一株油菜菌核病拮抗真菌的鉴定及生物学特性研究
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摘要
从兰科植物羊耳蒜根部分离并筛选对油菜菌核病菌Sclerotinia sclerotiorum具有较强拮抗作用的内生真菌,确定其分类地位,并对其体外培养特性进行研究。利用常规PDA培养法进行内生真菌的分离、培养,采用平板对峙法筛选具有较强抑菌活性的菌株并对其进行种类鉴定,同时设定不同碳源、氮源、温度和p H值条件研究该菌株的生物学特性。通过筛选获得1株对油菜菌核病菌具有较强抑制作用的真菌菌株,结合形态学特征和ITS分子序列分析将其鉴定为多节孢属Nodulisporium sp.。该菌株对碳源要求不严格,以麦芽糖、果糖、葡萄糖和蔗糖为碳源均能良好生长。氮源对该菌株生长具有显著影响,最适氮源为牛肉膏。其最适培养温度为25℃,且温度低于10℃或高于30℃时菌丝生长均受到明显抑制。同时该菌株能耐受较广的酸碱度范围,在p H5-9范围内均生长良好。
Our aims are to isolate and select antagonistic endophytic fungi against Sclerotinia sclerotionum from the roots of Liparis japonica(Orchidaceae),to identify their taxonomic status and study the characteristics of in vitro culturing it. Conventional PDA culturetechnology was applied to isolate and culture endophytic fungi and plate confrontation method to screen those strains with strong antagonisticactivities against S. sclerotionum and to identify their taxonomic status,as well as the biological characteristics were studied under differentcarbon sources,nitrogen sources,temperature and p H values. One strongly antagonistic endophytic fungus against S. sclerotionum wereobtained and identified as Nodulisporium sp. by its morphological characters and ITS molecular sequences. The carbon sources that thisfungus relied on were diverse and it grew well by any of maltose,fructose,glucose and sucrose as carbon resource. There existed significantdifferences among nitrogen sources for this fungus' growth,and the optimal nitrogen source was beef extract. The optimal temperature was25℃,and the hypha was inhibited significantly when the temperature fell below 10℃ or rose above 30℃. This fungus also tolerated very widerange of p H,and grew well under p H 5 to 9.
Our aims are to isolate and select antagonistic endophytic fungi against Sclerotinia sclerotionum from the roots of Liparis japonica(Orchidaceae),to identify their taxonomic status and study the characteristics of in vitro culturing it. Conventional PDA culturetechnology was applied to isolate and culture endophytic fungi and plate confrontation method to screen those strains with strong antagonisticactivities against S. sclerotionum and to identify their taxonomic status,as well as the biological characteristics were studied under differentcarbon sources,nitrogen sources,temperature and p H values. One strongly antagonistic endophytic fungus against S. sclerotionum wereobtained and identified as Nodulisporium sp. by its morphological characters and ITS molecular sequences. The carbon sources that thisfungus relied on were diverse and it grew well by any of maltose,fructose,glucose and sucrose as carbon resource. There existed significantdifferences among nitrogen sources for this fungus' growth,and the optimal nitrogen source was beef extract. The optimal temperature was25℃,and the hypha was inhibited significantly when the temperature fell below 10℃ or rose above 30℃. This fungus also tolerated very widerange of p H,and grew well under p H 5 to 9.
引文
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[10]任安芝,高玉葆.植物内生真菌——一类应用前景广阔的资源微生物[J].微生物学通报,2001,28(6):90-93.
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[15]杨润亚,冯培勇,李清.植物内生真菌农药活性的研究进展[J].农药,2006,45(7):440-444.
[16]范黎.植物内生真菌次级代谢产物抗菌机制分析[J].微生物学通报,2016,43(7):1660.
[17]Rodriguez RJ,Jr WJ,Arnold AE,et al.Fungal endophytes:diversity and functional roles[J].New Phytologist,2009,182(2):314.
[18]郑启昌,赵欢,陈国栋,等.多节孢属真菌次生代谢产物研究进展[J].国际药学研究杂志,2013,40(6):721-725.
[19]王高乾,赵琴,郑启昌,等.多节孢属Nodulisporium sp.真菌中一个新的生物碱类化合物[J].中国中药杂志,2015,40(13):2598-2601.
[20]Wu ZC,Li DL,Chen YC,et al.A new isofuranonaphthalenone andbenzopyrans from the endophytic fungus Nodulisporium sp.A4 fromAquilaria sinensis[J].Helvetica Chimica Acta,2010,93(5):920-924.
[2]张建忠,绍兴华,肖红艳.油菜菌核病的发生与防治研究进展[J].南方农业学报,2012,43(4):467-471.
[3]师俊玲,堵国成,陈坚.盾壳霉在油菜菌核病菌生物防治中的应用[J].中国生物工程杂志,2003,23(4):27-31.
[4]马炳田,文成敬.几种核盘菌菌核重寄生真菌生物防治潜能的研究[J].中国农学通报,2002,18(6):38-63.
[5]杨立,陈美兰,邵爱娟,等.生防菌对药用植物土传病害的抗病机制及应用探讨[J].中国中药杂志,2012,37(21):3188-3192.
[6]王艳红,吴晓民,朱艳萍,等.温郁金内生真菌Chaetomium globosum L18对植物病原菌的抑菌谱及拮抗机理[J].生态学报,2012,32(7):2040-2046.
[7]丁婷,孙微微,王帅,等.杜仲内生真菌中抗玉米纹枯病活性菌株的筛选[J].植物保护,2014(6):29-35.
[8]赵欢.药用植物内生真菌的研究进展[J].北方园艺,2017(13):170-175.
[9]文才艺,吴元华,田秀玲.植物内生菌研究进展及其存在的问题[J].生态学杂志,2004,23(2):86-91.
[10]任安芝,高玉葆.植物内生真菌——一类应用前景广阔的资源微生物[J].微生物学通报,2001,28(6):90-93.
[11]姜怡,杨颖,陈华红,等.植物内生菌资源[J].微生物学通报,2005,32(6):146-147.
[12]石晶盈,陈维信,刘爱媛.植物内生菌及其防治植物病害的研究进展[J].生态学报,2006,26(7):2395-2401.
[13]Cenis JL.Rapid extraction of fungal DNA for PCR amplification[J].Nucleic Acids Research,1992,20(9):2380.
[14]White TJ,Bruns T,Lee S,et al.Amplification and direct sequencingof fungal ribosomal RNA genes for phylogenetics[M]//Innis MA,Gelfand DH,Sninsky JJ,et al.PCR Protocols:a guide to methodsand applications.USA:Academic Press,1990:315-322.
[15]杨润亚,冯培勇,李清.植物内生真菌农药活性的研究进展[J].农药,2006,45(7):440-444.
[16]范黎.植物内生真菌次级代谢产物抗菌机制分析[J].微生物学通报,2016,43(7):1660.
[17]Rodriguez RJ,Jr WJ,Arnold AE,et al.Fungal endophytes:diversity and functional roles[J].New Phytologist,2009,182(2):314.
[18]郑启昌,赵欢,陈国栋,等.多节孢属真菌次生代谢产物研究进展[J].国际药学研究杂志,2013,40(6):721-725.
[19]王高乾,赵琴,郑启昌,等.多节孢属Nodulisporium sp.真菌中一个新的生物碱类化合物[J].中国中药杂志,2015,40(13):2598-2601.
[20]Wu ZC,Li DL,Chen YC,et al.A new isofuranonaphthalenone andbenzopyrans from the endophytic fungus Nodulisporium sp.A4 fromAquilaria sinensis[J].Helvetica Chimica Acta,2010,93(5):920-924.