Wenchan Chen

Alternative splicing (AS) of FgSdhC1 was identified in SDHI-sensitive (FgS) and intrinsically resistant (FgR) Fusarium graminearum isolates, generating four AS isoforms in each background. Isogenic replacement assays showed that individual isoforms differentially altered sensitivity to pydiflumetofen, boscalid, and fluopyram and affected multiple fitness-related traits. The average compound fitness index of FgR-derived isoform replacement strains was 0.54, compared with 0.20 for FgS-derived strains, and FgRAD2-C showed the highest value (1.43). FgSIR and FgSAD3 transcripts were significantly more abundant in FgS-C than the corresponding FgR-derived isoforms in FgR-C. Isoform transcript abundance, SDH activity, carbon source utilization, and responses to environmental stresses also differed among replacement strains. Transcriptomic and RT-qPCR analyses identified FgPRPF4B, FgDHX38/FgPRP16, and FgPPIL1 as differentially expressed candidate splicing-related genes. Collectively, these findings showed that FgSdhC1 alternative splicing isoforms differentially modulate intrinsic SDHI resistance and biological fitness in F. graminearum.

Fusarium graminearum causes Fusarium head blight and threatens cereal production through deoxynivalenol (DON) contamination. The biological role of FgSdhAF2 was characterized, and a potential antifungal pocket adjacent to the FgSdhAF2–FgSdhA interface was identified. Deletion of FgSdhAF2 markedly reduced mycelial growth, conidiation, virulence, DON production, and FgTri5 expression in SDHI-sensitive PH-1 and intrinsically resistant G1. The FgSdhAF2 E159Y substitution similarly reduced mycelial growth, conidiation, and virulence. Using this predicted pocket for structure-based virtual screening, approximately 1.6 million compounds were screened, yielding ten candidates. Compound 156758 inhibited PH-1, G1, and the highly pydiflumetofen-resistant FgSdhC1A83 V mutant, with EC50 values of 3.33–3.59 μg/mL, and reduced DON accumulation by 53.9–55.4%. It also showed broad-spectrum activity against eight additional phytopathogenic fungi. Collectively, these findings support this predicted interface as a potential antifungal target and 156758 as a lead compound for developing fungicides to manage SDHI-resistant F. graminearum populations.

Fusarium verticillioides is a major phytopathogenic fungus that causes maize Fusarium ear rot (FER), leading to severe yield losses and mycotoxin contamination worldwide. However, no fungicide has been registered in China for the control of FER. Pydiflumetofen (Pyd) is a new-generation succinate dehydrogenase inhibitor (SDHI) with strong fungicidal activity againstFusarium spp.. Here, the potential resistance risk of F. verticillioides to Pyd was evaluated. The baseline sensitivity of 183 F. verticillioides strains to Pyd was 0.10±0. 068 μg/mL, and the sensitivity distribution was unimodal. Thirteen Pyd-resistant mutants were generated by fungicide adaptation, and five genotype mutations were identified: FvSdhC1A83V, FvSdhDD133N, FvSdhC1A83VSdhDD133G, FvSdhC2A83VSdhDD133G and FvSdhDD133G. Notably, the mutation SdhDD133G associated with SDHI resistance in filamentous fungi is firstly reported. Furthermore, the FvSdhC1A83V, FvSdhC1A83VSdhDD133G and FvSdhC2A83VSdhDD133G mutants showed high resistance (259.83<RF<1175.03), whereas FvSdhDD133N mutants showed moderate resistance (64.94<RF<95.79). Mutants carrying FvSdhDD133G showed low resistance (20.79<RF<41.06). The Pyd-resistant mutants retained high biological fitness compared with the parental strain. Additionally, no cross-resistance was detected between Pyd and prochloraz, prothioconazole, phenamacril or pyraclostrobin. Together, these findings suggest a moderate to high risk that F. verticillioides will develop resistance to Pyd and provide essential data for monitoring and managing Pyd resistance in FER.

Leaf spots and blights, caused by filamentous fungus Alternaria alternata, are common diseases of numerous economic crops in China. Boscalid, a succinate dehydrogenase inhibitor, is widely used to control Alternaria leaf blights in China for decades. In current study, among the 114 isolates of A. alternata separated from broccoli and peanut grown in Yancheng City of Jiangsu province, the resistance frequency of boscalid is 4.39%, however, for the 54 isolates from Zoucheng City of Shandong province, the resistance frequency of boscalid is 5.56%. Compared to boscalid-sensitivity isolates, some boscalid-resistant isolates exhibited defects on fitness defined as mycelial growth, sporulation, spore germination and virulence. There was no cross-resistance between boscalid and thifluzamide, azoxystrobin, tebuconazole, fludioxonil or procymidone in the isolates of A. alternata from broccoli and peanut. Sequence alignment results of AaSDHB, AaSDHC or AaSDHD coding genes from eight field boscalid-resistant isolates showed that five broccoli-originated isolates possessed six types of amino acid substitutions in AaSDHB and AaSDHC (I21V, A28V, I52V, I62T, A118V and V169F), other three peanut-originated isolates possessed two types of amino acid substitutions in AaSDHD (V17A and A47T). Under the boscalid treatment the gene expressions of AaSDHB, AaSDHC or AaSDHD in boscalid-sensitivity isolate but not in boscalid-resistant isolates are increased, moreover, the succinate dehydrogenase (SDH) activity of the boscalid-sensitive isolate was lower than that of resistant isolates. Compared to the boscalid‑free condition, boscalid inhibited melanin synthesis in A. alternata, with down‑regulated expression of melanin synthesis genes. These results provide directions for controlling of leaf spots and blights in broccoli and peanut via scientifically using fungicide, and promote research development in fungicide resistance and fungi secondary metabolism.

Bipolaris maydis (B. maydis), the pathogen causing southern corn leaf blight, is spreading rapidly in China. New succinate dehydrogenase inhibitor (SDHI) fungicides pydiflumetofen (Pyd) and fluopyram (Flu) are potential control agents. Baseline sensitivities of B. maydis to Pyd and Flu were 0.0110 ± 0.0059 and 0.1211 ± 0.0377 μg/mL, respectively, by the mycelial growth method. In vitro, one Pyd-resistant mutant and two Flu-resistant mutants were generated via domestication of Pyd and Flu, respectively. Genetic validation confirmed that the BmSdhB^S231L and BmSdhC^R76C mutations individually confer differentiated resistance: high Flu/low Pyd resistance and low Flu/medium Pyd resistance, respectively. Fitness penalties were observed in some mutants, indicating a moderate to high resistance risk. Using AlphaFold3, the three-dimensional structure of BmSdh was modeled, showing that BmSdhB^S231L is adjacent to the Qp site (BmSDHB-Pro230) and BmSdhC^R76C is at the Qp site (BmSDHC-Arg76). Molecular docking revealed that these mutations reduce fungicide affinity. This study provides a basis for resistance management of B. maydis and insights into the SDHI's resistance mechanism.

Succinate dehydrogenase (SDH) is an integral component of the tricarboxylic acid cycle (TCA) and respiratory electron transport chain (ETC), targeted by succinate dehydrogenase inhibitors (SDHIs). Fusarium asiaticum is a prominent phytopathogen causing Fusarium head blight (FHB) on wheat. Here, we characterized the functions of the FaSdhA, FaSdhB, FaSdhC₁, FaSdhC₂, and FaSdhD subunits. Deletion of FaSdhA, FaSdhB, or FaSdhD resulted in significant growth defects in F. asiaticum. The FaSdhC₁ or FaSdhC₂ deletion mutants exhibited substantial reductions in fungal growth, conidiation, virulence, and reactive oxygen species (ROS). The FaSdhC₁ expression was significantly induced by pydiflumetofen (PYD). The ΔFaSdhC₁ mutant displayed hypersensitivity to SDHIs, whereas the ΔFaSdhC₂ mutant exhibited resistance against most SDHIs. The transmembrane domains of FaSdhC₁ are essential for regulating mycelial growth, virulence, and sensitivity to SDHIs. These findings provided valuable insights into how the two SdhC paralogues regulated the functional integrity of SDH, ROS homeostasis, and the sensitivity to SDHIs in phytopathogenic fungi.

Cytochrome P450 sterol 14α-demethylase (CYP51) is a key enzyme involved in the sterol biosynthesis pathway and serves as a target for sterol demethylation inhibitors (DMIs). In this study, the 3D structures of three CPY51 paralogues from Calonectria ilicicola (C. ilicicola) were first modeled by AlphaFold2, and molecular docking results showed that CiCYP51A, CiCYP51B, or CiCYP51C proteins individually possessed two active pockets that interacted with DMIs. Our results showed that the three paralogues play important roles in development, pathogenicity, and sensitivity to DMI fungicides. Specifically, CiCYP51A primarily contributed to cell wall integrity maintenance and tolerance to abiotic stresses, and CiCYP51B was implicated in sexual reproduction and virulence, while CiCYP51C exerted negative regulatory effects on sterol 14α-demethylase activity within the ergosterol biosynthetic pathway, revealing its genus-specific function in C. ilicicola. These findings provide valuable insights into developing rational strategies for controlling soybean red crown rot caused by C. ilicicola.

BACKGROUND: Garlic leaf spot (GLS) caused by Alternaria alternata is one of the main diseases in the garlic production areas, and its management heavily relies on dicarboximide fungicides. However, the efficacy of dicarboximides against the GLS disease has decreased year on year.RESULTS: In the present study, 10 of 148 A. alternata strains separated from Jiangsu Province were moderately resistant (MR) to a dicarboximide fungicide procymidone (Pro^MR). Positive cross‐resistance was observed between Pro and iprodione (Ipro) or fludioxonil (Fld), but not between Pro and fluazinam or azoxystrobin. Mutations at AaOS1, but not Aafhk1, were confirmed to confer the Pro resistance by constructing replacement mutants, whereas mutations at both AaOS1 and Aafhk1 decreased the gene expression level of AapksI, as well as the ability to produce mycotoxin AOH (polyketide‐derived alternariol) and virulence. Additionally, more genes (AaOS1 and Aafhk1) harboring the mutations experienced a larger biological fitness penalty.CONCLUSION: To our knowledge, this is the first report on Pro resistance selected in garlic fields, and mutations at AaOS1 of A. alternata causing a decreased ability to produce the mycotoxin AOH. © 2024 Society of Chemical Industry.

Heat-stable antifungal factor (HSAF) isolated from Lysobacter enzymogenes is considered a potential biocontrol agent. However, the target of HSAF in phytopathogenic fungi remains unclear. In this study, we investigated the target of HSAF in Valsa pyri that causes fatal pear Valsa canker. Thirty-one HSAF-binding proteins were captured and identified by surface plasmon resonance (SPR) and high-performance liquid chromatography-mass spectrometry (LC-MS/MS), and 11 deletion mutants were obtained. Among these mutants, only ΔVpVEB1 showed decreased sensitivity to HSAF. Additionally, ΔVpVEB1 exhibited significantly reduced virulence in V. pyri. Molecular docking and SPR results revealed that HSAF bound to threonine 569 and glycine 570 of VpVeb1, which are crucial for AAA ATPase activity. Another study showed that HSAF could decrease the ATPase activity of VpVeb1, leading to the reduced virulence of V. pyri. Taken together, this study first identified the potential target of HSAF in fungi. These findings will help us better understand the model of action of HSAF to fungi.