全 文 ：西北植物学报牞2007牞27牗1牘牶0121 - 0125
Acta Bot牣Boreal牣-Occident牣Sin牣
　　文章编号牶1000-4025牗2007牘01-0121-05 *
新疆乌恰县豆科植物根瘤菌数值分类研究
龚明福1牞2牞韦革宏1*牞曹　鹏1
牗1 西北农林科技大学生命科学学院牞西北农林科技大学 微生物研究中心牞陕西杨陵 712100牷2塔里木大学新疆生产建设兵团塔
里木盆地生物资源保护利用重点实验室牞新疆阿拉尔 843300牘
摘　要牶新疆乌恰县具有独特的地理环境与气候条件牞蕴藏着丰富的结瘤豆科植物资源牣本研究从乌恰县豆科植物
根瘤中分离到 40 株根瘤菌牞并进行唯一碳源利用 、唯一氮源利用 、对抗生素和染料的抗性 、耐盐性 、初始 pH 生长 、
生长温度范围及石蕊牛奶反应 、氧化酶 、过氧化氢酶 、脲酶共 118项生理生化性状测定牣结果表明牶乌恰县豆科植物
根瘤菌在碳氮源利用 、抗生素敏感性 、对染料抗性程度等方面存在着差异牣供试根瘤菌能耐受较低温度牞并具有较
强的耐盐 、碱能力牞均能在 pH 6～ 12 的 YM A培养基上生长牞有 60 %的菌株能耐受 0牣86 mol牤L 的 NaCl牣数值分类
结果表明牞在 84牣2 %的相似水平上 40 株供试菌株构成了 4 个新的表观群牣第Ⅰ 、Ⅱ 、Ⅲ和Ⅳ类群分别有 8 、9 、3 和 3
株菌牞中心菌株分别为 NWWQ73-2、NWWQ13-1 、NWWQ1-2 、NWWQ6-2牣此外牞乌恰县豆科植物根瘤菌与模式菌
株的相似性较低牞它们所形成的 4 个独立群可能有新种存在牣
关键词牶根瘤菌牷表型性状牷乌恰县牷数值分析
中图分类号牶Q939牣11 +4 文献标识码牶A
Numerical Classification of Rhizobia in Leguminous
Plants of Wuqia County牞Xinjiang
GONG M ing-fu1牞2牞WEI Ge-hong1*牞CAO Peng1
牗1 College of Life S cien ces牞Research Center of M icrobiology牞Northw es t S ci-Tech Universi ty of Agricul tu re and Forest ry牞Yan-
gling牞Shaanxi 712100牞Chin a牷2 Xin jiang Product ion & S tru ction C orps Key Laborato ry of Protect ion & Utili zation of Biological
Resources in Tarim Basin牞Tarim University牞Alar牞Xin jiang 843300牞China牘
Abstract牶Wuqia County of Xinjiang has abundant leguminous plants capable o f forming nit rogen-f ixing ro ot
nodules because of it s unique geog raphical and climatic condi tions牣This study isola ted fo rty st rains o f rhi-
zobia and conducted the tests about their 118 physio logical and biochemical paramete rs concerned w ith the
uses o f single carbon and nit rogen sources牞antibiotic and chemical dye resistance牞grow th at the ini tial pH牞
temperature range of grow th牞oxidase牞supe ro xidase and urease牞and li tmus and milk responses牣It w as
show n that the rhizobia in Wuqia County of Xinjiang dif fe red in carbon and nit rogen uses牞antibiot ic sensi-
tivity牞and chemical dye resistance牣The tested rhizobia could tolerate relatively low temperature牞had st rong
salt-and alkaline-to le ra ting capacit ies and w as capable of g row ing on Mannitol-yeast ex t ract medium牗
YEM牘w ith pH at w ithin 6 ～ 12牞and six ty percent o f them could tolerate 0牣86 mol牤L NaCl牣Numerical clas-
sification revealed that the tested fo rty st rains o f rhizobia formed four new pheno typic clusters牣with thei r
similarity at 84牣2%牣Clusters Ⅰ牞Ⅱ牞Ⅲ and Ⅳ separately contained 8牞9牞3 and 3 strains and thei r cent ral
st rains we re separately NWWQ73-2牞NWWQ13-1牞NWWQ1-2 and NWWQ6-2牣In addi tion牞the rhizobia in
* 收稿日期牶2006-05-30牷修改稿收到日期牶2006-09-27
基金项目牶全国优秀博士学位论文作者专项基金牗200254牘牷国家自然科学基金牗30470040牘牷教育部新世纪优秀人才支持计划牗NCET-04-
0953牘牷塔里木大学校长基金重点资金项目牗TDZKZD06001牞2004-01-02牘牷新疆生产建设兵团科委项目牗2006GG26牘
作者简介牶龚明福牗1970-牘牞男牗汉族牘牞在读博士牞副教授牞主要从事微生物资源研究牣
*通讯作者牶韦革宏牞博士牞教授牞博士生导师牞主要从事微生物多样性及分子系统学牣E-mai l牶weighong@yahoo牣com
the leguminous plants of Wuqia County w ere similar to the type strains and the new clusters that they
fo rmed probably contained new species牣
Key words牶rhizobium牷pheno typic t rai t牷Wuqia County牷numerical analy sis
　　Rhizobia a re soil bacte ria capable o f forming
nit rog en fixing nodules on roots and in some cases
on stems of plants o f the family Legumino sae牣Up
to now mo re than 40 species belonging to nine gen-
era have been described
犤1 ～ 3犦牞despite the fact that
there are 700 genera and 18 000 ～ 20 000 legume
species牞most of w hich are capable of symbio sis
wi th rhizobia
犤4犦牣
There are a lo t o f legumes in Wuqia county牞
Xinjiang Uygur A utonomous Region because of the
special geog raphical envi ronments and weather
condi tion in this region牣
This report covers our studies on rhizobia iso-
lated f rom leguminous plant grow n in Wuqia coun-
ty o f Xinjiang Uygur Autonomous Region牞thei r
phenotypic feature s牞and their taxonomic relat ion-
ship w ith the previously described species o f nodule
bacteria牣
1　Materials and methods
1牣1　Bacterial strains
Table 1 lists the sources of rhizobial st rains
used in this study牣
Table 1　The sources of rhizobial strains used in this study
S t rains Host S ources
NWWQ105 Mel i lotus a lba This s tudy
NWWQ107-1牞NWWQ111-2牞NWWQ51-1牞NWWQ57-1牞NWWQ71-1牞NWWQ71-2牞NWWQ73-2 Vicia f aba This s tudy
NWWQ65-2牞NWWQ78-1牞NWWQ90-1 Astraga lus libetanus This s tudy
NWWQ56-1牞NWWQ64-2牞NWWQ66-1牞NWWQ68-1牞NWWQ84-2 Meli lotuso f f icinal is This s tudy
NWWQ26-1牞NWWQ32-2牞NWWQ36-2牞NWWQ38-2牞NWWQ44-1牞NWWQ47-2牞NWWQ54牞NW-
WQ75-1牞NWWQ94-1 Thermop sis kax gar ica This s tudy
NWWQ103-1 H edysarum krasshovi i This s tudy
NWWQ100-2牞NWWQ62-2牞NWWQ104-1 Ammopip tanthus nanus This s tudy
NWWQ102-1牞NWWQ97-2 Glycyrrhiza uralensis This s tudy
NWWQ1-2牞NWWQ10-2牞NWWQ11-2牞NWWQ13-1牞NWWQ18-1牞NWWQ20牞NWWQ22-1牞NW-
WQ4-2牞NWWQ6-2 Med icago sat iva This s tudy
1牣2　Culture conditions
Mannitol-yeast ext ract medium 牗YEM牘 was
routinely used fo r isolation牞purification牞and main-
tenance of rhizobia
犤5犦牣Fo r some tests牞minimal
BS牏medium was used 犤6犦牣Bacterial cul tures w ere
incubated at 28℃牞unless o therwise stated牣The pu-
ri ty of the bacteria w as checked by repeated st reak-
ing as w ell as by microscopic examination牞and their
identity w as invest igated by plant infection tests on
the o riginal host plants牣
1牣3　Phenotypic tests
1牣3牣1　Morphology of cells and colonies　The st rains
were tested for Gram reaction牞cell shape牞colony struc-
ture牞color牞and presence of gummy substances 犤7犦牣
1牣3牣2　Growth characteristics　In o rder to deter-
mine the to le rance to sodium chlo ride of unknown
rhizobia牞thei r capacity to g row at different pHs
and temperatures牞the star ter cul tures w ere g row n
at 28℃ in YEM bro th for 18 h牞next diluted 10
t imes in f resh medium牗time 0牘牞and further incu-
bated as described below牣The pH range w as de-
fined in liquid YEM medium牗pH 4牞5牞8牞9牞10牞11
and 12牘牣Sodium chlo ride to lerance w as tested in
YEM medium supplemented w ith NaCl at a con-
centration of 1%牞2%牞3%牞4%牞5% and 6%牗W牤
V牘牣The range of g row th temperatures w as investi-
gated by incubation o f bacterial cul tures in YEM
medium at 4℃牞28℃牞40℃牣Control cul tures for all
tests w ere g row n in YEM medium under standard
conditions牗pH 7牞28℃牘牣The mo tility of bacteria
w as asse ssed in a semi-so lid YEM agar牗0牣3%牞W牤
V牘by their migrat ion aw ay from the spot of inocu-
lation牣Tests w ere done in t riplicate牣
1牣3牣3 　Antibiotic resistance 　Int rinsic antibio tic
resistance w as tested in YEM agar supplemented
w ith one of the follow ing antibio tics at one concen-
122 西　北　植　物　学　报　　　　　　　　　　　　　　　　　　　27 卷
t rat ion牗μg爛mL - 1牘 o f 5牞50牞100牞300牶albomycin牞
penici llin牞st reptomycin牞linneimycin牞lincomycin牞
kanamycin牞gentamycin牣The plates containing bas-
al YEM medium were used as controls牣Tests w ere
done in t riplicate牣
1牣3牣4　Tolerance to dyes　Resistance to dyes w as
investig ated in YEM medium containing the fol-
lowing dyes at one concentration 牗%牞W牤V牘 o f
0牣1牞0牣2牶deoxysodium cholate牞neutrall red牞me thyl
green牞methy l red牞methy l orange牞thymol blue牣
Plates containing basal YEM agar were used as
controls牣Tests w ere done in t riplicate牣
1牣3牣5　Biochemical tests　A cid or alkali produc-
tion w as dete rmined in YEM aga r medium w ith
bromo thymol blue indicato r 牗0牣002 5%牞W牤V牘牣
The uti lization of v arious carbon sources w as stud-
ied in BS agar w here mannitol was replaced by one
of the following compounds牗each at a concentra-
tion o f 0牣1%牞W牤V牘牶sucro se牞sodium hipporate牞in-
o sitol牞 ery thritol牞 so rbitol牞 sodium py ruvate牞
melibilse牞arabinose牞so rbo se牞rhamnose牞g lucogen牞
cerebro se牞D-xylopy ranose牞D-ribose牞sodium ci t-
rate牞gluco seamine hydrochlo ricde牞glucose牞malto-
biose牞lacto se牞D-levulose牞sodium aceta te牞sodium
succinate牞mannose牞meli to se牞ino sito l牣Utilization o f
lactamic acid牞L-no rleucine牞3牞5-Br-ty ro sine牞L-leu-
cine牞α-ornithine牞asparagines牞L-cy steine牞glycine牞
L-g lutamine牞L-ly sine牞L-arginine牞DL-histidine牞L-
t ryptophane牞L-pheny lalanine牞L-methionine牞L-cy s-
tine牗each a t a concentrat ion of 0牣1%牞W牤V牘was
investig ated in BS agar w ith tested substance in-
stead of NH 4Cl牣The g row th o f microo rganisms in
BS medium served as a po sitive control牣T he reac-
tion in li tmus milk and the activi ties of catalase牞u-
rease牞oxidase牞and nit rate reductase w ere investiga-
ted by the methods described by Gong犤8犦牣Tests
were done in t riplicate牣
1牣4　Numerical analysis
Fo r numerical analy sis牞the pheno typic fea-
tures of the studied bacteria w ere coded in the bi-
nary system牞and then the similarity of rhizobia w as
calculated 牗by simple matching coef ficient牘牞and
their clustering w as perfo rmed牗by the U PGMA
method牘with the so f tw are Mints package犤9犦牣
2　Results and discussion
We studied pheno typic propert ies of 40 st rains
iso lated from roo t nodules of leguminous plant
g row n in Wuqia county牞Xinjiang牞to determine
their diversity and relationship w ith the reference
st rains牣All studied st rains w ere Gram-negat ive牞
rod-shaped bacteria牞0牣6 ～ 0牣9 mm×1牣4 ～ 3 mm牞u-
sually containing g ranules o f poly β-hydro xybu-
tyrate牗data no t presented牘牣In YEM medium牞all
studied microsymbionts w ere synthe sized exopo-
ly saccharides characteristics fo r all rhizobia牣Nodu-
lators g rew at temperatures f rom 4℃ to 40℃w ith
an optimum o f 28℃～ 30℃牞and at pH from 6 to
12牣No strain tolerated pH 4牣There w ere 60%
st rains o f the nodule iso lates resisting to 6%NaC l牣
The int rinsic resistance of symbionts to the tested
antibiotics w as fai rly uni fo rm牣As a g roup they
showed a markedly high sensitivi ty to linneimycin牞
st reptomycin牞al though ammonium chloride w as a
bet ter nit rogen source for their grow th牗data not
present牘牣
Phenotypic features of unknown nodulators
and reference bacteria w ere subjected to computer
analy sis by Mints prog ram牣A numerical analy sis of
115 metabo lic propert ies al lowed us to group the
studied st rains into four phena and 17 separate lin-
eages a t a cor relation coef ficient of 84牣2%牣The re-
sults of strain clustering 牗UPGMA method牘 was
shown in Fig牣1牣
Cluster Ⅰ had 8 st rains and NWWQ73-2 w as
the central st rain牣Cluster Ⅱ had 9 st rains and NW-
WQ13-1 w as the central st rain牣Cluster Ⅲ had 3
st rains and NWWQ 1-2 w as the central strain牣C lus-
ter Ⅳ had 3 st rains and NWWQ6-2 w as the central
st rain牣Three clusters牗except cluster Ⅳ牘 contained
nodule bacteria isolated f rom more than tw o
plants牞whereas those st rains isolated f rom Medi-
cago sativa belonged to dif ferent cluster牣It w as hy-
pothesized tha t a high diversi ty of microo rganisms
might be the resul t of dif ferent plant o rigin牞di ffer-
ent g eographic o rigin牣The mechanism o f bacterium
heterog eneity proposed by Chen et al犤3犦牣It w as
suppo rted by us because same cluste r contained
1231 期　　　　　　　　　　　　龚明福牞等牶新疆乌恰县豆科植物根瘤菌数值分类研究牗英文牘
124 西　北　植　物　学　报　　　　　　　　　　　　　　　　　　　27 卷
nodule bacte ria o f different plant o rigin牞while the
rhizobia originated f rom same plant w ere belong to
dif ferent cluster o r same cluster牣
In taxonomic studies of nodule bacteria牞the
ho st specif icity is st ill used as one of the crite ria牞
although牞a cross-inoculation group concept as a
majo r principle fo r rhizobium classification w as a-
bandoned牣U nknown st rains o f rhizobia牞i solated
f rom roo t nodules o f leguminous deriving f rom
Wuqia county牞Xinjiang牞fo rmed effective symbiosis
w ith the original host牣
References
犤1犦　M A EK W牞SAJNAGA E牣Current taxonomy of the rhiz obia犤J犦牣A cta Microbiol牣Pol牣牞1999牞48牗1牘牶109- 122牣
犤2犦　V AN BERKUM P牞EARDLY BD牣Molecular evolutionary system at ics of th e Rhizobiaceae犤A犦牣In牶Spaink H牞Kondorosi A牞H ooykass PJJ牞
牗eds牘牞T he Rhizobiaceae犤M犦牣Dord rech t牶Kluwer Academic Publi shers牞1998牶1- 24牣
犤3犦　CHEN W X牗陈文新牘牞WANG E T牗汪恩涛牘牞CHEN W F牗陈文峰牘牣Th e relationship betw een the symbiotic p romiscui ty of rhizobia and
legumes and their geographical en vi ronm en ts犤J犦牣S cient ia A gr icu ltura S inica牗中国农业科学牘牞2004牞37牗1牘牞81 - 86牗in Chinese牘牣
犤4犦　A LLEN O N牞ALLEN E K牣The Legumin osae牶a source b ook of ch aracteri st ics牞u ses and nodulat ion犤M犦牣Madison牶The Universi ty of Wis-
con sin Pres s牞1981牣
犤5犦　VINCENT J牣A manual for the pract ical stu dy of root nodu le bacteria犤A犦牣In牶International biological p rog ramme handbook牗No牣15牘犤M犦牣
Oxford牶Blackw ell S ci牣Pub牣牞1970牣
犤6犦　SHERWOOD M T牣Im proved synthetic medium for the g row th of Rhizobium犤J犦牣J牣A ppl牣Bacter iol牣牞1970牞33牗2牘牶708- 713牣
犤7犦　SMIBERT R M牞KRIEG N牣Sys tematics牶general characterizat ion犤A犦牣In牶Gerhardt P牞Cos til ow RN牞Nester EW牞Wood WA牞K rieg NR牞
Phi llips GB牗eds牘M anu al of meth ods for general bacteriology犤M犦牣Washing ton牞DC牣牶American S ociety for M icrobiology Pres s牞1981牶409
- 443牣
犤8犦　GONG M F牗龚明福牘牞WEI G H牗韦革宏牘牞LIU J H牗刘江华牘牞CAO P牗曹　鹏牘牞SU P牗苏　鹏牘牣Numerical taxonomy of rhi zobia is olated
f rom the nodules of Sophora a lopecuroides f rom Xinjiang犤J犦牣B iod iver sity S cienc牗生物多样性牘牞2005牞13牗1牘牶75- 80牗in C hinese牘牣
犤9犦　M A J C牗马俊才牘牞ZHAO Y F牗赵玉峰牘牣The application of hierachical clustering meth od in numerical taxonomy of microorganism s犤J犦牣
Microbiolog y牗微生物学通报牘牞1986牞13牗1牘牶225- 228牗in C hinese牘牣
1251 期　　　　　　　　　　　　龚明福牞等牶新疆乌恰县豆科植物根瘤菌数值分类研究牗英文牘