农林生态区土壤中的可培养芽胞杆菌资源调查——以四川省和重庆市部分地区为例
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摘要
调查了四川和重庆农林生态区土壤中的芽胞杆菌资源种群分布,为芽胞杆菌功能资源挖掘和菌剂开发提供基础。采用可培养法从农林生态区土壤中分离获得芽胞杆菌,利用细菌16S rRNA基因初步确定其分类地位,并通过多样性指数分析芽胞杆菌种群结构。结果表明,共分离获得芽胞杆菌95株,经16S rRNA基因鉴定,7个属38个种,其中芽胞杆菌属(Bacillus)22个种、赖氨酸芽胞杆菌属(Lysinbacillus)4个种、类芽胞杆菌属(Paenibacillus)7个种、短芽胞杆菌属(Brevibacillus)2个种、嗜冷芽胞杆菌属(Psychrobacillus)1个种、鲁梅尔芽胞杆菌属(Rummeliibacillus)1个种和虚构芽胞杆菌属(Fictibacillus)1个种。其中,6株芽胞杆菌与其最相近模式菌株的16S rRNA相似性低于98.65%,为潜在新种。农林生态区土壤芽胞杆菌多样性指数香农指数大小依次为乐山林地>盐亭县农地>重庆林地>盐亭县林地。以芽胞杆菌种类为样本,含量为指标,当欧式距离λ=15时,芽胞杆菌种类可分为高含量分布广型和低含量分布不均匀型。综上所述,农林生态区土壤蕴藏着较丰富的芽胞杆菌种类,为芽胞杆菌资源开发和利用提供了资源保障。
To provide a foundation for new resource mining of the Bacillus-like species and development of microbial agents,we investigated the species distribution and diversity of Bacillus-like isolates from soil samples in the agroforestry biome of Sichuan province and Chongqing city. Isolates Bacillus were obtained from the soil samples of agroforestry biome by cultural method,and identified based on 16 S rRNA gene sequences. The community structure of the Bacillus-like bacteria was analyzed using the diversity index. A total of 95 Bacilluslike isolates were obtained from the 5 soil samples,and identified as 38 species belonging to 7 genera within Bacillaceae by 16 S rRNA gene sequences. The species number of each genus was 22 for Bacillus,4 for Lysinibacillus,7 for Paenibacillus,2 for Brevibacillus,and 1 for Psychrobacillus,Rummeliibacillus and Fictibacillus respectively. And 16 S rRNA similarities between 6 isolates and their most-closed type species were lower than 98.65 % of species defined threshold,indicating that these 6 isolates might be potential new Bacillus species. The order of Shannon index of Bacillus from the agroforestry biome was as forest land in Leshan > farmland in Yanting County > forest land in Zhong County > forest land Yanting County,respectively. According to Bacillus-like species and content,as Euclidean distance was about 15,Bacillus-like species could be divided into 2 clusters,i.e. high-content wide-distribution and low-content uneven-distribution. Thus,there are rich Bacillus-like species in the agroforestry biome,which offers the guarantees for the development and utilization of Bacillus-like resources.
To provide a foundation for new resource mining of the Bacillus-like species and development of microbial agents,we investigated the species distribution and diversity of Bacillus-like isolates from soil samples in the agroforestry biome of Sichuan province and Chongqing city. Isolates Bacillus were obtained from the soil samples of agroforestry biome by cultural method,and identified based on 16 S rRNA gene sequences. The community structure of the Bacillus-like bacteria was analyzed using the diversity index. A total of 95 Bacilluslike isolates were obtained from the 5 soil samples,and identified as 38 species belonging to 7 genera within Bacillaceae by 16 S rRNA gene sequences. The species number of each genus was 22 for Bacillus,4 for Lysinibacillus,7 for Paenibacillus,2 for Brevibacillus,and 1 for Psychrobacillus,Rummeliibacillus and Fictibacillus respectively. And 16 S rRNA similarities between 6 isolates and their most-closed type species were lower than 98.65 % of species defined threshold,indicating that these 6 isolates might be potential new Bacillus species. The order of Shannon index of Bacillus from the agroforestry biome was as forest land in Leshan > farmland in Yanting County > forest land in Zhong County > forest land Yanting County,respectively. According to Bacillus-like species and content,as Euclidean distance was about 15,Bacillus-like species could be divided into 2 clusters,i.e. high-content wide-distribution and low-content uneven-distribution. Thus,there are rich Bacillus-like species in the agroforestry biome,which offers the guarantees for the development and utilization of Bacillus-like resources.
引文
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[18]葛慈斌,郑榕,刘波,等.武夷山自然保护区土壤可培养芽胞杆菌的物种多样性及分布[J].生物多样性, 2016, 24(10):1164-1176.
[19]龚国淑,张世熔,唐志燕,等.土壤芽胞杆菌分离方法的比较——以成都郊区土壤为例[J].中国农业科学, 2008, 41(11):3685-3690.
[20]刘敏,崔莹,黄惠琴,等.东寨港红树林土壤芽胞杆菌分离及其多样性分析[J].微生物学杂志, 2014, 34(5):21-26.
[21]宋敏,黄惠琴,刘敏,等.永兴岛土壤芽胞杆菌多样性与抗线虫活性分析[J].基因组学与应用生物学, 2015, 34(3):535-540.
[22]吕刚.河南省土壤中芽孢杆菌多样性研究[D].南京:南京农业大学, 2012.
[23]Kim M, Oh HS, Park SC, et al. Towards a taxonomic coherence between average nucleotide identity and 16S rRNA gene sequence similarity for species demarcation of prokaryotes[J]. Int J Syst Evol Microbiol, 2014, 64(2):346-351.
[24]骆婷,夏虹,冯定胜,等.森林土壤中多功能降解菌的分离筛选及鉴定[J].西南农业学报, 2018(5):1032-1040.
[2]刘琴英,刘国红,王阶平,等.四川海螺沟冰川土样芽胞杆菌资源分析[J].微生物学通报, 2018, 45(6):1237-1249.
[3]葛慈斌,刘波,车建美,等.武夷山地衣表生和内生芽胞杆菌种群的多样性[J].微生物学报, 2015, 55(5):551-563.
[4]张华勇,李振高,王俊华,等.红壤生态系统下芽孢杆菌的物种多样性[J].土壤, 2003, 35(1):45-47.
[5]马健华.若尔盖高原土壤芽孢杆菌遗传多样性研究及三株苏云金芽孢杆菌的特性分析[D].雅安:四川农业大学, 2011.
[6]胡容平,龚国淑,张洪,等.四川甘孜州折多山与雀儿山地区土壤细菌的研究[J].西南农业学报, 2010, 23(5):1565-1570.
[7]黄春萍,吴福忠,张健,等.高寒森林溪流微生物群落结构的季节性变化[J].生态学报, 2018, 38(1):298-308.
[8]关鹏.四川蒙顶山苏云金芽胞杆菌cry基因鉴定及其新型模式cry基因的克隆与表达[D].雅安:四川农业大学, 2010.
[9]胡桦,陈强,李登煜,等. 38株紫色土硅酸盐细菌的遗传多样性和分类地位[J].武汉大学学报:理学版, 2008, 54(2):239-243.
[10]刘国红,朱育菁,刘波,等.玉米根际土壤芽胞杆菌的多样性[J].农业生物技术学报, 2014, 22:1367-1379.
[11]Liu B, Liu GH, Sengonca C, et al. Bacillus solani sp. nov., isolated from rhizosphere soil of potato field in Xinjiang of China[J]. Int J Syst Evol Microbiol, 2015, 65:4066-4071.
[12]Kim OS, Cho YJ, Lee K, et al. Introducing EzTaxon-e:a prokaryotic 16S rRNA Gene sequence database with phylotypes that represent uncultured species[J]. Int J Syst Evol Microbiol,2012, 62:716-721.
[13]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]. Nucleic Acids Research, 1997, 25:4876-4882.
[14]Tamura K, Stecher G, Peterson D, et al. MEGA 6:molecular evolutionary genetics analysis 6. 0[J]. Molecular Biology and Evolution, 2013, 30:2725-2729.
[15]Saitou N, Nei M. The neighbor-joining method:a new method for reconstructing phylogenetic trees[J]. Molecular Biology and Evolution, 1987, 4:406-425.
[16]Jukes TH, Cantor CR. Evolution of protein molecules[M]//HN.Munro. Mammalian Protein Metabolism. New York:Academic Press, 1969:21-132.
[17]Felsenstein J. Confidence limits on phylogenies:an approach using the bootstrap[J]. Evolution, 1985, 39:783-789.
[18]葛慈斌,郑榕,刘波,等.武夷山自然保护区土壤可培养芽胞杆菌的物种多样性及分布[J].生物多样性, 2016, 24(10):1164-1176.
[19]龚国淑,张世熔,唐志燕,等.土壤芽胞杆菌分离方法的比较——以成都郊区土壤为例[J].中国农业科学, 2008, 41(11):3685-3690.
[20]刘敏,崔莹,黄惠琴,等.东寨港红树林土壤芽胞杆菌分离及其多样性分析[J].微生物学杂志, 2014, 34(5):21-26.
[21]宋敏,黄惠琴,刘敏,等.永兴岛土壤芽胞杆菌多样性与抗线虫活性分析[J].基因组学与应用生物学, 2015, 34(3):535-540.
[22]吕刚.河南省土壤中芽孢杆菌多样性研究[D].南京:南京农业大学, 2012.
[23]Kim M, Oh HS, Park SC, et al. Towards a taxonomic coherence between average nucleotide identity and 16S rRNA gene sequence similarity for species demarcation of prokaryotes[J]. Int J Syst Evol Microbiol, 2014, 64(2):346-351.
[24]骆婷,夏虹,冯定胜,等.森林土壤中多功能降解菌的分离筛选及鉴定[J].西南农业学报, 2018(5):1032-1040.