泡桐根瘤内生细菌的分离及初步鉴定
|
摘要
【目的】为了探究非豆科植物泡桐与微生物共生固氮体系,对泡桐根瘤内生细菌的分离培养条件及其系统发育地位进行初步研究。【方法】对从广西钦州市采集的泡桐根瘤上分离纯化得到的根瘤内生细菌,进行传统生理生化研究和16S rDNA系统发育分析。【结果】经分离纯化得到9株根瘤内生细菌,大部分菌株能较好地利用8种碳源;78%的菌株能利用铵盐,89%的菌株能利用硝酸盐;所有的供试菌株在3-酮基乳糖和淀粉水解试验中呈阴性;89%的菌株能利用柠檬酸盐;B.T.B反应除菌株PG-7产碱外,其余都产酸;33%的菌株能使硝酸盐还原;有56%的菌株能在牛肉膏蛋白胨培养基上生长良好,多数菌株具备豆科植物根瘤菌的一般生理生化特征。通过16S rDNA全序列分析,确定9株供试菌株可分为4个菌属,其中菌株PG-1和PG-2共属于土壤杆菌属(Agrobacterium),PG-3、PG-5、PG-6、PG-8和PG-9等5株菌株共属于根瘤菌属(Rhizobium),PG-4隶属于草螺菌属(Herbaspirillum),PG-7隶属于伯克氏菌属(Burkholderia)。【结论】分离自同一寄主的根瘤内生细菌其生理生化特性方面存在较大的差异性,同时表现出了遗传的多样性。
【Objective】In order to explore the symbiotic nitrogen fixation system of non-leguminous plants Paulownia and microorganism, a preliminary study was carried out on isolated culture conditions and the phylogenetic position of endophyic bacteria in Paulowia root nodules.【Method】The endophytic bacteria isolated and purified from the root nodules of Paulownia collected from Qinzhou of Guangxi Province were subjected to traditional physiological and biochemical studies and 16 S rDNA phylogenetic analysis.【Result】In the nine strains of endophytic bacteria isolated and purified, most of the bacterial strains could make good use of 8 carbon sources; 78% of the bacterial strains could use ammonium salt, and 89% of the bacterial strains could use nitrate. All tested bacterial strains were negative in the 3-ketolactose and starch hydrolysis test; 89% of the strains could use citrate. Except that the bacterial strains PG-7 produced alkali, the rest of bacterial strains produced acid in the B.T.B reaction; 33% of the strains could reduce nitrate; 56% of the strains grew well on beef extract peptone medium, and most strains have the general physiological and biochemical characteristics of leguminous rhizobium after identification. According to 16 S rDNA complete sequence analysis, nine bacterial strains can be divided into four bacteria genera, among which the bacterial stains PG-1 and PG-2 belong to Agrobacterium, PG-3, PG-5, PG-6, PG-8 and PG-9 belong to Rhizobium, PG-4 belongs to Herbaspirillum and PG-7 belongs to Burkholderia.【Conclusion】The endophytic bacteria isolated from the same root nodule have great differences in their physiological and biochemical characteristics of endogenous bacteria, and showed genetic diversity.
【Objective】In order to explore the symbiotic nitrogen fixation system of non-leguminous plants Paulownia and microorganism, a preliminary study was carried out on isolated culture conditions and the phylogenetic position of endophyic bacteria in Paulowia root nodules.【Method】The endophytic bacteria isolated and purified from the root nodules of Paulownia collected from Qinzhou of Guangxi Province were subjected to traditional physiological and biochemical studies and 16 S rDNA phylogenetic analysis.【Result】In the nine strains of endophytic bacteria isolated and purified, most of the bacterial strains could make good use of 8 carbon sources; 78% of the bacterial strains could use ammonium salt, and 89% of the bacterial strains could use nitrate. All tested bacterial strains were negative in the 3-ketolactose and starch hydrolysis test; 89% of the strains could use citrate. Except that the bacterial strains PG-7 produced alkali, the rest of bacterial strains produced acid in the B.T.B reaction; 33% of the strains could reduce nitrate; 56% of the strains grew well on beef extract peptone medium, and most strains have the general physiological and biochemical characteristics of leguminous rhizobium after identification. According to 16 S rDNA complete sequence analysis, nine bacterial strains can be divided into four bacteria genera, among which the bacterial stains PG-1 and PG-2 belong to Agrobacterium, PG-3, PG-5, PG-6, PG-8 and PG-9 belong to Rhizobium, PG-4 belongs to Herbaspirillum and PG-7 belongs to Burkholderia.【Conclusion】The endophytic bacteria isolated from the same root nodule have great differences in their physiological and biochemical characteristics of endogenous bacteria, and showed genetic diversity.
引文
[1]常德龙.泡桐研究与全树利用[M].武汉:华中科技大学出版社,2016.CHANG D L.Pauiowia Research and Whole Tree Utilization[M].Wuhan:Huazhong University of Science and Technology Press,2016.
[2]王树芝.泡桐树的考古发现及其文化刍议[J].农业考古,2018(4):18-25.WANG S Z.On archaeological discoveries of the Paulownia tree and its cultural interpretations[J].Agricultural Archaeology,2018(4):18-25.
[3]常德龙,李芳东,胡伟华,谢青,黄文豪,张云岭.国内外泡桐木材市场分析与我国发展对策[J].世界林业研究,2018,31(1):57-62.CHANG D L,LI F D,HU W H,XIE Q,HUANG W H,ZHANG Y L.Analysis on international and national Paulownia wood market and its development strategy in China[J].World Forestry Research,2018,31(1):57-62.
[4]中国科学院.中国植物志[M].北京:科学出版社,2004.Chinese Academy of Sciences.Flora of China[M].Beijing:Science Press,2004.
[5]成俊卿.泡桐属木材的性质和用途的研究(一)[J].林业科学,1983(1):57-63,114-116.CHENG J Q.Study on the properties and uses of Paulownia wood(一)[J].Scientia Silvae Sinicae,1983(1):57-63,114-116.
[6]徐艳霞.环境异质性对罗汉松根瘤特性的影响[D].南宁:广西大学,2017.XU Y X.Effects of environmental heterogeneity on nodule characteristics of Podocarpus[D].Nanning:Guangxi University,2017.
[7]赵龙飞.我国大豆根瘤内生菌资源多样性研究[J].广东农业科学,2014,41(9):15-19.ZHAO L F.Research on resource diversity of soybean[Glycine max(L.)Merr]nodule endophytes in China[J].Guangdong Agricultural Sciences,2014,41(9):15-19.
[8]高晓杰,刁治民,范建平,高宁,侯颖春.青海省胡卢巴根瘤菌的抗逆性[J].湖北农业科学,2013,52(24):6020-6022.GAO X J,DIAO Z M,FAN J P,GAO N,HOU Y C.Stress resisitance of rhizobia in Trigonella foenum-grecum L.in Qinghai Province[J].Hubei Agricultural Sciences,2013,52(24):6020-6022.
[9]康文娟,姜哲浩,师尚礼.紫花苜蓿内生和非内生细菌遗传多样性及其分泌IAA和溶磷能力[J].草原与草坪,2018,38(6):34-47.KANG W J,JIANG Z H,SHI S L.Identification of the genetic diversity and the ability of secreting IAA and solubilizing phosphate of endophytic and non-endophytic bacteria from Medicago sativea[J].Grassland Turf,2018,38(6):34-47.
[10]黄宝灵,吕成群,唐东阶,柏学亮,熊英,韦源莲.罗汉松根瘤内生细菌的分离和特性[J].微生物学报,2002(5):620-624,651.HUANG B L,LYU C Q,TANG D J,BO X L,XIONG Y,WEI Y L.Isolation and characteristics of bacteria in the nodule structure from Podocarpus macrophyllus[J].Acta Microbiologica Sinica,2002(5):620-624,651.
[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[J].西南农业学报,2012,25(1):236-239.FENG J L,HE X H,LI Y W,LI F,QIN R Y.Isolation and cultural features of Frankia on root nodule of red bayberry in Guangxi[J].Southwest China Journal of Agricultural Sciences,2012,25(1):236-239.
[12]周德明,李遥.不同桤木根瘤内生菌的分离培养及生物学特性[J].中南林业科技大学学报,2010,30(7):78-84.ZHOU D M,LI Y.Isolation and biological characteristics of Frankia strains from different kinds of Alnus[J].Journal of Central South University of Forestry&Technology,2010,30(7):78-84.
[13]宋福强,牛丽纯,常伟,范晓旭,刘璇,高扬.盐碱地引种沙枣根瘤内生菌及根际土壤AM真菌的多样性[J].中国农学通报,2016,32(7):6-13.SONG F Q,NIU L C,CHANG W,FAN X X,LIU X,GAO Y.Diversity of root nodule endophytes from Elaeagnus angustifolia L.and Arbuscular Mycorrhizal Fungi from rhizosphere in saline-alkali soil[J].Chinese Agricultural Science Bulletin,2016,32(7):6-13.
[14]张爱梅,韩雪英,张世虎,孙坤,吴菊燕,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[J].生物资源,2018,40(2):114-119.ZHANG A M,HAN X Y,ZHANG S H,SUN K,WU J Y,YU J,GUO BM.Diversity of root nodule endophytic bacteria in Hippophae thibetana at different altitudes in Gannan area[J].Biotic Resources,2018,40(2):114-119.
[15]上海植物生理学会.植物生理学实验手册[M].上海:上海科学技术出版社,1985.Shanghai Plant Physiology Society.Experimental Manual of Plant Physiology[M].Shanghai:Shanghai Scientific&Technical Publishers,1985.
[16]中国科学院微生物研究所.一般细菌常用鉴定方法[M].北京:科学出版社,1978.Institute of Microbiology,Chinese Academy of Sciences.Identification Methods for Common Bacteria[M].Beijing:Science Press,1978.
[17]黄维南,蓝谷,黄绳全,邹小鲁,蔡龙祥,蔡克强.异叶银合欢根瘤菌的分离和特性[J].亚热带植物通讯,1997(2):12-17.HUANG W N,LAN G,HUANGY Q,ZOU X L,CAI L X.CAI K Q.Isolation and characterization of rhizobium from Leucaena diversifolia[J].Subtropical Plant Communication,1997(2):12-17.
[18]康文娟,师尚礼,王泽一,陈建纲,苗阳阳.3株紫花苜蓿内生根瘤菌功能差异性分析[J].草业科学,2018,35(7):1614-1623.KANG W J,SHI S L,WANG Z Y,CHEN J G,MIAO Y Y.Analysis of functional differences among three Medicago sativa endophytic rhizobial strains[J].Pratacultural Science,2018,35(7):1614-1623.
[19]高晓杰,刁治民,范建平.青海胡卢巴根瘤菌的生理生化特性研究[J].贵州农业科学,2010,38(6):85-89.GAO X J,DIAOZ M,FAN J P.Study on physiological and biochemical characteristics of rhizobium in Trigonella foenum-grecum in Qinghai Province[J].Guizhou Agricultural Sciences,2010,38(6):85-89.
[20]刘灵芝,陈玉玲,陈志刚,姜亮,吕晓锋.北方地区大豆根瘤菌的生物学特性[J].土壤通报,2007,38(1):141-144.LIU L Z,CHEN Y L,CHENZ G,JIANG L,LYU X F.Biological characteristics of indigenous rhizobia from soybean in the North Area[J].Chinese Journal of Soil Science,2007,38(1):141-144.
[21]韦原莲,黄宝灵,吕成群,张慧,樊利勤,李清香.马占相思根瘤菌生物学特性的研究[J].西北林学院学报,2006,21(5):120-124.WEI Y L,HUANG B L,LYU C Q,ZHANG H,FAN L Q,LI Q X.Biological characteristics of rhizobia in Acacia mangium[J].Journal of Northwest Forestry University,2006,21(5):120-124.
[22]吕成群,陆俊锟,黄宝灵,孙以树,李小华.喀斯特生境台湾相思根瘤菌的生物学特性研究[J].微生物学通报,2008,35(2):215-219.LYU C Q,LU J K,HUANG B L,SUN Y S,LI X H.Biological characteristics of the rhizobia isolated from Acacia confusa grew in the karst environment[J].Microbiology China,2008,35(2):215-219.
[23]刁治民,陈克龙,王文颖,孙苗苗,王恒生,李园媛.固氮微生物学[M].北京:科学出版社,2014.DIAO Z M,CHEN K L,WANG W Y,SUN M M,WANG H S,LI Y Y.Nitrogen Fixation Microbiology[M].Beijing:Science Press,2014.
[24]杨群.厚荚相思树种根瘤菌的多样性研究[D].南宁:广西大学.2010.YANG Q.Study on Diversity of Rhizobia Isolated From Root Nodules of Acacia crassicarpa[D].Nanning:Guangxi University,2010.
[25]王婷,杨升,陈亚雪,胡玲玲,屠琴,张丽,刘雪芹,王行国.两株茶树内生草螺菌的微生物学特性[J].微生物学报,2014,54(4):424-432.WANG T,YANG S,CHEN Y X,HU L L,TU Q,ZHANG L,LIU X Q,WANG H G.Microbiological properties of two endophytic bacteriaisolated from tea(Camellia sinensis L.)[J].Acta Microbiologica Sinica,2014,54(4):424-432.
[26]王秀呈,曹艳花,唐雪,马晓彤,高菊生,张晓霞.水稻内生固氮菌Herbaspirillum seropedicae DX35的筛选及其促生特性[J].微生物学报,2014,54(3):292-298.WANG X C,CAO Y H,TANG X,MA X T,GAO J S,ZHANG X X.Rice endogenous nitrogen fixing and growth promoting bacterium Herbaspirillum seropedicae DX35[J].Acta Microbiologica Sinica,2014,54(3):292-298.
[2]王树芝.泡桐树的考古发现及其文化刍议[J].农业考古,2018(4):18-25.WANG S Z.On archaeological discoveries of the Paulownia tree and its cultural interpretations[J].Agricultural Archaeology,2018(4):18-25.
[3]常德龙,李芳东,胡伟华,谢青,黄文豪,张云岭.国内外泡桐木材市场分析与我国发展对策[J].世界林业研究,2018,31(1):57-62.CHANG D L,LI F D,HU W H,XIE Q,HUANG W H,ZHANG Y L.Analysis on international and national Paulownia wood market and its development strategy in China[J].World Forestry Research,2018,31(1):57-62.
[4]中国科学院.中国植物志[M].北京:科学出版社,2004.Chinese Academy of Sciences.Flora of China[M].Beijing:Science Press,2004.
[5]成俊卿.泡桐属木材的性质和用途的研究(一)[J].林业科学,1983(1):57-63,114-116.CHENG J Q.Study on the properties and uses of Paulownia wood(一)[J].Scientia Silvae Sinicae,1983(1):57-63,114-116.
[6]徐艳霞.环境异质性对罗汉松根瘤特性的影响[D].南宁:广西大学,2017.XU Y X.Effects of environmental heterogeneity on nodule characteristics of Podocarpus[D].Nanning:Guangxi University,2017.
[7]赵龙飞.我国大豆根瘤内生菌资源多样性研究[J].广东农业科学,2014,41(9):15-19.ZHAO L F.Research on resource diversity of soybean[Glycine max(L.)Merr]nodule endophytes in China[J].Guangdong Agricultural Sciences,2014,41(9):15-19.
[8]高晓杰,刁治民,范建平,高宁,侯颖春.青海省胡卢巴根瘤菌的抗逆性[J].湖北农业科学,2013,52(24):6020-6022.GAO X J,DIAO Z M,FAN J P,GAO N,HOU Y C.Stress resisitance of rhizobia in Trigonella foenum-grecum L.in Qinghai Province[J].Hubei Agricultural Sciences,2013,52(24):6020-6022.
[9]康文娟,姜哲浩,师尚礼.紫花苜蓿内生和非内生细菌遗传多样性及其分泌IAA和溶磷能力[J].草原与草坪,2018,38(6):34-47.KANG W J,JIANG Z H,SHI S L.Identification of the genetic diversity and the ability of secreting IAA and solubilizing phosphate of endophytic and non-endophytic bacteria from Medicago sativea[J].Grassland Turf,2018,38(6):34-47.
[10]黄宝灵,吕成群,唐东阶,柏学亮,熊英,韦源莲.罗汉松根瘤内生细菌的分离和特性[J].微生物学报,2002(5):620-624,651.HUANG B L,LYU C Q,TANG D J,BO X L,XIONG Y,WEI Y L.Isolation and characteristics of bacteria in the nodule structure from Podocarpus macrophyllus[J].Acta Microbiologica Sinica,2002(5):620-624,651.
[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[J].西南农业学报,2012,25(1):236-239.FENG J L,HE X H,LI Y W,LI F,QIN R Y.Isolation and cultural features of Frankia on root nodule of red bayberry in Guangxi[J].Southwest China Journal of Agricultural Sciences,2012,25(1):236-239.
[12]周德明,李遥.不同桤木根瘤内生菌的分离培养及生物学特性[J].中南林业科技大学学报,2010,30(7):78-84.ZHOU D M,LI Y.Isolation and biological characteristics of Frankia strains from different kinds of Alnus[J].Journal of Central South University of Forestry&Technology,2010,30(7):78-84.
[13]宋福强,牛丽纯,常伟,范晓旭,刘璇,高扬.盐碱地引种沙枣根瘤内生菌及根际土壤AM真菌的多样性[J].中国农学通报,2016,32(7):6-13.SONG F Q,NIU L C,CHANG W,FAN X X,LIU X,GAO Y.Diversity of root nodule endophytes from Elaeagnus angustifolia L.and Arbuscular Mycorrhizal Fungi from rhizosphere in saline-alkali soil[J].Chinese Agricultural Science Bulletin,2016,32(7):6-13.
[14]张爱梅,韩雪英,张世虎,孙坤,吴菊燕,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[J].生物资源,2018,40(2):114-119.ZHANG A M,HAN X Y,ZHANG S H,SUN K,WU J Y,YU J,GUO BM.Diversity of root nodule endophytic bacteria in Hippophae thibetana at different altitudes in Gannan area[J].Biotic Resources,2018,40(2):114-119.
[15]上海植物生理学会.植物生理学实验手册[M].上海:上海科学技术出版社,1985.Shanghai Plant Physiology Society.Experimental Manual of Plant Physiology[M].Shanghai:Shanghai Scientific&Technical Publishers,1985.
[16]中国科学院微生物研究所.一般细菌常用鉴定方法[M].北京:科学出版社,1978.Institute of Microbiology,Chinese Academy of Sciences.Identification Methods for Common Bacteria[M].Beijing:Science Press,1978.
[17]黄维南,蓝谷,黄绳全,邹小鲁,蔡龙祥,蔡克强.异叶银合欢根瘤菌的分离和特性[J].亚热带植物通讯,1997(2):12-17.HUANG W N,LAN G,HUANGY Q,ZOU X L,CAI L X.CAI K Q.Isolation and characterization of rhizobium from Leucaena diversifolia[J].Subtropical Plant Communication,1997(2):12-17.
[18]康文娟,师尚礼,王泽一,陈建纲,苗阳阳.3株紫花苜蓿内生根瘤菌功能差异性分析[J].草业科学,2018,35(7):1614-1623.KANG W J,SHI S L,WANG Z Y,CHEN J G,MIAO Y Y.Analysis of functional differences among three Medicago sativa endophytic rhizobial strains[J].Pratacultural Science,2018,35(7):1614-1623.
[19]高晓杰,刁治民,范建平.青海胡卢巴根瘤菌的生理生化特性研究[J].贵州农业科学,2010,38(6):85-89.GAO X J,DIAOZ M,FAN J P.Study on physiological and biochemical characteristics of rhizobium in Trigonella foenum-grecum in Qinghai Province[J].Guizhou Agricultural Sciences,2010,38(6):85-89.
[20]刘灵芝,陈玉玲,陈志刚,姜亮,吕晓锋.北方地区大豆根瘤菌的生物学特性[J].土壤通报,2007,38(1):141-144.LIU L Z,CHEN Y L,CHENZ G,JIANG L,LYU X F.Biological characteristics of indigenous rhizobia from soybean in the North Area[J].Chinese Journal of Soil Science,2007,38(1):141-144.
[21]韦原莲,黄宝灵,吕成群,张慧,樊利勤,李清香.马占相思根瘤菌生物学特性的研究[J].西北林学院学报,2006,21(5):120-124.WEI Y L,HUANG B L,LYU C Q,ZHANG H,FAN L Q,LI Q X.Biological characteristics of rhizobia in Acacia mangium[J].Journal of Northwest Forestry University,2006,21(5):120-124.
[22]吕成群,陆俊锟,黄宝灵,孙以树,李小华.喀斯特生境台湾相思根瘤菌的生物学特性研究[J].微生物学通报,2008,35(2):215-219.LYU C Q,LU J K,HUANG B L,SUN Y S,LI X H.Biological characteristics of the rhizobia isolated from Acacia confusa grew in the karst environment[J].Microbiology China,2008,35(2):215-219.
[23]刁治民,陈克龙,王文颖,孙苗苗,王恒生,李园媛.固氮微生物学[M].北京:科学出版社,2014.DIAO Z M,CHEN K L,WANG W Y,SUN M M,WANG H S,LI Y Y.Nitrogen Fixation Microbiology[M].Beijing:Science Press,2014.
[24]杨群.厚荚相思树种根瘤菌的多样性研究[D].南宁:广西大学.2010.YANG Q.Study on Diversity of Rhizobia Isolated From Root Nodules of Acacia crassicarpa[D].Nanning:Guangxi University,2010.
[25]王婷,杨升,陈亚雪,胡玲玲,屠琴,张丽,刘雪芹,王行国.两株茶树内生草螺菌的微生物学特性[J].微生物学报,2014,54(4):424-432.WANG T,YANG S,CHEN Y X,HU L L,TU Q,ZHANG L,LIU X Q,WANG H G.Microbiological properties of two endophytic bacteriaisolated from tea(Camellia sinensis L.)[J].Acta Microbiologica Sinica,2014,54(4):424-432.
[26]王秀呈,曹艳花,唐雪,马晓彤,高菊生,张晓霞.水稻内生固氮菌Herbaspirillum seropedicae DX35的筛选及其促生特性[J].微生物学报,2014,54(3):292-298.WANG X C,CAO Y H,TANG X,MA X T,GAO J S,ZHANG X X.Rice endogenous nitrogen fixing and growth promoting bacterium Herbaspirillum seropedicae DX35[J].Acta Microbiologica Sinica,2014,54(3):292-298.