Preparation and antifungal activity evaluation of hinokitiol emulsifiable concentrate against Sclerotinia sclerotiorum.

Hinokitiol is a natural broad-spectrum antimicrobial monoterpenoid, which is widely used as an antiseptic in food, cosmetics and other products. In the present study, the toxic actions of hinokitiol to the plant pathogen Sclerotinia sclerotiorum were investigated. The EC50 value for mycelial growth inhibition was 2.63 µg/mL, and there was no positive or negative cross-resistance between hinokitiol and carbendazim. The emulsifiable concentrate of 30% hinokitiol was prepared, which has excellent application prospect in the prevention of sclerotinia and gray mould. Hinokitiol is a promising spray fungicide for stems and leaves rather than seeds and roots.

Facilitation of Sclerotinia sclerotiorum infestation by aphid feeding behaviour is not affected by aphid resistance in oilseed rape.

The relation between aphids and Sclerotinia stem rot (SSR) in oilseed rape is rarely examined because they are often studied alone. We have observed a significant correlation between the number of aphids and the occurrence of SSR in our field studies. Electropenetrography (EPG) was used to evaluate the effects of Brevicoryne brassicae (Linnaeus) on two oilseed rape cultivars while acquiring, vectoring and inoculating of Sclerotinia sclerotiorum Lib. (de Bary) ascospores. The results demonstrated that aphid feeding followed by the application of an ascospore suspension significantly increased S. sclerotiorum incidence. Aphids were capable of adhering to ascospores and carrying them to healthy plants, thereby causing diseases. The results of the EPG analysis indicated that aphid feeding behaviour was significantly altered in all leaf tissue levels following infection with S. sclerotiorum. Aphids initiated their first puncture significantly sooner than the control group, began probing mesophyll cells earlier, significantly increased the frequency of both short probes and intracellular punctures and had a significantly shorter pathway duration. On infected aphid-susceptible cultivars, aphids secreted more saliva but had reduced ingestion compared with aphids feeding on non-infected oilseed rape. In addition, ascospores can affect aphid feeding behaviour by adhering to aphids. Aphids carrying ascospores punctured cells earlier, with a significant increase in the frequency and duration of short probes and cell punctures, shortened pathway durations, increased salivation and reduced ingestion compared with aphids not carrying ascospores. On aphid-susceptible cultivars, aphids carrying ascospores delayed puncture onset, but on resistant cultivars, puncture onset was shortened. There is a correlation between aphids and S. sclerotiorum. The impact of S. sclerotiorum on aphid feeding behaviour is directional, favouring the spread of the fungus. This promotion does not appear to be altered by the aphid resistance of the cultivar.

Glutamate Receptor-like (GLR) Family in Brassica napus: Genome-Wide Identification and Functional Analysis in Resistance to Sclerotinia sclerotiorum.

Plant glutamate receptor-like channels (GLRs) are homologs of animal ionotropic glutamate receptors. GLRs are critical in various plant biological functions, yet their genomic features and functions in disease resistance remain largely unknown in many crop species. Here, we report the results on a thorough genome-wide study of the GLR family in oilseed rape (Brassica napus) and their role in resistance to the fungal pathogen Sclerotinia sclerotiorum. A total of 61 GLRs were identified in oilseed rape. They comprised three groups, as in Arabidopsis thaliana. Detailed computational analyses, including prediction of domain and motifs, cellular localization, cis-acting elements, PTM sites, and amino acid ligands and their binding pockets in BnGLR proteins, unveiled a set of group-specific characteristics of the BnGLR family, which included chromosomal distribution, motif composition, intron number and size, and methylation sites. Functional dissection employing virus-induced gene silencing of BnGLRs in oilseed rape and Arabidopsis mutants of BnGLR homologs demonstrated that BnGLR35/AtGLR2.5 positively, while BnGLR12/AtGLR1.2 and BnGLR53/AtGLR3.2 negatively, regulated plant resistance to S. sclerotiorum, indicating that GLR genes were differentially involved in this resistance. Our findings reveal the complex involvement of GLRs in B. napus resistance to S. sclerotiorum and provide clues for further functional characterization of BnGLRs.

Complete genome sequence of Bacillus velezensis strain Ag109, a biocontrol agent against plant-parasitic nematodes and Sclerotinia sclerotiorum.

Soybean is the main oilseed cultivated worldwide. Even though Brazil is the world's largest producer and exporter of soybean, its production is severely limited by biotic factors. Soil borne diseases are the most damaging biotic stressors since they significantly reduce yield and are challenging to manage. In this context, the present study aimed to evaluate the potential of a bacterial strain (Ag109) as a biocontrol agent for different soil pathogens (nematodes and fungi) of soybean. In addition, the genome of Ag109 was wholly sequenced and genes related to secondary metabolite production and plant growth promotion were mined. Ag109 showed nematode control in soybean and controlled 69 and 45% of the populations of Meloidogyne javanica and Pratylenchus brachyurus, respectively. Regarding antifungal activity, these strains showed activity against Macrophomia phaseolina, Rhizoctonia solani, and Sclerotinia sclerotiorum. For S. sclerotiorum, this strain increased the number of healthy plants and root dry mass compared to the control (with inoculation). Based on the average nucleotide identity and digital DNA-DNA hybridization, this strain was identified as Bacillus velezensis. Diverse clusters of specific genes related to secondary metabolite biosynthesis and root growth promotion were identified, highlighting the potential of this strain to be used as a multifunctional microbial inoculant that acts as a biological control agent while promoting plant growth in soybean.

Identification and expression profiling of GAPDH family genes involved in response to Sclerotinia sclerotiorum infection and phytohormones in Brassica napus.

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a crucial enzyme in glycolysis, an essential metabolic pathway for carbohydrate metabolism across all living organisms. Recent research indicates that phosphorylating GAPDH exhibits various moonlighting functions, contributing to plant growth and development, autophagy, drought tolerance, salt tolerance, and bacterial/viral diseases resistance. However, in rapeseed (Brassica napus), the role of GAPDHs in plant immune responses to fungal pathogens remains unexplored. In this study, 28 genes encoding GAPDH proteins were revealed in B. napus and classified into three distinct subclasses based on their protein structural and phylogenetic relationships. Whole-genome duplication plays a major role in the evolution of BnaGAPDHs. Synteny analyses revealed orthologous relationships, identifying 23, 26, and 26 BnaGAPDH genes with counterparts in Arabidopsis, Brassica rapa, and Brassica oleracea, respectively. The promoter regions of 12 BnaGAPDHs uncovered a spectrum of responsive elements to biotic and abiotic stresses, indicating their crucial role in plant stress resistance. Transcriptome analysis characterized the expression profiles of different BnaGAPDH genes during Sclerotinia sclerotiorum infection and hormonal treatment. Notably, BnaGAPDH17, BnaGAPDH20, BnaGAPDH21, and BnaGAPDH22 exhibited sensitivity to S. sclerotiorum infection, oxalic acid, hormone signals. Intriguingly, under standard physiological conditions, BnaGAPDH17, BnaGAPDH20, and BnaGAPDH22 are primarily localized in the cytoplasm and plasma membrane, with BnaGAPDH21 also detectable in the nucleus. Furthermore, the nuclear translocation of BnaGAPDH20 was observed under H2O2 treatment and S. sclerotiorum infection. These findings might provide a theoretical foundation for elucidating the functions of phosphorylating GAPDH.

Soil selenium enhanced the resistance of oilseed rape to Sclerotinia sclerotiorum by optimizing the plant microbiome.

Plant can recruit beneficial microbes to enhance their ability to resist disease. Selenium is well established as a beneficial element in plant growth, but its role in mediating microbial disease resistance remained poorly understood. Here, we investigated the correlation between selenium, oilseed rape rhizosphere microbes and Sclerotinia sclerotiorum. Soil application of 0.5 and 1.0 mg/kg selenium significantly increased the resistance of oilseed rape to Sclerotinia sclerotiorum compared with no selenium application, and the disease inhibition rate was higher than 20%. The disease resistance of oilseed rape was related to rhizosphere microorganisms, and beneficial bacteria isolated from the rhizosphere inhibited Sclerotinia stem rot. Burkholderia cepacia, and synthetic community enhanced plant disease resistance through transcriptional regulation and activated plant-induced systemic resistance to protect plants. Besides, inoculation of isolated bacteria optimized the bacterial community structure of leaves and enriched beneficial microorganisms such as Bacillus, Pseudomonas and Sphingomonas. Bacillus isolated from the leaves were sprayed on the detached leaves, and it also performed a significant inhibition effect on Sclerotinia sclerotiorum. Overall, our results suggested that selenium drive plant rhizosphere microorganisms to increase resistance to Sclerotinia sclerotiorum in oilseed rape.

Control of Sclerotinia sclerotiorum via an RNA interference (RNAi)-mediated targeting of SsPac1 and SsSmk1.

MAIN CONCLUSION: The study evaluates the potential of Spray-Induced Gene Silencing and Host-Induced Gene Silencing for sustainable crop protection against the broad-spectrum necrotrophic fungus Sclerotinia sclerotiorum. Sclerotinia sclerotiorum (Lib.) de Bary, an aggressive ascomycete fungus causes white rot or cottony rot on a broad range of crops including Brassica juncea. The lack of sustainable control measures has necessitated biotechnological interventions such as RNA interference (RNAi) for effective pathogen control. Here we adopted two RNAi-based strategies-Spray-Induced Gene Silencing (SIGS) and Host-Induced Gene Silencing (HIGS) to control S. sclerotiorum. SIGS was successful in controlling white rot on Nicotiana benthamiana and B. juncea by targeting SsPac1, a pH-responsive transcription factor and SsSmk1, a MAP kinase involved in fungal development and pathogenesis. Topical application of dsRNA targeting SsPac1 and SsSmk1 delayed infection initiation and progression on B. juncea. Further, altered hyphal morphology and reduced radial growth were also observed following dsRNA application. We also explored the impact of stable dsRNA expression in A. thaliana against S. sclerotiorum. In this report, we highlight the utility of RNAi as a biofungicide and a tool for preliminary functional genomics.

An effector SsCVNH promotes the virulence of Sclerotinia sclerotiorum through targeting class III peroxidase AtPRX71.

Many plant pathogens secrete effector proteins into the host plant to suppress host immunity and facilitate pathogen colonization. The necrotrophic pathogen Sclerotinia sclerotiorum causes severe plant diseases and results in enormous economic losses, in which secreted proteins play a crucial role. SsCVNH was previously reported as a secreted protein, and its expression is significantly upregulated at 3 h after inoculation on the host plant. Here, we further demonstrated that deletion of SsCVNH leads to attenuated virulence. Heterologous expression of SsCVNH in Arabidopsis enhanced pathogen infection, inhibited the host PAMP-triggered immunity (PTI) response and increased plant susceptibility to S. sclerotiorum. SsCVNH interacted with class III peroxidase AtPRX71, a positive regulator of innate immunity against plant pathogens. SsCVNH could also interact with other class III peroxidases, thus reducing peroxidase activity and suppressing plant immunity. Our results reveal a new infection strategy employed by S. sclerotiorum in which the fungus suppresses the function of class III peroxidases, the major component of PTI to promote its own infection.

Ca2+ affects the hyphal differentiation to sclerotia formation of Athelia rolfsii.

RNA-Sequencing (RNA-Seq) and transcriptomic analyses have become powerful tools to study the developmental stages of fungal structures scuh as sclerotia. While RNA-Seq experiments have been set up for many important sclerotia- and microsclerotia-forming fungi, it has not been implemented to study Athelia rolfsii, which is one of the earliest fungi used in literature to uncover the roles of reactive oxygen species (ROS) in stimulating sclerotia formation. This study applied RNA-Seq to profile gene expression in four developmental stages of A. rolfsii sclerotia. Surprisingly, gene ontology and expression patterns suggested that most ROS-scavenging genes were not up-regulated in the stages from hyphal differentiation to the initial sclerotia stage. Using antioxidant and oxidant-amended culture assay, the results suggested none of the ascorbic acid, dithiothreitol (DTT), H2O2, or superoxide dismutase inhibitors [diethyldithiocarbamate (DETC), NaN3, and sodium dodecyl sulfate] affected the sclerotia number. Instead, only glutathione reduced the sclerotia number. Because glutathione has also been suggested to facilitate Ca2+ influx, therefore, glutathione culture assays with the combination of CaCl2, Ca2+-chelator egtazic acid, DETC, and H2O2 were tested on A. rolfsii, as well as two other fungi (Sclerotinia sclerotiorum and Macrophomina phaseolina) for comparison. Although the addition of CaCl2 caused sclerotia or microsclerotia reduction for all three fungi, the CaCl2-ROS interaction was only observed for S. sclerotiorum and M. phaseolina, but not A. rolfsi. Collectively, this study not only pointed out a conserved function of Ca2+ in suppressing fungal sclerotia and microsclerotia formation but also highlighted sclerotia formation of A. rolfsii being only sensitive to Ca2+ and independent of ROS stimuli.IMPORTANCEManagement for plant diseases caused by soil-borne fungal pathogens is challenging because many soil-borne fungal pathogens form sclerotia for long-term survival. Advanced understanding of the molecular and cellular mechanisms of sclerotia formation may provide novel insights to prevent these fungal residues in fields. This study discovered that Ca2+ acts as a negative signal cue to suppress sclerotia and microsclerotia formation in three economically important fungal pathogens. Moreover, the southern blight fungus Athelia rolfsii appears to be only regulated by Ca2+ but not reactive oxygen species. Accordingly, A. rolfsii can be a useful system for studying the detailed mechanism of Ca2+, and the applicability of Ca2+ in reducing sclerotia could be further assessed for disease management.

Screening of Diverse Lupinus spp. Highlights New Resistances to Sclerotinia sclerotiorum.

Stem rot caused by Sclerotinia sclerotiorum is a serious, and sometimes devastating, disease of lupin (Lupinus spp.). Two hundred and thirty-six lupin accessions from across 12 Lupinus species were screened against the prevalent S. sclerotiorum isolate MBRS-1 (pathotype 76). L. angustifolius accession 21655 and L. albus var. albus accession 20589 showed immune and 'near-immune' responses, respectively. Thirteen accessions of L. angustifolius, three accessions each of L. albus and L. albus var. albus, and a single accession each of L. albus var. graecus, L. mutabilis, L. palaestinus and L. pilosus (totalling ~4%) showed a highly resistant (HR) response. A further 19 accessions of L. angustifolius, two accessions each of L. albus and L. pilosus, and a single accession of L. mutabilis (totalling ~10%) showed a resistant (R) response. The reactions of 16 (15 L. angustifolius, one L. digitatus) of these 236 accessions were also compared with their reactions to a different isolate, WW-3 (pathotype 10). Against this isolate, five L. angustifolius accessions showed a HR response and four showed a R response, and the L. digitatus accession showed a moderate resistance (MR) response. Overall, isolate WW-3 caused significantly (P<0.05) smaller lesions than MBRS-1 across tested accessions in common. In addition, 328 plants in a 'wild' naturalized field population of L. cosentini were screened in situ in the field against isolate MBRS-1. Five (~1.5%) of the 328 plants of wild lupin showed an immune response, 63 (~19%) showed a HR response, and 146 (~45%) showed a R response. We believe this is the first examination of diverse Lupinus spp. germplasm responses to a prevalent pathotype of S. sclerotiorum. Lupin genotypes exhibiting high level resistance to Sclerotinia stem rot identified in this study can now be used as parental lines for crosses in lupin breeding programs and/or directly as improved cultivars to reduce the adverse impact of this disease on lupin crops.

Genomic and biological control of Sclerotinia sclerotiorum using an extracellular extract from Bacillus velezensis 20507.

Introduction: Sclerotinia sclerotiorum is a known pathogen that harms crops and vegetables. Unfortunately, there is a lack of effective biological control measures for this pathogen. Bacillus velezensis 20507 has a strong antagonistic effect on S. Sclerotiorum; however, the biological basis of its antifungal effect is not fully understood. Methods: In this study, the broad-spectrum antagonistic microorganisms of B. velezensis 20507 were investigated, and the active antifungal ingredients in this strain were isolated, purified, identified and thermal stability experiments were carried out to explore its antifungal mechanism. Results: The B. velezensis 20507 genome comprised one circular chromosome with a length of 4,043,341 bp, including 3,879 genes, 185 tandem repeats, 87 tRNAs, and 27 rRNAs. Comparative genomic analysis revealed that our sequenced strain had the closest genetic relationship with Bacillus velezensis (GenBank ID: NC 009725.2); however, there were significant differences in the positions of genes within the two genomes. It is predicted that B. velezensis 20507 encode 12 secondary metabolites, including difficidin, macrolactin H, fengycin, surfactin, bacillibactin, bacillothiazole A-N, butirosin a/b, and bacillaene. Results showed that B. velezensis 20507 produced various antagonistic effects on six plant pathogen strains: Exserohilum turcicum, Pyricularia oryzae, Fusarium graminearum, Sclerotinia sclerotiorum, Fusarium oxysporum, and Fusarium verticillioides. Acid precipitation followed by 80% methanol leaching is an effective method for isolating the antifungal component ME80 in B. velezensis 20507, which can damage the membranes of S. sclerotiorum hyphae and has good heat resistance. Using high-performance liquid chromatography, and Mass Spectrometry analysis, it is believed that fengycin C72H110N12O20 is the main active antifungal substance. Discussion: This study provides new resources for the biological control of S. Sclerotiorum in soybeans and a theoretical basis for further clarification of the mechanism of action of B. velezensis 20507.

The APSES Transcription Factor SsStuA Regulating Cell Wall Integrity Is Essential for Sclerotia Formation and Pathogenicity in Sclerotinia sclerotiorum.

APSES (Asm1p, Phd1p, Sok2p, Efg1p, and StuAp) family transcription factors play crucial roles in various biological processes of fungi, however, their functional characterization in phytopathogenic fungi is limited. In this study, we explored the role of SsStuA, a typical APSES transcription factor, in the regulation of cell wall integrity (CWI), sclerotia formation and pathogenicity of Sclerotinia sclerotiorum, which is a globally important plant pathogenic fungus. A deficiency of SsStuA led to abnormal phosphorylation level of SsSmk3, the key gene SsAGM1 for UDP-GlcNAc synthesis was unable to respond to cell wall stress, and decreased tolerance to tebuconazole. In addition, ΔSsStuA was unable to form sclerotia but produced more compound appressoria. Nevertheless, the virulence of ΔSsStuA was significantly reduced due to the deficiency of the invasive hyphal growth and increased susceptibility to hydrogen peroxide. We also revealed that SsStuA could bind to the promoter of catalase family genes which regulate the expression of catalase genes. Furthermore, the level of reactive oxygen species (ROS) accumulation was found to be increased in ΔSsStuA. In summary, SsStuA, as a core transcription factor involved in the CWI pathway and ROS response, is required for vegetative growth, sclerotia formation, fungicide tolerance and the full virulence of S. sclerotiorum.

Bimetallic nanoparticles and biochar produced by Adansonia Digitata shell and their effect against tomato pathogenic fungi.

Adansonia digitata L. is a royal tree that is highly valued in Africa for its medicinal and nutritional properties. The objective of this study was to use its fruit shell extract to develop new, powerful mono and bimetallic nanoparticles (NPs) and biochar (BC) using an eco-friendly approach. Silver (Ag), iron oxide (FeO), the bimetallic Ag-FeO NPs, as well as (BC) were fabricated by A. digitata fruit shell extract through a reduction process and biomass pyrolysis, respectively, and their activity against tomato pathogenic fungi Alternaria sp., Sclerotinia sclerotiorum, Fusarium equiseti, and Fusarium venenatum were detected by agar dilution method. The Ag, FeO, Ag-FeONPs, and BC were characterized using a range of powerful analytical techniques such as ultraviolet-visible (UV-Vis) spectroscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier Transform-Infra Red (FT-IR), dynamic light scatter (DLS), and zeta potential analysis. The fabricated Ag, FeO and Ag-FeO NPs have demonstrated a remarkable level of effectiveness in combating fungal strains. UV-Vis spectra ofAg, FeO, Ag-FeONPs, and BC show broad exhibits peaks at 338, 352, 418, and 480 nm, respectively. The monometallic, bimetallic NPs, and biochar have indicated the presence in various forms mostly in Spherical-shaped. Their size varied from 102.3 to 183.5 nm and the corresponding FTIR spectra suggested that the specific organic functional groups from the plant extract played a significant role in the bio-reduction process. Ag and Ag-FeO NPs exhibited excellent antifungal activity against pathogenic fungi Alternaria sp., S. sclerotiorum, F. equiseti, and F. venenatum. The current study could be a significant achievement in the field of antifungal agents since has the potential to develop new approaches for treating fungal infections.

A Novel Strain of Bacillus cereus with a Strong Antagonistic Effect Specific to Sclerotinia and Its Genomic and Transcriptomic Analysis.

Sclerotinia, which is caused by Sclerotinia sclerotiorum, is a severe disease of oilseed rape, which is an important oil crop worldwide. In this study, we isolated a novel strain of Bacillus cereus, named B. cereus HF10, from the rhizosphere soil of the reed on the seaside of Yagzhou Bay, Sanya city, Hainan Province, China. HF10 exhibited a significant antagonistic effect on Sclerotinia sclerotiorum, with an inhibition rate of 79%, and to other species in Sclerotinia, but no antagonistic effect was found on various other fungi or bacteria. HF10 had an 82.3% inhibitory effect on the S. sclerotiorum infection of oilseed rape leaves and a 71.7% control effect on Sclerotinia infection in oilseed rape based on in vitro and in vivo experiments, respectively. The genomics and transcriptomics of HF10 and its loss of the antifungal function mutant Y11 were analyzed, and the results provided insight into potential antifungal substances. Our work provides a novel strain, HF10, for developing a promising biological control agent against Sclerotinia, which infects oilseed rape and other plants.

Antifungal Activity and Mechanism of Xenocoumacin 1, a Natural Product from Xenorhabdus nematophila against Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum (Lib.) de Bary, a polyphagous necrotrophic fungal pathogen, has brought about significant losses in agriculture and floriculture. Until now, the most common method for controlling S. sclerotiorum has been the application of fungicides. Xenocoumacin 1 (Xcn1) is a potential biopesticide having versatile antimicrobial activities, generated by Xenorhabdus nematophila. This study was intended to isolate Xcn1 from X. nematophila YL001 and clarify its efficacies for S. sclerotiorum control. Xcn1 demonstrated a wider antifungal spectrum against 10 plant-pathogenic fungi. It also exhibited a strong inhibitory effect on the mycelial growth of S. sclerotiorum with an EC50 value of 3.00 µg/mL. Pot experiments indicated that Xcn1 effectively inhibited disease extension on oilseed rape and broad bean plants caused by S. sclerotiorum. Morphological and ultrastructural observations revealed that the hyphae of S. sclerotiorum became twisted, shriveled, and deformed at the growing points after treatment with Xcn1 at 3.00 µg/mL and that the subcellular fractions also became abnormal concurrently, especially the mitochondrial structure. Moreover, Xcn1 also increased cell membrane permeability and decreased the content of exopolysaccharide as well as suppressing the activities of polygalacturonase and cellulase of S. sclerotiorum, but exerted no effects on oxalic acid production. This study demonstrated that Xcn1 has great potential to be developed as a new biopesticide for the control of S. sclerotiorum.

Aphids May Facilitate the Spread of Sclerotinia Stem Rot in Oilseed Rape by Carrying and Depositing Ascospores.

Aphids and Sclerotinia stem rot in oilseed rape are often studied in isolation, and their relationship is rarely explored. Our field studies have revealed a significant positive correlation between the number of aphids and the incidence of Sclerotinia stem rot. Hence, starting with the colonizing stages of the two pests, Breveroryne brassicae was assessed for its potential to acquire, transmit, and inoculate Sclerotinia sclerotiorum by being sprayed with an ascospore suspension. Moreover, distinctions in aphid feeding behavior were examined between aphids on inoculated/uninoculated winter and spring oilseed rape plants or aphids, both with and without S. sclerotiorum ascospores, using electropenetrography (EPG). The results showed that aphid feeding followed by dropping ascospore suspension significantly increased the incidence of S. sclerotiorum. Ascospores were able to adhere to aphids and were carried by aphids to healthy plants, causing disease. The results of the EPG analysis indicated that aphid feeding behavior was significantly altered in all leaf tissue levels following infection with S. sclerotiorum. Specifically, aphids initiated their first puncture significantly sooner, began probing mesophyll cells earlier, had a significantly shorter pathway duration, and secreted saliva more frequently but reduced salivation prior to feeding and ingestion compared to aphids feeding on uninfected oilseed rape. Additionally, the feeding behavior of aphids carrying ascospores was markedly different from that of aphids not carrying ascospores, implying that ascospores directly influence aphid feeding behavior but that this influence appeared to be beneficial only for S. sclerotiorum infection. Aphids carrying ascospores started to puncture cells more quickly, with a significant increase in the frequency and duration of short probes and cell punctures, shortened pathway durations, and reduced salivation before feeding compared to aphids not carrying ascospores. It is clear that there is an interaction between aphids and S. sclerotiorum. The impact of S. sclerotiorum on aphid feeding behavior is directional, favoring the spread of the fungus.

A Glycosyl Hydrolase 5 Family Protein Is Essential for Virulence of Necrotrophic Fungi and Can Suppress Plant Immunity.

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.

Characterization of SsHog1 and Shk1 Using Efficient Gene Knockout Systems through Repeated Protoplasting and CRISPR/Cas9 Ribonucleoprotein Approaches in Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum is the causal agent of sclerotinia stem rot in over 400 plant species. In a previous study, the group III histidine kinase gene of S. sclerotiorum (Shk1) revealed its involvement in iprodione and fludioxonil sensitivity and osmotic stress. To further investigate the fungicide sensitivity associated with the high-osmolarity glycerol (HOG) pathway, we functionally characterized SsHog1, which is the downstream kinase of Shk1. To generate knockout mutants, split marker transformation combined with a newly developed repeated protoplasting method and CRISPR/Cas9 ribonucleoprotein (RNP) delivery approach were used. The pure SsHog1 and Shk1 knockout mutants showed reduced sensitivity to fungicides and increased sensitivity to osmotic stress. In addition, the SsHog1 knockout mutants demonstrated reduced virulence compared to Shk1 knockout mutants and wild-type. Our results indicate that the repeated protoplasting method and RNP approach can generate genetically pure homokaryotic mutants and SsHog1 is involved in osmotic adaptation, fungicide sensitivity, and virulence in S. sclerotiorum.

The schizotrophic lifestyle of Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum is a cosmopolitan and typical necrotrophic phytopathogenic fungus that infects hundreds of plant species. Because no cultivars highly resistant to S. sclerotiorum are available, managing Sclerotinia disease caused by S. sclerotiorum is still challenging. However, recent studies have demonstrated that S. sclerotiorum has a beneficial effect and can live mutualistically as an endophyte in graminaceous plants, protecting the plants against major fungal diseases. An in-depth understanding of the schizotrophic lifestyle of S. sclerotiorum during interactions with plants under different environmental conditions will provide new strategies for controlling fungal disease. In this review, we summarize the pathogenesis mechanisms of S. sclerotiorum during its attack of host plants as a destructive pathogen and discuss its lifestyle as a beneficial endophytic fungus.

The Transcription Factor SsZNC1 Mediates Virulence, Sclerotial Development, and Osmotic Stress Response in Sclerotinia sclerotiorum.

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.