Biocontrol Science and Technology,

Vol. 18, No. 8, 2008, 809
828

Comparison of Metarhizium isolates for biocontrol of Helicoverpa armigera

(Lepidoptera: Noctuidae) in chickpea
Shuklangi A. Kulkarnia, Vandana Ghormadeb, Girish Kulkarnic, Manisha Kapoora,
Santosh B. Chavana, Armugam Rajendrana, Sanjay K. Patild, Yogesh Shouchec and
Mukund V. Deshpandea*
a
Biochemical Sciences Division, National Chemical Laboratory, Pune, India; bCentre for
Nanobioscience, Agharkar Research Institute, Pune, India; cMolecular Biology Unit, National Centre
for Cell Science, Pune, India; dMahatma Phule Krishi Vidyapeeth, Rahuri, Ahmednagar, India
(Received 22 February 2008; returned 1 April 2008; accepted 24 July 2008)

Metarhizium isolates from soil (53) and insect hosts (10) were evaluated for extracellular
production of cuticle degrading enzyme (CDE) activities such as chitinase, chitin
deacetylase (CDA), chitosanase, protease and lipase. Regression analysis demonstrated
the relation of CDE activities with Helicoverpa armigera mortality. On basis of this
relation, ten isolates were selected for further evaluation. Subsequently, based on LT50 of
the 10 isolates towards H. armigera, five isolates were selected. Out of these five isolates,
three were selected on the basis of higher conidia production (60
75 g/kg rice), faster
sedimentation time (ST50) (2.3
2.65 h in 0.1% (w/v) Tween 80) and lower LC50 (1.4

5.7 103 conidia/mL) against H. armigera. Finally, three Metarhizium isolates were
selected for the molecular fingerprinting using ITS sequencing and RAPD patterning.
All three isolates, M34412, M34311 and M81123, showed comparable RAPD patterns
with a 935G primer. These were further evaluated for their field performance against H.
armigera in a chickpea crop. The percent efficacies with the three Metarhizium isolates
were from 65 to 72%, which was comparable to the chemical insecticide, endosulfan
(74%).
Keywords: Metarhizium anisopliae; Helicoverpa armigera; chitinase; chitin deacetylase;
chitosanase; protease; lipase; appressorium formation

Introduction
Development of an effective mycoinsecticide depends on selection of an isolate that is
highly virulent for the target host and which is genetically and biologically stable (Milner,
Samson, and Bullard 2002). Recently, Metarhizium anisopliae M34412 has been identified
as a potential commercial strain for control of Helicoverpa armigera infestation on pulses
(Nahar et al. 2003, 2004b). Repeated in vitro sub-culturing on artificial media reduced the
virulence of M34412 towards H. armigera (Nahar et al. 2008). Although the virulence of
M. anisopliae was restored by four to five passages through H. armigera larvae, it was not
always feasible to maintain and revive cultures from the insect host. In addition, use of a
single strain for biocontrol could perhaps lead to development of resistance. Therefore, it
would be practical to identify better performing isolates for their use in the field after
generating all the concomitant safety data, as well as data on their molecular identity.

*Corresponding author. Email: mv.deshpande@ncl.res.in

ISSN 0958-3157 print/ISSN 1360-0478 online

# 2008 Taylor & Francis
DOI: 10.1080/09583150802366475
http://www.informaworld.com
810 S.A. Kulkarni et al.

It was reported that in fungus
insect interactions, different events such as germination

of conidia, formation of appressoria for penetration and yeast-like (unicellular) cells for
colonisation were important (Hajek and St. Leger 1994; Chavan, Ghormade, Nahar, and
Deshpande 2006). In addition, extracellular production of cuticle-degrading enzymes
(CDE) such as chitinase, chitin deacetylase (CDA), chitosanase, protease and lipase, as
well as non-enzymic killing components such as secondary metabolites to a certain extent,
play a significant role in pathogenesis (St. Leger, Cooper, and Charnley 1986; Krieger de
Moraes, Schrank, and Vainstein 2003; Nahar, Ghormade, and Deshpande 2004a). In
addition, Shakeri and Foster (2007) reported that entomopathogenicity of the mycopar-
asite Trichoderma harzianum towards Tenebrio molitor larvae was due to enzymes such as
chitinase and protease as well as the antibiotic, peptaibol.
In the present study, screening of 63 Metarhizium isolates including M. anisopliae
M34412 was carried out, first on the basis of CDE activities and virulence against
H. armigera using 1 107 conidia/mL and then on the basis of median lethal time (LT50),
productivity and settling time of conidia (ST50), median lethal concentration (LC50), and
the effect of malic acid (associated with chickpea leaf exudate) on conidia germination and
appressorium formation in a step-wise manner. Isolates selected from these screens were
further evaluated for their molecular characteristics and field performance.

Materials and methods

Isolation of Metarhizium isolates and their maintenance
The 63 Metarhizium isolates were obtained from various regions of Pune (18.318N,
73.558E), Maharashtra, India. Fifty-three of the strains were isolated from soil samples of
different fields using a soil dilution method according to Nahar et al. (2003), while10
strains were isolated from insect hosts. The insects showing abnormal behaviour with poor
coordination were collected from fields, kept at 288C until death and transferred to moist
chambers for further mycosis and sporulation, if any. Conidia from sporulating cadavers
were streaked on selective media containing (g/L): peptone, 10; glucose, 20; agar, 18;
streptomycin, 0.6; tetracycline, 0.05; cyclohexamide, 0.05; and dodine, 0.1 mL and pure
cultures were obtained by repeated subculturing (2
3 times) on the same media as
described by Keller, Kessler, and Schweizer (2003). After obtaining pure cultures, the
isolates were maintained on a potato dextrose agar (PDA) (g/L): peeled potato, 200;
dextrose, 20; agar, 20; pH 6, slants for complete sporulation at 288C at 70
80% RH for
7 days. Following sporulation, the mother cultures were maintained at 88C until use.

Production of cuticle degrading enzymes

The extracellular production of induced CDEs, i.e., chitinase (EC 3.2.1.14), chitosanase
(EC 3.2.1.132), protease (EC 3.4.21.62) and lipase (EC 3.1.1.3) for the 63 isolates was
studied in chitin containing medium (g/L): KH2PO4, 3; K2HPO4, 1; MgSO4, 0.7;
(NH4)2SO4, 1.4; NaCl, 0.5; CaCl2, 0.5; yeast extract, 0.5; bacto-peptone, 0.5; urea, 0.3;
oxgall, 1; trace metal solution, 1 mL (contained mg/mL: FeSO4, 5; MnSO4, 1.56; ZnSO4,
3.34; CoCl2, 2); chitin (Sigma, carbon source), 5; pH 6, as described by Vyas and
Deshpande (1989). The constitutively produced extracellular CDA (EC 3.5.1.41) was
studied in YPG medium containing (g/L): yeast extract, 3; peptone, 5; glucose, 10; pH 5.6.
For enzyme production, 500 mL of 1 107 conidia/mL from conidial suspension prepared
 Biocontrol Science and Technology 811

in 0.1% (w/v) Tween 80 from all the isolates was added in 50 mL YPG and chitin medium
and incubated at 288C for 72
96 h.
CDE production was carried out in three different flasks and enzyme assays were
carried out in triplicate for each flask. The experiment was conducted three times on
different occasions using freshly prepared medium constituting a randomised complete
block design (RCBD).
In all the experiments, the data were statistically examined for significant differences
among blocks. If there were no differences, the data from the blocks were pooled and
analysed as a completely random design in order to increase the power of the analysis.
In the case of CDE production, the data was subjected to analysis of variance and the
means were compared using F-test at a 0.05 level of critical difference as described by
Panse and Sukhatme (1989).

Enzyme assays
The chitinase, protease, lipase and chitosanase activities were measured after 96 h in chitin
medium grown culture filtrate while the CDA activity was measured after 72 h in YPG
grown culture filtrate.

Chitinase assay
Total chitinase activity in the culture supernatant was estimated colorimetrically using
acid-swollen chitin as a substrate, as described earlier (Vyas and Deshpande 1989). To
prepare acid-swollen chitin, 10 g chitin (purified powder from crab shells, Sigma) was
suspended in 300 mL O-phosphoric acid (88% w/v) and left at 48C for 1 h with occasional
stirring. The mixture was poured into ice-cold distilled water (4 L) and left for 30 min. The
swollen chitin was repeatedly washed with ice-cold distilled water, followed by a wash with
1% (w/v) NaHCO3 solution to adjust the pH 7. The swollen chitin was then dialysed at 48C
against distilled water. After homogenisation in a Waring blender for 1 min, the
concentration of acid swollen chitin was adjusted to 7 mg/mL by adding 50 mM acetate
buffer, pH 5.
The reaction mixture containing 1 mL 0.7% swollen chitin, 1 mL of 50 mM acetate
buffer, pH 5 and 1 mL of enzyme solution was incubated at 508C for 1 h. The N-
acetylglucosamine (GlcNAc) residues produced were estimated colorimetrically at 585 nm
with p-dimethylamino benzaldehyde (DMAB) as described by Reissig, Strominger, and
Leloir (1955). One international unit was defined as the activity which produced 1 mmol of
GlcNAc per min.

Chitosanase assay
Chitosanase activity was estimated using acid-swollen chitosan as a substrate. For
preparation of acid-swollen chitosan, crystalline chitosan was swollen with 10 N HCl.
The pH of the swollen chitosan was adjusted to 7 with 1 N NaOH. Repeated washing of
swollen chitosan was carried out with ice-cold distilled water by centrifugation at 10,000 
g for 10 min. The swollen chitosan was dialysed at 48C against distilled water. After
homogenisation in a Waring blender for 1 min, the concentration of swollen chitosan was
adjusted to 10 mg/mL by adding 50 mM acetate buffer, pH 5.
The assay mixture containing 1 mL acid-swollen chitosan (10 mg/mL), 1 mL of 50 mM
acetate buffer, pH 5, and 1 mL of enzyme was incubated at 508C for 1 h. The amount of
812 S.A. Kulkarni et al.

glucosamine produced was determined using the method of Reissig et al. (1955). One unit
of enzyme produced 1 mmol of glucosamine equivalents per min.

Chitin deacetylase assay

CDA activity was measured according to Nahar et al. (2004a), using acetylated ethylene
glycol chitosan as a substrate, prepared according to the method of Araki and Ito (1975).
For preparation of the substrate, ethylene glycol chitosan (40 mg) was treated at 48C with
400 mg of NaHCO3 and 200 mmol of acetic anhydride in a total volume of 4.5 mL and kept
at 48C. After 24 h, 200 mL of acetic anhydride were added and the mixture was allowed to
stand for further 24 h at 48C. After thorough dialysis, the product, acetylated ethylene
glycol chitosan (1 mg/mL) was used as a substrate for the assay of CDA.
The assay for CDA was carried out according to Kauss and Bausch (1988) with 100 mL
of 50 mM sodium tetraborate buffer, pH 8.5, 100 mL of 1 mg/mL acetylated ethylene glycol
chitosan, and 50 mL enzyme incubated at 378C for 30 min. The reaction was terminated
with addition of 250 mL of 5% (w/v) KHSO4. For colour development, 250 mL of 5% (w/v)
NaNO2 was added and allowed to stand for 15 min, and then 250 mL of 12.5% (w/v)
ammonium sulfamate (N2H6SO3) was added. After 5 min, 250 mL freshly prepared 0.5%
(w/v) 3-methyl-2-benzothiazoline hydrazone (MBTH) was added and the mixture was
heated in a boiling water bath for 3 min. The tubes were cooled under tap water and 250 mL
of freshly prepared 0.5% (w/v) FeCl3 was added and estimated spectrophotometrically at
650 nm. One unit of enzyme released 1 mmol of glucosamine from acetylated ethylene
glycol chitosan per min.

Protease assay
Protease activity was measured using Hammerstein casein as a substrate (Vyas and
Deshpande 1989). The reaction mixture contained 100 mL of suitably diluted enzyme
solution, 1 mL Hammerstein casein (1%) and 1 mL of 200 mM sodium carbonate buffer,
pH 9.7. Enzyme reaction was carried out at 358C for 20 min and terminated by the
addition of 3 mL trichloroacetic acid (TCA) (2.6 mL of 5% TCA0.4 mL of 3.3N HCl).
The absorbance of the TCA soluble fraction was measured at 280 nm. One unit of enzyme
liberated 1 mmol of tyrosine per min.

Lipase assay
Lipase activity was determined as described by Pignede et al. (2000). The substrate
emulsion was prepared with olive oil (50 mL) and gum arabic (50 mL, 10%, w/v, Sigma).
The reaction mixture contained 1 mL enzyme, 5 mL substrate emulsion, and 2 mL of 50
mM phosphate buffer, pH 6.8, and was incubated at 37 8C for 1 h under shaking (80 rpm).
The reaction was stopped with 4 mL of acetone
ethanol (1:1) containing 0.09%
phenolphthalein as an indicator. Enzyme activity was determined by titration of the fatty
acids released with 50 mM NaOH. One unit of lipase is the amount of enzyme that
released 1 mmol of fatty acids per min.

Insect rearing and bioassay

The initial colony of H. armigera was established by collecting larvae and pupae of
the insect from pulse fields. Larvae were reared individually in sterile polypropylene vials
 Biocontrol Science and Technology 813

(42 65 mm, 50 mL capacity; Laxbro Manufacturing Co., Pune, India) containing pieces
of okra (Abelmoschus esculentus) as diet. Okra was first disinfected for 10 min with 0.5%
sodium hypochlorite (Ignoffo, Futtler, Marston, Hostetter, and Dickerson 1975). Insect
eggs laid during rearing were also surface-sterilised with 0.5% sodium hypochlorite.
Conditions in the insect-rearing room were maintained at 25928C and 6595% RH.
Third instar larvae of H. armigera were used in bioassays of Metarhizium conidia. The
7-day-old sporulating cultures of PDA grown Metarhizium isolates were used to prepare
the conidial suspensions. Conidia were lightly scraped off the culture surface in 0.1% (w/v)
Tween 80 to prepare conidial suspensions (1 107conidia/mL). A set of 30 larvae with
three replications treated with 0.1% (w/v) Tween 80 in sterile distilled water served as
control. Larvae were dipped individually in 10 mL conidial suspensions of the isolates for
5 s. The experiment was conducted 3 times using freshly prepared conidial suspensions.
After treatment, each larva was individually transferred to a separate sterile vial containing
moist Whatman No. 1 filter paper and a piece of disinfected okra. The diet was changed
every other day and the larvae were kept at 25928C, 6595% RH and 16 h L:8 h D for 14
days. Dead larvae were transferred to sterile Petri plates containing moist cotton swabs and
kept at 28928C at 70
80% RH for at least 3
7 days to allow mycelial growth and conidia
formation over the cadavers. The data on percent mortality from three experiments were
pooled to get average values, which were corrected by Abbott’s formula (Abbott 1925).
The studies to determine median lethal time (LT50) were carried out using M2104,
M34311, M34412, M81123, M91427, M91629, M101133, M101335, M101537 and
M16760. The estimates of LT50 for mortality at 14 days post treatment were determined
using probit analysis (Throne, Weaver, Chew, and Baker 1995).
Median lethal concentration (LC50) studies for M34311, M34412, M81123, M91427
and M91629, were carried out with four concentrations of conidia: 1 103, 1 105, 1107
and 1109 conidia/mL to increase the possibility of identifying the difference in virulence
of isolates with high mortality values that might go undetected if only a single dose was
used. The experimental layout was a RCBD with each treatment containing a set of 30
larvae repeated 3 times. The estimates of LC50 were calculated manually using probit
analysis according to Finney (1981). For both LT50 and LC50, observed chi-square values
were compared with tabulated Chi-square values. SPSS (11) and SAS (9.1) were used for
the regression analysis, F-test and cluster analysis, respectively. In case of the regression
analyses the standardised b coefficients give a measure of the contribution of each variable
to the model. A large value indicates that a unit change in this predictor variable has a
large effect on the criterion variable. The t and significance (P) values give a rough
indication of the impact of each predictor variable
a big absolute t value and small
P value suggests that a predictor variable is having a large impact on the criterion variable.

Production of conidia
Conidia of M34311, M34412, M81123, M91427 and M91629, were produced by solid-state
fermentation. The inoculum was prepared by inoculating 2 107 conidia in 200 mL YPG
medium and incubating at 28928C under shaking (180 rpm) for 2 days. The unicorn-bags
(autoclavable, type/14 with single microvented filter of 0.2 mm, 2 kg capacity, 6436 cm,
Unicorn Imp & Mfg Corp, USA) filled with 2 kg of rice as a substrate were autoclaved at
1218C for 45 min and inoculated with 200 mL of mycelial inoculum (3593 g, wet wt). The
bags were incubated at 28928C and 70
80% RH for 14 days, after which they were dried at
37928C for 2 days to B20% moisture. The conidia were harvested with a Mycoharvester
(CABI Bioscience, UK), and yield (g/kg substrate) and percent viability of first quality
814 S.A. Kulkarni et al.

conidia (mesh size B100 mm) was determined from three different bags for each isolate.
For determining the percent viability, conidial suspensions were prepared in 0.1% (w/v)
Tween 80 and count was adjusted to 1 102 conidia/mL for inoculating PDA plates in
triplicate for each isolate. The plates were incubated at 28928C and 70
80% RH for 72 h
after which colonies were counted. The experiment was conducted 3 times. The percent
viability was transformed to arcsine values to improve the homogeneity of variances. The
data were then subjected to analysis of variance and the means were compared using F-test
at a 0.05 level of critical difference as described by Panse and Sukhatme (1989).

Conidial sedimentation time (ST50)

The conidial sedimentation rates for M34311, M34412, M81123, M91427 and M91629,
were determined as described by Jeffs and Khachatourians (1997). Conidial suspensions
(1.790.3 107 conidia/mL) were prepared in 0.2 M (NH4)2SO4 to obtain an initial
absorbance of 0.600 at 540 nm. The cuvettes were allowed to stand undisturbed for settling
of conidia for 6 h. The absorbance with an interval of 1 h was recorded for each isolate.
The experiment was conducted 3 times using freshly prepared conidial suspensions. The
sedimentation rate of conidia was also studied in 0.1% (w/v) Tween 80. The sedimentation
rate was expressed in percent and the time required for 50% sedimentation (ST50) was
calculated.

Effect of malic acid on conidial germination and appressorium formation

Conidial suspensions of M34311, M34412 and M81123, prepared in 0.1% (w/v) Tween 80
were inoculated (100 mL of 1 107 conidia/mL) separately in 5 mL YPG liquid medium
with and without addition of filter sterilised malic acid (1, 5, 10 and 15 mM/mL) and
incubated at 288C and 70
80% RH. Light microscopic observations were made every 2 h
until the germination was initiated, and then the nutrient source was depleted by
centrifugation at 10,000 g for 10 min as described by Xavier-Santos, Magalhaes, Elza,
and Luna-Alves (1999). Germinated conidia were washed twice with sterile distilled water
and the pellets were suspended in 100 mL sterile distilled water. These suspensions were
placed separately at the centre of polypropylene Petri plates, sealed with parafilm and
incubated at 288C and 70
80% RH. The plates were observed microscopically for
appressoria development periodically for 24 h. Counts for number of conidia (minimum
30 conidia/field) producing germ tubes and/or appressoria were taken every 2 h randomly
in 10 different fields per plate. The experiment was conducted thrice, each time arranged in
a RCBD, using freshly prepared suspensions. The data on percent appressoria formation
were subjected to arcsine transformation and subjected to analysis of variance. The means
were compared using F-test at a 0.05 level of critical difference as described by Panse and
Sukhatme (1989).

Genetic analysis
Conidial suspensions (1 107 conidia/100 mL of medium) of M34311, M34412 and
M81123, were inoculated in a semi-synthetic complete medium containing (g/L): KH2PO4,
0.36; Na2HPO4, 1.6; KCl, 1; MgSO4, 0.6; NH4NO3, 0.7; yeast extract, 5; glucose, 10; pH, 7
and incubated at 28928C under shaking conditions (180 rpm) for 48 h. The mycelium
from each flask was harvested separately by vacuum filtration and the genomic DNA was
extracted using Qiagen DNeasy Plant Mini kit.
 Biocontrol Science and Technology 815

The genomic DNA was amplified using ITS1 (5’-TCCGTAGGTGAACCTGCGG-3’)

and ITS4 (5’-TCCTCCGCTTATTGATATGC-3’) primers in PE 9700 thermocycler.
Amplification conditions were: initial denaturation at 948C for 2 min, followed by 35
cycles of denaturation at 948C for 1 min, annealing at 508C for 1 min and extension at 728C
for 1 min with final extension at 728C for 10 min. The PCR was carried out in 25 mL
reaction mixture consisting of 10 Taq polymerase buffer (New England Biolabs), 2 mM
dNTPs, 10 pM primers, 1 unit Taq polymerase (New England Biolabs), and 10 ng DNA.
The positive amplicons were purified by PCR Cleanup Qiagen Kit. Purified PCR products
were sequenced on both strands on an AB3730 DNA analyser using the Big Dye
terminator kit (Applied Biosystems, Inc., Foster City, CA). The sequences were analysed at
the National Center for Biotechnology Information (Bethesda, MD) (http//:
www.ncbi.nlm.nih.gov/BLAST) for close homology. Alignments of sequences with close
homology were carried out with Clustal using Bioedit program.
For RAPD analysis, six individual 10 nucleotide random primers were used for DNA
amplification (OPX03, 5’-TGGGGCAGTG-3’; 923GT, 5’-GGTGGTTGGG-3’; OPA03,
5’-AGTCAGCCAC-3’;OPA07, 5’-GAAACGGGTG-3’; OPA13, 5’-CACCACCCAC-3’;
935G, 5’-GGGTTGTGGG-3’). The reaction mixture (20 mL) consisted of 10Taq
polymerase buffer (New England Biolabs), 2 mM dNTPs, 10 pM primers, 1 unit Taq
polymerase (New England Biolabs), and 10 ng DNA. The RAPD protocols were: initial
denaturation at 948C for 4 min, followed by 40 cycles of denaturation at 948C for 1 min,
annealing at 508C for 1 min, extension at 728C for 1 min and final extension at 728C for 10
min. The RAPD-PCR amplicons were resolved in 2.5% agarose gel in 1 Tris borate
EDTA (TBE) buffer (pH 7.5) and stained with ethidium bromide and documented for
RAPD analysis. The RAPD gels were replicated thrice for determining the consistency of
results. Visual analysis of stained gel photographs was used to detect any change in
banding patterns. Similarity index was calculated from 2nxy/nxny, when nxy was the
number of common DNA bands in x and y isolates, while nx and ny were the total DNA
bands.

Field experiment
Performance of three M. anisopliae isolates was evaluated by applying conidia against
H. armigera in chickpea fields (variety Vishal) with the chemical insecticide endosulfan and
untreated plots as controls. Field experiments were conducted at the Agriculture
University, Rahuri, Ahmadnagar, Maharashtra (19820’N and 74835’E) for two successive
years during Rabi season (October
March) 2005
2006 and 2006
2007.The experimental
layout was a RCBD with five treatments and three replications. The plot size was 5 5 m.
The crop was sown during the first fortnight of October and was raised following normal
agronomical practices.
Based on the previous criteria, the three selected Metarhizium isolates M34311,
M34412 and M81123 with dosage of 51012 conidia/ha in 0.1% (w/v) Tween 80
suspension, and endosulfan at 350 g.a.i. were sprayed with a hand operated knapsack
sprayer. Control plots were sprayed with 0.1% (w/v) Tween 80. According to agricultural
practices for the chickpea crop, the insecticides were first sprayed 10
15 days after egg
laying and thereafter 2 more times with a 14-day interval. The spraying was carried out
between 16:00 and 18:00 h IST. Precautions such as monitoring wind direction and using
cloth curtains as necessary were taken to avoid wind drift to neighbouring plots.
Five plants per plot were randomly selected and tagged for recording observations. Live
larvae were counted before treatment and after each treatment on the 3rd, 7th and 10th
816 S.A. Kulkarni et al.

days. The percent efficacy was calculated for each treatment according to Henderson and
Tilton (1955). The observations on pod damage were recorded on five randomly selected
plants at the time of harvest by counting the total number of healthy and damaged pods.
The percent pod damage was calculated and the percentages were further transformed to
arcsine values for statistical analysis. The data on larval population for the 2 years of field
evaluations was converted to square root n0.5 transformation. The data was then
subjected to analysis of variance and the means were compared using F-test at a 0.05
level of a critical difference as described by Panse and Sukhatme (1989).

Results
Isolation of Metarhizium strains
The data in Table 1 shows the origin of 63 Metarhizium strains isolated from soil and insect
hosts of various regions in and around Pune, India.

Production of cuticle degrading enzymes and mortality against H. armigera

Enzyme activities of all 63 Metarhizium isolates ranged from 0.42
5.10 10 3 U/mL for
chitinase, 0.31
3.38 U/mL for protease, 0.083
0.996 U/mL for lipase, 0.20
6.48 10 3 U/
mL for CDA and 1.25
49.6 103 U/mL for chitosanase. Bioassays targeting H. armigera
showed mortality between 20 and 97% within 14 days.

Table 1. Origin of Metarhizium isolates.

Isolate Crop/Insect Total isolates

Source: Soil
M1311, M1322, M1333, M2104, M2305, M2416, Tomato 15
M2427, M2508, M42014, M45115, M45216,
M45317, M79120, M79221, M79322
M3419, M34210, M34311, M34412, M34513 Custard apple 5
M81123, M91124, M91225, M91326, M91528, Sugarcane 11
M91427, M91629, M91730, M91831, M91932, M111145
M101133, M101234, M101335, M101436, M101537, Brinjal 12
M101638, M101739, M101840, M101941, M102042,
M102143, M102244
M51118, M51219 Okra 2
M131150, M141151, M141252, M151153 Pigeon pea 4
M121146, M121247, M121348, M121449 Chickpea 4
Source: Insect hosts
M16255, M16356, M16457, M16558, M16659 Pigeon pea-Greasy 5
cutworm
M16154, M16760 Sugarcane-Mealybug 2
M16861 Sugarcane-White grub 1
M16962 Sugarcane-Beetle 1
M161063 Sugarcane-Pyrilla 1
perpussila
Isolate identity (I.D.) number for soil isolates is based on crop, geographical location, plot, sub-plot if any, sample
and isolate number. Isolate identity (I.D.) number for insect isolates is based on crop, geographical location, host
and isolate number.
 Biocontrol Science and Technology 817

Evaluation of the relationship between extracellular CDE activities and the pathogenic
potential of all 63 isolates was performed with regression analysis (Table 2). Multivariate
regression for effect of five enzyme activities on mortality generated a model with high
R value (0.814) that was significant (P B0.0001). However, as the regression coefficient for
lipase (0.994) and chitosanase (0.182) were not significant, the masking effect of chitinase
and protease was explored. Elimination of chitinase and protease singly and in
combination showed increase in the significance of lipase from 0.994, 0.913, 0.164 and
0.039 and chitosanase from 0.182, 0.104, 0.03 and 0.003, respectively.
As all five of the enzyme activities were important for initiating pathogenicity, they
were considered together with mortality data for cluster analysis. Cluster analysis of the
enzyme activities with mortality showed two major clusters that explained the variability in
the sample group of 63 isolates (Figure 1). Metarhizium isolates from the larger group were
further grouped into eight clusters based on the different levels of enzyme activities and the
mortality displayed by different strains. The average values for CDE activities and the
mortality of H. armigera for all nine clusters are given in the Table 3.
The eight potential isolates, namely M2104, M34311, M34412, M81123, M91427,
M91629, M101335, M16760, from cluster 8; and two isolates, M101133 and M101537,
from cluster 7 were selected for further studies. The isolates, M101133 and M101537,
produced high levels of chitinase, protease, CDA and lipase activities, and showed 90%
mortality of H. armigera in the bioassay; however, due to low chitosanase levels were
grouped in the seventh cluster. On the other hand, the two isolates, M2427 and M91124,
from cluster 8 were not selected because of their relatively lower chitinase, protease and
CDA activities and percent mortality of H. armigera as compared to other isolates from
the same group. The CDE activities of all the 10 selected isolates are given in Table 4. It can
be seen that, among the 10 isolates, M91629 produced highest levels of chitinase activity,
M34311 CDA activity and M2104 chitosanase activity. The protease and lipase activities
were high in M34412.

Determination of Median Lethal Time (LT50) against H. armigera

Day-wise mortality was recorded to determine the fastest kill time required for 10 M.
anisopliae isolates (Table 5). M. anisopliae M34412 recorded lowest LT50 (3.3 days) while
LT50 values were 3.3, 3.5, 3.6 and 4.1 days for M81123, M34311, M91629 and M91427,
respectively (Table 5). The remaining isolates had relatively higher LT50 values with
M16760 having the highest at 6.8 days. All Chi-square values were not significant (a 0.05)
indicating good fit of regression lines. Based on the LT50 results, M34311, M34412,
M81123, M91427 and M91629 were selected for conidia production on a solid substrate.

Production, viability and settling time for conidia of Metarhizium isolates

Conidia production was carried out with five selected isolates, M34311, M34412, M81123,
M91427 and M91629, using rice as a substrate. The conidial yields were not significantly
different from each other. The conidial yields from all five isolates were in the range of 60

75 g/kg substrate and the number of conidia/g of first quality spore powder obtained
as described under Materials and methods section, were in the range of 4.04 1010
4.4 1010. The total conidial count was 281011 from 70 g conidial powder recovered
from 1 kg of substrate on an average. The percent viability ranged from 92 to 97% (Table
6). The ST50 values determined using 0.2 M (NH4)2SO4 were in the range from 2.1 to 2.5 h.
For the bioassays as well as for spraying in the field, 0.1% (w/v) Tween 80 was used to
 818
Table 2. Regression analysis of cuticle degrading enzyme activities of Metarhizium isolates with Helicoverpa armigera percent mortality caused by the
isolates.

Regression coefficient Model summary

S.A. Kulkarni et al.

Chitinase CDA Chitosanase Protease Lipase
Model variables t b t b t b t b t b R r2 F P

1. All 5.949*** (0.440) 3.684** (0.238) 1.35 (0.100) 5.476*** (0.451) 0.007 (0.001) 0.902 0.814 49.8 (0.0001)
2. () Chitinase

3.849*** (0.309) 1.65 (0.154) 7.476*** (0.683) 0.109 (0.011) 0.835 0.698 33.5 (0.0001)
3. () Protease 7.936*** (0.632) 2.295* (0.179) 2.186* (0.194)

1.408 (0.128) 0.846 0.716 36.5 (0.0001)
4. () Chitinase

2.286* (0.254) 3.126** (0.383)

2.108* (0.269) 0.638 0.407 13.5 (0.0001)
and Protease
*Indicates significance of P value. ***P B 0.0001, **P B 0.001 and *P B 0.05. All: five enzyme variables: chitinase, protease, lipase, CDA, chitosanase
 Biocontrol Science and Technology 819

Figure 1. Average linkage cluster analysis of Metarhizium isolates based on the production of
extracellular cuticle degrading enzyme activities and percent corrected mortality of Helicoverpa
armigera in the bioassay.
820 S.A. Kulkarni et al.

Table 3. Cluster analysis of Metarhizium isolates based on the production of extracellular cuticle
degrading enzyme activities and the mortality of Helicoverpa armigera.

Average value

No. of Chitinase Protease Lipase CDA Chitosanase Mortality of

Cluster isolates ( 10
3 U/ml) (U/ml) (U/ml) (10
3 U/ml) (10
3 U/ml) H. armigera (%)

1 10 2.12 1.59 0.22 1.71 10.79 68.67

2 3 0.94 0.94 0.22 2.61 14.18 57.18
3 8 1.79 1.23 0.24 3.09 4.25 59.03
4 3 2.49 1.60 0.17 0.56 22.76 68.89
5 7 2.95 1.71 0.23 2.47 4.27 78.10
6 7 3.27 2.11 0.31 2.55 4.01 86.19
7 7 2.76 1.91 0.30 3.17 12.29 86.16
8 10 3.75 2.80 0.53 2.20 34.39 92.11
9 8 0.72 0.50 0.17 1.34 3.63 36.39

maintain homogenous conidial suspensions. It can be seen from Table 6 that in the
presence of Tween 80, the ST50 values were higher and in the range 2.3
3.1 h (Table 6).

Determination of median lethal concentration (LC50) against H. armigera

The number of conidia required to kill 50% (LC50) third instar larvae of H. armigera was
calculated for the selected five isolates at 1 103 conidia/mL (Table 7) as well as at 105, 107,
109 conidia/mL (data not shown). Isolate M34412 was more virulent than other isolates in
this experiment based on LC50 values. M34311 and M81123 displayed low LC50 values
(B6 103 conidia/mL) while M91427 and M91629 showed high LC50 values ( 16103
conidia/mL). All Chi-square values were not significant (a 0.05) indicating good fit of
regression lines (Table 7). Therefore, isolates, M34311, M34412 and M81123 were further
studied for conidial germination and appressorium formation with and without malic acid,
a chickpea exudate and for their performances in the field.

Table 4. The levels of extracellular cuticle degrading enzyme activities (mean9SD) produced by
Metarhizium isolates.

Chitinase CDA Chitosanase Protease Lipase

Isolate No. (10
3 U/ml) (10
3 U/ml) (10
3 U/ml) (U/ml) (U/ml)

M2104 3.290.2a 2.490.2b 49.692.0e 3.290.3b 0.590.04b

M34311 3.590.2a 3.290.2c 35.792.1d 3.390.2b 0.790.08d
M34412 3.990.1c 1.390.2a 32.491.1c 3.490.1c 1.090.04e
M81123 3.890.2c 2.590.2b 35.891.6d 3.390.2c 0.790.04d
M91427 3.990.2c 2.590.2b 25.191.1b 2.490.2a 0.590.04b
M91629 5.190.3d 2.390.2b 26.291.0b 3.290.2b 0.690.04c
M101133 3.390.3a 2.690.2b 12.591.4a 2.990.3b 0.390.04a
M101335 3.790.2c 2.590.2b 33.991.4c 3.090.2b 0.390.04a
M101537 3.490.3a 2.390.2b 12.291.0a 3.190.2b 0.390.04a
M16760 3.690.2a 2.790.2b 27.691.0b 2.490.3a 0.390.04a
Numbers followed by the same letter within a column are not statistically different. SD, Standard deviation.
 Biocontrol Science and Technology 821

Table 5. The Median Lethal Time (LT50) of third instar larvae of Helicoverpa armigera exposed to
isolates of Metarhizium anisopliae.

Isolate No. Chi-square value LT50 (days) Fiducial limit (days)

M2104 10.79 (12.59) 4.8 4.5
5.1

M34311 0.67 (18.31) 3.5 3.2
3.7
M34412 2.74 (14.07) 3.3 3.0
3.6
M81123 1.96 (15.51) 3.3 3.1
3.6
M91427 2.28 (15.51) 4.1 3.7
4.4
M91629 4.05 (15.51) 3.6 3.3
3.9
M101133 2.03 (15.51) 6.7 6.3
7.0
M101335 0.69 (15.51) 4.6 4.2
5.0
M101537 10.16 (15.51) 6.4 6.1
6.7
M16760 2.50 (15.51) 6.8 6.4
7.1
LT50, the median lethal time of conidia calculated to cause 50% mortality of H. armigera. Figures in parentheses
are tabular Chi-square (a 0.05)

Effect of malic acid

The effect of malic acid (1
15 mM) on the conidial germination and/or appressorium
formation on three M. anisopliae isolates showed that conidial germination as well as
appressorium formation were 90% even in the presence of 15 mM malic acid. In the
control experiment without malic acid conidial germination and appressoria formation
were 92% (data not shown).

Genetic analysis
The 438-base pair long ITS region sequences from the three isolates, M34311, M34412
and M81123 were aligned with M. anisopliae var anisopliae strain sequences deposited
in the NCBI database (EU307893 and AF135210). The ITS sequences were homolo-
gous (100% identical) to the M. anisopliae var anisopliae strain sequences. On the basis
of the RAPD banding pattern with primers 923G, OPA03, OPA13, OPX03 and OPA07,
the three isolates, M34412, M34311 and M81123 showed 100% similarity while with
primer 935G, isolate M34311 showed 71% similarity with isolate M34412 and M81123
(Figure 2).

Table 6. Production, viability and sedimentation time (ST50) for conidia of Metarhizium isolates.

Isolate Yield (g/kg rice) mean9SD Viability (%) mean9SD ST50 in T80 (h) Fiducial limit (h)

M34311 60.0092.64a 92.0092.64a 2.47 2.26
2.69

M34412 67.0093.46b 97.0091.73a 2.30 2.11
2.52
M81123 75.0093.60c 93.0091.73a 2.65 2.43
2.90
M91427 68.6692.08a 92.6696.11a 3.09 2.84
3.37
M91629 70.0093.00b 92.0091.00a 2.79 2.59
3.00
ST50, time required to sediment 50% conidia in 0.1% (w/v) Tween 80. Numbers followed by the same letter within
the column are not statistically different. SD, Standard deviation. T80, Tween 80 (0.1%, w/v).
822 S.A. Kulkarni et al.

Table 7. The Median Lethal Concentration (LC50) of third instar larvae of Helicoverpa armigera
exposed to isolates of Metarhizium anisopliae.

Chi-square LC50 Fiducial limit

Isolate No. value Slope SE of slope (103 Conidia/mL) (103 Conidia/mL)

M34311 1.47 (5.99) 0.299 0.0772 2.0 0.4
10.3

M34412 2.50 (5.99) 0.246 0.0762 1.4 0.1
1.9
M81123 2.95 (5.99) 0.281 0.0727 5.7 1.2
26.7
M91427 0.096 (5.99) 0.299 0.0726 35.8 9.1
140.7
M91629 3.31 (5.99) 0.266 0.0666 16.8 4.5
62.3
LC50, the Median Lethal Concentration of conidia calculated to cause 50% mortality of H. armigera after 14 days.
SE, Standard error. Figures in parentheses are tabular Chi-square values (a0.05).

Field experiment
Differences in the larval population due to different treatments were recorded up to 42
days (Figure 3). Larval survival was highest in the control. The mean number of surviving
larvae per plant were 1.6, 1.6, 2.1 and 2.1 after treatment with endosulfan, M34412,
M34311 and M81123, respectively, and 5.4 in the untreated control (Figure 3). The mean
percent efficacies with chemical insecticide endosulfan and M81123, M34311 and M34412
were in the range of 65
74%. The percent pod damage in the untreated control was 42%
while in the treated plots it was between 17 and 26% (Table 8). Accordingly, the grain yields
were also increased with different treatments (Table 8).

Discussion
Metarhizium anisopliae has been regarded as a mycoinsecticide since the time of
Metchkinikov (Lord 2005). The virulence factors such as CDE production, mainly of
chitinase and protease, toxic metabolite production, appressorium formation, hydropho-
bicity of conidia were found to be significant determinants of virulence (Hajek and St.
Leger 1994; Jeffs and Khachatourians 1997; Quesada-Moraga and Vey 2003; Murad,
Laumann, Mehta, Noronha, and Franco 2007). Nahar et al. (2008) reported earlier that
the reduction in virulence of M. anisopliae M34412 after 40 in vitro transfers on artificial
media was associated with the reduction in extracellular CDE activities including CDA,
chitosanase and lipase.
St. Leger et al. (1986) reported that the presence of an insect cuticle or chitin in the
medium induced sequential production of protease, chitinase and lipase activities in
response to the cuticular composition. Later, constitutively produced CDA and chitosa-
nase in YPG medium were also reported to be important in the fungus
insect interaction
(Nahar et al. 2004a). Modelling with regression analysis for effect of CDE activities on
virulence showed that lipase and chitosanase were not highly significant. However, with
removal of chitinase and protease from the model, the significance of lipase and
chitosanase increased. This could be attributed to the masking effect of the strong value
of chitinase and protease in relation to mortality. Removal of protease from the model had
more effect in increasing the effect of lipase (Table 2).
Earlier, St. Leger et al. (1986), St. Leger, Joshi, Bidochka, Rizzo, and Roberts (1996)
reported that chitinases and proteases were virulence factors for entomopathogenicity. In
the present investigations, chitinase and protease activities also showed high correlation
with mortality. Fang et al. (2005) reported that the endochitinase from M. anisopliae
 Biocontrol Science and Technology 823

Figure 2. Representative agarose gel electrophoresis of RAPD-PCR products for Metarhizium

anisopliae isolates M34412, M34311 and M81123, respectively. Lanes 1
3, Primer 923G; Lanes 4
6,
Primer OPA03; Lane 7, DNA ladder; Lanes 8
10, Primer OPA13; Lanes 11
13, Primer 935G; Lane
14; DNA ladder; Lanes 15
17; Primer OPX03; and Lanes 18
20, Primer OPA07.

showed only marginal correlation with virulence towards the aphid, Myzus persicae while
overproduction of endochitinase increased virulence of Beauveria bassiana towards aphids.
This variation could be attributed to the multiplicity of chitinases (Patil, Ghormade, and
Deshpande 2000). Hegedus and Khachatourians (1995) suggested that the significance of
any enzyme(s) was dependent upon the cuticle characteristics and physiological state of the
insect as well as the mechanism of invasion by the fungus. For instance, the lipase activity
contributed to the initial phase of infection when the germinating conidium had to break
the epicuticle layer to gain entry into the insect.
Recently, Sahayaraj and Borgio (2007) isolated M. anisopliae from soil samples from
different crop fields and tested their biocontrol potential on the cotton pest Dysdercus
cingulatus. Bidochka, Kamp, Lavender, Dekoning, and Amritha De Croos (2001) reported
that M. anisopliae could survive in the soil in absence of the insect host and that the habitat
could influence the evolution of fungus
host interaction. Recently, Hu and St. Leger (2002)
824 S.A. Kulkarni et al.

Figure 3. The effect of Metarhizium anisopliae and endosulfan on the larval population of
Helicoverpa armigera on chickpea under field conditions. (j
j), Endosulfan; Metarhizium anisopliae
isolates (%
%), M34311; (m
m), M34412; ('
'), M81123; and ("
"), untreated control.

demonstrated the rhizosphere competence of M. anisopliae in field studies with cabbage

plants up to a period of 1 year. Presumably Metarhizium species can very well survive and
grow saprophytically in the soil, suggesting that all the 63 isolates in the present study were
not expected to have an effective bio-control potential. Therefore, virulence factors such as
CDE activities were considered as an important parameter for the initial screening. All 63
isolates when subjected to cluster analysis using CDE activities and mortality of
H. armigera, clustered into nine groups. Interestingly, the correlation between CDE
activities and mortality was seen in all groups (Table 3). The 10 isolates from group 7 and 8
viz M2104, M34311, M34412, M81123, M91427, M91629, M101133, M101335, M101537,
M16760 were further studied. The levels of CDE activities again played a crucial role in the
selection of two isolates from group 7 (Figure 1, Table 3). Furthermore, on the basis of
LT50, five isolates, M34311, M34412, M81123, M91427, M91629, were selected (Table 5).
One of the isolates, M2104 possessing higher LT50 (4.8 days) than the 5 isolates (3.3
4.1
days) was also selected for further studies (Table 5) even though it had high chitosanase
activity (Table 4). It can be suggested that softening of the cuticle by the action of CDA to

Table 8. The evaluation of Metarhizium anisopliae isolates against Helicoverpa armigera

infestation on chickpea under field conditions.

Efficacy (%) Pod damage (%) Grain yield (kg/ha)

Treatments mean9SD mean9SD mean9SD

Endosulfan 74.095.9a 17.5910.6a 205392.2c

M34311 65.2921.1a 26.3910.6b 161096.4b
M34412 72.2920.5a 19.5910.6a 187894.2b
M81123 65.8919.9a 21.4910.6a 166293.2b
Untreated control
42.1910.6c 101393.6a
The percent efficacy was calculated using Henderson and Tilton (1955) over untreated control.
Numbers followed by the same letters within a column are not statistically different. SD, standard
deviation.
 Biocontrol Science and Technology 825

facilitate the penetration process could be more crucial than having synergisitic action of
CDA with chitosanase for cuticle degradation. The isolate from the mealybug, M16760 did
not qualify in the first five as the LT50 was high (6.8 days). This might be attributed to host
specificity of M. anisopliae isolate.
All five isolates were further evaluated for the parameters which are important in
conidia production. Yield, viability and sedimentation time of conidia were comparable for
all five isolates (Table 6). Jenkins, Heviefo, Langewald, Cherry, and Lomer (1998) reported
5.0 1010 conidia per g of pure conidial powder of M. flavoviride developed as a biocontrol
agent. Collection of conidia with the Mycoharvester gave an average yield of 1.5 109 per g
of rice substrate. In the present study, the yield per g of rice was 2.0 109 conidia. The
hydrophobicity of conidia is one of the essential characteristics for adhesion on the insect
cuticle. Metarhizium anisopliae strains M34311, M34412, M81123, M91427 and M91629
showed ST50 values in the range of 2.1
2.5 h in the presence of 0.2 M (NH4)2SO4. To
maintain the conidia in suspension, 0.1% (w/v) Tween 80 is often used. Therefore, the
sedimentation of conidia in 0.1% (w/v) Tween 80 was studied. As compared to the ST50
values in 0.2 M (NH4)2SO4 (2.1
2.5 h), ST50 values in 0.1% (w/v) Tween 80 (2.3
3.1 h) were
higher. Three isolates, M34311, M34412 and M81123 had LC50 in the range 1.4
5.7 103
conidia/mL. The LC50 value of the main isolate M34412 (1.4 103 conidia/mL) was
comparable to M34311 (2.04103 conidia/mL), while LC50 value for M81123 was 5.7 
103 conidia/mL. For isolates M91427 and M91629, the LC50 values were higher (16.8 
103 conidia/mL). While Nguyen, Borgemeister, Poehling, and Zimmermann (2007)
reported LC50 against third instar H. armigera as 6.0 105 conidia/mL, Kumar and
Chowdhury (2004) reported LC50 of 1.23 103 conidia/mL against second instars.
The acidic chickpea leaf exudate mainly consisted of malic acid (1.5
3 mmol/g of fresh
chickpea leaves) (Li and Copeland 2000). Nahar et al. (2003) reported that the acidic
exudates did not affect germination and performance of conidia of isolate M34412, per se
in the field. In case of M34311 and M81123, malic acid did not affect conidial germination.
The ITS and RAPD patterns of the three selected isolates were found to be similar to
M. anisopliae var. anisopliae strains reported by Driver, Milner, and Trueman (2000) and
Inglis, Duke, Goettel, and Kabaluk (2008). Bidochka, McDonald, St. Lager, and Roberts
(1994) reported the use of RAPD to differentiate species and strains of entomopathogenic
fungi. Milner et al. (2002) used eight primers and reported that one primer, F06, generated
differences in the RAPD banding pattern among 11 strains of M. anisopliae var. anisopliae
and proposed the use of RAPD pattern for ecological monitoring of released strains. In the
present study, RAPD analysis with primers 923G, OPA03, OPA13, 935G, OPX03 and
OPA07 did not display any significant differences in the banding pattern with the three
strains (Figure 2). However, primer 935G used for RAPD showed 71% similarity of
M34311 with the other two isolates while the other five primers used showed 100%
similarity. Neither RAPD polymorphism nor ITS sequence was able to discriminate the
three selected isolates which otherwise could be differentiated by other phenotypical
characteristics such as CDE production and mortality against insect host. However, the
additional molecular markers such as microsatellites, SCAR and SNPs can also be used.
All three M. anisopliae var. anisopliae strains, M34311, M81123 and M34412 were
studied for their field performance during 2005
2006 and 2006
2007. The percent efficacy
with the chemical insecticide, endosulfan was 74%. With the treatment of the three
Metarhizium isolates, the percent efficacy was from 65 to 72% (Figure 3, Table 8).
The three strains will be further studied for their non-target and toxicity data as a pre-
requisite for registration. These can further be tested singly and in IPM sequentially
826 S.A. Kulkarni et al.

against H. armigera and other insect hosts to increase the scope of mycoinsecticide use in
agriculture.

Acknowledgements
The authors gratefully acknowledge the critical evaluation by the two anonymous reviewers. The
authors are grateful to Indo-Swiss Collaboration in Biotechnology programme for financial support.
The authors thank Drs S. Rao, ARI, Pune, M.B. Rajarshi, Pune University, Pune, and Mr M. Kulye,
Jay Biotech, Pune, for their valuable help in analysing and interpreting the statistical data. SC and
SK are grateful to CSIR, New Delhi, for the Senior Research Fellowship.

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