透過基因體學與代謝組體學評估 Bacillus velezensis GFB08 在青蔥上的抑病機制

Abstract

青蔥是台灣重要作物，廣泛應用於蔬菜與香料。近年來，新興青蔥葉部病原菌如 Stemphylium vesicarium, Colletotrichum spaethianum 與 C. circinans 在台灣大量發生並造成顯著損失。在我們先前的研究中，從健康青蔥葉部中分離出一株Bacillus velezensis GFB08 菌株，具有在實驗室與溫室條件下降低青蔥葉部病害的能力。本篇研究的主要目標為透過基因體學與代謝組體學評估 B. velezensis GFB08 在青蔥上的抑病機制。透過結合illumina與Nanopore定序進行基因體組裝，B. velezensis GFB08具有單一3.89 mb的染色體，其中包含3,735編碼序列，藉由比較基因體分析，GFB08具有完整且高度保守的fengycin及bacillomycin D二次代謝物基因簇。代謝物純化與質譜分析進一步證實GFB08產生的fengycin對三種青蔥葉部病原菌皆具有廣效的抗真菌活性，而bacillomycin D 則表現出物種專一性的抗真菌活性，特別是fengycin與bacillomycin D 在抑制C. spaethianum上呈現出加成的抗菌效果。此外，GFB08產生的揮發性有機化合物（VOCs），如乙酸、乙偶姻、1-丁醇與3-甲基丁醇，均能顯著抑制C. spaethianum的菌絲生長。為了評估GFB08的實際應用潛力，本研究同時進行了植物生長促進試驗、發酵條件最佳化與田間防治試驗，結果顯示GFB08 僅在種子時期能夠顯著促進青蔥生長。5 L發酵試驗成果顯示，GFB08的最適產孢條件為1% 糖蜜, 0.5% 大豆粉, 28°C, pH 7, 與200 rpm，內生孢子濃度可達2×109 CFU/mL。田間防治試驗中，B. velezensis GFB08 稀釋液100倍可抑制青蔥黑腐病的罹病嚴重度。整體而言，本研究為生物防治菌株B. velezensis GFB08 提供了功能上的參考依據，並揭示其在永續農業上的應用潛力。

The Welsh onion is an essential crop in Taiwan, widely consumed as a vegetable or spice. In recent years, foliar fungal pathogens Stemphylium vesicarium, Colletotrichum spaethianum, and C. circinans have been identified as emerging threats, causing significant damage to the Welsh onion in Sangxing, Taiwan. In our recent study, Bacillus velezensis GFB08 isolated from healthy Welsh onion leaves demonstrated the ability to reduce foliar diseases under laboratory and greenhouse conditions. The main objective of this study was to elucidate the disease suppression mechanisms of B. velezensis GFB08 through genomic and metabolomic approaches. Using Illumina and Nanopore hybrid assembly, the present study found that B. velezensis GFB08 genome harbored a 3.89 mb chromosome with 3,735 coding sequences. Comparative genomic analysis revealed complete and highly conserved biosynthetic gene clusters (BGCs) for fengycin and bacillomycin D. Metabolite profiling confirmed that fengycin produced by GFB08 exhibited broad-spectrum antifungal activity against all three foliar pathogens, while bacillomycin D displayed species-specific effects. Notably, fengycin and bacillomycin D displayed additive antifungal activity against C. spaethianum. Additionally, volatile organic compounds (VOCs) such as acetic acid, 2-pentanone, acetoin, 1-butanol and 3-methyl- were identified as major antifungal VOCs from GFB08, significantly inhibiting C. spaethianum growth under in vitro. To facilitate the practical application of GFB08, plant growth promotion assays, fermentation optimization, and field evaluations were also conducted. GFB08 enhanced Welsh onion growth only during seed stage. Based on 5 L fermentation optimization, B. velezensis GFB08 achieved highest endospore production under the following conditions: 1% molasses, 0.5% soybean flour, 28°C, pH 7, and 200 rpm, reaching a concentration of 2×109 CFU/mL. Field trials demonstrated that a 100-fold dilution of B. velezensis GFB08 effectively reduced the disease severity of Welsh onion leave blight. Overall, this study provides a comprehensive functional characterization of the biocontrol agent B. velezensis GFB08 and highlight its potential for sustainable agriculture.