Effect of Chitosan from Black Soldier Fly Larvae Pupae Shells against Mycotoxin producing Aspergillus sp.

Commodities

Practices

The present work aims to test the effectiveness of insect (black soldier fly larvae) chitosan as an eco-friendly seed coating agent to combat Aspergillus sp., a common fungal pathogen affecting peanuts. Current seed treatment practices using commercial fungicides such as azoxystrobin are becoming increasingly ineffective due to the emergence of fungicide-resistant Aspergillus strains, as observed in recent random seed testing. Furthermore, the broad-spectrum nature of these fungicides adversely impacts beneficial soil microorganisms and non-target organisms such as earthworms. Hence, there is a need to develop an eco-friendly alternative. Chitosan, a biopolymer derived through the deacetylation of chitin, the second most abundant polysaccharide on earth found in the exoskeletons of insects, crustaceans, and fungal cell walls, has demonstrated significant antifungal properties against a wide range of phytopathogens. This study explores the synthesis of chitosan from BSFL pupal shells and evaluates its effectiveness as a seed treatment agent under controlled conditions. The antifungal potential of BSFL chitosan will be tested against Aspergillus sp. through in vitro inhibition assays and greenhouse trials, comparing its performance with commercial fungicides and untreated controls. The principal goal is to assess whether BSFL-derived chitosan can provide a cost-effective, environmentally responsible alternative for managing fungal infections in peanuts, while promoting sustainable agricultural practices through the valorization of insect biomass.

The objectives of this proposed work are as follows:

• Synthesize chitosan from the pupae of BSFL and characterize them to understand the physicochemical properties

This objective aims to synthesize chitosan from BSFL pupae shells (exoskeleton). The authors will use conventional organic solvents to synthesize chitosan from BSFL (following the protocol outlined in approach and methods section). The major outcome from this objective is to synthesize chitosan with a higher degree of deacetylation. Properties of the synthesized chitosan will then be compared against commercial crustacean chitosan (>85% deacetylated chitin).

• Investigate the effect of BSFL chitosan against aflatoxin producing Aspergillus invitro

This objective aims to perform invitro testing of BSFL chitosan against both toxin and non-toxin producing Aspergillus sp. BSFL chitosan in acetic acid will be prepared and added to liquid potato dextrose agar (PDA) medium (to achieve a final concentration of 0.1 to 1.0 % (v/v)) before solidification. Control (acidified water pH 5.5) will be used as a control in parallel. To evaluate the inhibition of hyphal growth circular plus (ca. 5mm) carrying mycelia of Aspergillus sp. will be placed at the center of the solidified BSFL chitosan added petri plate. The plates will be incubated for seven days at an optimal temperature of 28 ℃. Radical growth in (cm) for each plate will be measured daily. The inhibition of fungal growth in percentage will be calculated as follows: Inhibition (%) = [(Radical growth of Aspergillus sp. in control PDA plate - Radical growth of Aspergillus sp. in BSFL chitosan PDA plate)/ Radical growth of Aspergillus sp. in control PDA plate] x 100. This objective will help to determine the chitosan concentration used for seed coating (next objective).

• Coat the peanut seeds using BSFL chitosan and test its effectiveness against infection by Aspergillus upon real-time (4 ℃) and accelerated storage (37 ℃) conditions.

This objective aims to coat the peanut seeds using BSFL chitosan and assess its effectiveness against Aspergillus sp. infection under real-time and accelerated storage conditions. The germination ability of the peanuts coated using BSFL chitosan will be performed at regular intervals. For comparison, commercial fungicide coated peanuts (positive control) will be tested in parallel. Non-coated peanut seeds will be used as control for this coating study. This objective will help identify the shelf-life of seed coating using BSFL chitosan.

• Test the effectiveness of foliar application of BSFL chitosan in peanut plants to fight against Aspergillus infection in a controlled environment (greenhouse)

Peanut seeds showing good germination potential will be transferred to pots in a greenhouse. For this objective, experiments will be carried out using peanuts coated using BSFL chitosan and will be compared against commercial fungicide coated peanuts and non-seed coated peanuts. Foliar application will be carried out using BSFL chitosan, commercial fungicide and deionized water. Plants will be infected with spores of Aspergillus sp. to evaluate the ability of fungi to infect treated (chitosan and fungicide) and non-treated plants. This objective will demonstrate the efficacy of BSFL chitosan against phytopathogens in peanuts.