Biocontrol potential of indigenous fungi against mycotoxigenic fungi and mycotoxin contamination in grain maize agro-ecosystems in Terengganu, Malaysia

Biological control against mycotoxigenic fungi and the subsequent mycotoxins have been widely studied, and some are also commercially available in many countries, but the same cannot be said in Malaysia. Therefore, the present study aimed to identify novel indigenous fungal biocontrol agents against mycotoxigenic fungi and their subsequent mycotoxin production in pioneer grain maize farms in Malaysia. The objectives of the present study were: (1) to establish the mycodiversity of indigenous mycotoxigenic fungi and potential biocontrol agents from grain maize agro-ecosystem, (2) to molecularly and toxigenically differentiate the indigenous Aspergillus flavus isolates from grain maize agro-ecosystem, (3) to screen the antagonism of potential biocontrol agents against isolated indigenous mycotoxigenic fungi from grain maize agro- ecosystem, and (4) to determine effective biocontrol agents in controlling multi- mycotoxin production by indigenous mycotoxigenic fungi from grain maize agro-ecosystem using different pathogen:antagonist inoculum ratios. Fungi isolated from two pioneer grain maize farms in Terengganu were morphologically and molecularly identified. Mycotoxigenic potential of all mycotoxigenic isolates were also examined using high performance liquid chromatography (HPLC). Next, seven A. flavus isolates obtained from the farms were characterised molecularly and chemically by examining for the presence of aflatoxin biosynthesis genes, and confirmed their aflatoxigenicity using HPLC. Phylogenetic relationships of all A. flavus isolates were validated. Thereafter, dual-culture assay was conducted on grain maize agar (GMA) with 12 isolates of potential fungal antagonists against mycotoxigenic isolates producing aflatoxins, ochratoxin A, and fumonisins, respectively. Five pathogen:antagonist inoculum ratios – 100:0, 75:25, 50:50, 25:75, and 0:100, were then employed to examine mycotoxin reduction by selected fungal antagonists on GMA and grain maize kernels. Fungal isolation from two pioneer grain maize farms revealed among 30 species isolated, the mycotoxigenic Fusarium verticillioides was predominant. Other fungal isolates that could have potentials as biocontrol agents (e.g., Trichoderma spp.) were also isolated. The selected A. flavus, A. niger, F. verticillioides, and F. proliferatum isolates that could produce aflatoxins (AFBs), fumonisins (FBs), and ochratoxins A (OTA), respectively, were used for the subsequent experiments. Next, the identity of seven A. flavus isolates were taxonomically resolved. Among these isolates, five were aflatoxigenic and two were non- aflatoxigenic. Two non-aflatoxigenic isolates were examined for AFB1 reduction against the aflatoxigenic counterparts. Although both non- aflatoxigenic showed weak antagonistic activity against growth of the aflatoxigenic isolates, all could significantly (p < 0.05) reduce AFB1. Among 12 potential fungal antagonists, Trichoderma spp. exhibited the highest inhibitory activity against growth of the tested mycotoxigenic isolates. Against OTA and fumonisins, the efficacy of T. asperelloides Ta39KR was demonstrated for the first time. Against AFB1, the efficacy of Penicillium janthinellum Pj46SD was also demonstrated for the first time. The pathogen:antagonist inoculum ratios assay showed non-aflatoxigenic A. flavus Af1KD as the most effective against AFBs. T. asperelloides Ta39KR also effective against FBs. The indigenous P. janthinellum Pj46SD and T. asperelloides Ta39KR have potential to be developed as novel biocontrol agents against mycotoxigenic fungal growth and their subsequent aflatoxins, ochratoxin A, and fumonisins, respectively.

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