宜蘭三星蔥主要葉部病害生物防治菌之開發

Development of biological control agent against the major foliar pathogens of Welsh onion in Sanxing, Taiwan

王建元(Jian-Yuan Wang)

指導教授 ： 歐海仁

國立臺灣大學/生物資源暨農學院/植物病理與微生物學研究所/碩士(2022年)

https://doi.org/10.6342/NTU202201078

摘要

在台灣，青蔥(Allium fistulosum L.）作為重要蔬菜作物之一，長期受到由Stemphylium vesicarium 引起的黑腐病和Colletotrichum屬引起的炭疽病等葉部病害的影響。而當前以化學防治為主的方式卻不能有效地防治病原菌。近年來，植物內生菌因其潛在的防治效果成為了生物防治研究的焦點，但在台灣缺少青蔥相關的內生菌文獻。因此，本研究旨在識別與健康青蔥植物葉部和根部相關的內生菌，並篩選出具拮抗活性的有益微生物以作為青蔥葉部病害的防治手段。本研究從健康的青蔥葉子及根部中分離出150個細菌分離株和61個真菌菌株。基於真菌（ITS）和細菌(16S rRNA) 的 DNA序列，將211株分為16個真菌和47個細菌分類群。細菌大多屬於Bacillus、Burkholderia和Klebsiella三屬，而真菌則多隸屬於Fusarium、Colletotrichum和Chaetomium 三屬。透過體外和盆栽實驗進行初步評估，篩選出三個細菌菌株（GFB08，LFB28和Ba01）和兩個真菌菌株（GFF06和GFF08）。進一步利用gyrA、groEL、 polC、 purH、 rpoB 和 16S rRNA的序列對細菌及利用ITS、LSU和tef1-α對真菌內生菌進行的親緣關係分析，將這些菌株鑑定為Bacillus velezensis（GFB08、Ba01和 LFB28），Fusarium fujikuroi GFF06和 Chaetomium afropilosum GFF08。評估菌株的生物防治潛力上，包括定性的酵素測試(溶磷、蛋白酶，幾丁質酶，β-葡萄糖苷酶，澱粉酶，纖維素酶)，和在植物上拮抗病原菌Colletotrichum spaethianum SX15-2（Cs）和 Stemphylium vesicarium SX20-2 (Sv) 的能力。Bacillus velezensis GFB08在盆栽試驗中顯示出最好植物保護效果，因而選為本研究生物防治候選菌株。在防治機制的探討中，發現 B. velezensis具有至少三種抗菌胜肽的基因（srfAA, ituC, fenD），並可分泌纖維素酶和蛋白酶等胞外水解酶。透過接種後重新分離菌株的方式證明了GFB08的確可作為青蔥的葉部內生菌。最終抑制青蔥葉部病害的實驗中，發現預防性地施用 GFB08 對C. spaethianum 造成炭疽病具抑制效果，而預防性及治療性施用 GFB08 都對S. vesicarium造成的黑腐病有抑制效果。綜合以上，從青蔥中分離出具防治潛力的內生菌菌株，可望應用於後續田間的防治策略發展。

關鍵字

貝萊斯芽孢桿菌 ； 生物防治 ； 葉部病害防治 ； 葉部內生菌 ； 青蔥

並列摘要

Welsh onion (Allium fistulosum L.) is one of the important and oldest vegetable crops grown in Taiwan. However, Welsh onion leaves are subject to various fungal foliar diseases such as Stemphylium leaf blight caused by Stemphylium vesicarium and anthracnose caused by Colletotrichum species. Besides, the current approaches to control foliar disease such as chemical fungicides are not completely effective against foliar pathogens of Welsh onion. Recently, endophytes with biocontrol efficacy have drawn researchers’ attention, however, the information on endophytes associated with Welsh onions is lacking in Taiwan. Therefore, this study was designed to recognize cultivable endophytes associated with the phyllosphere and rhizosphere of healthy Welsh onion plants that are showing promising antagonistic activity against the major foliar pathogenic fungi. A total of 150 bacterial and 61 fungal strains were isolated from the root and leaf of healthy Welsh onion by using a culture-dependent approach DNA sequence data based on ITS (fungi) and 16S rRNA (bacteria) initially grouped the endophytes into 16 fungal and 47 bacterial taxa. The most abundant bacterial genera were Bacillus, Burkholderia, and Klebsiella, and the fungal genera were Fusarium, Colletotrichum, and Chaetomium. Evaluation of the endophytic isolates for biocontrol potential based on in vitro and pot experiments resulted in the selection of three bacterial strains (GFB08, LFB28, and Ba01) and two fungal strains (GFF06 and GFF08) as promising isolates for further evaluation. The selected strains were further identified up to the species based on Maximum likelihood phylogeny using combined gyrA, groEL, polC, purH, rpoB, 16S rRNA and ITS, LSU, plus tef1-α sequences data for bacteria and fungi, respectively. Based on the results of multi-gene phylogeny, three bacterial strains were identified as B. velezensis (GFB08, Ba01, and LFB28), while two fungal strains were recognized as Fusarium fujikuroi GFF06 and Chaetomium afropilosum GFF08, respectively. In addition, these five strains were screened for their biocontrol traits including extracellular enzyme activities phosphate solubilization, protease, chitinase, β-glucosidase, amylase, lipase, and antagonist ability under greenhouse conditions against two major foliar pathogens of Welsh onion Colletotrichum spaethianum (Cs) SX15-2 and Stemphylium vesicarium (Sv) SX20-2. Among the selected five strains, B. velezensis strain GFB08 exhibited the highest mycelial inhibition under in vitro conditions and provided the highest protection against foliar pathogens C. spaethianum SX15-2 and S. vesicarium. Therefore, B. velezensis GFB08 was further selected to understand the mechanism underlying the bio-controlling efficacy. The presence of three antimicrobial peptide genes (srfAA, ituC, fenD, bamC, bmyB, mycC, dfnA, mlna, and baer) and secretion of several cell wall degrading enzymes including cellulase and protease confirmed the antifungal nature of B. velezensis GFB08. Re-isolation experiment conducted using the resistant strain of B. velezensis GFB08 further confirms the endophytic nature of the B. velezensis GFB08 in healthy Welsh onion plants. The results of preventative and curative assays concluded that B. velezensis GFB08 showed preventive efficacy in terms of suppression of anthracnose caused by C. spaethianum while showing both preventive and curative effects on Stemphylium leaf blight caused by S. vesicarium. Overall, the current study indicated that the Welsh onion plant harbors various species of endophytic fungi and bacteria that possess excellent biocontrol activities. Hence, these isolates can be further formulated and used for field application.

並列關鍵字

Bacillus velezensis ； biological control ； foliar disease management ； foliar endophytes ； Welsh onion

參考文獻

Abdallah, D. B., Frikha-Gargouri, O., and Tounsi, S. (2018). Rizhospheric competence, plant growth promotion and biocontrol efficacy of Bacillus amyloliquefaciens subsp. plantarum strain 32a. Biolgical Control, 124. 61–67.

Adeleke, R. A., Nunthkumar, B., Roopnarain, A., and Obi, L. (2019). Applications of plant–microbe interactions in agro-ecosystems (pp. 1–34). In Kumar, V., Prasad, R., Kumar, M., Choudhary, D. K. (eds). Microbiome in Plant Health and Disease, Springer, Berlin, Germany.

Afzal, I., Shinwari, Z. K., Sikandar, S., and Shahzad, S. (2019). Plant beneficial endophytic bacteria: Mechanisms, diversity, host range and genetic determinants. Microbiological Research, 221, 36–49.

Ahluwalia, V., Kumar, J., Rana, V. S., Sati, O. P., and Walia, S. (2015). Comparative evaluation of two Trichoderma harzianum strains for major secondary metabolite production and antifungal activity. Natural Product Research, 29(10), 914–920.

Ahmed, E., and Holmström, S. J. (2014). Siderophores in environmental research: roles and applications. Microbial Biotechnology, 7(3), 196–208.