By Michael Fenton
(1994)
Dept of Microbiology
and Genetics, Massey University,
Palmerston North, NEW ZEALAND
ABSTRACT
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.
Microscopic
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 genes.
A Caulobacter
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.
INTRODUCTION
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, 1989).
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 micro-organism.
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.
DISCUSSION
1. Conjugation
in sterile soil microcosms.
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 convincing.
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 Non-Rhizobium
Species.
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 multivorum.
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 multivorum.
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 seedlings.
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).
4. Conclusion.
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.
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