马衔山中国沙棘根瘤内共生细菌多样性研究
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摘要
采集甘肃省兰州市马衔山中国沙棘(Hippophae rhamnoidoes)根瘤样品,采用高通量测序和纯培养方法,分析中国沙棘根瘤内定殖的内共生细菌的组成、相对丰度和物种多样性,并比较两种方法研究结果的差异。在不同的微生物分类单元,高通量测序检测到中国沙棘根瘤内共生细菌24门50纲90目167科215属;而纯培养方法仅检测到3门5纲7目8科8属。进一步分析沙棘根瘤内共生细菌主要类群的相对丰度,发现高通量测序与纯培养方法的结果有明显差异,在科和属的分类单元上差异尤其显著。两种方法都表明中国沙棘根瘤内共生细菌具有丰富的多样性,但高通量测序能够较为全面的反映中国沙棘根瘤内共生细菌的群落组成,而纯培养方法仅能够分离到部分可培养的中国沙棘根瘤内共生细菌,在很大程度上极可能低估中国沙棘根瘤内共生细菌的物种组成并高估其丰度。
Root nodules of Hippophae rhamnoides were collected in the Maxian Mountains. The plate culture method and high-throughput sequencing were used to study the diversity of endophytic bacteria in the root nodules of Hippophae rhamnoides. Microbial composition,abundance,and diversity of the nodule endophytic bacteria were analyzed. The differences in the results of the two techniques were compared. The results showed that 24 phyla,50 classes,90 orders,167 families,and 215 genera of nodule endophytic bacteria were detected by high-throughput sequencing,and 3 phyla,5classes,7 orders,8 families,and 8 genera were obtained by plate culture. The relative abundances of the dominant nodule endophytic bacteria revealed that the differences between the results of the two techniques were greater at lower classification levels,especially at the family and genus levels,than at higher levels. Both plate culture and high-throughput sequencing showed an abundant diversity of nodule endophytic bacteria. High-throughput sequencing provides a more comprehensive and accurate picture of microbial community structure than the plate culture method does. Plate culture can only detect a few dominant taxa of nodule endophytic bacteria. The results of this study indicated that plate culture greatly underestimates microbial community composition and overestimate their abundance.
Root nodules of Hippophae rhamnoides were collected in the Maxian Mountains. The plate culture method and high-throughput sequencing were used to study the diversity of endophytic bacteria in the root nodules of Hippophae rhamnoides. Microbial composition,abundance,and diversity of the nodule endophytic bacteria were analyzed. The differences in the results of the two techniques were compared. The results showed that 24 phyla,50 classes,90 orders,167 families,and 215 genera of nodule endophytic bacteria were detected by high-throughput sequencing,and 3 phyla,5classes,7 orders,8 families,and 8 genera were obtained by plate culture. The relative abundances of the dominant nodule endophytic bacteria revealed that the differences between the results of the two techniques were greater at lower classification levels,especially at the family and genus levels,than at higher levels. Both plate culture and high-throughput sequencing showed an abundant diversity of nodule endophytic bacteria. High-throughput sequencing provides a more comprehensive and accurate picture of microbial community structure than the plate culture method does. Plate culture can only detect a few dominant taxa of nodule endophytic bacteria. The results of this study indicated that plate culture greatly underestimates microbial community composition and overestimate their abundance.
引文
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[29]钟宇舟,余秀梅,陈强,谭渊,刘汉军,陈翠平,李艳梅,赵燕.四川盆地大豆根瘤内生细菌的分离鉴定及促生效果.应用与环境生物学报,2017,23(1):46-53.
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[2]黄贵修,刘先宝.植物内生菌.北京:中国农业科学技术出版社,2013:282-290.
[3] Santoyo G,Moreno-Hagelsieb G,Del Orozco-Mosqueda M,Glick B R. Plant growth-promoting bacterial endophytes. Microbiological Research,2016,183:92-99.
[4]王志伟,纪燕玲,陈永敢.植物内生菌研究及其科学意义.微生物学通报,2015,42(2):349-363.
[5]王鹏,陈波,张华.基于高通量测序的鄱阳湖典型湿地土壤细菌群落特征分析.生态学报,2017,37(5):1650-1658.
[6]张爱梅,韩雪英,张世虎,孙坤,吴菊艳,于杰,郭保民.甘南高寒地区不同海拔西藏沙棘根瘤内生菌多样性.生物资源,2018,40(2):114-119.
[7]徐瑞瑞.沙棘弗兰克氏和非弗兰克氏放线菌的分离、培养及分子鉴定[D].北京:中国林业科学研究院,2011:40-60.
[8]李琦,孙广宇.沙棘内生菌的分离与初步鉴定.中国农学通报,2006,22(10):300-302.
[9]张爱梅,牛世全,孙坤,曹文侠,达文燕.沙棘属植物结瘤特性及Frankia根瘤菌分离.草原与草坪,2010,30(2):43-46.
[10]史应武,娄恺,李春.天山北坡甜菜内生菌分离鉴定及其动态变化.生态学报,2009,29(5):2374-2382.
[11]冯健玲,何新华,李一伟,李峰,秦荣耀.广西杨梅根瘤Frankia菌的分离和培养特性研究.西南农业学报,2012,25(1):236-239.
[12]阮继生,黄英.放线菌快速鉴定与系统分类.北京:科学出版社,2011:60-62.
[13]东秀珠,蔡妙英.常见细菌系统鉴定手册.北京:科学出版社,2001:353-390.
[14]李振高,骆永明,滕应.土壤与环境微生物研究法.北京:科学出版社,2008:146-201.
[15]冯广达,陈美标,羊宋贞,朱红惠.用于PCR扩增的细菌DNA提取方法比较.华南农业大学学报,2013,34(3):439-442.
[16]徐平,李文均,徐丽华,姜成林.微波法快速提取放线菌基因组DNA.微生物学通报,2003,30(4):82-84.
[17]孔凡洲,于仁成,徐子钧,周名江.应用Excel软件计算生物多样性指数.海洋科学,2012,36(4):57-62.
[18]彭源东,柯丹兵,张忠泽. Frankia基因组DNA提取及其PCR带型.微生物学杂志,1997,17(3):1-15.
[19]李志真.福建弗兰克氏菌(Frankia)研究[D].福州:福建农林大学,2002:46-47.
[20]姬洪飞,王颖.分子生物学方法在环境微生物生态学中的应用研究进展.生态学报,2016,36(24):8234-8243.
[21]刘志强,张利平.沙棘和沙枣根瘤中固氮放线菌物种多样性研究.安徽农业科学,2010,38(11):5533-5534,5536-5536.
[22] Yang T,Chen Y,Wang X X,Dai C C. Plant symbionts:keys to the phytosphere. Symbiosis,2013,59(1):1-14.
[23]张爱梅,孙坤,达文燕.马衔山两种不同生境沙棘根际微生物研究.西北师范大学学报:自然科学版,2008,44(1):69-73.
[24]王元贞,潘廷国,柯玉琴.微生物与植物共生关系的研究及其利用.中国生态农业学报,2003,11(3):95-98.
[25]张小民,王岚,林美珍,张吉科.论沙棘根系与功能Ⅱ——Frankia菌侵染和结瘤.沙棘,2006,19(1):1-11.
[26]刘洪章,齐洁,李玉.沙棘根系分布及根瘤研究.吉林农业大学学报,2005,27(6):631-633,638-638.
[27]曲东明,王双,韩善华.沙棘共生固氮根瘤及其内生弗兰克氏菌.西北植物学报,1998,18(1):60-65.
[28]陈立红,闫伟,梁金慧,李鹏飞.内蒙古沙棘共生菌Frankia的遗传多样性.西北植物学报,2006,26(12):2458-2462.
[29]钟宇舟,余秀梅,陈强,谭渊,刘汉军,陈翠平,李艳梅,赵燕.四川盆地大豆根瘤内生细菌的分离鉴定及促生效果.应用与环境生物学报,2017,23(1):46-53.
[30] Liotti R G,Da Silva Figueiredo M I,Da Silva G F,De Mendon9a E A F,Soares M A. Diversity of cultivable bacterial endophytes in Paullinia cupana and their potential for plant growth promotion and phytopathogen control. Microbiological Research,2018,207:8-18.
[31] Abbamondi G R,Tommonaro G,Weyens N,Thijs S,Sillen W,Gkorezis P,Iodice C,De Melo Rangel W,Nicolaus B,Vangronsveld J. Plant growth-promoting effects of rhizospheric and endophytic bacteria associated with different tomato cultivars and new tomato hybrids. Chemical and Biological Technologies in Agriculture,2016,3:1.