Potato J (2016) 43 (2): 162-172

ISOLATION, CHARACTERIZATION AND

EVALUATION OF ENTOMOPATHOGENIC FUNGI
FROM BRAHMINA CORIACEA BEETLES
Anupam Sharma1, Desh Raj Thakur2 and Virender Kumar Chandla1
ABSTRACT: White grub, Brahmina coriacea is one of the most important pests of potato in North western hills of India. In
this study, B. coriacea beetles were trapped and investigated for fungal pathogens of this pest. Three fungi were consistently
isolated from the infected beetles. Based on partial sequencing of 18S rRNA, ITS1, 5.8S, ITS2 and a partial sequence of
28S rRNA, the isolates were identified as Beauveria bassiana strain CPRI16, Aspergillus flavus strain CPRI18 and Fusarium
oxysporum strain CPRI15. Additionally, insecticidal activity of these isolates was determined on B. coriacea grubs, eggs and
beetles by testing spore concentration of 1 √ 108 spores/ml. The highest mortality of second (92.59%) and third instar grubs
(81.85%) was obtained from B. bassiana after 60 days of treatment. All the isolates resulted in 100% mortality of adults. Egg
laying and percent hatching of eggs was observed minimum due to A. flavus.

KEYWORDS: Gene sequence, microbial pathogen, potato, rRNA, white grub

INTRODUCTION to be localized and unpredictable. Third

Brahmina coriacea (Hope) (Coleoptera:
Scarabaeidae) was reported for the first time
from Kullu valley of Himachal Pradesh (HP),
India feeding on pear, apple, plum, fig and
grapevine and later on noticed from other
mid-hills of HP (Fletcher, 1921; Beeson, 1941).
Both grub and adult of B. coriacea cause
severe damage especially in potato field and
apple orchards. Despite concerted efforts to
eradicate and limit its damage, unfortunately
B. coriacea progressively extended its range.
The pest appeared in epidemic form in Shimla
hills, causing losses up to 80% (Misra and
Chandla, 1989). Now it is the most widely
spread and destructive insect pest of potato
in mid and higher hills of Himachal Pradesh
(Chandla et al., 2001). The problem of white
grubs is quite serious in almost all the hilly
tracts where potato tubers are grown during
summer season as rain-fed crop under long
day conditions. Grub population densities
are typically aggregated and damage tends
1
CSKHPKV, Palampur-174023, Himachal Pradesh, India
Email: anupam_ihbt@hotmail.com
2
Himachal Pradesh Universities, Shimla-171005, Himachal Pradesh, India
instar grubs are most active and cause
severe damage by making large, shallow
and circular holes in potato tubers. They
remain concealed while feeding on tubers and
plants continue to grow normally without
any reflection of injury on aerial parts of the
plants. Because of this concealed behaviour
white grubs remain unnoticed in potato
during crop development.
At the time of harvesting, most of the
harvested tubers exhibit large, circular and
shallow holes which renders them unfit for
marketing (Misra and Chandel, 2003). A
number of approaches have been employed
for controlling the white grubs around the
world. As a means of soil pest control,
farmer use some of the dreaded chemicals
without considering pest biology, residual
effect of pesticides in the soil environment
and on the health of the consumers. Two
or three applications of the chemical in a
growing season are very common which

162 Potato J 43 (2): July - December, 2016

 Isolation, characterization and evaluation of entomopathogenic fungi
can eventually lead to the development
of insecticide resistance in the grubs and
also adversely affect the growth, microbial
diversity in the soil (Hussain et al., 2009;
Dutta et al., 2010). Moreover, the use of the
chemical insecticides is not advisable as the
potato tubers are used for the table purposes
(Ezekiel et al., 1999). Many cultural practices
have been used for controlling the white
grub (Misra and Chandla, 1989; Yubak Dhoj
and Keller 2002; Misra and Chandel, 2003)
without any effective management. These
factors have led to focus on the development
of alternative control measures. Therefore,
integrated pest management has placed great
hopes on bio-control agents. The best possible
and potential alternatives can be microbial
agents like fungi, bacteria, nematodes, viruses,
protozoa and plant products being compatible
with biological, toxicological, environmental
and social requirements (Pereira and Roberts
1991). Since, several fungal diseases have
been found to be associated with the white
grubs, there is a likelihood of discovering
useful novel strains. The knowledge of local
species or strains is very important if the
indigenous populations of entomopathogenic
fungi in soil are to be managed to facilitate
the control of pest insect populations (Meyling
and Eilenberg, 2006). Moreover, the isolation
of indigenous entomopathogenic fungi is
essential to provide insight into the naturally
occurring fungal biodiversity and to provide
potential biological control agents. Native
isolates will have ecological compatibility
with pest species and a reduced risk of
significant impact on non-target organism
when compared with exotic isolates (Banu
et al., 2004). Thus, this study was aimed at
isolating and identifying potential local fungal
pathogens by partial sequencing of 18S rRNA,
ITS1, 5.8S, ITS2 and a partial sequence of
28S rRNA for the management of B. coriacea.
Identification of three potential fungal isolates
from B. coriacea is reported in this paper.
Potato J 43 (2): July - December, 2016 163
MATERIALS AND METHODS
Lab rearing of B. coriacea
Beetles were collected with the help
of light traps from the different locations
around Shimla, HP, India and brought to the
laboratory with the help of portable insect
cages. Beetles were reared in 30 cm pots,
half-filled with mixture of fine soil and well
rotten Farm Yard Manure (FYM) (1:1). Fresh
leaves of robinia (Robinia pseudoacacia L.) were
kept in pots as a feed to the beetles and the
pots were covered with nylon nets. 10 pairs of
beetles per pot were reared. These pots were
checked after every 3-5 days interval for eggs.
Eggs were collected and kept collectively in
trays containing well rotten FYM (100 eggs/
tray). Newly hatched eggs were allowed to
grow in the same trays. After 20-25 days
of hatching, first instar grubs were shifted
to other trays with already grown maize
seedlings (20 grubs/tray). Second and third
instar grubs were kept individually in 15
and 25 cm pots, respectively, containing a
mixture of fine soil and well rotten FYM
(1:1) and fed with cut slices of potato/small
potato tubers. Simultaneously mortality of
beetles through pathogenic fungi was also
studied. Dead beetles were carefully separated
and transferred into sterilized vials by using
forceps, in order to know the mortality
whether caused by fungi or any other reason.
Beetles suspected to be affected by fungi
were kept at humid and damp condition to
enhance the fungal sporulation. The collected
cadavers were kept at 4°C until used for
fungal isolation.
Isolation and identification of fungi
Visible conidia from adult beetles were
harvested directly or scraped off on to media
plates. Alternatively grub cadavers without
external fungal growth were surface sterilized
for 2 min each with 70% ethanol, 1% sodium
Anupam Sharma, Desh Raj Thakur and Virender Kumar Chandla
hypochlorite and sterile water (2-3 washes)
and then homogenized. Small quantity of
homogenate was plated onto the SDAY media
(4% Sabouraud dextrose agar media + 2%
yeast extract + 0.1% streptomycin) (Papierok
and Hajek, 1997). Cultures were incubated at
25 ± 2°C for one week.
DNA sequencing and phylogenetic
analysis
The fungal DNA was extracted using
Qiagen Plant DNeasy Kit. Partial regions
of fungal rRNA including 18S rRNA,
ITS1, 5.8S, ITS2 and a partial sequence
of 28S rRNA were amplified using ITS1,
5’-TCCGTAGGTGAACCTGCGG-3’ and ITS4,
5’-TCCTCCGCTTATTGATATGC-3’ primers
(White et al., 1990). PCR was performed using
1 µl of the genomic DNA, 0.5 µmol each
primer, 10 mM Tris–HCl, pH 8.3, 50 mM
KCl, 1.5 mM MgCl2, 0.2 mM dNTPs and 1–2
U Taq polymerase (Qiagen, Germany) in a 50
µl reaction. PCR was performed in a thermal
cycler (Bio-Rad, USA) under conditions: initial
denaturation at 94°C for 3 min; 30 cycles of
94°C for 30 sec, 54°C for 40 sec and 72°C for
1 min followed by a final extension of 7 min
at 72°C. PCR products were analyzed on 1.5%
agarose gel with ethidium bromide staining.
The bands of desired size were cloned into
pGEM®-T easy vector (Promega, USA) and
sequenced. Sequencing was performed using
Big Dye® Terminator cycle sequencing kit
(Version 3.1, Applied Biosystems, USA) on
an automated DNA Sequencer (ABI Prism
3130xl, Applied Biosystems, USA). Sequences
were analyzed using BLASTN program
(Zhang et al., 2000) at NCBI (www.ncbi.
nlm.nih.gov). Phylogenetic and molecular
evolutionary analyses were performed using
MEGA 3.1 software (Kumar et al., 2004).
The phylogenetic tree was constructed by
the neighbor-joining method (Saitou and
Nei 1987) using the distance matrix from
the alignment and distances were calculated
164 Potato J 43 (2): July - December, 2016
by the Kimura 2-parameter (Kimura 1980).
The reliability of the tree was measured
by bootstrap analysis with 1,000 replicates
(Felsenstein 1985).
Bioassays of fungal isolates on B.
coriacea grubs, beetles and eggs
Bioassay tests of all the three isolates
obtained in this work were carried out
against second and third instar grubs of
B. coriacea as described by Goettel & Inglis
(1997). Each fungal isolate was grown on
media containg 4% Sabouraud dextrose agar
media, 1% yeast extract, 0.1% streptomycin
sulphate, tetracycline and cyclohexamide at
25 ± 2°C for one week (Papierok and Hajek,
1997). Spores from 7-10 day old sporulating
cultures were harvested in 0.01% Tween 80
and their final concentration was adjusted
to 1 √ 108 /ml using haemocytometer. A
mixture of soil and FYM (1:1) autoclaved at
15 psi and 121°C on three alternative days
was used as the substrate for testing isolates
against second and third instar grubs. Grubs
were immersed into spore suspension for 15-
20 seconds by holding them at the leg with
loose forceps. Excess liquid was dropped off
and the grubs were placed individually in
plastic pots (15 cm) containing 100 g soil and
FYM medium. Thirty grubs were taken for
each isolate (10 in each replication). Grubs for
control were dipped in sterile 0.01% Tween
80 and pots were placed in the experimental
room at a temperature of 25 ± 2°C. Second
and third instar grubs were fed with slices
of potatoes and small sized potato tubers,
respectively. Final soil moisture content of
15–20% was maintained by using sterile
distilled water throughout the bioassay period
by maintaining the initial gravimetric weight.
Grubs were checked for disease incidence on
every fifth day for eight weeks.
For testing these isolates on B. coriacea
beetles, a dose of 300 ml of fungal culture
 Isolation, characterization and evaluation of entomopathogenic fungi
(1 √108 spores/ml) was applied to 3 kg of
the soil: FYM medium, mixed thoroughly
and placed 300 g in each plastic pot (30 cm).
Two pairs of beetles were released in each
pot and fresh robinia leaves were provided
as feed to them. Pots were covered with
nylon nets and checked periodically for
further mortality and egg laying. For control,
beetles were maintained on sterile soil and
FYM mixture.
For testing these isolates on eggs, 200 g of
soil and FYM medium treated with respective
isolate (1 √ 108 spores/ml) as mentioned above
was placed in round boxes with perforated
lids. One day old ten eggs were placed in each
box (three replications). Eggs were checked
daily for hatching and fungal infection.
After hatching neonates were also checked
for further infection after every three days.
Similarly eggs were maintained on sterile soil
and FYM mixture as control group.
Mortality in control group (second and
third instar grubs) was corrected using
Abbott’s formula (Abbott, 1925). Data on
corrected accumulated mortality (grubs)
and accumulated mortality (beetles) were
statistically analyzed through ANOVA using
MSTAT-C software. The difference of two
means between treatments exceeding critical
differences value was taken as significant
Fig. 1. Beetles infected naturally with fungi, A. flavus (a) B. bassiana (b)
Potato J 43 (2): July - December, 2016 165
RESULTS
Identification of fungal isolates
In the present study, three fungi were
consistently isolated from the infected beetles.
Fig. 1 shows beetles found naturally infected
with these fungi. Based on partial sequencing
of 18S rRNA, ITS1, 5.8S, ITS2 and a partial
sequence of 28S rRNA, the isolates were
identified as A. flavus strain CPRI18, B. bassiana
strain CPRI16 and F. oxysporum strain CPRI15.
The 543 bp sequence containing ITS1 and
ITS2 sequences of Fusarium oxysporum strain
CPRI15 (GU565571) showed 100% identity
with Fusarium oxysporum strain UFMGCB
1333. The 569 bp sequence containing ITS1
and ITS2 sequences of Beauveria bassiana
strain CPRI16 (GU565572) showed 99%
identity with Beauveria bassiana strain B4. The
595 bp sequence containing ITS1 and ITS2
sequences of Aspergillus flavus strain CPRI18
18S (GU565573) showed 100% identity with
Aspergillus flavus strain HF5. All the sequences
were submitted to NCBI gene databank.
Based on genetic similarities, the isolates
were selected to construct the phylogenetic
tree (Fig. 2). The fungal genera analyzed
in this study were entomopathogenic and
phytopathogenic organisms. Besides being
phytopathogenic, A. flavus and B. bassiana
had earlier been isolated from other white
Anupam Sharma, Desh Raj Thakur and Virender Kumar Chandla

Fig. 2. Phylogenetic tree of fungal isolates obtained from the beetles. Isolate designations are shown in bold text. The optimal
tree with the sum of branch length = 0.60140602 is shown. The percentage of replicate trees in which the associated taxa
clustered together in the bootstrap test (1000 replicates) is shown next to the branches.

grub species (Gunde Cimerman et al., 1998;
Padmanaban et al., 2003; Hajek et al., 2006;
Bhattacharryya et al., 2008). F. oxysporum had
received considerable attention because of its
ability to cause wide range of plant diseases
(Kistler, 1997). Large numbers of Fusarium
spp. are also entomopathogenic and F.
oxysporum had also been isolated from other
insect species (Bai and Chen, 1991; Blodgett
et al., 2004).
Bioassays of fungal isolates on white
grub instars
Mortality caused by all the isolates in
second instar grubs was significantly different.
It was highest, 28.15% by B. bassiana strain
CPRI16 followed by A. flavus strain CPRI18
(21.48%) and F. oxysporum strain CPRI15
(14.07%) after 7 days of treatment. After
60 days of treatment, 92.59% mortality was
caused by B. bassiana strain CPRI16, 88.89%
by A. flavus strain CPRI18 and 85.19% by F.
oxysporum strain CPRI15 (Tables 1). Similar
trend was observed in case of third instar
grubs however, mortality in third instar was
slower as compared to second instar grubs
(Tables 2). Brahmina bassiana strain CPRI16
was again found to be highly pathogenic
among all the isolates in all the days after
treatment. After 60 days of treatment, 81.85%
mortality of third instar grubs was caused by
B. bassiana strain CPRI16. Further, emergence
of adults from treated third instar grubs
was highly affected due to all the isolates.
Adult emergence was least, 6.67% each in
B. bassiana strain CPRI16 and A. flavus strain
CPRI18 and 13.33% in F. oxysporum strain
CPRI15 as compared to 83.33% in control

Table 1. Effect of fungal isolates against second instar grubs.

Fungal treatment Corrected mortality% ± SE

(1 √ 108 spores ml-1)
7 DAT a
15 DAT 21 DAT 30 DAT 45 DAT 60 DAT
Baeuveria bassiana strain CPRI16 28.15 ± 5.92 45.93 ± 7.73 67.41 ± 7.06 78.15 ± 6.73 89.26 ± 0.37 92.59 ± 3.70
Aspergillus flavus strain CPRI18 21.48 ± 0.74 39.26 ± 3.22 60.74 ± 7.39 78.52 ± 6.46 85.56 ± 3.90 88.89 ± 0.0
Fusarium oxysporum strain CPRI15 14.07 ± 2.96 31.85 ± 5.18 56.67 ± 3.22 67.41 ± 7.06 81.85 ± 4.07 85.19 ± 3.70
CD (5%) 13.12 19.84 22.78 22.78 10.28 10.28
Each value is mean of three replications; CD critical difference, SE standard error, DAT days after treatment

166 Potato J 43 (2): July - December, 2016

 Isolation, characterization and evaluation of entomopathogenic fungi

Table 2. Effect of fungal isolates against third instar grubs and further emergence of adults.

Fungal treatment Corrected Mortality (%) ± SE Emergence Healthy

(1 √ 108 spores ml-1) of adults adults
7 DAT a
15 DAT 21 DAT 30 DAT 45 DAT 60 DAT 75 DAT
(%) ± SE (%) ± SE
B. bassiana strain 6.67 ± 26.67 ± 36.67 ± 51.48 ± 71.48 ± 81.85 ± 89.26 ± 6.67 ± 3.33 3.33 ±
CPRI16 3.33 3.33 3.33 4.55 3.29 4.07 0.37 3.33
A. flavus strain 3.33 ± 23.33 ± 26.67 ± 44.44 ± 64.44 ± 78.52 ± 89.26 ± 6.67 ± 3.33 6.67 ±
CPRI18 3.33 3.33 3.33 5.55 2.22 0.74 0.37 3.33
F. oxysporum strain 0.00 ± 10.00 ± 16.67 ± 34.44 ± 60.74 ± 71.48 ± 78.52 ± 13.33 ± 10.00 ±
CPRI15 0.00 3.33 3.33 2.93 3.22 3.29 0.74 3.33 0.00
Control 0.00 0.00 0.00 0.00 0.00 0.00 0.00 83.33 ± 83.33 ±
3.33 3.33
CD (5%) 7.45 9.42 9.99 10.70 9.67 9.21 1.05 9.99 9.42
Each value is mean of three replications; aDAT-Day after treatment; SE standard error

group. Further, survival of adults was also
significantly affected and it was least in case
of B. bassiana strain CPRI16 (3.33%) followed
by A. flavus strain CPRI18 (6.67%) and F.
oxysporum strain CPRI15 (10.00%) as compared
to 83.33% in control.
Present work indicates that all of the
three fungi isolated from B. coriacea adults
recovered from different localities in potato
growing regions where fungal epizootic
occurred, were highly pathogenic to this
insect host. All of the three strains of fungi
were entomopathogenic, indicating that
there are antagonistic microbial species
present against B. coriacea and it is possible
to control its infestation. High mortality
rates were caused in different developmental
stages of B. coriacea infected with B. bassaina
strain CPRI16 and A. flavus strain CPRI18.
B. bassiana is the most widely distributed
fungus in the world and it infects insects
in tropical, temperate, humid and desert
areas (Zimmermann, 2007). B. bassiana had
been found pathogenic against coleopteran
insects including various white grub species
(Poprawski and Yule, 1991; Bustillo et al.,
1999; Hajek et al., 2006; Bhattacharryya et
al., 2008). Several strains of B. bassiana had
been reported to cause mortality of grubs of
Phyllophaga species in laboratory with LC50
value 1.15 √ 109 spores/ml (Flores et al.,
2002). Morales-Rodriguez et al. (2009) had also
observed significant mortality in Amphimallon
majale (69.8%), Anomala orientalis (77.2%) and
Maladera castanea (75.6%) due to B. bassiana.
Hundred percent mortality in first, second
and third instar grubs of Holotrichia species
had been reported within 10, 12 and 16 days,
respectively when sprayed at 1 √ 108 spores
ml-1 of B. bassiana (Mohi-Ud-Din et al., 2007).
A. flavus was found to be second most
virulent isolate after B. bassiana. A. flavus is
an opportunistic plant pathogen (Plasencia,
2004) but can also infect a wide variety of
insect hosts (St. Leger et al., 2000). According
to earlier studies, chitinase producers can be
considered as potential pathogens (Sur et al.,
1999; Kumar et al., 2004). High chitinolytic
activities have been observed with A. flavus
on some insects (Gunde Cimerman et al.,
1998; Kumar et al., 2004). A. flavus had
been reported to be pathogenic to various
other insects including grubs of Leucopholis
burmesteri (Moraes et al., 2001b; Govindarajan
et al., 2005; Padmanaban et al., 2003). It
had been found that Fusarium species can
infect insects through circular wounds
(Vey, 1971). Gupta et al. (1991) isolated the
toxin beauvericin, a cyclodepsipeptide from
Fusarium and they showed its effect against

Potato J 43 (2): July - December, 2016 167

Anupam Sharma, Desh Raj Thakur and Virender Kumar Chandla

Leptinotarsa decemlineata. High mortality in (Table 3). Fig. 3 shows grub and beetle
several insect groups including Coleoptera mortality caused by different fungal isolates.
had been attributed to mycoses caused by F. All of the three isolates were able to cause
oxysporum (Perez et al., 1996; Torres Barragan 100% mortality of beetles of B. coriacea. Least
et al., 2004; Sun and Liu, 2008). number of eggs was laid by the beetles in A.
Bioassay on B. coriacea beetles flavus strain CPRI18 treatment and percent
hatching was also significantly affected.
After 7 days of treatment, A. flavus strain
Further survival of newly hatched grubs was
CPRI18 caused 25% mortality of beetles
followed by B. bassiana strain CPRI16 (16.67%)
which were significantly better over 8.33% in
control. All of the three isolates were able to
cause 100% mortality as compared to 58.33%
in control after 30 days of treatment. Only
2.33 numbers of eggs were obtained from
the beetles reared in A. flavus strain CPRI18
treated soil whereas it was 6.67 in B. bassiana
strain CPRI16 and 8.33 in F. oxysporum strain
CPRI15 which were significantly less as
compared to 11.00 eggs in control. Percent
hatching was also significantly affected and it
was 76.69% in A. flavus strain CPRI18, 77.15%
in B. bassaina strain CPRI16 and 85.31% in
F. oxysporum strain CPRI15 as compared to
92.81% in control. Further survival of newly
hatched grubs was also significantly less in
all the treatments. It was 22.92% in B. bassaina
strain CPRI16, 24.67% in A. flavus strain
Fig. 3. Second instar grub mortality caused by B. bassiana
CPRI18 and 26.13% in F. oxysporum strain (a) beetle mortality caused by A. flavus (b) B. bassiana (c)
CPRI15 as compared to 92.12% in control healthy beetle (d) healthy grub (e).

Table 3. Effect of fungal isolates on Brahmina coriacea beetles.

Fungal treatment Mortality ± SE Total number of eggs/2 pairs of Hatching Survival of

(1 √ 108 spores ml-1) beetles ± SE of eggs (%) neonates
± SE (%) ± SE
7 DATa 15 DAT 30 DAT 7 DAT 15 DAT 30 DAT
B. bassiana strain 16.67 ± 75.00 ± 0.0 100.0 ± 0.0 6.67 ± 0.33 9.00 ± 0.00 2.33 ± 0.33 77.15 ± 22.92 ±
CPRI16 8.33 1.84 2.93
A. flavus strain 25.00 ± 0.0 91.67 ± 100.0 ± 0.0 2.33 ± 0.33 8.33 ± 2.01 2.00 ± 0.57 76.69 ± 24.67 ±
CPRI18 8.33 1.80 6.81
F. oxysporum strain 8.33 ± 8.33 75.00 ± 0.0 100.0 ± 0.0 8.33 ± 1.20 9.33 ± 0.88 3.00 ± 0.00 85.31 ± 26.13 ±
CPRI15 3.77 3.70
Control 8.33 ± 8.33 41.67 ± 58.33 ± 11.00 ± 10.33 ± 4.33 ± 0.33 92.81 ± 92.12 ±
8.33 8.33 0.00 0.33 1.85 0.61
CD (5%) 18.61 16.65 14.42 3.03 5.19 1.91 9.21 14.51
Each value is mean of three replications; *Arcsine transformed; **√ x + 0.5 transformed values; aDAT-Day after treatment; SE
standard error

168 Potato J 43 (2): July - December, 2016

 Isolation, characterization and evaluation of entomopathogenic fungi
significantly less due to all the isolates when
were left in the same soil. In an earlier study,
B. bassiana had been found to be pathogenic
to adults of Anoplophora glabripennis with LT50
of 5.0 to 24.5 days (Dubois et al., 2008). B.
bassiana had been reported to be pathogenic
against Papuana uninodis adults with LT50
values of less than 6 weeks (Theunis and
Aloali, 1998). B. bassiana caused high mortality
in beetle, Ips typographus when tested at a
concentration of 1.0 × 107 conidia/ml (Kreutz
et al. 2004a). B. bassiana caused comparatively
slow mortality of adults and slow action in
New Zealand isolates (Townsend et al., 1995).
The slow infection processes of isolate on
adults in present study may be explained
either by inherent processes of the strain,
immunological characteristics of the insect
or both; the fungus develops slowly and
causes death of the host at its adult stage. It is
known that the youngest stages of insects and
instars are the most sensitive to the infection.
However, it was observed that fungi are able
to penetrate the membranous inter-segmental
regions having low amount of chitin, as well
as sclerotized areas, which are common in
adult coleopterans (Vey et al., 1982). In case
of beetles, mortality in control group was also
high because adult life cycle is small i.e. 11-21
days (Chandel and Kashyap, 1997) and 32.1
days (Sharma, 2011) as compared to larval
duration. Since, these fungal isolates were
able to cause mortalities in beetles, number
Table 4. Effect of fungal isolates on eggs of Brahmina coriacea.
Fungal treatment Hatching (%) ± SE
(1 ×√ 108 spores ml-1)
7 DAT a
B. bassiana strain CPRI16 26.67 ± 3.33
A. flavus strain CPRI18 6.67 ± 3.33
F. oxysporum strain CPRI15 33.33 ± 3.33
Control 40.00 ± 0.0
CD (5%) 11.09
Each value is mean of three replications; aDAT-Day after treatment; SE standard error
Potato J 43 (2): July - December, 2016 169
of eggs laid by the beetles were also reduced
which further reduced grub population.
Bioassay on B. coriacea eggs
A. flavus strain CPRI18 was found highly
pathogenic against B. coriacea eggs (Table
4). Percent hatching was 6.67% in A. flavus
strain CPRI18 and 26.67% in B. bassaina
treated eggs as compared to 40.00% in control
after 7 days of treatment. After 11 days of
treatment, minimum percent hatching was
76.67% in A. flavus strain CPRI18 and was
significantly better over 96.67% in control.
Further, least percent survival of grubs was
3.33% in case of B. bassaina strain CPRI16,
6.67% in A. flavus strain CPRI18 and 10.00%
in F. oxysporum strain CPRI15 as compared to
86.67% in control after 30 days of treatment.
There was significant effect on percent
hatching of eggs by A. flavus strain CPRI18.
Previously no such study of A. flavus had
been carried out. Further percent survival of
grubs was observed least in case of B. bassiana
strain CPRI16 followed by F. oxysporum
strain CPRI15. Earlier, B. bassiana had been
reported to cause mortality of the eggs of N.
bruchi, either sprayed or dipped in different
conidial concentrations (Chikwenhere and
Vestergaard, 2001). Lynch & Lewis (1978) had
frequently observed F. oxysporum invading
egg masses of the Ostrinia nubilalis (Hubner).
Though B. bassiana is already an established
microbial agent to control white grubs but it
is also a well known fact that native isolates
Survival of hatched grubs (%) ± SE
11 DAT 15 DAT 30 DAT
80.00 ± 0.0 6.67 ± 3.33 3.33 ± 3.33
76.67 ± 3.33 6.67 ± 3.33 6.67 ± 3.33
80.00 ± 0.0 13.33 ± 3.33 10.00 ± 0.0
96.67 ± 3.33 86.67 ± 3.33 86.67 ± 3.33
8.81 7.02 11.04
Anupam Sharma, Desh Raj Thakur and Virender Kumar Chandla
are always found to be more virulent when
compared to the exotic fungal isolates for
effective and sustainable control (Yubak Dhoj
et al., 2008). The knowledge of local species or
strains is an important aspect to manage the
local populations of entomopathogenic fungi
in soil and facilitate the insect pest control
(Meyling and Eilenberg, 2006).
Moreover, the isolation of indigenous
entomopathogenic fungi is essential to know
fungal biodiversity occurring naturally and so
to provide potential biological control agents.
Native isolates have ecological compatibility
with pest species and a reduced risk of
significant impact on non-target organism
when compared with exotic isolates (Banu et
al., 2004).
CONCLUSIONS
Three entomopathogenic fungi were
isolated from B. coriacea and tested against
the different stages of this pest for their
virulence. Based on their virulence, B.
bassiana strain CPRI16 and A. flavus strain
CPRI18 seem to be promising strain which
can be further developed as a potential
biological control agent locally against
white grubs in North western hills of India.
However, side effects and safety tests with
beneficial insects, mammals and human
cells need to be conducted. Further studies
are needed for fungal strain optimization
and development of better substrates for
their mass production. It also reveals that
opportunistic pathogens can also be tested
to prove their pathogenicity beside the well
known entomopathogens.
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MS received: 27 May 2016; Accepted: 09 November 2016

172 Potato J 43 (2): July - December, 2016