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Diversity and risk assessment of endophytic fungi from Cirsium kawakamii and the preliminary test on control of Fusarium wilt of lettuce
|關鍵字:||玉山薊;內生真菌;萵苣萎凋病;風險評估;Neofabraea屬;Fusarium屬;endophytic fungi;Cirsium kawakamii;Fusarium wilt of lettuce;Fusarium;Neofabraea||引用:||Aly, A.H., Debbab, A., and Proksch, P. 2011. Fungal endophytes: unique plant inhabitants with great promises. Applied Microbiology and Biotechnology 90:1829-1845. Anonymous. 2014. Agricultural statistics annual report. Taiwan Taipei: Council of Agriculture, Executive Yuan. Araújo, W.L., Maccheroni Jr, W., Aguilar-Vildoso, C.I., Barroso, P.A., Saridakis, H.O., and Azevedo, J.L. 2001. Variability and interactions between endophytic bacteria and fungi isolated from leaf tissues of citrus rootstocks. Canadian Journal of Microbiology 47:229-236. Arnold, A., Maynard, Z., Gilbert, G., Coley, P., and Kursar, T. 2000. Are tropical fungal endophytes hyperdiverse? Ecology Letters 3:267-274. Arnold, A.E. 2007. Understanding the diversity of foliar endophytic fungi: progress, challenges, and frontiers. Fungal Biology Reviews 21:51-66. 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Proceedings of the National Academy of Sciences 109:13022-13027.||摘要:||
本研究調查台灣特有種植物，玉山薊內生真菌多樣性與影響因子，並篩選具有防治萵苣萎凋病潛力之內生真菌及評估玉山薊中內生真菌具病原性之風險。自塔塔加、合歡山及埔里地區採集玉山薊，共分離出1,836株內生真菌，可分2個門、8個綱40個科68個屬，以子囊菌門，Sordariomycetes綱之Colletotrichum、Xylaria及Fusarium屬真菌為主要菌相。多樣性指數Fisher's α與Shannon指數分別為13.95與2.66，豐富度指數Chao 1、Chao 2、摺刀法 (First-order Jackknife richness)、二階摺刀法 (Second-order Jackknife richness)及Bootstrap值介於76.00 – 96.99。此外，以Bootstrap estimator所繪製出屬的累積曲線未達水平式漸進曲線，指出本研究實際所分離之玉山薊內生菌屬較預估可能含有屬數量少。綜合多樣性指數、豐富度指數及屬的累積曲線圖顯示，玉山薊具高多樣性與豐富度之內生菌相。比較合歡山、塔塔加與埔里等地區內玉山薊內生真菌之多樣性與豐富度指數，指出塔塔加地區內的菌相最高，合歡山地區內的菌相最低。分析上述三地區間內生真菌菌相之相似度，以塔塔加與埔里地區的菌相相似度最高，合歡山與埔里地區最低。比較埔里地區夏季與冬季之玉山薊內生真菌菌相，顯示夏季的菌相較多樣且豐富；於菌相相似度分析結果，兩季節之間相似度低於地域之間相似度，指出季節為主要影響內生真菌菌相之因子。篩選具有防治萵苣萎凋病潛力之內生真菌試驗中，測試之1,145株菌株中有174株菌株具有拮抗能力。進一步挑撰Fusarium屬中的CKK6059菌株進行萵苣萎凋病防治試驗，指出以浸根接種的方式將CKK6059菌株導入萵苣後，具延緩萵苣萎凋病的病勢進展的效果，然對萵苣生長會有抑制現象；反之，將CKK6059菌株接種於玉山薊後，可促進玉山薊之生長。測試玉山薊內生真菌中CKK6059菌株與Neofabraea屬真菌，對作物之病原性。指出CKK6059菌株可引起馬鈴薯乾腐病、蘋果果腐病及小麥赤黴病，且不同品種之蘋果對CKK6059菌株感病性不同，由高至低感受性之蘋果品種依序為史密斯蘋果、富士、五爪及加拉蘋果。以EF1-α核苷酸序列分析分子親緣性，指出與CKK6059菌株同屬Fusarium之其他內生真菌菌株，具有分子多樣性。Neofabraea屬真菌依形態學與ITS核苷酸序列分析顯示，Neofabraea屬真菌可分為三群，於分子群Ⅰ挑選CKK2013菌株進行初步接種測試，指出CKK2013菌株可引起蘋果與梨的牛眼病，進一步於三個分子群當中各挑兩株菌株，以史密斯青蘋果進行接種試驗，評估不同分子群間之毒力差異，接種試驗結果指出分子群Ⅰ毒力最強，分子群Ⅱ毒力最弱。
The objectives of this study were to A) analyze the diversity of endophytic fungi in Cirsium kawakamii Hayata, a herb indigenous in Taiwan, B) observe the effect of geographical and seasonal factors on endophytic fungi, C) screen the potential biocontrol agents of endophytic fungi in controlling the Fusarium wilt of lettuce and D) evaluate the risk of endophytic fungi as potential plant pathogens. In this study, a total 1836 endophytic fungi isolates were isolated from C. kawakamii plants collected from Puli Township, Tatachia, and Hehuanjian area. All these isolates were classified into 2 phylum, 8 classes, 40 families, 68 genera, and Colletotrichum, Xylaria, and Fusarium were the dominant genera, which belong to Sordariomycetes, Ascomycota. The EstimateS software was also used in this study to evaluate the diversity and richness of endophytic fungi in C. kawakamii. The Fisher's α and Shannon diversity indices were 13.95 and 2.66 respectively, and the richness indices of Chao 1, Chao 2, First-order Jackknife richness, Second-order Jackknife richness, and bootstrap value were between 76.00 to 96.99. Moreover, the genera accumulation curve remains non-asymptotic according to the bootstrape estimator, and the estimated richness significantly exceeds the richness captured by our sampling to date. Therefore, the endophytic fungi in C. kawakamii are highly diversity and richness, in addition, the diversity and richness of endophytic fungi showed the highest indices in Tatachia than the other two places, and the diversity of endophytic fungi in summer was higher than in winter at Puli Township area. The similarity indices of endophytic fungi showed the highest value between Puli Township and Tatachia. To evaluate the effect of geographical and seasonal factors on diversity and richness indices of endophytic fungi, the result indicated that the season was the key factor. The bioactive test showed that 174 of 1145 isolates showed antifungal ability against the mycelial growth of Fusarium oxysporum f. sp. lactucae race 3 Fo-10. Among these isolates, the Fusarium sp. was chosen for future study. The isolate CKK6059 showed the best inhibition ability in plate assay. Therefore, the isolate CKK6059 was used as a biocontrol agent in controlling Fusarium wilt of lettuce in greenhouse experiment. The result showed that the isolate CKK6059 could delay the disease progressing and decrease the disease severity with 18.4% at 28 days after inoculated into lettuce plants by root-dipping method. Moreover, the isolate CKK6059 showed the ability in promoting the plant growth in C. kawakamii, but with negative result in lettuce. In the risk assessment, the isolate CKK6059 and the isolates of Neofabraea spp. were chosen to evaluate the pathogenicity on crops. The results of pathogenicity test indicated that the CKK6059 could cause potato dry rot, apple fruit rot, and Fusarium head blight on wheat. Furthermore, the susceptibility of different apple cultivars from high to low was Granny Smith, Fuji, Red delicious, and Gala, respectively. The phylogeny analysis result indicated that the endophytic fungi of Fusarium sp, in C. kawakamii were divided into several groups and might belong to different species. On the other hand, the isolates of Neofabraea spp, could be separated into three groups based on the morphology and the phylogeny analysis results of ITS region. In preliminary test, the isolate CKK2013 (groupⅠ) was proved with the pathogenicity and caused the bull's rot disease on apple and pear. In addition, each two isolates form these three phylogeny groups were chosen randomly and for evaluating their virulence on Granny Smith apple. The results indicated that the groupⅠhad the highest virulence and groupⅡwas the lowest virulence on apple.
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