全 文 ：　文章编号 :100028551 (2000) 0520305206
Colonization of A lcaligenes f aecalis on the rice
roots under the salt st ress
H Wu1 　SZ Ping2 　KA Malik3 　M Lin2
(11 Southwest Nationalities College , Chengdu , S ichuan Prov . 　610041 ;
21 Instit ute f or A pplication of A tomic Energy , Beiji ng 　100094 , China ;
31 National Instit ute f or Biotechnology and Genetic Engineering , P. O . Box 577 , Faisalabad , Pakistan)
ABSTRAT:Alcaligenes f aecalis A 1501 could grow well in sol id of l iquid medium con2
taining 4 %NaCl. A 1501 could produce IAA under the salt stress and maintain eff i2
cient N2f ixing activity when NaCl concentration is lower than 4 %. Bacteria lost
chemotactic capabil ity when concentration of ammonia is higher than 310 %. A1501
colonized on root surface and root hairs , especially on zones of emergence of lateral
roots and eff iciently f ix nitrogen there under the salt stress. 015 % of NaCl enhanced
colonization of A 1501 on root surface. A1501 could enter the roots at points of emer2
gence of lateral roots.
Key words :colonization ; rice ; Alcaligenes faecalis ; the salt st ress
收稿日期 :2000206209
基金项目 :国家教委留学回国科研基金
作者简介 :伍红 (1965～) ,女 ,成都人。西南民族学院讲师。从事生物技术和生物化学方面的研究工作
This work is supported by National high technology development (863) plan (8632101203204201) and 948 Programme of Min2
istry of Agriculture (19482972086)
There are about 20 million hectares of saline land in North and East China ,which causes a
loss of approximately 30 million tons of crops annually. Some studies conducted in China showed
that biofertilizer associated with salt2tolerant nit rogen2fixing bacteria could promote growth of
plants and increase the yield of crops. In recent years , these bacteria which serve as both plant2
growth promoting rhizobacteria ( PGPR) and nit rogen2fixers have been att racted much more atten2
tion[2 ,3 ] . Effect of these kinds of bacteria on plant growth promotion is depend on its characters ,
such as motility and chemotaxis to root exudates , accumulation on root surface , production of
siderophore or bactericin , attachment and colonization to root surface , phytohormone secretion ,
efficiency of t ransfer of the fixed nit rogen to host plant etc. [5 ] . Among them the effective colo2
nization has been proposed to be one of the most essential factors , especially under the salt st ress.
The mechanism of attachment of A zospi rill um brasilense to wheat roots has been reported
by Michiels et al. using 3 H2labelling bacteria cells and root system without sheet . They proposed
two attachment modes :adsorption and anchoring[6 ] . Attachment of other bacteria ,especially Rhi2
503　核 农 学 报　2000 ,14 (5) :305～310Acta A gricult urae N ucleatae Sinica
zobi um bacteria to their specific legume host plant , also has been well described. It is thought to
be mediated by a root lectin that recognizes a specific receptor on the bacterial cell surface , and the
EPS or L PS(lipopolysaccharides) may also play important role in the attachment .
The salt2affected soils are not noly saline but also impermeable , poor2nit rogen and devoid of
organic matter. As establishment of associative symbiotic system in rice rhizosphere , diazotrophs
can utilize exudate f rom rice root and rice plant can uptake nit rogen fixed by diazotrophs. A lcali2
genes f aecalis are known to colonize the rhizosphere of rice plants , and part of the bacteria can
penetrated into root cells and fix dinit rogen there. But now , we have a little knowlegde about
mechanism of devolopment of benifit repationship between rice and A . f aecalis under the salt
st ress.
1 　Materials and methods
111 　Bacterial strains
The bacterial st rains used in this experiment are listed in Table 1.
Table 1 　Bacteria strains used
strains genotype or phenotype sources or references
A lcaligenes f aecalis
A1501 wild type Kms Qiu et al ,1981 [1 ]
A1531 exo - Kmr Lin and You ,1992 [5 ]
A1532 exo + + Kmr Lin and You ,1992 [5 ]
A1533 exo - che - Kmr Lin and You ,1992 [5 ]
A1541 che - Kmr Lin and You ,1992 [5 ]
A15H1 nif H2lacZ Kmr Ping et al ,1996 [7 ]
A15H1 nifA2lacZ Kmr Ping et al ,1996 [7 ]
A15L1 nifL2lacZ Kmr Ping et al ,1996 [7 ]
112 　Cultivation of rice seedl ings
Rice ( O ryz a sativa L . ) seeds cv. Yuefu were surface2sterilized with 011 % HgCl2 for 15 min
and then by 70 %(V/ V) ethanol for 3 min. Sterilized seeds were germinated and grown on steril2
ized sand at 25 ℃, 80 % relative humidity and a 16 h2day/ 8 h2night cycle[5 ] . 14 days old seedling
with approximately equally2sized root system were selected and washed free2of sand with sterilized
water , and then the seedlings were transferred to flasks (50 plants per flask) for further studies.
All procedure described above was under st rict aseptic condition.
113 　Bacteria attachment to rice roots
Single colony of A . f aecalis was picked up and inoculated in 50 ml LB medium containing
(15N H4) 2 SO4 (abundance 9517 %) ,which supplemented with 50μg/ ml kanamycin for mutants ,
respectively. Bacteria were grown aerobically. The 15 N2labelled bacteria were collected by cen2
t rifugation when their OD560 values were reached up to 110 , corresponding to 1 ×108 cells/ ml.
Then bacteria were wished twice in sterilized 85 % NaCl to remove unincorporated label , and re2
suspended to the original volume in rice N2f ree mineral medium ( RMM) containing 015 % NaCl.
603 核　农　学　报 14 卷
The abundance of bacteria obtained in this way was 4518 %. 015ml of labelled bacterial cells were
added to flasks containing 50 rice seedlings grown in RMM containing 015 % NaCl. Rice samples
were taken out at different time after inoculation , and then the roots were treated three times by
immersion in distilled water for 1 min without agitation or smoothly vortexing for 5 s or shaking
drastically with vortex for 10s[6 ] . Fresh roots with different t reatments was taken and 15N abun2
dance was measured by mass2spectrometer model MA T2250A according to the method of You[9 ]
and the adsorbed , anchored and colonized bacteria were calculated.
114 　Chemotaxis assay
The chemotaxis assay was performed as described by Adler [1 ] . About 109 motile cells were
deposited at the center of a plate containing 012 % agar. Measure was taken after 18 hours of in2
cubation at 37 ℃.
2 　Results and discussion
211 　Some properties of Alcaligenes f aecalis under the salt stress
A lcaligenes f aecalis A1501 , which was isolated from paddy soils in South China[8 ] ,could
grow well in solid or liquid medium containing 018mol/ L NaCl. Growth of bacteria was repressed
in solid medium containing 112mol/ L NaCl. A1501 grew very slowly even if NaCl concentration
reached 116mol/ L in liquid medium ( Table 2) . A1501 could produce IAA under the salt st ress
(Table 3) and maintain N2fixing activity till NaCl concentration was higher than 110mol/ L .
Southern hybridization of total DNA from A1501 grew under condition of different NaCl concen2
t ration with Klebsiella pneumoniae nif HD K DNA probe showed that homologous hybridization
bands under the normal condition was identical to that under the salt st ress. A study on chemo2
taxis of A1501 under the different concentrations of ammonia was made. The results showed that
bacteria lost chemotactic ability when concentration of ammonia was higher than 310 % ( Table.
4) .
Table 2 　Effect of different concentration of NaCl on growth of A. f aecalis grown in solid medium
Na + (mol/ L) 012 014 016 018 110 112
solid medium + + + + + + + + + + + -
　　Note : + + + 　grow well ; + + 　grow normally ;
+ 　grow slowly ; - 　No growth ;
Table 3 　IAA produced by A. f aecalis A1501 under the salt stress( 210 %NaCl)
time of incubation A1501 Klebsiella Sp . 101 Klebsiella NIAB2Ⅰ
24 + + + -
48 + + + -
72 + + + -
96 + + + -
120 + + + -
　　Note : + 　IAA production ; - 　No IAA production
703　5 期 盐胁迫下粪产碱菌在水稻根表的定殖
Table 4 　Chemotaxis of Alcaligenes f aecalis A1501 under the different concentrations of NaCl
concentration
of NaCl ( %) 010 0101 0105 110 210 214 218 310
diameter of
chemotactic cycle (cm) 515 616 713 513 411 310 110 010
212 　Adsorption of Alcaligenes f aecalis A1501 to rice roots under the salt stress
Attachment of A lcaligenes f aecalis A1501 to plant root surface has been reported previously ,
however , the mechanism and process remain unclear. Adsorption of A . f aecalis by rice root starts
immediately after inoculation (40min or less) and reaches a level of 310 % of the initial inoculant ,
corresponding to 310 ×106 bacteria per g fresh root . While E. coli HB101 almost can not be ad2
sorbed on rice root surface. Adsorbed A . f aecalis could be removed from root surface by vortexing
smoothly three times for 5s in water. These results demonstrated that the adhesion is rapid , but
interaction between host plant and bacteria is weak under the salt st ress.
Adsorption of Che2mutants is quite different f rom A . f aecalis A1501 and E. coil HB101 and
their ability of adsorption is lower than the wild type strain A1501 but higher than E. coli , which
reach maximal level (211 %) after 4 h of inoculation. These data indicated that the Che2mutants
only lost part of adsorbing ability , suggesting that the chemotaxis is just one of factors which af2
fect the adsorbing ability.
Scanning electron microscopy (SEM) of inoculated roots revealed massive adsorption of bacte2
ria on rice root surface , but no special site was shown. However , only a few bacteria could be
found on rice root cap , and heavy adsorption occurred on joint of main roots and lateral roots of
rice plants grown under the salt st ress.
213 　Anchoring of A . f aecalis A1501 to rice roots under the salt stress
A . f aecalis A1501 starts anchoring on the rice roots after 10 h of inoculation and reaches to
maximal level (19 %) after 22 h of inoculation. Anchored bacteria could not removed from root
surface by smoothly vortexing in water for 5s. E. coli HB101 can not anchor on rice roots. Part
of anchored A . f aecalis could be removed from rice root surface by drastically vortexing. The facts
suggested that the anchoring of bacteria on root surface be a rather st rong interaction between
bacteria and host plant .
Che2mutants A1501 show a weaker ability of adsorption than wild2type A1501 , but once
Che2mutants was adsorbed on rice root surface , they almost remained the same anchoring level as
wild type A1501. Exo2Chemutant A1533 is defective either in adsorption or in anchoring. But exo
mutants A1531 and A1532 are only detective in anchoring. Bacterial surface polysaccharides and
lipopolysaccharides are thought to help bacteria or plant pathogens to avoid plant defence responses
resulting in a compatible interaction. The observations described above suggest that exo mutants
are recognized in an early stage and subsequently colonizing during the interaction with their host
under the salt st ress.
214 　Colonization of A. f aecalis on the rice root under the salt stress
A part of anchored bacteria was dist ributed along the rice roots. In still closely attached to
803 核　农　学　报 14 卷
root surface after 22 h of inoculation and could not removed from the root system even after drasti2
cally vortexing for 10 s. 13 % of the total bacteria inoculated were colonized bacteria and they
grew and multiplied in the mucigel layer. All mutants show a weak ability of colonization. The re2
sults show that the adsorption and anchoring levels are direct factors contributing in the effective
colonization.
It can be concluded that the process of attachment of A . f aecalis could be divided in three
steps ,namely adsorption , anchoring and colonization. The three different states of bacterial cells
appeared on rice root surface after 24 h of inoculation made up 3 % ,19 % and 13 % of total amount
of bacteria inoculated , respectively.
SME of inoculated roots surface revealed that anchoring and colonization occurred on the sur2
face of main roots and lateral roots , but not on the root cal. A . f aecalis anchored and colonized
mainly on the surface of main roots , especially on the joint area of main root and lateral root ,
while anchored or colonized bacterial cells on the lateral roots surface was less.
Effective sites of attachment are also the sites of proton extrusion of rice roots. These sites
show activities of redox enzymes , A TPase , proton secretion etc , and a variable chemical , physical
and biological gradients are present . These gradients which are the result of passive and active se2
cretion of nutrients by the rice roots enhanced the attachment of bacteria. It is suggested that the
passive and active secretion of rice roots resulted in massive colonization on the surface of main
roots or joint area of main roots and lateral roots.
Inoculation with A1501 could enhance salt tolerance of host rice , promote growth of rice
plant and stimulate formation of lateral roots and root hairs under the salt st ress[7 ] . A1501 colo2
nized on root surface and root hairs , especially on zones of emergence of lateral roots and fix nit ro2
gen under the salt st ress. Associative N22fixing activities was much higher than activities of pure
culture when cultural medium contained 015 % NaCl ( Table 5) . At the same concentration of Na2
Cl , colonization of A1501 on root surface were greatly enhanced ( Table 6) . Study on colonization
of rice roots by histological assay indicated that 1) A1501 could enter the roots at points of emer2
gence of lateral roots. A . f aecalis nif H , A . brasilense nifA and A . vi nalandii nifL2lacZ genes fu2
sions could express in the high level on the surface or in the intercellular spaces between the epi2
dermis and the cortex or in the outer cortical layers ; 2) The sites and activities of expression of
three genes fusion were quite different in endorhizosphere of rice , which remained for further
study.
Table 5 　Associative nitrogen f ixation between host rice and A1501 under
the salt stress( nmol C2 H4 produced/ h·gFW roots)
concentration of NaCl ( %)
015 110 210 310
association between rice and A1501 98113 3 34114 10417 7811
pure culture of A1501 45611 18813 1710 317
903　5 期 盐胁迫下粪产碱菌在水稻根表的定殖
Table 6 　The Effect of NH+4 and NaCl on the colonization of
A1501 to rice roots
treatment CK3 15 mmol/ L NH +4 in RMM 015 %Na + in RMM
colonization (bacterial cells/ g fresh root) 315 ×109 219 ×108 417 ×1010
　　Note : 3 　RMM medium containing 0105 % NaCl
References :
[ 1 ] 　Adler J . Chemotaxis in bacteria. Science 1966 ,153 :708～716
[ 2 ] 　Okon Y. Azospirillum2plant associations. CRC Press Inc. ,Boca Raton 1994 ,1～175
[ 3 ] 　Okon Y. Root2associative A zospi rill um species can stimulate plants. ASM News. 63 :366～370
[4 ] 　Lin M , You CB. Advance in the study on mechanism of associative nitrogen fixation in endorhizosphere. Plant Physiology
Communication , 1992 ,28 :323～329
[ 5 ] 　Lin M , et al. Effect of inoculation with A lcaligenes f aecalis on proton efflux of rice roots and microecology in rhizosphere.
Acta Phytophysoil Sinica , 1992 ,18 :233～238
[6 ] 　Michiels K W , et al. Two different modes of attachment of A zospi rill um brasilense sp7 to wheat roots. J Gen Microbiol ,
1991 ,137 :2241～2246
[ 7 ] 　Ping SZ , Lin M , You CB ,Malik J . Salt tolerance of diazotrophic A lcaligenes f aecalis and its interaction with host rice under
the salt stress. J Agricul Biotech. 1996 ,1 :87～92
[ 8 ] 　Qiu YS , et al. Study of nitrogen fixation bacteria associated with rice root . Acta Microbiol Sin. 1981 ,21 :468～472
[ 9 ] 　You CB ,et al. Biological Nitrogen Fixation , Sciences Press , Beijing. 1987 ,224～245
盐胁迫下粪产碱菌在水稻根表的定殖
伍 　红1 　平淑珍2 　KA Malik3 　林 　敏2
(11 西南民族学院 ,四川成都　610041 ; 21 中国农业科学院原子能利用研究所 ,北京　100094 ;
31National Institute for Biotechnology and Genetic Engineering , P. O. Box 577 ,Faisalabad , Pakistan. )
摘 　要 :粪产碱菌 A1501 能在氯化钠浓度高达 4 %的固体和液体介质中良好生长 ,合成 IAA 和
固定空气中的氮素。当氯化钠浓度超过 3 %时 ,A1501 丧失运动和趋化能力。一定盐浓度胁
迫下 A1501 能定殖在水稻根表和根毛区和侧根伸出部位 ,表现出较高的联合固氮活性。
015 %的氯化钠能促进 A1501 在水稻根表的定殖能力。A1501 能通过侧根伸出部位侵入根
内。
关键词 :定殖 ;水稻 ;粪产碱菌 ;盐胁迫
013 Acta A gricult urae N ucleatae Sinica
2000 ,14 (5) :305～310