MADS-box proteins, a well-conserved family of transcription factors in eukaryotic organisms, specifically regulate a wide range of cellular functions, including primary metabolism, cell cycle, and cell identity. However, little is known about roles of the MADS-box protein family in the fungal pathogen Sclerotinia sclerotiorum. In this research, the S. sclerotiorum MADS-box gene SsMADS was cloned; it encodes a protein that is highly similar to Mcm1 orthologs from Saccharomyces cerevisiae and other fungi, and includes a highly conserved DNA-binding domain. MADS is a member of the MADS box protein SRF (serum response factor) lineage. SsMADS function was investigated using RNA interference. Silenced strains were obtained using genetic transformation of the RNA interference vectors pS1-SsMADS and pSD-SsMADS. SsMADS expression levels in silenced strains were analyzed using RT-PCR. The results showed that SsMADS mRNA expression in these silenced strains was reduced to different degrees, and growth rate in these silenced strains was significantly decreased. Infecting tomato leaflets with silenced strains indicated that SsMADS was required for leaf pathogenesis in a susceptible host. Our results suggest that the MADS-box transcription factor SsMADS is involved in S. sclerotiorum growth and virulence.

Summary: Botrytis cinerea is the causative agent of grey mould on over 1000 plant species and annually causes enormous economic losses worldwide. However, the fungal factors that mediate pathogenesis of the pathogen remain largely unknown. Here, we demonstrate that a novel B. cinerea ‐specific pathogenicity‐associated factor BcHBF1 ( h yphal b ranching‐related f actor 1), identified from virulence‐attenuated mutant M8008 from a B. cinerea T‐DNA insertion mutant library, plays an important role in hyphal branching, infection structure formation, sclerotial formation and full virulence of the pathogen. Deletion of BcHBF1 in B. cinerea did not impair radial growth of mycelia, conidiation, conidial germination, osmotic‐ and oxidative‐stress adaptation, as well as cell wall integrity of the ∆ Bchbf1 mutant strains. However, loss of BcHBF1 impaired the capability of hyphal branching, appressorium and infection cushion formation, appressorium host penetration and virulence of the pathogen. Moreover, disruption of BcHBF1 altered conidial morphology and dramatically impaired sclerotial formation of the mutant strains. Complementation of BcHBF1 completely rescued all the phenotypic defects of the ∆ Bchbf1 mutants. During young hyphal branching, host penetration and early invasive growth of the pathogen, BcHBF1 expression was up‐regulated, suggesting that BcHBF1 is required for these processes. Our findings provide novel insights into the fungal factor mediating pathogenesis of the grey mould fungus via regulation of its infection structure formation, host penetration and invasive hyphal branching and growth.

Cyclophilin (Cyp) and Ca²⁺/calcineurin proteins are cellular components related to fungal morphogenesis and virulence; however, their roles in mediating the pathogenesis of Botrytis cinerea, the causative agent of gray mold on over 1000 plant species, remain largely unexplored. Here, we show that disruption of cyclophilin gene BcCYP2 did not impair the pathogen mycelial growth, osmotic and oxidative stress adaptation as well as cell wall integrity, but delayed conidial germination and germling development, altered conidial and sclerotial morphology, reduced infection cushion (IC) formation, sclerotial production and virulence. Exogenous cyclic adenosine monophosphate (cAMP) rescued the deficiency of IC formation of the ∆Bccyp2 mutants, and exogenous cyclosporine A (CsA), an inhibitor targeting cyclophilins, altered hyphal morphology and prevented host-cell penetration in the BcCYP2 harboring strains. Moreover, calcineurin-dependent (CND) genes are differentially expressed in strains losing BcCYP2 in the presence of CsA, suggesting that BcCyp2 functions in the upstream of cAMP- and Ca²⁺/calcineurin-dependent signaling pathways. Interestingly, during IC formation, expression of BcCYP2 is downregulated in a mutant losing BcJAR1, a gene encoding histone 3 lysine 4 (H3K4) demethylase that regulates fungal development and pathogenesis, in B. cinerea, implying that BcCyp2 functions under the control of BcJar1. Collectively, our findings provide new insights into cyclophilins mediating the pathogenesis of B. cinerea and potential targets for drug intervention for fungal diseases.

Rice blast caused by Magnaporthe oryzae is one of the most serious rice diseases worldwide. Biological control is gaining popularity as a promising method for the control of this disease; however, more effective microbial strains with strong adaptability in rice fields need to be identified. Here, we report for the first time the successful identification of biocontrol bacterial strains from frozen soils of the soda saline-sodic land. We isolated 82 bacterial strains from rice fields in the western Songnen Plain of China, one of the three major soda saline soils in the world. Five of the isolated strains exhibited strong inhibition to M. oryzae growth. The potential strains were identified as Bacillus safensis JLS5, Pseudomonas koreensis JLS8, Pseudomonas saponiphila JLS10, Stenotrophomonas rhizophila JLS11 and Bacillus tequilensis JLS12, respectively, by 16s RNA gene sequence analysis. The antagonistic assay and the artificial inoculation tests showed that JLS5 and JLS12 could effectively inhibit conidial germination and pathogenicity of the rice blast fungus, both preventively and curatively. The suppression of pathogenicity was further confirmed by greenhouse experiments, showing the effectiveness of JLS5 and JLS12 as a potential biological control agents of M. oryzae. The potential application of these cold-tolerant strains for rice blast control in cold regions is discussed. Our data suggest that soda saline-sodic soils are a rich source for biocontrol strain isolation.