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.

The effect of application timing of metconazole on the control of Fusarium head blight (HFB) and mycotoxin accumulation in wheat and barley was investigated. Single and double applications of metconazole at different spray timings were performed in a wheat field trial. In single application plots, the mid-flowering stage was the optimal application timing of metconazole for controlling FHB symptom development, and the milking stage was optimal for reducing mycotoxin contamination. Similar efficacies were observed in all spray timings in double application plots, which included the mid-flowering stage and another spray timing. In the barley field trial, application at the mid-flowering stage controlled both FHB development and mycotoxin contamination with the highest efficacy. Similar efficacies for controlling the development of FHB in the barley field were observed with single and double applications of the fungicide. However, mycotoxin contamination was lower with double application than with single application of the fungicide.

The sensitivity of the Fusarium graminearum species complex, the causal fungus of Fusarium head blight, to metconazole was measured. The minimum inhibitory concentrations (MIC) of 101 isolates ranged from 0.20 to 6.25 mg/l with a single peak at 1.56 mg/l. The effective concentration for 50% growth inhibition (EC 50 ) was Ͻ0.1 mg/l in about 80% of isolates, and no isolate was significantly less sensitive to metconazole among this group. Among the F. graminearum species complex, F. asiaticum and F. graminearum s. str. were identified by PCR-RFLP. The trichothecene chemotypes were determined as 3ADON, 15ADON, or NIV by multiplex PCR. Although species-specific geographical distributions and mycotoxin production characteristics were found, the MIC values to metconazole of both species were distributed within a similar range, and no significant difference in sensitivity was observed between the species or trichothecene chemotypes. Next, CYP51 genes from isolates with different sensitivities to metconazole were amplified by PCR, and their sequences were compared. As a result, it was suggested that the differences in sensitivity to metconazole between the isolates were not due to the substitution of amino acids in CYP51, the target enzyme of DMI. Then, macrospores from isolates with various sensitivities to metconazole were sprayed onto wheat ears, and the efficacy of metconazole was examined. Metconazole showed high control activity against every isolate.

Various 2-N-acyl-5-methylisoxazolone derivatives were prepared, and their antifungal activities were evaluated in vitro with mycelial growth inhibition tests. In contrast with N-alkyl derivatives, the acyl compounds showed significant activity against Pyrenophora graminea, Fusarium graminearum, Alternaria alternata, Cercospora beticola, Rhynchosporium secalis, Septoria tritici, Microdochium nivale, Rhizoctonia solani and Gaeumannomyces graminis. Of note, cinnamoyl, 3-furan-3-ylacryloyland 3-thiophen-3-yl-acryloylamides, and t-butylacetyl and pivaloyl derivatives showed high inhibition rates at 25 mg/L against R. solani and G. graminis, respectively.

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