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

None judicious use of synthetic pesticides is a continuous threat to the environment and human health. Biocontrol using bacteria, viruses, and fungi is a suitable alternative to control different insect pests in agriculture. Unlike bacteria and viruses, insect pathogen fungi, they are effective by contact.

Metarhizium species is one of the most effective insect pathogen fungi. It is known to attack more than 200 insect pest species. We have developed a mycoinsecticide using spores of indigenous Metarhizium isolates.

This video will show you the protocols for isolation and identification of local fungal isolates, insect bioassay to screen and select more potent strains, the production of spores of selected Metarhizium strains, and the protocol to create a cost-effective formulation of the mycoinsecticide for field applications. Quality control measures to be taken during commercial production will also be highlighted. Isolation of the entomopathogenic fungi can be done by three methods.

First, from soils. Metarhizium is a naturally occurring fungus in most soils. To get a wide variety of isolates, soils from fields with different kinds of crops and from widely separated regions are collected.

The soil sample is taken from four to six inches below the surface. The samples from the field are collected in labeled bags, and later in the lab, 10 grams of each sample is transferred to vials. Metarhizium is isolated by the soil dilution plate method.

10 grams of the soil sample is transferred to 90 ml sterile 0.1%Tween 80. The slurry is stirred with a magnetic stirrer for 60 minutes. Meanwhile, 900 microliter aliquots of Tween 80 are prepared for filial dilution.

The supernatant from the slurry is now serially diluted in the Tween 80 aliquots. A hundred microliter aliquot from each sample is spread on the medium described by Keller and others. The plates are incubated at 28 degrees for three to seven days.

The individual sporulating colonies are later subcultured on the same medium to obtain pure cultures. To isolate insect pathogenic fungus, dead insects are collected from fields. The spores from mycose cadavers are separated by transferring them to Tween 80 and then by vortexing.

The spore suspension is then streaked on an agar plate. Pure cultures are obtained by further subculturing on the same medium. To bait entomopathogenic fungi from soil, rice malt larvae are used.

Four rice malt larvae are added to a vial containing 60 grams soil sample. The vials are incubated at 28 degrees. These larvae normally move deep into the soil, so the vials are turned upside down everyday for up to 14 days to facilitate contact with the larvae with fungal spores in the soil.

The dead larvae are taken out from time to time and kept for sporulation in a humidity incubator. The process for the isolation of the fungus is the same as the one used for insect cadavers. After obtaining pure cultures, the isolates are maintained on PDA slants, and the mother cultures are maintained at eight degrees centigrade until use.

For identification of the fungi, genomic DNA is extracted from the mycelial biomass. For the identification of Metarhizium isolates, the primers used are five prime, three prime, ITS1, and ITS4. The amplicons of expected size are gel eluted, quantified, and sequenced.

The sequences were then checked with the NCBI GenBank Database for species level identification of the isolates. Soil samples and dead insects collected from the Pune and Buldana Districts, Maharashtra, India gave 68 distinct isolates of Metarhizium. Out of these, 58 were isolated from soils and 10 from insects.

55 were Metarhizium anisopliae, eight were Metarhizium flavoviridae, and five, Metarhizium robertsii. The spores from the slants are added to 10 ml of Tween 80 and vortexed. For insect bioassay, the spore count is measured using a hemocytometer.

And the count is adjusted to 10 raised to seven spores for ml. A set of 30 larvae in triplicate are dipped individually in a 10 ml spore suspension of each Metarhizium isolate for five seconds. Late second or early third instar larvae of Helicoverpa armigera are used for insect bioassays.

After treatment, each larvae is transferred to a sterile vile containing moist Whatman filter paper number one and a piece of disinfected okra as feed. A set of 30 larvae in triplicate treated with 0.1%Tween 80 in sterile distilled water serve as control. The larvae are kept until they are dead, and monitored for up to 14 days.

These steps were repeated for all the 68 isolates. Percent mortality is calculated by counting dead and live larvae. The dead larvae are transferred to sterile Petri plates, containing most cotton swabs and kept at 28 degrees centigrade and 70 to 80%relative humidity for three to seven days for mycosis and sporulation.

The data on percent mortality from three experiments are pulled to get average values which are then corrected using Abbott's formula. 12 Metarhizium isolates which showed more than 90%mortality were selected for determining median lethal time against third instar larvae of Helicoverpa armigera. Based on LT 50 values, five isolates that are faster at killing insects were identified.

The median lethal concentrations for the identified five isolates were determined using four different concentrations of spore suspension. Based on LC 50 values, three Metarhizium isolates were selected for spore production by solid state fermentation. The YPD medium is prepared and sterilized by autoclaving at 121 degrees centigrade for 20 minutes.

Spores from the slants are transferred to 0.1%Tween 80. The spore suspensions of the three selected Metarhizium isolates are added to YPD medium. The flasks are then incubated on shaker.

Autoclavable Unicorn bags filled with two kilograms of rice are soaked overnight in one liter distilled water and then autoclaved at 121 degrees centigrade for 45 minutes. Solid state fermentation was tested on substrates such as rice, sorghum, corn, and wheat. Among the tested substrates, rice supported the maximum sporulation of Metarhizium isolates.

The bags are inoculated with 48-hour-old 10%mycelial biomass. This is mixed well and incubated for 14 days. The spores can be harvested using different methods.

For isolation using Mycoharvester and Vibrosifter, the bags containing sporulated mycelium are dried at 37 degrees centigrade for two days to reduce the moisture content to less than 20%Liquid extraction is a more efficient method. The rice with spores is put in large vessels. 0.1%Tween 80 is poured into the vessels, and the mixture is stirred well.

The supernatant is filtered to remove any rice particle. The spores are concentrated and separated by centrifugation. The spores are then dried at 37 degrees Celsius for two days.

These spores can be stored at minus 80 degrees centigrade until use. To increase shelf life during storage, talcum is added and mixed with the spores. To be effective, the spores have to be viable, so the isolate that gives the maximum number of viable spores needs to be identified.

The spore suspensions are prepares in 0.1%Tween 80 and the count is adjusted to 10 raised to seven spores per ml. 10 microliter spore suspension is spread on PDA slides in triplicate and then incubated. Within 12 hours, the viable spores start germinating.

After 12 hours, the germ tube forming spores are counted microscopically. The hydrophilicity of spores is important for the fungus to adhere to insect cuticle. A spore suspension in ammonium sulfate and Tween 80 is taken in a curette.

The other, without the spores, is taken as control. The count of spore suspension is adjusted to about seven to 10 raised to seven spores per ml so as to obtain an initial absorbance of 0.6 at 540 nanometers. The curettes are allowed to stand undisturbed for about six hours for settling of spores.

The absorbance is recorded and the time taken for 50%alimentation is calculated. Based on the results, one strain was selected for field applications. The other two served as backup strains.

The field performance of spores of selected Metarhizium isolate M34412 to control Helicoverpa armigera infestation on pigeon pea crop was carried out in a randomized block design with four replications. There are two different spray formulations. The formulation in 0.1%Tween 80 is sprayed with a knapsack sprayer.

An oil formulation of spores is sprayed with an ultra low volume sprayer. The spraying is done either in the morning or in the evening. The first spraying is done during the flowering state, and it is repeated two more times with a 14 day interval.

Helicoverpa armigera are collected on zero, three, five, seven, and 14 days after spraying and kept in a humidity chamber for mycosis. During the field trials for the control of Helicoverpa armigera in pigeon pea, more than 70%efficacy was obtained with Metarhizium anisopliae M34412. Pod damage in Metarhizium anisopliae treated plots was found to be the least compared to plots treated with chemical pesticide and untreated control plots.

The pigeon pea leaves are examined for any effects due to the diesel sunflower oil formulation of the mycoinsecticide. Soil dwelling arthropods and leaf inhabiting insects were collected from the plots treated with Metarhizium anisopliae. No harmful effect was observed on any non-target arthropods.

The demonstration trials were conducted in a village, doo-lally-pra-va-da, with a participation of 20 farmers. The formulation is effective against other insect pests such as greasy cutworm, spotted pod borer, mealy bug, wooly aphids, and mosquitoes. The experiments have been able to identify the essential quality control parameters in the production of mycoinsecticides.

The viability of spores measured as spore germination should be more than 90%on PDA after 16 hours at 28 degrees centigrade and 70 to 80%relative humidity. The mortality of Helicoverpa armigera should be more than 90%with 10 raised to seven spores in laboratory bioassay. While repeatedly subculturing on artificial laboratory medium, the organism loses its virulence.

After culturing the fungus in chitin medium for 72 hours, the activities of cuticle degrading enzymes such as chitinase, chitin deacetylase, chitosanase, protease, and lipase are measured. These serve as biochemical markers for virulence. Four different sets of three restriction enzymes were used for digestion of chitinase genes.

The isolates with more than 90%mortality showed the specific pattern for all three genes. These patterns show significant correlation with cuticle degrading enzyme profiles produced extracellularly by Metarhizium isolates. For the first time, we suggest that chitinase enzyme activity and PCR pattern of chitinase gene as virulence markers.

This can serve as quality indicators for commercial scale production. To make the product more cost-effective, we can use mycelium biomass core plant root promotion. It can also be used for chitosanase isolation for healthcare application.

This value added products will make this Metarhizium based mycoinsecticide more profitable.