山西大豆根瘤菌的分离、鉴定及共生匹配性筛选

doi:10.13560/j.cnki.biotech.bull.1985.2021-1315

摘要/Abstract

摘要：

山西作为大豆起源地之一,根瘤菌资源丰富。为了进一步挖掘山西省的大豆根瘤菌资源,筛选出与当地主栽大豆品种共生固氮效果好的根瘤菌。本实验在大同广灵、太原清徐、晋中太谷三地采集不同大豆品种的根瘤,经过多次划线,对分离纯化出的单菌落进行菌落形态、刚果红染色、BTB染色表型鉴定;16S rDNA、nodA和nifH基因型鉴定。利用BOX-PCR对所有根瘤菌进行电泳检测,去除条带完全一致的菌株,将目的条带不一致的菌在Bionumerics@7.5软件中聚类分析。选代表性菌株接种到晋大88号和科丰1号两个大豆材料,筛选匹配性好的菌株。共分离出203株菌,其中187株根瘤菌可以扩增出nodA、nifH基因,172株快生型根瘤菌,属于S. fredii;15株慢生型根瘤菌,分别属于B. diazoefficiens和 B. daqingense。BOX-PCR将85株条带不一致的根瘤菌分为四大类群,以70%为界限共分为16类。筛选出与晋大88号匹配性较好的根瘤菌GL11和GL43,其地上部分干重较接种USDA110分别增加38.5%、30.1%;根干重分别增加22.2%、27.8%;根瘤鲜重分别增加24.3%、41.5%;根瘤干重分别增加36.6%、31.0%。筛选出与科丰1号匹配性较好的根瘤菌TG37,其株高、地上干重、根干重、根瘤鲜重、根瘤干重和根瘤数较接种USDA110分别增加8.1%、15.0%、28.6%、27.3%、44.3%和60.6%。山西省根瘤菌资源丰富,种类多样,S. fredii为优势种,此外还分离出两类慢生型根瘤菌B. diazoefficiens和 B. daqingense,丰富了根瘤菌资源。筛选出共生匹配性好的根瘤菌为大豆生产实践奠定了物质基础和理论依据。

关键词: 根瘤菌, 分离与鉴定, BOX-PCR, 匹配性

Abstract:

As one of the origin of soybean,Shanxi is rich in rhizobia resources. In order to further excavate soybean rhizobia resources in Shanxi province and screen the rhizobia of having good symbiotic nitrogen fixation effect with the local main soybean varieties. In this experiment,root nodules of different soybean varieties were collected in Guangling of Datong,Qingxu of Taiyuan and Taigu of Jinzhong. After multiple streaking,the colony morphology,Congo red staining,BTB staining phenotype identification and 16S rDNA,nodA,nifH genotype identification of the isolated and purified single colonies were performed. BOX-PCR was used for electrophoresis detection of all rhizobia. The strains with completely consistent bands were excluded and the bacteria with inconsistent bands were clustered and analyzed in Bionumerics ^@ 7.5 software. Then the representative strains were selected and inoculated into two soybean materials Jinda 88 and Kefeng 1 to screen strains with good matching. A total of 203 strains were isolated,from 187 strains of them the nodA and nifH were amplified. While 172 fast-growing strains belonged to Sinorhizobium fredii and slowly-growing 15 strains belonged to Bradyrhizobium diazoefficiens and Bradyrhizobium daqingense,respectively. The 85 rhizobia strains with inconsistent bands were divided into 4 groups by BOX-PCR,which were divided into 16 subgroups with 70% as the limit. Rhizobium GL11 and GL43 were selected in better match with Jinda 88. Compared with USDA110 inoculation,the shoot dry weight increased by 38.5% and 30.1%,respectively;the root dry weight increased by 22.2% and 27.8%,respectively;the fresh weight of nodules increased by 24.3% and 41.5%,respectively;the dry weight of nodules increased by 36.6% and 31.0%,respectively. Rhizobium TG37 was selected as a good match with Kefeng 1 and its plant height,shoot dry weight,dry root weight,fresh nodule weight,dry nodule weight and number of nodules increased by 8.1%,15.0%,28.6%,27.3%,44.3% and 60.6%,respectively,compared with USDA110 inoculation. Shanxi province has rich resources of rhizobia. S. fredii is the dominant species. In addition,two types of slow-growing rhizobia B. diazoefficiens and B. daqingense were isolated,which enriches the rhizobia resources. The screening of rhizobia with good symbiosis and matching lays a material and theoretical basis for soybean production practice.

Key words: rhizobia, separation and identification, BOX-PCR, matching

王晓丽, 秦杰, 王敏, 王利祥, 杜维俊. 山西大豆根瘤菌的分离、鉴定及共生匹配性筛选[J]. 生物技术通报, 2022, 38(3): 59-68.

WANG Xiao-li, QIN Jie, WANG Min, WANG Li-xiang, DU Wei-jun. Isolation,Identification and Symbiotic Matching of Soybean Rhizobia from Shanxi Province[J]. Biotechnology Bulletin, 2022, 38(3): 59-68.

图/表 13

参考文献 27

[1]	Popp C, Ott T. Regulation of signal transduction and bacterial infection during root nodule symbiosis[J]. Curr Opin Plant Biol, 2011, 14(4):458-467. doi: 10.1016/j.pbi.2011.03.016 URL
[2]	柏宇, 关大伟, 李力, 等. 耐高氮优良大豆根瘤菌株的筛选与鉴定[J]. 大豆科学, 2014, 33(6):861-864.
	Bai Y, Guan DW, Li L, et al. Screening and characterization of superior nitrogen-tolerance soybean rhizobia[J]. Soybean Sci, 2014, 33(6):861-864.
[3]	Zhang XX, Guo HJ, Jiao J, et al. Pyrosequencing of rpoB uncovers a significant biogeographical pattern of rhizobial species in soybean rhizosphere[J]. J Biogeogr, 2017, 44(7):1491-1499. doi: 10.1111/jbi.2017.44.issue-7 URL
[4]	陈文峰. 根瘤菌系统学研究进展与展望[J]. 微生物学通报, 2016, 43(5):1095-1100.
	Chen WF. Progress and perspective of systematics of rhizobia[J]. Microbiol China, 2016, 43(5):1095-1100.
[5]	程涛, 焦如珍, 高俊莲. 对两个分离自海南尖峰岭的木本豆科植物根瘤菌菌株的多相鉴定[J]. 林业科学研究, 2015, 28(3):380-386.
	Cheng T, Jiao RZ, Gao JL. Polyphasic identification of two Rhizobium strains from woody legumes in Jianfengling of Hainan Province[J]. For Res, 2015, 28(3):380-386.
[6]	Mohammed M, Jaiswal SK, Dakora FD. Distribution and correlation between phylogeny and functional traits of cowpea(Vigna unguiculata L. Walp. )-nodulating microsymbionts from Ghana and South Africa[J]. Sci Rep, 2018, 8:18006. doi: 10.1038/s41598-018-36324-0 URL
[7]	Zhang YM, Tian CF, Sui XH, et al. Robust markers reflecting phylogeny and taxonomy of rhizobia[J]. PLoS One, 2012, 7(9):e44936. doi: 10.1371/journal.pone.0044936 URL
[8]	蒲强, 谭志远, 彭桂香, 等. 根瘤菌分类的新进展[J]. 微生物学通报, 2016, 43(3):619-633.
	Pu Q, Tan ZY, Peng GX, et al. Advances in rhizobia taxonomy[J]. Microbiol China, 2016, 43(3):619-633.
[9]	Borba MP, Ballarini AE, Witusk JPD, et al. Evaluation of BOX-PCR and REP-PCR as molecular typing tools for Antarctic Streptomyces[J]. Curr Microbiol, 2020, 77(11):3573-3581. doi: 10.1007/s00284-020-02199-6 URL
[10]	陈文新, 陈文峰. 发挥生物固氮作用减少化学氮肥用量[J]. 中国农业科技导报, 2004, 6(6):3-6.
	Chen WX, Chen WF. Exertion of biological nitrogen fixation in order to reducing the consumption of chemical nitrogenous fertilizer[J]. Rev China Agric Sci Technol, 2004, 6(6):3-6.
[11]	王金生, 丁宁, 吴俊江, 等. 大豆根瘤菌接种效应及竞争结瘤能力分析[J]. 大豆科学, 2017, 36(5):761-767.
	Wang JS, Ding N, Wu JJ, et al. Analysis of the inoculation effect of soybean rhizobia and the competitive nodulation ability[J]. Soybean Sci, 2017, 36(5):761-767.
[12]	Fan KK, Delgado-Baquerizo M, Guo XS, et al. Suppressed N fixation and diazotrophs after four decades of fertilization[J]. Microbiome, 2019, 7(1):143. doi: 10.1186/s40168-019-0757-8 URL
[13]	高俊莲, 孙建光, 陈文新. 斜茎黄芪根瘤菌结瘤基因nodA PCR扩增及PCR-RFLP分析[J]. 微生物学杂志, 2006, 26(4):1-5.
	Gao JL, Sun JG, Chen WX. PCR amplification and PCR-RFLP analysis of nodulation gene nodA of rhizobia from Astragalus adsurgens[J]. J Microbiol, 2006, 26(4):1-5.
[14]	Fredriksson NJ, Hermansson M, Wilén BM. The choice of PCR primers has great impact on assessments of bacterial community diversity and dynamics in a wastewater treatment plant[J]. PLoS One, 2013, 8(10):e76431. doi: 10.1371/journal.pone.0076431 URL
[15]	Wang JY, Wang R, Zhang YM, et al. Bradyrhizobium daqingense sp. nov., isolated from soybean nodules[J]. Int J Syst Evol Microbiol, 2013, 63(Pt 2):616-624. doi: 10.1099/ijs.0.034280-0 URL
[16]	Zhang YM, Li Y, Chen WF, et al. Biodiversity and biogeography of rhizobia associated with soybean plants grown in the North China plain[J]. Appl Environ Microbiol, 2011, 77(18):6331-6342. doi: 10.1128/AEM.00542-11 URL
[17]	葛诚, 徐玲玫, 樊惠. 超慢型大豆根瘤菌的分离和初步鉴定[J]. 中国农业科学, 1987, 20(4):96.
	Ge C, Xu LM, Fan H. Isolated and initially identified on extra slow-growing soybean rhizobia[J]. Sci Agric Sin, 1987, 20(4):96.
[18]	Han LL, Wang ET, Han TX, et al. Unique community structure and biogeography of soybean rhizobia in the saline-alkaline soils of Xinjiang, China[J]. Plant Soil, 2009, 324(1/2):291-305. doi: 10.1007/s11104-009-9956-6 URL
[19]	张璐. 不同品种大豆与土壤中根瘤菌的多样性研究[D]. 杨凌:西北农林科技大学, 2017.
	Zhang L. Diversity of Rhizobium from the roots of different soybeans varieties and soil[D]. Yangling:Northwest A & F University, 2017.
[20]	史清亮, 白成云, 温月香, 等. 山西省费氏中华根瘤菌数量分布及特性探析[J]. 中国生态农业学报, 2007, 15(3):120-122.
	Shi QL, Bai CY, Wen YX, et al. Quantitative distribution and character analysis of Sinorchizobium fredii in Shanxi province[J]. Chin J Eco Agric, 2007, 15(3):120-122. Chinese Journal of Eco-Agriculture, 2007(3):120-122.
[21]	张红侠. 黄土高原地区大豆根瘤菌遗传多样性、高效菌株筛选及应用效果的研究[D]. 北京:中国农业科学院, 2010.
	Zhang HX. Genetic diversity, screening and application effect of soybean rhizobia in Loess Plateau[D]. Beijing:Chinese Academy of Agricultural Sciences, 2010.
[22]	杨凤环, 李正楠, 姬惜珠, 等. BOX-PCR技术在微生物多样性研究中的应用[J]. 微生物学通报, 2008, 35(8):1282-1286.
	Yang FH, Li ZN, Ji XZ, et al. Applications of BOX-PCR technique in diversity study of microorganisms[J]. Microbiology, 2008, 35(8):1282-1286.
[23]	Loureiro MDF, Kaschuk G, Alberton O, et al. Soybean[Glycine max(L.)Merrill]rhizobial diversity in Brazilian oxisols under various soil, cropping, and inoculation managements[J]. Biol Fertil Soils, 2007, 43(6):665-674. doi: 10.1007/s00374-006-0146-x URL
[24]	王卫卫, 关大伟, 马鸣超, 等. 东北地区大豆根瘤菌遗传多样性与系统发育研究[J]. 大豆科学, 2013, 32(4):433-437.
	Wang WW, Guan DW, Ma MC, et al. Genetic diversity and phylogeny of soybean rhizobia isolated from northeast China[J]. Soybean Sci, 2013, 32(4):433-437.
[25]	张红侠, 冯瑞华, 关大伟, 等. 黄土高原地区优良大豆根瘤菌的筛选与接种方式研究[J]. 大豆科学, 2010, 29(6):996-1002.
	Zhang HX, Feng RH, Guan DW, et al. Screening of superior soybean rhizobial strains and analyzing of different inoculation methods in loess plateau region of China[J]. Soybean Sci, 2010, 29(6):996-1002.
[26]	蒋攀. 四川大豆根瘤菌高效菌株的初步筛选[D]. 雅安:四川农业大学, 2013.
	Jiang P. Preliminary screening of high efficient soybean rhizobia strains in Sichuan Province[D]. Ya’an:Sichuan Agricultural University, 2013.
[27]	伍惠, 钟喆栋, 樊伟, 等. 8株优良大豆根瘤菌与不同地区27个大豆主栽品种的匹配性研究[J]. 大豆科学, 2017, 36(3):405-418.
	Wu H, Zhong ZD, Fan W, et al. Symbiotic compatibility among eight elite soybean rhizobia strains and twenty-seven soybean cultivars from different planting regions[J]. Soybean Sci, 2017, 36(3):405-418.

Primer name	Primer sequence（5'-3'）	Literature source
nifH-F	AAAGGYGGWATCGGYAARTCCACCAC	[12]
nifH-R	TTGTTSGCSGCRTACATSGCCATCAT	[12]
nodA-F	TGCRGTGGAATNTRNNCTGGGAAA	[13]
nodA-R	GGNCCGTCRTCRAAWGTCATGTA	[13]
16S rDNA-F	AGAGTTTGATCCTGGCTCAG	[14]
16S rDNA-R	GGTTACCTTGTTACGACTT	[14]

Primer name	Primer sequence（5'-3'）	Literature source
nifH-F	AAAGGYGGWATCGGYAARTCCACCAC	[12]
nifH-R	TTGTTSGCSGCRTACATSGCCATCAT	[12]
nodA-F	TGCRGTGGAATNTRNNCTGGGAAA	[13]
nodA-R	GGNCCGTCRTCRAAWGTCATGTA	[13]
16S rDNA-F	AGAGTTTGATCCTGGCTCAG	[14]
16S rDNA-R	GGTTACCTTGTTACGACTT	[14]

Rhizobium	Jinda 88			Kefeng 1
Rhizobium	Plant height/cm	Shoot dry weight/g	Root dry weight/g	Plant height/cm	Shoot dry weight/g	Root dry weight/g
CK	26.65±4.78 c	0.23±0.07 c	0.19±0.06 abc	19.55±1.93 abcd	0.18±0.02 bcde	0.15±0.01 abc
USDA110	31.40±2.91 abc	0.26±0.04 bc	0.18±0.02 abc	19.83±0.45 abcd	0.20±0.02 abcd	0.14±0.00 bc
TG1	30.15±4.16 abc	0.28±0.05 abc	0.19±0.01 abc	21.80±2.17 a	0.20±0.03 abc	0.16±0.02 ab
TG14	33.02±3.04 abc	0.26±0.05 bc	0.20±0.02 abc	19.30±1.06 abcd	0.16±0.03 cde	0.12±0.02 c
TG20	32.10±5.05 abc	0.25±0.04 c	0.19±0.02 abc	16.40±1.67 d	0.15±0.02 de	0.12±0.01 c
TG37	32.73±3.83 abc	0.31±0.01 abc	0.23±0.03 a	21.43±2.35 ab	0.23±0.01 a	0.18±0.02 a
TG53	34.45±4.74 abc	0.28±0.04 abc	0.21±0.03 abc	20.37±1.29 abc	0.17±0.02 bcde	0.13±0.02 bc
TG79	28.68±1.55 bc	0.26±0.06 bc	0.19±0.04 abc	20.13±1.96 abc	0.18±0.04 bcde	0.14±0.03 bc
GL11	30.63±5.23 abc	0.36±0.02 a	0.22±0.02 ab	17.20±1.57 cd	0.19±0.01 abcde	0.14±0.00 bc
GL16	31.77±2.96 abc	0.25±0.03 c	0.16±0.00 bc	19.37±0.85 abcd	0.18±0.01 bcde	0.12±0.01 c
GL17	32.33±5.72 abc	0.31±0.07 abc	0.19±0.04 abc	18.23±2.41 bcd	0.17±0.01 bcde	0.12±0.02 c
GL19	33.03±1.76 abc	0.28±0.03 abc	0.22±0.01 ab	20.93±0.71 ab	0.18±0.02 bcde	0.13±0.01 bc
GL28	34.60±2.25 ab	0.30±0.02 abc	0.21±0.03 ab	19.67±1.11 abcd	0.20±0.02 abc	0.14±0.02 bc
GL40	28.87±8.18 bc	0.31±0.05 abc	0.15±0.07 c	16.97±2.30 cd	0.16±0.03 cde	0.13±0.01 c
GL43	33.67±4.53 abc	0.34±0.09 ab	0.23±0.06 a	19.47±2.39 abcd	0.21±0.01 ab	0.15±0.01 abc
GL67	35.53±4.24 ab	0.29±0.04 abc	0.21±0.01 ab	17.93±2.57 bcd	0.16±0.00 cde	0.14±0.02 bc
GL75	36.97±0.47 a	0.32±0.03 abc	0.21±0.03 ab	18.10±1.76 bcd	0.15±0.02 e	0.12±0.02 c
QX13	29.83±2.42 abc	0.26±0.06 bc	0.21±0.04 ab	20.43±2.15 abc	0.19±0.06 abcde	0.15±0.03 abc

Rhizobium	Jinda 88			Kefeng 1
Rhizobium	Plant height/cm	Shoot dry weight/g	Root dry weight/g	Plant height/cm	Shoot dry weight/g	Root dry weight/g
CK	26.65±4.78 c	0.23±0.07 c	0.19±0.06 abc	19.55±1.93 abcd	0.18±0.02 bcde	0.15±0.01 abc
USDA110	31.40±2.91 abc	0.26±0.04 bc	0.18±0.02 abc	19.83±0.45 abcd	0.20±0.02 abcd	0.14±0.00 bc
TG1	30.15±4.16 abc	0.28±0.05 abc	0.19±0.01 abc	21.80±2.17 a	0.20±0.03 abc	0.16±0.02 ab
TG14	33.02±3.04 abc	0.26±0.05 bc	0.20±0.02 abc	19.30±1.06 abcd	0.16±0.03 cde	0.12±0.02 c
TG20	32.10±5.05 abc	0.25±0.04 c	0.19±0.02 abc	16.40±1.67 d	0.15±0.02 de	0.12±0.01 c
TG37	32.73±3.83 abc	0.31±0.01 abc	0.23±0.03 a	21.43±2.35 ab	0.23±0.01 a	0.18±0.02 a
TG53	34.45±4.74 abc	0.28±0.04 abc	0.21±0.03 abc	20.37±1.29 abc	0.17±0.02 bcde	0.13±0.02 bc
TG79	28.68±1.55 bc	0.26±0.06 bc	0.19±0.04 abc	20.13±1.96 abc	0.18±0.04 bcde	0.14±0.03 bc
GL11	30.63±5.23 abc	0.36±0.02 a	0.22±0.02 ab	17.20±1.57 cd	0.19±0.01 abcde	0.14±0.00 bc
GL16	31.77±2.96 abc	0.25±0.03 c	0.16±0.00 bc	19.37±0.85 abcd	0.18±0.01 bcde	0.12±0.01 c
GL17	32.33±5.72 abc	0.31±0.07 abc	0.19±0.04 abc	18.23±2.41 bcd	0.17±0.01 bcde	0.12±0.02 c
GL19	33.03±1.76 abc	0.28±0.03 abc	0.22±0.01 ab	20.93±0.71 ab	0.18±0.02 bcde	0.13±0.01 bc
GL28	34.60±2.25 ab	0.30±0.02 abc	0.21±0.03 ab	19.67±1.11 abcd	0.20±0.02 abc	0.14±0.02 bc
GL40	28.87±8.18 bc	0.31±0.05 abc	0.15±0.07 c	16.97±2.30 cd	0.16±0.03 cde	0.13±0.01 c
GL43	33.67±4.53 abc	0.34±0.09 ab	0.23±0.06 a	19.47±2.39 abcd	0.21±0.01 ab	0.15±0.01 abc
GL67	35.53±4.24 ab	0.29±0.04 abc	0.21±0.01 ab	17.93±2.57 bcd	0.16±0.00 cde	0.14±0.02 bc
GL75	36.97±0.47 a	0.32±0.03 abc	0.21±0.03 ab	18.10±1.76 bcd	0.15±0.02 e	0.12±0.02 c
QX13	29.83±2.42 abc	0.26±0.06 bc	0.21±0.04 ab	20.43±2.15 abc	0.19±0.06 abcde	0.15±0.03 abc

Rhizobium	Jinda 88			Kefeng 1
Rhizobium	Fresh nodule weight/mg	Dry nodule weight/mg	Nodule number per plant	Fresh nodule weight/mg	Dry nodule weight/mg	Nodule number per plant
USDA110	88.15±15.17 ab	19.18±4.20 ab	45.83±16.10 a	46.73±11.90 abc	10.00±2.14 abc	22.00±8.54 abcd
TG1	116.07±34.50 a	30.02±8.30 a	43.67±12.50 a	53.73±34.00 abc	10.77±6.90 abc	26.67±26.80 abc
TG14	101.15±17.40 ab	23.15±5.20 ab	36.33±9.29 ab	68.70±4.25 abc	17.57±4.70 a	25.67±16.10 abc
TG20	87.73±14.20 ab	18.30±3.44 b	42.33±5.86 a	69.77±13.50 ab	16.33±4.70 ab	41.67±18.00 a
TG37	98.13±4.66 ab	22.43±2.10 ab	34.67±12.80 ab	59.47±27.90 abc	14.43±7.00 abc	35.33±23.60 abc
TG53	77.65±30.64 ab	19.97±5.30 ab	40.33±7.39 a	70.83±19.20 a	15.27±6.00 abc	28.67±6.11 abc
TG79	93.38±40.84 ab	22.40±9.42 ab	36.50±18.69 ab	39.80±20.08 bc	7.80±2.95 c	12.00±1.00 cd
GL11	109.53±57.90 ab	26.20±16.20 ab	37.33±20.80 ab	54.17±6.70 abc	13.03±1.90 abc	37.67±8.08 ab
GL16	32.77±14.45 cd	7.73±3.55 cde	28.67±6.51 abc	46.83±7.92 abc	9.70±2.54 abc	35.33±9.50 abc
GL17	84.97±20.66 ab	19.97±3.00 ab	46.33±5.86 a	42.40±12.88 abc	10.13±3.40 abc	24.00±12.12 abc
GL19	81.40±14.60 ab	17.50±2.01 bc	36.33±10.70 ab	41.37±3.35 abc	7.70±1.51 c	22.67±6.11 abcd
GL28	66.27±21.01 bc	15.13±5.20 bcd	17.33±1.53 bcd	44.40±29.99 abc	11.50±8.92 abc	17.67±6.81 bcd
GL40	95.55±39.39 ab	22.60±7.07 ab	47.50±24.75 a	63.20±9.21 abc	14.27±2.10 abc	33.67±13.00 abc
GL43	124.73±39.90 a	25.13±7.10 ab	40.67±9.29 a	53.80±9.36 abc	12.83±2.50 abc	21.67±9.87 abcd
GL67	94.57±11.71 ab	21.60±3.31 ab	41.33±6.11 a	47.47±6.53 abc	9.37±1.39 abc	19.00±2.65 abcd
GL75	106.27±2.25 ab	24.33±2.20 ab	43.33±3.06 a	59.73±11.60 abc	13.33±2.80 abc	25.67±13.30 abc
QX13	29.30±16.95 cd	6.13±3.11 de	14.67±9.61 cd	38.97±1.85 c	7.83±1.37 c	27.33±10.00 abc