Development and Comparison of Visual LAMP and LAMP-TaqMan Assays for Colletotrichum siamense.

Strawberry anthracnose caused by Colletotrichum spp. has resulted in significant losses in strawberry production worldwide. Strawberry anthracnose occurs mainly at the seedling and early planting stages, and Colletotrichum siamense is the main pathogen in North China, where mycelia, anamorphic nuclei, and conidia produced in the soil are the main sources of infection. The detection of pathogens in soil is crucial for predicting the prevalence of anthracnose. In this study, a visualized loop-mediated isothermal amplification (LAMP) assay and a loop-mediated isothermal amplification method combined with a TaqMan probe (LAMP-TaqMan) assay were developed for the ß-tubulin sequence of C. siamense. Both methods can detect Colletotrichum siamense genomic DNA at very low concentrations (104 copies/g) in soil, while both the visualized LAMP and LAMP-TaqMan assays exhibited a detection limit of 50 copies/µL, surpassing the sensitivity of conventional PCR and qPCR techniques, and both methods showed high specificity for C. siamense. The two methods were compared: LAMP-TaqMan exhibited enhanced specificity due to the incorporation of fluorescent molecular beacons, while visualized LAMP solely necessitated uncomplicated incubation at a constant temperature, with the results determined by the color change; therefore, the requirements for the instrument are relatively straightforward and user-friendly. In conclusion, both assays will help monitor populations of C. siamense in China and control strawberry anthracnose in the field.

Impact of tps1 Deletion and Overexpression on Terpene Metabolites in Trichoderma atroviride.

Terpenoids are structurally diverse natural products that have been widely used in the pharmaceutical, food, and cosmetic industries. Research has shown that fungi produce a variety of terpenoids, yet fungal terpene synthases remain not thoroughly explored. In this study, the tps1 gene, a crucial component of the terpene synthetic pathway, was isolated from Trichoderma atroviride HB20111 through genome mining. The function of this gene in the terpene synthetic pathway was investigated by constructing tps1-gene-deletion- and overexpression-engineered strains and evaluating the expression differences in the tps1 gene at the transcript level. HS-SPME-GC-MS analysis revealed significant variations in terpene metabolites among wild-type, tps1-deleted (Δtps1), and tps1-overexpressed (Otps1) strains; for instance, most sesquiterpene volatile organic compounds (VOCs) were notably reduced or absent in the Δtps1 strain, while nerolidol, ß-acorenol, and guaiene were particularly produced by the Otps1 strain. However, both the Δtps1 and Otps1 strains produced new terpene metabolites compared to the wild-type, which indicated that the tps1 gene played an important role in terpene synthesis but was not the only gene involved in T. atroviride HB20111. The TPS1 protein encoded by the tps1 gene could function as a sesquiterpene cyclase through biological information and evolutionary tree analysis. Additionally, fungal inhibition assay and wheat growth promotion assay results suggested that the deletion or overexpression of the tps1 gene had a minimal impact on fungal inhibitory activity, plant growth promotion, and development, as well as stress response. This implies that these activities of T. atroviride HB20111 might result from a combination of multiple metabolites rather than being solely dependent on one specific metabolite. This study offers theoretical guidance for future investigations into the mechanism of terpenoid synthesis and serves as a foundation for related studies on terpenoid metabolic pathways in fungi.

Antagonistic effects of Talaromyces muroii TM28 against Fusarium crown rot of wheat caused by Fusarium pseudograminearum.

Fusarium crown rot (FCR) caused by Fusarium pseudograminearum is a serious threat to wheat production worldwide. This study aimed to assess the effects of Talaromyces muroii strain TM28 isolated from root of Panax quinquefolius against F. pseudograminearum. The strain of TM28 inhibited mycelial growth of F. pseudograminearum by 87.8% at 72 h, its cell free fermentation filtrate had a strong antagonistic effect on mycelial growth and conidial germination of F. pseudograminearum by destroying the integrity of the cell membrane. In the greenhouse, TM28 significantly increased wheat fresh weight and height in the presence of pathogen Fp, it enhanced the antioxidant defense activity and ameliorated the negative effects of F. pseudograminearum, including disease severity and pathogen abundance in the rhizosphere soil, root and stem base of wheat. RNA-seq of F. pseudograminearum under TM28 antagonistic revealed 2,823 differentially expressed genes (DEGs). Most DEGs related to cell wall and cell membrane synthesis were significantly downregulated, the culture filtrate of TM28 affected the pathways of fatty acid synthesis, steroid synthesis, glycolysis, and the citrate acid cycle. T. muroii TM28 appears to have significant potential in controlling wheat Fusarium crown rot caused by F. pseudograminearum.