杨梅贮藏期真菌的分离鉴定及基于ITS序列的聚类分析
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摘要
目的分离幵鉴定杨梅贮藏期真菌。方法通过稀释涂布平板分离法以及划线纯化,从贮藏期杨梅果实中分离、纯化真菌菌种,幵结合传统形态学与ITS (internal transcribed spacer)全序列分析对其进行准确鉴定。结果最终鉴定出共14株真菌,其中1株毛霉(Mucorracemosus)、1株白地霉(Geotrichumcandidum)、1株腐皮壳属霉菌(Diaporthesp.)、1株好食链孢霉(Neurosporasitophila)、3株出芽短梗霉(Aureobasidium pullulans)、 4株酵母菌(Pichiakudriavzevii,Pichiaterricola,Hanseniasporaopuntiae,Saccharomycopsis crataegensis)以及3株青霉(Penicillium sp., Penicillium expansum, Penicillium herquei)。从进化关系上看有3类显示较近的亲缘关系,第1类为青霉属,第2类为出芽短梗霉,第3类为毕赤酵母。结论结合传统形态学与ITS全序列分析可对杨梅贮藏期真菌进行更为准确的鉴定。
Objective To separate and identify the fungi from bayberry in storage period. Methods The fungal strains were isolated and purified from bayberry during storage period by the spread plate method and streak plate method. And it was accurately identified by the traditional morphology and ITS(internal transcribed spacer) sequence analysis. Results A total of 14 strains of fungi were identified, including 1 strain of Mucor racemosus, 1 strain of Geotrichum candidum, 1 strain of Diaporthe sp., 1 strain of Neurospora sitophila, 3 strains of Aureobasidium pullulans, 4 strains of yeast(Pichia kudriavzevii, Pichia terricola, Hanseniaspora opuntiae, Saccharomycopsis crataegensis) and 3 strains of Penicillium(Penicillium sp., Penicillium expansum, Penicillium herquei). From the evolutionary relationship 3 categories showed close relationship. The first category was Penicillium, the second category was Aureobasidium pullulans, and the third was Pichia. Conclusion Combining traditional morphology and ITS full sequence analysis can more accurately identify fungi in storage of bayberry.
Objective To separate and identify the fungi from bayberry in storage period. Methods The fungal strains were isolated and purified from bayberry during storage period by the spread plate method and streak plate method. And it was accurately identified by the traditional morphology and ITS(internal transcribed spacer) sequence analysis. Results A total of 14 strains of fungi were identified, including 1 strain of Mucor racemosus, 1 strain of Geotrichum candidum, 1 strain of Diaporthe sp., 1 strain of Neurospora sitophila, 3 strains of Aureobasidium pullulans, 4 strains of yeast(Pichia kudriavzevii, Pichia terricola, Hanseniaspora opuntiae, Saccharomycopsis crataegensis) and 3 strains of Penicillium(Penicillium sp., Penicillium expansum, Penicillium herquei). From the evolutionary relationship 3 categories showed close relationship. The first category was Penicillium, the second category was Aureobasidium pullulans, and the third was Pichia. Conclusion Combining traditional morphology and ITS full sequence analysis can more accurately identify fungi in storage of bayberry.
引文
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[14]王琢,闫培生.真菌毒素产生菌的分子鉴定研究进展[J].中国农业科技导报,2010,12(5):42-50.Wang Z,Yan PS.Research Progress on molecular identification of mycotoxin producing fungi[J].J Agric Sci Technol,2010,12(5):42-50.
[15]李海波,吴学谦,王立武,等.青藏高原黄绿蜜环菌纯培养菌种的分离培养及分子鉴定[J].菌物学报,2008,27(6):873-883.Li HB,Wu XQ,Wang LW,et al.Pure culture isolation cultivation and molecular identification of Armillaria luteovirens from Tibet Plateau[J].Mycosystema,2008,27(6):873-883.
[16]王建波,张文驹,陈家宽.核rDNA的ITS序列在被子植物系统与进化研究中的应用[J].植物分类学报,1999,37(4):407-416.Wang JB,Zhang WJ,Chen JK.Application of ITS sequences of nuclear rDNA in phylogenetic and evolutionary studies of angiosperms[J].Acta Phytotax Sin,1999,37(4):407-416.
[2]袁兵兵,杨姗姗,陈静.微生物源槐糖脂对水果致腐真菌的抑制作用[J].应用与环境生物学报,2011,17(3):330-333.Yuan BB,Yang SS,Chen J.Antimicrobial activity of sophorolipids on pathogenic fungi isolated from fruits[J].Chin J Appl Environ Biol,2011,17(3):330-333.
[3]罗晓辉,朱心怡,陈晓青,等.不同成熟期杨梅果实微生物的多样性[J].浙江师范大学学报(自然科学版),2017,40(1):85-90.Luo XH,Zhu XY,Chen XQ,et al.The microbial diversity in different maturity stages of Chinese bayberry[J].J Zhejiang Norm Univ(Nat Sci Ed),2017,40(1):85-90.
[4]张宏,桂明英,张陶,等.云南杨梅贮藏期真菌病害研究[J].云南农业大学学报(自然科学版),2007,22(4):503-506.Zhang H,Gui MY,Zhang T,et al.Storage fungous diseases of Myrica rubra of Yunan[J].J Yunan Agric Univ,2007,22(4):503-506.
[5]戚行江,王连平,梁森苗,等.杨梅气调贮藏保鲜后果实真菌区系研究[J].浙江农业学报,2003,15(1):28-30.Qi XJ,Wang LP,Liang SM,et al.Study on the mycoflora in bayberry(Myrica rubra)fruits treated by controlled atmosphere storage[J].Acta Agric Zhejiangensis,2003,15(1):28-30.
[6]段爱莉,雷玉山,孙翔宇,等.猕猴桃果实贮藏期主要真菌病害的rDNA-ITS鉴定及序列分析[J].中国农业科学,2013,46(4):810-818.Duan AL,Lei YS,Sun XY,et al.Molecular identification and rDNA-ITSsequence analysis of fungal pahtogens in kiwifruit during storage[J].Sci Agric Sin,2013,46(4):810-818.
[7]周德庆.微生物学教程[M].北京:高等教育出版社,1993.Zhou DQ.Microbiology tutorial[M].Beijing:Higher Education Press,1993.
[8]沈萍,范秀容,李广武.微生物学实验[M].北京:高等教育出版社,1999.Shen P,Fan XR,Li GW.Experimental microbiology[M].Beijing:Higher Education Press,1999.
[9]江汉湖.食品微生物学[M].北京:中国农业出版社,2000.Jiang HH.Food of microbiology[M].Beijing:China Agriculture Press,2000.
[10]Thompson JD,Gibson TJ,Plewniak F,et al.The CLUSTAL-X windows interface:flexible strategies for multiple sequence alignment aided by quality analysis tools[J].Nucl Acid Res,1997,25(24):4876-4882.
[11]Koichiro T,Joel D,Masatoshi N,et al.MEGA4:molecular evolutionarygenetics analysis(MEGA)software version 4.0[J].Mol Biol Evolut,2007,24(8):1596-1599.
[12]魏景超.真菌鉴定手册[M].上海:上海科学技术出版社,1979.Wei JC.Handbook of identification of fungi[M].Shanghai:Shanghai Science and Technology Press,1979.
[13]Vakalounakis DJ,Fragkiadakis GA.Genetic diversity of Fusarium oxysporum isolates from cucumber:differentiation by pathogenicity,vegetative compatibility and RAPD fingerprinting[J].Phytopathology,1999,89(2):161-168.
[14]王琢,闫培生.真菌毒素产生菌的分子鉴定研究进展[J].中国农业科技导报,2010,12(5):42-50.Wang Z,Yan PS.Research Progress on molecular identification of mycotoxin producing fungi[J].J Agric Sci Technol,2010,12(5):42-50.
[15]李海波,吴学谦,王立武,等.青藏高原黄绿蜜环菌纯培养菌种的分离培养及分子鉴定[J].菌物学报,2008,27(6):873-883.Li HB,Wu XQ,Wang LW,et al.Pure culture isolation cultivation and molecular identification of Armillaria luteovirens from Tibet Plateau[J].Mycosystema,2008,27(6):873-883.
[16]王建波,张文驹,陈家宽.核rDNA的ITS序列在被子植物系统与进化研究中的应用[J].植物分类学报,1999,37(4):407-416.Wang JB,Zhang WJ,Chen JK.Application of ITS sequences of nuclear rDNA in phylogenetic and evolutionary studies of angiosperms[J].Acta Phytotax Sin,1999,37(4):407-416.