Baseline sensitivity and physiological characteristics of natural product hinokitiol against Sclerotinia sclerotiorum.

BACKGROUND: Sclerotinia sclerotiorum, a pathogenic fungus of oilseed rape, poses a severe threat to the oilseed rapeseed industry. In this study, we evaluated the potential of the natural compound hinokitiol against S. sclerotiorum by determining its biological activity and physiological characteristics. RESULTS: Our results showed that hinokitiol strongly inhibited the hyphae expansion of S. sclerotiorum, and its effective concentration of hyphae growing inhibition by 50% (EC50) against 103 S. sclerotiorum strains varied from 0.36 to 3.45 µg/mL, with an average of 1.23 µg/mL. Hinokitiol possessed better protective efficacy than therapeutic effects, and it exhibited no cross-resistance between carbendazim. After treatment with hinokitiol, many vesicular protrusions developed on the mycelium with rough surface and thickened cell wall. Moreover, the cell membrane permeability and glycerol content increased, while the oxalic acid declined after hinokitiol treatment. In addition, hinokitiol induced membrane lipid peroxidation and improved the production of reactive oxygen species (ROS) in S. sclerotiorum. Importantly, real-time quantitative polymerase chain reaction showed that cell wall and ROS synthesis-related genes were significantly up-regulated after hinokitiol treatment. CONCLUSION: This study revealed that hinokitiol has good biological activity against S. sclerotiorum and could be considered as an alternative bio-fungicide for the resistance management in controlling sclerotinia stem rot infected by S. sclerotiorum. These investigations provided new insights into understanding the toxic action of hinokitiol against pathogenic fungi. © 2024 Society of Chemical Industry.

Natural α-Methylene-γ-Butyrolactone Analogues: Synthesis and Evaluation of Isoaurone Derivatives as Antifungal Agents.

In order to develop novel, efficient and green fungicides, a series of novel isoaurone derivatives were designed and synthesized, which were characterized by 1H and 13C NMR, high-resolution mass spectra and melting points. The target compounds showed different inhibitory activities against seven plant pathogenic fungi. Compounds 1, 12, 17, 20, 22, 24 and intermediate A showed more than 90% inhibition rates against S. s at 50 mg/L. Interestingly, compound 22 and intermediate A showed the great inhibitory effect against S. s with EC50 values of 4.65 and 4.24 mg/L, which were better than the lead compound isoaurone (EC50 = 15.62 mg/L). The EC50 values of compounds 17 and 24 against B. c were 13.94 and 22.13 mg/L. Moreover, compound 19 displayed significant antifungal activity against G. g with the EC50 value of 11.88 mg/L. Theoretical calculations by DFT revealed that the α, ß-unsaturated carbonyl bond and the benzyl ring are very importantly linked to the strength of the fungicidal activity. Therefore, this study identified a valuable antifungal lead compound for further development of green fungicides.

A GDSL motif-containing lipase modulates Sclerotinia sclerotiorum resistance in Brassica napus.

Sclerotinia stem rot (SSR) caused by Sclerotinia sclerotiorum (Lib.) De Bary is a devastating disease infecting hundreds of plant species. It also restricts the yield, quality, and safe production of rapeseed (Brassica napus) worldwide. However, the lack of resistance sources and genes to S. sclerotiorum has greatly restricted rapeseed SSR-resistance breeding. In this study, a previously identified GDSL motif-containing lipase gene, Brassica napus GDSL LIPASE-LIKE 1 (BnaC07.GLIP1), encoding a protein localized to the intercellular space, was characterized as functioning in plant immunity to S. sclerotiorum. The BnaC07.GLIP1 promoter is S. sclerotiorum-inducible and the expression of BnaC07.GLIP1 is substantially enhanced after S. sclerotiorum infection. Arabidopsis (Arabidopsis thaliana) heterologously expressing and rapeseed lines overexpressing BnaC07.GLIP1 showed enhanced resistance to S. sclerotiorum, whereas RNAi suppression and CRISPR/Cas9 knockout B. napus lines were hyper-susceptible to S. sclerotiorum. Moreover, BnaC07.GLIP1 affected the lipid composition and induced the production of phospholipid molecules, such as phosphatidylethanolamine, phosphatidylcholine and phosphatidic acid, which were correlated with decreased levels of reactive oxygen species (ROS) and enhanced expression of defense-related genes. A B. napus bZIP44 transcription factor specifically binds the CGTCA motif of the BnaC07.GLIP1 promoter to positively regulate its expression. BnbZIP44 responded to S. sclerotiorum infection, and its heterologous expression inhibited ROS accumulation, thereby enhancing S. sclerotiorum resistance in Arabidopsis. Thus, BnaC07.GLIP1 functions downstream of BnbZIP44 and is involved in S. sclerotiorum resistance by modulating the production of phospholipid molecules and ROS homeostasis in B. napus, providing insights into the potential roles and functional mechanisms of BnaC07.GLIP1 in plant immunity and for improving rapeseed SSR disease-resistance breeding.

Fine-tuning of the dual-role transcription factor WRKY8 via differential phosphorylation for robust broad-spectrum plant immunity.

Plants perceive pathogen-associated molecular patterns (PAMPs) using plasma-membrane-localized pattern recognition receptors (PRRs) to activate broad-spectrum pattern-triggered immunity. However, the regulatory mechanisms that ensure robust broad-spectrum plant immunity remain largely unknown. Here, we reveal that the transcription factor WRKY8 has a dual role in the transcriptional regulation of PRR genes: repressing expression of the nlp20/nlp24 receptor gene RLP23 while promoting that of the chitin receptor gene CERK1. SsNLP1 and SsNLP2, two nlp24-type PAMPs from the destructive fungal pathogen Sclerotinia sclerotiorum, activate two calcium-elicited kinases, CPK4 and CPK11, which phosphorylate WRKY8 and thus release its inhibition on RLP23 to promote accumulation of RLP23 transcripts. Meanwhile, SsNLPs activate the RLCK-type kinase PBL19, which phosphorylates WRKY8 and thus enhances accumulation of CERK1 transcripts. Intriguingly, RLP23 is repressed at later stage by PBL19-mediated phosphorylation of WRKY8, thus avoiding excessive immunity and enabling normal growth. Our findings unveil a plant strategy of "killing two birds with one stone" to elicit robust broad-spectrum immunity. This strategy is based on PAMP-triggered fine-tuning of a dual-role transcription factor to simultaneously amplify two PRRs that recognize PAMPs conserved across a wide range of pathogens. Moreover, our results reveal a novel plant strategy for balancing the trade-off between growth and immunity by fine-tuning the expression of multiple PRR genes.

Rhamnolipids and fengycins interact differently with biomimetic lipid membrane models of Botrytis cinerea and Sclerotinia sclerotiorum: Lipidomics profiles and biophysical studies.

Rhamnolipids (RLs) and Fengycins (FGs) are biosurfactants with very promising antifungal properties proposed to reduce the use of synthetic pesticides in crops. They are amphiphilic molecules, both known to target the plasma membrane. They act differently on Botrytis cinerea and Sclerotinia sclerotiorum, two close Sclerotiniaceae phytopathogenic fungi. RLs are more efficient at permeabilizing S. sclerotiorum, and FGs are more efficient at permeabilizing B. cinerea mycelial cells. To study the link between the lipid membrane composition and the activity of RLs and FGs, we analyzed the lipid profiles of B. cinerea and S. sclerotiorum. We determined that unsaturated or saturated C18 and saturated C16 fatty acids are predominant in both fungi. We also showed that phosphatidylethanolamine (PE), phosphatidic acid (PA), and phosphatidylcholine (PC) are the main phospholipids (in this order) in both fungi, with more PA and less PC in S. sclerotiorum. The results were used to build biomimetic lipid membrane models of B. cinerea and S. sclerotiorum for all-atom molecular dynamic simulations and solid-state NMR experiments to more deeply study the interactions between RLs or FGs with different compositions of lipid bilayers. Distinctive effects are exerted by both compounds. RLs completely insert in all the studied model membranes with a fluidification effect. FGs tend to form aggregates out of the bilayer and insert individually more easily into the models representative of B. cinerea than those of S. sclerotiorum, with a higher fluidification effect. These results provide new insights into the lipid composition of closely related fungi and its impact on the mode of action of very promising membranotropic antifungal molecules for agricultural applications.

Fusiform nanoparticle boosts efficient genetic transformation in Sclerotinia sclerotiorum.

BACKGROUND: Sclerotinia sclerotiorum is a highly destructive phytopathogenic fungus that poses a significant threat to a wide array of crops. The current constraints in genetic manipulation techniques impede a thorough comprehension of its pathogenic mechanisms and the development of effective control strategies. RESULTS: Herein, we present a highly efficient genetic transformation system for S. sclerotiorum, leveraging the use of fusiform nanoparticles, which are synthesized with FeCl3 and 2,6-diaminopyrimidine (DAP). These nanoparticles, with an average longitude length of 59.00 nm and a positively charged surface, facilitate the direct delivery of exogenous DNA into the mycelial cells of S. sclerotiorum, as well as successful integration with stable expression. Notably, this system circumvents fungal protoplast preparation and tedious recovery processes, streamlining the transformation process considerably. Furthermore, we successfully employed this system to generate S. sclerotiorum strains with silenced oxaloacetate acetylhydrolase-encoding gene Ss-oah1. CONCLUSIONS: Our findings demonstrate the feasibility of using nanoparticle-mediated delivery as a rapid and reliable tool for genetic modification in S. sclerotiorum. Given its simplicity and high efficiency, it has the potential to significantly propel genetic research in filamentous fungi, offering new avenues for elucidating the intricacies of pathogenicity and developing innovative disease management strategies.

Preparation of microcapsules and evaluation of their biocontrol efficacy.

In this study, a combination of Serratia nematophila L2 and Bacillus velezensis W24 was used to biocontrol Sclerotinia sclerotiorum. When the mixed ratio of L2 to W24 was 1:1, the inhibition rate on the growth of S. sclerotiorum was 88.1 %. To gain a large number of bacteria, the culture medium and conditions were optimized. When the medium formula involved molasses (8.890 g/L), soy peptone (6.826 g/L), and NaCl (6.865 g/L), and the culture conditions were 32 °C, inoculum 4%, rotation speed 200 rpm, and pH 7, the maximum amounts of bacterial cells obtained. In order to prepare microcapsules, spray drying conditions were optimized. These conditions included the soluble starch concentration of 30 g/100 mL, the inlet air temperature of 160 °C, and the feed flow rate of 450 mL/h. Under these optimized conditions to prepare microcapsules, the mixed strain (L2 and W24) exhibited a survival rate of 93.9 ± 0.9% and a viable bacterial count of 6.4 × 1012 cfu/g. In addition, microcapsules (GW24Ms) which contained strains L2 and W24 had good storage stability. In the pot experiment, GW24Ms could effectively reduce the disease of soybean plants and the control effect was 88.4%. Thus, the microbial agent represents a promising biocontrol solution for managing Sclerotinia in soybean.

Sclerotinia rot of Zephyranthes candida caused by Sclerotinia sclerotiorum and Sclerotinia minor.

Sclerotinia rot is a serious disease that occurs on Zephyranthes candida. A thorough understanding of the pathogenic fungal species and understanding the biological characteristics are important for controlling sclerotinia. Fungal strains were isolated from the diseased leaves of Z. candida through tissue isolation. Koch's hypothesis screened pathogenic strains by pathogenicity of healthy leaves, including re-isolation and identification. A multigene phylogenetic tree was constructed by extracting genomic DNA from pathogenic strains and measuring the nucleotide sequences at four sites, including the internal transcribed spacer (ITS), RNA polymerase II second largest subunit (RPB2), glyceraldehyde-3-phosphate dehydrogenase (G3PDH), and heat shock protein 60 (HSP60). Morphological characteristics of the fungal structures were evaluated through microscopic analysis. The growth of pathogens was observed and recorded under different pH, different temperatures, different carbon sources and different nitrogen sources to clarify their biological characteristics. Representative strains D7, D13, X4, and X15 infected Z. candida and caused sclerotinia rot. At the beginning of the culture, white flocculent fungal hyphae appeared on the potato dextrose agar (PDA) medium, and black spherical to irregular-shaped sclerotia appeared at the edge of the colony after 7 days. The diameter of the sclerotia was 2.4-8.6 mm and 0.4-0.9 mm, respectively. One sclerotium was able to germinate from 1 to 5 apothecia. Ascus were cylindrical or spindle-shaped, with a size of 110.0-120.0 × 9.2-11.6 µm. One ascus contained eight colorless, oval ascospores, with a size of 8.4-12.0 × 4.5-5.5 µm. Based on the phylogenetic tree constructed with the gene sequences for ITS, G3PDH, HSP60, and RPB2, D7 and D13 shared 99% homology with sclerotinia sclerotiorum, whereas X4 and X15 shared 99% homology with sclerotinia minor. S. sclerotiorum growth was more suitable when the culture temperature was 15°C-25°C, pH 5.0, carbon source was maltose and nitrogen source was yeast powder. S. minor growth was more suitable when the culture temperature was 15°C, pH 5.0, the carbon source was glucose, and the nitrogen source was yeast powder. The results identified the pathogens as S. sclerotiorum and S. minor. To the best of our knowledge, this is the first report of S. sclerotiorum and S. minor causing sclerotinia rot on Z. candida. We herein aimed to identify the causal agent of sclerotinia rot of Z. candida in China based on morphological characteristics, molecular identification, and pathogenicity tests. Performed the experiments on the biological characteristics, to understand the law of disease occurrence. We also evaluated methods for the effective control of this disease. Our findings provide support for further studies on the pathogenesis mechanism of sclerotinia rot.

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.

Integrated Assays of Genome-Wide Association Study, Multi-Omics Co-Localization, and Machine Learning Associated Calcium Signaling Genes with Oilseed Rape Resistance to Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum (Ss) is one of the most devastating fungal pathogens, causing huge yield loss in multiple economically important crops including oilseed rape. Plant resistance to Ss pertains to quantitative disease resistance (QDR) controlled by multiple minor genes. Genome-wide identification of genes involved in QDR to Ss is yet to be conducted. In this study, we integrated several assays including genome-wide association study (GWAS), multi-omics co-localization, and machine learning prediction to identify, on a genome-wide scale, genes involved in the oilseed rape QDR to Ss. Employing GWAS and multi-omics co-localization, we identified seven resistance-associated loci (RALs) associated with oilseed rape resistance to Ss. Furthermore, we developed a machine learning algorithm and named it Integrative Multi-Omics Analysis and Machine Learning for Target Gene Prediction (iMAP), which integrates multi-omics data to rapidly predict disease resistance-related genes within a broad chromosomal region. Through iMAP based on the identified RALs, we revealed multiple calcium signaling genes related to the QDR to Ss. Population-level analysis of selective sweeps and haplotypes of variants confirmed the positive selection of the predicted calcium signaling genes during evolution. Overall, this study has developed an algorithm that integrates multi-omics data and machine learning methods, providing a powerful tool for predicting target genes associated with specific traits. Furthermore, it makes a basis for further understanding the role and mechanisms of calcium signaling genes in the QDR to Ss.

Colonization compatibility with Bacillus altitudinis confers soybean seed rot resistance.

The plant microbiome and plant-associated bacteria are known to support plant health, but there are limited studies on seed and seedling microbiome to reveal how seed-associated bacteria may confer disease resistance. In this study, the application of antibiotics on soybean seedlings indicated that seed-associated bacteria were involved in the seed rot resistance against a soil-borne pathogen Calonectria ilicicola, but this resistance cannot be carried to withstand root rot. Using PacBio 16S rRNA gene full-length sequencing and microbiome analyses, 14 amplicon sequence variants (ASVs) including 2 ASVs matching to Bacillus altitudinis were found to be more abundant in the four most resistant varieties versus the four most susceptible varieties. Culture-dependent isolation obtained two B. altitudinis isolates that both exhibit antagonistic capability against six fungal pathogens. Application of B. altitudinis on the most resistant and susceptible soybean varieties revealed different colonization compatibility, and the seed rot resistance was restored in the five varieties showing higher bacterial colonization. Moreover, quantitative PCR confirmed the persistence of B. altitudinis on apical shoots till 21 days post-inoculation (dpi), but 9 dpi on roots of the resistant variety TN5. As for the susceptible variety HC, the persistence of B. altitudinis was only detected before 6 dpi on both shoots and roots. The short-term colonization of B. altitudinis on roots may explain the absence of root rot resistance. Collectively, this study advances the insight of B. altitudinis conferring soybean seed rot resistance and highlights the importance of considering bacterial compatibility with plant varieties and colonization persistence on plant tissues.

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.

Selenium in soil enhances resistance of oilseed rape to Sclerotinia sclerotiorum by optimizing the plant microbiome.

Plants can recruit beneficial microbes to enhance their ability to resist disease. It is well established that selenium is beneficial in plant growth, but its role in mediating microbial disease resistance remains 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-1 selenium [selenate Na2SeO4, Se(VI) or selenite Na2SeO3, Se(IV)] significantly increased the resistance of oilseed rape to Sclerotinia sclerotiorum compared with no selenium application, with a disease inhibition rate higher than 20% in Se(VI)0.5, Se(IV)0.5 and Se(IV)1.0 mg kg-1 treatments. The disease resistance of oilseed rape was related to the presence of rhizosphere microorganisms and beneficial bacteria isolated from the rhizosphere inhibited Sclerotinia stem rot. Burkholderia cepacia and the synthetic community consisting of Bacillus altitudinis, Bacillus megaterium, Bacillus cereus, Bacillus subtilis, Bacillus velezensis, Burkholderia cepacia, and Flavobacterium anhui enhanced plant disease resistance through transcriptional regulation and activation of plant-induced systemic resistance. In addition, 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 detached leaves, and it also performed a significant inhibition effect on Sclerotinia sclerotiorum. Overall, our results indicate that selenium improves plant rhizosphere microorganisms and 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.

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.

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.). A total of 236 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 (totaling ∼4%) showed a highly resistant (HR) response. A further 19 accessions of L. angustifolius, 2 accessions each of L. albus and L. pilosus, and a single accession of L. mutabilis (totaling ∼10%) showed a resistant (R) response. The reactions of 16 (15 L. angustifolius, 1 L. digitatus) of these 236 accessions were also compared with their reactions to a different isolate, Walkaway-3 (WW-3; pathotype 10). Against this isolate, five L. angustifolius accessions showed an HR response and four showed an R response, and the L. digitatus accession showed a moderate resistance 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. cosentinii 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 an HR response, and 146 (∼45%) showed an 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 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.