Talaromyces sp. isolate KNB-422, isolated from a rice seedling, is a biofungicidal agent effective against several seedborne pathogens of rice including Gibberella fujikuroi, which causes Bakanae disease. Because the fungal mode of action (MOA) has not yet been clarified, we used the fluorescent protein markers GFP and RFP to visualize cell-cell interactions between the biocontrol agent and the pathogen G. fujikuroi. In slide culture, the hyphal cell wall of G. fujikuroi collapsed, and fluorescence of its cytoplasm disappeared 3 days after contact with hyphae of Talaromyces sp. On inoculated rice plants, both fungi occupied the same regions of coleoptiles and roots, where the biocontrol effect of Talaromyces sp. must be exerted. Our observations suggest that the MOA of Talaromyces sp. is mycoparasitic.

Bakanae disease, caused by Fusarium fujikuroi, is an economically important seed-borne disease of rice. F. fujikuroi is horizontally transmitted to rice flowers and vertically transmitted to the next generation via seeds. The fungus induces typical symptoms such as abnormal tissue elongation and etiolation. Sanitation of seed farms and seed disinfection are the only effective means to control bakanae disease at present; however, the efficacy of these methods is often insufficient. Therefore, alternative and innovative control methods are necessary. We developed a novel method for applying non-pathogenic fusaria as biocontrol agents by spraying spore suspensions onto rice flowers to reduce the incidence of seed-borne bakanae. We visualized the interaction between F. commune W5, a non-pathogenic fusarium, and F. fujikuroi using transformants expressing two different fluorescent proteins on/in rice plants. W5 inhibited hyphal extension of F. fujikuroi on/in rice flowers and seedlings, possibly by competing with the pathogen, and survived on/in rice seeds for at least six months. Importance We demonstrated that a spray-treatment of rice flowers with the spores of non-pathogenic fusaria mimicked the disease cycle of the seed-borne bakanae pathogen Fusarium fujikuroi and effectively suppressed the disease. Spray treatment of non-pathogenic fusaria reduced the degree of pathogen invasion of rice flowers and vertical transmission of the pathogen to the next plant generation via seeds, thereby controlling the bakanae disease. The most promising isolate, F. commune W5, colonized seeds and seedlings via treated flowers and successfully inhibited pathogen invasion, suggesting that competition with the pathogen was the mode of action. Seed-borne diseases are often controlled by seed treatment with chemical fungicides. Establishing an alternative method is a pressing issue from the perspectives of limiting fungicide resistance and increasing food security. This work provides a potential solution of these issues using a novel application technique to treat rice flowers with biocontrol agents.

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Talaromyces sp. KNB422, isolated from a rice seedling, is a fungus that is highly e ective against several rice seedling diseases. To clarify its mode of action, the interaction between KNB422 and Rhizopus oryzae, Pythium graminicola, and Gibberella fujikuroi was examined in vitro. In a PDA medium, KNB422 expanded its hyphae over the mycelia of the pathogens. e interaction between KNB422 and R. oryzae and P. graminicola was examined by scanning electron microscopy. e hyphae of KNB422 grew around and then penetrated the hyphae of the pathogens. Furthermore uorescence microscopy revealed that KNB422 grew toward, penetrated into and dissolved the G. fujikuroi hyphae. KNB422 has the ability to parasitize pathogens, such as R. oryzae, P. graminicola, and G. fujikuroi, to prevent their growth.

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