Monilinia fructicola is a fungal pathogen of worldwide significance that causes brown rot of stone fruits. There are only few reports related to the production of biologically active polyketides by this pathogen. In this study, we examined an atypical M. fructicola strain TW5-4 that shows strong antimicrobial activity against various plant pathogens. TW5-4 also displays sparse growth in culture, low virulence, and higher levels of melanin compared with its albino mutant, TW5-4WM, and a wild-type strain Mf13-81. Antifungal compounds were extracted from TW5-4 and purified by thin-layer chromatography following visualization with an on-the-chromatogram inhibition assay. The principal antifungal compound was identified by linear ion trap mass spectrometry, high-resolution electro-spray ionization mass spectrometry, and proton nuclear magnetic resonance analyses as the polyketide chloromonilicin. Multiple M. fructicola polyketide synthase (PKS) sequences were then cloned by degenerate PCR and inverse PCR. Sequence analyses support presence of a 10-member PKS gene family in the M. fructicola genome. Analyses of PKS gene expression found no strong correlation between chloromonilicin production in culture and transcript levels of any of the PKS gene family members in mycelium of strains TW5-4, TW5-4WM, and Mf13-81. However, MfPKS12, a homolog of BcPKS12 involved in biosynthesis of 1,8-dihydroxynaphthalene (DHN)-melanin in Botrytis cinerea, was strongly expressed in mycelia of TW5-4 and Mf13-81. An MfPKS12-silenced mutant accumulated significantly less melanin in mycelia, had lower resistance to polyethylene glycol-induced osmotic stress, and displayed reduced virulence on nectarine fruit. The results suggest that DHN-melanin is required for tolerance to osmotic stress and full virulence in M. fructicola.

Colletotrichum scovillei causes anthracnose of chili pepper in many countries. Three strains of this pathogen, Coll-524, Coll-153, and Coll-365, show varied virulence on chili pepper. Among the three strains, Coll-365 showed significant defects in growth and virulence. To decipher the genetic variations among these strains and identify genes contributing to growth and virulence, comparative genomic analysis and gene transformation to show gene function were applied in this study. Compared to Coll-524, Coll-153, and Coll-365 had numerous gene losses including 32 candidate effector genes that are mainly exist in acutatum species complex. A cluster of 14 genes in a 34-kb genomic fragment was lost in Coll-365. Through gene transformation, three genes in the 34-kb fragment were identified to have functions in growth and/or virulence of C. scovillei. CsPLAA encoding a phospholipase A2-activating protein enhanced the growth of Coll-365. A combination of CsPLAA with one transcription factor CsBZTF and one C6 zinc finger domain-containing protein CsCZCP was found to enhance the pathogenicity of Coll-365. Introduction of CsGIP, which encodes a hypothetical protein, into Coll-365 caused a reduction in the germination rate of Coll-365. In conclusion, the highest virulent strain Coll-524 had more genes and encoded more pathogenicity related proteins and transposable elements than the other two strains, which may contribute to the high virulence of Coll-524. In addition, the absence of the 34-kb fragment plays a critical role in the defects of growth and virulence of strain Coll-365.

Mango is an important tropic fruit, but its production is highly restricted by anthracnose diseases. Mango anthracnose development is related to the fruit-ripening hormone ethylene, but how the pathogen senses ethylene and affects the infection remains largely unknown. In this study, mango pathogen Colletotrichum asianum strain TYC-2 was shown to sense ethylene to enhance spore germination, appressorium formation and virulence. Upon further analysis of ethylene sensing signaling, three histidine kinase genes (CaHKs) and a G-protein gene (CaGα1) were functionally characterized. Ethylene upregulated the expression of the three CaHKs but had no influence on CaGα1 expression. No function in ethylene sensing was identified for the three CaHKs. Ethylene enhanced spore germination and multiple appressorium formation of the wild-type TYC-2 but not CaGα1 mutants. TYC-2 has extremely low germination in water, where self-inhibition may play a role in ethylene sensing via CaGα1 signaling. Self-inhibitors extracted from TYC-2 inhibited spore germination of TYC-2 and CaGα1 mutants, but ethylene could not rescue the inhibition, indicating that the self-inhibition was not mediated by CaGα1 and had no interactions with ethylene. Interestingly, spore germination of CaGα1 mutants was significantly enhanced in water on hydrophobic but not hydrophilic surfaces, suggesting that CaGα1 is involved in surface sensing. In the pathogenicity assay, CaGα1 mutants showed less virulence with delayed germination and little appressorium formation at early infection on mango leaves and fruit. Transcriptome and qRT–PCR analyses identified several pathogenicity-related genes regulated by ethylene, indicating that ethylene may regulate TYC-2 virulence partially by regulating the expression of these genes.

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