喷施菌剂后葡萄叶片真菌多样性的PCR-DGGE分析
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摘要
为探讨葡萄叶片喷施砖红微杆菌Y24菌剂和解淀粉芽胞杆菌N22菌剂后其表面真菌多样性的变化,本研究采用PCR-DGGE技术,以样品的基因组DNA为模板,扩增出大小约为400 bp的真菌18S rDNA高变区,DGGE电泳后进行图谱分析,并对标记条带进行切胶、回收、克隆和测序以研究真菌多样性相关的特征数和菌群组成。DGGE图谱分析表明,喷施砖红微杆菌Y24菌剂的叶片样品的丰富度指数S最高,香农-维纳指数H′最大,喷施解淀粉芽胞杆菌N22菌剂后的这两个指标与对照相比无明显变化。对DGGE图谱上标记出的25个条带进行测序比对,发现葡萄叶片上的真菌主要为非可培养链格孢(Uncultured Alternaria alternata)、枯叶格孢腔菌Pleospora herbarum、近平滑假丝酵母Candida parapsilosis和非可培养真核生物等。喷施砖红微杆菌Y24菌剂的特有的真菌类群包括非可培养真菌、隐球菌属Cryptococcussp.和非可培养真核生物。葡萄叶片喷施砖红微杆菌Y24菌剂后附生真菌种类变得更丰富,喷施解淀粉芽胞杆菌N22菌剂后与对照无显著差别。
PCR-DGGE technology was used to explore the variation of fungal diversity on the surface of grape leaves after spraying Microbacterium testaceum Y24 and Bacillus amyloliquefaciens N22 agents. The 18 S rDNA high variation area of the fungus with the size of 400 bp was amplified with the sample genome DNA as the template. After DGGE, the map and the marker strips were analyzed. The bands were cut, reclaimed, cloned and sequenced to determine the characteristic value and composition of fungal diversity. The DGGE map analysis showed that the species richness, Shannon Wiener index of the leaf samples spraying Y24 was the highest, and the two indexes of samples spraying N22 was not significantly changed compared with those of the control. The sequence alignment of 25 bands marked on the DGGE map showed that the fungi on the grape leaves were mainly unculturable Alternaria alternata, Pleospora herbarum, Candida parapsilosis and unculturable eukaryotes. The specific fungal groups of leaf spraying Y24 were unculturable fungi, Cryptococcus sp. and unculturable eukaryotes. The fungal diversity on leaves treated with Y24 was more abundant than the control and N22 agent, and there was no difference between N22 and control.
PCR-DGGE technology was used to explore the variation of fungal diversity on the surface of grape leaves after spraying Microbacterium testaceum Y24 and Bacillus amyloliquefaciens N22 agents. The 18 S rDNA high variation area of the fungus with the size of 400 bp was amplified with the sample genome DNA as the template. After DGGE, the map and the marker strips were analyzed. The bands were cut, reclaimed, cloned and sequenced to determine the characteristic value and composition of fungal diversity. The DGGE map analysis showed that the species richness, Shannon Wiener index of the leaf samples spraying Y24 was the highest, and the two indexes of samples spraying N22 was not significantly changed compared with those of the control. The sequence alignment of 25 bands marked on the DGGE map showed that the fungi on the grape leaves were mainly unculturable Alternaria alternata, Pleospora herbarum, Candida parapsilosis and unculturable eukaryotes. The specific fungal groups of leaf spraying Y24 were unculturable fungi, Cryptococcus sp. and unculturable eukaryotes. The fungal diversity on leaves treated with Y24 was more abundant than the control and N22 agent, and there was no difference between N22 and control.
引文
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[22]张世伟,陈曦,钟其顶.同品种酿酒葡萄表皮微生物群落多样性分析[J].生物技术通报,2017,33(3):128-137.
[23]张珣,李燕,刘晓.红地球葡萄健康叶片内生细菌多样性分析[J].植物病理学报,2017,47(1):107-116.
[24]马锋敏,杨利民,肖春萍.施用微生物菌剂对参后地土壤微生物功能多样性的影响[J].吉林农业大学学报,2015,37(3):317-322.
[25]雷先德,李金文,徐秀玲.微生物菌剂对菠菜生长特性及土壤微生物多样性的影响[J].中国生态农业学报,2012,20(4):488-494.
[26]刘长莉,李娜,王宝鑫.添加菌剂制作堆肥的肥效及菌群多样性分析[J].中国农学通报,2015,31(8):145-153.
[27]韩美哲,王小显,刘常宏.苏云金芽胞杆菌菌剂对棉花根际土壤细菌数量及多样性的影响[J].植物病理学报,2017,47(1):107-116.
[2]Susan S,Christen D.Bacteria in the leaf ecosystem with emphasis on pseudomonas syringae-apathogen,ice nucleus,and epiphyte[J].Microbiology and Molecular Biology Reviews,2000,64(3):624-653.
[3]Duarte S,B?rlocher F,Trabulo J,et al.Stream-dwelling fungal decomposer communities along a gradient of eutrophication unraveled by 454pyrosequencing[J].Fungal Divers,2015,70(1):127-148.
[4]Izhaki I,Fridman S,Gerchman Y J,et al.Variability of bacterial community composition on leaves between and within plant species[J].Current Microbiology,2013,66(3):227-235.
[5]国辉,毛志泉,刘训理.植物与微生物互作的研究进展[J].中国农学通报,2011,27(9):28-33.
[6]Spiegelman D,Whissell G,Greer C W.A survey of the methods for the characterization of microbial consortia and communities[J].Canadian Journal of Microbiology,2005,51(5):355-386.
[7]Matsumoto M,Sakamoto M,Hayashi H,et al.Novel phylogenetic assignment database for terminal-restriction fragment length polymorphism analysis of human colonic microbiota[J].Journal of Microbiology Methods,2005,61(3):305-319.
[8]Yong F W,Zhang F Q,Gu J D.Improvement of DGGE analysis by modifications of PCR protocols for analysis of microbial community members with low abundance[J].Applied Microbiology and Biotechnology,2014,98(12):5655-5663.
[9]Gianfranco R,Valeria M,Erica F,et al.Impact of alternative fungicides on grape downy mildew control and vine growth and development[J].Plant Disease,2016,100(4):739-748.
[10]蔡欣楠,李兴红,胡盼,等.几种杀菌剂和拮抗菌对葡萄白腐病的室内防治试验[J].中国农学通报,2014,30(16):295-298.
[11]王沛雅,王跃进,张建文,等.华东葡萄白河-35-1株系抗霜霉病基因cDNA文库构建及EST分析[J].农业生物技术学报,2009,17(2):294-300.
[12]陈浩,胡梁斌,唐春平,等.枯草芽胞杆菌B-FS01对葡萄霜霉病的防治效果[J].植物保护,2011,37(6):194-197.
[13]吉沐祥,姚克兵,缪康,等.生防菌剂对葡萄霜霉病菌抑菌活性及其田间防效评价[J].农业科学与技术,2016,17(7):1654-1657.
[14]郭继平,马光,朱会霞,等.葡萄霜霉病拮抗细菌N6的分离、筛选及鉴定[J].湖北农业科学,2014,53(17):4063-4065.
[15]崔贵青,王连君,姜楠,等.葡萄白腐病拮抗链霉菌G4的筛选、鉴定及发酵条件的优化[J].吉林农业大学学报,2012,34(2):147-151.
[16]齐牧遥,赵丹,王芬.混合拮抗菌防治葡萄白腐病培养基的优化[J].山西农业科学,2011,39(10):1102-1106.
[17]郭继平,马光,高小宽,等.葡萄霜霉病拮抗细菌N22的筛选鉴定及其防效初探[J].中国植保导刊,2016,36(4):9-14.
[18]Bloomfield S,Stewart G,Dodd C,et al.The viable but non-culturable phenomenon explained[J].Microbiology,1998,144(1):1-3.
[19]Rappe M S,Giovannoni S J.The uncultured microbial majority[J].Annual Review of Microbiology,2003,57:369-394.
[20]Steele H L,Streit W R.Metagenomics:advances in ecology and biotechnology[J].FEMS Microbiology Letters,2005,247(2):105-111.
[21]刘慧杰,杨彩云,田蕴.基于PCR-DGGE技术的红树林区微生物群落结构[J].微生物学报,2010,50(7):923-930.
[22]张世伟,陈曦,钟其顶.同品种酿酒葡萄表皮微生物群落多样性分析[J].生物技术通报,2017,33(3):128-137.
[23]张珣,李燕,刘晓.红地球葡萄健康叶片内生细菌多样性分析[J].植物病理学报,2017,47(1):107-116.
[24]马锋敏,杨利民,肖春萍.施用微生物菌剂对参后地土壤微生物功能多样性的影响[J].吉林农业大学学报,2015,37(3):317-322.
[25]雷先德,李金文,徐秀玲.微生物菌剂对菠菜生长特性及土壤微生物多样性的影响[J].中国生态农业学报,2012,20(4):488-494.
[26]刘长莉,李娜,王宝鑫.添加菌剂制作堆肥的肥效及菌群多样性分析[J].中国农学通报,2015,31(8):145-153.
[27]韩美哲,王小显,刘常宏.苏云金芽胞杆菌菌剂对棉花根际土壤细菌数量及多样性的影响[J].植物病理学报,2017,47(1):107-116.