Mass Production of Entomopathogenic Fungi, Metarhizium robertsii and Metarhizium pinghaense, for Commercial Application Against Insect Pests

This method provides new information on how to mass produce infective conidia of entomopathogenic fungi to control important insect pests in commercial agro-ecosystems, given the current situation where most insecticides are phased out due to concerns of their toxic effect on the environment and human health. The technique describes the method used to ensure the optimum yield of EPF conidia during mass production. The method is helpful for integrated pest management and biological control by providing insight into the effectiveness of mass produced conidia for large-scale management of damaging insecticide resistance insect pests in the agro-ecosystems.

This procedure was developed and demonstrated by Dr.Letodi Luki Mathulwe, Dr.Nomakholwa Faith Stokwe, and Professor Antoinette Paula Malan. Heat one liter distilled water and switch off the heat before reaching the boiling point. Now, add 30 grams of glucose, four grams of potassium phosphate dibasic, 20 grams of yeast extract, 15 milliliters of corn steep liquor, and bring the contents to a gentle boil for two to three minutes.

Pour a total of 100 milliliters of the medium into nine different 250-milliliter flasks. Place a cotton wool plug on each flask and cover the cotton wool with aluminum foil as a stopper. Autoclave the medium in the flasks for 55 minutes at 121 degrees Celsius.

Later, allow the medium in the flasks to cool and add 10 milligrams per milliliter of streptomycin to the medium in each flask. Collect two to three bacterial loops of fungal conidia from two-to three-week-old fungal culture plates for both the EPF isolates. Transfer the fungal conidia to each 100 milliliters of liquid media in the 250 milliliter flasks under sterile conditions and seal the flasks.

Incubate the liquid culture flasks at approximately 25 degrees Celsius on an orbital shaker at 140 RPM for three days and cease the incubation once the cultures show signs of high turbidity with fungal blastospore growth. To detect any possible bacterial contamination from the cultures, draw a 100-microliters sample from each liquid culture flask after 24 hours of incubation and plate on three SDA plates per isolate. Incubate the plates for 48 hours at a controlled temperature of plus or minus 25 degrees Celsius.

Use a small polyvinyl chloride waste pipe to create a neck for the fermentation bag at the open end of the autoclave bag and use autoclave tape to secure the pipe. Close the pipe with a sterile cotton wool plug to allow sufficient gas exchange during fermentation. Use parboiled long grain white rice as a solid substrate medium for the blastospores of both Metarhizium pinghaense and Metarhizium robertsii.

For each fermentation bag, add one kilogram of rice and 300 milliliters of sterile distilled water. Gently mix the contents of the fermentation bags. Place them in outer autoclave bags in an upright position and autoclave at 121 degrees Celsius for 55 minutes.

Following the autoclaving, allow the substrates to cool down for about 45 minutes under sterile conditions. Remove the closure of each of the liquid culture flasks of both Metarhizium pinghaense and Metarhizium robertsii under a laminar flow and flame the rim of each flask for 10 seconds. Pour 100 milliliters of liquid cultures into the fermentation bags by removing the cotton wool plugs from their necks.

Place the cotton plugs back on and cover the top of the bag's neck with surgical paper secured with a rubber band. Twist the top part of the bag and mix the bag's contents by shaking and manipulating the substrate by massage. Incubate the bags at about 25 degrees Celsius by flattening the substrate in the bags to prevent the formation of thick beds.

Two to three days after inoculation and incubation, when visible mycelial growth and the binding of the substrate by the fungus had occurred, break the substrate in the fermentation bags by massaging the bags'contents. Following fermentation, transfer the sporulated cultures into 26-to 30-kilogram brown paper bags and dry the fungal cultures for 10 to 12 days before their use in trials. To improve the tensile strength of the paper bags, horizontally cut off 1/3 of the top part of the bag and line the bottom of the bag.

Gently crumble the substrate in each fermentation bag. Cut off the corner of each bag and slowly transfer the whole culture to the paper bags through the space left by the cut off corner. Label each paper bag, fold over the top end of each bag twice, and close with staples to create a triangular tent structure.

Place the bags on wire drying racks to allow proper, even drying at a controlled temperature of about 25 degrees Celsius and low humidity of 30 to 40%Harvest fungal conidia from the cultures using three nested sieves mounted on a collection pan. Slowly load the dry culture sample on the ETS mesh number 35 and sieve. Place a lid on the sieve to prevent the release of fungal conidia into the air.

Add 10 to 12 glass marbles to the sieves to assist the passage of the fungal conidia through the mesh screens and avoid the retention of the conidia in the sieve, which can result in reduced spore recovery. Tape and seal the sieve joints using electrical tape. Place the sieves on a vibratory shaker fitted with a sticky pad to secure the collection pan and sieves for 20 to 25 minutes at a motion of 560 to 640 vibrations per minute.

Remove the test sieves from the collection pan, collect conidia, and store the collected conidia in airtight and water-impermeable zipper-lock bags for long-term storage. An average of approximately 1.83 grams of dry Metarhizium robertsii conidia was harvested from the flaked barley substrate, whereas zero Metarhizium pinghaense was harvested from the substrate. No dry fungal conidia were harvested from the flaked oat substrate for either Metarhizium species.

A significant difference was observed between the two species of Metarhizium in the estimated average conidia per gram harvested from the rice grains. Metarhizium robertsii had slightly higher average conidia count per gram than Metarhizium pinghaense. No significant difference was observed between the two species of Metarhizium in the estimated number of conidia present on the spent rice substrate.

However, Metarhizium pinghaense had slightly higher conidia on the rice substrate than Metarhizium robertsii No significant difference is estimated in the two species of Metarhizium in their overall conidia yield obtained from the rice substrate cultures. However, Metarhizium pinghaense produced a slightly higher conidia yield than did Metarhizium robertsii, which produced a lower conidial yield. The challenging part of this technique is the substrate contamination through inoculation with a contaminated blastospores inoculum.

Thus, always ensure sterility while working. This demonstration helps the students efficiently reproduce the technique without clumsy mistakes that can result in the termination of the entire process.