximum amount of dust deposition in the roadside plant was found to increase with time. The dust
particles settled in the leaf surface regularly, but deposition was not uniform in all the 15 selected plant species
in our study. Settlement of dust particles on the leaf surface mainly depends on the phyllotaxy of leaf,
smoothness of the leaf surface (i.e. presence of trichome), the shape of the leaf, petiole length as well as its
position on the plant (Davison & Blackmore 1976).
Continuous exposure to dust in leaf surface leading to the formation of dense dust layers, which reduce light
capturing capacity of plants and finally hamper plant photosynthetic activity (Prajapati & Tripathi 2008,
Pourkhabbaz et al. 2010). Decreased rate of photosynthesis and alteration of stomatal conductance are
responsible for the reduction of the stomatal index as well as dry matter content of leaf (Khan et al. 2015).
Reduction of the stomatal index 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 (Sharma et al. 2017).
The leaf is influenced by deposition of dust, which may lead to lower photosynthesis activity (Pourkhabbaz
et al. 2010). The present study shows that the chlorophyll content of all these selected plants was less in
comparison to the plants from the control site i.e. collection from University Campus. Degradation of
chlorophyll contents in the leaf is a common phenomenon from dust deposition on the leaf (Prajapati & Tripathi
2008). A plenty of studies reviewed declination of chlorophyll concentration due to deposition of dust in a
number of annual non- leguminous crop (Satao et al. 1993, Prusty et al. 2005, Gostin 2009).
The study revealed the definite correlation between the plant canopy and foliar morphological traits and
amount of dust captured by them. Morphological characteristics which have been found to play a significant role
in intercepting particulates are: orientation of leaf on the main axis, size (leaf area), shape, nature of the surface,
venation pattern, presence and absence of trichomes, morphology and frequency of trichomes, wax deposition,
etc.
CONCLUSION
Based on extensive field observations and laboratory investigations, a few plant species have been identified
which possess high dust filtering capacity. These species may be raised in green belts around granite mining
areas and both sides of the national highway to lessen the dust load of the surrounding environment. The species
with better dust filtering capacity are: Ficus hispida, Calotropis procera, Butea monosperma, Ficus
benghalensis, Datura metel, Ailanthus excelsa respectively and could be considered as first choice species.
Other species like- Phoenix dactylifera, Nerium oleander, Ficus religiosa, Cassia fistula, Millettia pinnata are
considered as second choice species and Psidium guajava, Ficus infectoria, Holoptelea integrifolia, Azadirachta
indica are considered as third choice species for the designing of green belt in and around mining industries as
well as beautification of roadsides.
Plants on both sides of a national highway or in the city not only have an ornamental function but they have
also an important role in mitigation of air pollution in terms of gaseous pollutants and filtration of air-borne
particles that allows characterizing air pollutants and determine the source of their elemental and mineral
profiles. Thus, plants with suitable traits for air pollution mitigation may be used for urban tree planting,
roadside plantation programs, green belt in an around mining areas to improve air quality and at the same time
such plants may be utilized for passive monitoring of air pollution.
ACKNOWLEDGEMENTS
The authors are thankful to staff of Forest Research Center, Bhagawantpura, Jhansi, Government of Uttar
Pradesh for their kind support during the identification of roadside plants.
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International Journal of Scientific Research 3: 1–12.
ISSN (E): 2349 – 1183
ISSN (P): 2349 – 9265
4(3): 468–470, 2017
DOI: 10.22271/tpr.2017.v4.i3.062
Research article
Solanum sisymbriifolium Lam. (Solanaceae): A new addition to

the flora of Odisha, India
S. C. Sahu, M. R. Mohanta* and A. K. Biswal
Department of Botany, North Orissa University, Baripada-757003, Odisha, India
*Corresponding Author: manasranjan.mrm@gmail.com [Accepted: 15 December 2017]
Abstract: During the exploration of invasive flora in the northern part of Odisha, Solanum
sisymbriifolium Lam. was collected from Rupsa of Balasore district. After reviewing its
distribution through flora and available literatures, the species is found to be a new one to the Flora
of Odisha. A detailed description with photographs, distribution, occurrence and phenology of the
taxon are provided for easy identification.
Keywords: Solanaceae - Solanum sisymbriifolium - New record - Odisha.
[Cite as: Sahu SC, Mohanta MR & Biswal AK (2017) Solanum sisymbriifolium Lam. (Solanaceae): A new
addition to the flora of Odisha, India. Tropical Plant Research 4(3): 468–470]
INTRODUCTION
Solanum L. is a large and diversified genus belonging to family Solanaceae possessing more than a thousand
numbers of species distributed throughout the world (Bohs & Olmstead 1997). Family Solanaceae has a number
of widely used drug plants such as Nicotiana tabacum L., Datura spp. and Atropa belladonna L. in India. The
genus Solanum also has global importance for its food crops such as Solanum melongena L., Solanum
tuberosum L., Solanum lycopersicum L. etc. This genus constitutes many economically important species
widely distributed throughout tropical and temperate regions, with centers of diversity in Central and South
America and Australia (Edmonds 1978, D’Arcy 1991). In India, the genus Solanum is represented by 42
species. A total of 9 wild and 3 cultivated species of Solanum have been reported in Flora of Orissa (Saxena &
Brahmam 1994–96). S. sisymbriifolium is distributed mostly in the southern part of India and also it has been
reported in Jharkhand (Panda et al. 2014), Uttar Pradesh (Srivastava et al. 2015), Delhi (Mishra 2015), Bihar
(Mishra & Kumar 1992), Rajasthan (Yadav & Menna 2007) and Tripura (Saha & Datta 2013). However,
Solanum sisymbriifolium has not been reported so far from the state of Odisha. With the addition of this newly
recorded species, the total number of Solanum wild species in Odisha is increased to ten.
Figure 1. Map showing the collection site in Odisha.
Sahu et al. (2017) 4(3): 468–470
During July 2017, few specimens belonging to genus Solanum are collected near Rupsa railway station,
Balasore district of Odisha (Fig. 1). Rupsa railway station is situated at 21° 37 21 N longitude and 87° 01 09 E
latitude. After critical identification and consultation with the Flora of Orissa (Saxena & Brahmam 1994–1996),
it was found different from the Solanum species recorded for the state. The phenology, distribution, habitat,
ecology are observed at the same locality. The specimens were verified with other published Solanum literatures
(Deb 1983, Hooker 1885, Prain 1903, Kanjilal et al. 1939, Shetty & Singh 1991, Almeida 2001). Finally, the
specimens are identified as Solanum sisymbriifolium Lam. with consultation of herbarium at CAL (Central
National Herbarium, Kolkata). The voucher specimens are preserved in the herbarium of Botany Department,
North Orissa University, Baripada.
Figure 2. Solanum sisymbriifolium Lam.: A, Plant in natural habitat (Habit); B, A flower twig; C, Unripe berry (fruit); D,
Ripe berry and prickles.
DESCRIPTION OF THE SPECIES
Solanum sisymbriifolium Lam., Tabl. Encycl. 2: 25. 1794. (Fig. 2)
Solanum sisymbriifolium f. lilacinum Kuntze
Perennial under shrub with maximum height of 2 meter. Stem sub erect or scandent with glandular hairs and
prickled throughout. Prickles up to 15mm long, yellow in colour and sharped. Leaf alternate, lamina ovate-
oblong, 9–16 × 4–11 cm, deeply lobed, pinnatified with many prickles along main veins on both surfaces with
sparsely stellate hairs, wavy margin. Inflorescence racemose, 5–10 flowered. Peduncle ca. 20 cm long, hairs
glandular and simple. Pedicel ca. 1 cm long, slender and slightly prickly. Flower white, small, actinomorphic.
Sahu et al. (2017) 4(3): 468–470
Calyx cup-shaped, 9 × 3 mm, deeply 5-parted, green, prickly. Corolla white or slightly bluish, 35–45 mm in
diameter, rotate, stellate, lobes triangular. Stamens 5, exserted, yellow, filaments reduced, slender, glabrous, ca.
0.2 cm long, anthers 0.7–0.8 cm long attached to petals. Ovary ovoid, 0.2 cm, superior, glabrous; style 1.3 cm
long. Fruiting pedicels deflexed with acrescent calyx, densely glandular- pilose and viscid, fruiting calyx
enlarged 1.1–1.3 × 0.4–0.7 cm, enveloping most berry. Berry bright red, globose, 0.6–1.4 cm. diameter. Seeds
reniform, ca. 0.2 cm diameter.
Flowering & Fruiting: June–September.
Habitat: The species grows on waste places, roadsides, landfills and ploughed fields.
Voucher specimen: India, Balasore dist., Rupsa railway station, 12 m, N 21º 37 21 E 87º 01 09 , 14.07.2017,
M.R. Mohanta et al.1562 (NOU).
Global distribution: This species is a native of Central and South America (Argentina, Southern Brazil,
Paraguay, Uruguay, Bolivia and Colombia) and introduced in North America (Canada, Mexico, the United
States), Europe (Spain, the Netherlands), Asia (India, China, Taiwan), Africa (South Africa, Congo, Swaziland),
and Australasia (Australia, New Zealand).
Indian distribution: Andhra Pradesh, Assam, Bihar, Kerala, Karnataka, Maharashtra, Manipur, Orissa,
Jharkhand, Punjab, Sikkim, Tripura Uttar Pradesh and West Bengal.
ACKNOWLEDGEMENT
The authors are thankful to the Head of Department Botany, North Orissa University, Baripada for providing
infrastructure facility.
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Journal of Economic & Taxonomic Botany 31(3): 749–753.
ISSN (E): 2349 – 1183
ISSN (P): 2349 – 9265
4(3): 471–479, 2017
DOI: 10.22271/tpr.2017.v4.i3.063
Research article
Macrophyte - environment relationships

during a monospecific and a multispecific massive invasion
in a fishpond
Lúcia H. Sipaúba-Tavares1*, Cecilia B. Anatriello1, Ana Milstein2,
Rodrigo N. Millan3 & Bruno Scardoeli-Truzzi1
1
Aquaculture Center, Univ. Estadual Paulista - UNESP, Jaboticabal, São Paulo, Brazil
2
Agricultural Research Organization, Fish and Aquaculture Research Station Dor,
M.P. Hof HaCarmel, 30820 Israel
3
Universidade do Estado de Minas Gerais - UEMG, Department of Science,
Frutal, Minas Gerais, 38200-000 Brazil
*Corresponding Author: sipauba@caunesp.unesp.br [Accepted: 18 December 2017]
Abstract: The current study evaluated the water quality and zooplankton community in a shallow
fish pond during two different macrophyte invasions, one of Salvinia auriculata only and the other
of several aquatic plants. The results showed that the macrophyte covering of the pond surface led
to anoxic conditions, not desirable in fish culture ponds. This negative effect occurred in winter
and in summer, and under monospecific and multispecific macrophyte mass developments. The
multispecific macrophyte invasion provided a more complex habitat structure that allowed higher
zooplankton diversity and species richness but lower zooplankton density than the monospecific
invasion. High organic loading may influence the mass development of floating macrophytes in
one pond or another. To prevent macrophyte invasions it would be advisable to reduce organic
loading in the pond system, which is a fish farm is rather difficult. But if a macrophyte invasion
event occurs, proper management practices of plant removal can be applied to solve the immediate
problem and to avoid sporocarps dissemination that could launch future such events. The
management practices in the fish farm must be adequate to keep good water quality for the
production of good market products.
Keywords: Salvinia auriculata - Sediment - Zooplankton community - Water quality.
[Cite as: Sipaúba-Tavares LH, Anatriello CB, Milstein A, Millan RN & Scardoeli-Truzzi B (2017) Macrophyte
- environment relationships during a monospecific and a multispecific massive invasion in a fishpond. Tropical
Plant Research 4(3): 471–479]
INTRODUCTION
Macrophytes have a crucial role in the metabolism of shallow aquatic ecosystems. They exert considerable
influence on the nutrient dynamics of water and sediment due to water shadowing and induction of particle
sedimentation. They affect the habitat of the plankton community, particularly as a food source directly or
indirectly through the periphyton growing on the macrophytes. Macrophyte species have specific and divergent
traits that result from details in plant architecture, allelopathic activity, palatability for herbivores and
association with certain organisms that may attract or repel zooplankton species (Van Onsem et al. 2010). The
micro-invertebrate community associated to macrophytes consists of planktonic, phytophylus and micro-faunal
species (Zrum & Hann 1997). Dense beds of macrophytes are favorable to zooplankton development owing to
the protection that plants provide against fish predation (Raut & Pejaver 2013), and to increased water residence
time that allows more time for the development of the zooplankton community (Basu et al. 2000).
Macrophyte presence in fish ponds remove nutrients and provide a large number of habitats for micro-
invertebrate species. However, if managed inadequately, invasive macrophytes may inflict harmful ecological
and economic impacts on these systems (Chatterjee & Dewanji 2014). Invasive macrophytes in fish ponds are
Sipaúba-Tavares et al. (2017) 4(3): 471–479
not common, but when they massively occur they cause changes in biotic and abiotic water conditions that
affect the whole ecosystem, causing serious problems in the use of ponds.
In southeastern Brazil, continuous water flow fish culture systems are used, where the water from one
fishpond enters directly into the next one without previous treatment. Eventually, the water characteristics of the
first fishpond may affect the following ones, increasing nutrients load. In this kind of system, the aquatic plants
tend to propagate quickly and to cover the entire surface of the pond, mainly due to lack of adequate
management (Sipaúba-Tavares et al. 2003).
Despite the widespread occurrence of macrophytes in shallow ecosystems in neotropical regions and their
potentially harmful impacts on the environment, state-of-the-art information on invasive macrophyte effects
with regard to water conditions and zooplankton community in fish ponds is still rare. The current study
evaluated two different massive macrophyte invasions, one only with Salvinia auriculata Aublet (monospecific)
and other with several aquatic plants (multispecific) that developed in the same fish pond, and their influence on
the zooplankton community and water column.

Study Area
The current study was performed in a fish pond of the aquaculture farm of the University of São Paulo State
(UNESP) at Jaboticabal (SP, Brazil) (S 21°11’; W 48°18’) (Fig. 1). Water and zooplankton samples were
collected during two macrophyte invasion periods: one in summer 2009 when a monospecific invasion of
Salvinia auriculata (SA) developed, and the other in autumn/winter 2010 when an invasion of multiple
macrophytes species (MS) occurred. The fish pond under analysis has a surface area of 4,268 m2, a maximum
depth of 1.3 m, with continuous water flow and water renewal equivalent to 5% of its total volume/day
(calculated by discharge volume). It is the fourth in a sequential series of six similar size fish ponds (areas
ranging between 2,036 m2 and 8,067 m2), each directly and/or indirectly receiving water from the previous one
through an underground pipe grid (Fig. 1). This fish pond has two water inlets at its northern end, receiving
water from the previous fish ponds in the line and from a total of forty-nine small fish ponds with areas between
45 m2 and 400 m2, as well as from a frog culture sector. The water outlet lies at the south end of the fish pond.
Figure 1. Diagram of the fish pond studied. Inset A: shaded area indicates southeastern Brazil (the state of São Paulo). Inset
B: aquaculture farm of the Univ. Estadual Paulista. Inset C: fishpond studied with sampling sites (CI, FP, and WO) and _-_-
= frog culture sector.
The fish species present in the pond during the Salvinia auriculata invasion were Oreochromis niloticus,
Ctenopharyngodon idella, Colossoma macropomum and Piaractus mesopotamicus at a total density of
approximately 0.5 kg.m-2. During the second invasion (multispecific) the fish pond had about 400
Ctenopharyngodon idella individuals. Fish were fed during both invasions with a supplementary diet at a rate of
3% of their weight per day.
Macrophytes Handling and Sampling
The initial monospecific Salvinia auriculata invasion in 2009 covered the total surface area of the fish pond.
The species was harvested by several people that entered into the pond, removed the plants by hand and
deposited them on the pond banks. After the physical removal of S. auriculata, the fish pond was drained,
refilled and fish were stocked in the fish pond.
Macrophyte sampling at the end of the second invasion period was performed using a 1 m2 quadrat. All
macrophyte materials collected in 6 quadrants taken throughout the pond were carefully identified and measured
to determine the coverage (%) of each species as indicated by Thomaz et al. (2004).
Environmental Parameters Sampling
Environmental parameters were determined always in the morning, at about 8:00. Surface water was weekly
sampled with a 1L Van Dorn bottle at three sampling sites, CI = close to water inlets; FP = deep water site; WO
= water outlet (Fig. 1). Sampling was performed from a boat taking care not to disturb sediments in the
sampling point. Water temperature (Temp), dissolved oxygen (DO), conductivity (Cond) and pH were measured
in situ with a Horiba U-10 multi-sensor. Total phosphorus (TP) and nitrogen compounds were quantified by
spectrophotometer following Golterman et al. (1978) and Koroleff (1976). Alkalinity (Alk) was determined as
described by Mackereth et al. (1978) and chlorophyll-a (Chlor-a) was extracted with alcohol 90% and quantified
at 663 nm and 750 nm (Nusch 1980). Total suspended solids (TSS), total dissolved solids (TDS) and 5-day
biochemical oxygen demand (BOD5) were determined according to Boyd & Tucker (1992). Water samples for
microbiological analysis using the multiple-tube methods were collected in 500 mL flasks and taken to the
laboratory in an isothermal container. The material for microbiological analysis and thermotolerant coliforms
(TC) was sterilized prior to use (APHA 1995).
Zooplankton sampling was weekly performed filtering 10 L of water through a 58 µm pore net. The
collected material was concentrated to 50 ml and formaldehyde was added to reach 4% of final concentration.
Net samples were observed under optical microscope for preliminary taxonomical identification. Copepoda and
Cladocera species were counted in a reticulated acrylic chamber under a stereoscopic microscope (40 X) and
Rotifera were analyzed by counting organisms with a Sedgewick-Rafter chamber under a Leitz microscope (100
X). Taxonomic identification followed specialized literature (Koste 1978, Reid 1985, Elmoor-Loureiro 1997).
Data Analysis
All water parameters underwent one-way analysis of variance (ANOVA) and post-hoc Tukey’s HSD
multiple comparison test with Statistica 8.0 to compare sites (CI, FP and WO) and macrophyte invasion periods
at a P<0.05 significance level. In all cases, ANOVA was preceded by a test of variance homogeneity (Levene’s
test). All results were expressed as Means ± Standard Deviation (SD). Analyses of zooplankton species
dominance (d) and abundance (a) were also conducted. Species were considered dominant when their density
was higher than 50% of the total number of specimens present in each sampling site and invasion period; they
were abundant when the number of specimens was higher than the mean density of all occurring species (Lobo
& Leighton 1986). The Shannon-Wiener (H’) and richness (total number of species) indexes were calculated
from the total number of zooplankters present in each sampling site and invasion period. Sites were grouped by
cluster analysis (unweighted pair group average linkage, UPGMA) based on Bray Curts Index, using abundance
log10 (x + 1) transformed data of all zooplankton (Valentin 2000).
RESULTS
The initial monospecific macrophyte invasion in summer 2009 by the free-floating fern Salvinia auriculata
covered in two months the total surface area of the fish pond. During the second invasion period in autumn-
winter 2010, multiple macrophytes species occurred: the small free-floating plants S. auriculata and Lemna
minor L. covered 49% of the occupied pond area, the bigger free-floating plant Eichhornia crassipes (Mart.) a
further 33%, the ground and floating grass Panicum repens L. and the prostrate herb Polygonum capitatum
another 10%, and the rooted species Typha domingensis Pers. and Cyperus rotundus L. an additional 8%.
Starting with S. auriculata development, the multispecies invasion covered in two months the total surface of
the fish pond.
Table 1. Water variables and thermotolerant coliforms (Mean ± Standard deviation during the sampling period) in
the fish pond under the invasions of Salvinia auriculata (SA) and multiple macrophytes species (MS) in three
sampling sites: CI = Close to water inlets; FP = Deep-water; WO = Water outlet.
SA (summer) MS (autumn-winter)
Variables
CI FP WO CI FP WO
Temp (C) 27±1a 26±0.4ab 26±0.5b 20±1c 20±1c 19±1c
pH 9±0.2a 9±0.2a 9±0.1a 6±0.4b 6±1b 6±1b
Cond (µS.cm-1) 116±5a 121±9 a
120±5a 87±16 b
95±6 b
90±7b
Alk (mg.L-1) 115±21a 119±30a 116±30a 105±6a 107±16a 105±11a
DO (mg.L-1) 5±0.2a 1±0.1 bc
1±0.2c 4±1 a
2±1 b
1.4±1bc
TP (µg.L-1) 207±46a 203±69ab 189±56ab 82±47b 85±42b 210±145a
TAN (µg.L-1) 526±38b 440±51 bc
262±50c 1,089±243 955±221 1,034±146a
a a
Nitrite (µg.L-1) 22±5a 21±3a 13±2b 20±5a 22±10a 15±8b

Nitrate (µg.L-1) 224±63c 467±150 b
327±85bc 374±55 bc
720±51 a
392±242bc
BOD5 (mg.L-1) 7±0.6a 4.5±1.5b 4.8±1.5b 7±1a 5±1b 5±1b
TSS (mg.L-1) 11±3a 12±7 a
10±9a 9±1 a
13±1 a
16±3a
TDS (mg.L-1) 100±21a 83±21a 88±17a 98±15a 82±29a 106±27a
Chlor-a (µg.L-1) 43±10b 45±15b 37±8b 96±23a 67±45ab 55±14b
TC (MPN.100mL-1) 1,259±173a 1,009±394 967±352a
a
33±21 b
22±18 b
14±8b
Note: Values in the same row with different superscripts are significantly different (p<0.05); TAN = total ammonia
nitrogen.
The ANOVA results of the water variables measured in each macrophyte invasion period and sampled site
are presented in table 1. As expected, temperature was significantly higher during the summer than during the
winter. During the summer S. auriculata invasion period were recorded significantly lower TAN and
significantly higher pH, conductivity and thermotolerant coliforms than in the multispecific winter invasion
period. Thermotolerant coliforms were 2 orders of magnitude lower during the multiple species invasion period.
Some variables presented differences among sampling sites in one or both invasion periods. In both periods
dissolved oxygen and BOD5 were significantly higher close to the water inlets nitrate was significantly higher in
the center of the pond, while nitrite was significantly higher in the output area. During the summer period there
were decreasing temperature and TAN gradients from the water input to the water output, while during the
winter period both parameters were homogeneous in the whole pond. Total phosphorus was homogeneously
high in the whole pond during the SA invasion, while during the MS invasion it was significantly lower in the
inlet and central areas. No differences among sites and periods were recorded in alkalinity, TSS and TDS.
Figure 2. Zooplankton during the sampling period, under the invasions with Salvinia auriculata (SA) and multiple
macrophyte species (MS) in different sampling site (CI = close to water inlets; FP = deep-water sites; WO = water outlet): A,
Mean relative abundance (%); B, Density (individuals.L-1).
Zooplankton was composed by Rotifera, Cladocera and Copepoda. Rotifera constituted most of the
zooplankton (70–90 %) and had higher density (3,200–10,300 ind.L-1) during the SA invasion, while during the
MS invasion their density was lower (1,600–2,200 ind.L-1) and they were the most abundant group only near the
inlets (74% in CI vs. 30% in FP and WO) (Fig. 2). Copepoda was the second most abundant group, representing
55–60 % of total zooplankton only during the MS invasion in the FP and WO sites, with similar densities but
different spatial distributions within the pond in both invasion periods (Fig. 2). Cladocera was the least abundant
group, with just a few organisms during the SA invasion and up to 700 ind.L-1 (10% of zooplankton), during the
MS invasion (Fig. 2).
Table 2. Specific composition, abundance and ecological indexes (diversity and species richness) of zooplankton in the
fish pond under the invasions of Salvinia auriculata (SA) and of multiple macrophytes species (MS), in three sampling
sites: CI = close to water inlets; FP = deep-water site; WO = water outlet.
SA MS
Taxa
CI FP WO CI FP WO
Diversity (H’) 0.33 0.60 0.61 0.62 0.94 0.88
Species richness 27 20 20 32 23 22
Cladocera
Alona monacantha (Sars, 1901) + + + + a a
Bosmina hagmanni (Stingelin, 1904) + + + - - -
Diaphanosoma birgei (Korineck, 1981 + + + + + +
Copepoda
Argyrodiaptomus furcatus (Sars, 1901) + + + + + +
Nauplii + + - + + a
Thermocyclops decipiens (Lowndes, 1934) + + + + a a
Nauplii a a a a a a
Harpacticoida - + + - + +
Rotifera
Ascomorpha agilis (Zacharias, 1893) + + + + - -
Ascomorpha ecaudis (Perty, 1850) + - - + - -
Asplanchna sp. (Guerne, 1888) - + - - - -
Asplanchnopus sp. (Guerne, 1888) + + + + + -
Brachionus calyciflorus (Pallas, 1766) + + + a + -
Brachionus caudatus (Barrois & Daday, 1894) + + + - - -
Brachionus falcatus (Zacharias, 1898) + + + + - -
Brachionus havanaensis (Rousselet, 1911) + + + - - -
Brachionus quadridentatus (Hermann, 1783) + - + - - -
Brachionus patulus (Muller, 1786) + - - - + +
Cephalodella remanei (Wiszniewski, 1934) + - - - - +
Colurella uncinata (Ehrenberg, 1832) + + - + - -
Euchlanis arenosa (Myers, 1936) + + - + + +
Filinia terminalis (Plate, 1886) + + a + - -
Keratella cochlearis (Gosse, 1851) + + + + + -
Keratella serrulata (Ehrenberg, 1838) a a + a - -
Keratella tropica (Apstein, 1907) + - + a + -
Lecane bula (Gosse, 1851) + - - + + +
Lecane elsa (Hauer, 1931) + - - - - -
Lecane proiecta (Hauer, 1956) + - - - - -
Lecane submagna (De Ridder, 1960) - - - + - +
Lepadella heterostyla (Murray, 1913) - - - + + -
Lepadella ovalis (Müller, 1786) - - - + + +
Platyias quadricornis (Ehrenberg, 1832) - - - - - +
Polyarthra dolichoptera (Idelson, 1925) a a a a + +
Proales doliaris (Rousselet, 1895) a + + a a +
Proales globulifera (Hauer, 1921) + - + + + +
Proales sordida (Gosse, 1886) - - - + - -
Proalinopsis caudatus (Collins,1873) + - - + - -
Synchaeta stylata (Wierzejski, 1893) - - - - - +
Testudinella mucronata (Gosse, 1886) + - - - - +
Trichocerca longiseta (Schrank, 1802) a a a a + +
Note: a, Abundance; +, Present; -, Absent.
Zooplankton species diversity and richness were higher during the multispecies macrophyte invasion.
Together with this, in both period diversity was lower and richness was higher near the inlets than in the center
and outlet areas (Table 2). Cluster analysis based on zooplankton composition and densities showed that during
the SA invasion the inlets and outlet areas were similar and different from the pond center, while during the MS
invasion the inlets area was markedly different from the other sites (Fig. 3).
Figure 3. Cluster analysis of total average densities of zooplankton (individuals.L-1) in each sampling site under the
invasions with macrophyte: A, Salvinia auriculata (SA); B, Multiple species (MS).
Overall, in the zooplankton occurred 32 species of Rotifera, 3 of Cladocera and 3 of Copepoda (Table 2).
None of the recorded species was dominant in any site or invasion period. Two species of Copepoda, 2 of
Cladocera, and 3 of Rotifera (Poliarthra dolichoptera, Proales doliaris, Trichocerca longiseta) occurred in all
sampled sites in both invasion periods. The Cladocera Bosmina hagmanni and 6 Rotifera species (Asplanchna
sp., Brachionus caudatus, B. havanaensis, B. quadridentatus, Lecane elsa, L. proiecta) were recorded only
during the SA invasion period. No Crustacea and 6 Rotifera species (Lecane submagna, Lepadella heterostyla,
L. ovalis, Platyias quadricornis, Proales sordida, Synchaeta stylata) were recorded only during the MS invasion
period. Five Rotifera species (Ascomorpha ecaudis, Lecane elsa, L. proiecta, Proales sordida, Proalinopsis
caudatus) only occurred in the water inlet area. During the SA invasion period the abundant taxa in the whole
pond were nauplii of the Copepoda Thermocyclops decipiens and the Rotifera Polyarthra dolichoptera and
Trichocerca longiseta. Abundant in the inlet and/or center areas were also Keratella serrulata and Proales
doliaris, and in the outlet area Filinia terminalis. During the MS invasion period only nauplii of the Copepoda
T. decipiens were abundant in the entire pond. In the inlet area were also abundant the Rotifera Brachionus
calyciflorus, Keratella serrulata, K. tropica, Polyarthra dolichoptera, Trichocerca longiseta, and Proales
doliaris that also was abundant in the center of the pond. In contrast, in the central and outlet areas the abundant
taxa were crustaceans: the Cladocera Alona monacantha and all developmental forms of the Copepoda T.
decipiens. Nauplii of the Copepoda Argyrodiaptomus furcatus were abundant only in the outlet area.
DISCUSSION
Macrophyte presence in this aquaculture farm is rather common, mainly in the six larger ponds. Although
this presence generally does not compromise the well-functioning of the system, it represents a potential threat
as a source for an aggressive macrophyte development. When a macrophyte invasion occurs it is important to
study such events in order to acquire knowledge for the better management of the ecosystem. At the time of our
studies the aggressive development occurred only in this pond, not in the previous or posterior ones, indicating
that appropriate conditions for such developments were rather localized. The invasions occurred during different
seasons, which introduce some degree of confounding between seasonal and macrophyte effects on the observed
differences between both periods. This could not be avoided since the invasions naturally occurred (as opposed
to experimentally induced) in such periods. We do not know which, were the conditions that led to the
monospecific Salvinia auriculata invasion, but we have a hypothesis to explain the second multispecific
invasion. The hypothesis is that there is a link between the incorrect plant removal in the summer 2009 and the
multispecies invasion in the autumn-winter 2010. During that removal operation, the workers walking in the
pond disrupted pond bottom, water column and S. auriculata integrity, so that sporocarps and broken parts of
the plants should have remained in the pond. Sporocarps and plant material should have also entered the pond as
rains washed the removed macrophyte biomass left on the pond banks. These sporocarps allowed the
appearance of S. auriculata the next year, which became a substrate for the natural growth of other macrophytes
ending up in the multispecies invasion. The multispecies invasion was removed by a professional team that
pushed nets from the banks without disturbing the pond bottom, and did not leave the removed material on the
banks. No further macrophyte invasion was recorded the following years in this pond.
Both studied invasions were only or largely dominated by floating macrophyte species. In small water
bodies, where water flow or winds cannot wipe them out, dense beds of free-floating plants are a symptom of
high-nutrient loading. This is because, having no direct access to the sediment pool of nutrients, floating plants
depend on high nutrient concentrations in the water for their growth (Scheffer et al. 2003). In our study,
effluents of fish ponds and frog culture facilities are the main source of organic and inorganic matter entering
the pond. Fish and frog culture activities are more intense during the summer (higher density, feeding rate, etc),
hence the higher concentrations of salts (conductivity), total phosphorus and thermotolerant coliforms observed
during this season. Summer is also the rainy season, clouds reducing light for phytoplankton photosynthesis in
fish ponds without macrophyte cover, hence reducing the amount of planktonic chlorophyll entering our
macrophyte covered pond. In contrast to those variables whose winter-summer differences were mostly related
to the water quality entering the pond, TAN concentration is also strongly determined by processes such as
decomposition, absorption and nitrification occurring within the pond, which led to lower TAN levels during the
warm season. TAN release into the water column through organic matter decomposition seems to have been
similar during both studied periods, as indicated by the similar BOD5, TSS and TDS levels measured in both
periods. But TAN removal from the water column through absorption may account for its lower concentration in
summer than in winter, since under higher temperature the increased metabolism of the large macrophyte
biomass present in the pond should have absorbed/removed more of it. TAN removal from the water column
through nitrification occurred in both periods, as indicated by the presence of nitrate together with the almost
absence of nitrite; the higher TAN availability as substrate for nitrification in winter would explain the overall
higher nitrate levels observed during that period.
Floating plants capacity to remove nutrients may be used to improve water quality. Specifically for Salvinia
auriculata and Eichhornia crassipes, the main floating macrophytes in our study, in another pond of the same
farm it was found that they efficiently reduced levels of nitrate, total phosphorus and orthophosphate (Sipaúba-
Tavares et al. 2003). However, when free-floating plants form dense extensive mats, they have adverse effects
on freshwater ecosystems because they create anoxic conditions, which strongly reduce animal biomass and
diversity and hamper fish production (Scheffer et al. 2003, Chatterjee & Dewanji 2014). This may have
occurred in our study, since oxygen concentrations were under 1.5 mg.L-1 during all SA invasion and most of
the time during the MS invasion. Only in the inlet area, where the entering water falls from a height of 1 m over
the pond surface strongly mixing the water, oxygen was over 4 mg.L-1 during both invasion periods.
In a flow-through system, macrophyte reduce water flow rate within the pond promoting particle
sedimentation, as indicated by the low sub-surface amount of suspended particles (TSS) observed throughout
the pond during both macrophyte covered periods.
The abundance of the mesotrophic-eutrophic environments copepod bio-indicator Thermocyclops decipiens
(Tibúrcio et al. 2015) and the large amounts of Rotifera observed in both studied periods respond to the high
nutrient and organic loadings always entering the pond. The differences in zooplankton species composition
observed in both invasion periods seem not to be largely related to season, since 38 out of the 40 taxa in table 2
have been recorded during the rainy summer and during the dry winter either in this or in previous macrophyte
studies conducted in this farm (Travaini-Lima et al. 2016, Sipaúba-Tavares et al. 2017). The exceptions are the
Rotifera Brachionus havanaensis and B. quadridentatus that here and in the work of Travaini-Lima et al. (2016)
where observed only during the rainy summer. Thus, the higher zooplankton diversity and species richness
during the multispecies invasion may be considered an indication of the more complex habitat structure
provided by several floating and rooted macrophytes as compared to the simpler habitat structure provided only
by one macrophyte free-floating species.
Summing up, macrophyte covering of the pond surface led to anoxic conditions, not desirable in fish culture
ponds. This negative effect occurred in winter and in summer, and under monospecific and multispecific
macrophyte mass developments. The multispecific macrophyte invasion provided a more complex habitat
structure that allowed higher zooplankton diversity and species richness but lower zooplankton density than the
monospecific invasion. High organic loading may influence the mass development of floating macrophytes in
one pond or another. To prevent macrophyte invasions it would be advisable to reduce organic loading in the
pond system, which in a fish farm is rather difficult. But if a macrophyte invasion event occurs, proper
management practices of plant removal can be applied to solve the immediate problem and to avoid sporocarps
dissemination that could launch future such events. The management practices in the fish farm must be adequate
to keep good water quality for the production of good market products.
ACKNOWLEDGEMENTS
The authors would like to thank the Brazilian Council for Scientific and Technological Development (CNPq
– PQ II) and the Foundation for Research Support of the State of São Paulo (FAPESP) for the scholarship
awarded to the second author (09/51852-1 and 12/06382-0) and for funding (12/09884-4).
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ISSN (E): 2349 – 1183
ISSN (P): 2349 – 9265
4(3): 480–485, 2017
DOI: 10.22271/tpr.2017.v4.i3.064
Research article
In vitro propagation of Pueraria tuberosa (Roxb. ex Willd.) DC.

Bindu T. K.1*, Sheema Dharmapal P.1, P. S. Udayan1, A. V. Raghu2 and Rahul R. Nair3
1
Post Graduate Department of Botany & Research Centre, Sree Krishna College, Guruvayur,
Ariyannur P. O., Thrissur-680102, Kerala, India
2
Kerala Forest Research Institute, Peechi, Thrissur-680653, Kerala, India
3
Aushmath Biosciences, Vadavalli, Coimbatore-641046, Tamil Nadu, India
*Corresponding Author: bindutksahadevan@gmail.com [Accepted: 20 December 2017]
Abstract: Pueraria tuberosa, commonly known as Indian kudzu or 'Vidari' is an important
medicinal plant belonging to the family Fabaceae. The tubers of this plant are an important
constituent in many Ayurvedic formulations as a restorative tonic, immune booster and antiageing.
Annual demand of Vidari by the Ayurvedic industry is 135 tonnes and industry is facing a severe
scarcity of this raw material. Micropropagation technology offers large-scale production of
disease-free, quality planting materials for pharmaceutical industries. A successful protocol for the
in vitro propagation of P. tuberosa has been achieved by using nodal segments as explants.
Multiple shoot induction and proliferation was obtained in Murashige and Skoog medium
supplemented with 0.5 mg.L-1 BAP, 0.5 mg.L-1 KN and 2% glucose. MS medium with 1.5 mg.L-1
KN favoured maximum shoot elongation. A maximum number of roots with the highest
percentage of rooting was observed on half strength MS media supplemented with 0.5 mg.L-1 IBA.
Elongated shoots in half strength basal MS medium induced roots in 14 days of culture.
Regenerated shoots with well-developed roots were successfully hardened with 80% survival.
Keywords: Multiple shoots - Nodal explants - Antioxidants - Vidari.
[Cite as: Bindu TK, Sheema Dharmapal P, Udayan PS, Raghu AV & Nair RR (2017) In vitro propagation of
Pueraria tuberosa (Roxb. ex Willd.) DC. Tropical Plant Research 4(3): 480–485]
INTRODUCTION
Pueraria tuberosa (Roxb. ex Willd.) DC. known as 'Mile a minute vine' is a rapidly growing perennial
woody climber, distributed throughout tropical parts of India, mostly in moist regions, monsoon-forests and
coastal tracts (Chopra et al. 1956). The rejuvenating drug 'Vidari' is prepared from its tuberous roots which act
as a galactagogue, stimulant and emollient (Warrier et al. 1995). It has been reported to have an extensive range
of medicinal properties and exhibits effective role in the treatment of leprosy, spermatorrhoea,
hepatosplenomegaly, tuberculosis and cough. In ethnomedicine, the edible tubers are used to treat various
ailments such as chest pain, rheumatism and fever (Jain 1991). Pueraria species are also popular for its
hypoglycemic (Raghuwanshi & Jain 2012), fibrinolysis enhancing (Verma et al. 2009), antioxidant (Pandey et
al. 2007), hypolipidemic (Tanwar et al. 2008), and antimicrobial (Ratnam & Raju 2009) properties. Some of the
isoflavonoids present in the P. tuberosa are puerarin, daidzein, genistein and genistin (Goyal & Ramawat 2007).
Studies proved that genistein reduces systolic blood pressure and enhances aortic relaxation to acetylcholine
(Vera et al. 2007). The isolated antioxidant tuberosin exhibited a variety of biological responses including
influence on inflammatory pathways (Pandey & Tripathi 2010). Dietary antioxidants lower the risk of heart
diseases and neurodegenerative diseases caused by the free radicals or reactive oxygen species (ROS) generated
during metabolism (Sulaiman et al. 2014).
The tubers of P. tuberosa are widely used in various formulations in the Indian system of Ayurvedic
medicine (Goyal & Ramawat 2007). The widespread harvesting of the medicinal plants as a source of the drug
has restricted its reproduction, regeneration and survival. The growth of Pharmaceutical industries accompanied
by unscientific and destructive collection, threaten the existence of many rare species. Micropropagation
protocols offer an alternate method to propagate medicinal plants and produce the compounds of interest in a
short period of time, without sacrifice of natural populations.
Bindu et al. (2017) 4(3): 480–485

Plant sample and experiment designing
The nodal segments of Pueraria tuberosa were collected from 10 months old plant maintained in pots.
Nodes were washed in running tap water followed by soap water treatment for 15 minutes. Nodal segments (2
cm length) were immersed in Bavistin (15 g.L-1), cefotaxime (200 mg.L-1 and tetracycline (200 mg.L-1) for 40
minutes and thoroughly washed with distilled water. These explants were surface sterilized with 0.1% (w/v)
HgCl2 for four minutes followed by wash with sterile distilled water. The explants were cultured on Murashige
and Skoog (Murashige & Skoog 1962) medium containing 0.8% (w/v) agar with 2% (w/v) sucrose and growth
regulators. The pH of the media was adjusted and maintained to 5.7. Growth regulators like 6-
Benzylaminopurine -BAP (0.5 to 1 mg.L-1) and Kinetin –KN (0.5 to 1.0 mg.L-1) were experimented with MS
medium at different combinations or alone for multiple shoot induction. The shoot proliferation in different
combinations was recorded in the present study. The effect of Kinetin on in vitro shoot elongation was observed
by inoculating the in vitro shoots on MS medium supplemented with various concentrations of KN (0.25, 0.5,
1.0, 1.5, 2.0 mg.L-1). The in vitro regenerated shoots were transferred to full strength MS and half MS with
Indole Butyric Acid-IBA (0.25, 0.5, 1.0 mg.L-1) for rooting. For hardening, two to three weeks old rooted shoots
were removed from the culture tubes, wash thoroughly and transferred to polycups containing soil and
vermiculite in the ratio 1:2 and kept in mist chamber for acclimatization.
Statistical analysis
All experiments were performed with three replications, having 30 samples each. The effect of various
treatments on selected growth parameters was measured quantitatively and statistically tested using analysis of
variance (ANOVA) using SPSS (Statistical Package for the Social Sciences) version 11.0. The significance of
the mean values of various treatments was assessed by Duncan’s New Multiple Range Test (DMRT) at p < 0.05.
RESULTS
Table 1. Effect of BAP and Kinetin on shoot induction of Pueraria tuberosa.
MS + Growth regulators % explants showing Number of days for
Treatments
BAP (mg.L-1) KN (mg.L-1) shoot formation shoot induction
T0 0.0 0.0 0.000f 0.000f
T1 1.0 0.5 74.00±1.05b 9.82±0.10a
T2 0.5 0.5 83.00±2.10a 6.05±0.05e
T3 0.5 1.0 48.00±3.16c 9.35±0.05b
T4 - 1.0 31.40±2.06e 7.50±0.11d
T5 1.0 - 40.00±0.05d 8.14±0.05c
Note: Level of significance was measured at p < 0.05. Column values with same superscript are not differing
significantly (P>0.05).
Table 2. Effect of BAP and Kinetin on shoot multiplication of Pueraria tuberosa after 40 days of culture.
MS+ Growth regulators Number of multiple
Treatments
BAP (mg.L-1) KN (mg.L-1) shoots per explants
T0 0.0 0.0 00.000f
T1 1.0 0.5 09.85±0.47b
T2 0.5 0.5 17.95±0.48a
T3 0.5 1.0 08.84±0.21c
T4 - 1.0 06.25±0.16e
T5 1.0 - 08.36±0.20d
Note: Level of significance was measured at p < 0.05. Column values with same superscript are not
differing significantly (P>0.05).
Morphological changes of the nodal explants were noticed within 7 days of inoculation. No shoots were
developed in explants grown on the control medium. All the plant growth regulators used induced multiple
shoots singly or in combination with variable response. Subcultures were done at every 15 days interval into
fresh nutrient media with same nutrient composition (Table 1). MS medium supplemented with 0.5 mg.L-1 BAP
with 0.5 mg.L-1 KN was found to be the most suitable medium for shoot induction from nodal explants of
Pueraria tuberosa. 83 % of bud initiation was observed within 6 days of culture. Increasing the concentrations
of growth regulators in culture medium resulted in callus formation at the basal part of the shoots. The same
hormonal combination gave the maximum number of multiple shoots in 40 days of culture (Table 2; Figs. 1 &
Bindu et al. (2017) 4(3): 480–485
2). Multiple shoot formation with the rate of 17.95±0.48 were observed in concentrations of BAP (0.5 mg.L -1)
with KN (0.5 mg.L-1) and it can be attributed to its synergistic effect of the combination. Shoot bud elongation
occurred when the shoots were placed on MS medium supplemented with different concentrations of KN (0.5,
1.0, 1.5, 2.0, 2.5 mg.L-1. MS supplemented with 1.5 mg.L-1 KN was found to be the single best treatment to
achieve maximum shoot length (6.21±0.98 cm) and a number of leaves (5.94±0.05) on cultures within 10 days.
It is also evident from the results that the shoot length of P. tuberosa increases with increasing KN concentration
up to 1.5 mg.L-1 (Table 3). In the present study, a further increase in KN concentration (2.0, 2.5 mg.L-1) had a
negative effect on shoot length.
Figure 1. A, Shoot bud initiation of Pueraria tuberosa from nodal explants on MS medium supplemented with 0.5 mg.L -1
BAP and 0.5 mg.L-1 KN; B–C, Multiple shoot induction after 40 days of culture on 0.5 mg.L-1 BAP and 0.5 mg.L-1 KN; D,
Shoot elongation on MS medium with 1.5 mg.L-1 KN; E, In vitro rooting on half srength MS with 0.5 mg.L-1 IBA; F,
Acclimatization.
Bindu et al. (2017) 4(3): 480–485
Figure 2. A, Multiple shoot induction after 40 days of culture in 0.1 mg.L-1 KN; B, Multiple shoot induction after 40 days of
culture in 0.1 mg.L-1 BAP.
Table 3. Effect of KN on shoot elongation after 10 days of culture.
Treatments MS+KN (mg.L-1) Shoot length (cm) Number of leaves
T0 0.0 0.000f 0.000f
T1 0.5 2.89±0.14e 2.35±0.05e
T2 1.0 3.38±0.22d 3.35±0.10c
T3 1.5 6.21±0.98a 5.94±0.05a
T4 2.0 4.76±0.61b 4.05±0.05b
T5 2.5 4.13±0.04c 3.15±0.05d
Note: Level of significance was measured at p < 0.05. Column values with same superscript are not
differing significantly (P>0.05).
To induce rooting, in vitro shoots were cultured on half strength MS media alone and in combination with
various concentrations of growth regulator IBA. None of the in vitro shoots differentiated roots in full strength
MS media without hormones. A number of roots, length of roots and number of days for root induction were
observed and recorded. The differentiation of root buds varied with the growth regulator combination of the half
MS (Table 4). A number of days for root induction differed significantly among the IBA concentrations in half
strength MS. The number of roots and length of roots were significantly reduced on media with two levels of
IBA (0.25, 1.0 mg.L-1). Among the different concentrations of IBA studied half strength MS media with 0.5
mg.L-1. IBA induced an optimum number of robust, healthy roots within 14 days. The micro shoots excised
from in vitro cultures were successfully planted out to the soil in polypots with 80% survival. They were later
planted in the field after about a month where they established well.
Table 4. Effect of Media and IBA on in vitro rooting.
Number of days for
Treatments Media MS IBA (mg.L-1) Number of roots Root length (cm)
root induction
T0 Full 0.0 0.000c 0.000d 0.000d
T1 Half 0.25 1.18±0.40b 0.81±0.07c 18.36±0.50a
T2 Half 0.5 3.54±0.52a 2.96±0.10a 14.18±0.42c
T3 Half 1.0 1.36±0.50b 1.14±0.11b 16.63±0.51b
Note: Level of significance was measured at p < 0.05. Column values with same superscript are not differing significantly
(P>0.05).
DISCUSSION
After 6 weeks of culture, a maximum number of in vitro shoots (17.95±0.48) was obtained on MS medium
containing 0.5 mg.L-1 BAP with 0.5 mg.L-1 KN which differed significantly from other concentrations and
combinations of BAP and KN tested as well as in the control (Fig. 1). The cytokinins BAP and KN were
effective in terms of shoot induction from nodal explants of Stereospermum suaveolens (G. Don) DC. (Trivedi
& Joshi 2014) and Costus speciosus (Raghu et al. 2006). The combinations of 0.5 mg.L-1 BA and 0.5 mg.L-1 KN
proved to be most effective for culture initiation with healthy shoots (Shubha et al. 2016). It was reported that in
vitro cultures of P. tuberosa showed high shoot proliferation in KN supplemented media (Rathore & Shekhawat
2009). It has been reported that in vitro shoot induction and multiplication are largely based on media
Bindu et al. (2017) 4(3): 480–485
composition containing cytokinins as major plant growth regulators (Afshin et al. 2011). Multiple shoot
formation clumps having 6–7 shoots per explants have been observed at synergistic combinations of BAP and
KN on in vitro cultures of Achyranthes aspera (Fazlima et al. 2008). The use of comparatively lower
concentration of growth regulator in present protocol is an important factor worth mentioning, as it minimizes
the risk of producing genetically altered individuals (Raghu et al. 2007). It is seen that treatment with KN alone
significantly inhibited shoot production compared to treatments with BAP. It was reported that KN was less
effective on multiple shoot induction of Gentiana kurroo as compared to BAP (Sharma et al. 1993). In several
medicinal plant species, BAP enhances shoot multiplication of in vitro cultures (Lakshmi & Mythili 2003),
which is in conformity with the result of our study.
Shoot length was enhanced in the presence of all tested concentrations of KN compared to control and there
was a significant difference among the KN concentrations. The shoot length differed significantly among the
KN concentrations. In the present protocol, shoot bud elongation was maximum when the multiple shoots were
placed on MS medium supplemented with 1.5 mg.L-1 KN. These results correspond to other reports where the
best shoot length and a maximum number of nodes of Matthiola incana resulted in KN enriched media (Afshin
et al. 2011). There are reports which state that KN promotes elongation of buds in Vigna radiata (L.) Wilczek
(Chandra & Pal 1995). The length of shoots was observed to increase with increasing concentration of Kinetin
(0.5–1.5 mg.L-1). Similar finding was observed in Stereospermum suaveolens (Trivedi & Joshi 2014) when KN
supplemented medium with higher concentrations form long healthy shoots with large leaves. It is evident from
the study that micro shoots failed to exhibit high shoot elongation on media with two different levels of KN
(2.0, 2.5 mg.L-1).
In the present study in vitro shoots rooted in different concentrations of IBA in half MS. Among the different
concentrations of IBA tested, 0.5 mg.L-1 IBA significantly favoured maximum rooting, where the average root
number increased to 3.54±0.52 with highest root induction response. These strong, short, pointed and healthy
roots originated with an average length of 2.96±0.10 cm showed high survival rate during hardening. Half
strength MS media with two levels of IBA (0.25, 0.1 mg.L-1) developed slim and tender roots that were
damaged during transfer. In vitro shoots of P. tuberosa rooted in IBA supplemented half strength MS media
(Rathore & Shekhawat 2009). It has been reported that in vitro rooting in Paulownia elongata exhibited a
maximum number of roots and 100% rooting on half strength MS medium supplemented with 0.5 mg.L-1 IBA
(Zayova et al. 2014). The number of roots and its length decreased with 1.0 mg.L-1 IBA supplemented
media. Earlier, Nayak & Kalidass (2016) reported similar observations in Blepharispermum subsessile.
CONCLUSION
This protocol for regeneration of Pueraria tuberosa through nodal explants is highly effective for clonal
multiplication and conservation. In vitro regeneration holds tremendous potential to select, multiply and
conserve medicinally important genotypes, which are a potential resource of bioactive compounds.
ACKNOWLDGEMENTS
The authors are thankful to Dr. G. Jayakrishnan, Head of the Department of Botany, Sree Krishna College,
Guruvayur and Dr. E.M. Muraleedharan, Scientist, Kerala Forest Research Institute (KFRI), Peechi for
providing all the necessary facilities and encouragement during the work.
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ISSN (E): 2349 – 1183
ISSN (P): 2349 – 9265
4(3): 486–495, 2017
DOI: 10.22271/tpr.2017.v4.i3.065
Research article
Analysis of medicinal and economic important plant species of

Hollongapar Gibbon wildlife sanctuary, Assam, northeast India
Moumita Sarkar and Ashalata Devi*
Department of Environmental Science, Tezpur University, Tezpur-784028, Sonitpur, Assam, India
*Corresponding Author: ashalatadevi12@gmail.com [Accepted: 23 December 2017]
Abstract: An investigation has been made to recognise the medicinal and economic potential of
plant species occurred in the semi-evergreen forest of Hollongapar Gibbon wildlife sanctuary,
Assam using semi-structured questionnaire. In the present study, the importance of plant species
recorded in this semi-evergreen forest is analysed and assessed in terms of their medicinal and
economic values. A total of 157 plant species belonging to 136 genera and 78 families were having
medicinal and economic values. These include 69 trees (55 genera and 39 families), 17 shrubs (15
genera and 14 families), 58 herbs (57 genera and 37 families), 5 lianas (5 genera and 5 families)
and 8 bamboo/cane/palm (5 genera and 2 families). The study revealed 78% of plant species were
having significant values either in terms of medicinal or economic and both which make the plant
diversity of the sanctuary a vital source for resource supply. Majority of the recorded medicinal
plants were used for the treatment of some common health problems such as fever, cough, cold,
skin diseases, jaundice, dysentery, etc. Non Timber Forest products consist of wild edible
vegetables, resins, gums, fire woods, fodder, wild edible fruits, bamboo, canes, etc.
Keywords: Plant diversity - Economic plants - Medicinal plants - Anthropogenic activities -
Conservation.
[Cite as: Sarkar M & Devi A (2017) Analysis of medicinal and economic important plant species of
Hollongapar Gibbon wildlife sanctuary, Assam, northeast India. Tropical Plant Research 4(3): 486–495]
INTRODUCTION
Tropical forests are rich centres of species diversity having high productivity which results in a great supply
of resources to mankind. The services provided by tropical forests are numerous and taken as granted.
Contribution to human welfare with their distinctive medicinally and economically important plants is worth
mentioning. In general, forests provide a variety of resources which may be of direct or indirect use to human
life and regarded as a vital source of livelihood. The fundamental social, ethical, cultural and economic values
of humans have directly or indirectly revolved around biological resources since the earliest date of historical
record (Ramesh 2003, Pandey & Pandey 2016). People have traditionally and substantially depended upon the
resources in the wild for their sustenance (Gadgil 1989, Mehra et al. 2014, Bajpai et al. 2016).
The northeast region of India is a centre of rich biodiversity in the Indian sub-continent with a significant
proportion of flora and fauna having medicinal and economic values. Assam a state of northeast India is a
constituent unit of the Eastern Himalayan Biodiversity Region which is one of the three biodiversity hotspots in
the country. In Assam, there are 23 protected areas represented by 5 national parks and 18 wildlife sanctuaries.
The total area under national parks and wildlife sanctuaries in 2012 is 3925 Km2. contributing 5% of state‟s
geographical area (PCCF 2012). Several studies have been undertaken in different protected areas likewise in
the Hollongapar Gibbon wildlife sanctuary, Assam. However, most of the study undertaken in the sanctuary was
found to primarily focus on the primate species (Tilson 1979, Chetry 2001, Hazarika & Gupta 2005, Chetry et
al. 2007, Chetia & Kalita 2012). Bujarbarua (2002) carried out an ecological study of the sanctuary. A study on
Poaceae family of the sanctuary was done by Bujarbarua & Sarma (2006) where a total number of 37 species
belonging to 22 genera were collected. Verma et al. (2012) enumerated liverwort and hornwort flora of the
sanctuary. An analysis of tree species diversity, population dynamics and assessment of their regeneration
Sarkar & Devi (2017) 4(3): 486–495
pattern was studied by Sarkar & Devi (2014a) while, Borah & Devi (2014) carried out the ecological study on a
critically endangered tree species (Vatica lanceaefolia Bl.) in the sanctuary. However, there is no record of
assessment of the potential value of plant species diversity occurred in the sanctuary in terms of medicinal and
economic importance and their subsequent utilization. Therefore, an attempt has been made to investigate the
potential value of plant species diversity occurred in Hollongapar Gibbon wildlife sanctuary, Assam.

The Hollongapar Gibbon wildlife sanctuary (HGWLS), popularly known as Gibbon wildlife sanctuary, is an
isolated forest patch surrounded by human population and tea gardens from all sides. It is located at N 26°40'–
26°45' and E 94°20'–94°25' at an altitudinal range of 100–120 m amsl, in Mariani, Jorhat district of upper
Assam. It covers an area of 20.98 Km2 and the sanctuary has been divided into five compartments. The HGWLS
falls under “North East Biogeographic Zone (9)” and “N.E. Brahmaputra Valley Biogeographic Province (9A)”
as per Rodgers & Panwar (1988) and the forest type is “Eastern Alluvial Secondary Semi-Evergreen Forest
(1/2/2B/2S2)” under Moist Tropical Forests of India (Champion & Seth 1968). The climate in the region may be
classified as sub-tropical humid type (monsoonal climate) having four seasons in a year: pre-monsoon (March to
May), monsoon (June to September), post-monsoon (October to November) and winter (December to February).
The location of this sanctuary and accessibility by the local inhabitants of the fringe area for resource utilization
has made this site important for ecological studies.
Importance of plant species occurred in HGWLS was investigated by collecting relevant data using informal
semi-structured questionnaire from the people in terms of medicinal and economic importance. The local people
inhabited in and around the sanctuary were interview through informal conversation and any information on
uses of the plant in different proposes was recorded using the questionnaire and other relevant information were
also noted. Prior Information Consent (PIC) was taken before the conversation/interview. Literature like „Flora
of Assam‟ (Kanjilal & Bor 2005) was also consulted for any relevant information to be incorporated.
RESULTS
A total of 100 individuals/villagers inhabited in and around the sanctuary, belonging to the age group of 30–
80, were interviewed for collecting the information on the importance of plant species. Analysing the collected
data it was found that several plant species having medicinal and economic values were collected from the
sanctuary by the villagers for their subsistence. A total of 157 species belonging to 136 genera and 78 families
were found having ethnobotanical values which includes 69 trees (55 genera and 39 families), 5 lianas (5 genera
and 5 families), 17 shrubs (15 genera and 14 families), 58 herbs (57 genera and 37 families), and 8 species under
bamboo, cane and palm (5 genera and 2 families). Detail of these recorded ethnobotanical important plant
species in terms of medicinal and economic values is given in table 1. It was estimated that 78% of plant species
were having certain significant values either medicinal or economic or both which make the plant diversity of
the sanctuary a vital source of resource supply to the nearby inhabitants. Out of the 157 species, 52 species were
exclusively used for a medicinal purpose which belonged to 51 genera and 34 families. Out of these 52 species,
3 species were trees (2 genera and 2 families), 9 species were shrubs (9 genera and 8 families) and 40 species
were herbs (40 genera and 25 families). These plants like Dillenia indica, Macaranga denticulata, Terminalia
chebula, Clerodendrum infortunatum, Phlogacanthus thyrsiformis, Cheilocostus speciosus, Cissampelos
pareira, Commelina benghalensis, Paederia scandens, Phyllanthus fraternus, etc. are widely used for the
treatment of some common health problems such as fever, cough, cold, skin diseases, jaundice, dysentery, etc.
While, 36 species belonging to 33 genera and 23 families were having economic values. Among the 36 species,
21 species were trees (20 genera and 19 families), 1 species was shrub, 3 species were herbs (3 genera and 3
families), 4 species were lianas (4 genera and 4 families), 3 species were bamboo (3 genera and 1 family), 3
species were cane (1 genus and 1 family) and 1 species was palm. 15 species were found to be used exclusively
for timber. These plants are important and found to be used extensively as wild edible vegetables, resins, gums,
fire woods, fodder, wild edible fruits, etc. by the local people inhabited in and around the sanctuary to meet their
economic subsistence.

The sanctuary harbours a rich diversity of medicinal and economic important plants which adds the potential
value of the sanctuary. The commercially important plant species enumerated in the study area supports
Sarkar & Devi (2017) 4(3): 486–495
Table 1. List of medicinal and economically important plant species recorded in Hollongapar Gibbon wildlife sanctuary.
S.N. Name of Species Family Ver. name Importance Uses
Tree
1 Aglaia spectabilis (Miq.) S.S. Jain & Bennet Meliaceae Amari - E Timber
2 Ailanthus integrifolia Lam. Simaroubaceae Borpaat - E Timber
3 Albizia lebbek (L.) Benth. Mimosaceae Shirish - E Timber
4 Alseodaphne petiolaris Hook. f. Lauraceae Gojua M - Jaundice
5 Alstonia scholaris (L.) R. Br. Apocynaceae Sotiona M E Timber
6 Altingia excelsa Noronha Altingiaceae Jutuli M E Headache, allergy, boils; timber
7 Aquilaria malaccensis Lam. Thymelaeaceae Agaru M E Skin diseases; resin, oil, highly scented wood
8 Artocarpus chaplasha Roxb. Moraceae Samkothal M E Diarrhoea, sores; timber, fruit edible
9 Artocarpus lakoocha Roxb. Moraceae Bohot M E Skin diseases; fruit and flower head edible,dye
10 Baccaurea ramiflora Lour. Phyllanthaceae Leteku M E Stomach ache, toothache; fruit edible, fish farming
11 Balakata baccata (Roxb.) Esser Euphorbiaceae Seleng - E Timber, wood
12 Barringtonia acutangula (L.) Gaertn. Lecythidaceae Paani amara M E Diarrhoea, syphilis, leprosy; timber, bark for
intoxicating fish
13 Canarium bengalense Roxb. Burseraceae Dhuna M E Rashes, snake bite; resin, air purifier
14 Carallia brachiata (Lour.) Merr. Rhizophoraceae Maahithekera M E Stomach disorder; Timber, fruit edible
15 Castanopsis indica (Roxb. ex Lindl.) A.DC. Fagaceae Hingori - E Leaves used for cigarettes, fruit edible, timber
16 Castanopsis tribuloides (Sm.) A.DC. Fagaceae Phoolhingori M E Cough, goitre, indigestion; Timber, fruit edible
17 Chukrasia tabularis A.Juss. Meliaceae Bogapoma M E Stomach ache, diarrhoea, dysentery; timber
18 Cinnamomum glaucescens (Nees) Hand.-Mazz. Lauraceae Gonsoroi - E Timber
19 Cyathea gigantea (Wall. ex Hook.) Holtt. Cyatheaceae Tree fern M E Anti-inflammatory; stem edible
20 Dillenia indica L. Dilleniaceae Outenga M E Stomach ache, jaundice, diarrhoea, dysentery,
dandruff; timber, fruit edible
21 Dipterocarpus retususBl. Dipterocarpaceae Holong - E Timber
22 Drimycarpus racemosus (Roxb.) Hook.f. ex Marchand. Anacardiaceae Aamsia - E Timber
23 Duabanga grandiflora (DC.) Walp. Lythraceae Khakan - E Timber
24 Dysoxylum gotadhora (Buch.-Ham.) Mabb. Meliaceae Bandardima M E Diarrhoea, dysentery; timber
25 Elaeocarpus serratus L. Elaeocarpaceae Rudraksh - E Cultural and religious use
26 Eurya acuminata DC. Pentaphylacaceae Murmura M E Diarrhoea; fuelwood, dye
27 Evodia meliaefolia Benth. Rutaceae Maiphak - E Timber
Sarkar & Devi (2017) 4(3): 486–495
28 Ficus benghalensis L. Moraceae Borgoch M E Rheumatism, diarrhoea, dysentery, diabetes; timber,
fodder
29 Ficus benjamina L. Moraceae Jorigoch M - Stomach disorder
30 Ficus fistulosa Reinw. ex Bl. Moraceae Kathiadimoru M E Post-natal treatment, diaphoretic; firewood
31 Ficus lamponga Miq. Moraceae Dimoru M - Jaundice
32 Ficus racemosa L. Moraceae Jagya Dimoru M E Diabetes, liver disorders, diarrhoea, urinary diseases;
fruit edible
33 Ficus religiosa L. Moraceae Ahot M E Dysentery, fever, scabies, piles, skin diseases,
gonorrhoea; religious, cultural, Timber, fodder, fruit
edible
34 Garcinia morella (Gaertn.) Desr. Clusiaceae Kujithekera M E Diarrhoea, leprosy, ulcers; resin, oil, dye, fruit edible
35 Garcinia pedunculata Roxb. ex Buch.-Ham. Clusiaceae Borthekera M E Diarrhoea, dysentery, jaundice; fruit edible
36 Gmelina arborea Roxb. Lamiaceae Gamari M E Stomach disorder; timber, fruit and flower edible
37 Hydnocarpus kurzii (King) Warb. Achariaceae Saalmugura M E Leprosy; oil
38 Ilex godajam Coleb. ex Hook.f. Aquifoliaceae Haatikerepa - E Firewood
39 Kydia calycina Roxb. Malvaceae Pisola M E Skin diseases, wounds, cuts, boils, veterinary
medicine; timber, fibrous bark
40 Lagerstroemia speciosa (L.) Pers. Lythraceae Azar M E Diarrhoea, dysentery, jaundice; timber, cultivated for
flowers
41 Litsea monopetala (Roxb.) Pres. Lauraceae Sualu M E Stomach ache; mugasilk worm reared on leaves
42 Macaranga denticulata (Blume) Müll.Arg. Euphorbiaceae Moralia M E Skin diseases, cuts, wounds; firewood, fodder
43 Magnolia champaca (L.) Baill. ex Pierre Magnoliaceae Titasopa M E Chronic gastritis, cough, fever; timber
44 Magnolia hodgsonii (Hook. f. & Th.) H. Keng Magnoliaceae Borhomothuri M E Tooth and gum; firewood, dye, timber
45 Magnolia hookeri (Cubitt& Smith) Raju & Nayar Magnoliaceae Paansopa - E Timber
46 Magnolia oblonga (Wall. ex Hook.f. & Thomson) Figlar Magnoliaceae Borsopa - E Timber
47 Mallotus nudiflorus (L.) Kulju & Welzen Euphorbiaceae Bhelkar - E Timber
48 Mangifera sylvatica Roxb. Anacardiaceae Bon-aam M E Gastrointestinal disorder; unripe fruit for pickles,
jelly and tarts
49 Mesua ferrea L. Calophyllaceae Nahor M E Fever, vomiting, urinary tract disorders, migraine;
oil, timber
50 Morinda angustifolia Roxb. Rubiaceae Aasugoch M E Body pain, cough, cracked feet; dye
51 Neolamarckia cadamba (Roxb.) Bosser Rubiaceae Kadam M E Blood purifier, antidiuretic, abortifacient; timber,
fruit edible
52 Olea dioica Roxb. Oleaceae Poreng M E Skin diseases, fever; fuelwood, charcoal
53 Palaquium obovatum (Griff.) Engl. Sapotaceae Kotholua - E Yields an inferior kind of Gutta percha
Sarkar & Devi (2017) 4(3): 486–495
54 Premna bengalensis Cl. Lamiaceae Gohora - E Timber
55 Pterospermum acerifolium (L.) Willd. Malvaceae Mota-marulia M E Glandular swelling of neck and ears; fodder, roofing
material
56 Saurauia roxburghii Wall. Saurauiaceae Bonposola M E Constipation; fruit edible, fodder, country liquor
57 Spondias mombin L. Anacardiaceae Khamolimola - E Bark chewed as substitute for areca nut, fruit edible
58 Spondias pinnata (L.f.) Kurz. Anacardiaceae Amara M E Dysentery; fruits and flower buds edible
59 Sterculia villosa Roxb. Malvaceae Udal M E Dysentery; timber,fibrous bark, seeds edible
60 Stereospermum chelonoides (L.f.) DC. Bignoniaceae Paroli M E Skin diseases, cough, arthritis; timber
61 Symplocos ferruginea Roxb. Symplocaceae Motabhomloti - E Fruits used for rosaries
62 Syzygium kurzii (Duthie) Balakr. Myrtaceae Bogijamuk M E Stomach trouble; timber
63 Terminalia catappa L. Combretaceae Kaathbadam - E Kernel edible, cultivated for fruits, dye
64 Terminalia chebula Retz. Combretaceae Hilikha M E Diarrhoea, dysentery, bleeding gums, conjunctivitis,
appetizer; timber, tanning
65 Terminalia myriocarpa Van Heurck & Müll. Arg. Combretaceae Halakh M E Urinary disorder; Timber, charcoal
66 Tetrameles nudiflora R. Br. Tetramelaceae Bheleu - E Timber
67 Vatica lanceaefolia Bl. Dipterocarpaceae Morsal M E Dysentery; firewood, resin, oil
68 Vernonia arborea Buch.-Ham. Asteraceae Maskoita - E Bark chewed as substitute for betel leaf
69 Walsura robusta Roxb. Meliaceae Lali M E Antibacterial, antioxidant; timber
Shrub
70 Clerodendrum glandulosum Lindl. Lamiaceae Nephaphu M - High blood pressure, hypertension
71 Clerodendrum infortunatum L. Lamiaceae Dhapatphool M E Skin diseases, bee’s sting; roots used for fermenting
liquor
72 Clerodendrum japonicum (Thunb.) Sweet Lamiaceae Dhapattita M E Febrile and catarrhal affection; leaves edible
73 Croton joufra Roxb. Euphorbiaceae Mahudi M - Skin diseases, anticancer, antioxidant
74 Dendrocnide sinuata (Blume) Chew Urticaceae Churaat M - Jaundice, urogenital disorder, toothache, dysentery
75 Grewia multiflora Juss. Malvaceae Kukurhuta M E Dysentery, fruit edible, lac insect reared on this plant
76 Ixora acuminata Roxb. Rubiaceae Borpotia M - Jaundice
77 Lantana camara L. var. aculeata (L.) Mold. Verbenaceae Bon baahaar M E Tetanus; insect repellent
78 Leea indica (Burm.f.) Merr. Vitaceae Kukurathengia M E Digestive disorders; fruit edible, dye
79 Melastoma malabathricum L. Melastomataceae Phutuka M E Cuts, wounds, tooth and gum diseases; fruit
edible,stem used as toothbrush
80 Mussaenda frondosa L. Rubiaceae Chubaiata M - Jaundice, ulcer, fever, wounds, asthma
81 Pandanus odorifer (Forssk.) Kuntze Pandanaceae Keyakothal M - Pneumonia
Sarkar & Devi (2017) 4(3): 486–495
82 Phlogacanthus thyrsiformis (Roxb. ex Hardw.) Mabb. Acanthaceae Titaphool M E Cough, fever, abdominal pain; flowers edible
83 Rauvolfia serpentina (L.) Benth. ex Kurz Apocynaceae Sarpagandha M - Snake bite, insomnia, stomach ache
84 Syzygium fruticosum DC. Myrtaceae Katahijamuk M - Blood dysentery
85 Viburnum colebrookeanum Wall. Caprifoliaceae Mezenga M - Sores
86 Ziziphus funiculosa Buch.-Ham. Rhamnaceae Bonbogori - E Fruits edible
Herb
87 Abrus precatorius L. Papilionaceae Latumoni M - Abortifacient, induce sterility in women
88 Ageratum conyzoides L. Asteraceae Gendheli bon M - Cuts, wounds, jaundice
89 Alocasia forniculata (Roxb.) Schott. Araceae Adoliakochu M - Crack of heels
90 Alpinia nigra (Gaertn.) Burtt. Zingiberaceae Tora/Bogitora M - Bone weakness, irregular menstruation
91 Begonia roxburghii A. DC. Begoniaceae Begonia tenga M - Skin diseases
92 Cheilocostus speciosus (J.König) C.Specht Costaceae Jamlakhuti M - Jaundice, insect bite
93 Chromolaena odorata (L.) R.M. King & H. Rob. Asteraceae Germany bon M - Cuts, wounds
94 Chrysopogon aciculatus (Retz.) Trin. Poaceae Bonguti M - Arthritis, rheumatism,antidote (sting)
95 Cissampelos pareira L. Menispermaceae Tubukilata M Fever, headache
96 Commelina benghalensis L. Commelinaceae Kanasimolu M E Snake bite, leprosy, skin inflammations; fodder
97 Curcuma aromatica Salisb. Zingiberaceae Bonhaladi M - Blood purification, constipation
98 Cuscuta reflexa Roxb. Convolvulaceae Raghumala M - Induce sterility in women, purgative, veterinary
medicine
99 Cynodon dactylon (L.) Pers. Poaceae Dubori bon M - Headache, irregular menstruation, tonic
100 Cyperus rotundus L. Cyperaceae Keyabon M - Stomach ache, purgative
101 Dicranopteris linearis (Burm.f.) Underw. Gleicheniaceae Kap-dhekia M E Indigestion, asthma, women’s sterility, fever,
anticancer; vascular bundles from the stalks woven to
make products
102 Dioscorea bulbifera L. Dioscoreaceae Aalulata M - Dysentery, indigestion
103 Diplazium esculentum (Retz.) Sw. Woodsiaceae Dhekiasak M E Constipation, young fronds edible
104 Eleusine indica Gaertn. Poaceae Bobosabon M - Post partum aidto mother, effective in fracture of
bones of hen and duck
105 Emilia sonchifolia DC. Asteraceae Bonkopahua M - Eye inflammation, febrifuge
106 Eragrostis unioloides Nees ex Steud. Poaceae - M - Asthma, wounds
107 Evolvulus nummularius L. Convolvulaceae - M - Burns, cuts, wounds
108 Floscopa scandens Lour. Commelinaceae - M - Sore eyes, fractured bone
109 Gonostegia hirta (Blume ex Hassk.) Miq. Urticaceae Sialkotahi - E Roots are used as hair wash, stem and leaves edible
Sarkar & Devi (2017) 4(3): 486–495
110 Hodgsonia macrocarpa (Bl.) Cogn. Cucurbitaceae Thebou M E Antifertility; seed kernels edible, silk worms are fed
on the leaves
111 Homalomena aromatica (Spreng.) Schott Araceae Gankochu M E Liver and kidney disorder; essential oil
112 Impatiens balsamina L. Balsaminaceae Demdeuka M - Fever, sterility
113 Lasia spinosa Thw. Araceae Chengmora M - Piles, sore throat, irregular menstruation
114 Leuca splukenetii Spreng. Lamiaceae Durun M E Piles, tonsillitis, stomach trouble, nasal haemorrhage;
young shoots edible
115 Lindernia crustacea (L.) F. Muell. Linderniaceae Kaachidoria M - Diabetes, dysentery, boils, ringworm infection
116 Ludwigia octovalvis (Jacq.) P.H. Raven Onagraceae Longbon M - Fever, dysentery, jaundice, cancer
117 Lygodium microphyllum (Cav.) R.Br. Schizaeaceae Kopoudhekia M E Dysentery, skin diseases; basket making and plaiting
from old stems, young stems edible
118 Mikania scandens (L.) Willd. Asteraceae - M - Antifungal
119 Millettia pachycarpa Benth. Papilionaceae Bokolbihlata M E Skin infection; fish poison
120 Mimosa pudica L. Mimosaceae Nilajibon M - Toothache, skin diseases, piles, boils
121 Murdannia nudiflora (L.) Brenan Commelinaceae - M - Asthma, leprosy, piles
122 Oxalis debilis var. corymbosa (DC.) Lour. Oxalidaceae Bor-tengechi M - Dysentery
123 Paederia scandens (Lour.) Merr. Rubiaceae Bhedailata M - Diarrhoea, dysentery, rheumatism
124 Persicaria barbata (L.) H. Hara Polygonaceae - M - Ulcer, purgative, tonic
125 Phrynium pubinerve Bl. Marantaceae Koupat M - Skin diseases, boils, leprosy
126 Phyllanthus fraternus Webster Phyllanthaceae Bon aamlokhi M - Dysentery, urinary trouble
127 Piper longum L. Piperaceae Pipoli M E Cough, cold, loss of appetite; condiment
128 Piper thomsonii (C. DC.) Hook.f. Piperaceae Auni pan M E Cough, cold; leaves chewed raw
129 Polygonum microcephalum D. Don Polygonaceae Madhuxuleng M - Dysentery
130 Pteris ensiformisBurm. f. Pteridaceae - M E Dysentery; tender frond edible
131 Rhynchotechum ellipticum (Wall. ex D. Dietr.) A. DC. Gesneriaceae - M E Cough, boils; edible
132 Scoparia dulcis L. Plantaginaceae Bondhania M - Cough, bronchitis, kidney trouble
133 Sida cordifolia L. Malvaceae Hunborial M E Post delivery trouble; yields fibre
134 Smilax perfoliata Lour. Smilacaceae Baaghaasuralata M - Wounds
135 Solanum ferox L. Solanaceae Katahibegena M - Appetizer, antiasthmatic
136 Spilanthes paniculata Wall. ex DC. Asteraceae Suhoni M - Cough, toothache, constipation
137 Stenochlaena palustris (Burm. f.) Bedd. Blechnaceae Dhekialata M E Fever, skin diseases; tender shoots edible, rhizomes
used in binding fish traps, anchor ropes, making
baskets
Sarkar & Devi (2017) 4(3): 486–495
138 Tetracera sarmentosa (L.) Vahl Dilleniaceae Oulata - E Leaves used as substitute for sand paper for
polishing, stem gives copious and potable water
when cut
139 Tetrastigma thomsonianum Planch. Vitaceae Naltenga - E Tender stem edible
140 Tinospora sinensis (Lour.) Merr. Menispermaceae Hoguni lot M - Gonorrhoea
141 Uncaria sessilifructus Roxb. Rubiaceae Barakhialata M Nervous disorder, hypertension
142 Urena lobata L. Malvaceae Hunborolua M E Diuretic, dysentery, rheumatism;
yields fibre, stem & branches used as tooth brush
143 Vitis planicaulis Hook. f. Vitaceae Panilata M - Fever, sore throat
144 Zingiber purpureum Rosc. Zingiberaceae Bon aada M - Sprain, inflammation, paralysis
Liana
145 Byttneria aspera Collebr. ex Wall. Malvaceae Tikonibarualata M E Fever; young parts and bark used as hair wash
146 Combretum roxburghii Spreng. Combretaceae Lotachali - E Bark chewed with/as betel nut
147 Connarus paniculatus Roxb. Connaraceae Makoilata - E Oil (soap making)
148 Dalbergia pinnata (Lour.) Prain Papilionaceae Laalengsali - E Bark chewed with betel leaf
149 Uvaria macrophylla Roxb. Annonaceae - - E Ripe carpels edible
Bamboo, cane and palm
150 Bambusa pallida Munro Poaceae Bijulibaah - E Edible young shoots, culms for making hut, baskets,
mats etc.
151 Calamus erectus Roxb. Arecaceae Jeng bet M E Antidiabetic, antioxidant, dyspepsia; leaves for
roofing materials, cane, etc.
152 Calamus flagellum Griff. ex Mart. Arecaceae Raaidang bet - E Cane is used for various purposes
153 Calamus floribundus var. depauperatus Becc. Arecaceae Lejaai bet - E Fruits edible, stem for making basket
154 Calamus tenuis Roxb. Arecaceae Jati bet - E Cane is used for various purposes
155 Pinanga gracilis Bl. Arecaceae GerukaTamul - E Fruits used as substitute of betel nut
156 Pseudostachyum polymorphum Munro Poaceae Bojaalbaah - E The rhizomes are used for weaving sieves for
selecting young fish, the culms are split for weaving
fences, etc.
157 Schizostachyum dullooa (Gamble) R. B. Majumder Poaceae Dolubaah - E Fencing, roofing, making baskets, mats, small boxes,
etc.
Note: M, Medicinal; E, Economical; Ver. name, Vernacular name in Assamese.
Sarkar & Devi (2017) 4(3): 486–495
economic condition of the local inhabitants residing in and around the sanctuary. Local people utilized various
plant resources in terms of medicine, timber and NTFP like, firewood, resin, gum, oil, dye, wild edibles, etc.
The ethnobotanical importance of the various plant species enumerated in this sanctuary has been well
documented by several workers (Kanjilal & Bor 2005, Purkayastha et al. 2005, Purkayastha & Nath 2006, Patiri
& Borah 2007, Das et al. 2008, Barbhuiya et al. 2009, Nath et al. 2010, Barukial & Sarmah 2011, Sarkar &
Devi 2014b, Dutta et al. 2016) from different areas of the state. Plant species like Dillenia indica, Macaranga
denticulata, Terminalia chebula, Clerodendrum infortunatum, Phlogacanthus thyrsiformis, Cheilocostus
speciosus, Cissampelos pareira, Commelina benghalensis, Paederia scandens, Phyllanthus fraternus, etc. are
well known to cure common health ailments such as fever, cough, cold, skin diseases and dysentery. Timber
yielding species included trees such as Albizia lebbek, Dipterocarpus retusus, Magnolia hookeri, Premna
bengalensis, etc. Few NTFP yielding species recorded from the study area includes Aquilaria malaccensis
(resin/oil/highly scented wood), Canarium bengalense (resin), Morinda angustifolia (dye yielding stem and
root), Melastoma malabathricum (stem and edible fruits), Diplazium esculentum (edible young and tender
shoot), Connarus paniculatus (oil), Pinanga gracilis (edible fruits), Schizostachyum dullooa (bamboo),
Calamus floribundus (cane), etc. A critically endangered and dominant tree of the study site (Sarkar & Devi
2015), Vatica lanceaefolia, is a multipurpose plant species and was found to be collected frequently by the
fringe people for fuel-wood. This critically endangered multipurpose tree species yields good quality firewood,
resin as well as medicine. Calamus floribundus var. depauperatus is threatened to the northeastern region of
India (Basu 1992) which is used for furniture. Artocarpus chaplasha and Ficus lepidosa are dominant food tree
species of the H. hoolock inhabited in the sanctuary and contributed about 10–11 % to their annual diet (Borah
2016).
Anthropogenic activities like illegal felling (personal observation during study period) can exert disturbance
to the survival and existence of these important species, and their structure and composition in the sanctuary.
Illegal activities like tree felling have also been reported in the study area by other workers like Chakraborty &
Gupta (2005) and Sharma et al. (2010). If such interference continues or increases in future, it may pose a
serious threat to the existence of the plant species diversity in this region. Nevertheless, quantification of the
degree of disturbance exerted by biotic or abiotic factors is always necessary to understand the pressure and
consequent effect on plant species richness and diversity of a particular area. Quantification of such parameters
is also very essential for wise management of resources and to formulate conservation strategies accordingly.
ACKNOWLEDGEMENTS
Authors are grateful to the P.C.C.F (Wildlife) and D.F.O, Jorhat Division (Forest Department, Assam), for
granting permission to carry out the work in Hollongapar Gibbon wildlife sanctuary. The kind assistance
received from Deben Borah, other staff of Meleng Beat office and local residents during the field work is
sincerely acknowledged. We also thank the staff of BSI, Arunachal Pradesh Circle, Itanagar for help and
suggestions.
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ISSN (E): 2349 – 1183
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4(3): 496–513, 2017
DOI: 10.22271/tpr.2017.v4.i3.066
Research article
Phytoecological studies of some protected and degraded forest

areas of lowland humid forest, Ondo state, Nigeria:
a comparative approach
Iyagin F. O.* and Adekunle V. A. J.
Department of Forestry and Wood Technology, Federal University of Technology Akure,
P.M.B 704, Ondo State, Akure, Nigeria
*Corresponding Author: fiyagin@yahoo.com [Accepted: 24 December 2017]
Abstract: This study compared the phytoecological characteristics of some protected areas [Strict
nature reserve (SNR), Oluwa natural forest (ONF) and permanent sample plot 29 aimed at
biodiversity conservation and a degraded forest not under reservation] in the lowland humid forest,
Ondo state, Nigeria. Data were collected using Systematic Line Transect method with two parallel
transects of 200 m apart for plot location. Four sample plots (25 m × 25 m) were laid alternately on
each transect in each of the forests. In each plot, trees with DBH ≥ 10 cm were measured
(diameters at the base, middle, top and total height), identified and classified into families and
frequency of occurrence to ascertain the present status of the protected areas in term of tree
diversity, abundance and yield. A total of 411 stems.ha-1 from 78 species and 30 families was
recorded in the study sites. Celtis zenkeri, Cola gigantea and Funtumia elastica were common to
all the forests. The DBH distribution curve of the species followed inverse J-shaped pattern with
49.4% of trees falling within the lowest diameter class (10–20 cm) while only 7.14% falls in the
highest diameter class (>80 cm). SNR and ONF have emergent trees whose heights were above 30
m (18.7% and 7.02% of the total number of trees in the Sterculaceae and Meliaceae families,
respectively) whereas 14.90 m was the highest height recorded in the free forest. On biovolume
yield, ONF has the highest volume per hectare (141.06 m3) while the least (14.56 m3) was from the
forest not under reservation. The Shannon-Weiner index, Pielou’s Evenness index and Margalef's
index for all the forest reserves are 5.11, 0.69 and 12.79 respectively with the highest for ONF.
These indices indicated that the protected areas are mature and foster in-situ conservation of tree
species especially the keystone species.
Keywords: Biodiversity - Protected areas - Conservation - Deforestation - Forest structure.
[Cite as: Iyagin FO & Adekunle VAJ (2017) Phytoecological studies of some protected and degraded forest
areas of Lowland Humid forest, Ondo state Nigeria: a Comparative approach. Tropical Plant Research 4(3):
496–513]
INTRODUCTION
Biodiversity has been defined as the variety and variability of life forms on earth. The "biological diversity"
typically measures variation at the genetic, the species, and the ecosystem level, though not evenly distributed
on earth but richest in the tropics (UNEP 2011). Flora and fauna diversity and the activities vary from one
ecological zone to the other in the tropic. This could be attributed to the differences in climate and weather
condition of the ecosystems. The tropical rainforest, which is located in the southwest and southeast geopolitical
zones of Nigeria, is highly complex and is known for its astonishing wealth of plant species because of the usual
favorable climate (Adekunle et al. 2007). Forest biodiversity provides wide variety of goods and services that
support the existence of humans on earth. These services include the provision of services which involve the
production of renewable resources such as food, wood, fertile land, wildlife, and fresh water, regulating
services. These are those that are responsible for environmental changes (e.g. climate regulation and mitigate,
biodiversity conservation, combating desertification encroachment, pest/disease control) and cultural services
Iyagin & Adekunle (2017) 4(3): 496–513
representing human values and beliefs (e.g. landscape aesthetics, cultural heritage, outdoor recreation and
spiritual significance) (Behera et al. 2012, Cardinale et al. 2012, Daniel et al. 2012). According to Phillips et al.
(2003) and Royal Society (2003), biodiversity assessment was recognized by international policy process such
as the convention on biological diversity, as inevitable tool to guide conversation. The importance of
Biodiversity can be related to the phytosociology of a community (Bajpai et al. 2015).
As stated by Dengler (2017), Phytosociology is an aspect of science which study vegetation in terms of plant
assemblages, classification and characterization of the vegetation types based on the floristic composition in the
plant community. Phytosociological studies are essential to characterize and classify plant community (trees) in
term of their structure and composition for protecting the natural plant environment and proper management of
the forest resources. Understanding and appreciating the need for biodiversity conservation in Nigeria is of great
worth (Aju & Ezeibekwe 2010) as this could help in reducing the continuous external threat imposed on
protected areas and the restoration of loss that has taken place in the ecosystems. More than half of Nigeria’s
primary forest has been lost to deforestation through: urbanization, over exploitation of timber, subsistence
agriculture, and increase in the demand for fuel wood in the last decade (FAO 2006). Other factors against
biological diversity conservation in the tropics include explosive growth in human population, poverty, and
failure to implement the methods or approaches aimed at sustainable agriculture and forestry practices (Ekpo et
al. 2011).
Protected Areas (PAs) are potentially beneficial for carbon sink and environmental conservation. Adekunle
et al. (2014), defined Protected Areas as geographical space, recognised, dedicated and managed by means of
legal or other effective strategies to achieve the continuing conservation of nature with associated ecosystem
services and cultural values. Protected areas were categorised into: the Strict Nature Reserves (strict protection),
National Parks (Ecosystem Conservation and Protection), Natural Monuments (conservation of natural
features), Habitat/Species Management Area, Protected landscape/seascape (Landscape/Seascape conservation
and recreation) and Managed Resource Protected Area (Sustainable use of natural resources) (IUCN 2008).
Every PA, irrespective of the management strategy should have the following objectives: conservation of the
composition, structure, function and evolutionary potential of biodiversity; contributions to regional
conservation strategies; maintenance of diversity of landscape or habitat and of associated species and
ecosystems; long-term maintenance of the specified conservation targets; maintain the values for which it was
assigned in perpetuity; be operating under the guidance of a management plan and possess a clear and equitable
governance system (IUCN 2008, Adekunle et al. 2014). Also, it should be able to conserve significant
landscape features, geomorphology and geology; provide regulatory ecosystem services, including buffering
against the impacts of climate change and recreational benefits; provide for cultural, spiritual, educational
opportunities and scientific research purposes and deliver benefits to resident and local communities.” These
losses are unfavourable to the continued existence of animals, plants, human beings, and the general
environment because biodiversity conservation is recognized as a global life support system (Isichei 1995).
Tree canopy of the tropical rainforest ecosystem is made up of the upper layer (at about 30–40 m), the
second layer (between 23–30 m) and the lower layer which is made up of saplings of a number of species
(Bourgeron 1983). However, the high species diversity and the notable number of goods and services obtainable
from the rainforest ecosystem are partly responsible for the pressure to which it has been subjected for centuries;
and is presently on the increase (Onyekwelu et al. 2005) thereby increasing deforestation and forest degradation.
These, have been reported as the principal causes of forest cover change (Sedano et al. 2016) which is
responsible for the high percentage of the global carbon emission today (Van der werf et al. 2009).
Deforestation has been defined as the unchanging or long-term conversion of forest land use to other non-forest
uses thereby, causing the sudden and rapid change in land cover; consequently resulting in forest degradation,
contributing to the build-up of the carbon dioxide content in the atmosphere (GOFC-GOLD 2009, van der werf
et al. 2009). Other direct drivers of deforestation include agricultural activities, infrastructure development and
settlements (Halperin & Turner 2013). Forest degradation has caused lots of havoc among which are; colossal
reduction or total loss of forest land due to several human activities (Chadman 2008, Mackey et al. 2008, Simula
2009, FAO 2011) and reduction in forested landscape carbon stocks in relation to its natural carbon carrying
capacity (Mackey et al. 2008). This research therefore established the current status of selected protected areas
in the tropical rainforest ecosystem of southwest Nigeria that could enhance sustainable forest management
ecosystem restoration, thereby increasing the biodiversity efforts of all forest stakeholders.
Iyagin & Adekunle (2017) 4(3): 496–513

Study areas
This study was carried out at three PAs and an adjoining free forest area to one of the selected PAs (Fig. 1).
The PAs are a Natural Strict Reserve within Akure Forest Reserve, Oluwa Forest Reserve and a Permanent
Sample Plot (PSP 29). The free forest area is adjacent to the PSP 29.
Figure 1. Map of Ondo state showing the location of the forest reserves.
Location, climate and vegetation
Akure Forest Reserve (Aponmu) is one of the Strict Nature Forest Reserves in Nigeria located in the tropical
rainforest in Ondo State. It covers an area of about 32 hectares (Adeduntan & Olusola 2013) and lies between
latitude 06.59718° N and longitude 004.49199º E. In this protected area, the mean annual rainfall is about 1700
m and the temperature ranges from 20.6–33.5 ºC. Oluwa natural forest is within Oluwa Forest Reserve. This
forest lies between latitude 06.59718º N and longitude 004.49199º E and covers an area of 87, 816 ha
(Adeduntan 2009). The average rain fall of this reserve is 1700 mm, Relative Humidity of 80%, an annual
temperature of 26ºC with an average elevation of 100 m (Adekunle & Dafiwhare 2011). Akure/ Owena
Permanent Sample plot (PSP 29) is situated along Akure-Ondo road and about 1km away from Cocoa Research
Institute of Nigeria (CRIN) Owena substation. It covers an area of 65.93 km and falls within the high forest
Zone of Nigeria between longitude 005.02911º E and latitude 07.20109º N (Pelemo et al. 2011). In this reserve,
the relative humidity during the raining season ranges between 85 and 100% but less than 60% during the dry
season (Fasinmirin & Oguntuase 2008). It has a mean annual rainfall between (1300–1600 mm) and average
temperature of 27ºC. This area was demarcated and being managed by the Federal Research Institute of Nigeria
(FRIN).The Raining season in these locations starts from March and ends November with dry season starting
from December and ends in February. The soil in these PAs is typical of the soil found in the rainforest region
of the south western part of Nigeria. The soil texture is sandy-loamy but gradually becomes heavier in depth.
The ferrric luvisol soils are formed as a result of continuous weathering of the crystalline rock which feature
mostly in the typical upland soils in many parts of South-Western Nigeria (FAO 1988).
Data collection
I. Plot demarcation: The systematic line transects was employed in laying of plots for data collection, two
parallel transects of 200 m apart were laid in each of the study sites after a 50 meter distance has been taken
from the edge of each forest. Thereafter, four sample plots of equal size (25 m × 25 m) were laid alternately on
each transect.
II. Measurement of tree growth variables: All trees with Diameter at Breast Height (DBH) ≥ 10 cm
encountered in each sample plot were tagged identified and measured (Okali & Ola-Adams, 1987, Chavan &
Rasal 2010). The diameters at the base, middle and top and the total height of all the trees (DBH ≥ 10 cm) in
Iyagin & Adekunle (2017) 4(3): 496–513
each plot were measured.

III. Tree species identification: The scientific names of all the tree species encountered on each field plot were
recorded. Local names were used for tree species whose scientific names were not known immediately on the
field and their parts (such as leaves, barks and fruits) were collected and taken to the herbarium for
identification. Such species were temporarily referred to as unknown but were subsequently assigned their
scientific name immediately after identification. All the species were classified into families and their frequency
of occurrence were obtained to ascertain tree species diversity and abundance. The trees were also grouped into
diameter distribution classes based on the DBH measurement taken on the field.
Data analysis
I. Basal Area computation: The total basal area for each of the sample plot was obtained by summing up all
the Basal Area of the individual trees in the plots while the basal area per hectare was obtained by multiplying
the mean Basal Area per plot with 16 (the number of 25 m X 25 m plots in one hectare).
D 2
BA  ……………………………………………………....(1)
4
Where, BA = Basal area (m2), D = Diameter at breast height (cm) and  = 3.142 or 22/7
II. Volume Estimation: The volume of individual trees was estimated using the Newton formula (Husch et al.
2003). Volume per hectare was obtained by multiplying the mean volume per plot with the number of 25 m × 25
m plots in a hectare (16).
V 
h
24
D b
2 2 2
 4 Dm  Dt …………………………….…….(2)
Where, V = Volume of tree (m3), Db = Diameter at the base (cm), Dm = Diameter at the middle (cm), Dt =
Diameter at the top (cm), H = height (m)
III. Biodiversity Indices and Tree Species Classification:
a) Species relative density was computed following Brashears et al. (2004)
ni
RD   100 …………………………………..(3)
N
Where, RD (%) = species relative density; ni = number of individuals of species i; N = total number of all
tree species in the entire community
b) Species relative dominance (RDo (%)) was computed using Aidar et al. (2001) equation:
RDO 
 Ba  100 ……………...…………………………..(4)
i
 Ba n
Where: Bai = basal area of individual tree belonging to species i and Ba n = stand basal area.
c) The maximum diversity index was determined using the Shannon–Wiener diversity index (Kent & Coker
1992, Guo et al. 2003). This was because it takes into account the richness and abundance of each species in
different ecosystems (Price 1997).
S
H '    p i ln( p i ) ……………………………………………(5)
i 1
Where, H’ = Shannon diversity index, S = the total number of species in the community, p i = proportion S
(species in the family) made up of the ith species and Ln = natural logarithm.
d) To determine the Species evenness (E), in each community, Shannon's equitability equation was adopted
(Kent & Coker 1992):
S
H'
 P ln( P )
i 1
i i
EH   ………….………………………(6)
H Max ln( S )
e) Importance Value Index (IVI): The Importance Value Index for each species were obtained by summing
up the RD and RDo divided by 2 (RD x RDo/2) (Brashears et al. 2004). This was used to express the share of
Iyagin & Adekunle (2017) 4(3): 496–513
each species in the tree community.

f) Family Importance Value (FIV): This is the sum of the relative dominance (RDm), relative density (RD)
and relative frequency (RF).
RDm = (Total Basal area for a family ÷ Total Basal area of all families) X 100
RD = (Number of individual ‘a’ of family ‚ Total number of all individual) X 100
RF = (Frequency ‘a’ of family ‚ Sum frequencies all of s families) X 100
Therefore, the FIV is calculated as: RDm+ RD+ RF ………….(7)
Number 1 of Hill diversity index
N1=exp(-Σpi(lnpi))…………………………...….……………….(8)
pi = ni / N …………………………………..…………………………….(9)
Where: pi: is the proportional abundance of ith species, ni: number of individuals of ith species, N: total
number of individuals.
Number 2 of Hill diversity index
N2: Reciprocal of Simpson’s dominance Index;
N2= 1/Σ pi2 ……………………………………………….….... (10)
g) Shannon max diversity index was also determined using
Hmax= LnS……………………………………………………..(11)
Where, S = the total number of species in the community
h) Sorensen’s species similarity index was used to compare species diversity among the selected sites:
2C
SI= X 100………………….…..……………....(12)
abcd
Where, C is the total number of species in four communities (i.e. aggregate of all species encountered in the
entire study area); while a, b, c, & d are the number of species at communities 1, 2, 3 & 4 respectively.
RESULTS
Tree species diversity and abundance
This study revealed that Oluwa Forest Reserve has the highest number of species ha-1 (170 stems.ha-1)
distributed into 23 and 54 families and species respectively. Monodora myristica (Gaertn.) Dunal, of
Annonaceae family has the highest number of stem per hectare (19 spp.ha -1) with a relative density of 11.18.
This is followed by Buchholzia coriacea Engl.of Capparaceae family with 13 spp.ha-1 and relative density of
7.65 and Diospryos dendo Welw. ex Hiern (11 stems.ha-1) with relative density of 6.47. In Permanent Sample
Plot 29, Celtis zenkeri Engl. of Ulmaceae family has the highest number of stems (15 stems.ha -1) and next in
abundance is Sterculia rhinopetala K. Schum, of the family Sterculiaceae, and Funtumia elastica (Preuss)
Stapf of Apocynaceae family (14 spp.ha-1 and 12 spp.ha-1 respectively) in the Strict Nature Reserve of Akure
forest reserve. The tree species (Celtis zenkeri Engl., Cola gigantean A.Chev. and Funtumia elastica (Preuss)
Stapf) are common to all the four forest sites assessed.
The tree species diversity is represented in table 1. The Strict Nature Reserve has a total of 88 stems.ha -1,
distributed among 25 species and 15 different families. Sterculia rhinopetala K. Schum, has the highest number
of stems (14 stems.ha-1). This is followed closely by Funtumia elastica (Preuss) Stapf (12 stems.ha-1). A total of
99 stems.ha-1 which spans through 27 different species from 14 families were obtained at the PSP 29. In this
location, Celtis zenkeri Engl. has the highest occurrence (15 stems.ha-1) and Relative density of 15.15. The free
forest area adjoining PSP 29 has a total of 54 stems.ha -1. The most abundant species in the free area is Celtis
zenkeri Engl. (11 stems.ha-1) with relative density of 20.37. The total number of families and species that were
encountered in the site is 12 and 23 respectively. From these results, about 39% of all the species were
represented by single individual per ha.
The Families Importance Value for the selected forests is presented in table 2. The results revealed that 30
different trees families were encountered in the four forest types. Sterculiaceae family has the highest Family
Importance Value (FIV) of 39.10%. This was followed by Ulmaceae (24.93 %) while Ochnaceae family has the
least Family Importance Value of 0.49%. The highest volume per hectare was also recorded for Papilionoideae
family (3.14 m3), followed by Myristicaceae family (2.92 m3) and the least by Guttiferae family (0.10 m3). The
highest RD and RDo were recorded for Sterculiaceae (19.46%) and Bignoniaceae (0.13%).
Iyagin & Adekunle (2017) 4(3): 496–513
Table 1. Tree Species abundance per ha, diversity indices and tree growth variables of the selected forest reserves.
MDBH Ht BA VOL RDO
Sites S.N. Family Species nha-1 PiLnPi RD IVI
(cm) (m) (m2) (m3) (%)
SNR 1 Annonaceae Monodora myristica (Gaertn.) Dunal 3 22.03 22.43 0.05 0.34 -0.12 3.41 1.35 2.30
2 Apocynaceae Alstonia boonei De Wild. 1 16.40 15.00 0.02 0.20 -0.05 1.14 0.61 0.34
3 Apocynaceae Funtumia elastica (Preuss) Stapf 12 22.38 20.90 0.07 0.43 -0.27 13.64 1.93 13.15
4 Burseraceae Canarium schweinfurthii Engl. 5 59.24 33.62 0.31 2.71 -0.16 5.68 8.90 25.29
5 Ebenaceae Diospyros mespiliformis Hochst. 1 22.00 15.30 0.04 0.29 -0.05 1.14 1.09 0.62
6 Euphorbiaceae Macaranga hurifolia Beille 1 17.60 29.00 0.02 0.22 -0.05 1.14 0.70 0.40
7 Lecythidaceae Petersianthus macrocarpum (P. Beauv) 3 55.87 31.53 0.25 1.93 -0.12 3.41 7.26 12.37
8 Meliaceae Cedrela odorata Linn. 3 91.33 41.40 0.68 4.92 -0.12 3.41 19.39 33.05
9 Meliaceae Trichilia heudelottii Planch. ex. Oliv. 5 27.66 33.16 0.06 0.90 -0.16 5.68 1.86 5.28
10 Meliaceae Trichilia prieuriana A. Juss 3 25.60 32.37 0.07 0.64 -0.12 3.41 1.95 3.32
11 Moraceae Trilepisium madagascariense Dc. Fl. Cam. 5 25.24 24.48 0.06 0.50 -0.16 5.68 1.84 5.22
12 Olacaceae Strobosia pustulata Oliv. 3 14.10 17.97 0.02 0.10 -0.12 3.41 0.46 0.78
13 Rutaceae Fagara leprieurii Engl. 1 28.00 23.70 0.06 0.35 -0.05 1.14 1.77 1.00
14 Rutaceae Fagara macrophylla (Oliv.) Engl 1 35.80 26.10 0.10 0.66 -0.05 1.14 2.89 1.64
15 Sapotaceae Chrysopyllum albidum G Don. 1 33.00 35.30 0.09 1.07 -0.05 1.14 2.46 1.40
16 Sapotaceae Chrysopyllum perpulchrum Mildbr. ex 2 27.90 29.25 0.06 0.72 -0.09 2.27 1.81 2.06
Hutch. & Dalziel
17 Sterculiaceae Cola gigantean A.Chev. 4 41.10 32.63 0.22 2.62 -0.14 4.55 6.24 14.17
18 Sterculiaceae Mansonia altissima A. Chev. 10 36.57 30.00 0.13 1.02 -0.25 11.36 3.60 20.47
19 Sterculiaceae Pterygota macrocarpa K Schum. 4 31.38 32.15 0.10 1.12 -0.14 4.55 2.99 6.79
20 Sterculiaceae Sterculia rhinopetala K. Schum. 14 30.21 27.53 0.09 0.84 -0.29 15.91 2.65 21.07
21 Sterculiaceae Triplochiton scleroxylon K. Schum. 2 98.50 37.00 0.77 5.59 -0.09 2.27 22.13 25.15
22 Surmardaceae Pierradendron africanum Hook . f 1 43.10 58.00 0.15 2.06 -0.05 1.14 4.19 2.38
23 Ulmaceae Celtis zenkeri Engl. 1 15.40 14.00 0.02 0.14 -0.05 1.14 0.53 0.30
24 Verbaneaceae Gmelina arborea Roxb. 2 23.20 36.20 0.05 0.66 -0.09 2.27 1.41 1.60
Total 88 3.48 30.02 -2.82
PSP 29 1 Annonaceae Anogeissus leiocarpa (DC) Guill&Perr. 1 14.00 18.50 0.02 0.14 -0.05 1.01 0.54 0.27
2 Annonaceae Cleistopholis patens (Benth.) Engl. & Diels 1 8.50 9.50 0.01 0.03 -0.05 1.01 0.20 0.10
Iyagin & Adekunle (2017) 4(3): 496–513
5 Apocynaceae Tabernaemontana pachysiphon Stapf 1 96.00 26.50 0.72 7.44 -0.05 1.01 25.33 12.79
6 Boraginaceae Cordia millenii Bak 2 57.75 17.65 0.39 1.74 -0.08 2.02 13.51 13.64
7 Bombacaceae Bombax buonopozense P Beauv 2 23.10 14.85 0.06 0.30 -0.08 2.02 1.97 1.99
8 Caesalpinioideae Afzelia Africana Sm. 2 24.40 17.15 0.05 0.46 -0.08 2.02 1.72 1.74
9 Caesalpinioideae Anthonotha macrophylla P. Beauv. 8 13.80 10.66 0.02 0.11 -0.20 8.08 0.53 2.14
10 Caesalpinioideae Brachystegia nigerica Hoyle &A.P.D. Jones 2 75.63 27.00 0.48 4.92 -0.08 2.02 16.86 17.03
11 Combretaceae Terminalia ivorensis Chev. 5 48.56 18.96 0.27 2.23 -0.15 5.05 9.30 23.48
12 Ebenaceae Diospryos dendo Welw. ex Hiern 2 19.95 10.10 0.04 0.20 -0.08 2.02 1.45 1.47
13 Moraceae Myrianthus arboreus P. Beauv 4 23.48 12.88 0.06 0.45 -0.13 4.04 2.13 4.31
14 Moraceae Trilepisium madagascariense DC. 2 18.35 11.55 0.03 0.18 -0.08 2.02 1.04 1.05
15 Moreceae Antiaris toxicaria Lesch. subsp. 1 15.90 8.00 0.02 0.07 -0.05 1.01 0.69 0.35
17 Papilionaceae Pterocarpus osun Craib 1 52.00 27.20 0.21 2.28 -0.05 1.01 7.43 3.75
18 Sapindataeae Lecaniodiscus cupanioides Planch. 1 15.70 14.00 0.02 0.17 -0.05 1.01 0.68 0.34
19 Sapotaceae Malacantha alnifolia (Baker) Pierre. 1 13.65 15.50 0.01 0.13 -0.05 1.01 0.51 0.26
20 Sterculiaceae Cola acuminata (P.Beauv.) Schott & Endl. 2 14.45 12.90 0.02 0.09 -0.08 2.02 0.57 0.58
22 Sterculiaceae Mansonia altisima A. Chev. 5 19.16 11.74 0.03 0.20 -0.15 5.05 1.14 2.87
23 Sterculiaceae Pterygota macrocarpa K Schum. 2 40.40 17.95 0.20 2.08 -0.08 2.02 6.89 6.96
24 Sterculiaceae Sterculia tragacantha K Schum. 6 16.08 14.58 0.02 0.17 -0.17 6.06 0.86 2.60
25 Ulmaceae Celtis philippensis Bl. var. wightii 9 18.49 13.82 0.03 0.16 -0.22 9.09 1.07 4.84
(Planch.) Soepadmo
26 Ulmaceae Celtis zenkeri Engl 15 15.51 11.33 0.02 0.14 -0.29 15.15 0.73 5.53
27 Ulmaceae Holoptelea grandis Hutch .Mildbr 2 15.50 16.50 0.02 0.17 -0.08 2.02 0.76 0.77
Total 99 2.86 24.56 -2.96
FREE 1 Apocynaceae Funtumia elastica (Preuss) Stapf 1 16.50 13.35 0.02 0.17 -0.07 1.85 1.81 1.68
FOREST 2 Apocynaceae Rauvolfia vomitoria Afzel. 1 10.50 11.25 0.01 0.04 -0.07 1.85 0.73 0.68
AREA
3 Boraginaceae Cordia millenii Bak. 3 20.83 9.77 0.03 0.16 -0.16 5.56 2.91 8.10
4 Bignoniaceae Newbouldia laevis (P. beauv) 2 13.45 12.58 0.02 0.06 -0.12 3.70 1.28 2.37
5 Caesalpiniaceae Brachystegia eurycoma Harms 1 18.80 13.30 0.03 0.16 -0.07 1.85 2.35 2.18
6 Euphorbiaceae Antidesma sp. 1 15.50 12.25 0.02 0.13 -0.07 1.85 1.60 1.48
Iyagin & Adekunle (2017) 4(3): 496–513
7 Euphorbiaceae Bridelia micrantha (Hochst.) Baill. 5 20.26 10.99 0.04 0.18 -0.22 9.26 3.04 14.10
8 Euphorbiaceae Macaranga barteri Müll.-Arg. 1 9.40 13.40 0.01 0.04 -0.07 1.85 0.59 0.54
9 Meliaceae Trichilia heldelotii Planch. ex. Oliv. 2 16.75 11.38 0.02 0.13 -0.12 3.70 1.90 3.52
10 Mimoceae Albizia adianthifolia (Schumach.) Wightii 1 12.00 13.45 0.01 0.09 -0.07 1.85 0.96 0.89
11 Mimosaceae Albizia zygia (DC.) J. F. Macbr. 4 14.00 12.49 0.02 0.09 -0.19 7.41 1.35 5.00
12 Moraceae Ficus exasperate Vahl 1 12.70 12.55 0.01 0.08 -0.07 1.85 1.07 0.99
13 Moreaceae Milicia excels (Welw.) C. Berg 2 54.00 10.08 0.31 0.81 -0.12 3.70 26.68 49.40
14 Rutaceae Zanthoxylum leprieurii Guill. & Perr 1 13.40 11.60 0.01 0.08 -0.07 1.85 1.20 1.11
15 Sapindaceae Blighia sapida K Konig 1 38.50 14.90 0.12 0.50 -0.07 1.85 9.87 9.14
17 Sterculiaceae Sterculia rhinopetela K. Schum. 1 28.90 14.20 0.07 0.29 -0.07 1.85 5.56 5.15
19 Sterculiaceae Theobroma cacao L. 1 12.00 5.90 0.01 0.04 -0.07 1.85 0.96 0.89
20 Sterculiaceae Triplochiton scleroxylon K Schum. 5 35.78 11.87 0.12 0.63 -0.22 9.26 10.55 48.84
21 Ulmaceae Celtis philippensis Bl. var. wightii 5 14.30 10.00 0.02 0.08 -0.22 9.26 1.37 6.36
23 Verbenaceae Gmelina arborea Roxb 2 47.25 13.75 0.18 0.69 -0.12 3.70 15.09 27.95
Total 54 1.18 4.95 -2.79
OLUWA 1 Anacardiaceae Lannea welwitschii (Hiern) Engl. 2 19.00 13.05 0.03 0.31 -0.05 1.18 0.81 0.47
2 Annonaceae Cleistopholis patens (Benth.) Engl. & Diels 1 17.20 11.50 0.02 0.25 -0.03 0.59 0.64 0.19
3 Annonaceae Enantia chlorantha Oliv. 1 22.50 9.50 0.04 0.27 -0.03 0.59 1.10 0.32
4 Annonaceae Monodora myristica (Gaertn.) Dunal 19 21.50 15.10 0.05 0.61 -0.24 11.18 1.25 6.98
7 Apocynaceae Hunteria umbellate K. Schum 4 13.68 12.54 0.02 0.18 -0.09 2.35 0.43 0.50
8 Apocynaceae Picralima nitida (Stapf) T. Durand & H. 10 19.62 9.99 0.03 0.22 -0.17 5.88 0.88 2.59
Durand
9 Apocynaceae Rauvolfia vomitoria Afzel. 1 16.40 11.40 0.02 0.20 -0.03 0.59 0.58 0.17
10 Boraginaceae Cordia millenii Bak. 1 44.30 16.00 0.15 1.35 -0.03 0.59 4.26 1.25
11 Bignoniaceae Newbouldia laevis (P. beauv) 1 19.70 10.00 0.03 0.17 -0.03 0.59 0.84 0.25
12 Burseraceae. Canarium schweinfurthii Engl. 2 73.30 26.10 0.42 4.51 -0.05 1.18 11.66 6.86
13 Caesalpinioideae Anthonatha macrophylla P. Beauv. 3 12.03 10.90 0.01 0.09 -0.07 1.76 0.32 0.28
Iyagin & Adekunle (2017) 4(3): 496–513
14 Capparaceae Buchholzia coriacea Engl. 13 29.28 16.90 0.12 2.60 -0.20 7.65 3.23 12.34
15 Clusiaceae Allanblackia floribunda Oliv. 1 12.70 12.90 0.01 0.14 -0.03 0.59 0.35 0.10
16 Clusiaceae Garcinia afzeli Engl. 2 19.75 10.25 0.03 0.22 -0.05 1.18 0.87 0.51
17 Ebenaceae Diospyros barteri Hiern. 6 18.72 17.78 0.03 0.38 -0.12 3.53 0.84 1.48
18 Ebenaceae Diospyros dendo Welw. ex Hiern 11 18.65 10.30 0.04 0.33 -0.18 6.47 1.17 3.78
19 Ebenaceae Diospyros mespiliformis Hochst. ex A.DC. 6 21.10 11.24 0.04 0.44 -0.12 3.53 1.14 2.01
20 Euphorbiaceae Antidesma sp. 2 11.40 10.95 0.01 0.08 -0.05 1.18 0.28 0.17
21 Euphorbiaceae Bridelia grandis Pierre ex. Hutch 1 50.00 34.40 0.20 4.51 -0.03 0.59 5.43 1.60
22 Euphorbiaceae Croton sp. 2 15.65 15.65 0.02 0.35 -0.05 1.18 0.54 0.32
23 Euphorbiaceae Ricinodendron heudelotii (Ball.) Pierre 3 46.20 21.98 0.28 3.88 -0.07 1.76 7.66 6.76
24 Lecythidaceae Napoleonaea vogelii Hook. & Planch. 1 22.70 8.90 0.04 0.14 -0.03 0.59 1.12 0.33
25 Meliaceae Trichilia emetica Vahl 1 13.50 14.22 0.01 0.74 -0.03 0.59 0.40 0.12
26 Meliaceae Trichilia heudelottii Planch. ex. Oliv. 9 23.94 13.09 0.06 0.71 -0.16 5.29 1.59 4.21
27 Meliaceae Trichilia prieuriana A. Juss 3 19.17 19.73 0.03 0.36 -0.07 1.76 0.86 0.76
28 Mimosoideae Albizia zygia (DC.) J. F. Macbr. 1 14.70 15.90 0.02 0.27 -0.03 0.59 0.47 0.14
29 Mimosoideae Pentaclethra macrophylla Benth. 1 11.11 9.60 0.01 0.05 -0.03 0.59 0.27 0.08
30 Moraceae Antiaris toxicaria Lesch. subsp. 2 44.30 20.00 0.16 1.06 -0.05 1.18 4.30 2.53
welwitschii (Engl.) C.C Berg
31 Moraceae Ficus exasperate Vahl 2 23.50 17.45 0.05 0.46 -0.05 1.18 1.29 0.76
32 Moraceae Ficus sur Thunb. 1 17.50 17.90 0.02 0.32 -0.03 0.59 0.66 0.20
33 Moraceae Milicia excels (Welw.) C. Berg 1 10.70 18.80 0.01 0.08 -0.03 0.59 0.25 0.07
34 Moraceae Myrianthus arboreus P. Beauv. 5 20.16 12.13 0.04 0.53 -0.10 2.94 1.17 1.72
35 Moraceae Trilepisium madagascariense Dc. Fl. Cam. 1 37.30 8.00 0.11 0.39 -0.03 0.59 3.02 0.89
36 Myristicaceae Pycnanthus angolensis (Welw.) Warb. 1 58.20 26.90 0.27 5.42 -0.03 0.59 7.35 2.16
37 Myristicaceae Staudtia stipitata Warb. 1 24.60 14.40 0.05 0.42 -0.03 0.59 1.31 0.39
38 Ochnaceae Barteria fistulosa Mast. 1 16.70 24.50 0.02 0.30 -0.03 0.59 0.61 0.18
40 Papilionoideae Amphimas pterocarpoides Harms 1 23.90 21.10 0.04 0.68 -0.03 0.59 1.24 0.36
41 Papilionoideae Baphia pubescens Hook. f. 1 27.80 12.30 0.06 0.42 -0.03 0.59 1.68 0.49
42 Papilionoideae Dalbergia sp. 1 35.80 32.00 0.10 1.78 -0.03 0.59 2.78 0.82
43 Papilionoideae Pterygota macrocarpa K Schum. 2 16.75 29.70 0.02 0.72 -0.05 1.18 0.66 0.39
44 Rubiaceae Massularia acuminate 1 12.20 7.20 0.01 0.04 -0.03 0.59 0.32 0.10
Iyagin & Adekunle (2017) 4(3): 496–513
45 Rubiaceae Pausinystalia talbotii Wernham. 2 29.35 15.65 0.07 0.80 -0.05 1.18 1.92 1.13
46 Sapindaceae Lecaniodis cuscupanioides Planch. 1 26.40 23.50 0.05 1.17 -0.03 0.59 1.51 0.44
47 Sapotaceae Chrysophyllum albidum G Don. 1 47.50 23.70 0.18 2.20 -0.03 0.59 4.90 1.44
48 Sapotaceae Malacantha alnifolia (Baker) Pierre. 3 14.53 12.03 0.02 0.19 -0.07 1.76 0.47 0.41
49 Sterculiaceae Sterculiar hinopetala K. Schum. 6 23.58 14.27 0.05 0.49 -0.12 3.53 1.34 2.37
50 Sterculiaceae Cola gigantea A.Chev. 1 17.90 13.00 0.03 0.23 -0.03 0.59 0.70 0.20
51 Sterculiaceae Cola heterophylla (P.Beauv.) Schott & Endl 5 18.84 15.05 0.03 0.43 -0.10 2.94 0.93 1.37
52 Sterculiaceae Cola nigerica Brenan & Keay 2 13.80 8.65 0.01 0.06 -0.05 1.18 0.41 0.24
Total 170 - - 3.62 46.95 -3.55 - - -
Note: nha-1- number of stem per hectare, MDBH - Mean Diametre at breast height (cm), M Ht -Mean height (m), BA - Basal area per hectare (m2), VOL - Volume per hectare (m3), RD -
Spp. Relative density, RDO - Spp. Relative dominance.
Iyagin & Adekunle (2017) 4(3): 496–513
Table 2. Families’ importance index for the study areas.
S.N. Families BA/ha VOL/ha RF RD (%) RDO (%) FIV%
1 Anacardiaceae 0.03 0.31 0.49 0.49 0.01 0.98
2 Annonaceae 0.10 1.01 6.33 6.33 0.03 12.71
3 Apocynaceae 0.21 1.68 11.92 11.92 0.05 23.95
4 Bignoniaceae 0.50 2.62 2.19 2.19 0.13 4.52
5 Bombacaceae 0.06 0.30 0.49 0.49 0.01 0.99
6 Burseraceae 0.31 2.71 1.22 1.22 0.08 2.52
7 Caesalpinioideae 0.14 1.22 3.89 3.89 0.03 7.84
8 Capparaceae 0.12 2.60 3.16 3.16 0.03 6.37
9 Clusiaceae 0.03 0.24 0.49 0.49 0.01 0.98
10 Combretaceae 0.27 2.23 1.22 1.22 0.07 2.51
11 Ebenaceae 0.12 0.86 6.33 6.33 0.03 12.71
12 Euphorbiaceae 0.19 2.50 3.89 3.89 0.05 7.85
13 Guttiferae 0.02 0.10 0.24 0.24 0.01 0.49
14 Lecythidaceae 0.29 2.07 0.97 0.97 0.07 2.02
15 Malvaceae 0.03 0.18 0.73 0.73 0.01 1.47
16 Meliaceae 0.30 2.68 6.33 6.33 0.08 12.76
17 Mimosoideae 0.03 0.25 1.70 1.70 0.01 3.42
18 Moraceae 0.39 1.92 6.57 6.57 0.10 13.27
19 Myristicaceae 0.16 2.92 0.49 0.49 0.04 1.01
20 Ochnaceae 0.02 0.30 0.24 0.24 0.01 0.49
21 Olacaceae 0.20 2.38 1.95 1.95 0.05 3.95
22 Papilionoideae 0.26 3.14 1.46 1.46 0.07 2.99
23 Rubiaceae 0.05 0.55 0.73 0.73 0.01 1.48
24 Rutaceae 0.10 0.59 0.73 0.73 0.02 1.49
25 Sapindaceae 0.19 1.84 0.73 0.73 0.05 1.51
26 Sapotaceae 0.13 1.45 1.95 1.95 0.03 3.94
27 Sterculiaceae 0.32 2.55 19.46 19.46 0.08 39.10
28 Surmardaceae 0.15 2.06 0.24 0.24 0.04 0.52
29 Ulmaceae 0.21 1.62 12.41 12.41 0.05 24.93
30 Verbenaceae 0.23 1.34 0.97 0.97 0.06 2.01
Total 5.13 46.23
Biodiversity Indices
The summary of the abundance, level of diversity and evenness of all the tree species encountered with the
various biodiversity indices is presented in table 3. These indices were used to compare tree species diversity of
the selected forest communities. Oluwa Forest Reserve has the highest Shannon-Weiner index (3.55), indicating
that it has the highest species richness when compared with the other locations. The Pielou’s evenness
(equitability index) and Margalef's index value of 0.69 and 10.12 respectively were also obtained for this forest
reserve and 34.74 and 11.11 were obtained respectively for number 1 and number 2 of Hill’s diversity indices.
The free forest area adjoining PSP 29 has the lowest Shannon-wiener diversity index (2.79), Margalef's index
value of 5.51 but, the highest Pielou's evenness index value of 0.70.
Table 3. Summary of the tree species diversity indices in the selected areas.
Locations
Variables Oluwa All
SNR PSP (29) Free Area
FR locations
No of trees/ ha 88 99 54 170 411
No of Families (NF) 15 14 12 23 30
No of Species (NS) 24 27 23 53 78
Shannon- Wieners (H1) 2.82 2.96 2.79 3.55 5.11
Pielou’s spp evenness index (E ) 0.63 0.64 0.70 0.69 0.69
Simpson’s concentration (λ) 0.07 0.07 0.18 0.09 0.03
N1 of hill diversity (N1) 16.81 19.29 16.23 34.74 35.40
N2 of hill diversity (N2) 14.28 14.28 5.55 11.11 33.33
Margalef’s index of Spp. richness (M) 5.13 5.65 5.51 10.12 12.79
Tree growth variables in the selected forest reserves
The summary of the tree growth variables according to the location is shown in table 4. Oluwa forest
Iyagin & Adekunle (2017) 4(3): 496–513
Table 4. Tree growth variables for the selected area.
Oluwa
Variables SNR PSP 29 Free Area All locations
FR
2
Basal area per ha (m ) 12.31 6.73 3.40 10.54 48.48
Volume per ha (m3) 104.76 53.44 14.56 141.06 123.10
Mean DBH (cm) 35.15 27.15 20.48 24.57 25.06
Dominant DBH (cm) 98.50 96.00 54.00 73.30 98.50
Dominant height (m) 58.00 27.20 14.90 34.40 58.00
Mean height (m) 28.1 14.92 12.09 15.87 17.09
reserve has the highest tree volume per hectare of 141.06 m3 while the free Forest has the lowest tree volume per
hectare (14.56 m3). The highest dominant DBH is 98.50 cm in the SNR, followed by PSP 29 (96.00 cm) while
the lowest dominant DBH of 54 cm was obtained in the free forest area. The highest mean DBH value of 35.15
cm was also recorded in the SNR. The basal area/ha ranged between 3.40 m2 and 12.31 m2, with the least
obtained at the disturbed free area and the highest at the SNR. Similarly, the dominant height and Mean height
followed the same trend. The dominant height value ranged between 14.90 m and 58.00 m while the value for
mean height ranged from 12.09 to 28.1 respectively.
Diameter and height Distribution
Table 5. Species diversity, abundance, basal area and volume distribution of the study area into diameter classes.
Location DBH Class (cm) NS NF Ni Ba.h-1 (m2) Vol.h-1 (m3)
SNR 0–20 17 11 34 0.61 5.47
21–40 15 10 27 1.81 17.68
41–60 7 5 13 2.45 21.06
61–80 5 4 7 2.47 21.79
81–100 5 4 5 2.92 24.84
>100 2 2 2 1.95 13.92
Total 24 15 88 12.21 104.76
Oluwa Forest Reserve 0–20 38 21 94 1.53 14.43
21–40 29 17 60 3.80 43.80
41–60 9 9 10 1.92 28.38
61–80 3 3 3 1.01 11.09
81–100 2 2 2 1.29 15.27
>100 1 1 1 0.99 28.09
Total 50 23 170 10.54 141.06
PSP 29 0–20 24 13 67 1.00 6.05
21–40 13 8 22 1.28 8.21
41–60 3 3 3 0.57 5.23
61–80 3 3 3 1.01 8.71
81–100 4 4 4 2.87 25.24
Total 27 14 99 6.73 53.44
Free Forest Area 0–20 19 10 40 0.69 3.93
21–40 7 6 7 0.45 1.96
41–60 4 3 5 1.03 5.00
61–80 2 2 2 1.23 3.67
Total 23 12 54 3.40 14.56
Note: NF - Number of Family, NS - Number of species, Ni - Number of individual species.
The diameter distribution of the study areas is presented in tables 5. This shows that 49.4% of trees falls
within the lowest diameter class (10–20 cm) while only 7.14% falls in the highest diameter class (>80cm). The
highest Volume per hectare (141.06 m3) was recorded in Oluwa forest while the lowest (14.56 m3) was from the
free Forest. The same trend was noted in the distribution of the individual species and families into diameter
classes. Figure 2 was used to describe the tree species population structure based on DBH (cm) classes with
frequency and volume per hectare (m3). As one of the peculiar features of a mature natural forest ecosystem, the
DBH distribution curve followed inverse J-shaped pattern (Fig. 3). Table 6 revealed that 49.4% of the tree
species was in the height class of between 11–20 m in Oluwa Forest reserve. Only the SNR and Oluwa forest
reserve have trees with height above 30 m (18.7% and 7.02% respectively). These are trees that can be regarded
as emergent. Significant differences (P- value < 0.05) exist in all the tree growth variables of the study sites
when compared with one-way ANOVA (Table 7). Table 8 revealed the results of the mean separation of tree
Iyagin & Adekunle (2017) 4(3): 496–513
growth variables of the forests with Duncan Multiple Range Test (DMRT). The Mean values in the same row
followed by the same letter(s) are not significantly different at 0.05 level of significance.
Figure 2. Tree species population structure of the study areas based on DBH (cm) classes with frequency and volume (m 3)
(A, Oluwa Forest Reserve; B, SNR Aponmu; C, PSP 29; D, Free Forest).
Figure 3. Diameter distribution curve for the selected Protected Areas and the free area.
Table 6. Species diversity, basal area and volume distribution of the study area into Height classes.
Height (m) NS NF Ni Ba.h-1 (cm2) Vol.h-1 (cm3)
SNR <10 1 1 1 0.03 0.06
11–20 14 10 24 1.08 7.21
21–30 13 9 28 1.9 14.65
31–40 12 6 20 4.62 38.54
41–50 12 6 15 4.59 44.3
Total 88 12.22 104.76
Oluwa <10 23 19 47 1.17 5.34
11–20 39 20 92 4.01 41.27
21–30 16 15 21 3.37 45.83
31–40 7 7 8 0.96 19.2
41–50 2 1 2 1.02 29.41
Total 170 10.53 141.05
Iyagin & Adekunle (2017) 4(3): 496–513
PSP 29 <10 18 10 32 0.4 1.87

11–20 18 12 52 2.16 12.36
21–30 12 8 15 4.18 39.22
Total 99 6.74 53.45
Free <10 8 5 13 1.51 4.63
Forest 11–20 22 11 41 1.9 9.95
area Total 54 3.41 14.58
Table 7. ANOVA table for comparing differences in tree growth variables among the study sites.
Source of
SS Df MS F Sig
variation
Vol. Location 2418.06 3 806.02 5.70 0.01
Error 1695.62 12 141.30
Total 4113.69 15
BA Location 11.99 3 4.00 3.76 0.04
Error 12.76 12 1.06
Total 24.75 15
DBH Location 1090061.90 3 363353.97 9.14 0.00
Error 476814.23 12 39734.52
Total 1566876.14 15
Ht Location 679483.31 3 226494.44 22.75 0.00
Error 119471.91 12 9955.99
Total 798955.22 15
Nha Location 1782.69 3 594.23 17.45 0.00
Error 408.75 12 34.06
Total 2191.44 15
Table 8. Comparison of growth variables in the forests with Duncan multiple range test (DMRT).
Properties SNR Oluwa Free Forest PSP 29
Volume 26.19 ± 11.62ab 35.96 ± 20.02a 3.64 ± 0.83c 13.36 ± 5.34bc
Basal Area 3.06 ± 1.31a 2.68 +±1.34a 0.85 ± 0.17b 1.68 ± 0.81ab
DBH 759.97 ± 227.62ab 1012.22 ± 295.55a 303.37 ± 51.98c 553.67 ± 130.64bc
Height 625.56 ± 145.23a 649.13 ± 121.70a 155.25 ± 51.95c 336.90 ± 34.93b
N/ha 22.0 ± 3.91b 42.50 ± 8.38a 13.50 ± 4.79c 24.75 ± 5.25bc
DISCUSSION
There are two different conservation methods of natural resources in Nigeria; the in-situ and ex situ.
Different types of PAs aimed at conserving the natural resources in their natural habitat, such as those used in
this study, are examples of an in-situ conservation method. The tropical rainforest ecosystem of southwest
Nigeria is noted for its high diversity in terms of species, genetic materials and ecological processes in
comparison to other ecosystems (Adekunle et al. 2013). This assertion is supported by the results of this present
study where 78 species in 30 families were encountered selected PAs. When these different forest communities
were compared, the undisturbed PAs (PSP 29; SNR, and Oluwa Forest Reserve) were more diverse than the free
forest area that has witnessed human activities. The low number of stems.ha -1 recorded in the free area,
compared to the PAs, is an indication of an ecosystem that has been disturbed and greatly degraded. The
number of species encountered in the sample survey plots can be used as a substitute for the exact species
richness in the study area. Though, varying degree of degradation through illegal logging, forest conversion to
Agricultural uses among others has resulted to unsustainable management of the lowland humid forest. The
tropical forests are very vulnerabile to deforestation and degradation (FAO 2001, FAO 2006, Onyekwelu et al.
2005, Lafrankie et al. 2006). As a result, there is reduction in tree species richness, abundance and evenness of
the ecosystem (Lafrankie et al. 2006) which is not creditable to biodiversity conservation. The tree species
distribution into families agrees with the work of Adekunle et al. (2010) who indicated that the tropical
rainforest ecosystem of southwest Nigeria is dominated by some specific families such as the Sterculiaceae,
Meliaceae, Moraceae and Ebeneceae. Isichei (1995) also reported that Nigerian rainforest ecosystem is
dominated by members of Sterculiaceae, Moraceae, Ulmaceae and Meliaceae families. The dominant families
represented in this study sites differ from the families (Euphorbiaceae, Mimosoideae, Rubiaceae and Guttiferae)
reported by Ifo et al. (2016) in their study of a similar ecosystem.
Iyagin & Adekunle (2017) 4(3): 496–513
This study revealed a decrease in species richness and diversity from the undisturbed forests to the disturbed
forest. The number of trees per hectare (170, 99, 88 and 54 for Oluwa forest, PSP 29, SNR, and the free forest
area, respectively) are similar to the number of tree per hectare recorded for the tropical natural forest by
Parthasarathy (2001) but was far below the values obtained for Kalapahad (579 ha-1) and Macarthy Valley (732
ha-1) in the giant evergreen forest of Andaman and Nicobar Islands as reported by Rajkumar & Parthasarathy
(2008). The highest number of tree species (50 species from 23 families) was recorded in Oluwa Forest
(undisturbed), indicating richness in species while the lowest number of species (23 species from 12 families)
was recorded in the free forest site (disturbed site). This reveals a lower level of species richness (Table 5).
Simillar trend as this was reported by Borah et al. (2014). This is due to more frequent and heavy extraction of
forest resources such as collection of fuel wood and NTFPs by the Local people. The number of family (30),
number of Species (78) and Species Evenness (0.69) in this study corresponds to the results of Adekunle &
Olagoke (2008) as well as Onyekwelu et al. (2007) but lesser when compared to the studies of Lu et al. (2010),
Rajkumar & Parthasarathy (2008). These researchers reported a total of 95 species and 105 species in
Xishuangbana, Chain tropical rainforest and India evergreen forest of Andaman Giant respectively. Various
researchers have considered the use of Shannon wiener diversity index for the determination of forest
community diversity in the tropics (Onyekwelu et al. 2007, Adekunle & Olagoke 2008, Borah et al. 2014,
Boboye & Jimoh 2016). Shannon Diversity Index values for this study ranged between 2.79 and 3.55. These
values fall within the range (between 0.70 and 3.57) reported for tropical forests (Bhuyan et al. 2003, Bajpai et
al. 2012, Borah et al. 2014, Sarkar & Devi 2014, Bajpai et al. 2015, Vinayaka & Krishnamurthy 2016).
However, the overall Shannon-Weiner diversity index for the whole study areas (5.11) is higher than what was
obtained by Onyekwelu et al. (2007) and Kent & Coker (1992). The Pielou’s species evenness of the entire
study areas (0.69) is higher than 0.66 and 0.55 reported by Onyekwelu et al. (2005) and Adekunle (2006)
respectively but less than the 0.82 that was recorded by Adekunle et al. (2013) when he examined the efficacy
of Strict Nature Reserves (SNR) as a means of biodiversity conservation. This finding indicated that forest
exploitation could lead to a drastic reduction in species diversity but might increase stand density in terms of
number of tree stems per hectare.
As one of the peculiar features of a mature natural forest ecosystem, the DBH distribution curve of tree
species followed inverse J-shaped pattern (Figs. 2 & 3). The same was reflected in the study of Adekunle et al.
2013. Highest proportion of the trees were within the lowest diameter class (10–20 cm) and the least percentage
in the highest diameter class (>80cm). In comparison, Huang et al. (2003) and Lu et al. (2010) reported a lesser
percentage (4.5% and 3.5%) for trees that falls within the highest diameter class in forests of similar ecosystem
as the study area.
Similarly, the same trend was noticed in the distribution of the individual species and families into diameter
classes. Oluwa forest recorded the highest Vol.ha-1 (141.06 m3) and the lowest value (14.56 m3) was from the
free forest area adjoining Permanent Sample Plot 29, representing a degraded forest. Only the SNR and Oluwa
forest reserve have trees with height above 30m (18.7% and 7.02% respectively). These are trees that can be
regarded as emergent. Going by the recommended value of 25 m2.ha-1 for mean basal area of a well-stocked
forest (Alder & Abayomi 1994), the mean basal areas obtained in some of the forest reserves in the study areas
shows that the forest reserves are not yet well stocked. The variation in the basal area of the forests (ranged from
3.40 to 12.31 m2.ha-1) is significantly lower to the recommended basal area.
CONCLUSION AND RECOMMENDATION

The results of this study revealed the present phytosociological properties of selected PAs in the humid
forest ecosystem in terms of tree species diversity, evenness, and stand bio-volume. The study also
demonstrated the importance of in-situ conservation method and the detrimental effect of forest degradations to
tree species diversity and abundance. The biodiversity indices were high and the forest structure shows a
continuous growth feature until they reach maturity and a stable state. Therefore, logging, hunting, farming and
human settlement should stop in the free forest areas and be delineated as PAs while the existing in-situ
conserved areas should be strictly monitored against any form of anthropogenic activities.
For greater production, conservation and sustainability of the protected areas, the government and non-
governmental organizations should provide farmers with incentives as motivation tool to enhance the forest and
its resources. More attention should therefore be paid to species with narrow range and those threatened with
extinction in the ecosystem through the conservation of their genetic material such as seed ex-situ conservation.
Iyagin & Adekunle (2017) 4(3): 496–513
ACKNOWLEDGEMENTS
We wish to appreciate the Director of Forestry in Ondo State and the Forestry Research Institute of Nigeria
(FRIN) for giving us permit to access the Protected Areas within their Jurisdictions where data was collected for
this work.
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ISSN (E): 2349 – 1183
ISSN (P): 2349 – 9265
4(3): 514–517, 2017
DOI: 10.22271/tpr.2017.v4.i3.067
Mini review
Pepino (Solanum muricatum Ait.): A potential future crop

for subtropics
Ashok Kumar*, Tarun Adak and S. Rajan
ICAR-Central Institute for Subtropical Horticulture, Rehman Khera, P.O. Kakori,
Lucknow-226101, Uttar Pradesh, India
*Corresponding Author: ashokhort@gmail.com [Accepted: 26 December 2017]
Abstract: Pepino (Solanum muricatum) is an Andean region’s crop, originated from South
America. The crop has medicinal values and underutilized for its cultivation. It has a wider
adaptability across the different locations of Spain, New Zealand, Turkey, Israel, USA, Japan etc.
The crop can be grown under diverse soil and climatic conditions in India also. Its fruits are juicy,
mild-sweet, sub-acidic and aromatic berry which are rich in antiglycative, antioxidant, dietary
fibres and low calorific energy. Fruit is visually attractive with golden yellow colour with purple
stripes. The crop was evaluated for its growth and development at ICAR-Central Institute for
Subtropical Horticulture, Rehmankhera, Lucknow, Uttar Pradesh, India (planted in the month of
October, 2014). The results of the study exhibited its adaptation to climatic conditions of
subtropics with higher yield and acceptable fruit quality.
Keywords: Solanum muricatum - Pepino - Subtropic - Adaptation.
[Cite as: Kumar A, Adak T & Rajan S (2017) Pepino (Solanum muricatum Ait.): A potential future crop for
subtropics. Tropical Plant Research 4(3): 514–517]
INTRODUCTION
Introduced crops have a vital role in the progress of mankind; on any region of the world, many most
important crops did not originate there but were new crops at the time of their introduction. Nonetheless, many
underutilized or neglected crops and nondomesticated species that could develop as new crops in many regions
worldwide are to be explored. The diversification and introduction of these new crops are growing interest as
they can result in an increase of income for farmers. It can contribute to a more environment-friendly
horticulture, minimize the risk of crop failure and increase ethnobotanical knowledge. For a successful
establishment, a new crop must adapt to the new agro-ecological and production conditions, as well as to the
demands of the markets and consumers. Introduction and adaptation of new crops have historically been one of
the greatest technological challenges in agriculture, as it transfers cultivated species from their places of origin
to new environmental conditions. The success in the introduction of new crops has contributed to the spread and
success of agriculture and to the production of an ample and wide diversity of plant products. Most of the
cultivated crops in a given region of the World were not domesticated there and once were new crops like
wheat, potato and tomato in India. Unlike in former centuries, in which exploration of new areas of the world
allowed the discovery and exchange of potential new crops, nowadays attempts of introducing new crops to
exotic regions is mainly based on already existing, mostly neglected, crops. In this respect, there are many “lost”
crops, which in some cases have the local importance that could be of interest for introduction in other areas of
the world.
About the Crop

One of the crops of Solanaceae family is Pepino (Solanum muricatum Aiton), which is a neglected crop that
was very important in the Andean region during pre-Columbian times. It flowers between the months of
December to May (Fig. 1A). The Pepino is an herbaceous crop which yields fleshy berries which weigh between
100 and 700 g, and are round, ovate or elongated, with a normally golden yellow skin (Fig. 1B). The flesh is
yellow, juicy and has a sub-acid, mild flavor (Levy et al. 2006). It is visually attractive; the skin is commonly
Kumar et al. (2017) 4(3): 514–517
golden yellow and is covered with purple stripes (IPGRI & COMAV 2004). Aroma of the fruit resembles with
muskmelon. The Pepino is entirely edible, including skin and seeds and hence tastes like a cross between a pear
and banana. Although, it is sexually fertile and highly heterozygous by seed propagation, but can also propagate
vegetatively (normally by cuttings). Though the crop originated in South America, it has domesticated in other
parts of the world like Netherland, New Zealand and Spain (Blanca et al. 2007).
Figure 1. Pepino (Solanum muricatum Aiton) under Lucknow conditions: A, Flowering; B, Fruiting.
Exotic but can be grown under Indian climatic conditions

Solanum muricatum is of temperate, mountain and coastal climates but can be grown under tropical climates
also. In the Andean region, cultivation takes place in the inter-Andean valleys and on the western slopes from
900 to approximately 2800 m amsl. These boundaries are set within the lower limit (18°C) and the upper limit
24°C at, with an annual precipitation of between 500 and 800 mm. The climatic characteristics described
correspond to the high part of the subtropical dry forest and the low dry mountain forest or to the high yungas
and quechua of Peru. Coastal cultivation takes place south of lat. S 7°. During the autumn and winter when the
temperature fluctuates between 21–17°C and atmospheric humidity increases as a result of mists and drizzle.
The original cultivation of S. muricatum extended along the Andes, from southern Colombia to Bolivia and the
Peruvian coast.
Fruit of medicinal importance

It’s also prized for its medicinal applications. Aqueous extract of its fruit could attenuate the progression of
diabetes due to its anti-inflammatory, antiglycative and antioxidant effects (Hsu et al. 2011). A medium serving
(~100 g) of its fruit provides 80 calories of energy and 5 g of dietary fibres similar to oatmeal, which helps to
lower cholesterol and it’s easy to digest. The fibre also helps with constipation and it tends to sooth away gastric
ulcers too. The fruit is rich in minerals and vitamin C but low in starch, sugars and free from oxalates. The
minerals contained in Pepino fruits are Fe, Zn, Cu, Mn, Ca & P. It has been observed that level of glucose and
fructose decreases during ripening, whereas, sucrose concentration increases as the ripening progresses. A
discernible reduction has also been noticed in contents of protein and fat as the fruit turns from raw to mature
(Huyskens-Keil et al. 1999). Pepino is known as a source of beta-carotene, 27 mg per 100 grams of fruit flesh.
The crop is also considered as a sucrose accumulator during final ripening stage. Fruits picked when immature
are flavorless and non-aromatic.
Soil and climatic requirement

The crop can be grown in different types of soil, preferably low in fertility as higher fertility may hamper
reproductive growth. It performs better in soil pH 6.0–7.5 with well-drained soil. Pepino has also wider soil
adaptability even under salinity conditions of 8 dS m-1. A vast area in India having salt-affected soils including
the coastal area where the scope for cultivation may be explored. The crop has wider adaptability across
temperate, tropics and subtropical conditions but the fruit set is very much influenced by temperature, the
optimum range being reported between 12–25 °C. The crop is often considered as non-climacteric fruit but
some cultivars also behave as climacteric. The crop generally prefers warmth climatic conditions however if cut
back even at a suboptimal temperature (< -3°C), it can survive (Prohens et al. 1996). The analysis of the weather
Kumar et al. (2017) 4(3): 514–517
parameters during its growth and development periods (2014–15 and 2015–16) were recorded at the established
agro-meteorological observatory. Monthly average minimum and maximum temperatures varied between 6.2–
25.8 °C and17.2–39.8 °C, respectively. The monthly average relative humidity ranged between 25.1–89.7 %.
Most importantly higher pan evaporation up to 11.4 mm per day during summer months was recorded while in
winter months as low as 2.3 mm per day was observed. Uneven distribution of rainfall was noticed. A sum of
732.2 and 553.2 mm of rainfall was recorded during 2014–15 and 2015–16 respectively. This rainfall is lower
than the average of around 800–1000 mm. Unseasonal rainfall of 87.2 mm was also recorded during Jan.–Feb.
months of the 2015–16 season. A bright sunshine 2.8–9.8 hour and wind velocity of 1.1–4.3 km.h-1 was
observed. Profuse growth and higher productivity of Pepino under such variations in weather parameters under
subtropical climatic conditions of Lucknow region were observed.
Potential in subtropics
Keeping in view the potentiality of the crop that might undergo adaptability in a subtropical environment,
Pepino cuttings were planted at ICAR-Central Institute for Subtropical Horticulture, Rehmankhera, Lucknow,
Uttar Pradesh, India during winter months (October, 2014). The plant survived during harsh summer conditions
of 2015 (Fig. 2). The survived plants were multiplied and planted in the field conditions for performance
evaluation. Fruit harvest started from March onwards and continued until the month of May. The variations in
different parameters viz., fruits weight (150–350 g), yield (4–5 kg per plant), moisture content (93.6%), Vitamin
C (37.17 mg per 100 g), Acidity (0.13–0.14 %) and TSS (4.9–5.5 %) was recorded (Kumar 2016).
Figure 2. Crop growth and adaptation under subtropics (ICAR-CISH, Lucknow, India).
Figure 3. Profuse rooting of cutting.

Kumar et al. (2017) 4(3): 514–517
How to multiply
The Pepino can be grown from seeds but is usually propagated vegetatively to maintain the original quality
from semi-hardwood cuttings. In the subtropical conditions, the mother plants need attention during rainfall i.e.
covering of the mother plants with ventilated plastic rain shelter from July–September. The cuttings should be
made during the month of October for maximum survival in the subtropics (Fig. 3). Cuttings are easy to root
and treatment with growth regulator is not mandatory.
ACKNOWLEDGEMENT
Authors duly acknowledged the facilities and support extended by ICAR-CISH for conducting the
experiment.
REFERENCES
Blanca JM, Prohens J, Anderson GJ, Zuriaga E, Cañizares J & Nuez F (2007) AFLP and DNA sequence
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