Name: development of metarhizium anisopliae as a mycoinsecticide: from isolation to field performance
Uploaded: 2017-07-30T15:00:00
Description: Watch this Scientific Journal Video about Development of Metarhizium anisopliae as a Mycoinsecticide: From Isolation to Field Performance at JoVE.com

The authors acknowledge the contribution of collaborators from the Indo-Swiss Collaboration in Biotechnology (ISCB) program of the Department of Biotechnology, New Delhi and the Swiss Agency for Development and Cooperation, Berne, Switzerland. The contributions of project students and staff involved in the development of the mycoinsecticide, including Vandana Ghormade, Pallavi Nahar, Priya Yadav, Shuklangi Kulkarni, Manisha Kapoor, Santosh Chavan, Ravindra Vidhate, Shamala Mane, and Abhijeet Lande, are acknowledged. EKP and SGT thank the University Grants Commission, India and the Council of Scientific and Industrial Research (CSIR), India, respectively, for research fellowships. MVD acknowledges the support from the Council of Industrial and Scientific Research, New Delhi for the Emeritus Scientist Scheme. The authors are grateful to the Department of Biotechnology, New Delhi, India for the financial support under the ISCB and SBIRI programs. We are thankful to reviewers for their inputs.

1. Isolation of Entomopathogenic Fungi
<ol>
	<li>Soil dilution method
	<ol>
		<li>Collect the soil samples and mycosed insects from different crop fields (Table 1). Isolate the entomopathogenic fungi from soil samples using the soil dilution plating method8. 
		Note: In this study, samples were collected from the Pune (18&#176;31&#39;13&#39;&#39;N; 73&#176;51&#39;24&#39;&#39;E) and Buldhana (19&#176;58&#39;36&#39;&#39;N 76&#176;30&#39;30&#39;&#39;E) distric...

<ol>
	<li>Aktar, M. W., Sengupta, D., Chowdhury, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+pesticides+use+in+agriculture:+their+benefits+and+hazards.">Impact of pesticides use in agriculture: their benefits and hazards.</a> Interdisciplinary Toxicology. 2 (1), 1-12 (2009).</li><li>Dhaliwal, G. 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V., MV, D. e. s. h. p. a. n. d. e., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+evaluation+of+indigenous+fungal+isolates,+Metarhizium+anisopliae+M34412,+Beauveria+bassiana+B3301+and+Nomuraea+rileyi+N812+for+the+control+of+Helicoverpa+armigera+(H&#252;b.)+on+pulses.">Comparative evaluation of indigenous fungal isolates, Metarhizium anisopliae M34412, Beauveria bassiana B3301 and Nomuraea rileyi N812 for the control of Helicoverpa armigera (H&#252;b.) on pulses.</a> Proceeding of the international workshop on entomopathogenic fungi - a valuable alternative to fight against insect pests. , 51-59 (2004).</li><li>Roberts, D. W., Hajek, A. E., Leathan, G. 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During the 1880s, the first attempt was made to use Metarhizium to control the scarab beetle, Anisoplia austriaca, and the sugar beet curculio, Cleonis punctiventris21. In this protocol, one of the prerequisites was to isolate a virulent strain, either from the soil or from infected insects. Indeed, other parameters, such as LC50, LT50, and ST50, significantly contributed to the cost-effectiveness of the product22<...

From the introduction of organochlorine pesticides in the 1940s in India, the use of pesticides has increased many fold1, with crop pests still costing billions of rupees2 annually in terms of yield loss in agricultural production. The widespread and non-judicious use of synthetic pesticides is a continuous threat to the environment and human health1. The indiscriminate use of pesticides leads to residues in the soil and the depletion of natural pest predators. It also serves as a powerful selection pressure for altering the genetic makeup of a pest population, leading to the development of resistance1. Despite the enormous benefits of the green revolution, which required high inputs, like fertilizers and pesticides, pests continue to be a major biotic constraint. A general estimate of recorded annual crop losses in India and worldwide are USD 12 billion2 and USD 2,000 billion3, respectively.
When chemical pesticides have detrimental effects when used to control insect pests, it becomes imperative to search for alternative methods that are ecologically sound, reliable, economical, and sustainable. Biological control offers a suitable alternative and includes the use of parasites, predators, and microbial pathogens4. Fungi, for instance, are known to infect a broad range of insect pests, including lepidopterans, hymenopterans, coleopterans, and dipterans, often resulting in natural epizootics. Furthermore, unlike other bacterial and viral insect control agents, the mode of action of insect pathogenic fungi is by contact5. These fungi comprise a heterogenous group of over 100 genera, with approximately 750 species reported among different insects. The important fungal pathogens are: Metarhizium sp., Beauveria sp., Nomuraea rileyi, Lecanicillium lecanii, and Hirsutella sp., to name a few6. M. anisopliae (Metchnikoff) Sorokin is the second most widely used entomopathogenic fungus in biocontrol. It is known to attack over 200 species of insects7.
In this study, different stages involved in the knowledge-based development of a mycopesticide using M. anisopliae are presented. This includes: 1) the identification of a source (i.e., either soil or mycosed insects) for virulent entomopathogens, 2) entomopathogen identification and selection, 3) strategies to maintain their virulent nature and effectiveness in the laboratory bioassay and in the field, 4) the cost-effective formulation of infective propagules, 5) the development of unique quality-control parameters for virulent preparation, and 6) bioprospecting and value addition.

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			<td height="21" style="height:21px;width:216px;">Agar</td>
			<td style="width:115px;">Hi-Media</td>
			<td style="width:119px;">RM666</td>
			<td style="width:167px;">Reagent</td>
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			<td height="21" style="height:21px;width:216px;">Ammonium sulphate&#160;</td>
			<td style="width:115px;">Thomas Baker</td>
			<td style="width:119px;">11645</td>
			<td>Reagent</td>
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			<td height="42" style="height:42px;width:216px;">DNA analyzer&#160;</td>
			<td style="width:115px;">Applied biosystem</td>
			<td>ABI prism 3730&#160;&#160;</td>
			<td>Instrument</td>
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			<td height="21" style="height:21px;">DNA islation kit</td>
			<td style="width:115px;">Qiagen</td>
			<td style="width:119px;">69104</td>
			<td>Reagent</td>
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			<td height="21" style="height:21px;">Dodine</td>
			<td style="width:115px;">Sigma</td>
			<td style="width:119px;">45466</td>
			<td>Reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gel extraction kit</td>
			<td style="width:115px;">Qiagen</td>
			<td style="width:119px;">28604</td>
			<td>Reagent</td>
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			<td height="21" style="height:21px;width:216px;">Glucose</td>
			<td style="width:115px;">Hi-Media</td>
			<td style="width:119px;">GRM077</td>
			<td>Reagent</td>
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			<td height="21" style="height:21px;width:216px;">Knapsac sparyer</td>
			<td style="width:115px;">Kaypee</td>
			<td style="width:119px;">HY-16L (1004)</td>
			<td>Instrument</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Peptone</td>
			<td style="width:115px;">Hi-Media</td>
			<td style="width:119px;">RM006-500G</td>
			<td>Reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polypropylene vials&#160;</td>
			<td style="width:115px;">Laxbro</td>
			<td style="width:119px;">SV-50</td>
			<td>Plasticware</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potato dextrose agar (PDA)&#160;</td>
			<td style="width:115px;">Hi-Media</td>
			<td style="width:119px;">M096-500G</td>
			<td>Reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tween-80</td>
			<td style="width:115px;">SRL</td>
			<td style="width:119px;">28940</td>
			<td>Reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ultra low volume sparyer</td>
			<td style="width:115px;">Matabi</td>
			<td style="width:119px;">INSECDISK</td>
			<td>Instrument</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Unicorn-bags&#160;</td>
			<td style="width:115px;">Unicorn</td>
			<td style="width:119px;">UP-140024-SMB</td>
			<td>Autoclavalbe bag for SSF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Yeast extract</td>
			<td style="width:115px;">Hi-Media</td>
			<td style="width:119px;">RM027-500G</td>
			<td>Reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chromas 2.1</td>
			<td style="width:115px;"></td>
			<td style="width:119px;"></td>
			<td>software</td>
		</tr>
	</tbody>
</table>

development of metarhizium anisopliae as a mycoinsecticide: from isolation to field performance

A major concern when developing commercial mycoinsecticides is the kill speed compared to that of chemical insecticides. Therefore, isolation and screening for the selection of a fast-acting, highly virulent entomopathogenic fungus are important steps. Entomopathogenic fungi, such as Metarhizium, Beauveria, and Nomurea, which act by contact, are better suited than Bacillus thuringiensis or nucleopolyhedrosis virus (NPV), which must be ingested by the insect pest. In the present work, we isolated 68 Metarhizium strains from infected insects using a soil dilution and bait method. The isolates were identified by the amplification and sequencing of the ITS1-5.8S-ITS2 and 26S rDNA region. The most virulent strain of Metarhizium anisopliae was selected based on the median lethal concentration (LC50) and time (LT50) obtained in insect bioassays against III-instar larvae of Helicoverpa armigera. The mass production of spores by the selected strain was carried out with solid-state fermentation (SSF) using rice as a substrate for 14 days. Spores were extracted from the sporulated biomass using 0.1% tween-80, and different formulations of the spores were prepared. Field trials of the formulations for the control of an H. armigera infestation in pigeon peas were carried out by randomized block design. The infestation control levels obtained with oil and aqueous formulations (78.0% and 70.9%, respectively) were better than the 63.4% obtained with chemical pesticide.

During the investigations, different strains of Metarhizium, Beauveria, and Nomuraea were isolated by various isolation methods (data not shown)6,14 As Metarhizium strains were found to be more effective at controlling H. armigera, a dreadful pest in pulses6,14, further isolations were targeted to isolate Metarhizium strains from different crop fields and insec...

Watch this Scientific Journal Video about Development of Metarhizium anisopliae as a Mycoinsecticide: From Isolation to Field Performance at JoVE.com

Development of Metarhizium anisopliae as a Mycoinsecticide: From Isolation to Field Performance

Here, we report the different stages involved in the knowledge-based development of an effective mycoinsecticide, including the isolation, identification, screening, and selection of the "best-fit" entomopathogenic fungus, Metarhizium anisopliae, for the control of insect pests in agriculture.

Development of Metarhizium anisopliae as a Mycoinsecticide: From Isolation to Field Performance

A major concern when developing commercial mycoinsecticides is the kill speed compared to that of chemical insecticides. Therefore, isolation and ...

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