Wenyong Shao

Neopestalotiopsis is one of the three genera within the Pestalotiopsis-like fungi that infect tea plants, causing severe diseases. Carbendazim (MBC) is a broad-spectrum, systemic benzimidazole fungicide widely used to control various phytopathogenic fungi. In this study, a 20% MBC resistance frequency was observed among the Neopestalotiopsis strains collected from Jiangsu Province, eastern China. The molecular mechanism of this resistance was identified; specifically, amino acid substitutions (E197K, E197A, F199Y) within the ß2-tubulin conferred high-level resistance (RF > 200, MIC ≥ 200 μg/mL) of Neopestalotiopsis sp. against MBC, which was further confirmed by isogenic replacement experiments. The MBC-resistant strains exhibited no fitness costs in terms of mycelial growth, conidiation and pathogenicity. Furthermore, no cross-resistance was observed with phenamacril, prochloraz, fludioxonil or tebuconazole. These findings reveal that the control of Neopestalotiopsis spp. with MBC faces significant challenges due to the emerging resistance, while alternative fungicides can be integrated into management strategies.

Fusarium falciforme, one of the predominant causal agents of Fusarium root rot (FRR), poses a severe threat to sustainable soybean production worldwide. Fludioxonil (Flu) exhibits highly antifungal activity against F. falciforme. However, the molecular mechanisms underlying Flu-resistance remain elusive. In this study, the baseline sensitivity of 60 wild-type isolates to Flu was established at 0.0401 ± 0.0114 μg/mL, exhibiting as a biased curve. Three FluHR mutants generated via in vitro repetitive exposure to Flu (mutation frequency: 0.6%) were stable. All the FluHR mutants exhibited severe biological fitness penalties. Sequence alignment results showed that two novel amino acid substitutions (FfOs1A576T and FfOs1M626I) located at the highly conserved HAMP5 domain, and independently conferred high-level Flu resistance (RF > 2,000) by validation of genetic method. Molecular docking results revealed that the binding affinities of Flu to the FfOs1A576T or FfOs1M626I were significantly reduced when compared with the corresponding FfOs1 from FluS parental strain. Furthermore, no cross-resistance was observed between Flu and other fungicide with different modes of action. All the FluR mutants displayed pronounced hypersensitivity to osmotic stress agents and a significant reduction in intracellular glycerol accumulation. Collectively, this study is the first to identify two novel substitutions, A576T and M626I, in the HAMP5 domain of FfOs1 that individually confer Flu resistance while substantially impairing asexual reproduction, virulence, and osmoregulatory capacity in F. falciforme.

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.

BACKGROUND: Fusarium head blight (FHB) caused by Fusarium graminearum species complex (FGSG) remains a major challenge to cereal crops and resistance to key fungicides by the pathogen threatens control efficacy. Pydiflumetofen, a succinate dehydrogenase inhibitor, and phenamacril, a cyanoacrylate fungicide targeting myosin I, have been applied to combat this disease. Nonetheless, emergence of pydiflumetofen resistance in a subset of field isolates alongside laboratory‐induced facile generation of phenamacril‐resistant isolates signals a critical danger of resistance proliferation.RESULTS: Our study investigates the development of dual resistance to these fungicides in F. graminearum. Utilizing pydiflumetofen‐resistant (Py^R) and ‐sensitive (Py^S) isolates, we obtained dual‐resistant (Py^RPh^R) and phenamacril‐resistant (Py^SPh^R) mutants on potato sucrose agar containing phenamacril. Mutation rates for phenamacril resistance were comparable between pydiflumetofen‐resistant and ‐sensitive isolates, implying independent pathways for resistance development. The mutants compromised in fungal growth, competitive viability and deoxynivalenol production, suggesting fitness penalties for the dual‐resistant mutants. However, no cross‐resistance was found with tebuconazole or fludioxonil. In addition, we characterized four critical amino acid changes (S217L, C423R, K537T, E420G) in the Myo1 that were verified to confer phenamacril resistance in F. graminearum.CONCLUSION: This research indicates the possibility of resistance development for both pydiflumetofen and phenamacril in F. graminearum and emphasizes the need for fungicide resistance management for FHB. © 2024 Society of Chemical Industry.

Eukaryotic genomes are spatially organized within the nucleus in a nonrandom manner. However, fungal genome arrangement and its function in development and adaptation remain largely unexplored. Here, we show that the high-order chromosome structure of Fusarium graminearum is sculpted by both H3K27me3 modification and ancient genome rearrangements. Active secondary metabolic gene clusters form a structure resembling chromatin jets. We demonstrate that these jet-like domains, which can propagate symmetrically for 54 kb, are prevalent in the genome and correlate with active gene transcription and histone acetylation. Deletion of GCN5, which encodes a core and functionally conserved histone acetyltransferase, blocks the formation of the domains. Insertion of an exogenous gene within the jet-like domain significantly augments its transcription. These findings uncover an interesting link between alterations in chromatin structure and the activation of fungal secondary metabolism, which could be a general mechanism for fungi to rapidly respond to environmental cues, and highlight the utility of leveraging three-dimensional genome organization in improving gene transcription in eukaryotes.

Fungicide treatments are often essential for maintaining healthy crops and to achieve reliable and high-quality yields. However, continued use of fungicides with the same modes of action can lead to development of fungicide resistance, which has emerged in various plant pathogens and is a serious threat to effective crop protection. Exploration of resistance mechanisms is critical for resistance monitoring and management. This brief review summarizes advances during the past five decades in understanding the molecular resistance mechanisms of plant pathogenic fungi and oomycetes to major classes of fungicides, including benzimidazoles, myosin inhibitors, sterol demethylation inhibitors, quinone outside inhibitors, succinate dehydrogenase inhibitors, anilinopyrimidines, carboxylic acid amides, and oxysterol-binding protein homolog inhibitors. Based on known resistance mechanisms, PCR- and loop-mediated isothermal amplification-based approaches have been developed to allow high-throughput monitoring and early/rapid detection of emerging resistance. Classical principles in fungicide resistance management are also summarized, including using different modes of action of fungicides, limiting the number of applications of the chemicals with site-specific modes of action, and avoidance of their eradicant use. Future studies on novel strategies of disease management, including development of epigenetics- and RNA-based fungicides, will provide valuable knowledge for management of fungicide resistance.

Summary: SUMOylation as one of the protein post‐translational modifications plays crucial roles in multiple biological processes of eukaryotic organisms. Botrytis cinerea is a devastating fungal pathogen and capable of infecting plant hosts at low temperature. However, the molecular mechanisms of low‐temperature adaptation are largely unknown in fungi. Combining with biochemical methods and biological analyses, we report that SUMOylation regulates pathogen survival at low temperature and oxidative DNA damage response during infection in B. cinerea. The heat shock protein (Hsp70) BcSsb and E3 ubiquitin ligase BcRad18 were identified as substrates of SUMOylation; moreover, their SUMOylation both requires a single unique SUMO‐interacting motif (SIM). SUMOylated BcSsb regulates β‐tubulin accumulation, thereby affecting the stability of microtubules and consequently mycelial growth at low temperature. On the contrary, SUMOylated BcRad18 modulates mono‐ubiquitination of the sliding clamp protein proliferating cell nuclear antigen (PCNA), which is involved in response to oxidative DNA damage during infection. Our study uncovers the molecular mechanisms of SUMOylation‐mediated low‐temperature survival and oxidative DNA damage tolerance during infection in a devastating fungal pathogen, which provides novel insights into low‐temperature adaptation and pathogenesis for postharvest pathogens as well as new targets for inhibitor invention in disease control.