| Research Article |
Open Access |
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| Biopesticidal Formulation of Beauveria Bassiana Effective against Larvae of
Helicoverpa Armigera |
| Agarwal Ritu1*, Choudhary Anjali1, Tripathi Nidhi1, Patil Sheetal2 and Bharti Deepak1 |
| 1Assistant Professor, Department of Biotechnology and Biochemistry, Career College, Bhopal 462 023, M.P, India |
| 2Trainee student, Department of Biotechnology and Biochemistry, Career College, Bhopal, 462 023, M.P, India |
| *Corresponding author: |
Ritu Agarwal
Department of Biotechnology
Career College
Bhopal 462 023
M.P, India
Ph: +91-9893835225
E-mail: ritu.agarwal@rediffmail.com |
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| Received June 30, 2012; Accepted June 19, 2012; Published June 21, 2012 |
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Citation: Ritu A, Anjali C, Nidhi T, Sheetal P, Deepak B (2012) Biopesticidal
Formulation of Beauveria Bassiana Effective against Larvae of Helicoverpa
Armigera. J Biofertil Biopestici 3:120.
doi:10.4172/2155-6202.1000120 |
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| Copyright: © 2012 Ritu A, et al. This is an open-access article distributed under
the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and
source are credited. |
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| Abstract |
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| The present study was to emphasize entomopathogens in pest management of cash crops over chemical
pesticides, optimization of media for growth of Beauveria bassiana and bioassay of different formulations for
their efficacy as marketable and easily applicable biopesticide. Beauveria bassiana (Order: Hypocreales, Family:
Cordycipitaceae) popularly known as white muscardine entomogenous fungi was isolated from soyabean fields
of Misrod, Bhopal. Media for optimal growth of fungus were standardized. Its different formulations viz. carrier
based powder formulation, oil based formulation and bentonite oil based formulation using homogenizers were
prepared. These formulations were bioassayed against Helicoverpa armigera (Order: Lepidoptera, Family:
Noctuidae), the most destructive pest in soyabean. Bentonite based liquid formulation was observed to be most
effective as determined by measuring larval mortality as well as viability of fungal spores and ease of applicability. |
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| Keywords |
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| Biopesticide; Beauveria bassiana; Helicoverpa armigera;
Formulations; Bentonite |
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| Abbreviations |
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| BOBLF: Bentonite Oil-Based liquid Formulation; OBLF: Oil-Based
Liquid Formulation; CBPF: Carrier Based Powder Formulation |
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| Introduction |
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| Over reliance on broad spectrum pesticides has been severely
condemned in different parts of the world after International
Conference on Chemicals Management. Since then, an alternative eco
friendly strategy for the management of noxious insect pests has been
explored to trim down the harmful effects of chemical insecticides on
humanity. Studies of biodiversity in agro ecosystems and the delivery
of ecosystem services to agricultural products have usually ignored the
contribution of entomopathogens in the regulation of pest populations
(Tscharntke et al.). |
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| In recent years, crop protection based on biological control of crop
pests with microbial pathogens like virus, bacteria, fungi and nematodes
has been recognized as a valuable tool in pest management [1,2]. The
appropriate use of eco friendly microbial biopesticide can be engaged
in recreation of sustainable organic crop production by providing
a stable pest management program. In light of this understanding,
extensive work has been done on various species of bacteria and fungi
(especially entomopathogenic that parasitize insects) implicated as
effective biocontrol agents. |
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| Till date, various entomopathogenic fungi such as Lecanicillium sp.
[3,4], Beauveria bassiana [5-7] and Metarhizium anisopliae [8-11] have
been effectively used to control aphids, lepidopteron larvae and other
pests. |
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| Sixteen different mycotoxins have been analysed of which
the cyclodepsipeptidic mycotoxin, beauvericin, produced by
Beauveria bassiana has been documented to be most effective for its
larvicidal properties. Sowjanya Sree and Padmaja [10] reported the
ultrastructural effects of crude beauvericin on the salivary glands of
9-day-old S. litura larva after 24 h of treatment with the mycotoxin at a
dosage of 0.147 μg/g body wt. (LD50). Helicoverpa armigera (Hubner) is
commonly known as the gram pod borer as it is a serious pest on pulses (Nahar et al., David and Ananthakrishnan). It has been reported that
M.anisopliae is effective against H. armigera [12]. |
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| The present investigation was carried out to highlight the
significance of entomopathogens in pest management of cash crops,
optimization of media for growth of Beauveria bassiana and bioassay
of different formulations for their efficacy as marketable and easily
applicable biopesticide. The investigation established the role of
Beauveria bassiana bentonite formulation as an effective biocontrol
agent against the most destructive pest Helicoverpa armigera. |
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| Materials and Methods |
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| Chemicals |
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| Sabouraud-Dextrose media, Czapek Dox media, Yeast Extract and
Bentonite were purchased from HiMedia Biosciences Mumbai, India.
Corn oil, gum and glycerine were purchased from local market. Maize
flour was prepared by grinding maize grains in laboratory. Ponds
talcum powder was used as one of the carriers. |
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| Culture |
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| Beauveria bassiana employed in this study was isolated from soil
collected from soyabean field of Misrod, Bhopal (Rice technology
bulletin, 2003 [13,14]. Beauveria bassiana was grown on Czapek Dox
media modified with maize flour as per findings of Ramle et al. [15]
and on Sabouraud-dextrose media with yeast extract [16] for growth
optimization of the entomopathogenic fungus. Once the fungus was
identified [17], pure cultures were obtained followed by flask cultures
to develop the fungal mat. |
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| Formulations |
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| For preparing the formulations viz. carrier based powder
formulation (CBPF) using talcum powder, glycerine and gum, oilbased
liquid formulation (OBLF) using corn oil, gum and glycerin,
bentonite oil-based liquid formulation (BOBLF) using corn oil, gum,
glycerin and bentonite [6] using a knife mill grindomix (Remi, India).
Fungal spore suspensions in different types of formulations were
prepared from the fungal mats. |
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| Viability of spores was assessed before and after preparation of
formulations by serial dilution test in modified Czapek-Dox broth
and after six months, viability of spores was again carried out in the
prepared formulations as per Maheshwari [14]. Spore count of each
formulation was done using Neubaeur chamber [18]. A range of
concentration from 10% to 100% of each formulation was also prepared
using distilled water and assayed at laboratory scale in triplicate and the
optimal concentration for effective infection was screened for assay at
the levels of pot culture and field assay. Reproducibility of each assay
was established in triplicate. |
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| Laboratory Bioassay |
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| The fungal formulations of Beauveria bassiana were assayed against
larvae of Helicoverpa armigera in the laboratory. Eggs of Helicoverpa
armigera collected from the fields and were kept in optimum condition
maintained at 23 ± 2°C and 35 ± 5% relative humidity under a 16:8
(L:D) cycle in a growth chamber. The larvae obtained from these eggs
were fed with soya beans and sugar beet roots and leaves. The three
days old larvae were divided into four groups of control and treated
with different formulations and subsequently examined for symptoms
of infection and mortality at regular intervals of 24 hours (Table 1). |
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| Pot culture assay |
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| In pot culture experiments, Beauveria bassiana formulations were evaluated against first and third instar larvae at 60% concentration of
formulations with dosage equivalent to 2.1 × 104 spores ml-1, calibrated on the surface area of the pot. As a control, contents of formulation were
sprayed excluding Beauveria bassiana. The larvae were released in pots
pre planted with sugar beet plants and allowed to settle for 24 hrs. In
the test against larvae, different formulations carrying the fungal spores
were applied to pots in replicates of four with seven larvae each. The
pots were watered immediately. Larvae were examined for infection
due to Beauveria bassiana at weekly intervals [19]. The experiments
were repeated three times. |
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| Field evaluation |
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| Beauveria bassiana was tested at 2.1 × 104 spores ml-1 in a
randomized block design (RBD) and plot size of 24 sq. m. in triplicate in soyabean fields of Misrod, Bhopal having four month old stalks.
Spray method was used in the toxicity assays and as a control, contents
of formulation was sprayed excluding Metarhizium anisopliae. First
instar larvae were collected from the experimental plots about a month
later by completely uprooting the plants or digging deep on either side
of the rows. These larvae were brought to the laboratory in individual
boxes filled with moist soil and reared on sugar beet and soya bean
roots and leaves, which were changed every week. The proportion of
larvae that showed symptoms of fungal attack after treatments with
optimal concentration of all three formulations, were recorded as per
protocol of Samuel et al. [20]. |
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| Statistical analysis |
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| Each assay was established in triplicate for each parameter. The
statistical analysis was performed using mean as a base for central
tendency followed by calculation of deviation using standard error.
Statistical significance was drawn by comparing the p value from students “t” test table. Significantly different from the control if p<0.05,
significant if p<0.01, highly significantly if p<0.001. |
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| Results and Discussion |
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| The fungus, Beauveria bassiana showed fast growth on a medium
consisting of yeast extract i.e Sabouraud-dextrose-yeast extract media
which exhibited a shorter fungal mat development period of 15 days as
compared to 20 days on Czapek Dox media. |
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| Among the three different formulations, it was found that the
bentonite based liquid formulation exhibited the highest efficacy of
infection against Helicoverpa armigera. A range of concentration
from 10% to 100% of each formulation was assayed in the laboratory
bioassay and it was found that a 60% concentration of each formulation
showed optimum results (Table 1 and Figure 1). |
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Table 1: Infection rate of different formulations of Beauveria bassiana without dilution
on larvae of Helicoverpa armigera. |
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Figure 1:Showing number of days required by different concentration of formulations
of Beauveria bassiana to infect larvae of Helicoverpa armigera in laboratory
assay. |
|
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| Thereafter, for pot culture assay and field trial, the concentration
of formulations was assayed at 60% as in laboratory assay the result
obtained from 60% to 100% were nearby. Preparation of varied
formulations did not hamper the viability of spores as was assessed
before and after, by serial dilution test in modified Czapek-Dox broth
and provided an easy applicability in the liquid bentonite formulation.
After six months, the viability of spores was again assessed in the
formulations and it was found that the spores were still viable, although
better result was seen in bentonite formulation as compared to the
other two. |
|
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| Beauveria bassiana is cited to be highly active against more than
hundreds of insect pests and highly selective in its parasitisation [21].
The fungus, Beauveria bassiana was cultured with excellent results
on a medium consisting of yeast extract i.e Sabouraud-dextrose-yeast
extract [15]. Among the three different formulations, it was found that
the bentonite based liquid formulation had the highest efficacy. |
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| Anand et al. [1] has reported that the fungal pathogen Beauveria
bassiana infects pupae of Spodoptera litura in a dose–dependent manner
for each of the formulations investigated which corroborates the
findings of the present investigation. They also reported that Beauveria
bassiana was more infective and resulted in maximum average percent
mortality among the three species viz. Metarhizium anisopliae,
Beauveria bassiana and Lecanicillium sp. under investigation. |
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| In the lab assay, 52% of larvae treated with 2.7 × 109 spores per ml of
Beauveria bassiana developed infection in 7 days as against no infection
in control, a finding also published by Chen et al. [22] during their
work on Metarhizium anisopliae. Laboratory bioassay by Chandler and
Davidson [8], documented higher mortality in M. anisopliae (ARSEF
7487) than L. muscarium (ARSEF 7037) in all soil based studies. |
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| In pot culture assay, Beauveria bassiana caused low level of infection
in larvae at the dosage equivalent to 2.1 × 104 spores ml-1 hectare since
the fungus required 30-35 days incubation period to produce disease
symptoms. In test, with a higher dosage range (1 × 106 – 108 spores
per ml) observations made at monthly intervals, showed similar dosage
dependent infection rates. |
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| In the field trials with Beauveria bassiana, infection rates in larvae
collected from the experimental plot were remarkably higher at
the higher dosage of 2.1 × 104 spores ml-1 than at the lower dosage.
The larvae collected 25 days after treatment developed infection in a
maximum period of 32 days after collection from the field. This was in
accordance with the field bioassay of Anand et al. [1]. |
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| Conclusion |
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| Formulations of Beauveria bassiana can thus serve as an effective
broad spectrum biocontrol agent for soyabean and various other cash
crops. As also evident from the study, Bentonite is an effective carrier of
the Beauveria bassiana in terms of being economical, maintaining the
biological activity and increasing the ease of application. This carrier
can also serve to be good medium for other fungal biopesticides for
instance Beauveria bassiana, further studies on which are in progress. |
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| Acknowledgements |
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| The authors are grateful to their academic councils for support in the form of
infrastructural facilities made available for undertaking the present study. |
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