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中国农业微生物菌种保藏管理中心大豆根瘤菌精准评价  PDF

  • 韩嘉诚 1,2,3
  • 朱宏图 1,3
  • 杨芾 1,3
  • 郭捷 1,3
  • 马晓彤 1,3 ✉
  • 张晓霞 1,3,4 ✉
1. 中国农业科学院农业资源与农业区划研究所，北方干旱半干旱耕地高效利用全国重点实验室，北京； 2. 西北农林科技大学 生命科学学院，陕西 杨凌； 3. 国家农业微生物种质资源库，北京； 4. 国家盐碱地综合利用技术创新中心，山东 东营

最近更新：2025-04-09

DOI： 10.13343/j.cnki.wsxb.20240811

CSTR： 32112.14.j.AMS.20240811

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EN 引
目录contents

摘要

大豆(Glycine max)是世界上重要的粮食作物和油料作物。农业供给侧结构性改革需要增加优质食用大豆种植面积，但由于我国耕地资源的固有特性，大豆的国内产量远不能实现自给自足，亟需提高国内大豆种植面积和产量，摆脱对进口的依赖。根瘤菌(rhizobia)是研发最早的微生物肥料，但在我国的接种面积极低。

目的

本研究拟从库藏根瘤菌中筛选出大豆促生结瘤菌株，为缓解粮食问题提供种质资源保障。

方法

以中国农业微生物菌种保藏管理中心数十年收集的大豆根瘤菌为实验材料，结合16S rRNA基因和recA序列分析，复核菌种保藏信息；通过水培结瘤实验，综合考虑结瘤率、根瘤数量和根瘤质量以及植株株高和干重，评价菌株的结瘤和促生能力。

结果

共活化获得213株大豆根瘤菌菌株，其中156株隶属于慢生根瘤菌(Bradyrhizobium)、48株隶属于中华根瘤菌属(Sinorhizobium)、9株隶属于根瘤菌属(Rhizobium)。在这些菌株中，149株能与本研究选用的大豆品种匹配结瘤，43株能够显著促进大豆植株生长。

结论

本研究进一步明确了库藏大豆根瘤菌的分类地位，评价了其结瘤促生能力，为大豆根瘤菌菌剂的开发提供了丰富的菌质资源。

微生物将N2还原为NH3的过程称为生物固氮(biological nitrogen fixation, BNF)。据固氮微生物(nitrogen-fixing microorganisms)与其他生物的关系，固氮微生物可分为：自生固氮菌(free-living nitrogen-fixer)、共生固氮菌(symbiotic nitrogen-fixer)以及联合固氮菌(associative nitrogen-fixer)。根瘤菌(rhizobia)和豆科(Leguminosae)植物的固氮体系是共生固氮的典型代表。全球每年生物固氮量约为2.28×108 t，是工业固氮的1.52倍[

1-2]，其中豆科植物与根瘤菌共生固氮量约2.15×107 t[3-4]，占根瘤共生固氮量的83.01%，占陆地生态系统参与的固氮量的24.43%[5]。

根瘤菌多指广泛存在于土壤中，能与豆科植物共生固氮的革兰氏阴性细菌[

6]。绝大多数具有结瘤固氮能力的根瘤菌属于α-变形菌纲(Alphaproteobacteria)，包括根瘤菌属(Rhizobium)、剑菌属(Ensifer)、中华根瘤菌属(Sinorhizobium)[7]、异根瘤菌属(Allorhizobium)、副根瘤菌属(Pararhizobium)、新根瘤菌属(Neorhizobium)、申氏菌属(Shinella) (之前称为Crabtreella)、中慢生根瘤菌属(Mesorhizobium)、胺杆菌属(Aminobacter)、叶杆菌属(Phyllobacterium)、布鲁氏菌属(Brucella) (之前称为Ochrobactrum)、甲基杆菌属(Methylobacterium)[8]、微枝形杆菌属(Microvirga)[9]、慢生根瘤菌属(Bradyrhizobium)、固氮根瘤菌属(Azorhizobium)和德沃斯氏菌属(Devosia)。此外也有分类于β-变形菌纲[10](Betaproteobacteria)的副伯克霍尔德氏菌属(Paraburkholderia)和贪铜菌属(Cupriavidus)[11]，以及隶属于γ-变形菌纲(Gammaproteobacteria)的假单胞菌属(Pseudomonas)[12]。根瘤菌的多样性体现出共生固氮体系在自然界的广泛性，是研究豆科植物-根瘤菌共生体系进化、选育高效固氮系统以提高农业粮食产量的宝贵资源。

豆科植物与根瘤菌的共生固氮作用发生在植物根系。根瘤菌响应豆科植物根释放的黄酮类(flavonoids)物质，经过一系列生理反应后[

13-14]与植物共生形成根瘤[15-16]。成熟根瘤在低氧微环境[17-18]中将N2还原为NH3供植物利用，植物则为根瘤菌提供营养物质实现共生。根瘤菌只能响应少数黄酮类物质，因此大多数根瘤菌具有较强的宿主特异性。除了共生固氮作用外，根瘤菌还具有各种植物根际促生菌(plant growth promoting rhizobacteria, PGPR)的能力，如溶磷[19-20]、解钾[21-22]，产生植物激素[23-27]、胞外多糖、铁载体[28]和生防植物病原体等[29-31]。

根瘤菌菌剂是最早研发的生物肥料产品，是世界上公认效果最稳定的微生物肥料，已在全世界范围内推广应用。据统计，根瘤菌在美国、巴西等大豆种植的主要国家接种率达到95%以上，然而我国主要豆科作物的根瘤菌接种面积极低，仅为1%-2%。

为了更好地配合大豆等油料作物的种植，有必要针对大豆等主要种植区的根瘤菌资源进行精准评价，以服务农业生产。中国农业微生物菌种保藏管理中心(Agricultural Culture Collection of China, ACCC)在根瘤菌研究方面已有60余年的积累，保藏根瘤菌菌株约3 700株，基本涵盖了能与我国播种豆科作物共生结瘤的优良菌种，并在20世纪70-90年代的生产实践中广泛应用。然而，因化肥的大量施用，从20世纪90年代起根瘤菌的应用面积骤减，导致对根瘤菌的研究主要关注基础理论，忽略了应用研究。由于ACCC保藏的菌株历史较长，大部分根瘤菌分类鉴定基于较为传统的互接种族和生理生化特性，且存在结瘤能力退化的风险。因此，本研究对1982年以来保藏的大豆根瘤菌采用现代分类方法和体系进行重新鉴定，并对其结瘤及促生功能进行评价，以期为大豆根瘤菌菌剂的开发提供种质资源。

1 材料与方法

1.1 供试作物和菌株

大豆种子购自哈尔滨团贸食品有限公司。基于以往农业生产实践经验筛选出241株供试菌株，均保藏于中国农业微生物菌种保藏管理中心，保藏信息详见表1。

表1  库藏根瘤菌保藏信息
Table 1  Preservation information of rhizobium in store
NumberTimeSource of strainNumberTimeSource of strain
E. fredii ACCC 15176# 2012.04 Changsha, Hunan, China
ACCC 15061 1989.05 No record ACCC 15177 No record Changsha, Hunan, China
ACCC 15067# 1989.04 America ACCC 15178 2017.12 Changsha, Hunan, China
ACCC 15068# 1989.04 Shangdong, China ACCC 15179 1994 Changsha, Hunan, China
ACCC 15069# 2021.10 America ACCC 15180# 1994 Changsha, Hunan, China
ACCC 15070# 1989.04 Henan, China ACCC 15182# No record Changsha, Hunan, China
ACCC 15071# 1989.04 Henan, China ACCC 15184 No record Changsha, Hunan, China
ACCC 15072 1989.04 Henan, China ACCC 15185# 2018.02 Changsha, Hunan, China
ACCC 15075 1989.04 Henan, China ACCC 15186# 1996 Changsha, Hunan, China
ACCC 15076# 1989.04 Henan, China ACCC 15187# 1994 Changsha, Hunan, China
ACCC 15077 1989.04 Shanxi, China ACCC 15189# 1995 Jingong, Jiangxi, China
ACCC 15082# 1989.04 Jiangsu, China ACCC 15190# 1995 Jingong, Jiangxi, China
ACCC 15084# 1989.04 No record ACCC 15191# 1995 Jingong, Jiangxi, China
ACCC 15085# 1989.04 Changping, Beijing, China ACCC 15192# 1995 Jingong, Jiangxi, China
ACCC 15086# 2003.01 Changping, Beijing, China ACCC 15193# 1995 Changsha, Hunan, China
ACCC 15090# 1989.05 Lingxian, Shandong, China ACCC 15194# 1995 Changsha, Hunan, China
ACCC 15101# 2003.01 Changping, Beijing, China ACCC 15195# 1995 Changsha, Hunan, China
ACCC 15102# 1989.05 Lingxian, Shandong, China ACCC 15196# 1995 Changsha, Hunan, China
ACCC 15104# 2003.01 Manasi, Xinjiang, China ACCC 15197# 1995 Changsha, Hunan, China
ACCC 15106# 2003.01 Ningxia, China ACCC 15198# 1995 Nanchang, Jiangxi, China
ACCC 15107# 1990.11 Lingxian, Shandong, China ACCC 15200 1995 Nanchang, Jiangxi, China
ACCC 15108# 1989.03 Jiaxiang, Shandong, China ACCC 15201 1995 Nanchang, Jiangxi, China
ACCC 15109# 2003.01 Lingxian, Shandong, China ACCC 15202# 1995 Jingong, Jiangxi, China
ACCC 15117# 1989.05 Changping, Beijing, China ACCC 15203# No record Jingong, Jiangxi, China
ACCC 15118# 2003.01 Changping, Beijing, China ACCC 15204# 1995 Jingong, Jiangxi, China
ACCC 15119# 2002.12 Changping, Beijing, China ACCC 15205 1995 Jingong, Jiangxi, China
ACCC 15120# 2002.12 Changping, Beijing, China ACCC 15206# 1995 Futian, Fujian, China
ACCC 15121# 2002.12 Changping, Beijing, China ACCC 15212# 1995 Futian, Fujian, China
ACCC 15123# 2003.01 Changping, Beijing, China ACCC 15214# 1995 Futian, Fujian, China
ACCC 15125# 2003.01 Changping, Beijing, China ACCC 15215# 1995 Futian, Fujian, China
ACCC 15126# 1989.05 Lingxian, Shandong, China ACCC 15216# 1995 Futian, Fujian, China
ACCC 15127# 1989.05 Lingxian, Shandong, China ACCC 15217# 1995 Futian, Fujian, China
ACCC 15129# 1989.05 Lingxian, Shandong, China ACCC 15218# 1995 Futian, Fujian, China
ACCC 15130# 1989.05 Lingxian, Shandong, China ACCC 15220# 2010.10 Huiyang, Guangdong, China
ACCC 15131# 1989.05 Lingxian, Shandong, China ACCC 15221# 2020.01 Huiyang, Guangdong, China
ACCC 15132 No record Lingxian, Shandong, China ACCC 15222# 1995 Huiyang, Guangdong, China
ACCC 15133# 1988.11 Manasi, Xinjiang, China ACCC 15223# 1995 Huiyang, Guangdong, China
ACCC 15139# 1989.05 Manasi, Xinjiang, China ACCC 15224 1995 Huiyang, Guangdong, China
ACCC 15140# 2003.01 Manasi, Xinjiang, China ACCC 15225# 1995 Huiyang, Guangdong, China
ACCC 15142# 1988.11 Manasi, Xinjiang, China ACCC 15226# 1995 Huiyang, Guangdong, China
ACCC 15143# 1989.05 Manasi, Xinjiang, China ACCC 15228# 1995 Huiyang, Guangdong, China
ACCC 15145# 1989.03 Jiaxiang, Shandong, China ACCC 15229# 1995 Huiyang, Guangdong, China
ACCC 15147# 2003.01 Shanghai, China ACCC 15230 1995 Huiyang, Guangdong, China
B. japonicum ACCC 15230# 1995 Huiyang, Guangdong, China
ACCC 15005# 2003.01 America ACCC 15231# 1995 Huiyang, Guangdong, China
ACCC 15006# 1994 Heze, Shandong, China ACCC 15235# 1995 Huiyang, Guangdong, China
ACCC 15007# 2003.01 No record ACCC 15238# 1995 Huiyang, Guangdong, China
ACCC 15018# 1990.11 Shenyang, Liaoning, China ACCC 15239# 1995 Huiyang, Guangdong, China
ACCC 15020# 1990.11 Guizhou, China ACCC 15241# 2017.05 Huiyang, Guangdong, China
ACCC 15021# 1991.01 Shangdong, China ACCC 15242 1995 Ningming, Guagnxi, China
ACCC 15022-1# 1982.12 Shangdong, China ACCC 15243# 1995 Ningming, Guagnxi, China
ACCC 15022-2# 1982.12 Shangdong, China ACCC 15245# 1995 Meizhou, Guangdong, China
ACCC 15023# 1985.04 Guizhou, China ACCC 15246# 1995 Meizhou, Guangdong, China
ACCC 15027# 1982.12 America ACCC 15247# 1995 Meizhou, Guangdong, China
ACCC 15028# 2003.03 No record ACCC 15248# 1995 Guangxi, China
ACCC 15032# 1990.11 America ACCC 15250# 1995 Fujian, China
ACCC 15033# 2003.01 America ACCC 15251# 1995 Fujian, China
ACCC 15034# 2003.01 America ACCC 15252# 1995 Fujian, China
ACCC 15035# 2003.01 America ACCC 15254# 1995 No record
ACCC 15036# 2003.01 America ACCC 15255# 1995 Changsha, Hunan, China
ACCC 15037 2003.01 America ACCC 15256# 1995 Nanchang, Jiangxi, China
ACCC 15038# 1990.11 America ACCC 15257# 1995 Futian, Fujian, China
ACCC 15039 1990.11 America ACCC 15258# 1995 Ningming, Guagnxi, China
ACCC 15041 2001.04 America ACCC 15259# 1995 Meixian, Guangdong, China
ACCC 15042# 1991.03 America ACCC 15260# 1995 Changsha, Hunan, China
ACCC 15043# 2003.01 Australia ACCC 15262# 1995 Changsha, Hunan, China
ACCC 15043 2003.01 Australia ACCC 15263# 1995 Changsha, Hunan, China
ACCC 15044 1990.11 America ACCC 15264# 1995 Changsha, Hunan, China
ACCC 15045# 1985.04 America ACCC 15269# 1995 Changsha, Hunan, China
ACCC 15046# 2003.01 Argentina ACCC 15273# 1995 Nanchang, Jiangxi, China
ACCC 15047# 1991.01 America ACCC 15275# 1995 Nanchang, Jiangxi, China
ACCC 15049 1990.11 America ACCC 15276# 1995 Nanchang, Jiangxi, China
ACCC 15051 2020.06 America ACCC 15277# 1995 Jingong, Jiangxi, China
ACCC 15052 1990.11 Yulin, Guangxi, China ACCC 15279# 2003.01 Futian, Fujian, China
ACCC 15053 2003.03 Wuhan, Hubei, China ACCC 15280# 2003.01 Futian, Fujian, China
ACCC 15055# 1982.12 Wuhan, Hubei, China ACCC 15281# 2003.01 Futian, Fujian, China
ACCC 15057# 1991.01 Argentina ACCC 15282# 2003.01 Futian, Fujian, China
ACCC 15058# 1991.01 Argentina ACCC 15283# 2003.01 Futian, Fujian, China
ACCC 15059# 1990.11 America ACCC 15284# 2003.01 Futian, Fujian, China
ACCC 15060# 1990.11 New Zealand ACCC 15285# 2003.01 Futian, Fujian, China
ACCC 15062 2003.01 America ACCC 15289# 2003.01 Ningming, Guagnxi, China
ACCC 15063 1990.11 Liaoning, China ACCC 15291# 2003.01 Ningming, Guagnxi, China
ACCC 15064# 1982.12 India ACCC 15293# 2003.01 Meixian, Guangdong, China
ACCC 15065# 1990.11 America ACCC 15402# No record Guangzhou, Guangdong, China
ACCC 15081# 2003.01 No record ACCC 15601# 2001.04 America
ACCC 15083# 2003.01 No record ACCC 15603# 1991.01 America
ACCC 15095# 1990.11 America ACCC 15605# 1991.04 America
ACCC 15096# No record America ACCC 15606# 2003.01 America
ACCC 15097# 1991.01 America ACCC 15608# 1990.11 No record
ACCC 15150# 1996.06 Wuxuan, Guangxi, China ACCC 15610# 2003.01 America
ACCC 15156# 1994 Nanchang, Jiangxi, China ACCC 15611# 1982.12 No record
ACCC 15157# No record Nanchang, Jiangxi, China ACCC 15617 2006.12 Inner Mongolia, China
ACCC 15158# No record Nanchang, Jiangxi, China ACCC 15618 2006.12 Inner Mongolia, China
ACCC 15159# 1994 Jingong, Jiangxi, China ACCC 15619# 2006.12 Inner Mongolia, China
ACCC 15161# No record Futian, Fujian, China ACCC 15620# No record Inner Mongolia, China
ACCC 15162# 2018.01 Futian, Fujian, China ACCC 15621# No record Inner Mongolia, China
ACCC 15163# 1994 Futian, Fujian, China ACCC 15622# 2017.12 Inner Mongolia, China
ACCC 15164 No record Huiyang, Guangdong, China ACCC 15623# 2006.12 Inner Mongolia, China
ACCC 15165# 1994 Huiyang, Guangdong, China ACCC 15624# 2006.12 Inner Mongolia, China
ACCC 15166# 1994 Ningming, Guagnxi, China ACCC 15627# No record Inner Mongolia, China
ACCC 15167# 1994 Ningming, Guagnxi, China ACCC 15630# 2006.12 Inner Mongolia, China
ACCC 15168# 1994 Nanning, Guagnxi, China ACCC 15631# 2006.12 Inner Mongolia, China
ACCC 15169# 1994 Nanning, Guagnxi, China ACCC 15632# 2006.12 Inner Mongolia, China
ACCC 15170# 1994 Nanning, Guagnxi, China ACCC 15633# 2006.12 Inner Mongolia, China
ACCC 15171# 1994 Meixian, Guangxi, China ACCC 15634# 2006.12 Inner Mongolia, China
ACCC 15172# No record Changsha, Hunan, China S. meliloti
ACCC 15173# No record Changsha, Hunan, China ACCC 15094# 1990.11 Gagong, Xizang, China
ACCC 15175 2018.04 Changsha, Hunan, China ACCC 15660 No record Zhenyuan, Gansu, China

(待续)

The strains marked with “#” were conducted in nodule experiments.

1.2 菌种活化

在超净工作台中用浸过75%乙醇的脱脂棉球将干燥安瓿管表面擦净，用酒精灯外焰加热安瓿管顶端，将冷的无菌水滴至热的安瓿管顶端使其玻璃碎裂，用无菌镊子敲开安瓿管顶端。

慢生根瘤菌接种于豆芽汁培养基(bean sprouts extract medium, BSE)[

32]：甘露醇/蔗糖10.0 g，MgSO4·7H2O 0.2 g，NaCl 0.1 g，K2HPO4 0.5 g，CaCl2 0.1 g，1% H3BO3 2.0 mL，1% Na2MoO4 2.0 mL，豆芽汁(500 g黄豆芽/绿豆芽用1 500 mL水煮沸30 min，四层纱布过滤2次定容至1 000 mL) 1 000 mL，琼脂20.0 g，自然pH，115 ℃灭菌20 min。快生根瘤菌接种于甘露醇酵母汁琼脂培养基(yeast extract mannitol agar, YMA)：甘露醇/蔗糖10.0 g，酵母粉1.0 g，MgSO4·7H2O 0.2 g，NaCl 0.1 g，K2HPO4 0.5 g，CaCO3 3.0 g，1% H3BO3 2.0 mL，1% Na2MoO4 2.0 mL，琼脂20.0 g，自然pH，115 ℃灭菌20 min。用无菌滴管吸取适量无菌水于安瓿管内，缓慢吹打使冻干菌体悬浮。另取无菌滴管吸取适量菌悬液接种于新鲜的培养基，涂匀，置于30 ℃恒温培养箱避光培养3-5 d，直至出现菌苔。挑取适量菌苔划线纯化，直至出现单菌落。将纯培养的菌株接种到BSE/YMA斜面中，4 ℃冰箱保存备用。

1.3 菌株信息复核

采用细菌基因组DNA提取试剂盒[天根生化科技(北京)有限公司]提取活化菌株的基因组。

PCR反应体系(50 μL)：DNA模板6 μL，上、下游引物(10 μmol/L)各2 μL，ddH2O 15 μL、2×Taq PCR StarMix [康润景星(苏州)生物科技有限公司] 25 μL。用通用引物27F (5′-AGAGTTTGATCMTGGCTCAG-3′)和1492R (5′-GGTTACCTTGTTACGACTT-3′)扩增16S rRNA基因，扩增程序：94 ℃预变性10 min；94 ℃变性30 s，55 ℃退火60 s，72 ℃延伸90 s，循环30次；72 ℃终延伸10 min。用特异性引物41F (5′-TTCGGCAAGGGMTCGRTSATG-3′)和640R (5′-ACATSACRCCGATCTTCATGC-3′)扩增recA，扩增程序：95 ℃预变性2 min；95 ℃变性45 s，58 ℃退火30 s，72 ℃延伸90 s，循环35次；72 ℃终延伸7 min。

扩增产物送至生工生物工程(上海)股份有限公司测序。16S rRNA基因序列提交至EzBioCloud数据库(https://www.ezbiocloud.net/)进行序列比对，recA序列提交至NCBI中的GenBank数据库(https://blast.ncbi.nlm.nih.gov/Blast.cgi)进行BLAST分析比对，对比结果在中国典型培养物保藏中心(http://cctcc.whu.edu.cn/portal/dictionary/index)查询菌株中文名。复核无误后的菌株保藏至4 ℃ YMA斜面和-80 ℃甘油管备用。利用MEGA-X和邻接法(neighbor-joining method)构建系统发育树。

1.4 结瘤实验

1.4.1 松本哲良营养液[

33]

组分A (g/L)：KH2PO4 8.8，KCl 62.0，MgSO4·7H2O 100.0。组分B (g/L)：CaCl2 86.0。组分C (g/L)：柠檬酸铁12.0。组分D (g/L)：NaNO3 12.0，MnSO4·H2O 0.4，ZnSO4 0.1，CuSO4·5H2O 0.1，H3BO3 0.1，Na2MoO4 0.02。使用时4个组分等量混合后稀释200倍，自然pH。

1.4.2 培养体系准备

采用滤纸桥法进行结瘤实验[

34]，每个处理设置3次重复。将滤纸裁剪成350 mm×40 mm滤纸条，纵向对折后再横向对折，横向折痕处向内翻折150 mm制成“M”型，中间凹处根据种子大小剪出“V”型小孔，将滤纸桥放入25 mm×200 mm试管(图1)。每支试管加入适量松本哲良营养液，液面约与“M”型桥底部平齐，用棉塞封口，121 ℃灭菌30 min备用。

fig

图1  滤纸桥法大豆结瘤实验示意图

Figure 1  Schematic diagram of soybean nodulation in filter paper bridge.

1.4.3 种子表面消毒

选取饱满、均一、健壮的大豆种子，放入干燥的500 mL或1 000 mL锥形瓶中(根据种子量选择)，加入浓硫酸没过种子表面，精确处理3 min，其间不断摇晃。倒去浓硫酸，用无菌水冲洗至少10次，直至清洗废液经pH试纸检测呈中性。

1.4.4 种子准备

消毒后的种子用无菌水浸泡6-8 h，无菌水的体积约为种子总体积的250%-300%。吸水膨胀的种子播于铺有4层无菌湿润纱布的铝盒内，避光置于25 ℃恒温培养箱内催芽24-48 h，种子胚根伸长约0.5-1.0 cm为宜，其间保持萌发床湿润。

1.4.5 菌悬液准备

结合菌株入库时的菌株登记信息、农业生产应用效果以及菌种复核结果，选择190株菌株进行结瘤试验，菌株信息见表1。供试菌株斜面长出大面积菌苔后，加入5 mL无菌水，用无菌接种环刮洗菌苔，涡旋仪振荡20 s，制成菌悬液。

1.4.6 播种

挑选萌发状态相似的种子放入无菌培养皿中，倒入菌悬液，使种子均匀附着菌液，浸泡30 min后播种。

最先播种对照组。长柄镊子经火焰消毒后，用无菌水冷却，将种子播种于滤纸桥“M”型凹槽内，胚根嵌入“V”型小孔，菌悬液均匀加入每支试管，棉塞封口。在室温25-28 ℃，14 h/10 h光照培养箱中培养，待幼苗长出几片真叶后摘除棉塞，其间根据实际情况补充营养液。30 d后统计实验结果。

1.4.7 植株结瘤情况和农艺性状调查

结瘤实验结束后，计算结瘤率，统计植株结瘤总数，并迅速摘下根瘤，在分析天平上称量鲜重。结瘤率计算如公式(1)所示。

结瘤率(%)=结瘤植株数量/实验植株总数×100 (1)

以第一子叶叶痕作为划分地上和地下部分的标准，测量地上部分茎尖生长点以下的长度作为株高；测量后将地上部分装入信封，置于烘箱中105 ℃杀青2 h，再80 ℃烘干至恒重后称量干重。计算株高和干重的增长率，如公式(2)所示。

增长率(%)=(处理组平均指标-CK组平均指标)/CK组平均指标×100 (2)

1.5 数据分析

原始数据经过Excel预处理后，用R和GraphPad进行显著性分析及图示，显著性水平设定为α=0.05。

2 结果与分析

2.1 根瘤菌菌种信息复核

本研究对库藏根瘤菌进行了信息复核。213株经16S rRNA基因序列比对为根瘤菌(图2)：156株隶属慢生根瘤菌属(Bradyrhizobium)，占比73.24%；48株隶属中华根瘤菌属(Sinorhizobium)，占比22.54%；9株隶属根瘤菌属(Rhizobium)，占比4.22%。结合recA序列比对，慢生根瘤菌属中，57株鉴定为埃氏慢生根瘤菌(B. elkanii)，占比36.54%；34株鉴定为高效固氮慢生根瘤菌(B. diazoefficiens)，占比21.79%；23株鉴定为甜菜慢生根瘤菌(B. betae)，占比14.74%；19株鉴定为日本慢生根瘤菌(B. japonicum)，占比12.18%；8株鉴定为渥太华慢生根瘤菌(B. ottawaense)，占比5.13%；其余分别为8株黄淮海慢生根瘤菌(B. huanghuaihaiense)、7株圆明慢生根瘤菌(B. yuanmingense)、1株大庆慢生根瘤菌(B. daqingense)和1株辽宁慢生根瘤菌(B. liaoningense)。中华根瘤菌属中，有45株弗氏中华根瘤菌(S. fredii)，占比93.75%，2株萨赫里中华根瘤菌(S. saheli)以及1株圣丰中华根瘤菌(S. shofinae)。根瘤菌属中，有5株线状根瘤菌(R. viscosum)和4株内蒙古根瘤菌黄土亚种(R. mongolense subsp. loessense)。菌株分类信息已在中国农业微生物菌种保藏管理中心官网(http://www.accc.org.cn/)更新。

fig

图2  基于本研究中部分根瘤菌16S rRNA基因序列构建的系统发育树

Figure 2  Neighbour-joining phylogenetic tree based on nearly complete 16S rRNA gene sequences of a part of rhizobium in this study.

213株根瘤菌具有来源多样性(表1)，其中17.37%的菌株来源于海外，剩余菌株主要来源于我国广东(11.27%)、湖南(11.27%)、福建(9.86%)、江西(9.39%)、山东(8.45%)、广西(6.57%)、内蒙古(6.57%)、北京(4.69%)等19个省(自治区、直辖市)。

2.2 与大豆匹配结瘤的根瘤菌筛选

本研究根据菌株信息复核结果以及前期实验结果，共选出186株根瘤菌进行结瘤实验：慢生根瘤菌属138株、中华根瘤菌属43株、根瘤菌属5株。实验规模较大，分为两部分完成，第一部分共109株，第二部分共77株。

在两部分实验中，选择根瘤数量和根瘤质量均排名前10的菌株，结瘤差异统计见表2。第一部分实验筛选出ACCC 15033、ACCC 15065、ACCC 15083、ACCC 15263和ACCC 15611等5株菌，根瘤数量(P>0.23)和根瘤质量(P>0.34)之间均无显著差异；第二部分实验筛选出ACCC 15023、ACCC 15090、ACCC 15254、ACCC 15276、ACCC 15279和ACCC 15282等6株菌，根瘤数量之间无显著差异(P>0.45)，ACCC 15254的根瘤质量最大，ACCC 15282次之，但两者之间无显著差异(P=0.141)，ACCC 15254的根瘤质量与除ACCC 15282外的菌株有显著差异(P<0.12)，ACCC 15282的根瘤质量与其他菌株之间无显著差异(P>0.10)。11株菌的结瘤率均为100%。

表2  高效结瘤根瘤菌的筛选结果
Table 2  Screening results of efficient nodulation rhizobium
Preservation numberStrainAverage number of root nodulesAverage weight of root nodules (g)
ACCC 15033 B. elkanii 10.33±3.68a 0.078±0.009a
ACCC 15065 B. diazoefficiens 11.67±5.44a 0.092±0.028a
ACCC 15083 S. fredii 14.00±2.16a 0.090±0.008a
ACCC 15263 B. elkanii 9.67±3.09a 0.079±0.006a
ACCC 15611 B. japonicum 9.67±0.94a 0.082±0.002a
ACCC 15023 B. japonicum 14.67±7.32a 0.015±0.007b
ACCC 15090 S. shofinae 16.00±1.41a 0.017±0.002b
ACCC 15254 B. diazoefficiens 12.67±6.65a 0.060±0.017a
ACCC 15276 B. diazoefficiens 12.00±3.27a 0.020±0.007b
ACCC 15279 B. diazoefficiens 19.00±15.12a 0.018±0.011b
ACCC 15282 B. diazoefficiens 17.67±12.28a 0.039±0.024ab

Perform univariate ANOVA on the data after passing the Shapiro-Wilk test, n=3, multiple comparisons by the LSD. The data were mean±SE, different lowercases represent significant differences in the treatment groups (α=0.05).

2.3 根瘤菌对大豆植株农艺性状的影响

在第一部分植株样本中，仅ACCC 15611 (0.254±0.007) g处理的干重高于CK (0.236±0.042) g，但差异不显著(P=0.667)。在第二部分植株样本中，ACCC 15108 (0.414±0.010) g、ACCC 15273 (0.409±0.178) g、ACCC 15118 (0.373±0.055) g等34株菌处理的大豆植株干重高于CK (0.255±0.019) g，但差异均不显著(P>0.06)。有27株菌处理的大豆植株株高高于CK (20.87±1.89) cm，其中ACCC 15055、ACCC 15243、ACCC 15197、ACCC 15176、ACCC 15206和ACCC 15611与CK差异显著(表3)，其中ACCC 15055极显著地促进植株生长(P<0.001)，相比CK株高增长了48.81% (表3，图3A)。

表3  高效促生根瘤菌对大豆株高的影响
Table 3  Effects of efficient growth promoting rhizobium on soybean height
Preservation numberStrainStem length (cm)Growth rate (%)
ACCC 15055 B. yuanmingense 30.63±0.82a 48.81***
ACCC 15176 B. diazoefficiens 28.35±0.35a 35.86**
ACCC 15197 B. elkanii 28.63±3.31a 37.22**
ACCC 15206 B. betae 26.00±1.42a 24.60*
ACCC 15243 B. elkanii 29.40±2.28a 40.89**
ACCC 15611 B. japonicum 25.80±2.05a 23.64*
ACCC 15006 B. diazoefficiens 36.46±1.67bcd 21.90*
ACCC 15007 B. elkanii 36.51±1.10bcd 22.09*
ACCC 15085 S. fredii 38.70±4.05abcd 29.42**
ACCC 15086 S. fredii 36.24±5.49bcd 21.18*
ACCC 15101 S. fredii 41.45±0.11abcd 38.61***
ACCC 15108 S. fredii 40.32±1.08abcd 34.81**
ACCC 15118 S. fredii 37.41±0.44abcd 25.11*
ACCC 15119 S. fredii 36.99±0.22abcd 23.69*
ACCC 15123 S. fredii 37.22±0.00abcd 24.44*
ACCC 15131 S. fredii 36.93±2.79abcd 23.50*
ACCC 15143 S. fredii 37.61±1.59abcd 25.76*
ACCC 15147 S. fredii 37.09±4.30abcd 24.03*
ACCC 15191 B. ottawaense 35.84±3.41d 19.86*
ACCC 15194 B. elkanii 43.00±2.80ab 43.80***
ACCC 15222 B. diazoefficiens 42.02±1.17abcd 40.51***
ACCC 15225 B. elkanii 35.92±2.81d 20.10*
ACCC 15250 B. diazoefficiens 39.80±1.35abcd 33.10**
ACCC 15252 B. diazoefficiens 42.83±2.81abc 43.22***
ACCC 15254 B. diazoefficiens 37.76±1.53abcd 26.27**
ACCC 15262 B. elkanii 38.26±2.31abcd 27.93**
ACCC 15264 B. elkanii 39.18±0.58abcd 31.03**
ACCC 15273 B. ottawaense 40.31±1.23abcd 34.80**
ACCC 15275 B. ottawaense 39.88±1.33abcd 33.37**
ACCC 15276 B. diazoefficiens 40.64±1.35abcd 35.89***
ACCC 15279 B. diazoefficiens 39.76±2.26abcd 32.94**
ACCC 15282 B. diazoefficiens 39.57±2.06abcd 32.31**
ACCC 15285 B. elkanii 37.18±4.53abcd 24.32*
ACCC 15291 B. elkanii 38.23±3.83abcd 27.85**
ACCC 15402 B. diazoefficiens 38.04±4.80abcd 27.19**
ACCC 15615 B. japonicum 43.37±0.65a 45.03***
ACCC 15622 S. fredii 38.90±0.83abcd 30.08**
ACCC 15623 S. fredii 40.30±2.12abcd 34.77**
ACCC 15624 S. fredii 38.02±2.56abcd 27.12**
ACCC 15630 B. elkanii 37.38±1.14abcd 24.98*
ACCC 15631 R. mongolense subsp. loessense 36.04±2.82cd 20.50*
ACCC 15632 R. mongolense subsp. loessense 35.88±2.89d 19.98*
ACCC 15634 R. mongolense subsp. loessense 36.16±1.82bcd 20.93*

Perform univariate ANOVA on the data after passing the Shapiro-Wilk test, n=3, multiple comparisons by the LSD. The data were mean±SE, different lowercases represent significant differences in the treatment groups (α=0.05), * represents significant differences between treatment group and CK. *: α<0.05; **: α<0.01; ***: α<0.001.

fig

图3  接种高效促生根瘤菌的大豆长势

Figure 3  Soybean growth inoculated with efficient growth promoting rhizobium. A: ACCC 15055; B: ACCC 15615; C: ACCC 15194; D: ACCC 15252; E: ACCC 15101; F: ACCC 15222; G: ACCC 15276.

在第二部分实验中，39株菌处理的植株均高于CK，除ACCC 15117 (35.42±4.13) cm (P=0.065)和ACCC 15619 (35.20±1.77) cm (P=0.076)处理的植株外，所有植株株高相较于CK均有显著差异(表3)，其中ACCC 15615、ACCC 15194、ACCC 15252、ACCC 15222、ACCC 15101和ACCC 15276对植株的促生作用极显著(P<0.001)，相比CK分别增加了45.03%、43.80%、43.22%、40.51%、38.61%、35.89% (表3，图3B-3G)。

2.4 根瘤菌的结瘤和促生特性差异

所有菌株的结瘤率见表4。根据菌株生长速率不同，将慢生根瘤菌属归类为慢生型根瘤菌(slow-growing rhizobium)，中华根瘤菌属和根瘤菌属菌株归类为快生型根瘤菌(fast-growing rhizobium)。根据结瘤率将根瘤菌分成4类，以此进行列联分析，探究根瘤菌间的结瘤功能差异，分析结果见图4A。快生型根瘤菌整体结瘤率较低，能够结瘤的菌株占比33.33%，66.67%的快生型根瘤菌未检测到根瘤；而未与大豆共生形成根瘤的慢生型根瘤菌仅占6.52%，55.07%的慢生型根瘤菌结瘤率达到100%。卡方检验(Chi-square test)表明根瘤菌类型与结瘤率之间不相互独立(P<0.001)，Pearson相关系数为-0.632 (P<0.001)，即慢生型根瘤菌相比快生型根瘤菌对宿主的选择性更低，能够更广谱地与宿主共生结瘤。菌株保藏时间(P=0.225)或菌株来源(P=0.520)与结瘤率互相独立，无相关性。

表4  结瘤实验结瘤率汇总
Table 4  Summary of nodulation rate in nodulation experiment
Nodulation rate (%)Treatment
0 ACCC 15005 ACCC 15067 ACCC 15068 ACCC 15069 ACCC 15070 ACCC 15071
ACCC 15076 ACCC 15082 ACCC 15102 ACCC 15108 ACCC 15109 ACCC 15118
ACCC 15120 ACCC 15123 ACCC 15125 ACCC 15126 ACCC 15127 ACCC 15129
ACCC 15130 ACCC 15131 ACCC 15133 ACCC 15139 ACCC 15140 ACCC 15142
ACCC 15143 ACCC 15291 ACCC 15402 ACCC 15615 ACCC 15621 ACCC 15622
ACCC 15623 ACCC 15624 ACCC 15631 ACCC 15632 ACCC 15633 ACCC 15634
ACCC 15064# ACCC 15170# ACCC 15196# ACCC 15259# ACCC 15281#
33 ACCC 15007 ACCC 15101 ACCC 15104 ACCC 15106 ACCC 15119 ACCC 15145
ACCC 15147 ACCC 15273 ACCC 15280 ACCC 15284 ACCC 15610 ACCC 15619
ACCC 15620 ACCC 15627 ACCC 15630 ACCC 15022-1# ACCC 15022-2# ACCC 15027#
ACCC 15042# ACCC 15157# ACCC 15158# ACCC 15159# ACCC 15163# ACCC 15215#
ACCC 15241# ACCC 15603# ACCC 15606#
67 ACCC 15006 ACCC 15018 ACCC 15034 ACCC 15085 ACCC 15094 ACCC 15096
ACCC 15121 ACCC 15147 ACCC 15165 ACCC 15185 ACCC 15204 ACCC 15264
ACCC 15283 ACCC 15285 ACCC 15601 ACCC 15020# ACCC 15028# ACCC 15032#
ACCC 15035# ACCC 15046# ACCC 15059# ACCC 15081# ACCC 15097# ACCC 15150#
ACCC 15162# ACCC 15168# ACCC 15176# ACCC 15180# ACCC 15189# ACCC 15190#
ACCC 15193# ACCC 15195# ACCC 15198# ACCC 15238# ACCC 15248# ACCC 15257#
ACCC 15258# ACCC 15293#
100 ACCC 15023 ACCC 15033 ACCC 15057 ACCC 15065 ACCC 15083 ACCC 15084
ACCC 15086 ACCC 15090 ACCC 15095 ACCC 15107 ACCC 15117 ACCC 15169
ACCC 15173 ACCC 15182 ACCC 15191 ACCC 15194 ACCC 15203 ACCC 15222
ACCC 15222 ACCC 15223 ACCC 15225 ACCC 15226 ACCC 15228 ACCC 15229
ACCC 15230 ACCC 15245 ACCC 15246 ACCC 15246 ACCC 15247 ACCC 15250
ACCC 15252 ACCC 15254 ACCC 15255 ACCC 15256 ACCC 15262 ACCC 15263
ACCC 15269 ACCC 15275 ACCC 15276 ACCC 15277 ACCC 15279 ACCC 15282
ACCC 15611 ACCC 15021# ACCC 15036# ACCC 15038# ACCC 15043# ACCC 15045#
ACCC 15047# ACCC 15055# ACCC 15058# ACCC 15060# ACCC 15156# ACCC 15161#
ACCC 15166# ACCC 15167# ACCC 15171# ACCC 15172# ACCC 15186# ACCC 15187#
ACCC 15192# ACCC 15197# ACCC 15202# ACCC 15206# ACCC 15212# ACCC 15214#
ACCC 15216# ACCC 15217# ACCC 15218# ACCC 15220# ACCC 15221# ACCC 15231#
ACCC 15235# ACCC 15239# ACCC 15243# ACCC 15251# ACCC 15260# ACCC 15289#
ACCC 15605# ACCC 15608#

The strains marked with “#” were inoculated in the first part, while the left was inoculated in the second. The nodulation rate in CK was 0 in both parts.

fig

图4  不同类型根瘤菌结瘤促生差异比较。A：结瘤率；B：促生效果显著性。

Figure 4  Comparison of nodulation and growth promotion between different kinds of rhizobia. A: Nodulation rate; B: Significance of growth promotion effect.

结瘤实验结果表明，供试根瘤菌对大豆植株地上部分的干物质积累无显著影响，但共有93株根瘤菌能够增加大豆株高，其中41株菌有显著的促生效果(P<0.05)。根据P值将菌株分成4类进行列联分析，探究不同类型根瘤菌间促生特性的差异，分析结果见图4B。快生型根瘤菌中能够显著促进植株生长的菌株占38.46%：10.26%促生效果非常显著(0.001≤P<0.01)，5.13%促生效果极显著(P<0.001)；48.15%的慢生型根瘤菌能显著促进植株生长：促生效果非常显著的菌株占比20.37%，促生效果极显著的菌株占比14.81%。虽然慢生型根瘤菌中促生效果非常显著和极显著的菌株均比快生型根瘤菌多，但卡方检验结果表明根瘤菌类型与促生能力之间相互独立(P=0.162)，即不同类型根瘤菌的促生能力无相关性。

3 讨论与结论

微生物菌种资源的复核是针对那些被正式收录和保藏的菌株所进行的纯度、分类地位和功能特性的核查。本研究基于16S rRNA基因和recA序列，对241株库藏大豆根瘤菌进行了信息复核，明确了其系统分类地位，并对其与大豆的共生结瘤活性进行了检测。

16S rRNA基因序列比对与进化分析是研究原核生物系统发育地位的最常用方法。16S rRNA基因在细菌中普遍存在，其基因中同时存在高度保守的区域和高度可变的区域，因此适合作为衡量细菌生命进化过程中亲缘关系的标准。如果2个菌株的16S rRNA基因相似性>99.0%，则被认为属于同一种；若<98.7%，则可能为一个新种。因此，16S rRNA基因序列常用于根瘤菌的分类鉴定[

35-36]，有些研究也会选择23S rDNA[37-38]或16S-23S rDNA内转录间隔区[39-41](internal transcribed spacer, ITS)。然而，由于16S rRNA基因的高度保守性以及基因片段的互相转移现象[42-43]，仅基于16S rRNA基因的根瘤菌系统发育体系并不十分准确。根瘤菌中具有特殊功能的基因，如nodA[44-46]和nifH[47-48]，以及一些保守性较强的蛋白编码持家基因，如dnaK[49-50]、glnⅡ[51-53]、gyrB[39,54]和recA[55-56]等可作为16S rRNA基因的替代或补充，用于在种属水平对细菌进行精准鉴定。本研究结合了16S rRNA基因与recA基因序列进行系统分类鉴定。

最初能与大豆结瘤的根瘤菌均被分类为B. japonicum。Hollis等[

57]通过DNA/DNA杂交技术将其分类为3个类群，其中Group Ⅱ被定义为一个新种，命名为B. elkanii[58]。随着B. japonicum[59]和B. huanghuaihaiense[60]相继从我国大豆根瘤中分离，B. yuanmingense[61]、B. liaoningense[59]以及R. leguminosarum[47,62]等菌种也被发现具有与大豆结瘤固氮的能力。根瘤菌与宿主的共生关系具有选择性[63]。马中雨等[64]的研究发现，慢生型根瘤菌比快生型根瘤菌对宿主的选择性更为松弛，这一点与本研究的结论一致。尽管土壤中根瘤菌广泛分布，但其中大部分为无效或低效菌株，仅有一小部分能与大豆结瘤，且多集中在慢生根瘤菌属和中华根瘤菌属。因此，选择能有效与大豆匹配结瘤的根瘤菌是推广根瘤菌菌剂的前提。本研究筛选出一批能有效与大豆匹配结瘤的根瘤菌，其中包括ACCC 15023、ACCC 15033、ACCC 15065、ACCC 15083、ACCC 15090、ACCC 15254、ACCC 15263、ACCC 15276、ACCC 15279、ACCC 15282、ACCC 15611等11株能与大豆高效结瘤的菌株，也包含了宿主选择性严苛的快生型根瘤菌。此外，ACCC 15055、ACCC 15101、ACCC 15194、ACCC 15222、ACCC 15252、ACCC 15276、ACCC 15615等43株根瘤菌能够显著促进大豆植株生长，为后期菌剂研发和功能菌株筛选创造了条件。

由于本研究的局限性，所使用的菌株在地理和时间维度上并未表现出显著的结瘤差异，未来可针对性地从不同来源以及不同保藏时间的根瘤菌中筛选出有应用前景的菌株，在丰富大豆根瘤菌种质资源库的同时，了解农耕环境和自然环境对根瘤菌种群数量、遗传背景以及结瘤固氮能力的影响。在根瘤菌分离鉴定过程中，可检验菌株与目前主要栽培大豆品种的结瘤能力，以完善菌种信息。此外，由于不同品种大豆的遗传差异，能够招募共生的根瘤菌各不相同。未来可将本研究筛选出的高效菌株与我国不同地区种植的大豆品种进行交叉结瘤实验，以筛选出更有应用前景的菌株，为育种以及微生物肥料的研发和推广提供参考。

作者贡献声明

韩嘉诚：水培实验，调查大豆植株农艺性状以及菌株结瘤形状，数据分析，撰写文章；朱宏图：负责菌株整理、活化与鉴定，完成水培实验；杨芾：协助数据处理；郭捷：协助完成实验；马晓彤：指导并协助完成水培实验，设计、指导实验；张晓霞：指导实验设计，提供研究所需的资源与材料，修订文章。

利益冲突

作者声明不存在任何可能会影响本文所报告工作的已知经济利益或个人关系。

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