Deoxynivalenol (DON) is a mycotoxin produced in cereal crops infected with Fusarium graminearum. DON poses a serious threat to human and animal health, and is a critical virulence factor. Various environmental factors, including reactive oxygen species (ROS), have been shown to interfere with DON biosynthesis in this pathogen. The regulatory mechanisms of how ROS trigger DON production have been investigated extensively in F. graminearum. However, the role of the endogenous ROS-generating system in DON biosynthesis is largely unknown. In this study, we genetically analysed the function of leucine zipper-EF-hand-containing transmembrane 1 (LETM1) superfamily proteins and evaluated the role of the mitochondrial-produced ROS in DON biosynthesis. Our results show that there are two Letm1 orthologues, FgLetm1 and FgLetm2, in F. graminearum. FgLetm1 is localized to the mitochondria and is essential for mitochondrial integrity, whereas FgLetm2 plays a minor role in the maintenance of mitochondrial integrity. The ΔFgLetm1 mutant demonstrated a vegetative growth defect, abnormal conidia and increased sensitivity to various stress agents. More importantly, the ΔFgLetm1 mutant showed significantly reduced levels of endogenous ROS, decreased DON biosynthesis and attenuated virulence in planta. To our knowledge, this is the first report showing that mitochondrial integrity and endogenous ROS production by mitochondria are important for DON production and virulence in Fusarium species.

Inspired by commercially established fluxapyroxad as the lead compound of novel efficient antifungal ingredients, novel pyrazole carboxylate derivatives containing a flexible thiazole backbone were successfully designed, synthesized, and detected for their in vitro and in vivo biological activities against eight agricultural fungi. The antifungal bioassay results showed that compound 24 revealed excellent bioactivities against Botrytis cinerea and Sclerotinia sclerotiorum, with median effective concentrations (EC 50 ) of 0.40 and 3.54 mg/L, respectively. Compound 15 revealed remarkable antifungal activity against Valsa mali, with an EC 50 value of 0.32 mg/L. For in vivo fungicide control against B. cinerea and V. mali, compounds 3 and 24 at 25 mg/L, respectively, displayed prominent efficacy on cherry tomatoes and apple branches. Molecular docking results demonstrated that compound 15 could form an interaction with several crucial residues of succinate dehydrogenase (SDH), and the in vitro enzyme assay indicated that the target compound 15 displayed an inhibitory effect toward SDH, with an IC 50 value of 82.26 μM. The experimental results indicated that phenyl pyrazole carboxylate derivatives displayed a weak antifungal property and low activity compared to the other title substituent pyrazole carboxylate derivatives. Compounds 3, 15, and 24 are promising antifungal candidates worthy of further fungicide development due to their prominent effectiveness.

The zinc finger transcription factor Crz1 is an important downstream regulator of calcium-dependent signal transduction pathways in many organisms. The function of Crz1 in the wheat-head blight pathogen Fusarium graminearum remains unclear. In this study, we identified and functionally characterised FgCrz1A, a potential ortholog of yeast Crz1. The deletion mutant ΔFgCrz1A exhibited slower hyphal growth on basic medium, and conidia formation and sexual reproduction were completely blocked. ΔFgCrz1A also displayed increased sensitivity to metal cations Ca, Mg, Mn and Li, but decreased sensitivity to Zn. Unexpectedly, the deletion mutant was more resistant to osmotic stress and cell wall-damaging agents than the wild-type fungus. Pathogenicity assays showed that virulence of the mutant was dramatically decreased on flowering wheat heads and corn silks, consistent with the observed reduction in deoxynivalenol production. Moreover, GFP-fused FgCrz1A was mainly localised in the nucleus, and was required for transcriptional induction of abaA and wetA that are involved in conidiogenesis, as well as genes of the MAT locus during sexual reproduction, and TRI genes responsible for deoxynivalenol biosynthesis. Taken together, the results indicate that FgCrz1A plays critical roles not only in regulating fungal development, secondary metabolism and virulence in F. graminearum, but also in multiple stress responses.

Plant pathogenic fungi seriously threaten agricultural production. There is an urgent need to develop novel fungicides with low toxicity and high efficiency. In this study, we designed and synthesized 44 pyrazolo­[3,4-d]­pyrimidin-4-one derivatives and evaluated them for their fungicidal activities. The bioassay data revealed that most of the target compounds possessed moderate to high in vitro antifungal activities. Especially compound g22 exhibited remarkable antifungal activity against Sclerotinia sclerotiorum with an EC50 value of 1.25 mg/L, close to that of commercial fungicide boscalid (EC50 = 0.96 mg/L) and fluopyram (EC50 = 1.91 mg/L). Moreover, compound g22 possessed prominent protective activity against S. sclerotiorum in vivo for 24 h (95.23%) and 48 h (93.78%), comparable to positive control boscalid (24 h (96.63%); 48 h (93.23%)). Subsequent studies indicated that compound g22 may impede the growth and reproduction of S. sclerotiorum by affecting the morphology of mycelium, destroying cell membrane integrity, and increasing cell membrane permeability. In addition, the application of compound g22 did not injure the growth or reproduction of Italian bees. This study revealed that compound g22 is expected to be developed for efficient and safe agricultural fungicides.