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Abstract Non-self recognition is a fundamental aspect of life, serving as a crucial mechanism for mitigating proliferation of molecular parasites within fungal populations. However, studies investigating the potential interference of plants with fungal non-self recognition mechanisms are limited. Here, we demonstrate a pronounced increase in the efficiency of horizontal mycovirus transmission between vegetatively incompatible Sclerotinia sclerotiorum strains in planta as compared to in vitro. This increased efficiency is associated with elevated proline concentration in plants following S. sclerotiorum infection. This surge in proline levels attenuates the non-self recognition reaction among fungi by inhibition of cell death, thereby facilitating mycovirus transmission. Furthermore, our field experiments reveal that the combined deployment of hypovirulent S. sclerotiorum strains harboring hypovirulence-associated mycoviruses (HAVs) together with exogenous proline confers substantial protection to oilseed rape plants against virulent S. sclerotiorum. This unprecedented discovery illuminates a novel pathway by which plants can counteract S. sclerotiorum infection, leveraging the weakening of fungal non-self recognition and promotion of HAVs spread. These promising insights provide an avenue to explore for developing innovative biological control strategies aimed at mitigating fungal diseases in plants by enhancing the efficacy of horizontal HAV transmission.

ABSTRACT Ascospores, forcibly released into the air from perithecia, are the primary inoculum for Fusarium head blight. In Fusarium graminearum, the biological functions of four RNA-dependent RNA polymerases (RdRPs) (Fgrdrp1–4) have been reported, but their regulatory mechanisms are poorly understood and the function of Fgrdrp5 is still unknown. In this study, we found that in addition to Fgrdrp1 and Fgrdrp2, Fgrdrp5 also plays an important role in ascospore discharge, and they all participate in the generation of turgor pressure in a polyol-dependent manner. Moreover, these three genes all affect the maturation of ascospores. Deep sequencing and co-analysis of small RNA and mRNA certified that Fgrdrp1, Fgrdrp2, and Fgrdrp5 partly share their functions in the biogenesis and accumulation of exonic small interference RNA (ex-siRNA), and these three RdRPs negatively regulate the expression levels of ex-siRNA corresponding genes, including certain genes associated with ascospore development or discharge. Furthermore, the differentially expressed genes of deletion mutants, those involved in lipid and sugar metabolism or transport as well as sexual development-related transcription factors, may also contribute to the defects in ascospore maturation or ascospore discharge. In conclusion, our study suggested that the components of the dicer-dependent ex-siRNA-mediated RNA interference pathway include at least Fgrdrp1, Fgrdrp2, and Fgrdrp5.IMPORTANCEWe found that in addition to Fgrdrp1 and Fgrdrp2, Fgrdrp5 also plays important roles in ascospore maturation and ascospore discharge of Fusarium graminearum. These three RNA-dependent RNA polymerases participate in the biogenesis and accumulation of exonic small interference RNA and then regulate ascospore discharge.

Abstract Apoptosis‐like programmed cell death is associated with fungal development, ageing, pathogenicity and stress responses. Here, to explore the potential of Botrytis cinerea type II inhibitor of apoptosis (IAP) BcBIR1 in elevating the biocontrol efficacy of Coniothyrium minitans, the BcBIR1 gene was heterologously expressed in C. minitans. Results indicated that the strains expressing BcBIR1 had higher rates of conidiation, mycelial growth and biomass growth than the wild‐type strain. Moreover, BcBIR1 was found to inhibit apoptosis, indicating its role as an IAP in C. minitans. Under various abiotic stresses, the growth rates of BcBIR1‐expressing strains were significantly higher than that of the wild‐type strain. Moreover, the conidial survival rate of the BcBIR1‐expressing strains treated with ultraviolet irradiation was enhanced. In antifungal activity assay, the culture filtrates of BcBIR1‐expressing strains displayed a stronger inhibitory effect on B. cinerea and Sclerotinia sclerotiorum than the wild‐type strain. The study also found that BcBIR1 expression increased the mycoparasitism against the sclerotia, but not the hyphae of S. sclerotiorum. Taken together, these results suggest that BcBIR1 enhances vegetative growth, conidiation, anti‐apoptosis activity, abiotic stress resistance, antifungal activity and mycoparasitism in C. minitans. As an IAP, BcBIR1 may improve the control capacity of C. minitans against S. sclerotiorum.

ABSTRACTChitinase plays an important role in plant resistance against chitin-containing pathogens through hydrolysis of chitin. Clubroot caused by Plasmodiophora brassicae is a major disease for cruciferous crops and vegetables worldwide. The cell wall of P. brassicae resting spores contains chitin. Chitinase is regarded as capable of improving plant resistance to fungal diseases. However, there has been no report about the function of chitinase in P. brassicae. Here, wheat germ agglutinin staining and commercial chitinase treatment demonstrated that chitin is a functional component in P. brassicae. In addition, Chitinase PbChia1 was identified by chitin pull-down assay combined with LC-MS/MS. PbChia1 was found to be a typical secreted chitinase, which could bind chitin with chitinase activity in vitro. PbChia1 could significantly decrease the resting spores of P. brassicae and therefore relieve the severity of clubroot symptom, with a biocontrol effect of 61.29%. Overexpression of PbChia1 in Arabidopsis thaliana improved its resistance to P. brassicae, increased host survival rate and seed yield, enhanced PAMPs-triggered reactive oxygen species burst, MAPK activation and expression of immune-related genes. PbChia1 transgenic plants also showed resistance to other pathogens, such as biotrophic bacterium Pst DC3000, necrotrophic fungi Sclerotinia sclerotiorum and Rhizoctonia solani. These findings indicate that chitinase PbChia1 is a candidate gene that can confer broad-spectrum disease resistance in breeding.

The seed microbiota is an important component given by nature to plants, protecting seeds from damage by other organisms and abiotic stress. However, little is known about the dynamic changes and potential functions of the seed microbiota during seed development. In this study, we investigated the composition and potential functions of the seed microbiota of rapeseed (Brassica napus). A total of 2496 amplicon sequence variants (ASVs) belonging to 504 genera in 25 phyla were identified, and the seed microbiota of all sampling stages were divided into three groups. The microbiota of flower buds, young pods, and seeds at 20 days after flowering (daf) formed the first group; that of seeds at 30 daf, 40 daf and 50 daf formed the second group; that of mature seeds and parental seeds were clustered into the third group. The functions of seed microbiota were identified by using PICRUSt2, and it was found that the substance metabolism of seed microbiota was correlated with those of the seeds. Finally, sixty-one core ASVs, including several potential human pathogens, were identified, and a member of the seed core microbiota, Sphingomonas endophytica, was isolated from seeds and found to promote seedling growth and enhance resistance against Sclerotinia sclerotiorum, a major pathogen in rapeseed. Our findings provide a novel perspective for understanding the composition and functions of microbiota during seed development and may enhance the efficiency of mining beneficial seed microbes.

Phytopathogenic fungi normally secrete large amounts of CWDEs to enhance infection of plants. In this study, we identified and characterized a secreted glycosyl hydrolase 5 family member in Sclerotinia sclerotiorum (SsGH5, Sclerotinia sclerotiorum Glycosyl Hydrolase 5). SsGH5 was significantly upregulated during the early stages of infection. Knocking out SsGH5 did not affect the growth and acid production of S. sclerotiorum but resulted in decreased glucan utilization and significantly reduced virulence. In addition, Arabidopsis thaliana expressing SsGH5 became more susceptible to necrotrophic pathogens and basal immune responses were inhibited in these plants. Remarkably, the lost virulence of the ΔSsGH5 mutants was restored after inoculating onto SsGH5 transgenic Arabidopsis. In summary, these results highlight that S. sclerotiorum suppresses the immune responses of Arabidopsis through secreting SsGH5, and thus exerts full virulence for successful infection.

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 notorious phytopathogenic fungus that harbors diverse mycoviruses. A novel positive-sense single-stranded RNA virus, Sclerotinia sclerotiorum alphaflexivirus 2 (SsAFV2), was isolated from the hypovirulent strain 32–9 of S. sclerotiorum, and its complete genome was determined. The SsAFV2 genome contains 7,162 nucleotides (nt), excluding the poly (A) structure, and is composed of four open reading frames (ORF1–4). ORF1 encodes a polyprotein that contains three conserved domains: methyltransferase, helicase, and RNA-dependent RNA polymerase (RdRp). The ORF3 putative encodes coat proteins (CP), with ORF2 and ORF4 encoding hypothetical proteins of unknown functions. Phylogenetic analysis revealed that SsAFV2 clustered with Botrytis virus X (BVX) based on multiple alignments of helicase, RdRp, and CP, but the methyltransferase of SsAFV2 was most closely related to Sclerotinia sclerotiorum alphaflexivirus 1, suggesting that SsAFV2 is a new member of the Botrexvirus genus within the Alphaflexiviridae family, and also revealed the occurrence of potential inter-species horizontal gene transfer events within the Botrexvirus genus during the evolutionary process. Our results contribute to the current knowledge regarding the evolution and divergence of Botrexviruses.

Abstract Over 110 million tons of nitrogen fertilizer every year is used for crop production. Scientists have dreamed of enabling rhizobial nitrogen fixation in non-leguminous crops to mitigate the increasing demand for nitrogen fertilizer. However, despite decades of research, rhizobial nitrogen fixation in non-host plants has not been demonstrated. Here, we reported that an N-fixing rhizobium and a clubroot pathogen Plasmodiophora brassicae exhibited a synergistic effect on fixing nitrogen in cruciferous plants. Rhizobia were found to invade P. brassicae-infected rapeseed (Brassica napus) roots in the field. The colonization of rhizobium on rapeseed roots was confirmed by co-inoculating Mesorhizobium huakuii with P. brassicae under controlled laboratory conditions. M. huakuii infection could alleviate clubroot symptoms and promote the growth of diseased rapeseeds. M. huakuii could fix nitrogen in P. brassicae-infected plants based on the results of 15N isotope dilution tests. The expression of homologs of legume genes required for symbiosis and early-nodulin genes was significantly upregulated in Arabidopsis during early infection by P. brassicae. More importantly, M. huakuii could even fix nitrogen in P. brassicae-resistant rapeseed cultivar and promote plant growth when co-inoculated with P. brassicae. Our findings provide a new avenue to understand the interaction of rhizobia with non-host plants, stimulate the exploration of fixing nitrogen in non-leguminous plants by nitrogen-fixing rhizobia, and develop a strategy for both disease control and nitrogen fixation on non-host crops.

ABSTRACT Glycosylphosphatidylinositol (GPI) anchoring of proteins is a conserved posttranslational modification in eukaryotes. GPI-anchored proteins are widely distributed in fungal plant pathogens, but the specific roles of the GPI-anchored proteins in the pathogenicity of Sclerotinia sclerotiorum, a devastating necrotrophic plant pathogen with a worldwide distribution, remain largely unknown. This research addresses SsGSR1, which encodes an S. sclerotiorum glycine- and serine-rich protein named SsGsr1 with an N-terminal secretory signal and a C-terminal GPI-anchor signal. SsGsr1 is located at the cell wall of hyphae, and deletion of SsGSR1 leads to abnormal cell wall architecture and impaired cell wall integrity of hyphae. The transcription levels of SsGSR1 were maximal in the initial stage of infection, and SsGSR1-deletion strains showed impaired virulence in multiple hosts, indicating that SsGSR1 is critical for the pathogenicity. Interestingly, SsGsr1 targeted the apoplast of host plants to induce cell death that relies on the glycine-rich 11-amino-acid repeats arranged in tandem. The homologs of SsGsr1 in Sclerotinia, Botrytis, and Monilinia species contain fewer repeat units and have lost their cell death activity. Moreover, allelic variants of SsGSR1 exist in field isolates of S. sclerotiorum from rapeseed, and one of the variants lacking one repeat unit results in a protein that exhibits loss of function relative to the cell death-inducing activity and the virulence of S. sclerotiorum. Taken together, our results demonstrate that a variation in tandem repeats provides the functional diversity of GPI-anchored cell wall protein that, in S. sclerotiorum and other necrotrophic pathogens, allows successful colonization of the host plants. IMPORTANCE Sclerotinia sclerotiorum is an economically important necrotrophic plant pathogen and mainly applies cell wall-degrading enzymes and oxalic acid to kill plant cells before colonization. In this research, we characterized a glycosylphosphatidylinositol (GPI)-anchored cell wall protein named SsGsr1, which is critical for the cell wall architecture and the pathogenicity of S. sclerotiorum. Additionally, SsGsr1 induces rapid cell death of host plants that is dependent on glycine-rich tandem repeats. Interestingly, the number of repeat units varies among homologs and alleles of SsGsr1, and such a variation creates alterations in the cell death-inducing activity and the role in pathogenicity. This work advances our understanding of the variation of tandem repeats in accelerating the evolution of a GPI-anchored cell wall protein associated with the pathogenicity of necrotrophic fungal pathogens and prepares the way toward a fuller understanding of the interaction between S. sclerotiorum and host plants.

ABSTRACT Sclerotinia sclerotiorum, a widespread pathogen of dicotyledons, can grow endophytically in wheat, providing protection against Fusarium head blight and stripe rust and enhancing wheat yield. In this study, we found that wheat seed treatment with strain DT-8, infected with S. sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1) and used as a “plant vaccine” for brassica protection, could significantly increase the diversity of the fungal and bacterial community in rhizosphere soil, while the diversity of the fungal community was obviously decreased in the wheat root. Interestingly, the relative abundance of potential plant growth-promoting rhizobacteria (PGPR) and biocontrol agents increased significantly in the DT-8-treated wheat rhizosphere soil. These data might be responsible for wheat growth promotion and disease resistance. These results may provide novel insights for understanding the interaction between the schizotrophic microorganism and the microbiota of plant roots and rhizosphere, screening and utilizing beneficial microorganisms, and further reducing chemical pesticide utilization and increasing crop productivity. IMPORTANCE Fungal pathogens are seriously threatening food security and natural ecosystems; efficient and environmentally friendly control methods are essential to increase world crop production. S. sclerotiorum, a widespread pathogen of dicotyledons, can grow endophytically in wheat, providing protection against Fusarium head blight and stripe rust and enhancing wheat yield. In this study, we discovered that S. sclerotiorum treatment increased the diversity of the soil fungal and bacterial community in rhizosphere soil, while the diversity of the fungal community was obviously decreased in the wheat root. More importantly, the relative abundance of potential PGPR and bio-control agents increased significantly in the S. sclerotiorum-treated wheat rhizosphere soil. The importance of this work is that schizotrophic S. sclerotiorum promotes wheat growth and enhances resistance against fungal diseases via changes in the structure of the root and rhizosphere microbiome.

Abstract Rice sheath blight pathogen, Rhizoctonia solani, produces numerous sclerotia to overwinter. As a rich source of nutrients in the soil, sclerotia may lead to the change of soil microbiota. For this purpose, we amended the sclerotia of R. solani in soil and analyzed the changes in bacterial microbiota within the soil at different time points. At the phyla level, Proteobacteria, Acidobacteria, Bacteroidetes, Actinobacteria, Chloroflexi and Firmicutes showed varied abundance in the amended soil samples compared to those in the control. An increased abundance of ammonia-oxidizing bacterium (AOB) Nitrosospira and Nitrite oxidizing bacteria (NOB) i.e., Nitrospira was observed, where the latter is reportedly involved in the nitrifier denitrification. Moreover, Thiobacillus, Gemmatimonas, Anaeromyxobacter and Geobacter, the vital players in denitrification, N2O reduction and reductive nitrogen transformation, respectively, depicted enhanced abundance in R. solani sclerotia-amended samples. Furthermore, asymbiotic nitrogen-fixing bacteria, notably, Azotobacter as well as Microvirga and Phenylobacterium with nitrogen-fixing potential also enriched in the amended samples compared to the control. Plant growth promoting bacteria, such as Kribbella, Chitinophaga and Flavisolibacter also enriched in the sclerotia-amended soil. As per our knowledge, this study is of its kind where pathogenic fungal sclerotia activated microbes with a potential role in N transformation and provided clues about the ecological functions of R. solani sclerotia on the stimulation of bacterial genera involved in different processes of N-cycle within the soil in the absence of host plants.

Abstract Background Enhancers are cis-regulatory elements present in eukaryote genomes, which constitute indispensable determinants of gene regulation by governing the spatiotemporal and quantitative expression dynamics of target genes, and are involved in multiple life processes, for instance during development and disease states. The importance of enhancer activity has additionally been highlighted for immune responses in animals and plants; however, the dynamics of enhancer activities and molecular functions in plant innate immunity are largely unknown. Here, we investigated the involvement of distal enhancers in early innate immunity in Arabidopsis thaliana. Results A group of putative distal enhancers producing low-abundance transcripts either unidirectionally or bidirectionally are identified. We show that enhancer transcripts are dynamically modulated in plant immunity triggered by microbe-associated molecular patterns and are strongly correlated with open chromatin, low levels of methylated DNA, and increases in RNA polymerase II targeting and acetylated histone marks. Dynamic enhancer transcription is correlated with target early immune gene expression patterns. Cis motifs that are bound by immune-related transcription factors, such as WRKYs and SARD1, are highly enriched within upregulated enhancers. Moreover, a subset of core pattern-induced enhancers are upregulated by multiple patterns from diverse pathogens. The expression dynamics of putative immunity-related enhancers and the importance of WRKY binding motifs for enhancer function were also validated. Conclusions Our study demonstrates the general occurrence of enhancer transcription in plants and provides novel information on the distal regulatory landscape during early plant innate immunity, providing new insights into immune gene regulation and ultimately improving the mechanistic understanding of the plant immune system.

Plants produce polygalacuturonase-inhibiting proteins (PGIPs) to counteract cell wall degradation by pathogenic microbes. Here the authors show that Sclerotinia sclerotiorum, a fungal pathogen that causes stem rot disease, secretes a PGIP-inactivating effector to diminish plant resistance.

Rhizosphere microbiota is important for plant growth and health. Domestication is a process to select suitable plants to satisfy the needs of humans, which may have great impacts on the interaction between the host and its rhizosphere microbiota. Rapeseed (Brassica napus) is an important oilseed crop derived from the hybridization between Brassica rapa and Brassica oleracea ~7500 years ago. However, variations in rhizosphere microbiota along with rapeseed domestication remain poorly understood. Here, we characterized the composition and structure of the rhizosphere microbiota among diverse rapeseed accessions, including ten B. napus, two B. rapa, and three B. oleracea accessions through bacterial 16S rRNA gene sequencing. B. napus exhibited a higher Shannon index and different bacterial relative abundance compared with its wild relatives in rhizosphere microbiota. Moreover, artificial synthetic B. napus lines G3D001 and No.2127 showed significantly different rhizosphere microbiota diversity and composition from other B. napus accessions and their ancestors. The core rhizosphere microbiota of B. napus and its wild relatives was also described. FAPROTAX annotation predicted that the synthetic B. napus lines had more abundant pathways related to nitrogen metabolism, and the co-occurrence network results demonstrated that Rhodoplanes acted as hub nodes to promote nitrogen metabolism in the synthetic B. napus lines. This study provides new insights into the impacts of rapeseed domestication on the diversity and community structure of rhizosphere microbiota, which may highlight the contribution of rhizosphere microbiota to plant health.

Sclerotinia sclerotiorum is an ascomycetous fungus and hosts various mycoviruses. In this study, a novel fungal alphaflexivirus with a special genomic structure, named Sclerotinia sclerotiorum alphaflexivirus 1 (SsAFV1), was cloned from a hypovirulent strain, AHS31. Strain AHS31 was also co-infected with two botourmiaviruses and two mitoviruses. The complete genome of SsAFV1 comprised 6939 bases with four open reading frames (ORFs), a conserved 5′-untranslated region (UTR), and a poly(A) tail in the 3′ terminal; the ORF1 and ORF3 encoded a replicase and a coat protein (CP), respectively, while the function of the proteins encoded by ORF2 and ORF4 was unknown. The virion of SsAFV1 was flexuous filamentous 480–510 nm in length and 9–10 nm in diameter. The results of the alignment and the phylogenetic analysis showed that SsAFV1 is related to allexivirus and botrexvirus, such as Garlic virus X of the genus Allexivirus and Botrytis virus X of the genus Botrevirus, both with 44% amino-acid (aa) identity of replicase. Thus, SsAFV1 is a novel virus and a new genus, Sclerotexvirus, is proposed to accommodate this novel alphaflexivirus.

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.

Abstract Sclerotial development is a vital stage in the life cycles of many fungal plant pathogens. In this study, the protein Ss-CAD, which contains three conserved domains of cinnamyl alcohol dehydrogenase (CAD), was found to be required for sclerotial development in Sclerotinia sclerotiorum. Ss-CAD was significantly upregulated during early stage of sclerotial development. In Ss-CAD-silenced strains, sclerotial development was abnormal. In these silenced strains, formation of sclerotia was delayed or sclerotia yield was reduced, whereas hyphal growth and virulence were normal. Nox1, Nox2, and NoxR, which encode reactive oxygen species (ROS)-generating NADPH oxidases, were downregulated in Ss-CAD-silenced strains. NoxR-silenced strains displayed similar defects during sclerotial formation as Ss-CAD-silenced strains. Treatment of Ss-CAD-silenced strains with exogenous oxidants or NADPH restored normal sclerotial development. Sclerogenesis in Ss-CAD-silenced strains could also be recovered through Nox1 overexpression. The results suggest that Ss-CAD is linked to the NADPH oxidase pathways to affect sclerotial development in S. sclerotiorum.

Although endophytes are widespread in nature, the interactions between endophytes and their hosts are still not fully understood. Members of a unique class of endophytes, the virus-mediated endophytic fungi, are continuously being discovered and have received wide attention. In this study, we investigated the interaction between a mycovirus-mediated endophytic fungus and its host rapeseed by using dual-UMI RNA-seq.

Mycoviruses are an important component of the virosphere, but our current knowledge of their genome organization diversity and evolution remains rudimentary. In this study, the mycovirus composition in a hypovirulent strain of Sclerotinia sclerotiorum was molecularly characterized. Nine mycoviruses were identified and assigned into eight potential families. Of them, six were close relatives of known mycoviruses, while the other three had unique genome organizations and evolutionary positions. A deltaflexivirus with a tripartite genome has evolved via arrangement and horizontal gene transfer events, which could be an evolutionary connection from unsegmented to segmented RNA viruses. Two mycoviruses had acquired a second helicase gene by two different evolutionary mechanisms. A rhabdovirus representing an independent viral evolutionary branch was the first to be confirmed to occur naturally in fungi. The major hypovirulence-associated factor, an endornavirus, was finally corroborated. Our study expands the diversity of mycoviruses and potential virocontrol agents, and also provides new insights into virus evolutionary modes including virus genome segmentation.

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.

Codon usage bias (CUB) could reflect co-evolutionary changes between viruses and hosts in contrast to plant and animal viruses, and the systematic analysis of codon usage among the mycoviruses that infect plant pathogenic fungi is limited. We performed an extensive analysis of codon usage patterns among 98 characterized RNA mycoviruses from eight phytopathogenic fungi. The GC and GC3s contents of mycoviruses have a wide variation from 29.35% to 64.62% and 24.32% to 97.13%, respectively. Mycoviral CUB is weak, and natural selection plays a major role in the formation of mycoviral codon usage pattern. In this study, we demonstrated that the codon usage of mycoviruses is similar to that of some host genes, especially those involved in RNA biosynthetic process and transcription, suggesting that CUB is a potential evolutionary mechanism that mycoviruses adapt to in their hosts.

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.

Abstract Plasmodiophora brassicae is an important biotrophic eukaryotic plant pathogen and a member of the rhizarian protists. This biotrophic pathogen causes clubroot in cruciferous plants via novel intracellular mechanisms that are markedly different from those of other biotrophic organisms. To date, genomes from six single spore isolates of P. brassicae have been sequenced. An accurate description of the evolutionary status of this biotrophic protist, however, remains lacking. Here, we determined the draft genome of the P. brassicae ZJ-1 strain. A total of 10,951 protein-coding genes were identified from a 24.1 Mb genome sequence. We applied a comparative genomics approach to prove the Rhizaria supergroup is an independent branch in the eukaryotic evolutionary tree. We also found that the GPCR signaling pathway, the versatile signal transduction to multiple intracellular signaling cascades in response to extracellular signals in eukaryotes, is significantly enriched in P. brassicae-expanded and P. brassicae-specific gene sets. Additionally, treatment with a GPCR inhibitor relieved the symptoms of clubroot and significantly suppressed the development of plasmodia. Our findings suggest that GPCR signal transduction pathways play important roles in the growth, development, and pathogenicity of P. brassicae.

In pursuing higher rice production, we have often jeopardized soil at an alarming rate. It is hypothesized that intensive farming practices degrade soil health and increase the abundance of rice diseases while fallowing increases the abundance of biocontrol agents. In this study, the bacterial community was monitored in the paddy and fallow soil. Proteobacteria, Bacteroidetes, and Actinobacteria were abundant in the fallow soil, whereas Acidobacteria, Chloroflexi, and Gemmatimonadetes were more abundant in the paddy soil. Among the potential biocontrol agents, Bacillus, Thiobacillus, Rhizobium, Massilia, Rhizobacter, Streptomyces, Micromonospora, and Pseudonocardia were more abundant in the fallow soil, while Pseudomonas and Burkholderia were more abundant in the paddy soil. The possible rice pathogens, i.e., Xanthomonas and Erwinia, were more abundant in the paddy soil. The alpha diversity was higher in paddy soil than in fallow soil. Additionally, the principal coordinate analysis based on UniFrac distances revealed distinct clusters of the soils. Moreover, the functional analysis suggested that the fallow soil was abundant in genes associated with the biosynthesis of siderophores and secondary metabolites. In contrast, the paddy soil was abundant in genes related to plant-pathogen interactions. In conclusion, these results highlight the significance of fallowing to improve soil health.

Endornaviruses are capsidless linear (+) ssRNA viruses in the family Endornaviridae. In this study, Scelrotinia sclerotiorum endornavirus 11 (SsEV11), a novel endornavirus infecting hypovirulent Sclerotinia sclerotiorum strain XY79, was identified and cloned using virome sequencing analysis and rapid amplification of cDNA ends (RACE) techniques. The full-length genome of SsEV11 is 11906 nt in length with a large ORF, which encodes a large polyprotein of 3928 amino acid residues, containing a viral methyltransferase domain, a cysteine-rich region, a putative DEADc, a viral helicase domain, and an RNA-dependent RNA polymerase (RdRp) 2 domain. The 5’ and 3’ untranslated regions (UTR) are 31 nt and 90 nt, respectively. According to the BLAST result of the nucleotide sequence, SsEV11 shows the highest identity (45%) with Sclerotinia minor endornavirus 1 (SmEV1). Phylogenetic analysis based on amino acid sequence of RdRp demonstrated that SsEV11 clusters to endornavirus and has a close relationship with Betaendornavirus. Phylogenetic analysis based on the sequence of endornaviral RdRp domain indicated that there were three large clusters in the phylogenetic tree. Combining the results of alignment analysis, Cluster I at least has five subclusters including typical members of Alphaendornavirus and many unclassified endornaviruses that isolated from fungi, oomycetes, algae, and insects; Cluster II also has five subclusters including typical members of Betaendornavirus, SsEV11, and other unclassified viruses that infected fungi; Cluster III includes many endorna-like viruses that infect nematodes, mites, and insects. Viruses in Cluster I and Cluster II are close to each other and relatively distant to those in Cluster III. Our study characterized a novel betaendornavirus, SsEV11, infected fungal pathogen S. sclerotiorum, and suggested that notable phylogenetic diverse exists in endornaviruses. In addition, at least, one novel genus, Gammaendornavirus, should be established to accommodate those endorna-like viruses in Cluster III.

Abstract Background The eukaryotic unicellular protist Plasmodiophora brassicae is an endocellular parasite of cruciferous plants. In host cortical cells, this protist develops a unicellular structure that is termed the plasmodium. The plasmodium is actually a multinucleated cell, which subsequently splits and forms resting spores. The mechanism for the growth of this endocellular parasite in host cell is unclear. Results Here, combining de novo genome sequence and transcriptome analysis of strain ZJ-1, we identified top five significant enriched KEGG pathways of differentially expressed genes (DEGs), namely translation, cell growth and death, cell communication, cell motility and cancers. We detected 171 proto-oncogenes from the genome of P. brassicae that were implicated in cancer-related pathways, of which 46 were differential expression genes. Three predicted proto-oncogenes (Pb-Raf1, Pb-Raf2, and Pb-MYB), which showed homology to the human proto-oncogenes Raf and MYB, were specifically activated during the plasmodial growth in host cortical cells, demonstrating their involvement in the multinucleate development stage of the unicellular protist organism. Gene networks involved in the tumorigenic-related signaling transduction pathways and the activation of 12 core genes were identified. Inhibition of phosphoinositol-3-kinase relieved the clubroot symptom and significantly suppressed the development process of plasmodia. Conclusions Proto-oncogene-related regulatory mechanisms play an important role in the plasmodial growth of P. brassicae.

Recently, an increasing number of ourmia-like viruses have been found in fungi; however, the features of these viruses remain unknown. Here, we report a novel ourmia-like virus isolated from Sclerotinia sclerotiorum. This virus, named S. sclerotiorum ourmia-like virus 4 (SsOLV4), has a genome 2,982 nt in length with a G-pentamer (GGGGG) at the 5′-terminus and a C-pentamer (CCCCC) at the 3′-terminus. The SsOLV4 genome has only one large putative open reading frame (ORF) predicted with both standard codes and mitochondrial codes and encodes an RNA-dependent RNA polymerase (RdRp). SsOLV4 is closely phylogenetically related to Pyricularia oryzae ourmia-like virus 1, with 42% identity between the RdRp amino acid sequences. We constructed full-length cDNA of SsOLV4 and synthesized RNA in vitro using the T7 RNA polymerase. The synthesized RNA could transfect S. sclerotiorum protoplasts efficiently. We further found that viral RNA could infect mycelia when mixed with PEG buffer. Our study suggests that a novel genus in family Botourmiaviridae should be established for SsOLV4 and other related viruses and demonstrates that one single-stranded RNA segment encoding RdRp is sufficient for ourmia-like viruses in fungi.

Mycoparasite Coniothyrium minitans parasitizes specifically the mycelia or sclerotia of Sclerotinia sclerotiorum, a worldwidely spread plant fungal pathogen causing serious diseases on crops. The interaction of C. minitans with S. sclerotiorum remains reciprocal and complex and little is known, especially on the side of the host (S. sclerotiorum). In this study, the early transcriptional response of S. sclerotiorum to the mycoparasitism by C. minitans was explored and the differentially expressed genes (DEGs) were analyzed. Based on GO ontology, KEGG pathway and fungal categories database, 887 up-regulated DEGs were enriched in the growth related function (i.e., rRNA processing, ribosome biogenesis, binding and transport), while the 546 down-regulated DEGs were enriched in the stress-related functions (i.e., oxidoreductase, response to stress and heat and the chorismate biosynthetic process). The expression of shikimate pathway and the biosynthesis of phenylalanine involving genes was significantly suppressed. Furthermore, 581 unenriched DEGs were explored in the parasitizing process and were mapped on the Pfam domains of redox enzymes, Alpha/Beta hydrolase, haloacid dehalogenase, and other universal conserved domain containing proteins. Thirty-two DEGs encoding candidate effectors, with 16 up-regulated and 16 down-regulated, were observed with diverse function. SS1G_11912 (encoding SsNEP2) was significantly up-regulated and may function in the parasitism. The involving of the shikimate pathway of phenylalanine biosynthesis and effector candidates were discussed. The results provide a basal understand on the interaction of S. sclerotiorum and C. minitans.

Following the publication of this article [1], the authors noted the following errors.

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.

Sclerotinia sclerotiorum, an important phytopathogenic fungus, harbors rich diversity of mycoviruses. Lately, more mycoviruses can be successfully and accurately discovered by deep sequencing, especially those that could not be detected by traditional double-stranded RNA (dsRNA) extraction. Previously, we reported that the hypovirulent S. sclerotiorum strain SZ-150 is coinfected by Sclerotinia sclerotiorum hypovirus 1 (SsHV1) and its related satellite RNA. Here, aside from SsHV1, we detected two other mycoviruses, Sclerotinia sclerotiorum botybirnavirus 3 (SsBV3/SZ-150) and Sclerotinia sclerotiorum mycotymovirus 1 (SsMTV1/SZ-150), coinfecting strain SZ-150, by deep sequencing and assembly of mycovirus-derived small RNAs and determined their full-length genomes. The genome of SsBV3/SZ-150 was found to be composed of two linear dsRNA segments, 6,212, and 5,880 bp in size, respectively. Each dsRNA segment of SsBV3/SZ-150 contains a large open reading frame (ORF) encoding RNA-dependent RNA polymerase (RdRp) and a hypothetical protein. The whole genome of SsBV3/SZ-150 shares more than 95% sequence identity with Botrytis porri botybirnavirus 1 (BpBV1) at the nucleotide (nt) or amino acid level. Thus, SsBV3/SZ-150 was assumed to be a strain of BpBV1. The genome of SsMTV1/SZ-150 consists of 6,391 nt excluding the poly(A) tail. SsMTV1/SZ-150 was predicted to contain a large ORF that encodes a putative replication-associated polyprotein (RP) with three conserved domains of viral RNA methyltransferase, viral RNA helicase, and RdRp. Phylogenetic analyses suggest that SsMTV1/SZ-150 is related, albeit distantly, to members of the family Tymoviridae. Analysis of the small RNAs derived from SsBV3/SZ-150 and SsMTV1/SZ-150 revealed that small-RNA lengths mainly range from 20 to 24 nt, with a peak at 22 nt, and the most abundant 5′-terminal nucleotide is uridine, suggesting that the Dicer 2 and Argonaute 1, two key components in the RNA inference pathway, may play important roles in the resistance to mycoviral infection in S. sclerotiorum. Neither SsBV3/SZ-150 nor SsMTV1/SZ-150 is a causal agent of hypovirulence in strain SZ-150.

Coniothyrium minitans is a sclerotial parasite, which has been investigated for commercial control of crop diseases caused by Sclerotinia sclerotiorum. Previously, we obtained a T-DNA insertional mutant, ZS-1TN24363, which did not produce melanin during conidiation. To understand the function of melanin in C. minitans, we cloned the gene that was disrupted by the T-DNA insertion, and found that this gene, called CmMR1, encoded a putative protein of 1,011 amino acids, which is a homolog of the transcription factor MR. Full-length CmMR1 contains 3,167 bp, with three exons and two introns. To confirm that the disrupted gene is responsible for the melanin-deficiency of the mutant, CmMR1 was disrupted and three targeted knockout mutants were obtained. Biological assays showed that the phenotype of the targeted knockout mutants was similar to that of the T-DNA insertional mutant. Furthermore, gene complementation confirmed that CmMR1 is responsible for the mutant phenotype. CmMR1 disruption did not affect hyphal growth, conidiation, and parasitization of C. minitans, however, the ROS accumulation increased and tolerance to UV light decreased significantly in the mutants. Our result may enhance the understanding of melanin in the ecology of C. minitans on molecular level.

Considering the huge economic loss caused by postharvest diseases, the identification and prevention of citrus postharvest diseases is vital to the citrus industry. In 2018, 16 decayed citrus fruit from four citrus varieties—Satsuma mandarin (Citrus unshiu), Ponkan (Citrus reticulata Blanco cv. Ponkan), Nanfeng mandarin (Citrus reticulata cv. nanfengmiju), and Sugar orange (Citrus reticulata Blanco)—showing soft rot and sogginess on their surfaces and covered with white mycelia were collected from storage rooms in seven provinces. The pathogens were isolated and the pathogenicity of the isolates was tested. The fungal strains were identified as Lasiodiplodia pseudotheobromae based on their morphological characteristics and phylogenetic analyses using the internal transcribed spacer regions (ITS), translation elongation factor 1-α gene (TEF), and beta-tubulin (TUB) gene sequences. The strains could infect wounded citrus fruit and cause decay within two days post inoculation, but could not infect unwounded fruit. To our knowledge, this is the first report of citrus fruit decay caused by L. pseudotheobromae in China.

Via virome sequencing, six viruses were detected from Magnaporthe oryzae strains YC81-2, including one virus in the family Tombusviridae, one virus in the family Narnaviridae and four viruses in the family Botourmiaviridae. Since the RNA-dependent RNA polymerase (RdRp) of one botourmiavirus show the highest identity (79%) with Magnaporthe oryzae ourmia-like virus 1 (MOLV1), the virus that was grouped into the genus Magoulivirus was designated as Magnaporthe oryzae botourmiavirus 2 (MOBV2). The three other novel botourmiaviruses were selected for further study. The complete nucleotide sequences of the three botourmiaviruses were determined. Sequence analysis showed that virus 1, virus 2, and virus 3 were 2598, 2385, and 2326 nts in length, respectively. The variable 3′ untranslated region (3′-UTR) and 5′-UTR of each virus could be folded into a stable stem-loop secondary structure. Each virus consisted of a unique ORF encoding a putative RdRp. The putative proteins with a conserved GDD motif of RdRp showed the highest sequence similarity to RdRps of viruses in the family Botourmiaviridae. Phylogenetic analysis demonstrated that these viruses were three distinct novel botourmiaviruses, clustered into the Botourmiaviridae family but not belonging to any known genera of this family. Thus, virus 1, virus 2, and virus 3 were designated as Magnaporthe oryzae botourmiavirus 5, 6, and 7 (MOBV5, MOBV6, and MOBV7), respectively. Our results suggest that four distinct botourmiaviruses, MOBV2, MOBV5, MOBV6, and MOBV7, co-infect a single strain of Magnaporthe oryzae, and MOBV5, MOBV6, and MOBV7 are members of three unclassified genera in the family Botourmiaviridae.

We previously identified Sclerotinia sclerotiorum negative-stranded virus 1 (SsNSRV-1), the first (−) ssRNA mycovirus, associated with hypovirulence of its fungal host Sclerotinia sclerotiorum. In this study, functional analysis of Open Reading Frame Ι (ORF Ι) of SsNSRV-1 was performed. The integration and expression of ORF Ι led to defects in hyphal tips, vegetative growth, and virulence of the mutant strains of S. sclerotiorum. Further, differentially expressed genes (DEGs) responding to the expression of ORF Ι were identified by transcriptome analysis. In all, 686 DEGs consisted of 267 up-regulated genes and 419 down-regulated genes. DEGs reprogramed by ORF Ι were relevant to secretory proteins, pathogenicity, transcription, transmembrane transport, protein biosynthesis, modification, and metabolism. Alternative splicing was also detected in all mutant strains, but not in hypovirulent strain AH98, which was co-infected by SsNSRV-1 and Sclerotinia sclerotiorum hypovirus 1 (SsHV-1). Thus, the integrity of SsNSRV-1 genome may be necessary to protect viral mRNA from splicing and inactivation by the host. Taken together, the results suggested that protein ORF Ι could regulate the transcription, translation, and modification of host genes in order to facilitate viral proliferation and reduce the virulence of the host. Therefore, ORF Ι may be a potential gene used for the prevention of S. sclerotiorum.

Botrytis cinerea, a ubiquitous necrotrophic plant-pathogenic fungus, is responsible for grey mold and rot disease in a very wide range of plant species. Subtilisin-like proteases (or subtilases) are a very diverse family of serine proteases present in many organisms and are reported to have a broad spectrum of biological functions. Here, we identified two genes encoding subtilisin-like proteases (Bcser1 and Bcser2) in the genome of B. cinerea, both of which contain an inhibitor I9 domain and a peptidase S8 domain. The expression levels of Bcser1 and Bcser2 increased during the sclerotial forming stage, as well as during a later stage of hyphal infection on Arabidopsis thaliana leaves, but the up-regulation of Bcser1 was significantly higher than that of Bcser2. Interestingly, deletion of Bcser1 had no effect on the fungal development or virulence of B. cinerea. However, deletion of Bcser2 or double deletion of Bcser1 and Bcser2 severely impaired the hyphal growth, sclerotial formation and conidiation of B. cinerea. We also found that ∆Bcser2 and ∆Bcser1/2 could not form complete infection cushions and then lost the ability to infect intact plant leaves of Arabidopsis and tomato but could infect wounded plant tissues. Taken together, our results indicate that the subtilisin-like protease Bcser2 is crucial for the sclerotial formation, conidiation, and virulence of B. cinerea.

Infection by diverse mycoviruses is a common phenomenon in Sclerotinia sclerotiorum. In this study, the full genome of a single-stranded RNA mycovirus, tentatively named Hubei sclerotinia RNA virus 1 (HuSRV1), was determined in the hypovirulent strain 277 of S. sclerotiorum. The HuSRV1 genome is 4492 nucleotides (nt) long and lacks a poly (A) tail at the 3ˊ- terminus. Sequence analyses showed that the HuSRV1 genome contains four putative open reading frames (ORFs). ORF1a was presumed to encode a protein with a conserved protease domain and a transmembrane domain. This protein is 27% identical to the P2a protein encoded by the subterranean clover mottle virus. ORF1b encodes a protein containing a conserved RNA-dependent RNA polymerase (RdRp) domain, which may be translated into a fusion protein by a -1 ribosome frameshift. This protein is 45.9% identical to P2b encoded by the sowbane mosaic virus. ORF2 was found to encode a putative coat protein, which shares 23% identical to the coat protein encoded by the olive mild mosaic virus. ORF3 was presumed to encode a putative protein with an unknown function. Evolutionary relation analyses indicated that HuSRV1 is related to members within Sobemovirus, but forms a unique phylogenetic branch, suggesting that HuSRV1 represents a new member within Solemoviridae. HuSRV1 virions, approximately 30 nm in diameter, were purified from strain 277. The purified virions were successfully introduced into virulent strain Ep-1PNA367, resulting in a new hypovirulent strain, which confirmed that HuSRV1 confers hypovirulence on S. sclerotiorum.

Ascospores act as the primary inoculum of Fusarium graminearum, which causes the destructive disease Fusarium head blight (FHB), or scab. MicroRNAs (miRNAs) have been reported in the F. graminearum vegetative stage, and Fgdcl2 is involved in microRNA-like RNA (milRNA) biogenesis but has no major impact on vegetative growth, abiotic stress or pathogenesis. In the present study, we found that ascospore discharge was decreased in the Fgdcl1 deletion mutant, and completely blocked in the double-deletion mutant of Fgdcl1 and Fgdcl2. Besides, more immature asci were observed in the double-deletion mutant. Interestingly, the up-regulated differentially expressed genes (DEGs) common to ΔFgdcl1 and ΔFgdcl1/2 were related to ion transmembrane transporter and membrane components. The combination of small RNA and transcriptome sequencing with bioinformatics analysis predicted 143 novel milRNAs in wild-type perithecia, and 138 of these milRNAs partly or absolutely depended on Fgdcl1, while only 5 novel milRNAs were still obtained in the Fgdcl1 and Fgdcl2 double-deletion mutant. Furthermore, 117 potential target genes were predicted. Overall, Fgdcl1 and Fgdcl2 genes were partly functionally redundant in ascospore discharge and perithecium-specific milRNA generation in F. graminearum, and these perithecium-specific milRNAs play potential roles in sexual development.

Sclerotinia sclerotiorum is a devastating plant pathogen that attacks numerous economically important broad acre and vegetable crops worldwide. Mycoviruses are widespread viruses that infect fungi, including S. sclerotiorum. As there were no previous reports of the presence of mycoviruses in this pathogen in Australia, studies were undertaken using RNA_Seq analysis to determine the diversity of mycoviruses in 84 Australian S. sclerotiorum isolates collected from various hosts. After RNA sequences were subjected to BLASTp analysis using NCBI database, 285 contigs representing partial or complete genomes of 57 mycoviruses were obtained, and 34 of these (59.6%) were novel viruses. These 57 viruses were grouped into 10 distinct lineages, namely Endornaviridae (four novel mycoviruses), Genomoviridae (isolate of SsHADV-1), Hypoviridae (two novel mycoviruses), Mononegavirales (four novel mycovirusess), Narnaviridae (10 novel mycoviruses), Partitiviridae (two novel mycoviruses), Ourmiavirus (two novel mycovirus), Tombusviridae (two novel mycoviruses), Totiviridae (one novel mycovirus), Tymovirales (five novel mycoviruses), and two non-classified mycoviruses lineages (one Botrytis porri RNA virus 1, one distantly related to Aspergillus fumigatus tetramycovirus-1). Twenty-five mitoviruses were determined and mitoviruses were dominant in the isolates tested. This is not only the first study to show existence of mycoviruses in S. sclerotiorum in Australia, but highlights how they are widespread and that many novel mycoviruses occur there. Further characterization of these mycoviruses is warranted, both in terms of exploring these novel mycoviruses for innovative biocontrol of Sclerotinia diseases and in enhancing our overall knowledge on viral diversity, taxonomy, ecology, and evolution.

Coniothyrium minitans is an important mycoparasite of Sclerotinia sclerotiorum. In addition, it also produces small amounts of antifungal substances. ZS-1TN1812, an abnormal mutant, was originally screened from a T-DNA insertional library. This mutant showed abnormal growth phenotype and could significantly inhibit the growth of S. sclerotiorum when dual-cultured on a PDA plate. When spraying the filtrate of ZS-1TN1812 on the leaves of rapeseed, S. sclerotiorum infection was significantly inhibited, suggesting that the antifungal substances produced by this mutant were effective on rapeseed leaves. The thermo-tolerant antifungal substances could specifically suppress the growth of S. sclerotiorum, but could not significantly suppress the growth of another fungus, Colletotrichum higginsianum. However, C. higginsianum was more sensitive to proteinous antibiotics than S. sclerotiorum. The T-DNA insertion in ZS-1TN1812 activated the expression of CmSIT1, a gene involved in siderophore-mediated iron transport. It was also determined that mutant ZS-1TN1812 produced hypha with high iron levels. In the wild-type strain ZS-1, CmSIT1 was expressed only when in contact with S. sclerotiorum, and consistent overexpression of CmSIT1 showed similar phenotypes as ZS-1TN1812. Therefore, activated expression of CmSIT1 leads to the enhanced antifungal ability, and CmSIT1 is a potential gene for improving the control ability of C. minitans.

Recent studies have demonstrated that a diverse array of mycoviruses infect the plant pathogenic fungus Sclerotinia sclerotiorum. Here, we report the molecular characterization of a newly identified mycovirus, Sclerotinia sclerotiorum fusarivirus 1 (SsFV1), which was isolated from a sclerotia-defective strain JMTJ14 of S. sclerotiorum. Excluding a poly (A) tail, the genome of SsFV1 comprises 7754 nucleotides (nts) in length with 83 and 418 nts for 5'- and 3'-untranslated regions, respectively. SsFV1 has four non-overlapping open reading frames (ORFs): ORF1 encodes a 191 kDa polyprotein (1664 amino acid residues in length) containing conserved RNA-dependent RNA polymerase (RdRp) and helicase domains; the other three ORFs encode three putative hypothetical proteins of unknown function. Phylogenetic analysis, based on RdRp and Helicase domains, indicated that SsFV1 is phylogenetically related to Rosellinia necatrix fusarivirus 1 (RnFV1), Fusarium graminearum virus-DK21 (FgV1), and Penicillium roqueforti RNA mycovirus 1 (PrRV1), a cluster of an independent group belonging to a newly proposed family Fusarividae. However, SsFV1 is markedly different from FgV1 and RnFV1 in genome organization and nucleotide sequence. SsFV1 was transmitted successfully to two vegetatively incompatible virus-free strains. SsFV1 is not responsible for the abnormal phenotype of strain JMTJ14.

Clubroot disease is a serious threat to cruciferous plants worldwide, especially to oilseed rape. However, knowledge on pathogenic molecular mechanisms and host interaction is limited. We presume that the recognition between Arabidopsis thaliana and Plasmodiophora brassicae occurs at the early stage of infection and within a relatively short period. In this study, we demonstrated changes on gene expression and pathways in A. thaliana during early infection with P. brassicae using transcriptome analysis. We identified 1,903 and 1,359 DEGs at 24 and 48 h post-inoculation (hpi), respectively. Flavonoids and the lignin synthesis pathways were enhanced, glucosinolates, terpenoids, and proanthocyanidins accumulated and many hormonal- and receptor-kinase related genes were expressed, caused by P. brassicae infection during its early phase. Therefore, the early interaction between A. thaliana and P. brassicae plays an important role in the entire infection process. The results provide a new contribution to a better understanding of the interaction between host plants and P. brassicae, as well as the development of future measures for the prevention of clubroot.

Non-self recognition is a common phenomenon among organisms; it often leads to innate immunity to prevent the invasion of parasites and maintain the genetic polymorphism of organisms. Fungal vegetative incompatibility is a type of non-self recognition which often induces programmed cell death (PCD) and restricts the spread of molecular parasites. It is not clearly known whether virus infection could attenuate non-self recognition among host individuals to facilitate its spread. Here, we report that a hypovirulence-associated mycoreovirus, named Sclerotinia sclerotiorum mycoreovirus 4 (SsMYRV4), could suppress host non-self recognition and facilitate horizontal transmission of heterologous viruses. We found that cell death in intermingled colony regions between SsMYRV4-infected Sclerotinia sclerotiorum strain and other tested vegetatively incompatible strains was markedly reduced and inhibition barrage lines were not clearly observed. Vegetative incompatibility, which involves Heterotrimeric guanine nucleotide-binding proteins (G proteins) signaling pathway, is controlled by specific loci termed het (heterokaryon incompatibility) loci. Reactive oxygen species (ROS) plays a key role in vegetative incompatibility-mediated PCD. The expression of G protein subunit genes, het genes, and ROS-related genes were significantly down-regulated, and cellular production of ROS was suppressed in the presence of SsMYRV4. Furthermore, SsMYRV4-infected strain could easily accept other viruses through hyphal contact and these viruses could be efficiently transmitted from SsMYRV4-infected strain to other vegetatively incompatible individuals. Thus, we concluded that SsMYRV4 is capable of suppressing host non-self recognition and facilitating heterologous viruses transmission among host individuals. These findings may enhance our understanding of virus ecology, and provide a potential strategy to utilize hypovirulence-associated mycoviruses to control fungal diseases.

Clubroot caused by Plasmodiophora brassicae, is a severe disease of cruciferous crops that causes large hypertrophic galls in the roots. The plant microbiome is important for growth promotion and disease suppression. In this study, using 16S rRNA and internal transcribed spacer (ITS) sequencing techniques, we compared the endosphere microbiome of symptomatic and asymptomatic B. napus roots infected with P. brassicae collected from the same natural clubroot field. The results showed that the microbial population and its relative abundance in the asymptomatic roots was far higher than that in the symptomatic roots, and that many microorganisms in asymptomatic roots have biological control and plant growth promotion functions that may be related to clubroot symptoms. These results suggest the importance of the endosphere microbiome in clubroot disease and provide potential bio-control resources for its prevention.

To decipher the mechanism of pathogenicity in Sclerotinia sclerotiorum, a pathogenicity-defective mutant, Sunf-MT6, was isolated from a T-DNA insertional library. Sunf-MT6 could not form compound appressorium and failed to induce lesions on leaves of rapeseed though it could produce more oxalic acid than the wild-type strain. However, it could enter into host tissues via wounds and cause typical necrotic lesions. Furthermore, Sunf-MT6 produced fewer but larger sclerotia than the wild-type strain Sunf-M. A gene, named Ss-caf1, was disrupted by T-DNA insertion in Sunf-MT6. Gene complementation and knockdown experiments confirmed that the disruption of Ss-caf1 was responsible for the phenotypic changes of Sunf-MT6. Ss-caf1 encodes a secretory protein with a putative Ca2+-binding EF-hand motif. High expression levels of Ss-caf1 were observed at an early stage of compound appressorium formation and in immature sclerotia. Expression of Ss-caf1 without signal peptides in Nicotiana benthamiana via Tobacco rattle virus-based vectors elicited cell death. These results suggest that Ss-caf1 plays an important role in compound appressorium formation and sclerotial development of S. sclerotiorum. In addition, Ss-Caf1 has the potential to interact with certain host proteins or unknown substances in host cells, resulting in subsequent host cell death.

Botrytis-induced kinase1 (BIK1), a receptor-like cytoplasmic kinase, plays an important role in resistance against pathogens and insects in Arabidopsis thaliana. However, it remains unknown whether BIK1 functions against Plasmodiophora brassicae, an obligate biotrophic protist that attacks cruciferous plants and induces gall formation on roots. Here, we investigated the potential roles of receptors FLS2, BAK1 and BIK1 in the infection of P. brassicae cruciferous plants. Wild-type plants, fls2 and bak1 mutants showed typical symptom on roots, and the galls were filled with large quantities of resting spores, while bik1 mutant plants exhibited strong resistance to P. brassicae. Compared with that of the wild-type plants, the root hair and cortical infection rate of bik1 mutant were significantly reduced by about 40-50%. A considerable portion of bik1 roots failed to form typical galls. Even if some small galls were formed, they were filled with multinucleate secondary plasmodia. The bik1 plants accumulated less reactive oxygen species (ROS) at infected roots than other mutants and wild-type plants. Exogenous salicylic acid (SA) treatment alleviated the clubroot symptoms in wild-type plants, and the expression of the SA signaling marker gene PR1 was significantly increased in bik1. Both sid2 (salicylic acid induction-deficient 2) and npr1-1 (non-expresser of PR genes that regulate systemic acquired resistance (SAR)) mutants showed increased susceptibility to P. brassicae compared with wild-type plants. These results suggest that the resistance of bik1 to P. brassicae is possibly mediated by SA inducible mechanisms enhance the resistance to clubroot disease.

Histone methylation is widely present in animals, plants and fungi, and the methylation modification of histone H3 has important biological functions. Methylation of Lys9 of histone H3 (H3K9) has been proven to regulate chromatin structure, gene silencing, transcriptional activation, plant metabolism and other processes. In this work, we investigated the functions of a H3K9 methyltransferase gene BcDIM5 in Botrytis cinerea, which contains a PreSET domain, a SET domain and a PostSET domain. Characterization of BcDIM5 knockout transformants showed that the hyphal growth rate and production of conidiophores and sclerotia were significantly reduced, while complementary transformation of BcDIM5 could restore the phenotypes to the levels of wild type. Pathogenicity assays revealed that BcDIM5 was essential for full virulence of B. cinerea. BcDIM5 knockout transformants exhibited decreased virulence, down-regulated expression of some pathogenic genes and drastically decreased H3K9 trimethylation level. However, knockout transformants of other two genes heterochromatin protein 1 (HP1) BcHP1 and DNA methyltransferase (DIM2) BcDIM2 did not exhibit significant change in the growth phenotype and virulence compared with the wild type. Our results indicate that H3K9 methyltransferase BcDIM5 is required for H3K9 trimethylation to regulate the development and virulence of B. cinerea.

We previously determined that virions of Sclerotinia sclerotiorum hypovirulence associated DNA virus 1 (SsHADV-1) could directly infect hyphae of Sclerotinia sclerotiorum, resulting in hypovirulence of the fungal host. However, the molecular mechanisms of SsHADV-1 virions disruption of the fungal cell wall barrier and entrance into the host cell are still unclear. To investigate the early response of S. sclerotiorum to SsHADV-1 infection, S. sclerotiorum hyphae were inoculated with purified SsHADV-1 virions. The pre- and post-infection hyphae were collected at one–three hours post-inoculation for transcriptome analysis. Further, bioinformatic analysis showed that differentially expressed genes (DEGs) regulated by SsHADV-1 infection were identified in S. sclerotiorum. In total, 187 genes were differentially expressed, consisting of more up-regulated (114) than down-regulated (73) genes. The identified DEGs were involved in several important pathways. Metabolic processes, biosynthesis of antibiotics, and secondary metabolites were the most affected categories in S. sclerotiorum upon SsHADV-1 infection. Cell structure analysis suggested that 26% of the total DEGs were related to membrane tissues. Furthermore, 10 and 27 DEGs were predicted to be located in the cell membrane and mitochondria, respectively. Gene ontology enrichment analyses of the DEGs were performed, followed by functional annotation of the genes. Interestingly, one third of the annotated functional DEGs could be involved in the Ras-small G protein signal transduction pathway. These results revealed that SsHADV-1 virions may be able to bind host membrane proteins and influence signal transduction through Ras-small G protein-coupled receptors during early infection, providing new insight towards the molecular mechanisms of virions infection in S. sclerotiorum.