Ying-Hong Lin

Plant-parasitic nematodes, particularly the root-knot nematode Meloidogyne incognita, severely threaten agricultural production in tropical and subtropical regions. Although nematode-trapping fungi have been considered environmentally friendly alternatives to chemical nematicides, their field performance often remains inconsistent due to limited ecological adaptation. This study combined a field survey of crop-associated nematode communities with laboratory and greenhouse evaluation of selected nematode-trapping fungal isolates under subtropical conditions. Field surveys across multiple crops identified M. incognita as the dominant plant-parasitic nematode species. Five fungal isolates with different trapping mechanisms were compared for growth under contrasting nutrient conditions and nematode-trapping efficiency. Among them, Drechslerella brochopaga (NTF2), which forms constricting rings, showed superior growth stability under nutrient-limited conditions and the highest nematode mortality in vitro. In greenhouse experiments, NTF2 reduced root gall formation in two of three independent trials, indicating potential but variable suppression of M. incognita on pumpkin. These findings highlight the importance of ecological adaptation and trapping strategy in determining the effectiveness of nematode-trapping fungi for sustainable nematode management.

Bursaphelenchus rainulfi is a pine-associated, non-pathogenic nematode that serves as a motile comparative species for evaluating nematophagous fungi. We investigated the in vitro biocontrol activity of Purpureocillium takamizusanense strain PMEPF27, originally isolated from insect cadavers in Taiwan, against mixed motile stages of B. rainulfi. Identity of the fungus was confirmed by morphology and ITS/EF-1α sequencing. Nematodes were exposed in liquid suspension to PMEPF27 conidia, with sterile water as the negative control and fluopyram as the positive control. Mortality was monitored over 7 days, and scanning electron microscopy was used to observe fungus–nematode interactions. PMEPF27 caused significantly higher mortality than the water control, reaching ~80% by day 7, and showed surface disruption of nematode cuticles, although no direct spore penetration was observed. These findings expand the nematophagous profile of P. takamizusanense beyond egg and sedentary stages, validate B. rainulfi as a motile comparative species in pine-nematode bioassays, and highlight PMEPF27 as a promising candidate for biocontrol development.

Fusarium oxysporum plays crucial roles as both a damaging plant pathogen and a model for studying host-pathogen interactions. It is well established that systematic functional genomics approaches are vital for advancing agricultural biotechnology and developing biological agents. Creating efficient gene-editing systems can help elucidate virulence mechanisms and enable rapid production of modified strains for practical use. However, current transformation methods face major challenges, such as high enzyme costs, and CRISPR/Cas9 systems are limited by PAM sequence availability and by blunt-end DNA cleavage, which hampers homologous recombination. Furthermore, complex guide RNA scaffolds complicate large-scale functional studies with traditional methods. To address these challenges, we have developed a streamlined CRISPR/Cpf1 (Cas12a) platform that combines small-scale protoplast preparation with significantly reduced enzyme use, exploiting Cpf1's unique features, such as flexible PAM recognition, staggered DNA cuts that promote recombination, improved target specificity, and simpler guide RNA design. This platform can also accelerate the development of biological agents and support high-throughput screening applications essential to the progress of agricultural biotechnology.

SUMMARY: Fusarium oxysporum is a significant threat to agriculture and One Health, requiring advanced molecular tools for functional genomics analyses and biological control agent development. Existing gene-editing methods are hampered by costly protoplast preparation protocols and by CRISPR/Cas9 limitations, such as restricted PAM sequences and complex guide RNA requirements. We engineered an efficient CRISPR/Cpf1 system that overcomes these issues through three main innovations: small-scale protoplast generation using novel filter columns that greatly reduces enzyme consumption while simplifying workflows, a CRISPR/Cpf1 system with flexible PAM recognition and staggered DNA cleavage to promote homologous recombination, and minimal homology arm strategies that significantly decrease cloning complexity. Extensive validation confirms successful gene targeting with molecular verification and functional analysis via standardized pathogenicity assays. This integrated platform offers affordable, accessible tools for systematic F. oxysporum research, enhancing fundamental understanding of plant-pathogen interactions and supporting high-throughput screening vital for agricultural biotechnology and biological agent development. MOTIVATION: Fusarium oxysporum plays crucial roles as both a damaging plant pathogen and a model for studying host-pathogen interactions. It is well established that systematic functional genomics approaches are vital for advancing agricultural biotechnology and developing biological agents. Creating efficient gene-editing systems can help elucidate virulence mechanisms and enable rapid production of modified strains for practical use. However, current transformation methods face major challenges, such as high enzyme costs, and CRISPR/Cas9 systems are limited by PAM sequence availability and by blunt-end DNA cleavage, which hampers homologous recombination. Furthermore, complex guide RNA scaffolds complicate large-scale functional studies with traditional methods. To address these challenges, we have developed a streamlined CRISPR/Cpf1 (Cas12a) platform that combines small-scale protoplast preparation with significantly reduced enzyme use, exploiting Cpf1’s unique features, such as flexible PAM recognition, staggered DNA cuts that promote recombination, improved target specificity, and simpler guide RNA design. This platform can also accelerate the development of biological agents and support high-throughput screening applications essential to the progress of agricultural biotechnology. HIGH LIGHTS: Reduced enzyme costs by 95% through small-scale protoplast preparation CRISPR/Cpf1 system established for efficient gene editing in Fusarium oxysporum Streamlined workflow enables routine gene targeting and rapid mutant screening Complete workflow validated with EGFP-marked pathogenicity

Efficient and sustainable food production is crucial in global agricultural development. Overuse of chemical fertilizers leads to soil acidification, destruction of soil properties, and harm to soil micro-organisms. Plant growth-promoting rhizobacteria (PGPR) have emerged as a solution, enhancing soil fertility and crop yields while reducing chemical fertilizer dependency and disease occurrence. In this study, Bacillus strains KHC2, KHH5, and KHH13, isolated from organic rice field soils in Taiwan, were identified through molecular techniques as B. velezensis (KHC2, KHH13) and B. amyloliquefaciens (KHH5). The strains exhibited various hydrolytic enzymes (including protease, cellulase, amylase, and lecithinase), with KHH13 showing the highest phosphate solubilization (2186.1 µg mL⁻¹ day⁻¹) and indole-3-acetic acid (IAA) production (63.067 ± 0.595 ppm mL⁻¹). These properties indicate KHH13’s potential as a bio-enhancer for plant growth. Therefore, we hypothesized that KHH13 can enhance plant growth and control soil-borne diseases. A greenhouse experiment demonstrated that KHH13, KHC2, and KHH5 effectively promoted the growth of red lettuce, with KHH13 showing superior efficacy. The study also found KHH13’s treatment enhanced the growth of various vegetables, including tomato, cucumber, and red lettuce. In terms of disease control, KHH13 significantly reduced Fusarium wilt in cucumbers, as evidenced by the reduction in disease index from 74.33% to 41.67% after KHH13 treatment. The treatment group displayed better plant growth, including plant height and fresh weight, compared to the control group in the greenhouse experiment. Furthermore, oral and pulmonary acute toxicity analysis in rats showed no adverse effects on rat weight or mortality, indicating KHH13’s safety for mammalian use. These findings suggest B. velezensis KHH13 as a safe, effective, and sustainable biological agent for enhancing vegetable growth and controlling soil-borne diseases, with potential applications in sustainable agriculture.

Melon (Cucumis melo L.) is a global commercial crop that is sensitive to seed-borne wilt infections caused by Fusarium oxysporum f. sp. melonis (Fom). To address the challenge of detecting Fom contamination, we designed a probe-based real-time PCR method, TDCP2, in combination with rapid or column-based DNA extraction protocols to develop reliable molecular detection methods. Utilizing TDCP2, the detection rate reached 100% for both artificially Fom-inoculated (0.25–25%) and pod-inoculated melon seeds in conjunction with DNA samples from either the rapid or column-based extraction protocol. We performed analyses of precision, recall, and F1 scores, achieving a maximum F1 score of 1 with TDCP2, which highlights the robustness of the method. Additionally, intraday and interday assays were performed, which revealed the high reproducibility and stability of column-based DNA extraction protocols combined with TDCP2. These metrics confirm the reliability of our developed protocols, setting a foundation for future enhancements in seed pathology diagnostics and potentially broadening their applicability across various Fom infection levels. In the future, we hope that these methods will reduce food loss by improving the control and management of melon diseases.

Red crown rot (RCR), caused by the soilborne fungus Calonectria ilicicola, is an emerging soybean disease in Taiwan, and fungicide screening is desired to identify effective management for C. ilicicola. This study screened 11 fungicides, including azoxystrobin, boscalid, cyprodinil, cyprodinil + fludioxonil, difenoconazole, fluopyram, flutolanil, mancozeb, prochloraz, pyraclostrobin, and tebuconazole, for their inhibitory effects on the mycelial growth of 10 C. ilicicola field isolates. Subsequently, a microplate-based high-throughput screening (MHTS) method was established to measure the fungicide sensitivity in a population composed of 80 C. ilicicola isolates to three effective fungicides, cyprodinil + fludioxonil, fluopyram, and tebuconazole. The MHTS was optimized for multiple factors, including the optical scanning pattern, absorption wavelength, conidial concentration, and measurement timing based on the quality controls of Z′ factor and the log-phase growth curve. The population mean EC₅₀ estimated by MHTS were 0.14, 2.34, and 2.46 ppm to cyprodinil + fludioxonil, fluopyram, and tebuconazole, respectively. In addition to the in vitro assessment, fungicide efficacy was evaluated by coating cyprodinil + fludioxonil, fluopyram, or tebuconazole on soybean seeds in the pot assay. The results showed that cyprodinil + fludioxonil significantly reduced both postemergence damping-off and disease severity, while fluopyram and tebuconazole reduced only the postemergence damping-off but not disease severity. Based on the MHTS and the pot assay results, this study demonstrated cyprodinil + fludioxonil to be a potential fungicide to manage soybean RCR.

Brown blight, a destructive foliar disease of tea, has become a highly limiting factor for tea cultivation in Taiwan. To understand the population composition of the causal agents (Colletotrichum spp.), the fungal diversity in the main tea-growing regions all over Taiwan was surveyed from 2017 to 2019. A collection of 139 Colletotrichum isolates was obtained from 14 tea cultivars in 86 tea plantations. Phylogenic analysis using the ribosomal internal transcribed spacer, glutamine synthetase gene, Apn2-Mat1-2 intergenic spacer, β-tubulin, actin, calmodulin, and glyceraldehyde-3-phosphate dehydrogenase genes together with morphological characterization revealed three species associated with brown blight of tea; namely, Colletotrichum camelliae (95.6% of all isolates), C. fructicola (3.7%), and C. aenigma (0.7%). This is the first report of C. aenigma in Taiwan. The optimal growth temperatures were 25°C for C. camelliae and 25 and 30°C for C. fructicola and C. aenigma. Although C. fructicola and C. aenigma were more adapted to high temperature, C. camelliae was the most pathogenic across different temperatures. Regardless of whether spore suspensions or mycelial discs were used, significantly larger lesions and higher disease incidences were observed for wounded than for nonwounded inoculation and for the third and fourth leaves than for the fifth leaves. Wounded inoculation of detached third and fourth tea leaves with mycelial discs was found to be a reliable and efficient method for assessing the pathogenicity of Colletotrichum spp. within 4 days. Preventive application of fungicides or biocontrol agents immediately after tea pruning and at a young leaf stage would help control the disease.