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Sclerotinia sclerotiorum is a fungal pathogen with a broad range of hosts, which can cause diseases and pose a great threat to many crops. Fungal-specific Zn2Cys6 transcription factors (TFs) constitute a large family prevalent among plant pathogens. However, the function of Zn2Cys6 TFs remains largely unknown. In this study, we identified and characterized SsZNC1, a Zn2Cys6 TF in S. sclerotiorum, which is involved in virulence, sclerotial development, and osmotic stress response. The expression of SsZNC1 was significantly up-regulated in the early stages of S. sclerotiorum infection on Arabidopsis leaves. The target deletion of SsZNC1 resulted in reduced virulence on Arabidopsis and oilseed rape. In addition, sclerotial development ability and growth ability under hyperosmotic conditions of SsZNC1 knockout transformants were reduced. A transcriptomic analysis unveiled its regulatory role in key cellular functions, including cellulose catabolic process, methyltransferase activity, and virulence, etc. Together, our results indicated that SsZNC1, a core regulatory gene involved in virulence, sclerotial development and stress response, provides new insight into the transcription regulation and pathogenesis of S. sclerotiorum.

Sclerotinia sclerotiorum is a broad-spectrum necrotrophic phytopathogen that can infect many plant species worldwide. The application of fungicides is a common measure for controlling Sclerotinia sclerotiorum. Due to the risk of developing resistance to fungicides, it is imperative to find ways to be environmentally friendly and even effective. Using bioactive compounds in plants to reduce the amounts of fungicides has become a clean and sustainable strategy of controlling Sclerotinia sclerotiorum. Our study found that selenium in soil mediated the phenylacetic acid-related metabolic pathway in oilseed rape and reduced the incidence rate of Sclerotinia sclerotiorum. The growth-inhibition rates of Sclerotinia sclerotiorum were observed at 25.82%, 19.67%, and 52.61% for treatments of 0.8 mg·L−1 dimethachlon, 0.1 mg·mL−1 phenylacetic acid, and dimethachlon (0.8 mg·L−1) + phenylacetic acid (0.1 mg·mL−1), respectively. Phenylacetic acid reduced the application amount of dimethachlon and enhanced the inhibition effect for Sclerotinia sclerotiorum. Results also suggested that phenylacetic acid severely damaged the morphological structure, changed the electrical conductivity, and reduced the capacity of acid production and oxalic acid secretion of Sclerotinia sclerotiorum mycelium. Further studies revealed that phenylacetic acid increased the gene-expression level of Ssodc1, Ssodc2, CWDE2 and CWDE10 in mycelium while decreasing the expression level of SsGgt1, and phenylacetic acid + dimethachlon reduced the relative expression level of SsBil. These findings verified that phenylacetic acid could partially replace the amount of dimethachlon, as well as enhance the prevention of Sclerotinia sclerotiorum by dimethachlon, which provides evidence for developing an environment-friendly method for Sclerotinia sclerotiorum control.

Heredity and variation are inherent characteristics of species and are mainly reflected in the stability and variation of the genome; the former is relative, while the latter is continuous. However, whether life has both stable genomes and extremely diverse genomes at the same time is unknown. In this study, we isolated Sclerotinia sclerotiorum strains from sclerotium samples in Quincy, Washington State, USA, and found that four single-sclerotium-isolation strains (PB4, PB273, PB615, and PB623) had almost identical genomes to the reference strain 1980 isolated in the west of Nebraska 40 years ago. The genome of strain PB4 sequenced by the next-generation sequencing (NGS) and Pacific Biosciences (PacBio) sequencing carried only 135 single nucleotide polymorphisms (SNPs) and 18 structural variations (SVs) compared with the genome of strain 1980 and 48 SNPs were distributed on Contig_20. Based on data generated by NGS, three other strains, PB273, PB615, and PB623, had 256, 275, and 262 SNPs, respectively, against strain 1980, which were much less than in strain PB4 (532 SNPs) and none of them occurred on Contig_20, suggesting much closer genomes to strain 1980 than to strain PB4. All other strains from America and China are rich in SNPs with a range of 34,391–77,618 when compared with strain 1980. We also found that there were 39–79 SNPs between strain PB4 and its sexual offspring, 53.1% of which also occurred on Contig_20. Our discoveries show that there are two types of genomes in S. sclerotiorum, one is very stable and the other tends to change constantly. Investigating the mechanism of such genome stability will enhance our understanding of heredity and variation.

Transcriptional activity is mediated by chromatin remodeling, which in turn is affected by post-translational modifications, including histone acetylation. Histone acetyltransferases (HATs) are capable of promoting euchromatin formation and then activating gene transcription. Here, we characterize the Elp3 GNAT family HAT, which is also a subunit of Elongator complex, in the environmentally and economically important fungal insect pathogen, Beauveria bassiana. BbElp3 showed high localization levels to mitochondria, with some nuclear and cytoplasmic localization also apparent. Targeted gene knockout of BbElp3 resulted in impaired asexual development and morphogenesis, reduced tolerances to multiple stress conditions, reduced the ability of the fungus to utilize various carbon/nitrogen sources, increased susceptibility to rapamycin, and attenuated virulence in bioassays using the greater wax moth, Galleria mellonella. The ΔBbElp3 mutant also showed disrupted cell cycle, abnormal hyphal septation patterns, and enlarged autophagosomes in vegetative hyphae. Transcriptome analyses revealed differential expression of 775 genes (DEGs), including 336 downregulated and 438 upregulated genes in the ΔBbElp3 strain as compared to the wild type. Downregulated genes were mainly enriched in pathways involved in DNA processing and transcription, cell cycle control, cellular transportation, cell defense, and virulence, including hydrophobins, cellular transporters (ABC and MFS multidrug transporters), and insect cuticular degrading enzymes, while upregulated genes were mainly enriched in carbohydrate metabolism and amino acid metabolism. These data indicate pleiotropic effects of BbElp3 in impacting specific cellular processes related to asexual development, cell cycle, autophagy, and virulence.

Tomato cultivation is seriously affected by infection from Botrytis cinerea. The safe and effective control of tomato gray mold remains elusive. Plant-related microbial communities regulate not only plant metabolism but also plant immune systems. In this study, we observed that Selenium application in soil combined with foliar spraying of methyl jasmonate could reduce Botrytis cinerea infection in tomato fruits and leaves and improve tomato fruit quality. The infection rate of leaves decreased from 42.19% to 25.00%, and the vitamin C content increased by 22.14%. The bacterial community structure of the tomato was studied by using amplicon sequencing technology. The leaf bacterial alpha diversity of tomatoes treated with Se plus methyl jasmonate was significantly higher than that of the control. Then we isolated five strains antagonistic to Botrytis cinerea in vitro from tomato leaves in the treatment of Se plus methyl jasmonate. The antagonistic strains were identified as Bacillus subtilis and Bacillus velezensis. Spraying mixed antagonistic strain suspension significantly inhibited the diameter of Botrytis cinerea with an inhibition rate of 40.99%. This study revealed the key role of plant-beneficial bacteria recruited by Se combined with methyl jasmonate in improving tomato plant disease resistance. These findings may benefit our understanding of the new regulation of microorganisms on Botrytis cinerea.

Clubroot, a soil-infective disease caused by Plasmodiophora brassicae, is a serious disease affecting cruciferous plants around the world. There is no effective control measure to completely remove this pathogen from fields after infection. Here, we screened and identified two strains (Hz36, Trichoderma guizhouense; Hk37, Trichoderma koningiopsis) of Trichoderma from the gall of clubroot in rapeseed fields with biocontrol potential for clubroot. The fermentation broth of Hz36 could significantly inhibit the germination of resting spores of P. brassicae, and promote the seed germination and root growth of rapeseed. The biocontrol efficiency of Hz36 strain on clubroot for rapeseed and Arabidopsis thaliana was 44.29% and 52.18%, respectively. The qPCR results revealed that strain Hz36 treatment could significantly reduce the content of P. brassicae in root cells, and paraffin section analysis revealed that it could delay the development of P. brassicae. Strain Hk37 showed similar effects to strain Hz36, whose biocontrol efficiency of clubroot could reach 57.30% in rapeseed and 68.01% in A. thaliana. These results indicate that strains Hz36 and Hk37 have the potential for the biocontrol of clubroot.

Mycoviruses are ubiquitous in pathogenic fungi including Sclerotinia sclerotiorum. Using RNA sequencing, more mycoviruses have been identified in individual strains, which were previously reported to be infected by a single mycovirus. A hypovirulent strain of S. sclerotiorum, HC025, was previously thought to harbor a single mitovirus, Sclerotinia sclerotiorum mitovirus 1 (SsMV1), based on the analysis of the conventional dsRNA extraction method. We found HC025 to be co-infected by five mycoviruses. In addition to SsMV1, four mycoviruses were identified: Sclerotinia sclerotiorum narnavirus 4 (SsNV4), Sclerotinia sclerotiorum negative-stranded RNA virus 1 (SsNSRV1), Sclerotinia sclerotiorum ourmia-like virus 14 (SsOLV14), and SsOLV22. Three mycoviruses including SsNV4, SsNSRV1, and SsOLV14 share high replicase identities (more than 95%) with the previously reported corresponding mycoviruses, and SsOLV22 shows lower identity to the known viruses. The complete genome of SsOLV22 is 3987 nt long and contains a single ORF-encoded RdRp, which shares 24.84% identity with the RNA-dependent RNA polymerase (RdRp) of Hubei narna-like virus 10 (query coverage: 26%; e-value: 8 × 10−19). The phylogenetic tree of RdRp suggests that SsOLV22 is a new member within the family Botourmiaviridae. All of the mycoviruses except for SsNSRV1 could horizontally co-transfer from HC025 to the virulent strain Ep-1PNA367 with hypovirulent phenotypes, and converted a later strain into a hypovirulent strain. In summary, we molecularly characterized the hypovirulent strain HC025 and identified five RNA mycoviruses including a new member within Botourmiaviridae.

The plant cuticle is one of the protective layers of the external surface of plant tissues. Plants use the cuticle layer to reduce water loss and resist pathogen infection. Fungi release cell wall-degrading enzymes to destroy the epidermis of plants to achieve the purpose of infection. Sclerotinia sclerotiorum secretes a large amount of cutinase to disrupt the cuticle layer of plants during the infection process. In order to further understand the role of cutinase in the pathogenic process of S. sclerotiorum, the S. sclerotiorum cutinsae 1 (SsCut1) gene was cloned and analyzed. The protein SsCut1 contains the conserved cutinase domain and a fungal cellulose-binding domain. RT-qPCR results showed that the expression of SsCut1 was significantly upregulated during infection. Split-Marker recombination was utilized for the deletion of the SsCut1 gene, ΔSsCut1 mutants showed reduced cutinase activity and virulence, but the deletion of the SsCut1 gene had no effect on the growth rate, colony morphology, oxalic acid production, infection cushion formation and sclerotial development. Complementation with the wild-type SsCut1 allele restored the cutinase activity and virulence to the wild-type level. Interestingly, expression of SsCut1 in plants can trigger defense responses, but it also enhanced plant susceptibility to SsCut1 gene knock-out mutants. Taken together, our finding demonstrated that the SsCut1 gene promotes the virulence of S. sclerotiorum by enhancing its cutinase activity.

The infection by a single-stranded DNA virus, Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1), causes hypovirulence, a reduced growth rate, and other colony morphological changes in its host Sclerotinia sclerotiorum strain DT-8. However, the mechanisms of the decline are still unclear. Using digital RNA sequencing, a transcriptome analysis was conducted to elucidate the phenotype-related genes with expression changes in response to SsHADV-1 infection. A total of 3110 S. sclerotiorum differentially expressed genes (DEGs) were detected during SsHADV-1 infection, 1741 of which were up-regulated, and 1369 were down-regulated. The identified DEGs were involved in several important pathways. DNA replication, DNA damage response, carbohydrate and lipid metabolism, ribosomal assembly, and translation were the affected categories in S. sclerotiorum upon SsHADV-1 infection. Moreover, the infection of SsHADV-1 also suppressed the expression of antiviral RNA silencing and virulence factor genes. These results provide further detailed insights into the effects of SsHADV-1 infection on the whole genome transcription in S. sclerotiorum.