盘龙参内生真菌多样性及其拮抗作用研究
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摘要
为研究盘龙参内生真菌的多样性,并筛选具有抑菌活性的内生真菌。通过组织分离法分离重庆合川野生盘龙参内生真菌,利用ITS序列分析进行其多样性研究,并选用3种重要农作物病原菌进行平板对峙试验,最后对拮抗效果较好的菌株进行鉴定。结果表明:从盘龙参的根、茎、叶和花序中共分离出内生真菌48株,从中挑选出17株代表菌株,经ITS序列分析其分属于10个属,其中假埃希氏菌属真菌Pseudallescheria minutispora对番茄灰霉病菌有较好的拮抗作用,抑菌带达7.1 mm。本研究为盘龙参内生真菌的开发利用提供了基础资料,首次在国内详细描述了P. minutispora的形态学特征,为P. minutispora的分类鉴定提供依据。
This study aimed at the diversity and antifungal activity of endophytic fungi in wild Spiranthes sinensis(Pers.)Ames from Hechuan,Chongqing,China.The endophytic fungi from Spiranthes sinensis were col?lected with tissue isolation.The diversity was evaluated based on ITS sequence analysis.All isolates were tested for antagonistic effect on three major crop pathogens.The isolates with great antagonistic activity were identified.A total of 48 isolates were isolated from the root,stem,leaf and inflorescence of the plants.17 representative iso?lates were selected and found belonging to 10 fungal genera based on ITS sequences.The isolates of Pseud?allescheria minutispora showed the highest antagonistic activity against Botrytis cinerea from tomato(the zone of inhibition up to 7.1 mm).The diversity provides basic data for the utilization of endophytic fungi of Spiranthes si?nensis(Pers.)Ames.This study is the first description of P. minutispora with morphological characteristics in China,which lays a foundation for the classification and identification of P. minutispora.
This study aimed at the diversity and antifungal activity of endophytic fungi in wild Spiranthes sinensis(Pers.)Ames from Hechuan,Chongqing,China.The endophytic fungi from Spiranthes sinensis were col?lected with tissue isolation.The diversity was evaluated based on ITS sequence analysis.All isolates were tested for antagonistic effect on three major crop pathogens.The isolates with great antagonistic activity were identified.A total of 48 isolates were isolated from the root,stem,leaf and inflorescence of the plants.17 representative iso?lates were selected and found belonging to 10 fungal genera based on ITS sequences.The isolates of Pseud?allescheria minutispora showed the highest antagonistic activity against Botrytis cinerea from tomato(the zone of inhibition up to 7.1 mm).The diversity provides basic data for the utilization of endophytic fungi of Spiranthes si?nensis(Pers.)Ames.This study is the first description of P. minutispora with morphological characteristics in China,which lays a foundation for the classification and identification of P. minutispora.
引文
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[9]刘紫英.产黄酮成分的绶草内生真菌的鉴定[J].菌物学报,2011,30(1):133-137.Liu Z Y.Endophytic fungi producing flavonoids from Spiranthes sinensis[J].Institution of Microbiogy,2011,30(1):133-137.
[10]高宁,毕珊珊,胡凤,等.盘龙参内生真菌抑菌活性的初步研究[J].广东药学院学报,2011,27(4):367-369.Gao N,Bi S S,Hu F,et al.Antibacterial activity of endophytic fungi isolated from Sprianthes sinensis(Pers.)Ames[J].Journal of Guangdong College of Pharmacy,2011,27(4):367-369.
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[12]刘少华,陆金萍,朱瑞良,等.一种快速简便的植物病原真菌基因组DNA提取方法[J].植物病理学报,2005(4):362-365.Liu S H,Lu J P,Zhu R L,et al.A rapid and simple extraction method for plant pathogenic fungi[J].Acta Phytopathologica Sinica,2005(4):362-365.
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[14]伍晓丽,宋旭红,杨宏国,等.玄参内生真菌分离、鉴定及系统发育分析[J].中国中药杂志,2017,42(5):902-911.Wu X L,Song X H,Yang H G,et al.Identification and phylogenetic analysis of endophytic fungi isolated from Scrophularia ningpoensis[J].China Journal of Chinese Materia Medica,2017,42(5):902-911.
[15]Lahlali R,Hijri M.Screening,identification and evaluation of potential biocontrol fungal endophytes against Rhizoctonia solani AG3 on potato plants[J].Fems Microbiology Letters,2010,311(2):152-159.
[16]Ko W,Tsou Y,Ju M,et al.Production of a fungistatic substance by Pseudallescheria boydii isolated from soil amended with vegetable tissues and its significance[J].Mycopathologia,2010,169(2):125-131.
[17]王红,万喆,余进.波氏假阿利什霉复合种对4种抗真菌药物的敏感性研究[J].中国真菌学杂志,2015,10(2):96-99.Wang H,Wan Z,Yu J.In vitro susceptibility test of clinical isolates of Pseudallescheria boydii complex to four anti-fungus agents[J].Chinese Journal of Mycology,2015,10(2):96-99.
[18]张军林.小囊菌目和蛇口壳目真菌分类研究[D].杨凌:西北农林科技大学,2007.Zhang J L.Classification of microascales and ophiostomatales in China[D].Yanglin:Northwest A&F University,2007.
[19]Lackner M,de Hoog G S,Yang L,et al.Proposed nomenclature for Pseudallescheria,Scedosporium and related genera[J].Fungal Diversity,2014,67:1-10.
[2]国家中医药管理局《中华本草》编委会.中华本草[M].上海:上海科学技术出版社,1999.Editorial board of Traditional Chinese Medicine State Administration of Chinese Materia Medica.Chinese materia medica[M].Shanghai:Shanghai Science and Technology Press,1999.
[3]崔红花,罗兰,王淑美,等.盘龙参化学成分预实验[J].时珍国医国药,2012,23(2):359-361.Cui H H,Luo L,Wang S M,et al.Preliminary test for chemical components of Spiranthes sinensis[J].Lishizhen Medicine and Materia Medica Research,2012,23(2):359-361.
[4]崔红花,尹永芹,王淑美,等.盘龙参药材的质量标准研究[J].中国药房,2012,23(35):3310-3312.Cui H H,Yin Y Q,Wang S M,et al.Study on the quality standard of Spiranthes sinensis[J].China Pharmacy,2012,23(35):3310-3312.
[5]周秀玲,李家敏.药用植物盘龙参种子形态、萌发及贮藏的初步研究[J].种子,2012,31(6):79-82.Zhou X L,Li J M.Preliminary studies on shapes,germination and storage of Spiranthes sinensis seeds[J].Seed,2012,31(6):79-82.
[6]邢晓科.药用植物内生真菌资源:一个亟待开发的宝库[J].菌物学报,2018,37(1):14-21.Xing X K.Endophytic fungal resource of medicinal plants:a treasure need to be developed urgently[J].Institute of Microbiogy,2018,37(1):14-21.
[7]高宁.盘龙参内生真菌的分离鉴定及其化学成分相关性的研究[D].哈尔滨:黑龙江中医药大学,2009.Gao N.The study on identification of endophytic fungi isolated from Spiranthes sinensis(Pers)Ames and the chemical relationship among the fungi[D].Harbin:Heilongjiang University of Chinese Medicine,2009.
[8]刘紫英.濒危药用植物盘龙参根际真菌与内生真菌的多样性研究[J].宜春学院学报,2010,32(12):120-121.Liu Z Y.Study on the diversity of the rhizosphere fungi and the endophytic fungi of endangered and officinal plant Spiranthes sinensis(Pers)Ames[J].Journal of Yichun University,2010,32(12):120-121.
[9]刘紫英.产黄酮成分的绶草内生真菌的鉴定[J].菌物学报,2011,30(1):133-137.Liu Z Y.Endophytic fungi producing flavonoids from Spiranthes sinensis[J].Institution of Microbiogy,2011,30(1):133-137.
[10]高宁,毕珊珊,胡凤,等.盘龙参内生真菌抑菌活性的初步研究[J].广东药学院学报,2011,27(4):367-369.Gao N,Bi S S,Hu F,et al.Antibacterial activity of endophytic fungi isolated from Sprianthes sinensis(Pers.)Ames[J].Journal of Guangdong College of Pharmacy,2011,27(4):367-369.
[11]Paul N C,Deng J X,Sang H K,et al.Distribution of antifungal activity of endophytic fungi in different growth stages of chili pepper(Capsicum annuum L.)in Korea[J].The Plant Pathology Journal,2012,28(1):10-19.
[12]刘少华,陆金萍,朱瑞良,等.一种快速简便的植物病原真菌基因组DNA提取方法[J].植物病理学报,2005(4):362-365.Liu S H,Lu J P,Zhu R L,et al.A rapid and simple extraction method for plant pathogenic fungi[J].Acta Phytopathologica Sinica,2005(4):362-365.
[13]Gilgado F,Cano J,Gene J,et al.Molecular phylogeny of the Pseudallescheria boydii species complex:proposal of two new species[J].Journal of Clinical Microbiology,2005,43(10):4930-4942.
[14]伍晓丽,宋旭红,杨宏国,等.玄参内生真菌分离、鉴定及系统发育分析[J].中国中药杂志,2017,42(5):902-911.Wu X L,Song X H,Yang H G,et al.Identification and phylogenetic analysis of endophytic fungi isolated from Scrophularia ningpoensis[J].China Journal of Chinese Materia Medica,2017,42(5):902-911.
[15]Lahlali R,Hijri M.Screening,identification and evaluation of potential biocontrol fungal endophytes against Rhizoctonia solani AG3 on potato plants[J].Fems Microbiology Letters,2010,311(2):152-159.
[16]Ko W,Tsou Y,Ju M,et al.Production of a fungistatic substance by Pseudallescheria boydii isolated from soil amended with vegetable tissues and its significance[J].Mycopathologia,2010,169(2):125-131.
[17]王红,万喆,余进.波氏假阿利什霉复合种对4种抗真菌药物的敏感性研究[J].中国真菌学杂志,2015,10(2):96-99.Wang H,Wan Z,Yu J.In vitro susceptibility test of clinical isolates of Pseudallescheria boydii complex to four anti-fungus agents[J].Chinese Journal of Mycology,2015,10(2):96-99.
[18]张军林.小囊菌目和蛇口壳目真菌分类研究[D].杨凌:西北农林科技大学,2007.Zhang J L.Classification of microascales and ophiostomatales in China[D].Yanglin:Northwest A&F University,2007.
[19]Lackner M,de Hoog G S,Yang L,et al.Proposed nomenclature for Pseudallescheria,Scedosporium and related genera[J].Fungal Diversity,2014,67:1-10.