PHILIPP AGRIC SCIENTIST ISSN 0031-7454

Vol. 97 No. 2, 199203

June 2014

Susceptibility of the Legume Pod Borer, Maruca vitrata, to Microbial

Control Agents
R. Srinivasan1,*, S. K. Ghosh2, Nguyen Thi Thanh Hien3, M. Y. Lin1, Vu Thi Thuy Trang3, Le
Duc Khanh3 and Manh Hai Vu4
1
AVRDC The World Vegetable Center, Shanhua, Tainan 74151, Taiwan
2
Bio-Control Research Laboratories, Bangalore 561203, Karnataka, India
3
Plant Protection Research Institute, Dong Ngac, Tu Liem, Ha Noi, Vietnam
4
Vietnam Academy of Agricultural Sciences, Thanh Tri, Ha Noi, Vietnam
Author for correspondence; Mail address: AVRDC The World Vegetable Center, 60 Yi-Min Liao, Shanhua, Tainan
74151, Taiwan; Tel: +886 6 5837801; Fax: +886 6 5830009; e-mail: srini.ramasamy@worldveg.org

Baseline susceptibility of Maruca vitrata to Bacillus thuringiensis and entomopathogenic fungi (EPF)
formulations and/or isolates was assessed under laboratory conditions in Taiwan, Vietnam and India.
In Taiwan, Xentari (B. thuringiensis subsp. aizawai) was more toxic to M. vitrata. In Vietnam, M.
vitrata was highly susceptible to Zitarback F.C. (B. thuringiensis subsp. aizawai); it was highly
susceptible to the EPF formulation Luc cuong (Metarhizium anisopliae). M. anisopliae isolate
BCRLMa-3 was highly toxic to M. vitrata in India.

Key Words: Bacillus thuringiensis, baseline susceptibility, entomopathogenic fungi, Maruca vitrata

INTRODUCTION exclusively on the application of chemical

Vegetable legumes are important crops in South and
Southeast Asia. They are important sources of plant
proteins in the human diet. Yard-long bean is the
most popular vegetable legume in Southeast Asia,
with a cultivated area of more than 130,000 ha in
Indonesia, Thailand and Vietnam (Benchasri and
Bairaman 2010; Kuswanto and Waluyo 2011).
Vegetable legumes are highly susceptible to insect
pests and diseases. Among the documented pests,
legume pod borer, Maruca vitrata (F.) (syn. M.
testulalis) (Lepidoptera: Pyralidae), is considered
the most serious pest of food legumes (Sharma
1998; Ulrichs et al. 2001; Soeun 2001). Its larvae
attack floral buds, flowers and pods, and damage
several plant parts throughout the year, especially
during dry seasons (Sharma 1998). Severe damage
from M. vitrata has been reported on several food
legumes. For example, cowpea (Vigna unguiculata)
pod yield losses range from 17% to 53% in Taiwan
(Liao and Lin 2000). Up to 68% pod damage in
pigeonpea (Cajanus cajan) has been reported in
India (Sunitha et al. 2008a). Farmers rely almost
insecticides to combat M. vitrata, but without
satisfactory results. In Bangladesh, country bean
(Lablab purpureus) is sprayed at weekly or
biweekly intervalssometimes every dayto
control M. vitrata (Hoque et al. 2001). Sustainable
management strategies for M. vitrata are needed to
curtail pesticide misuse on vegetable legumes in
tropical and subtropical Asia.
Earlier studies have documented the
susceptibility of M. vitrata to Bacillus thuringiensis
-endotoxins and/or formulations, and entomo-
pathogenic fungi (EPF) in a few countries in
tropical Asia and sub-Saharan Africa (AVRDC
1996 & 1997; Machuka 2002; Ekesi et al. 2002;
Srinivasan 2008; Srinivasan et al. 2009; Yule and
Srinivasan 2013). However, the susceptibility of M.
vitrata to newer bio-pesticide formulations and
isolates of EPF has not been documented in India,
Taiwan and Vietnam. This study was conducted to
assess the baseline susceptibility of M. vitrata to
selected bio-pesticide formulations and/or EPF
isolates under laboratory conditions.

The Philippine Agricultural Scientist Vol. 97 No. 2 (June 2014) 199

Susceptibility of the Legume Pod Borer to Microbial Control Agents
MATERIALS AND METHODS
Insects
An M. vitrata colony was established at the
Insectary of AVRDC The World Vegetable
Center in southern Taiwan in May 2010 from field
populations that had not been sprayed with any bio-
pesticides. The larvae had no contact with bio-
pesticides in rearing facilities and they were reared
on a meridic diet of a polyphagous insect, beet
armyworm (Spodoptera exigua Hbner, [Bio-Serve
French Town, NJ, USA]) modified with cowpea
powder, at 27 1C, and 70 10% relative
humidity (RH), photoperiod 14:10 h (Light:Dark)
until pupation. On pupation, they were placed in
acrylic cylinders (30 cm long and 15 cm diameter),
whose ends were covered with nylon nets, and
emerged adults were used for multiplication. Adults
were maintained on a 10% (w/v) sugar solution
dispensed on cotton wool placed inside the
cylinders, for mating and egg laying. Eggs were
collected from nylon nets covering the cylinder
ends. When the eggs hatched, a proportion of the
neonate larvae were transferred to plastic containers
with the diet for multiplication and the remaining
larvae were used for bioassays. A similar M. vitrata
colony was established at the Plant Protection
Research Institute (PPRI), Hanoi, Vietnam using the
field-collected insect population. Instead of the
meridic diet, a modified artificial diet based on
soybean powder and Sesbania cannabina leaf and
seed powders was used to feed the larvae (Yule and
Srinivasan 2013). The diet was prepared from 50 g
soybean powder, 50 g S. cannabina leaf powder and
60 g S. cannabina seed powder in 400 mL distilled
water, 10 g brewer's yeast, 3 g L-ascorbic acid, 0.6 g
sorbic acid, and 1.2 g methyl-p-hydroxybenzoate in
150 mL distilled water and 12.5 g agar in 250 mL
distilled water. At the Bio-Control Research
Laboratories (BCRL), Karnataka, India the M.
vitrata colony was maintained on a combination of
both natural and artificial diets. Pieces of Phaseolus
vulgaris bean were used to rear the neonate to
second instar larvae, and then the larvae were
allowed to feed on an artificial diet containing
cowpea leaf and seed powder until pupation.
Laboratory Bioassays
Commercially available bio-pesticide formulations
based on B. thuringiensis such as Crymax, strain
200
R. Srinivasan et al.
E-911 and Redcat (B. thuringiensis subsp. kurstaki),
Xentari and Zitarback F.C. (B. thuringiensis subsp.
aizawai), EPF such as Luc cuong (Metarhizium
anisopliae) and Bach cuong (Beauveria bassiana)
were procured. Redcat, Zitarback F.C., Luc cuong
and Bach cuong were used in Vietnam, whereas
only Crymax, strain E-911 and Xentari were
used in Taiwan. Selected isolates of B. bassiana
(BCRLBb-4, BCRLBb-16 and BCRLBb-18) and M.
anisopliae (BCRLMa-3 and BCRLMa-6) were used
in India. Four widely spaced concentrations (10,
100, 1000 and 10000 ppm for each bio-pesticide or
EPF isolate) were used in the preliminary range-
finding tests. At least five concentrations for each
bio-pesticide or EPF isolate that could cause 10%
90% mortality according to the preliminary tests
plus an untreated control were included in each
bioassay. Insect bioassays were performed by
treating the artificial diet with selected
concentrations of each bio-pesticide. Ten early
second-instar larvae were introduced into each
plastic cup (4.5 cm high and 4 cm wide) containing
treated artificial diet that served as an experimental
unit. The cups were covered with lids. The
experiment had five replicates. For EPF isolate, 3
L of the selected conidial concentrations were
deposited on the pronotum of individual larvae.
Twenty-five larvae were treated with each
concentration and the larvae were placed
individually in the multi-cavity trays containing
pieces of cleaned field beans 2 cm in length as a
food source; the beans were replaced on alternate
days. The cups or trays were incubated at 27 1C,
and 70 10% RH, photoperiod 12:12 h
(Light:Dark). Larval mortality was recorded after 4
or 5 d. The lethal concentrations causing 50%
mortality (LC50), 90% mortality (LC90), their 95%
fiducial limits (FL) and the slope value of probit
line were assessed using the statistical program LdP
line (Ehab Mostofa Bakr, Cairo, Egypt).
RESULTS AND DISCUSSION
The efficacy of different bio-pesticides against
second instar larvae of M. vitrata is presented in
Table 1. In Taiwan, both LC50 and LC90 values have
confirmed that Xentari (B. thuringiensis subsp.
aizawai) was more toxic than Crymax and Strain
E-911 (B. thuringiensis subsp. kurstaki), since the
The Philippine Agricultural Scientist Vol. 97 No. 2 (June 2014)
Susceptibility of the Legume Pod Borer to Microbial Control Agents R. Srinivasan et al.

Table 1. Toxicity of different bio-pesticides and/or entomopathogenic fungi to Maruca vitrata larvae in Taiwan, Vietnam
and India.
Fiducial Fiducial
Bio-pesticide / LC50 Slope
Country Limits LC90 (ppm) Limits 2 Df p
Entomopathogen (Isolate) (ppm) (SE)
(95%) (95%)
Xentari (B. thuringiensis (68.78 (394.36 1.88
Taiwan 102.64 a 492.09 a 2.16 3 0.34
subsp. aizawai) 132.63) 682.20) (0.26)

Crymax (B. thuringiensis (309.71 (1794.86 1.62
371.62 b 2309.57 b 9.03 5 0.06
subsp. kurstaki) 437.34) 3213.14) (0.13)

Strain E-911 (B. (1188.78 (7042.91 1.47
1432.51 c 10677.65 c 7.69 4 0.05
thuringiensis subsp. kurstaki) 1764.90) 19583.16) (0.15)

Zitarback F.C. (B. (39.80 (242.79 2.09

Vietnam 60.58 AB 249.03 A 12.06 4 0.007
thuringiensis subsp. aizawai) 124.68) 1648.92) (0.20)
Redcat (B. thuringiensis (178.93 (2735.47 1.04
238.34 C 4024.67 B 6.61 5 0.16
subsp. kurstaki) 307.60) 6658.85) (0.09)
Luc cuong (Metarhizium (33.66 (218.47 2.57
56.48 A 178.37 A 16.84 4 0.001
anisopliae) 112.16) 1345.51) (0.23)
Bach cuong (Beauveria (119.61 (81542.89 0.40
272.90 BC 414230 C 6.39 5 0.17
bassiana) 497.29) 9,040,500) (0.07)

Metarhizium anisopliae (7.66 (3.7 x 103) 0.43

India 20.96 a 1.90 x 104 a 9.39 4 0.05
(BCRLMa-3) 55.78) (2.62 x 105) (0.06)
(59.94 (7.41 x 103) 0.52
M. anisopliae (BCRLMa-6) 140.70 b 3.76 x 104 ab 6.40 4 0.17
400.64) (5.34 x 105) (0.07)
Beauveria bassiana (39.88 (3.60 x 104) 0.36
120.18 ab 3.85 x 105 ab 4.00 4 0.41
(BCRLBb-4) 465.99) (2.65 x 107) (0.06)
(809.61 (3.94 x 105) 0.37
B. bassiana (BCRLBb-16) 3390.58 c 8.65 x 106 b 2.75 4 0.60
36022.85) (3.80 x 109) (0.07)
(567.34 (4.99 x 104) 0.56
B. bassiana (BCRLBb-18) 1268 bc 2.35 x 105 b 4.81 4 0.31
3584.05) (2.72 x 106) (0.07)
Toxicity values in a column followed by the same letter(s) are not significantly different based on 95% overlapping fiducial limits.

fiducial limits are not overlapping. In Vietnam, M.
vitrata was more susceptible to Zitarback F.C. (B.
thuringiensis subsp. aizawai) than Redcat (B.
thuringiensis subsp. kurstaki) and was more
susceptible to Luc cuong (M. anisopliae) than Bach
cuong (B. bassiana). In addition, the toxicity of Luc
cuong (M. anisopliae) was similar to that of
Zitarback F.C. since their fiducial limits for LC50
overlapped. Similarly, the toxicity of Redcat was
similar to that of Bach cuong (B. bassiana), because
of their overlapping fiducial limits for LC50. Based
on the LC90 values, Zitarback F.C. and Luc cuong
(M. anisopliae) showed a similar trend in their
toxicities, and Redcat was found to be better than
Bach cuong (B. bassiana) based on the LC90 values.
Among the EPF isolates tested in India, M.
vitrata was more susceptible to M. anisopliae
isolate BCRLMa-3 because of its significantly
lower LC50 and LC90 values compared with
BCRLMa-6 (Table 1). The toxicity of B. bassiana
isolate BCRLBb-4 was intermediate to those of
BCRLMa-3 and BCRLMa-6. B. bassiana isolate
BCRLBb-18 was moderately toxic to M. vitrata,
whereas BCRLBb-16 was least effective. The
toxicity of B. bassiana isolate BCRLBb-4 was not
significantly different from other two isolates
(BCRLBb-16 and BCRLBb-18) because of their
overlapping fiducial limits for LC90 values. M.
anisopliae isolate BCRLMa-3 was the most toxic to
M. vitrata.
To our knowledge, there has been no prior
report documenting the susceptibility of M. vitrata
to different B. thuringiensis and EPF bio-pesticides
in Vietnam. However, different B. thuringiensis -
endotoxins and commercial formulations already
have been evaluated against M. vitrata in Taiwan.
Among the different B. thuringiensis commercial
formulations tested against M. vitrata, Dipel (B.
thuringiensis subsp. kurstaki) and Florbac (B.
thuringiensis subsp. aizawai) had the lowest LC50
values (AVRDC 1996 & 1997). In subsequent
laboratory bioassays in Taiwan, the toxin Cry1Ab
was the most potent, followed by Cry1Ca to M.
vitrata (Srinivasan 2008). A recent study found that
M. vitrata was more susceptible to B. thuringiensis
subsp. kurstaki than B. thuringiensis subsp. aizawai

The Philippine Agricultural Scientist Vol. 97 No. 2 (June 2014) 201

Susceptibility of the Legume Pod Borer to Microbial Control Agents
in Thailand (Yule and Srinivasan 2013). It must be
noted that Cry1A is found in some of the B.
thuringiensis subsp. kurstaki formulations, whereas
Cry1Ca is found in B. thuringiensis subsp. aizawai
formulations (Hofte and Whiteley 1989). Thus,
most of the earlier studies have confirmed the
toxicity of B. thuringiensis subsp. kurstaki
formulations and the major toxin(s) that are present
in B. thuringiensis subsp. kurstaki against M. vitrata
in Taiwan and Thailand. However, the current study
recorded a contrary result, in which the B.
thuringiensis subsp. aizawai formulation was highly
toxic to M. vitrata in both Taiwan and Vietnam.
Since Cry1Ca is the major toxin in B. thuringiensis
subsp. aizawai formulations, and the M. vitrata
population in Taiwan was also shown to be
susceptible to Cry1Ca next to Cry1Ab, it is possible
that the M. vitrata population in Taiwan is
susceptible to Xentari. It is also possible that the
M. vitrata in the Hanoi region of Vietnam could
follow a similar susceptibility pattern to Cry1C
toxin, and hence demonstrated enhanced
susceptibility to Zitarback F.C. (B. thuringiensis
subsp. aizawai) in this study. However, the
susceptibility of M. vitrata in Hanoi must be
confirmed with individual B. thuringiensis -
endotoxins.
Although several studies under field conditions
have shown different responses of EPF formulations
against M. vitrata (Sreekanth and Seshamahalakshmi
2012; Yule and Srinivasan 2013), very few studies
have demonstrated the efficacy of EPF isolates.
Isolates of B. bassiana and M. anisopliae were
found to be highly pathogenic to eggs of M. vitrata,
causing 89%100% mortality in Africa. These
isolates also caused high M. vitrata larval mortality
(Ekesi et al. 2002). In the current study, M.
anisopliae isolate BCRLMa-3 was highly toxic,
whereas another M. anisopliae isolate, BCRLMa-6,
and B. bassiana isolate BCRLBb-4 were moderately
toxic to M. vitrata larvae at BCRL in India. Luc
cuong (M. anisopliae) was as toxic as Zitarback
F.C. (B. thuringiensis subsp. aizawai) against M.
vitrata at PPRI, Vietnam. However, a study by
Sunitha et al. (2008b) found that bio-pesticides
based on B. thuringiensis and M. anisopliae were
moderately effective against third instar M. vitrata
larvae under laboratory conditions. A recent study
has revealed that M. anisopliae and B. bassiana
formulations were least effective against second
202
R. Srinivasan et al.
instar M. vitrata larvae in Thailand (Yule and
Srinivasan 2013). Another study by Sreekanth and
Seshamahalakshmi (2012) confirmed that only a B.
bassiana SC formulation at a rate of 300 mg L-1
slightly reduced pigeon pea pod damage by M.
vitrata, compared with B. thuringiensis. The
efficacy of EPF formulations may vary due to the
differences in the formulations, local climatic
conditions (especially temperature and humidity),
age of the larvae, etc. (Tsai et al. 2006; Srinivasan
2012). The efficacy of the formulations must be
validated before EPF can be recommended to
manage M. vitrata on vegetable legumes.
Although the current study confirmed the
effectiveness of B. thuringiensis, EPF isolates and
formulations against M. vitrata populations in
Taiwan, Vietnam and India, the individual
performance of these bio-pesticides against M.
vitrata under field conditions may vary, as
demonstrated in a recent study by Yule and
Srinivasan (2013). Hence, the microbial pesticides
have to be combined with compatible chemical
pesticides and/or botanical pesticides to achieve
additive or synergistic effects. Thus, additional
studies should be carried out with combinations of
microbial, botanical and chemical pesticides rather
than a single bio-pesticide per treatment, in Taiwan,
Vietnam and India to manage M. vitrata on
vegetable legumes in field conditions.
ACKNOWLEDGMENT
This study was supported with funds from the
Federal Ministry for Economic Cooperation and
Development, Germany.
REFERENCES CITED
AVRDC. 1996. AVRDC 1995 Report. Asian Vegetable
Research and Development Center, Shanhua, Tainan,
Taiwan. p. 187.
AVRDC. 1997. AVRDC 1996 Report. Asian Vegetable
Research and Development Center, Shanhua, Tainan,
Taiwan. p. 172.
BENCHASRI S, BAIRAMAN C. 2010. Evaluation of
yield, yield components and consumers satisfaction
towards yard-long bean and cowpea in agricultural
organic system. Bulg J Agric Sci 16: 705712.
The Philippine Agricultural Scientist Vol. 97 No. 2 (June 2014)
Susceptibility of the Legume Pod Borer to Microbial Control Agents
EKESI S, ADAMU RS, MANIANIA NK. 2002.
Ovicidal activity of entomopathogenic hyphomycetes
to the legume pod borer, Maruca vitrata and the pod
sucking bug, Clavigralla tomentosicollis. Crop Prot
21(7): 589595.
HOFTE H, WHITELEY HR. 1989. Insecticidal crystal
proteins of Bacillus thuringiensis. Microbiol Rev 53:
242255.
HOQUE M, MOLLIK SR, NAZIMUDDIN M, ALAM
SN, KHORSHEDUZZAMAN M, JASMINE HS,
SULTANA NA, RAHMAN M, AHMED B,
RAJOTTE E, LUTHER G. 2002. Survey of Bean
Pod Borer (Maruca testulalis), whitefly and aphids
and their natural enemies on country bean (Lablab
purpureus). Tenth Annual Report of the Integrated
Pest Management Collaborative Research Support
Program (IPM CRSP), http://www.oired.vt.edu/
ipmcrsp/communications/annrepts/annrep03/
Bangladesh/8BangladeshY_10_ANNUAL_REPORT
1.pdf#page=10. p. 123124.
KUSWANTO, WALUYO B. 2011. The adaptation test
on yard-long bean lines tolerant to aphids and high
yield. AGRIVITA 33(2): 182187.
LIAO CT, LIN CS. 2000. Occurrence of the Legume Pod
Borer, Maruca testulalis Geyer (Lepidoptera:
Pyralidae) on Cowpea (Vigna nguiculata Walp) and
its insecticides application trial. Plant Prot Bull
(Taiwan) 42: 213222.
MACHUKA J. 2002. Potential role of transgenic
approaches in the control of cowpea insect pests. In:
Fatokun CA, Tarawali SA, Singh BB, Kormawa PM,
Tamo M, editors. Proc. World Cowpea Conference
III, Challenges and Opportunities for Enhancing
Sustainable Cowpea Production, September 47,
2000, International Institute of Tropical Agriculture,
Ibadan, Nigeria. p. 213232.
SHARMA HC. 1998. Bionomics, host plant resistance,
and management of the legume pod borer, Maruca
vitrata a review. Crop Prot 17: 373386.
SOEUN M. 2001. Legumes in rice-based cropping
systems in Cambodia: Constraints and opportunities.
In: Gowda CLL, Ramakrishna A, Rupela OP, Wani
SP, editors. Legumes in Rice-based Cropping
Systems in Tropical Asia: Constraints and
Opportunities, ICRISAT, India. p. 410.
The Philippine Agricultural Scientist Vol. 97 No. 2 (June 2014)
R. Srinivasan et al.
SREEKANTH M, SESHAMAHALAKSHMI M. 2012.
Studies on relative toxicity of biopesticides to
Helicoverpa armigera (Hubner) and Maruca vitrata
(Geyer) on pigeonpea (Cajanus cajan L.). J Biopest 5
(2): 191195.
SRINIVASAN R. 2008. Susceptibility of legume pod
borer (LPB), Maruca vitrata to -endotoxins of
Bacillus thuringiensis (Bt) in Taiwan. J Invertebr
Pathol 97: 7981.
SRINIVASAN R. 2012. Integrating biopesticides in pest
management strategies for tropical vegetable
production. J Biopest 5: 3645.
SRINIVASAN R, TAMO M, LEE ST, LIN MY,
HUANG CC, HSU YC. 2009. Towards developing a
biological control program for legume pod borer,
Maruca vitrata. In: Gupta S, Ali M, Singh BB,
editors. Grain Legumes: Genetic Improvement,
Management and Trade. Kanpur, India: Indian
Society of Pulses Research and Development. p. 183
196.
SUNITHA V, RANGA RAO GV, VIJAYA LAKSHMI
K, SAXENA KB, RAMESHWAR RAO V, REDDY
YVR. 2008a. Morphological and biochemical factors
associated with resistance to Maruca vitrata
(Lepidoptera: Pyralidae) in short-duration pigeonpea.
Int J Trop Insect Sci 28: 4552.
SUNITHA V, VIJAYA LAKSHMI K, RANGA RAO
GV. 2008b. Laboratory evaluation of certain
insecticides against pigeonpea pod borer, Maruca
vitrata Geyer. J Food Legume 21(2): 137139.
TSAI YS, YANG PS, HUNG TH, KAO SS. 2006.
Biological characterization of Beauveria bassiana
isolates from Taiwan. Plant Prot Bull (Taipei) 48:
117128.
ULRICHS C, MEWIS I, SCHNITZLER WH,
BURLEIGH JR. 2001. Effectivity of synthetic
insecticides against Maruca vitrata F. and the
parasitoid Bassus asper CHOU & SHARKEY in the
Philippines. Mitt DGAAE 13(16): 279282.
YULE S, SRINIVASAN R. 2013. Evaluation of bio-
pesticides against legume pod borer, Maruca
vitrata Fabricius (Lepidoptera: Pyralidae), in
laboratory and field conditions in Thailand. J Asia-
Pacific Entomol 16(4): 357360
203