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Penicillium digitatum, causing citrus green mold, is one of the most devastating pathogenic fungi for postharvest fruits. The disease control is becoming less efficient because of the dispersal of fungicide-resistant strains. However, genome-scale analyses of its resistance mechanism are scarce. In this work, we sequenced the whole genome of the R1 genotype strain Pd01- ZJU and investigated the genes and DNA elements highly associated with drug resistance. Variation in DNA elements related to drug resistance between P. digitatum strains was revealed in both copy number and chromosomal location, indicating that their recent and frequent translocation might have contributed to environmental adaptation. In addition, ABC transporter proteins in Pd01- ZJU were characterized, and the roles of typical subfamilies ( ABCG, ABCC, and ABCB) in imazalil resistance were explored using real-time PCR. Seven ABC proteins, including the previously characterized PMR1 and PMR5, were induced by imazalil, which suggests a role in drug resistance. In summary, this work presents genome information of the R1 genotype P. digitatum and systematically investigates DNA elements and ABC proteins associated with imazalil resistance for the first time, which would be indicative for studying resistant mechanisms in other pathogenic fungi. [ABSTRACT FROM AUTHOR]

Abstract: The Ca2+/calmodulin-dependent pathway is a critical signal transduction pathway that mediates various cellular responses in multiple organisms. Calcineurin acts through zinc finger transcription factor Crz1 in fungi. In the present study, we identified an ortholog of Crz1, PdCrz1, in Penicillium digitatum, the most important pathogen of postharvest citrus. To determine the function(s) of PdCrz1, a gene-disruption mutant was generated using an Agrobacterium tumefaciens-mediated transformation (ATMT) procedure. The PdCrz1 disruption (ΔPdCrz1) mutant was defective in conidiation, cell wall integrity and virulence, and was hypersensitivity to stresses caused by Ca2+, H2O2 or 14α-demethylation inhibitor (DMI) fungicides. Expressions of cell wall synthase genes (CHS2, CHS3 and FKS1) and P-type ATPase genes (PMR1 and PMC1) were defective in the ΔPdCrz1 mutant when compared with the wild type. Calcineurin inhibitors were shown to enhance the effect of imazalil on P. digitatum. In contrast, Ca2+ (≤0.2moll−1) could alleviate the antifungal activity by imazalil. These results indicate that PdCrz1 has important roles in conidiation, virulence and DMI resistance in P. digitatum. In addition, this study also indicates that specific calcium signaling pathway inhibitors could be used as adjuvants in the conventional DMI fungicides. [Copyright &y& Elsevier]

Previously, we found a 199 bp element which inserted into the promoter of PdCYP51B gene in Penicillium digitatum, was associated with the overexpression of this gene and DMI fungicides resistance. However, the mechanism how this 199 bp element upregulate the expression of downstream gene was completely unknown. In the current study, we confirmed that this 199 bp element was a MITE-like element, designated as PdMLE1. blast searching and Southern blot showed that this 199 bp element was unique to P. digitatum. Genome-wide localization of PdMLE1 showed that it preferentially inserted into A + T rich regions, and several copies localized at the coding or regulation regions of genes were found. Penicillium digitatum mutant harbouring the PdMLE1 fused GFP gene showed the strong green fluorescence, indicating the powerful promoter activity of PdMLE1. By promoter deletion method, we identified a 20 bp core sequence in PdMLE1 which was associated with its promoter activity. In addition, we also limited the core element of PdCYP51B promoter to a 368 bp region. Collectively, we proposed a model that PdMLE1 acted as a powerful promoter and most likely recruited the transcription factor(s), therefore led to the overexpression of PdCYP51B gene and conferred P. digitatum with DMI resistance. This is the first regulation model of transposon resulted fungicide resistance proved in plant pathogens. [ABSTRACT FROM AUTHOR]

[Display omitted] A series of (-)-borneol derivatives containing 2-aryl-thiazole scaffold were designed, synthesized, and characterized by 1H NMR, 13C NMR, and HRMS. The fungicidal activities of these novel compounds against Fusarium oxysporum, Magnaporthe grisea, Botrytis cinerea, and Penicillium digitatum were evaluated. The results indicated that (1S,2R,4S)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-yl(Z)-4-oxo-4-(((2-phenylthiazol-4-yl)methyl)amino)but-2-enoate (6a) displayed potential fungicidal activities with broad spectrum. Especially, 6a exhibited an IC 50 value of 48.5 mg/L against P. digitatum, which has higher fungicidal activity than commercial products hymexazol and amicarthiazol. Moreover, (1S,2R,4S)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-yl-4-oxo-4-(((2-phenylthiazol-4-yl)methyl)amino)butanoate (5a) possesses an IC 50 value of 24.3 mg/L against B. cinerea, comparable to hymexazol and far superior to amicarthiazol. Furthermore, the superficial structure–activity relationship was discussed, which might be helpful for discovering novel fungicides. [ABSTRACT FROM AUTHOR]