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Abstract Phytopathogens often secrete effectors to enhance their infection of plants. In the case of Sclerotinia sclerotiorum, a necrotrophic phytopathogen, a secreted protein named SsPEIE1 (Sclerotinia sclerotiorum Plant Early Immunosuppressive Effector 1) plays a crucial role in its virulence. During the early stages of infection, SsPEIE1 is significantly up-regulated. Additionally, transgenic plants expressing SsPEIE1 exhibit increased susceptibility to different phytopathogens. Further investigations revealed that SsPEIE1 interacts with a plasma membrane protein known as hypersensitive induced reaction (HIR) that dampens immune responses. SsPEIE1 is required for S. sclerotiorum virulence on wild-type Arabidopsis but not on Arabidopsis hir4 mutants. Moreover, Arabidopsis hir2 and hir4 mutants exhibit suppressed pathogen-associated molecular pattern-triggered reactive oxygen species (ROS) bursts and salicylic acid (SA)-associated immune gene induction, all of which are phenocopied by the SsPEIE1 transgenic plants. We find that the oligomerization of AtHIR4 is essential for its role in mediating immunity, and that SsPEIE1 inhibits its oligomerization through competitively binding to AtHIR4. Remarkably, both Arabidopsis and rapeseed plants overexpress AtHIR4 display significantly increased resistance to S. sclerotiorum. In summary, these results demonstrate that SsPEIE1 inhibits AtHIR4 oligomerization-mediated immune responses by interacting with the key immune factor AtHIR4, thereby promoting S. sclerotiorum infection.

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.

Summary: The Ralstonia solanacearum species complex causes bacterial wilt in a variety of crops. Tomato cultivar Hawaii 7996 is a widely used resistance resource; however, the resistance is evaded by virulent strains, with the underlying mechanisms still unknown. Here, we report that the phylotype Ⅱ strain ES5-1 can overcome Hawaii 7996 resistance. RipV2, a type Ⅲ effector specific to phylotype Ⅱ strains, is vital in overcoming tomato resistance. RipV2, which encodes an E3 ubiquitin ligase, suppresses immune responses and Toll/interleukin-1 receptor/resistance nucleotide-binding/leucine-rich repeat (NLR) (TNL)-mediated cell death. Tomato helper NLR N requirement gene 1 (NRG1), enhanced disease susceptibility 1 (EDS1), and senescence-associated gene 101b (SAG101b) are identified as RipV2 target proteins. RipV2 is essential for ES5-1 virulence in Hawaii 7996 but not in SlNRG1-silenced tomato, demonstrating SlNRG1 to be an RipV2 virulence target. Our results dissect the mechanisms of RipV2 in disrupting immunity and highlight the importance of converged immune components in conferring bacterial wilt resistance.

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.

Strain IBc-114 was isolated from a gray mold lesion and was identified as the fungus Schizophyllum commune. In this strain, two mycoviruses, Schizophyllum commune RNA virus 1 (ScRV1, C_AA053475.1) and Botrytis cinerea mitovirus 9 strain IBc-114 (BcMV9/IBc-114, C_AA053476.1), were isolated and characterized. ScRV1 has flexuous filamentous particles about 20 ± 2.1 nm in diameter and 1000 ± 94.2 nm in length. The genome of ScRV1 is 7370 nt in length and contains two open reading frames (ORFs) which encode a polyprotein and a coat protein, respectively. The polyprotein has 1967 aa, including a helicase domain and an RdRp domain which has the highest identity of 28.21% with that of Entomophthora benyvirus E (EbVE). The coat protein has 241 aa which is mostly phylogenetically close to the coat proteins of Alphatetraviridae. Based on the phylogenetic analysis of ScRV1 and viruses selected, ScRV1 might represent a new family (temporarily named Mycobenyviridae) of the order Hepelivirales. The genome of BcMV9/IBc-114 that infects S. commune is 2729 nt in length and has only one ORF encoding an RdRp protein with 719 aa. BcMV9/IBc-114 has the highest identity of 98.61% with Botrytis cinerea mitovirus 9 (BcMV9) (MT089704). ScRV1, but not BcMV9/IBc-114, has certain effects on the host growth of S. commune. Furthermore, BcMV9/IBc-114 has been demonstrated to replicate in the ascomycetous fungi Botrytis cinerea and Sclerotinia sclerotiorum, and it negatively affects the growth and pathogenicity of B. cinerea, but it does not affect S. sclerotiorum. This is the first report of mycoviruses in S. commune and cross-phyla transmission of mitovirus in nature.

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.

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.

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 Phase‐change random‐access memory (PCRAM) devices suffer from pronounced resistance drift originating from considerable structural relaxation of phase‐change materials (PCMs), which hinders current developments of high‐capacity memory and high‐parallelism computing that both need reliable multibit programming. This work realizes that compositional simplification and geometrical miniaturization of traditional GeSbTe‐like PCMs are feasible routes to suppress relaxation. While to date, the aging mechanisms of the simplest PCM, Sb, at nanoscale, have not yet been unveiled. Here, this work demonstrates that in an optimal thickness of only 4 nm, the thin Sb film can enable a precise multilevel programming with ultralow resistance drift coefficients, in a regime of ≈10−4–10−3. This advancement is mainly owed to the slightly changed Peierls distortion in Sb and the less‐distorted octahedral‐like atomic configurations across the Sb/SiO2 interfaces. This work highlights a new indispensable approach, interfacial regulation of nanoscale PCMs, for pursuing ultimately reliable resistance control in aggressively‐miniaturized PCRAM devices, to boost the storage and computing efficiencies substantially.

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.

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.

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.

The concentration of selenium (Se) in agricultural products primarily depends on the concentration of Se in soil and the ability of plants to accumulate Se. Selenium deficiency not only leads to decreased body resistance, but also increases the risk of cancer. The form and concentration of bioavailable Se is important for diet. The present study was carried out via field experiment with wheat and broad beans in soil of different Se concentrations (0, 1.12, and 11.2 kg·ha−1), which was determined based on the national standard and the team’s previous experience. Results indicated that the concentration of Se in the edible organs of wheat was higher than in broad bean, while the enriched Se concentration in the root of broad bean was more than twice and three times higher than that of wheat at medium and high levels of Se, respectively. Selenomethionine, which accounted for over half of the total Se speciations, was the dominant species in the edible parts of the two crops, followed by Selenocystine and methylselenocysteine. Through the analysis of the rhizosphere soil, it was found that Fe-Mn oxide-bound Se exceeded 80% of the total Se. Additionally, there was a significant linear correlation between the Se content in the edible parts of the two crops and the Se content in the soil. Findings suggested that wheat was more favorable than broad beans as Se supplement crops in a Se-supplied field.

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.

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.

Tomato, which is regarded as an important worldwide crop, is susceptible to gray mold caused by Botrytis cinerea. Selenium and methyl jasmonate can act as antifungal agents against pathogenic infections. To clarify the effect of selenium and methyl jasmonate on the fungal pathogen, the spore germination and mycelial growth of B. cinerea were investigated in vitro using the growth rate method. Additionally, the electrical conductivity, soluble protein content, malondialdehyde content and oxalic acid secretion of B. cinerea mycelium were also determined to further explore the antifungal mechanism of selenium and methyl jasmonate. The results showed that selenium application significantly increased cell membrane permeability and malondialdehyde content, and methyl jasmonate treatment decreased the soluble protein content in mycelium of B. cinerea. Furthermore, supplementation of the medium with both selenium and methyl jasmonate effectively inhibited spore germination and colony growth of B. cinerea by compromising membrane integrity, and significantly reduced soluble protein content and the oxalic acid secretion of hypha. The resulting incidence of postharvest tomato gray mold with the combination of selenium and methyl jasmonate was 34.7%, which was approximately half of that of the control. To sum up, the combined use of selenium and methyl jasmonate inhibited the normal physiological activity and pathogenicity of B. cinerea, which suggests that selenium and methyl jasmonate have the potential for controlling gray mold disease caused by B. cinerea in postharvest fruits and vegetables. These findings may offer a promising and eco-friendly strategy to control gray mold disease in postharvest fruits and vegetables.

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.

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.