Xinqiu Tan

Persea americana (avocado) is a fruit rich in nutrients; however, its industry is facing major threats from pathogen infection. Here, we clearly identified Colletotrichum fructicola as the pathogen causing avocado diseases in Pu’er City, Yunnan Province. However, the biological characteristics, genetic transformation system, and early cell cycle regulation of this pathogen remained unclear. In this study, C. fructicola exhibited a maximum growth rate on complete medium (CM), with the conidial yield reaching 2 × 10⁵ conidia/mL after 24 h in liquid CM. Conidia of C. fructicola had nearly fully germinated at 4 h post-inoculation (hpi), with the appressorium formation rate exceeding 95% at 12 hpi. We also established a PEG-CaCl₂-mediated genetic transformation system. The GFP-tagged transformants showed no significant differences in core biological function from the wild type. Using eGFP labeling, we visually elucidated the early cell cycle regulation of C. fructicola. Furthermore, cell cycle inhibitor assays demonstrated that C. fructicola conidial germination is independent of nuclear division and relies on cytoskeletal modulation, whereas appressorium formation and mycelial expansion require functional cell cycle regulation. This is probably the first study to systematically elucidate the cell cycle regulatory characteristics of C. fructicola isolated from avocado, and to successfully develop its genetic transformation system. These results provide important theoretical and technical support for the formulation of integrated control strategies against C. fructicola, as well as facilitating the sustainable development of the avocado industry.

Colletotrichum gloeosporioides is a major phytopathogen responsible for anthracnose in Capsicum annuum (pepper) which leads to significant yield losses. At present, the molecular mechanism of C. gloeosporioides pathogenesis is not very clear. In this study, we focused on the MET30 protein and its key WD40 domain, with an emphasis on its role in the biological functions of C. gloeosporioides. Bioinformatics analysis revealed that the MET30 protein contains a conserved F-box domain and multiple WD40 repeats, which interact with other proteins to participate in various cellular processes, including nutrient acquisition, stress responses, and pathogenicity. Gene knockout and complementation experiments demonstrated that deleting the MET30 protein or its WD40 domain significantly reduced the rates of spore production and hyphal growth while increasing tolerance to environmental stresses such as high salinity and oxidative stress. Furthermore, pathogenicity assays revealed that the WD40 domain of the MET30 protein is crucial for regulating fungal pathogenicity, as mutants lacking WD40 domains presented increased virulence on pepper leaves. These findings suggest that the WD40 domain, in synergy with the MET30 protein, regulates the pathogenicity and stress response of C. gloeosporioides, provides new insights into the molecular mechanisms of anthracnose, and offers potential strategies for effective disease control.

Fungal diseases of plants are one of the key factors causing global crop losses. In this study, we isolated a Bacillus velezensis strain VC3, which was found to have a broad-spectrum inhibitory effect on a variety of phytopathogenic fungi through in vitro and in planta experiments, especially on Magnaporthe oryzae and Colletotrichum gloeosporioides. Further genomic and transcriptomic analyses revealed that the B. velezensis VC3 has multiple functional gene clusters encoding for the synthesis of a variety of antifungal secondary metabolites, including antimicrobial peptides (AMPs) and lipopeptides (LPs). In addition, AMPs and LPs were isolated and purified from B. velezensis VC3 fermentation broth and their antifungal activities were verified in this study. AMPs and LPs significantly inhibited spore germination, appressorium formation, and disease development, and AMPs have a better potential for controlling M. oryzae and C. gloeosporioides than LPs. These findings open new avenues for utilizing B. velezensis VC3 as biocontrol agents, providing potential sustainable solutions for agricultural production.

Pepper anthracnose caused by Colletotrichum gloeosporioides infection is an important fungal disease and represents a serious threat to pepper yield and quality. At present, the pathogenic molecular mechanism of C. gloeosporioides is not very clear. In our study, we characterized the function of C. gloeosporioides CgNis1, a homolog of Magnaporthe oryzae MoNis1. We found that the ∆Cgnis1 mutant reduced the growth rate and was defective in conidiation. Although the rate of appressorium formation was unaffected, the germ tube was found to be abnormal. CgNis1 was shown to be involved in the H₂O₂ stress response and maintaining cell membrane permeability. The pathogenicity assays performed in this study indicated that the deletion of CgNIS1 is associated with virulence. Our results indicate that CgNis1 is necessary for the growth, development, and pathogenicity of the fungus. This work provides an in-depth analysis of the Nis1 protein, helps to enhance studies on pathogen-related molecular mechanisms, and provides a theoretical basis for the prevention and control of C. gloeosporioides in peppers.

Previous research has shown that the pathogenicity and appressorium development of Magnaporthe oryzae can be inhibited by the ATP synthase subunit beta (Atp2) present in the photosynthetic bacterium Rhodopseudomonas palustris. In the present study, transgenic plants overexpressing the ATP2 gene were generated via genetic transformation in the Zhonghua11 (ZH11) genetic background. We compared the blast resistance and immune response of ATP2-overexpressing lines and wild-type plants. The expression of the Atp2 protein and the physiology, biochemistry, and growth traits of the mutant plants were also examined. The results showed that, compared with the wild-type plant ZH11, transgenic rice plants heterologously expressing ATP2 had no significant defects in agronomic traits, but the disease lesions caused by the rice blast fungus were significantly reduced. When infected by the rice blast fungus, the transgenic rice plants exhibited stronger antioxidant enzyme activity and a greater ratio of chlorophyll a to chlorophyll b. Furthermore, the immune response was triggered stronger in transgenic rice, especially the increase in reactive oxygen species (ROS), was more strongly triggered in plants. In summary, the expression of ATP2 as an antifungal protein in rice could improve the ability of rice to resist rice blast.

Endophytes can colonize a host plant without inducing obvious disease symptoms. Elicitors from endophytes may play important roles in balancing endophyte colonization. We identified a novel endophytic elicitor from Colletotrichum tofieldiae , Ct RALF, that has structural and functional characteristics similar to those of rapid alkalinization factor (RALF) peptides in Arabidopsis thaliana . We found that Ct RALF can directly interact with the host plant receptor‐like kinase FERONIA (FER). The colonization of FER mutant plants by hyphae of C. tofieldiae was obviously reduced compared with that on wild‐type plants. Furthermore, the interaction of Ct RALF with FER regulated symbiotic signals by triggering calcium waves, inhibiting reactive oxygen species bursts, increasing mitogen‐activated protein kinase phosphorylation and reducing the stabilization of MYC2. Collectively, these results suggested that the RALF homologue of endophytes may improve their symbiosis by interacting with host plant‐encoded FER.

Antimicrobial peptides (AMPs) from black solider flies (Hermetia illucens, BSF) exhibiting broad-spectrum antimicrobial activity are the most promising green substitutes for preventing the infection of phytopathogenic fungi; therefore, AMPs have been a focal topic of research. Recently, many studies have focused on the antibacterial activities of BSF AMPs against animal pathogens; however, currently, their antifungal activities against phytopathogenic fungi remain unclear. In this study, 7 AMPs selected from 34 predicted AMPs based on BSF metagenomics were artificially synthesized. When conidia from the hemibiotrophic phytopathogenic fungi Magnaporthe oryzae and Colletotrichum acutatum were treated with the selected AMPs, three selected AMPs—CAD1, CAD5, and CAD7—showed high appressorium formation inhibited by lengthened germ tubes. Additionally, the MIC₅₀ concentrations of the inhibited appressorium formations were 40 μM, 43 μM, and 43 μM for M. oryzae, while 51 μM, 49 μM, and 44 μM were observed for C. acutatum, respectively. A tandem hybrid AMP named CAD-Con comprising CAD1, CAD5, and CAD7 significantly enhanced antifungal activities, and the MIC₅₀ concentrations against M. oryzae and C. acutatum were 15 μM and 22 μM, respectively. In comparison with the wild type, they were both significantly reduced in terms of virulence when infection assays were performed using the treated conidia of M. oryzae or C. acutatum by CAD1, CAD5, CAD7, or CAD-Con. Meanwhile, their expression levels of CAD1, CAD5, and CAD7 could also be activated and significantly increased after the BSF larvae were treated with the conidia of M. oryzae or C. acutatum, respectively. To our knowledge, the antifungal activities of BSF AMPs against plant pathogenic fungi, which help us to seek potential AMPs with antifungal activities, provide proof of the effectiveness of green control strategies for crop production.