By Michael Fenton (1994)
Dept of Microbiology and Genetics, Massey
University, Palmerston North, NEW ZEALAND
Rhizobium leguminosarum biovar
trifolii strain ICMP2163::Tn5 was able to spontaneously transfer its
pSym to the non-nodulating Rhizobium loti soil isolate NR40 in
sterile soil microcosms containing Ramiha hill soil or Ashurst silt
loam soil at pH 6.0 or higher. In sterile soil microcosms at pH 6.0
containing sterile ryegrass or white clover plants the frequency of
NR40 transconjugants was higher than in microcosms containing soil
alone. The survival of the parent strains decreased in soil with a pH
of 5.5 or less, and no transconjugant NR40 bacteria were detectable.
Southern blots of the genomic digests
probed with nodA DNA confirmed that transconjugant NR40 contained
symbiotic genes. On artificial media strain ICMP2163::Tn5 transferred
its symbiotic plasmid, by conjugation, to Sphingobacterium
multivorum, an organism that can be found in soil. The
transconjugant bacteria were able to nodulate
white clover seedlings but were unable to fix nitrogen.
examination revealed that the root nodule structure, and
bacteroid formation, were abnormal. The bacteria occupying the nodules
were isolated and the total DNA extracted. The partial 16S RNA gene
sequence from a transconjugant derived from a nodule was shown to be
identical with that of the recipient S. multivorum. Southern
blots of the genomic digests probed with
nodA DNA confirmed that the transconjugant contained symbiotic
sewage isolate was also able to induce a tumour- like growth on white
clover seedlings after receiving the pPN1 co-integrate plasmid from E.coli
strain PN200. Eckhardt gel
analysis confirmed that the transconjugant Caulobacter carried
the R68.45:pSym co-integrate plasmid. Bacteroids were absent but Caulobacter
cells were found in the outer two or three layers of the growth and the
plant cells in this region had degenerated.
Sequence data was obtained for a 260 bp
fragment of the 16S rRNA gene from Sphingobacterium multivorum
and Caulobacter crescentus corresponding to postions 44 to 360
on the Escherichia coli genome. A distance matrix was
constructed showing the relationship between S. multivorum, C.
crescentus, Rhizobium, and related bacteria and neighbor-joining
was used to construct a tree.
From the tree given it is concluded that
the ability to carry or express symbiotic genes is not dependant on
having a phylogenetic relationship with Rhizobium.
1. The significance of the genus Rhizobium.
Pasture growth is limited by the quantity
of fixed nitrogen available when other soil nutrient deficiencies have
been corrected by top-dressing. Nitrogen fixation is the process by
which atmospheric nitrogen gas is made available for incorporation into
organic compounds. Only certain bacteria are capable of carrying out
this process, the genus Rhizobium being the most common (Raven
et al., 1981). Members of this genus are Gram negative aerobic rods
that occur free-living in soil or as micro-symbionts in root nodules of
leguminous plants (Jordan, 1984). Rhizobia in root nodules are
estimated to carry out 50-70% of the world biological nitrogen fixation
(Quispel, 1974), reducing approximately 20 million tonnes of
atmospheric nitrogen to ammonia (Beringer et al.,1980). Biological
nitrogen fixation is of particular importance to New Zealand
agriculture, providing 1 million tonnes of nitrogen annually (Ball and
Field, 1985). Compared to the 26,373 tonnes (Douglas and Cochrane,
1989) of nitrogenous fertiliser used by New Zealand farmers this is
more than 97% our annual requirements.
Although this process is free,
self-sustaining and non-polluting, it does not necessarily operate with
optimum efficiency. Although New Zealand pasture soils contain high
numbers (e.g. 10,000 to 1 million per gram of soil) of indigenous
clover rhizobia (Bonash and MacFarlane, 1987) the introduction of
superior nitrogen fixing strains is still considered an important
management practice. However, the inoculant strains may be prone to
loss of symbiotic traits such as infectiveness and effectiveness
(O'Hara, 1985), and may not be competitive with the indigenous strains
already present in the soil (Rhys and Bonish, 1984). The recommended
inoculum for white clover consists of a mixture of three strains of Rhizobium
leguminosarum biovar trifolii which includes strain ICMP2163,
ICMP2666, and ICMP2668. Stock cultures are maintained by the Plant
Diseases Division of the Crown Research Institute, Auckland (Bianchin,
2. The Biology of Nitrogen Fixation.
Rhizobium bacteria are able to
invade the root hairs of leguminous plants via an infection thread
formed by the plant cells. The plant cells then respond by undergoing
rapid cortical division to form either a tumour or a refined structure
called a nodule. The genetic requirements for nodulation are divided
between the Rhizobium bacteria and the host plant. Both contain
genes that are only expressed in the presence of the other. The process
is reviewed in Djordjevic et al. (1987). Flavanoids, excreted by the
plant, activate nodulation (nod) genes carried by the bacteria. The
nodAB genes on the Rhizobium symbiotic plasmid may produce a
low molecular weight substance that induces plant cell divsion (John et
al., 1988). Attachment of the bacterial cell to the root hair is
proposed to be mediated by binding to lectin. Rhizobia appear to attach
in an end-on fashion followed by involution of the plant cell wall to
form an infection thread. As the infection thread grows through 3 to 6
layers of root outer cortex cells, meristematic activity is initiated
in a small group of root cortical cells directly in front of the tip of
the infection thread. Growth of the infection thread continues into
this meristematic region where rhizobia are released into the inner
most cells, where the bacteria continue to divide until the cytoplasm
is filled with bacteroids (Robertson and Farnden, 1980).
The nodules formed on clover are called
indeterminate nodules. The infection threads continue to penetrate the
plant cortical cells in the nodule meristem, providing a continuous
release of rhizobia into the plant cells as the nodule increases in
size (Beringer et al., 1979). In the process of nodule development, the
bacteria undergo morphological and physiological changes that lead to
the formation of bacteroids (Irigoyen et al., 1990). Free living
rhizobia are not capable of fixing atmospheric nitrogen as oxygen
inactivates the nitrogenase enzyme that converts nitrogen to ammonia
and blocks the transcription of nitrogenase genes. The atmosphere in
the nodule environment is micro-aerophilic due to high concentrations
of the plant protein leghaemoglobin. This protein plays a role in the
transport of oxygen by maintaining a sufficiently high pO2 in the plant
cytoplasm for oxidative phosphorylation, while providing a sustained
low level of oxygen to the bacteroids (Verna and Long, 1983).
In this environment, bacteroids are able to
supply the plant with ammonia which is assimilated into glutamate,
glutamine and other translocatable products. In return, the bacteria is
supplied with an abundance of carbon compounds such as sugars, and is
provided with a protected environment from the outside world. An
ineffective nodule which is not able to fix nitrogen may be formed if
the plant is infected by a Rhizobium strain with a mutation in
the nitrogen fixing (fix) genes.
3. Taxonomy of Rhizobium.
Until recently, the rhizobia that infect
beans, peas, and clovers were clustered in a single species, Rhizobium
leguminosarum (Jordan, 1984), which had three biovars; Rhizobium
leguminosarum bv phaseoli, Rhizobium leguminosarum bv
viceae, and Rhizobium leguminosarum bv trifolii. The artificial
nature of this simplistic classification scheme is becoming more
evident as knowledge is acquired and new species discovered. Currently
three species, Rhizobium leguminosarum bv phaseoli, R. etli
bv phaseoli, and R. tropici, two new Rhizobium genomic species,
and other unclassified genotypes have been isolated from nodules of Phaseolus
vulgaris (Laguerre et al., 1994).
It appears that there may be a greater
diversity of bacteria capable of nodulating legumes than was previously
recognised (Laguerre et al., 1994). Within R. leguminosarum
biovar trifolii there is considerable phenotypic variability (Dughri
and Bottomley, 1984; Harrison et al., 1987), reflected by the genetic
diversity observed (Jarvis et al., 1980; Crow et al., 1981). Jarvis et
al. (1980) compared reference DNA from clover inoculant strains NZP561
and TAI with DNAs from 18 other R. leguminosarum bv trifolii
strains. The range of DNA-relatedness and DTm(e) values with strains
NZP561 and TAI was 61 - 91% and 0 - 8.2oC and 49 - 94% and 1.3 - 7.0oC
respectively. DTm(e) is a statistic which expresses the base sequence
homology in the fraction of DNA which hybridises. Each 1oC represents a
1% miss-match in the hybridising sequences (Jarvis et al., 1991). The
values quoted extend well beyond the phylogenetic limits for a
bacterial species as proposed by Wayne et al., (1987). It is concluded
that, Rhizobium leguminosarum bv trifolii may not be a single
species but a group of inter-related species capable of expressing the
appropriate symbiotic genes.
Normally the primary isolation of Rhizobium
strains is from nodulated legumes (Schofield et al., 1987; Vincent,
1970; Young, 1985) and this has made it difficult to define
phylogenetic relationships with other bacteria in the soil. However,
the ability to nodulate leguminous plants is regarded as the
characteristic function of the genus Rhizobium with nitrogen
fixation a normal but not essential consequence of nodulation (Jordan,
1984). The nodulation and nitrogen fixation genes are usually located
on a symbiotic plasmid (pSym), that encodes distinct nodulation
specificities (Johnston et al., 1978; Hirsch et al., 1980). The plasmid
may be lost under certain environmental conditions, so that soil
bacteria lacking this plasmid cannot be classified as rhizobia although
they may be able to express the symbiotic genes. Strains of bacteria
exist that fail to satisfy Jordan's definition but are clearly rhizobia
lacking the symbiotic plasmid (Scott and Ronson, 1982; Soberon-Chavez
and Najera, 1988; Segovia et al, 1991).
Another difficulty arises from the ability
of the symbiotic plasmid to be transferred from one strain of Rhizobium
to another. This may change the strains host specificity or lead to the
loss of the ability to nodulate. It has been shown that pSym genes can
be expressed to a limited degree in Agrobacterium species
(Hooykass et al. 1981; Kondorosi et al., 1982; O'Connell et al., 1987),
Pseudomonas aeruginosa and Lignobacter
species (Plazinski and Rolfe, 1985). Jarvis et al. (1989) suggested
that Rhizobium classification should be defined in terms of
DNA-DNA or rRNA-DNA homology to accepted reference bacteria. In
addition, it may be useful to use the 16S ribosomal DNA sequence to
determine what is a 'true' rhizobia. PCR-RFLP analysis has been
described as a rapid method for the identification of nodule isolates
and new taxa (Laguerre et al., 1994). The use the fatty acid
composition profiles has also been described as another reliable means
of rapid identification (Jarvis and Tighe, 1994).
4. The Symbiotic Plasmid.
The nodulation and nitrogen fixation genes
are usually located on large (>100 kb) symbiotic plasmids (pSym or
Sym plasmid), some of which can be transferred to other bacteria via
conjugation (Djordjevic et al., 1983; Johnston et al., 1978). There is
evidence that pSym transfer occurs in natural field populations.,
Schofield et al., (1987) studied 16 soil isolates of Rhizobium
leguminosarum and observed similar Sym plasmids in different host
chromosomal backgrounds and different Sym plasmids in similar host
chromosomal backgrounds, as well as the presence of a putative
recombinant Sym plasmid. Jarvis et al., (1985) reported the isolation
of soil bacteria that showed DNA homology to Rhizobium leguminosarum
but were unable to nodulate white clover. Transconjugation experiments
with the co-integrate plasmid pPN1 (Scott and Ronson, 1982) showed that
these bacteria could express symbiotic genes from clover rhizobia.
Plasmid transfer in non-sterile soil has been demonstrated between Rhizobium
fredii and a pSym cured Rhizobium leguminosarum (Kinkle and
Schmidt, 1991) and between Rhizobium leguminosarum and Enterobacter
(Dohler and Klingmuller, 1988).
Indigenous soil bacteria, including native
rhizobia, are well adapted to survive in the absence of a host plant.
Potential competitors may not initially be able to nodulate crop plants
but may be enabled to by obtaining the appropriate symbiotic plamid
(Dowling and Broughton, 1986). If complemented by a Sym plasmid from an
introduced Rhizobium strain, the indigenous soil bacteria will
compete for nodulation sites and may form the majority of nodules on
the host plant (Meade et al., 1985; Weaver & Frederick, 1974a,
1974b). The inoculant strain may need to be supplied at 1000X the level
of the indigenous Rhizobium population in order to form 50% of
the nodules. For the inoculation industry this may yield unexpected
benefits if it were possible to isolate indigenous soil bacteria able
to nodulate and fix nitrogen better than the commercial Rhizobium
inoculant. However, it becomes a problem when the indigenous soil
bacteria form ineffective nodules incapable of nitrogen fixation. In
this instance, increasing the inoculum added to the soil is simply
adding more DNA for the competitors to pick up. There may also be
important consequences for the release of genetically engineered
However, many factors influence the
competitive ability of a Rhizobium strain, and any factor which
adversely effects plant growth will also profoundly effect competition
for nodulation. Phosphorous limitation has been shown to be exacerbated
by low pH and the combination of low pH and phosporous levels can have
a strong influence on competition for nodulation (Dowling and
Broughton, 1986). Most soils in New Zealand are moderately acidic,
having a pH between 5.0 and 6.5. It appears that an acidity of pH
5.8-6.0 is considered ideal for the legume to prevent aluminium and
manganese toxicity, but the other partner in the symbiotic relationship
appears to have been overlooked. Other environmental factors such as
soil type, temperature, and moisture also affect the outcome of
competition. Biological factors, such as bacteriophage effects,
epiphtyic bacteria, mycorrhizal effects predation by protozoa should
all be considered when applying laboratory results outside. It is
concluded that symbiotic plasmid transfer occurs between Rhizobium
strains and other bacteria in soil but the nature and diversity of the
recipient remains unclear.
1. Conjugation in sterile soil
1.1 Strains used :
Rhizobium leguminosarum bv trifolii
strain ICMP2163 is used as a white clover inoculant strain in New
Zealand. The symbiotic plasmid was labelled by insertion of the
transposon Tn5 which confers neomycin resistance to its host (Rao et
al., 1994). The Sym plasmid from strain ICMP2163::Tn5 used in this
study was shown to be fully effective for strain PN165, a pSym cured
derivative of strain ICMP2163. This demonstrated that the pSym was
self-transmissible and that insertion of Tn5 did not affect the
expression of nodulation genes. Our laboratory had been investigating
the transfer of pSym from New Zealand inoculant strains to native
non-nodulating soil isolates. The soil isolate NR40 was identified by
its fatty acid profile, a method shown to be reliable (Jarvis and
Tighe, 1994), as Rhizobium loti. Growth requirements, cell
morphology and colony morphology of NR40 on TY agar are consistant with
those of R.loti, however a detailed study comparable to Segovia
et al., (1991) would confirm this identification. The observation that
transconjugant NR40 forms ineffective nodules on white clover seedlings
would not be surprising for R. loti transconjugants.
1.2 Factors affecting soil rhizobia.
A number of factors affect the survival of
micro-organisms in soil. Investigators tend to concentrate on factors
that lend themselves to study in the laboratory, such as temperature,
pH, moisture content and organic matter content (reviewed by Dowling
and Broughton, 1986). Rhizobium strains vary in their acid
tolerance. In soil, pH not only directly affects the growth of
micro-organisms, but also affects the solubility of many cations which
may indirectly alter growth patterns. A strain of Rhizobium fredii
did not to survive in soil below pH 5.25 (Richaume, 1989). Dughri and
Bottomley (1984) were able to alter the outcome of competition between
indigenous rhizobia in the soil by changing the acidity. Rhizobium
leguminosarum bv trifolii strain ICMP2163::Tn5 was able to transfer
its Tn5-marked symbiotic plasmid to the pSym deficient Rhizobium
soil isolate NR40, in sterile soil at a pH greater than 5.5. NR40 was
not able to incite root nodule formation but transconjugant bacteria
were able to form ineffective nodules on white clover seedlings. The
parent strains survived for a maximum of 21 days in soil at pH 5.5 or
less. It is concluded that the the inoculant strain ICMP2163 would not
be suitable for use in acidic soils. There was a significant increase
in the frequency of plasmid transfer in the presence of ryegrass or
white clover plants; from 1 X 10-6 to 3 X 10-6. This may be due to
stimulating factors associated with the rhizosphere. Bacteria
associated with the rhizosphere will have access to attachment sites,
nutrients and minerals at high concentrations and as a consequence,
will be metabolically more active than their free-living counterparts.
Overall, there will be mucher greater
opportunity for genetic exchange to occur. In sterile Ramiha hill soil
at 50% water holding capacity (pH 6.0) with clover seedlings present,
the number of transconjugants present per gram of soil increased
10-fold over an 18 day period; from 4 CFU/g to 44 CFU/g. The
significance of these results are two-fold. Firstly, Theis et al.,
(1919a,1991b) have shown that as few as 50 indigenous rhizobia per gram
of soil eliminated the inoculum response to 1million to 10 million
rhizobia per seed. The inoculant strain ICMP2163 has the potential to
transfer its pSym to indigenous soil bacteria at a high enough
frequency to eliminate future inoculum responses. Secondly, in order to
get meaningful results, laboratory simulations should be as close to
conditions in natural environments as possible. These experiments
involved one potential recipient strain. In non-sterile soil there are
a great number of species that could be involved, although there is no
indication as to how many that may be. There appeared to be no
significant difference in using Ramiha hill soil or Ashurst silt loam
soil in the above mentioned experiments. This implies that what happens
in one soil type may well occur in others. There may be no need to
taylor bacteria for specific soil types if this is true. However, the
experiments carried out so far are rather simplistic, looking at a few
of the variables associated with soil. Experiments with non-sterile
soil, in the manner of Kinkle and Schmit (1991), would be more
1.3 Significance of plamid transfer in soil.
Rhizobium strains lacking
symbiotic plasmids in soil may act as biological sinks for the
symbiotic plasmids from inoculant strains. Strains of bacteria exist
that fail to satisfy Jordan's definition but are clearly rhizobia
lacking the symbiotic plasmid (Scott and Ronson, 1982; Soberon-Chavez
and Najera, 1988; Segovia et al, 1991). This could explain the temporal
loss of inoculant strains in the field and the appearance of indigenous
rhizobia where there was no evidence of previous Rhizobium
populations (Roughley et al., 1976). It is now well established that
self-transmissible symbiotic plasmids can be exchanged between strains
of Rhizobium on artificial media and there is evidence that
this exchange occurs in the natural field populations. Two independent
studies, one involving Rhizobium leguminosarum bv trifolii
(Schofield et al., 1987), and the other involving Rhizobium
leguminosarum bv viceae (Young and Wexler, 1988), have reported
that similar symbiotic plasmids could be found in genetically unrelated
isolates. Kinkle and Schmit (1991) observed the transfer of the
symbiotic plasmid pJB5JI between strains of Rhizobium in
sterile and non-sterile soil. It is concluded that the improved use of Rhizobium
seed inoculants will require further study of plasmid transfer
mechanisms between the inoculant bacteria and the other soil bacteria.
2. Expression of Symbiotic Genes by
The recognition that Rhizobium
strains lacking Sym plasmids exist in soil has meant that soil is often
screened for new Rhizobium strains. Isolates are usually
selected on the basis of having particular growth characteristics and
colony morphology on solid media compared to known reference strains.
It was in this manner that NR40 was isolated. It can take some time for
an isolate to be completely characterised and accurately identified.
One of the soil isolates used in our laboratory (NR64) was thought to
be a Sphingobacterium (Bianchin, 1989) but was later shown to
be a strain of Rhizobium. A strain of Sphingobacterium
was obtained for furthur study. The approach used was to cross Rhizobium
leguminosarum bv trifolii strain ICMP2163::Tn5 with a number of
known non-Rhizobium strains. In this manner it was hoped to gain
some insight as to the distance that symbiotic genes could travel.
2.1 Transfer of pSym to Sphingobacterium
Sphingobacterium multivorum is an
organism that can be found in soil. It is able to grow agar at 37 oC,
and is sometimes isolated from clinical samples. As strain NZRM1228 was
spontaneously resistant to neomycin it was not possible to use the Tn5
antibiotic resistance marker to select for transconjugants retaining
pSym on artificial media. The symbiotic plasmid was lost from nodule
isolates after 2 - 3 rounds of single colony purification. Luria
Rif.Neo.Str agar ensured that only the Sphingobacterium would
grow for use in plant inoculation tests. The use of white clover
seedlings to select for transconjugants and the isolation of the
bacteria from the root nodules was the most effective way to obtain
enough bacteria for study. This suggests that as long as the
appropriate selection pressure is applied the Sphingobacterium
transconjugants would continue to nodulate other clover seedlings.
Comparison of the electron micrographs in indicates that the nodule
occupant of the MF100 plant was different from the Rhizobium
donor strain. Total genomic digest profiles of the nodule isolate were
the same as strain NZRM1228 and Southern blots probed with nodA DNA
confirmed that the nodule isolate contained symbiotic genes. 16S rDNA
sequence analysis identified the nodule isolate as Sphingobacterium
multivorum recipient strain NZRM1228. This is the first report of
the spontaneous transfer of the symbiotic plasmid from an inoculant
strain of Rhizobium leguminosarum bv trifolii to Sphingobacterium
2.2 Expression of pSym Genes in Caulobacter.
E. coli strain PN200, carrying the
co-integrate plasmid pPN1, and Caulobacter strain MCDF23 was
crossed in an alternative method to test the ability to express Sym
plasmid genes. Scott and Ronson (1982) obtained the 770 Mda pPN1 by
co-integrating the Sym plasmid from Rhizobium leguminosarum bv
trifolii strain NZP514 (pRtr514) with the broad-host-range plasmid
R68.45. The plasmid confers neomycin resistance to its host.
Caulobacter belongs to the budding and prosthecate group of
organisms and is found in soils and waterways. A transconjugant Caulobacter
isolate MCDF100 containing the co-integrate plasmid pPN1 (Scott and
Ronson, 1982) was able to induce a tumour-like growth within 12 days of
inoculation onto sterile 3 day old white clover seedlings. Attempts to
isolate the nodule occupants were unsuccessful. Examination by electron
microscope showed that the growth did not appear to be invaded by
bacteria. A few bacteria were found within the intercellular spaces of
the outermost cells of the structure and the plant cells in this region
had degenerated. Plazinski and Rolfe (1985) reported similar results
with Pseudomonas strain PAO5. Expression of the Sym plasmid
genes carried on pPN1 may have been affected by the RP4 tra genes in
the R68.45 section of the co-integrate plasmid (Hynes and O'Connell,
1988). This is the first report of Caulobacter carrying
symbiotic plasmid genes and causing tumour-like growths on white clover
3. Consequences for Taxonomy.
The ability to nodulate leguminous plants
is regarded as the characteristic function of the genus Rhizobium
with nitrogen fixation a normal but not essential consequence of
nodulation (Jordan, 1984). There are a number of Rhizobium
species that carry the nodulation and nitrogen fixation genes on
plasmids which may be transferred by conjugation. Soil bacteria other
than rhizobia could be involved in the dissemination of symbiotic
genes, perhaps acting as temporary hosts before passing the genes back
to an appropriate Rhizobium strain. The existance of species of
soil bacteria, outside of Rhizobium, capable of expressing Sym
plasmid genes may have been overlooked because of the screening method
or media used. Possible candidates are Agrobacterium (Hooykass
et al. 1981; Kondorosi et al., 1982; O'Connell et al., 1987), Enterobacter
(Dohler and Klingmuller, 1988), Pseudomonas and Lignobacter
(Plazinski and Rolfe, 1985), Caulobacter, and
Sphingobacterium multivorum. Caulobacter is distantly
related to Rhizobium, and Sphingobacterium is the most
distantly relaled. Schematic 2D models of the 16S RNA molecule indicate
that Sphingobacterium is quite unrelated to Rhizobium.
It is concluded that the ability to express or carry symbiotic plasmid
genes is not dependant on having a phylogenetic relationship to Rhizobium.
Clearly, it is undesirable for there to be this confusion in a
classification scheme. A number of authors have suggested revised
descriptions of the genus Rhizobium and Agrobacterium
based on 16S rDNA sequences and DNA/DNA homology studies (Willems and
Collins, 1993; Sawada et al., 1993; Yanagi and Yamasato, 1993).
This study reports the isolation of a
strain of Sphingobacterium multivorum that is able to nodulate
white clover seedlings and would fit Jordan's definition of a Rhizobium
species. It is concluded that taxonomic relationships based on
characteristics carried on plasmids may not reflect real relationships
amongst micro-organisms. It would be preferable to identify known Rhizobium
species from their fatty acid profiles or specific DNA probes and
define new species in terms of their 16S rRNA gene sequence and DNA-DNA
relatedness with recognised reference strains. Further work needs to be
done in looking at the factors affecting pSym transfer in soil,
preferably using non-sterile soil. Non-Rhizobium strains should
continue to be tested to see if they are able to receive the Sym
plasmid from New Zealand inoculant strains. This may result in new
inoculant strains becoming available with unique characteristics.
Ausubel, F.M., R. Brent, R.E. Kingston,
D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl. 1991. Current
protocols in molecular biology 2. The Polymerase Chain Reaction 15.0.3
Altschul, S.F., W. Gish, W.Miller, E.W.
Myers, and D.J Lipman. 1990. Basic local alignment search tool. Journal
of Molecular Biology. 215:403- 410.
Ball, P.R., and T.R.O. Field. 1985.
Productivity and economics of legume- based pastures and grass swards
receiving fertilizer nitrogen in New Zealand. In Forage legumes for
energy efficient animal production. Edited by R.F. Barnes et al.
Grasslands Divison, DSIR, Palmerston North, New Zealand.
Bailey, N.T.J. 1981. Statistical methods in
biology. 2nd ed. Hodder and Stroughton, Great Britain.
Beringer, J.E. 1974. R-factor transfer in
Rhizobium leguminosarum. J. Gen. Microbiol. 84:188-198.
Beringer, J.E., N.J. Brewin and A.W.B.
Johnston, H.M. Schulman, and D.A Hopwood. 1979. The Rhizobium-legume
symbiosis. Proc. R. Soc. Lond. B., 204: 219-233.
Beringer, J.E., N.J. Brewin and A.W.B.
Johnston. 1980. Invited review; The genetic analysis of Rhizobium in
relation to symbiotic nitrogen fixation. Heredity, 45(2): 161-186.
Bianchin, M.B. 1989. Competition on white
clover between a nodulating soil bacterium and a strain of Rhizobium
leguminosarum biovar trifolii. B.Sc.Hon. thesis, Massey University,
Bonish, P.M., and M.J. MacFarlane. 1987.
Nodulation of introduced white clovers by naturalised soil clover
rhizobia: symbiotic effectiveness and host-strain compatability. N.Z J.
Ag. Res. 30: 273-280.
Brosius, J., T.J. Dull, D.D. Sleeter, and
H.F. Noller. 1981. Gene organisation and primary structure of a
ribosomal RNA operon from Escherichia coli. J. Mol. Biol. 148:107-127.
Buchanan-Wollaston, A.V., J.E Beringer,
N.J. Brewin, P.R. Hirsch and A.W.B Johnston. 1980. Isolation of
symbiotically defective mutants in Rhizobium leguminosarum by insertion
of the transposon Tn5 into a transmissible plasmid. Mol. Gen. Genet.
Cowie, J.D. 1972. Soil map and extended
legend of Kairanga county, North Island, New Zealand. N.Z. Soil Bureau
538. Department of Scientific and Industrial Research, Wellington.
Crow, V.L., B.D.W. Jarvis, and R.M.
Greenwood. 1981. Deoxyribonucleic acid homologies among acid-producing
strains of Rhizobium. Int. J. Syst. Bacteriol. 31:152-172.
Devereux, J., P. Haeberly, and O. Smithies.
1984. A comprehensive set of sequence analysis programs for the VAX
computer. Nucleic Acids Res. 12: 387-395.
Djordjevic, M. A., W. Zurkowski, J. Shine,
and B.G . Rolfe. 1983. Sym plasmid transfer to various symbiotic
mutants of Rhizobium trifolii, R. leguminosarum, and R. meliloti. J.
Djordjevic, M. A., J. W. Redmond, M.
Batley, and B.G . Rolfe. 1987. Clovers secrete specific phenoic
compounds which either stimulate or repress nod gene expression in
Rhizobium trifolii. EMBO Journal, 6(5): 1173- 1179.
Dohler, K. and W. Klingmuller. 1988.
Genetic interaction of Rhizobium leguminosarum biovar viceae with
Gram-negative bacteria. In W. Klingmuller (ed.) Risk assessment for
deliberate releases, Springer-Verlag, Berlin.
Douglas, J. and J. Cochrane. 1989. A review
of nitrogen in New Zealand and overseas. Nitrogen in New Zealand
agriculture and horticulture. Edited by R.E. White and L.D. Currie.
Dowling, D.N., and W.J. Broughton. 1986.
Competition for nodulation of legumes. Ann. Rev. Microbiol. 40:131-157.
Dughri, M.H., and P.J. Bottomley. 1984.
Soil acidity and the compositon of an indigenous population of
Rhizobium trifolii in nodules of different cultivars of Trifolium
subterraneum. Soil Biol. and Biochem. 16:405-411.
Eckhardt, T. 1978. A rapid method for the
identification of plasmid DNA in bacteria. Plasmid, 1:584-588.
Felsenstein, J. 1982. Numerical methods for inferring evolutionary
trees. Q. Rev. Biol. 57:379-404.
Harrison, S.P., J.P.W. Young, and D.G.
Jones. 1987. Rhizobium population genetics: effect of clover variety
and inoculum dilution on the genetic diversity sampled from natural
populations. Plant and soil. 103:147-150.
Hirsch, P.R., M. van Montagu, A.W.B.
Johnston, N.J. Brewin, and J.Schell. 1980. Physical identifcation of
the bacteriocinogenic, nodulation and other plasmids in strains of
Rhizobium leguminosarum. J.Gen. Microbiol. 120:403-412.
Hoagland, D. R., and D.T. Arnon. 1938. The
water culture method for growing plants without soil. University of
California Agriculture Experiment Station Circulation no. 347.
Hooykaas, P.J.J., A.A.N. van Brussel, H.
den Dulk-Ras, G.M.S. van Slogteren, and R.A. Schilperoort. 1981. Sym
plasmid of Rhizobium trifolii expressed in different rhizobial species
and Agrobacterium tumefaciens. Nature (London) 291:351-353.
Hynes, M.F., and M.P. O'Connell. 1988.
Influence of RP4 on nodulation and nitrogen fixation by strains of
Rhizobium leguminosarum biovar viceae. Abstr. VIb-1 4th International
Symposium on the Molecular Genetics of Plant-Microbe Interactions,
Irigoyen, J.J., M. Sanchez-Diaz and D.W.
Emerich. 1990. Carbon metabolism enzymes of Rhizobium meliloti cultures
and bacteriods and their distribution within alfalfa nodules.
Jarvis, B.D.W., A.G. Dick, and R.M.
Greenwood. 1980. Deoxyribonucleic acid homologies among strains of
Rhizobium trifolii and related species. Int. J. Syst. Bacteriol.
Jarvis, B.D.W., M. Gillis, and J. de Ley.
1985. Intra- and intergeneric similarities between 23S ribosomal RNA
cistrons from Rhizobium and related bacteria. In Nitrogen Fixation
Research Progress (H.J.Evans, P. J. Bottomley and W. E. Newton (Eds.)
pp.149- Martinus Nijhoff, Dordrecht, The Netherlands.
Jarvis, B. D. W., L. J. H. Ward and E. A.
Slade. 1989. Expression by soil bacteria of nodulation genes from
Rhizobium leguminosarum biovar trifolii. Appl. Environ. Microbiol.
Jarvis, B.D.W., G. Ionas, and J.C. Clarke.
1991. Quantatative DNA:DNA hybridisation and hydroxyapatite elution. In
Molecular techniques in taxonomy (G.M. Hewitt, A.W.B. Johnston and
J.P.W. Young, Eds), pp 379-383. Springer-Verlag, Berlin.
Jarvis, B.D.W., H.L. Downer, and J.P.W.
Young. 1992. Phylogeny of fast- growing soybean nodulating rhizobia
supports synonymy of Sinorhizobium and Rhizobium and assignment to
Rhizobium fredii. Int. J. Syst. Bacteriol. 42: 93-96.
Jarvis, B.D.W., and S. Tighe. 1994. Rapid
identification of Rhizobium species based on cellular fatty acid
analysis. Plant and Soil (in press).
John, M., J. Scmidt, U. Weineke, H.D.
Krussmann, and J. Schell. 1988. Transmembrane orientation and
receptor-like structure of the Rhizobium meliloti common nodulation
protein NodC. EMBO Journal., 7(3): 583-588.
Johnston, A.W.B., J.L. Beynon, A.V.
Buchanan-Wollaston, S.M. Setchell, P.R. Hirsch, and J.E. Beringer.
1978. High frequency transfer of nodulating ability between strains and
species of Rhizobium. Nature (London) 276:634-636.
Jordan, D.C. 1984. Family III Rhizobiaceae
Conn. 1983, p234-256. In N. R. Kreig and J. G. Holt (ed.), Bergey's
manual of systematic bacteriology. Vol. 1. Williams & Williams Co.,
Jukes, T.H., and C.R. Cantor. 1969.
Evolution of protein molecules, p.21- 132. In H.N. Munro (ed.),
Mammalian protein metabolism. Academic Press, New York.
Kinkle, K.B., and E. L. Schmidt. 1991.
Transfer of the pea symbiotic plasmid pJB5JI in non-sterile soil. Appl.
Environ. Microbiol. 57:3264-3269.
Kondorosi, A., E. Kondorosi, C.E.
Pankhurst, W.J. Broughton, and Z. Banfalvi. 1982. Mobilisation of a
Rhizobium meliloti megaplasmid carrying nodulation and nitrogen
fixation genes into other rhizobia and Agrobacterium. Mol. Gen. Genet.
Laguerre, G., M. Allard, F. Revoy, and N.
Amarger. 1994. Rapid identification of Rhizobia by restriction fragment
length polymorphism analysis of PCR-amplified 16S rRNA genes. Appl.
Environ. Microbiol. In press.
Maniatis, T., E. F. Fritsch, and J.
Sambrook. 1982. Molecular cloning: a laboratory manual. Cold Spring
Harbor Laboratory, Cold Spring Harbor, N. Y.
Marshall, R.B., B.E Wilton, and A.J.
Robinson. 1981. Identification of Leptospirosa serovars by
restriction-endonuclease analysis. J. Med. Microbiol. 14:163-166.
Meade, J., P. Higgins, and F. O'Gara. 1985.
Studies on the inoculation and competitiveness of a Rhizobium
leguminosarum strain in soils containing indigenous rhizobia. Appl.
Environ. Microbiol. 49:899-903.
Metson, A.J. 1961. Methods for the chemical
analysis of soil survey samples. Soil Bureau Bulletin 12. New Zealand
Government Printer, Wellinton. Miller, J.H. (Ed.) 1972. Assay of
b-galactosidase. Experiments in molecular genetics (exp 48 pp352-355).
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y.
O'Connell, M.P., M.F. Hynes, and A. Puhler.
1987. Incompatibility between a Rhizobium Sym plasmid and a Ri plasmid
of Agrobacterium. Plasmid 18:156-163.
O'Hara, M.J. 1985. Investigation of the
genetic changes in inoculant strains of Rhizobium trifolii isolated
from the soil. M.Sc. thesis, Massey University, Palmerston North.
Pankhurst, C. E., A.S. Craig, and W.T.
Jones. 1979. Effectiveness of Lotus root nodules. I Morphology and
flavolan content of nodules formed on Lotus pedunculatus by
fast-growing Lotus rhizobia. J. Exp. Bot. 30:1085-1093.
Plazinski, J., and B.G. Rolfe. 1985. Sym
Plasmid Genes of Rhizobium trifolii expressed in Lignobacter and
Pseudomonas strains. J. Bact. Vol 162 3:1261-1269.
Quispel, A. 1974. In A. Quispel (Ed.) The
Biology of Nitrogen Fixation. North-Holland Publishiing Company,
Amsterdam, Netherlands. Raven, P.H., R.F. Evert, and H. Curtis (Eds.).
1981. Biology of Plants (3rd Ed.). New York: Worth Publishers Inc.
Rao, J.R., M. Fenton, and B.D.W. Jarvis.
1994. Symbiotic plasmid transfer in Rhizobium leguminosarum biovar
trifolii and competition between the inoculant strain ICMP2163 and
transconjugant soil bacteria. Submitted for publication.
Richaume, A., J. Angle , and M. Sadowsky.
1989. Influence of soil variables on in situ plasmid transfer from
Escherichia coli to Rhizobium fredii. Appl. Environ. Microbiol. 55:
Robertson, J.G. and K.J.F. Farnden. 1980.
Ultrastructure and metabolism of the developing legume root nodule. In
Stumpt, P.K., and Cohn, E.E. (eds). The biochemistry of plants: a
comprehensive treatise, Vol 5:pp65- 113). New York: Academic Press.
Rossen, L., A.W.B. Johnston, and J.A
Downie. 1984. DNA sequence of the Rhizobium leguminosarum nodulation
genes nodAB and C required for root hair curling. Nucl. Acids. Res. 12:
Roughley, R.J., W.M. Blowes, and D.F.
Herridge. 1976. Nodulation of Trifolium subterraneum by introduced
rhizobia in competition with naturalized strains. Soil Biol. and
Rhys, G.J., and P.M. Bonish. 1984.
Influence of inoculation and additional nutrients on establishment of
the white clover symbiosis in a cultivated soil. N.Z. J. Exp. Ag.
Saito, N., and M. Nei. 1987. The
neighbor-joining method: A new method for reconstructing phylogenetic
trees. Mol. Biol. Evol. 4:406-425.
Sambrook, J. 1989. Molecular cloning: a
laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor,
Sanger, F., S. Nicklen and A.R. Coulson.
1977. DNA sequencing with chain terminating inhibitors. Proc. Natl.
Acad. Sci. USA. 74: 5463-5467.
Sawada, H., H. Ieki, H. Oyaizu, and S.
Matsumoto. 1993. Proposal for the rejection of Agrobacterium
tumefaciens and revised descriptions for the genus Agrobacterium and
for Agrobacterium radiobacter and Agrobacterium rhizogenes. Int. J.
Sys. Bacteriol. 43:694-702.
Schofield, P.R., A.H. Gibson, W.F. Dudman,
and J.M. Watson. 1987. Evidence for the genetic exchange and
recombination of Rhizobium symbiotic plasmids in a soil population.
Appl. Environ. Microbiol. 53: 2942-2947.
Scott, D.B., and C.W. Ronson. 1982.
Identification and mobilisation by cointegrate formation of a
nodulation plasmid in Rhizobium trifolii. J. Bact. Vol 151 3:36-43.
Segovia, L., D. Pinero, R. Palacious, and
E. Martinez-Romero. 1991. Genetic structure of a soil population of
non-symbiotic Rhizobium leguminosarum. Appl. Environ. Microbiol.
Soberon-Chavez, G., and R. Najera. 1989.
Isolation from soil of Rhizobium leguminosarum lacking symbiotic
information. Can. J. Microbiol. 35: 464-468.
Southern, E.M. 1975. Detection of specific
sequences among DNA fragments seperated by gel electrophoresis. J. Mol.
Stackebrandt, E, and M. Goodfellow (Eds).
1991. Nucleic acid techniques in bacterial systematics.
Theis, J.E., P.W. Singleton, and B.B.
Bohlool. 1991a. Influence of the size of indigenous rhizobial
populations on establishment and symbiosis performance of introduced
rhizobia on field-grown legumes. Appl. Env. Microbiol. 57:19-28.
Theis, J.E., P.W. Singleton, and B.B.
Bohlool. 1991b. Modelling symbiotic performance of introduced rhizobia
in the field by use of indices of indigenous population size and
nitrogen status of the soil. Appl. Env. Microbiol. 57:29-37.
Thornton, H.G. 1930. The early development
of the root nodule of Incerne (Medicago sativa L.). Ann. Bot.
Verma, D.P.S., and S. Long. 1983. The
molecular biology of Rhizobium- legume symbiosis. International review
od cytology, supplement, 14:pp211-245. New York: Academic Press.
Vincent, J.M. 1970. A manual for the study
of root nodule bacteria. Blackwell Scientific Publications, Oxford,
Wayne, L.G., D.J. Brenner, R.R. Colwell,
P.A.D. Grimont, O. Kandler, M.I. Krichevsky, L. H. Moore, W.E.C. Moore,
R.G.E. Murray, E. Stackebrandt, M.P. Starr, and H.G. Truper. 1987.
Report of the ad hoc committee on reconciliation of approaches to
bacterial systematics. Int. J. Syst. Bacteriol. 37:463-464.
Weaver, R.W., and L.R. Frederick. 1974a.
Effect of inoculum rate on competitive nodulation of Glycine max. L.
Merrill. II. Greenhouse studies. Agron. J. 66:229-232.
Weaver, R.W., and L.R. Frederick. 1974b.
Effect of inoculum rate on competitive nodulation of Glycine max. L.
Merrill. II. Field studies. Agron. J. 66:233-236.
Willems, A., and M.D. Collins. 1993.
Phylogenetic analysis of rhizobia and agrobacteria based on 16S rRNA
gene sequences. Int. J. Sys. Bacteriol. 43:305-313
Yanagi, M., and K. Yamasato. 1993.
Phylogentic analysis of the family Rhizobiaceae and related bacteria by
sequencing of 16S rRNA gene using PCR and DNA sequencer. FEMS
Microbiology Letters 107 115- 120.
Young, J.P.W. 1985. Rhizobium population
genetics: enzyme polymorphism in isolates from peas, clover, beans, and
lucerne grown at the same site. J. Gen. Microbiol. 131:2399-2408.
Young, J.P.W., and M. Wexler. 1988. Sym
plasmid and chromosomal genotypes are correlated in field populations
of Rhizobium leguminosarum. J. Gen. Bact. 134: 2731-2739.
Young, J.P.W., H.L. Downer, and B.D.
Eardlyl. 1991. Phylogeny of the phototrophic Rhizobium strain BTAi1 by
polymerase chain reaction based sequencing of a 16S rRNA gene segment.
J. Bact. 173: 2271- 2277.
(Reproduced with permission)