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

Summary

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.

Abstract

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.

Introduction

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 resist....

Protocol

1. Isolation of Entomopathogenic Fungi

1. Soil dilution method
   1. 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°31'13''N; 73°51'24''E) and Buldhana (19°58'36''N 76°30'30''E) districts, Maharashtra, India.
   2. Weigh 10 g of each soil sample and add them separately to 90 mL of sterile 0.1% (w/v) Tween-80.
   3. Thorough....

Results

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 pulses^6,14, further isolations were targeted to isolate Metarhizium strains from different crop fields and insec.......

Discussion

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<.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Agar	Hi-Media	RM666	Reagent
Ammonium sulphate	Thomas Baker	11645	Reagent
DNA analyzer	Applied biosystem	ABI prism 3730	Instrument
DNA islation kit	Qiagen	69104	Reagent
Dodine	Sigma	45466	Reagent
Gel extraction kit	Qiagen	28604	Reagent
Glucose	Hi-Media	GRM077	Reagent
Knapsac sparyer	Kaypee	HY-16L (1004)	Instrument
Peptone	Hi-Media	RM006-500G	Reagent
Polypropylene vials	Laxbro	SV-50	Plasticware
Potato dextrose agar (PDA)	Hi-Media	M096-500G	Reagent
Tween-80	SRL	28940	Reagent
Ultra low volume sparyer	Matabi	INSECDISK	Instrument
Unicorn-bags	Unicorn	UP-140024-SMB	Autoclavalbe bag for SSF
Yeast extract	Hi-Media	RM027-500G	Reagent
Chromas 2.1			software