分離自華蕉品種的內生細菌和真菌作為拮抗香蕉黃葉病菌熱帶型四號生理小種的生物防治劑

Abstract

香蕉（Musa spp.），是世界上最為重要的經濟和農業作物之一，然而幾種病害的發生，大大降低其產量。香蕉黃葉病，由土壤傳播病原菌Fusarium oxysporum f. sp. cubense（Foc）引起，為目前對香蕉生產最具威脅的病害。因對環境友善防治方法的需求與日俱增，尋找非化學的替代防治方法，如利用內生微生物作為生物防治製劑，為時勢所趨。因此，本研究旨在分離香蕉根部組織中的內生細菌和真菌，評估其作為香蕉黃葉病生物防治劑的潛力。從栽植對香蕉黃葉病菌熱帶型四號生理小種（Tropical race 4, Foc TR4）呈現中等抗性的兩華蕉品種「寶島蕉（Formosana）」及「台蕉5號（Tai-Chiao No. 5）」的有機香蕉園植株根部組織中，分離得到193株內生細菌和124株內生真菌，其中17株細菌和28株真菌分離株對Foc TR4具有拮抗活性。16株內生細菌經鑑定為Pseudomonas mosselii，P. chlororaphis， Serratia marcescens，及Bacillus mycoides；其中5株內生真菌經鑑定為Ceratobasidium sp.，Fusarium nematophilum，Trichoderma spirale，T. virens，以及Sarocladium kiliense 。進一步篩選這些內生菌產生吲哚-乙酸（IAA）及螯鐵蛋白（siderophore）的能力，顯示P. chlororaphis C3H2-L具有最高的活性，其次分別為P. mosselii AH2-E1，P. mosselii AH1-E1和S. marcescens AH2-B1，而B. mycoides C3H1-I及內生真菌則未測到IAA活性；而P. mosselii AH2-E1，S. marcescens AH2-B1、P. mosselii AH1-E1、P. chlororaphis C3H2-L，及S. kiliense C3H2-8，亦可檢測到螯鐵蛋白活性。此外，將10株內生菌接種至組織培養蕉苗，經由再分離確認其組織內生生長的能力；並且進一步利用GFP螢光質體標定P. mosselii AH1-E1及P. chlororaphis C3H2-L，確認其定殖香蕉根部組織的能力。為了評估這些內生菌生物防治的能力，選取7株內生菌接種至感病華蕉品種北蕉蕉苗根部，然後接種黃葉病菌，結果顯示內生細菌S. marcescens和Pseudomonas spp. 具有不錯的防治效果。總結，本研究發現具防治香蕉黃葉病潛力的內生細菌和真菌，未來可進一步評估其實際田間防治能力，以期應用於香蕉的商業化生產。

Banana (Musa spp.) is one of the most important economic and agricultural crops in the world. However, there are several diseases that can reduce the yield greatly. Panama disease or Fusarium wilt, caused by the soilborne pathogen Fusarium oxysporum f. sp. cubense (Foc), is one of the most destructive diseases that can reduce banana production drastically. Demand for eco-friendly safe control measures has encouraged researchers to find non-chemical alternatives, for example using endophytic microbes as biological control agents. Therefore, this study aims to investigate potential endophytic bacteria and fungi from banana plants and evaluate their potentials as biocontrol agents for Panama disease caused by tropical race 4 of Foc (Foc TR4). A total of 193 endophytic bacteria and 124 endophytic fungi were isolated from the root tissues of banana plants of two moderately resistant cultivars, Formosana and Tai-Chiao No. 5, at two organic fields, Pingtung, Taiwan. Among these endophytes, 17 bacterial isolates and 28 fungal isolates showed antagonistic activity against the growth of Foc TR4. Sixteen endophytic bacteria were identified and belonged to Pseudomonas mosselii, P. chlororaphis, Serratia marcescens and Bacillus mycoides; whereas 5 endophytic fungi were identified as Ceratobasidium sp. , Fusarium nematophilum, Trichoderma spirale, T. virens and Sarocladium kiliense. Ten endophytic isolates were further screened for their ability to produce indole-acetic acid (IAA) and siderophore. P. chlororaphis C3H2-L showed the highest activity in producing IAA and followed by P. mosselii AH2-E1, P. mosselii AH1-E1, and S. marcescens AH2-B1. We failed to detect IAA activity in B. mycoides C3H1-I and endophytic fungi. On the other hand, siderophore activities were positively detected in P. mosselii AH2-E1, S. marcescens AH2-B1, P. mosselii AH1-E1 and P. chlororaphis C3H2-L and S. kiliense C3H2-8. Furthermore, these 10 endophytes were inoculated onto banana tissue plantlets and re-isolation was conducted to confirm their ability of endophytic growth inside banana root tissue. In addition, GFP-tagged P. mosselii AH1-E1 and GFP-tagged P. chlororaphis C3H2-L were further created and used to monitor their abilities to colonize banana root tissue. To evaluate the biocontrol activities, 7 endophytes were selected, inoculated onto the roots of tissue culture plantlets of susceptible Cavendish banana cultivar Pei-Chiao, and then challenged with Foc TR4. Bacterial endophytes such as S. marcescens and Pseudomonas spp. showed promising protection results. In conclusion, our studies found candidate endophytic bacteria and fungi with biocontrol potential for Panama disease and field tests are needed to further evaluate their practical potentials for commercial banana production.