ABSTRACTPostharvest fungal diseases cause substantial losses in horticultural crops and are commonly managed using chemical fungicides, which raise concerns regarding resistance development and environmental impact. Plant‐derived antifungal peptides represent a promising alternative for sustainable disease control. Here, we evaluated the antifungal activity and mode of action of three nodule‐specific cysteine‐rich (NCR) peptides—NCR044, NCR192 and NCR13_PFV2—against major postharvest fungal pathogens. Among the peptides tested, NCR13_PFV2 exhibited the strongest inhibitory activity against Alternaria alternata, Colletotrichum gloeosporioides, Lasiodiplodia theobromae and Penicillium expansum, although activity against C. gloeosporioides was predominantly fungistatic rather than fungicidal. Mechanistic analyses using A. alternata as a model pathogen revealed that NCR13_PFV2 rapidly associates with fungal membranes, induces membrane permeabilization and exhibits selective binding to anionic phospholipids and phosphoinositides. Peptide treatment was associated with mitochondrial depolarization, elevated reactive oxygen species accumulation, reduced cellular respiration and disruption of vacuolar integrity. These intracellular effects were accompanied by loss of turgor pressure and hyphal collapse. Importantly, application of NCR13_PFV2 to wounded bell pepper fruit significantly reduced lesion development caused by A. alternata in a concentration‐dependent manner. Together, these findings demonstrate that NCR13_PFV2 suppresses postharvest fungal infection through a multi‐target mechanism that disrupts fungal cellular homeostasis, highlighting its potential as a peptide‐based alternative to synthetic fungicides.

ABSTRACTFlavescence dorée (FD), caused by FD phytoplasma (FDp) and transmitted by the leafhopper Scaphoideus titanus, is a quarantine disease that seriously threatens viticulture across Europe. Research on resistance or tolerance to FDp in grapevine is limited by the perennial nature of the host, high cultivar variability and the univoltine life cycle of the insect vector. To overcome these constraints, we employed Arabidopsis thaliana as a model host to identify genes conferring resistance to FDp. RNA sequencing (RNA‐seq) was first used to identify genes deregulated in A. thaliana during infection by either the woody host pathogen FDp or the herbaceous host pathogen Chrysanthemum yellows phytoplasma (CYp). A subset of these genes was then validated by monitoring their expression in the FDp–A. thaliana pathosystem at various time points during infection. Five genes were consistently deregulated upon infection, and functional analysis of the corresponding mutants revealed two lines with significantly reduced susceptibility to FDp compared with the wild type. The double mutant combining these two genes exhibited a similar phenotype, suggesting functional convergence. Electrical penetration graph (EPG) analysis indicated that this resistance is independent of vector feeding behaviour, pointing to a genetically determined defence mechanism rather than antixenosis. These findings uncover novel components of plant defence against phytoplasma infection and establish A. thaliana as a valuable system for dissecting the molecular basis of tolerance to FDp. We discussed how the genes we identified represent promising targets for developing sustainable, phytoplasma‐resistant grapevine varieties.

Fusarium head blight (FHB), caused by Fusarium asiaticum, is a significant wheat disease in China, reducing yields and contaminating grain with deoxynivalenol (DON). Due to the absence of highly resistant wheat varieties, chemical control remains the primary method for managing FHB. This study identified two homologous SDHC genes in F. asiaticum, namely FaSDHC1 and FaSDHC2. Deletion of FaSDHC2 impaired fungal sporulation, virulence and DON production, whereas the loss of FaSDHC1 increased DON production. Both genes were found to regulate sensitivity to SDHI fungicides-ΔFaSDHC1 showed decreased sensitivity, while ΔFaSDHC2 exhibited increased sensitivity. To explore the regulatory role of the SdhC subunit in SDHI fungicides sensitivity in F. asiaticum, this study obtained 11 boscalid-resistant mutants from ΔFaSDHC2 through chemical taming. Genetic analysis identified six mutation genotypes, including two previously reported in Fusarium species (FaSdhB-H248Y and FaSdhD-E166K) and four novel mutations in the Fusarium genus (FaSdhC1-H144Y, FaSdhC1-H144N, FaSdhD-H122Y and FaSdhD-D133N). The site-directed mutation verification and sensitivity assays revealed that mutants harbouring FaSdhC1-H144Y/N, FaSdhD-H122Y, FaSdhD-D133N or FaSdhD-E166K exhibited resistance to multiple SDHI fungicides (boscalid, fluopyram, pydiflumetofen, isopyrazam and benzovindiflupyr). In contrast, FaSdhB-H248Y mutants remained sensitive to fluopyram while showing resistance to other SDHI fungicides. This study elucidates the important regulatory role of SdhC genetic differentiation to SDHI fungicide sensitivity in F. asiaticum. Furthermore, it confirms that specific variations within distinct subunits of the SDH complex endow F. asiaticum with resistance to SDHI fungicides. The findings advance understanding of SDHI fungicides resistance mechanisms and provide a basis for developing FaSdhC-specific inhibitors and resistance management strategies.

Ferroptotic death of the developing conidia is important for pathogenicity of the blast fungus Magnaporthe oryzae, yet its regulatory mechanism is not fully elucidated in fungal cell. In this study, we found that elevated indole-3-acetic acid (IAA) promoted iron and lipid peroxide accumulation, leading to a higher degree of ferroptotic death of conidia. By characterising the IAA biosynthesis mutant, tam1Δ, we found that conidial death was positively correlated with levels of endogenous IAA. Furthermore, we found an IAA-dependent lipid metabolism gene mutant ppoaΔ displayed deficiencies in conidial death and the consequent delay in appressorium formation, and reduced pathogenicity. Exogenous addition of two types of phosphatidylethanolamines (PEs), DOPE and SLPE, could at least partially restore such defects in both tam1Δ and ppoaΔ, indicating that IAA promotes ferroptosis by affecting lipid metabolism and/or peroxidation. Additionally, we found TAM1 affected autophagy by modulating transcription of the ATG8 gene. Overall, our study reveals that M. oryzae produces and uses IAA to promote pathogenicity through triggering ferroptotic conidial death during pathogenic development. Our results provide a theoretical basis for blast disease control using IAA synthesis inhibitors.

Quinofumelin, a quinoline-based fungicide, demonstrated potent antifungal activity against Fusarium graminearum, the pathogen responsible for Fusarium head blight (FHB) in wheat. Previously, we elucidated that quinofumelin targets dihydroorotate dehydrogenase (DHODH), an indispensable enzyme in the de novo pyrimidine biosynthesis pathway. To explore its resistance mechanism, five FgDHODHII site-directed mutants with distinct genotypes were generated using the homologous recombination strategy. Sensitivity assays identified varying resistance levels: FgDHODHII-A94V remained sensitive to quinofumelin, FgDHODHII-D155T conferred low-level resistance, FgDHODHII-N281A conferred moderate-level resistance, and FgDHODHII-V179E/V179D conferred high-level resistance. Quinofumelin inhibition was reversed by the addition of uridine monophosphate (UMP), uridine or uracil. Although the mutants did not exhibit growth defects, quinofumelin-resistant mutants displayed reduced sporulation and virulence. Quinofumelin showed cross-resistance to ipflufenoquin, but no cross-resistance to other common fungicides (phenamacril, carbendazim, tebuconazole, pydiflumetofen, prothioconazole, fluopyram, benzovindiflupyr, pyraclostrobin and azoxystrobin). Molecular docking, molecular dynamics (MD) simulations and microscale thermophoresis (MST) revealed that D155T, V179E, V179D and N281A mutations altered the binding mode of quinofumelin, resulting in diminished binding affinity. This study provides key insights into its mode of action and resistance mechanism of quinofumelin, facilitating its application in sustainable FHB management.

Functional validation of NLR genes is critical for confirming their specific roles and developing durable disease-resistant crops. The Fom-1 gene of Cucumis melo, controlling resistance to races 0 and 2 of F. oxysporum f. sp. melonis (FOM), had been identified by map-based cloning as MELO3C022146, which encodes a TIR-NBS-LRR (TNL) protein. It resides in a head-to-head orientation adjacent to another TNL gene, Prv, controlling resistance to papaya ring spot virus (PRSV). In this study we validated the function of Fom-1 in mediating FOM resistance by applying CRISPR/Cas9 editing to the resistant cultivar, Védrantais. Two gRNAs were designed to target exons 1 and 2, respectively, and mutations were introduced at both target sites, resulting in truncated open reading frames in both alleles. Inoculation assays of T1 plants with FOM races 2 and 0 revealed a breakdown of resistance, manifested by leaf necrosis and wilting, and susceptibility was stably inherited in the T2 generation. This proved that MELO3C022146, the candidate gene for Fom-1, is responsible for resistance in melon cultivar Védrantais. Future studies will address the molecular functions of this gene as well as possible interactions between Fom-1 and its neighbour R-gene, Prv.

Fusarium oxysporum f. sp. pseudostellariae (Fop) is the major causal agent of root rot in Pseudostellaria heterophylla. This study focused on VeA, a conserved pathogenicity regulator in Fop. RNA interference constructs targeting the Velvet domain (VeA-1) and nuclear localization signal (NLS) region (VeA-2) were generated to assess their effects on fungal growth, virulence and host responses. Phylogenetic and structural analyses showed that Fop VeA contains a canonical Velvet domain and NLS motif conserved across filamentous fungi. Silencing of VeA significantly impaired colony growth and conidiation, indicating its positive regulatory role in development. Pathogenicity assays revealed markedly reduced virulence of ΔVeA-1 and ΔVeA-2 strains, with alleviated root rot symptoms in P. heterophylla. Biochemical assays showed that wild-type infection induced strong antioxidant enzyme activities (peroxidase, catalase, superoxide dismutase), whereas these responses were attenuated under VeA silencing, corresponding to reduced oxidative stress. Reverse transcription-quantitative PCR confirmed that VeA modulates host defence gene expression (PR2, PR4, PR5). Transcriptomic enrichment further indicated activation of immune-related pathways during infection. Collectively, VeA acts as a key regulator coordinating fungal development and virulence while indirectly manipulating host oxidative and immune responses, providing a potential molecular target for biological control of root rot in P. heterophylla.

Sugarcane mosaic virus (SCMV) causes substantial yield losses worldwide, yet the molecular basis underlying resistance and susceptibility in sugarcane remains incompletely understood. Here, we performed time-resolved transcriptome profiling of two contrasting sugarcane genotypes, the SCMV-susceptible cultivar Badila and its resistant somatic mutant FG1, across five infection stages. Absolute quantification revealed rapid viral RNA replication in Badila, whereas FG1 showed early suppression followed by SCMV clearance. Comparative transcriptomic analyses showed that FG1 mounted a rapid and sustained defence-associated transcriptional response, whereas Badila displayed delayed, predominantly repressive gene expression changes. Weighted gene co-expression network analysis identified gene modules strongly correlated with viral RNA levels and highlighted the small heat shock protein gene ScHSP17.5 as a central hub associated with susceptibility. Protein-protein interaction assays demonstrated that ScHSP17.5 and ScHSP17.9A specifically interact with the SCMV movement protein P3N-PIPO, but not with P3 or the coat protein. Functional assays in Nicotiana benthamiana further showed that overexpression of either ScHSP enhanced SCMV RNA replication, with co-expression producing a synergistic effect. Together, these results support a model in which SCMV exploits host small heat shock proteins via P3N-PIPO to promote viral accumulation, whereas early redox- and signalling-associated responses restrict infection in resistant sugarcane. This study provides mechanistic insight into SCMV-host interactions and identifies candidate targets for resistance breeding.

MicroRNAs (miRNAs) are pivotal regulators of plant immunity. While our prior work implicated zma-miR169s in maize defence against Bipolaris maydis, its upstream regulation and downstream signalling mechanisms remained elusive. Here, we decipher a complete signalling pathway that confers resistance to B. maydis. We show that the transcription factor ZmWRKY92 directly binds to the promoter of zma-miR169s to suppress its transcription. Consistent with its role as a positive regulator, loss of ZmWRKY92 function increased maize susceptibility to the pathogen. We further delineate downstream of this pathway, demonstrating that ZmNF-YA13, a nuclear-localised target of zma-miR169s induced upon infection, is a positive defence regulator. Overexpression of ZmNF-YA13 enhanced resistance, whereas knockout mutants were more susceptible. Integrated multi-omics analysis further revealed that ZmNF-YA13 activates the flavonoid biosynthesis pathway, promoting the accumulation of antimicrobial compounds like gallocatechin, quercetin and chalcone. Collectively, our work establishes the ZmWRKY92-miR169s-ZmNF-YA13-flavonoid module as a key signalling pathway in maize antifungal immunity, providing novel targets for maize disease resistance improvement.