Monilinia fructicola is a devastating pathogen on stone fruits, causing blossom blight and fruit rot. Little is known about pathogenic mechanisms in M. fructicola and related Monilinia species. In this study, five endopolygalacturonase (endo-PG) genes were cloned and functionally characterized in M. fructicola. Quantitative reverse-transcriptase PCR (qRT-PCR) revealed that the five MfPG genes are differentially expressed during pathogenesis and in culture under various pH regimes and carbon and nitrogen sources. MfPG1 encodes the major endo-PG and is expressed to significantly higher levels compared to the other four MfPGs in culture and in planta. MfPG1 function during pathogenesis was evaluated by examining the disease phenotypes and gene expression patterns in M. fructicola MfPG1-overexpressing strains and in strains carrying the β-glucuronidase (GUS) reporter gene fused with MfPG1 (MfPG1-GUS). The MFPG1-GUS reporter was expressed in situ in conidia and hyphae following inoculation of flower petals, and qRT-PCR analysis confirmed MfPG1 expression during pathogenesis. MfPG1-overexpressing strains produced smaller lesions and higher levels of reactive oxygen species (ROS) on the petals of peach and rose flowers than the wild-type strain, suggesting that MfPG1 affecting fungal virulence might be in part resulted from the increase of ROS in the Prunus–M. fructicola interactions.

The MfCUT1 gene encoding the major cutinase of Monilinia fructicola, a causal agent of blossom blight and fruit rot of stone fruits, is a virulence factor of the pathogen. The pathogen remains quiescent on stage II fruit, which contain high levels of chlorogenic acid, a quinate ester of caffeic acid (CA). A medium shift system was established to show that MfCUT1 expression is down-regulated by CA, consistent with previous findings in continuous culture, and by the antioxidants glutathione (GSH), N-acetyl-L-cysteine and ascorbic acid. However, MfCUT1 expression is up-regulated by the superoxide-generating oxidant menadione and by the GSH synthesis inhibitor buthionine sulphoximine (BSO). The changes in MfCUT1 transcript levels induced by CA or BSO are related to the levels of intracellular GSH and H 2 O 2 . In addition, GSH reductase activity was increased by CA. Monilinia fructicola genes encoding enzymes that may contribute to redox homeostasis (GSH reductase and GSH peroxidase) were cloned and their expression was found to be slightly affected following exposure of cultures to CA. The hybrid tea rose cv. Flaming Peace was identified as an alternative host to complement pathogenicity assays of M. fructicola on stone fruits, which are seasonally restricted. Pathogenicity assays on detached peach fruit and rose petals revealed that CA suppresses and H 2 O 2 enhances formation of brown rot lesions. The results indicate that changes in cellular redox status impact MfCUT1 expression and virulence in M. fructicola, and suggest that redox cycling of GSH is related to this regulation.

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.