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PhD Project - Specificity of Symbioses in New Zealand Rhizobia

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Introduction

In Chapter 3 it was established that native legumes of New Zealand are nodulated by a diverse range of Mesorhizobium strains, and Rhizobium leguminosarum. It was also found that introduced legume weeds---Acacia, broom, and gorse---are nodulated by Bradyrhizobium species. In this chapter, these relationships are investigated further by examining the specificity of these rhizobia--host relationships.

Specificity varies greatly in rhizobia--legume relations. Historically it was assumed that one rhizobial species corresponded to one legume genus or species, for example Rhizobium trifoliiNow classified as Rhizobium leguminosarum biovar trifolii. was considered specific to clover (Trifolium spp.) Dangeard26,Hirsch01. Many other Rhizobium and Mesorhizobium species appear to be more or less `promiscuous', nodulating more than one plant genus, and most nodulate with two or more plant genera Chen05a,Kuykendall05. Rhizobium galegae was thought specific to only goat's rue (Galega officinalis); however it is now known to nodulate Galega orientalis (as a separate biovar) Radeva01, Astragalus cruciatus, Lotus creticus and Anthyllis henoniana Zakhia04, and possibly more Wei03. At the extreme end of the scale, Ensifer fredii strain NGR234 nodulates 232 species in 112 genera Hirsch01,Pueppke99,Saldana03. Generally, rhizobia of herbaceous host species are reported to be more promiscuous than those of woody legumes Perret00. Despite more than a century of research, host ranges for rhizobial species are known for only a few hundred legume species (out of 18000), most being crop, forage or grain legumes Kuykendall05,Chen05a, predominately from the Northern Hemisphere.

Given New Zealand's geographical and temporal isolation from the rest of the world, it is hypothesised that New Zealand legumes have evolved independently and may be `symbiotically isolated' from other species. Evidence in Chapter 3 that host legumes are nodulated by a variety of strains (e.g. Carmichaelia nodulated by three genomic groups), indicates that relationships within New Zealand legumes are probably not specific. However, there may be some specificity related to nod genes (see Chapter 4). To resolve this, host-range testing was carried out with the aim of determining the specificity of the symbioses, including the status of the Rhizobium leguminosarum isolates.

Methods

Legumes were inoculated with various strains of rhizobia, and grown under controlled conditions, for several weeks. The presence of an effective nitrogenase enzyme was determined by acetylene reduction, then plants were harvested and the presence of nodules determined. Complete methods are described in the methods chapter in section s-Host-range.

Specificity of Mesorhizobium to native legumes

Little research on effective nodulation by rhizobia on New Zealand legumes has been done, apart from a few studies in the 1960's and `70's by R.M.Greenwood. In his Greenwood69 publication, there appears within a discussion of Lotus in New Zealand pastures, a note that strains isolated from native legumes cross-nodulated with either Carmichaelia, Sophora, and Clianthus, were effective with Sophora alone; or were effective with Carmichaelia and Clianthus but ineffective with Sophora. Unfortunately complete results of this work are not available, apart from those few listed in the strain information sections of the ICMP database. In a later study, strain isolated from Sophora spp.were found to nodulate Sophora spp.effectively, but Carmichaelia and Clianthus ineffectively Crow81.

In Pueppke99's extensive Pueppke99 study, broad-host-range Ensifer fredii strains NGR234 and USDA257 were unable to form nodules on New Zealand Sophora. Other New Zealand native legumes were not examined.

Experimental design

Ten Mesorhizobium strains isolated from New Zealand legumesStrains of Mesorhizobium spp.isolated from New Zealand native legumes will be referred to as Meso-NZL for simplicity of prose. were selected from previously described strains (ICMP numbers 14330, 11719, 15054, 12685, 11721, 11726, 11541, 12680, 12690, and 13190). These strains represent diversity in genomic grouping, original host legume, and nodA type.

Five native legumes (Sophora microphylla, Sophora tetraptera, Carmichaelia australis, Carmichaelia stevensonii, and Clianthus puniceus) were selected as hosts. Two Sophora and two Carmichaelia species were used to access any interspecific variation. Montigena seeds were not available in sufficient numbers for these experiments. Plants were inoculated and grown as described in the methods chapter (section s-Host-range).

Results

sidewaystable Mesorhizobium strains nodulating native legumes tabularlccccccc 3*tabularc Mesorhizobium strain tabular & 3*tabularc Genomic group tabular & 3*tabularc nodA type tabular & 5cNod/Fix response on inoculated native legume 4-8 & & & Sophora & Sophora & Carmichaelia & Carmichaelia & Clianthus & & & microphylla & tetraptera & australis & stevensonii & puniceus 14330 (Sophora) & A & 3 & Nod Fix & Nod Fix & Nod Fix & Nod Fix & Nod Fix 11719 (Sophora) & A & 3 & Nod Fix & Nod Fix & Nod Fix & Nod Fix & Nod Fix 15054 (Carmichaelia) & A & 1 & Nod Fix & Nod Fix & Nod Fix & Nod Fix & Nod Fix 12685 (Montigena) & B & 1 & Nod Fix & Nod Fix & Nod Fix & Nod Fix & Nod Fix 11721 (Clianthus) & C & 1 & Nod Fix & Nod Fix & Nod Fix & Nod Fix & Nod Fix 11726 (Clianthus) & C & 1 & Nod Fix & Nod Fix & Nod Fix & Nod Fix & Nod Fix 11541 (Clianthus) & D & 2 & Nod Fix & Nod Fix & Nod Fix & Nod Fix & Nod Fix 12680 (Clianthus) & D & 1 & Nod Fix & Nod Fix & Nod Fix & Nod Fix & Nod Fix 12690 (Montigena) & D & 2 & Nod Fix & Nod Fix & Nod Fix & Nod Fix & Nod Fix 13190 (Carmichaelia) & D & 1 & Nod Fix & Nod Fix & Nod Fix & Nod Fix & Nod Fix tabular

t-Meso-native Accession numbers in the ICMP culture collection, original host in parenthesis. Grouping according to 16S rRNA gene. Grouping according to nodA gene. Presence of nodules: Nod, absence: Nod. Presence of nitrogenase activity: Fix, absence: Fix.

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Five native legume species from three genera were inoculated with by ten Mesorhizobium strains originally isolated from these species. Isolates were able to nodulate their host legume, and further nodulate other tested legumes. The ability of each association to form nodules and fix nitrogen (reduce acetylene) is presented in Table t-Meso-native.

The most notable feature is the extent of nodulation. In all cases, strains were able to nodulate the genus from which they were isolated. Additionally, there was extensive nodulation outside the host of isolation, for example, all strains formed effective nodules on Clianthus (Nod Fix), and four strains (15054, 11721, 11726, and 12680) were able to nodulate all five legumes effectively.

There were exceptions however to this widespread nodulation. No nodules (Nod Fix) were formed on either Sophora species by three strains: 12685 (Montigena), 12690 (Montigena), 13190 (Carmichaelia). There were also limited cases of ineffective nodulation; strains 14330 (Sophora) and 11719 (Sophora) formed ineffective nodules (Nod Fix) on both Carmichaelia species. Strain 11541 (Clianthus) formed ineffective nodules on both Sophora species.

There was a weak correlation between widespread nodulation ability and nodA gene type. Four strains capable of nodulating all five legumes had the nodA type 1 geneSee Fig p-nodA-MB for an explanation of nodA gene types.. However other strains with the nodA type 1 gene were unable to nodulate Sophora. Both isolates with the nodA type 3 gene had identical nodulation patterns (nodulating all legumes, but ineffective on Carmichaelia).

The presence of the original inoculant in the root nodules was confirmed by UARR PCR amplification and DNA sequencing of an isolate from each replicate (see Section UARR). Controls for each legume species, consisting of uninoculated plants, did not form nodules.

Discussion

A primary aim of this research was to determine the extent of specificity among the Mesorhizobium species nodulating the native legumes of New Zealand. In experiments where native plants were inoculated with Mesorhizobium isolates, there was a distinct lack of specific rhizobia--host interactions, indeed the results show a high level promiscuity amongst the native legumes. Every isolate tested had the ability to effectively nodulate Clianthus. Sophora, although seemingly only nodulated by Group A Mesorhizobium in the field, was also nodulated in these experiments by Mesorhizobium groups C and D (grouping based on the 16S rRNA gene). Four strains were capable of nodulating all native legumes tested. There was no apparent intraspecific variation in nodulation within a legume genus, as the results for two species of Sophora and two of Carmichaelia were the same to each strain. Nevertheless, there are eight Sophora and 22 Carmichaelia species in New Zealand and it is therefore possible there may be some intraspecific variation among these species. Few host-range studies have been made of native legumes in other countries, so it remains to be seen if this pattern of high promiscuity among native legumes with native rhizobia is generally applicable.

The extent of promiscuity was not universal, there were some symbiotic combinations that did not nodulate or produced ineffective nodules. All these cases were in some way linked to Sophora species. Strains 12685, 12690, and 13190 were unable to form nodules on two Sophora species and strain 11541 produced ineffective nodules. More intriguingly, strains 14330 and 11719 (isolated from Sophora species) produced ineffective nodules on Carmichaelia. This latter example is the only one in Table t-Meso-native where a pattern in nodulation is reflected in a corresponding relationship in the genomic or nodA genes. In this case, only nodA type 3 (in genomic group A) caused ineffective nodules on Carmichaelia.

In Chapter 4, a clear relationship was seen between nodA gene and host legume. However in this host-range testing it was found that some strains with type 3 (`Sophora') nodA genes could nodulate Carmichaelinae members, and nodA types `Carmichaelinae 1' and `Carmichaelinae 2' could nodulate Sophora. This apparent incongruence between field isolations, and actual host-range ability may result from sample bias in collection. Alternatively, the results from the field may reflect the most effective nitrogen fixers selected for by the host. Although the acetylene reduction assays were not statistically designed to be quantitative, the assay does provide a quantitative result (pmoljarmin). From this it was determined that strains with type 3 (`Sophora') nodA genes were approximately ten times more effective when inoculated on Sophora, than on other species (data not shown). However, rigorous statistically-sound further experiments would be necessary to confirm this.

Exotic legumes associated with Mesorhizobium

Background

On the basis of data showing broad indigenous host-ranges of native Mesorhizobium, further experiments were designed to test these species against a range of exotic legumes, those not naturally found in New Zealand. The compatibility with invasive woody weeds (gorse, broom, Acacia) was tested in section s-Meso-Brady. In this experiment, the legumes most likely compatible with native rhizobia were chosen vis.those that typically nodulate with Mesorhizobium species in their country of origin. Table t-Meso-hosts lists type strains of Mesorhizobium and the legumes they are able to effectively nodulate (as determined by original publication, see Table t-Rhizobia-species). The actual host-range is certainly larger than shown, for example M.amorphae strains, other than the type, can nodulate all native legumes (genomic group C, in Table t-Meso-native), as well as Sophora viciifoliaNow classified as Sophora davidii., Robinia pseudoacacia, Lotus oroboides, Astragalus canadensis, Dalea purpurea and various Amorpha species Tan99,Ulrich00,Qian02,Tlusty05.

table Host plants of Mesorhizobium type strains tabularlll Type strains & Genus and [Tribe] of host legume M.amorphae & Amorpha [Amorpheae] M.chacoense & Prosopis [Mimoseae] M.ciceri & Cicer [Cicereae] M.huakuii & Astragalus [Galegeae], Acacia [Acacieae] M.loti & Lotus [Loteae] M.mediterraneum & Cicer [Cicereae] M.plurifarium & Prosopis, Leucaena [Mimoseae], Acacia [Acacieae] M.septentrionale & Astragalus [Galegeae] M.temperatum & Astragalus [Galegeae] M.tianshanense & Glycyrrhiza, Swainsona, Caragana [Galegeae], & Glycine [Phaseoleae], Sophora [Sophoreae] tabular

t-Meso-hosts Host genus in italics, tribe in square brackets. References for hosts are original publications, see Table t-Rhizobia-species.

table

Experimental design

Seven Mesorhizobium strains isolated from New Zealand legumes were selected from previously described strains (ICMP numbers 14330, 11719, 15054, 12685, 11726, 11541, and 13190). These strains represent the diversity in genomic grouping, original host legume, and nodA type.

Five exotic Mesorhizobium-associated legume species were selected as hosts: Astragalus membranaceus (milk vetch), Lotus tetragonolobus (asparagus pea), Cicer arietinum (chick pea), Styphnolobium japonicumFormerly Sophora japonica. (Japanese pagoda tree), and Glycine max (soybean). These species were chosen based on tribal diversity and the availability of sufficient seed. Insufficient Glycine max plants survived for analysis. Plants were inoculated and grown as described in the methods chapter.

Results

sidewaystable Mesorhizobium strains nodulating exotic legumes tabularlcccccccc 3*tabularc Mesorhizobium strain tabular & 3*tabularc Genomic group tabular & 3*tabularc nodA type tabular & 4cNod/Fix response on inoculated legume species 4-7 & & & Astragalus & Lotus & Cicer & Styphnolobium & & & membranaceus & tetragonolobus & arietinum & japonicum 14330 (Sophora) & A & 3 & Nod Fix & Nod Fix & Nod Fix & Nod Fix 15054 (Carmichaelia) & A & 1 & Nod Fix & Nod Fix & Nod Fix & Nod Fix 11719 (Sophora) & A & 3 & Nod Fix & Nod Fix & Nod Fix & Nod Fix 12685 (Montigena) & B & 1 & Nod Fix & Nod Fix & Nod Fix & Nod Fix 11726 (Clianthus) & C & 1 & Nod Fix & Nod Fix & Nod Fix & Nod Fix 11541 (Clianthus) & D & 2 & Nod Fix & Nod Fix & Nod Fix & Nod Fix 13190 (Carmichaelia) & D & 1 & Nod Fix & Nod Fix & Nod Fix & Nod Fix tabular

t-Meso-exotic Accession numbers in the ICMP culture collection, original host in parenthesis. Grouping according to 16S rRNA gene. Grouping according to nodA gene. Presence of nodules: Nod, absence: Nod. Presence of nitrogenase activity: Fix, absence: Fix.

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When four exotic legumes---typically associated with Mesorhizobium species---were challenged by seven native Mesorhizobium strains, only Astragalus membranaceus was effectively nodulated (Tablet-Meso-exotic).

Effective nodules (Nod Fix) were formed by all strains inoculated on Astragalus membranaceus irrespective of genomic group or nodA type. No nodules were formed on Lotus tetragonolobus, Cicer arietinum, or Styphnolobium japonicum. No results for Glycine max were obtained as too few plants survived, possibly due to poor quality seed.

The presence of the original inoculant in the root nodules was confirmed by UARR PCR amplification and DNA sequencing of an isolate from each replicate (see Section UARR). Controls for each legume species, consisting of uninoculated plants, did not form nodules.

Discussion s-Exotic-meso

An important issue in the understanding of rhizobia--legume ecology is the ability of rhizobia to nodulate legumes isolated by geographical boundaries or evolutionary distance. In New Zealand, legumes have been isolated---presumably along with their symbiotic bacteria---for millions of years. In this study, an experiment was designed to investigate if the New Zealand Mesorhizobium isolates can nodulate legumes that they have been separated from, by thousands of kilometres of ocean and for millions of years.

It was hypothesised that if the Meso-NZL isolates were able to nodulate other species, the most likely compatible legumes would be those that typically nodulate with Mesorhizobium species in their country of origin. In this study four exotic legume species that nodulated with Mesorhizobium were tested (Astragalus membranaceus, Lotus tetragonolobus, Cicer arietinum and Styphnolobium japonicum). The results were unambiguous, only A.membranaceus formed nodules with all strains, which in all cases were effective, reflecting the results seen for Clianthus which also nodulated with all strains. It is interesting that some of the Mesorhizobium strains (14330, 11719, 12685, 11541, and 13190) were able to from effective nodules on an exotic legume, yet failed to nodulate some native legumes effectively, notably members of the Sophora genus.

Styphnolobium japonicum did not form nodules in this experiment. This possibly indicated incompatibility with New Zealand rhizobia. However the literature suggests it may not be able to form nodules at all. Styphnolobium japonicum, the Japanese pagoda tree, was known as Sophora japonica until it was reclassified in 1993 Sousa93,Santamour97. There have been conflicting reports over the ability of S.japonicum to nodulate Foster98. Early work suggested that it nodulated Wilson39b,Asai44,Ishizawa53,Allen81,Sutherland94. However the absence of nodulation reported in Batzli91,Santamour97, and the comprehensive Foster98 study, presented strong evidence to the contrary. Nodulation was once thought common in legumes, but in subfamilies Caesalpinioideae, Mimosoideae, and Papilionoideae; 71, 10, and 3 of the genera respectively are reported to not form root nodules Bryan96. It is possible that Styphnolobium is a non-nodulating legume, and that prior reports of nodulation are due to nodule or plant misidentification. Nevertheless, New Zealand has a large population of Sophora species (kowhai), in Sophoreae---the same tribe as Styphnolobium. No isolates from New Zealand were tested in the studies mentioned above, so there remained the possibility that nodulation could occur with kowhai-adapted rhizobia.

However, the results of this thesis show that none of the Meso-NZL isolates were able to form nodules on S. japonicum, supporting its status as a non-nodulating legume. This is verified by molecular studies that show that Sophoreae is actually polyphyletic, and S.japonicum is distinct from the Sophora sector Edwardsia (southern hemisphere species), and more similar to other non-nodulators Calia and Cladrastis Kass97. Additionally, studies on the legume early nodulation gene ENOD2, show that S. japonicum may lack a component of the signal transduction pathway leading to nodule organogenesis Foster00.

There has been some previous research on the exotic host-range of Meso-NZL strains. All prior knowledge comes from work by R.M.Greenwood and colleagues Greenwood77,Greenwood78a,Crow81, and additional unpublished work by Greenwood that is recorded in the strain information section of the ICMP database. As a component of their research, Meso-NZL isolates were tested against exotic legumes. Other exotic genera (and tribe in square brackets) they report to nodulate are: Lessertia [Galegeae] (ICMP), Sutherlandia [Galegeae] (ICMP), Swainsona [Galegeae]Now [Carmichaelinae]. Greenwood78a, Colutea [Galegeae] (ICMP), and Onobrychis [Hedysareae] Greenwood77,Greenwood78a. Meso-NZL isolates were found to be unable to nodulate Leucaena [Mimoseae], Lotus [Loteae], Trifolium [Trifolieae], Phaseolus [Phaseoleae], Pisum [Vicieae], Vigna [Phaseoleae], and Canavalia [Phaseoleae] Greenwood78a,Crow81. In this thesis it was found that Meso-NZL isolates nodulated Astragalus [Galegeae], but not Lotus [Loteae], or Cicer [Vicieae].

From these data an interesting pattern emerges; all of the nodulations by Meso-NZL rhizobia were confined to the Galegeae, Hedysareae, and Carmichaelinae tribes (Carmichaelinae contains the New Zealand natives---with the exception of Sophora Wagstaff99,Wojciechowski04). These tribes are closely related in phylogenies Doyle00,Sanderson96,Lavin04. It may be that symbiotic barriers restrict the symbiosis of Meso-NZL isolates to these few closely related legume tribes

The next obvious step was to test Mesorhizobium strains isolated in other countries on native legumes. However under New Zealand lawSpecifically the Biosecurity Act 1993, and the Hazardous Substances And New Organisms Act 1996. these exotic strains are restricted, would require permits, and need to be conducted under strict containment, which was not readily available. Fortunately such experiments were conducted by other researchers before the introduction of these laws.

It was found that members of the Carmichaelinae can effectively nodulate with rhizobia isolated from other countries, generally with strains isolated from related tribes. Carmichaelia nodulated when planted in overseas soil (England, France, South Africa), although it is unclear if these nodulations were effective Dawson00,Milovidov28,Grobbelaar74. Crow81 also determined that rhizobia isolated from Galegeae and Hedysareae members were able to nodulate native legumes. Carmichaelia odorataAs Carmichaelia angustata in publication. or Clianthus puniceus were effectively nodulated by strains from Onobrychis viciifolia [Hedysareae], Caragana arborescens [Galegeae], Leucaena leucocephala [Mimoseae], Astragalus onobrychis [Galegeae], and Caragana chamlagu [Galegeae]. Ineffective nodules were formed by many other strains, and for some no nodulation was found Crow81.

Sophora microphylla was nodulated by the same exotic rhizobia as members of the Carmichaelinae, and also rhizobia isolated from Securigera variaAs Coronilla varia in publication. [Loteae], Sophora formosa [Sophoreae], Sophora secundiflora [Sophoreae], Parochetus communis [Trifolieae], and Mimosa invisa [Mimoseae] Crow81. Strains isolated from two native Sophora species were only effective on other Sophora spp., whilst forming ineffective or no nodules on Carmichaelinae members and related species Crow81.

Thus it is clear that rhizobia isolated from phylogenetically similar legumes can effectively cross nodulate, but rhizobia from more distant legumes are usually ineffective.

Rhizobium leguminosarum

An exception to the observation of Mesorhizobium isolates nodulating the native legumes, was the identification of four Rhizobium leguminosarum strains, isolated from Carmichaelia, Clianthus, and Sophora.

Rhizobium leguminosarum was the first rhizobial species named Frank79,Frank89 and is well studied. It has three infrasubspecific variants called biovars (trifolii, phaseoli, viciae) that are considered specific to the Trifolium, Phaseolus and Pisum/Vicia/Lathyrus/Lens genera respectively (Table taxbox-Crop). These biovars have been used extensively world-wide as commercial inoculants to improve the growth of these crop and pasture plants Hastings66.

table Legumes nodulated by Rhizobium leguminosarum tabularcccc Kingdom: & 3cPlantae Division: & 3cMagnoliophyta Class: & 3cMagnoliopsida Order: & 3cFabales Family: & 3cFabaceae Subfamily: & 3cFaboideae Tribe: & Trifolieae & Phaseoleae & Vicieae Genus: & Trifolium & Phaseolus & Pisum, Vicia, Lathyrus, Lens tabular

taxbox-Crop Note: The three biovars trifolii, phaseoli, viciae are considered to specifically nodulate genera in their respective tribes: Trifolieae, Phaseoleae, and Vicieae. table

R.leguminosarum has been infrequently recorded nodulating other plant species. Bergeys84 lists R.leguminosarum bv.phaseoli as occasionally nodulating Macroptilium atropurpureum [Phaseoleae], with the nodules commonly ineffective. R.leguminosarum bv.phaseoli strain CFN299, nodulates Phaseolus vulgaris and Leucaena esculenta [Mimoseae] effectively, and when its plasmids were transferred to Rhizobium radiobacterAs Agrobacterium tumefaciens in the publication. strain GM19023, this bacterium gained the ability to nodulate Phaseolus and Leucaena Martinez87 effectively. Tlusty05 indicated that nodule isolates from Dalea purpurea [Amorpheae] in Iowa and Minnesota were R.leguminosarum, however critical analysis of their data places these isolates into a wider cluster including R.tropici and R.leguminosarum.

The host-range of R.leguminosarum has also been extended by genetic engineering techniques. Laboratory mutants of nodD, possessing inducer-independent ability to activate nod gene expression, were capable of extending the host-range of R.leguminosarum bv.trifolii to the non-legume Parasponia (in the elm family) McIver89. Transconjugant R.leguminosarum strains containing the nodZ gene of Bradyrhizobium extended the host-range of R.leguminosarum bv.viciae to include Macroptilium atropurpureum [Phaseoleae], Glycine sojaNow classified as Glycine max. [Phaseoleae], Vigna unguiculata [Phaseoleae], and Leucaena leucocephala [Mimoseae]. The nodules induced on M. atropurpureum were ineffective Lopez-Lara96.

Greenwood69 wrote that ``[native legumes] will readily form ineffective nodules with a range of introduced rhizobia including clover rhizobia. This was unexpected, as clover rhizobia are considered to nodulate Trifolium species only, apart from occasional ineffective nodules on pea and vetch''.

Hence the discovery of naturally occurring R.leguminosarum isolates in the root nodules of New Zealand native legumes is interesting, and presents a novel expansion of the nodulating host-range of this bacterium. It is possible however, that these strains were isolated from misidentified legumes or are otherwise erroneous. Only four strains (11542, 11727, 12687, 14642) out of the 22 isolated from native legumes were identified as R.leguminosarum by gene sequences and fatty acid profiles. Additionally only a single strain (14642) was isolated as part of this thesis research---the other strains were obtained from the ICMP, and it is possible that errors may have been made during deposition and transfer from the previous collection (The New Zealand Department of Scientific and Industrial Research, Applied Biochemistry Division collection -- NZP). Thus it was important that these strains are verified for their nodulation and fixation capacity.

Experimental design

The experiment was designed to investigate if R.leguminosarum strains 11542, 11727, 12687, and 14642 nodulate New Zealand native legumes, and if the nodulation is effective.

In the experiment, three representative species of native legumes were inoculated with four strains of R.leguminosarum (11542, 11727, 12687, and 14642) isolated from New Zealand legumes.

As a control, the native legumes were also inoculated with the three recognised biovars of R.leguminosarum, to determine if nodulation of New Zealand native legumes is a general property of all R.leguminosarum strains, or if the New Zealand isolates are unique. These biovar strains are ICMP 2668 (bv.trifolii), ICMP 2672 (bv.phaseoli), ICMP 5943 (bv.viciae). Controls included the three conventional host legumes (pea, bean, clover) which were inoculated with all seven strains above. In the case of the three biovar strains, these legumes act as positive controls. In the case of the New Zealand isolates, this would determine if these strains have retained their conventional host-range nodulation capacity, and thus to assign the strains to a biovar type. Plants were inoculated and grown as described in the methods chapter.

To determine if the isolation of R.leguminosarum from native legumes was a rare or common event, other rhizobial isolates collected in the vicinity of strain 14642 were sequenced (16S rRNA) to determine their identity.

Results

sidewaystable Rhizobium leguminosarum host-range tabularlccccccc 3*tabularc Rhizobium leguminosarum strain tabular & 3*tabularc nodA type tabular & 6cNod/Fix response on inoculated legume species 3-8 & & Sophora & Carmichaelia & Clianthus & Trifolium & Phaseolus & Pisum & & microphylla & australis & puniceus & repens & vulgaris & sativum 11542 (Clianthus) & 4 & Nod Fix & Nod Fix & Nod Fix & Nod Fix & Nod Fix & Nod Fix 11727 (Carmichaelia) & 4 & Nod Fix & Nod Fix & Nod Fix & Nod Fix & Nod Fix & Nod Fix 12687 (Carmichaelia) & 4 & Nod Fix & Nod Fix & Nod Fix & Nod Fix & Nod Fix & Nod Fix 14642 (Sophora) & 5 & Nod Fix & Nod Fix & Nod Fix & Nod Fix & Nod Fix & Nod Fix 1-8 2668 (trifolii) & 4 & Nod Fix & Nod Fix & Nod Fix & Nod Fix & Nod Fix & Nod Fix 2672 (phaseoli) & 6 & Nod Fix & Nod Fix & Nod Fix & Nod Fix & Nod Fix & Nod Fix 5943 (viciae) & 5 & Nod Fix & Nod Fix & Nod Fix & Nod Fix & Nod Fix & Nod Fix tabular

t-R.leg-nod-nif Accession numbers in the ICMP culture collection, original host (upper section) or biovar (lower section) in parenthesis. Grouping according to nodA gene. Native legumes in left section, introduced crop legumes in right section. Presence of nodules: Nod, absence: Nod. Presence of nitrogenase activity: Fix, absence: Fix. Inconsistency: 50 of replicates were positive.

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Patterns of nodulation and nitrogen fixation (acetylene reduction) are presented in Table t-R.leg-nod-nif. Both Carmichaelia and Clianthus were nodulated by all four R.leg-NZL strainsThe strains of Rhizobium leguminosarum isolated from New Zealand native legumes will be referred to as R.leg-NZL for simplicity of prose., but in all cases the nodules were ineffective. Nodulation of C.australis with strain 14642 (Sophora) was variable. R.leguminosarum bv.trifolii also produced ineffective nodules on those species. In contrast, Sophora was only nodulated, albeit ineffectively, by the strain that was previously isolated from a Sophora root nodule.

The R.leg-NZL strains, shown above to form ineffective nodules on native legumes, also retained their ability to nodulate---effectively in most cases---the conventional host plants of R.leguminosarum: pea, bean and clover (see upper right section of Table t-R.leg-nod-nif).

This result confirms the expansion of the known nodulating host-range to include some New Zealand native legumes, although in all cases the symbiosis is ineffective.

To compare the R.leg-NZL strains with typical R.leguminosarum strains, the same six legume species (native and exotic) used above, were challenged by three strains representing the different biovars of R.leguminosarum (see lower right section of Table t-R.leg-nod-nif). These strains effectively nodulated their respective host legume.

When the native New Zealand legumes were challenged by the three biovar strains, some ineffective nodules were formed, but to a lesser extent than with the R.leg-NZL strains (see lower left section of Table t-R.leg-nod-nif). None of the biovar strains were able to form nodules on S.microphylla. Strain 2672 (bv.phaseoli) did not form nodules on any of the native legumes.

Controls for each legume species consisting of uninoculated plants did not form nodules for any of the experiments above.

To determine if the isolation of R.leguminosarum from native legumes was a rare chance event, further 16S rRNA genes were sequenced from other isolates from Sophora chathamica plants in the same vicinity as that from which strain 14642 was isolated. These isolates were all obtained from different nodules. Sequences of a 400-bp section (UARRUniversal Amplified Ribosomal Repeat, see Section UARR) of the 16S rRNA gene from strains 14643, 14644, and 14645; revealed that these were also R.leguminosarum.

Discussion

Characterisation of strains

Host-range studies showed that nodules were formed on the native legumes by several strains of R.leguminosarum, although this symbiosis was ineffective. This discovery extends the known host-range of R.leguminosarum to include New Zealand native legumes---albeit ineffectively.

Not only did R.leguminosarum strains, isolated from root nodules of native plants, re-nodulate native plants in this experiment, but two biovar strains (trifolii, and viciae) also had this ability. This implies that there may be nothing unique about the four R.leg-NZL strains and that other R.leguminosarum strains have the ability to nodulate native legumes. None of the biovar strains nodulated Sophora, and the phaseoli strain did not nodulate any of the native legumes. The viciae strain did nodulate Clianthus but only half of the replicates had nodules. This difference between R.leg-NZL and biovar strains may result from selection for better nodulation that occurs in `wild' strains, or be a representation of strain variation.

In the previous chapter it was determined that the nodA gene from the R.leg-NZL strains match those of the biovar strains (Fig.p-nodA-MB), and those of R.leguminosarum strains in other countries Schofield86. Through linkage with the nodA gene it may be inferred that the other nodulation genes also match those of R.leguminosarum, although this remains to be confirmed. Thus it appears that these R.leg-NZL strains are typical R.leguminosarum strains without acquired accessory genetic elements. The fact that all R.leg-NZL isolates also retained their ability to nodulate their typical host species of pea, bean, and clover further supports this idea. This implies that the set of nodulation and fixation genes, as well as accessory symbiosis components such as exopolysaccharides, lipopolysaccharides, and cyclic--glucans, are that of R.leguminosarum.

Biovar assignment

It is unclear to which biovar the R.leg-NZL isolates should be assigned. The nodA gene phylogeny (Fig.p-nodA-MB) shows that R.leguminosarum biovars may be able to be distinguished by nodA gene sequence. Biovar trifolii is type 4, bv.phaseoli is type 6, and bv.viciae is type 5. The R.leg-NZL strains 11542, 11727, 12687 all are grouped with type 4 (the trifolii type), except for 14642 which is type 5. These gene sequences alone are not enough to assign biovar type, as it is a phenotype and should be assigned on nodulation ability. Although three of the R.leg-NZL strains have the trifolii nodA type, their nodulation ability is quite different from that expected of a trifolii biovar---only one strain actually formed nodules on Trifolium repens. However, they do elicit nodules on Phaseolus vulgaris and Pisum sativum. Strain 14642 could possibly be assigned to bv.viciae as it nodulates Phaseolus vulgaris as expected, however further testing would be necessary to confirm this.

The verified biovar strains used as controls nodulated their respective legume hosts effectively, although in some cases they also nodulated outside of the normal range. However, this is not unexpected as biovars are not as tightly specific as their names suggest. Table t-Biovar, adapted from Bergeys84, shows that biovars are capable of nodulating other legumes to a lesser extent. The biovar strains used in this study were selected by DSIRDepartment of Scientific and Industrial Research. scientists in the 1970s for use as commercial inoculants, and thus may have a higher degree of nodulation capacity than standard Rhizobium leguminosarum strains.

table [Cross inoculation in R.leguminosarum biovars]Cross inoculation in R.leguminosarum biovars tabularlccc & 3cR.leguminosarum 2-4 Host Plant & biovar & biovar & biovar & viciae & trifolii & phaseoli Pisum sativum & + & & Phaseolus vulgaris & () & () & + Trifolium repens & & + & () tabular

t-Biovar Adapted from Table 4.54 in Bergeys84. Symbols: +: generally nodulates; : sometimes nodulates, nodules commonly ineffective; (): rarely nodulates, nodules commonly ineffective. table

Host-range of R.leguminosarum

It is probable that R.leguminosarum can nodulate legumes other than its typical hosts and New Zealand native legumes, but its nodulation ability does not appear to have been investigated thoroughly. Originally the biovars of R.leguminosarum were considered separate species (Rhizobium trifolii, Rhizobium phaseoli), and were assigned primarily on their ability to nodulate a particular legume. It was not until recently that bacterial species could be accurately identified using molecular methods. It is probable that in the past if R.leguminosarum was isolated from a non-target legume it would not be recognised, and would be listed as `Rhizobium sp.' along with thousands of other poorly characterised strains. With the rise of molecular techniques and the expanding examination of non-commercial and historically under-examined legumes, the accepted nodulating host-range of R.leguminosarum---and other species of rhizobia could be expanded even further.

Specificity of Bradyrhizobium to introduced legume weeds

Background

In Chapter 3 it was established that the introduced woody legume weeds---gorse (Ulex), broom (Cytisus), and Acacia---were solely nodulated by diverse Bradyrhizobium species. Given the diversity of Bradyrhizobium and the widespread nodulation of legume weed species in New Zealand, it would be informative to know if there was any specificity in these relationships, or if cross nodulation between different weed legumes is possible.

Gorse and broom arrived in New Zealand from Europe, but most Acacias in New Zealand are from Australia. This difference in origins and presumably evolutionary history may have resulted in different nodulation ability. The nodA gene data (see Fig. p-nodA-MB) showed that nodA type 7 was found in gorse and broom isolates and nodA type 8 was found in Acacia isolates. Studies were therefore undertaken to determine whether the nodA patterns also reflected host-range associations for gorse, broom, and Acacia.

All previously published literature reports that Cytisus (broom) species are nodulated by Bradyrhizobium Greenwood77,Sajnaga01b,Perez03,Rodriguez-Echeverria03. Surprisingly, given the serious weed status of gorse, there are only two modern studies identifying gorse rhizobia. One is based on Chapter 1 of this thesis Weir04. The other study Leary06, found that gorse invading volcanic sites in Hawaii also nodulates with Bradyrhizobium spp.

Ulex and Cytisus form ineffective nodules with the exceptionally broad host-range rhizobia Ensifer fredii NGR234 Pueppke99. Australian Acacia have been reported to nodulate dominantly with Bradyrhizobium and also with Rhizobium, and Mesorhizobium and Lafay01.

Experimental design

Four Bradyrhizobium strains isolated from introduced legumes were selected from the previously described strains (ICMP numbers: 12674, 14291, 14533, and 14755). Three introduced legume species representing species from which the test strains were isolated, were selected as hosts: Cytisus scoparius, Ulex europaeus, and Acacia longifolia. Each legume was inoculated with each strain. Plants were inoculated and grown as described in the methods chapter.

Results

table Bradyrhizobium strains nodulating woody weed legumes tabularlccccc 3*tabularc Bradyrhizobium strain tabular & 3*tabularc Genomic group tabular & 3*tabularc nodA type tabular & 3cNod/Fix response 4-6 & & & Cytisus & Ulex & Acacia & & & scoparius & europaeus & longifolia 12674 (Ulex) & G & 7 & Nod Fix & Nod Fix & Nod Fix 14291 (Cytisus) & H & 7 & Nod Fix & Nod Fix & Nod Fix 14533 (Ulex) & F & 7 & Nod Fix & Nod Fix & Nod Fix 14755 (Acacia) & F & 8 & Nod Fix & Nod Fix & Nod Fix tabular

t-Brady-cross Accession numbers in the ICMP culture collection, original host in parenthesis. Grouping according to 16S rRNA gene. Grouping according to nodA gene. Presence of nodules: Nod, absence: Nod. Presence of nitrogenase activity: Fix, absence: Fix. Inconsistency: 50 of replicates were positive.

table

Results of nodule formation and nitrogen fixation (acetylene reduction) are presented in Table t-Brady-cross. All strains formed nodules with all three plant species, and these were shown to be effective in both Cytisus and Ulex. Acacia only formed effective nodules with the strain which was originally sourced from this host (14755).

This pattern of nodulation correlates with nodA type in that nodA type 7 strains nodulated all legumes tested, but were ineffective on Acacia. nodA type 8 effectively nodulated all legumes, although only a single nodA type 8 strain was tested.

The presence of the original inoculant in the root nodules was confirmed by UARR PCR amplification and DNA sequencing of an isolate from each replicate (see Section UARR). Controls for each legume species, consisting of uninoculated plants, did not form nodules.

Discussion

Gorse, broom and Acacia are serious invasive weeds in New Zealand. Several characteristics contribute to invasiveness, including high levels of seed production, long-term seed survival, mature plant longevity, high density seedling rejuvenation, and nitrogen fixation with symbiotic bacteria. The latter trait, notably the ability to nodulate with rhizobia in nitrogen deficient soils, confers a tremendous competitive advantage over other plants.

The results presented here indicate that gorse and broom can effectively cross nodulate with the same bradyrhizobia, and even bradyrhizobia isolated from Acacia, further enhancing their potential for successful establishment in new areas. Acacia is less successful, forming ineffective nodules with all but its own rhizobia. This correlates with the nodA gene data showing that gorse and broom have a different type of nodA gene than Acacia (Fig.p-nodA-MB). More extensive experiments with more strains and additional Acacia species would be required to confirm these results.

New Zealand currently has 19 known species of Acacia NZPlant, all of which are exotic species arriving since the mid nineteenth century from Australia and other counties. However, the fossil record shows that during the Neogene (23 mya -- present) New Zealand had a native Acacia population, which then became extinct during the last ice age (ten thousand years ago) Stevens95,Lee01,Raine05. It would seem reasonable to suggest that these Acacia had a complementary population of symbiotic Bradyrhizobium spp., these Bradyrhizobium spp.may have existed in soils since that time as autochthons, unable to nodulate with the existing legumes, until the recent introduction of compatible legumes. Another plausible explanation is that the Acacia bradyrhizobia arrived with their hosts during human colonisation.

The origin of the gorse and broom bradyrhizobia is less clear, although the different nodA gene (type 7) present in strains which do not fix nitrogen with Acacia, would indicate a different origin. It is apparent more work needs to be done in this area, including investigating the presence of Bradyrhizobium in undisturbed New Zealand soils. This is investigated later in section s-soils.

Verification of host-range of Mesorhizobium and Bradyrhizobium s-Meso-Brady

Background

The identification of rhizobial isolates in Chapter 3 indicated a distinct partitioning of Mesorhizobium (and R.leguminosarum) isolated from root nodules of native legumes, and Bradyrhizobium isolated from the root nodules of the introduced weeds. However these data indicate the host-range and specificity of these isolates seen in the field, and may not fully represent actual nodulation ability. Further host-range tests were therefore conducted to determine whether effective cross-species/strain nodulation was possible.

Experimental design

Four Bradyrhizobium strains (ICMP numbers: 12674, 14291, 14533, and 14755) were inoculated on to three native legumes (Sophora microphylla, Carmichaelia australis and Clianthus puniceus). At the same time the introduced legumes Cytisus scoparius, Ulex europaeus, and Acacia longifolia were inoculated with Mesorhizobium strains 11726, 12685, 14330, and 15054. Strains were selected to represent diversity in genomic grouping, original host legume, and nodA type. Plants were inoculated and grown as described in the methods chapter.

Results and discussion

sidewaystable Mesorhizobium / Bradyrhizobium incompatibility tabularlcccccc 3*tabularc Rhizobia strain tabular & 6cNod/Fix response on inoculated legume species 2-7 & Sophora & Carmichaelia & Clianthus & Acacia & Cytisus & Ulex & microphylla & australis & puniceus & longifolia & scoparius & europaeus Meso 14330 & --- & --- & --- & Nod Fix & Nod Fix & Nod Fix Meso 11726 & --- & --- & --- & Nod Fix & Nod Fix & Nod Fix Meso 15054 & --- & --- & --- & Nod Fix & Nod Fix & Nod Fix Meso 12685 & --- & --- & --- & Nod Fix & Nod Fix & Nod Fix Brady 14291 & Nod Fix & Nod Fix & Nod Fix & --- & --- & --- Brady 14755 & Nod Fix & Nod Fix & Nod Fix & --- & --- & --- Brady 12674 & Nod Fix & Nod Fix & Nod Fix & --- & --- & --- Brady 14291 & Nod Fix & Nod Fix & Nod Fix & --- & --- & --- tabular

t-Meso-Brady-Cross 3cm Accession numbers in the ICMP culture collection, original host in parenthesis. 3cm Presence of nodules: Nod, absence: Nod. 3cm Presence of nitrogenase activity: Fix, absence: Fix. 3cm Note: Data indicated by `---' is presented in Tables t-Meso-native and t-Brady-cross. sidewaystable

Results of nodule formation and nitrogen fixation (acetylene reduction) are presented in Table t-Meso-Brady-Cross. None of the native legumes tested were nodulated with Bradyrhizobium strains. Likewise none of the exotics were nodulated with Mesorhizobium, confirming that the host ranges indicated by isolations. Positive controls for this experiment were the plants in Tables t-Meso-native and t-Brady-cross which were performed concurrently with this experiment.

The presence of the original inoculant in the root nodules was confirmed by UARR PCR amplification and DNA sequencing of an isolate from each replicate (see Section UARR). Controls for each legume species, consisting of uninoculated plants, did not form nodules.

These results confirm that Bradyrhizobium is symbiotically incompatible from at least these native New Zealand legumes, and that Mesorhizobium species found nodulating native legumes are symbiotically isolated from woody weed legumes. These results reflect those seen in the field, and that of nodA gene type differences between Mesorhizobium and Bradyrhizobium.

Presence of rhizobia in pristine New Zealand soils s-soils

Background

One objective of this research was to establish if Bradyrhizobium nodulating introduced weed legumes in New Zealand are cosmopolitan or introduced. DNA sequencing of four housekeeping genes in Chapter 3 indicate that both Mesorhizobium spp.and Bradyrhizobium spp.are diverse but related to species found in other countries. The Bradyrhizobium nodA genes are very similar to international sequences, which may indicate that they have been introduced from overseas. The introduction may have occurred naturally or by human activity.

If Bradyrhizobium spp.were introduced recently by human activities, then differences might be expected in the distribution of these strains in New Zealand soils. Gorse and broom commonly grow in disturbed forest and pastures, which have high human activity and introduction of foreign materials. If effective Bradyrhizobium spp.were introduced along with the weeds then one would expect no nodulation to occur in areas with little human activity such as the inner areas of the protected National Parks.

New Zealand has 14 National Parks covering more than five million hectares---a third of New Zealand's surface area DoC06. These are monitored for invasions, and some areas have remained relatively pristine since New Zealand was colonised. Soil from such areas would be relatively unaltered by human activity, but would still be subject to natural means of bacterial dispersal, such as wind and water flow.

Two previous studies have investigated rhizobia in pristine New Zealand soils. Greenwood78a found that ``rhizobia were not detected in soil samples taken from unmodified natural habitats where legumes were not present''. In another study, Lotus corniculatus planted in Otago soil was unable to nodulate. When a Mesorhizobium strain capable of nodulating Lotus was introduced to the soil, the nodulation and fixation genes of the introduced strain were acquired by extant native Mesorhizobium strains in the soil Sullivan95. This indicates that Mesorhizobium spp.are found in pristine soils---at least in this area. It is unknown if these extant rhizobia were able to nodulate native legumes, or if they had lost (or never had) nodulation genes.

If native and exotic legumes were planted in pristine soil, there are several possible outcomes:

itemize High levels of effective nodulation on both native and exotic legumes. This would indicate that Mesorhizobium and Bradyrhizobium have a wide-spread distribution and harbour effective nodulation genes.

High levels of nodulation on native legumes only. This would indicate that there is no indigenous effective Bradyrhizobium population and these strains must have been introduced into disturbed areas, where host species are now found.

Both native and exotic legumes are poorly nodulated. This scenario might indicate a patchy distribution of rhizobia which could possibly be associated with active legume populations. itemize

An experiment was conducted to test these possibilities.

Experimental design

figure [width=12cm]soil-map [Geographical location of soil samples]Map of New Zealand showing location of soil samples used in this study. Codes are, UNP: Urewera National Park, WNP: Whanganui National Park, NLNP: Nelson Lakes National Park, OLM: Otago lammermoors. p-Soil-map figure

Soil samples were collected by local Department of Conservation (DoC) field staff according to strict conditions detailed in the methods chapter (see Section soil-method). Soil was collected from pristine areas (indicated on the map in Figure p-Soil-map) in National Parks away from the presence of all legumes, and areas of human influence. One sample (OLM) was taken in the vicinity of the Sullivan95 study, where Mesorhizobium are known to be present. Soil samples were posted to Auckland at ambient temperature and stored under refrigeration as detailed in Section soil-method.

Seedlings from three native legume species (Sophora microphylla, Carmichaelia australis, Clianthus puniceus) and three introduced legume species (Ulex europaeus, Cytisus scoparius, Acacia longifolia) were planted in this soil as `bait legumes' or `trap hosts' Mercante98 to determine if nodulating rhizobia were present.

Because of the weight limitations of postal mail, only a limited supply of soil from each site was available. Because of this, only 150cm of soil was used in each jar, equivalent to the volume of vermiculite used in previous experiments, and often only one or two replicates could be used for each legume species. To partially compensate, four seedlings were planted per jar to ensure that their roots extended through as much soil as possible.

To ensure that nodulation could occur in these soils, and was not inhibited by some factor, positive controls were included. These consisted of known effective nodulating strains (Mesorhizobium sp. 15054 and Bradyrhizobium sp. 14291) which were inoculated on to the soil of some jars. Plants were grown as described in the methods chapter.

Results

table Bait legumes planted in pristine soils tabularlcccccc 3*tabularl Soil sample location tabular & 6cBait legume nodulation response 2-7 & Sophora & Carmichaelia & Clianthus & Ulex & Cytisus & Acacia & microphylla & australis & puniceus & puniceus & scoparius & longifolia Lammermoors & Nod & Nod & Nod & Nod & Nod & Nod Nelson Lakes 1 & Nod & Nod & Nod & Nod & Nod & Nod Nelson Lakes 2 & Nod & Nod & Nod & Nod & Nod & Nod Nelson Lakes 3 & Nod & Nod & Nod & Nod & Nod & Nod Nelson Lakes 4 & Nod & Nod & Nod & Nod & Nod & Nod Urewera 1 & Nod & Nod & Nod & Nod & Nod & Nod Urewera 2 & Nod & Nod & Nod & Nod & Nod & Nod Whanganui & Nod & Nod & Nod & Nod & Nod & Nod tabular

t-Soils Presence of nodules: Nod, absence: Nod. Inconsistency: 50 of replicates were positive.

table

The results of nodulation success are presented in Table t-Soils. The extent of nodulation was very low, with most plants failing to yield any nodules.

Three nodules were found on the roots of only one of the four Cytisus plants in UNP2 soil. In soil from the Nelson Lakes (NLNP4), a total of twelve nodules were found on four plants in a single jar. No nodules were found on replicates of these jars. In all cases, positive controls consisting of inoculated soils produced nodules. Non-nodulated plants generally grew well, presumably due to adequate nutritional conditions of the soils.

DNA sequences of the 16S rRNA gene were obtained for one isolate from the Cytisus nodules and two from the Clianthus nodules, following previously mentioned protocols. The Cytisus isolate was a Bradyrhizobium strain as expected from previous work. The isolates from the Clianthus nodules in NLNP4 soil were R.leguminosarum and Paenibacillus sp.

Although R.leguminosarum has been shown in the previous experiments to nodulate Clianthus, the genus Paenibacillus has never been recorded to nodulate any legume. Since nodule contamination was suspected, an attempt was made to amplify the nodA gene (which is not present in non-rhizobia). A strong product did amplify, and DNA sequencing placed it into the type 4 nodA grouping (`Trifolii') (see Fig.p-nodA-MB).

Discussion s-pristine-soil

An objective of this research was to establish if Bradyrhizobium nodulating introduced weed legumes in New Zealand are cosmopolitan or introduced. To this end, eight soil samples from remote pristine areas of New Zealand were planted with legume species to act as trap hosts. The results show a very low number of positive nodulation, with only two jars containing nodules. This supports the third hypothesis listed in the introduction that effective rhizobial populations are patchy in distribution and most likely found in detectable numbers near active legume populations. It may be that there are low numbers of rhizobia outside of a legume rhizosphere, or the effective rhizobia are simply absent from certain areas. Alternatively, bacteria of `rhizobial genera' may be present but not have the appropriate symbiosis genes.

The low nodulation rate raises the possibility that the conditions were not suitable for nodulation. However, all positive controls consisting of soils inoculated with strains Mesorhizobium sp.15054 and Bradyrhizobium sp.14291 nodulated well, indicating permissive growth conditions.

It is possible that the nodulation could be due to external contaminants, especially since no nodulation was seen in replicates of positive jars. This possibility is difficult to entirely eliminate, but does not change the result significantly.

Sequences of the 16S gene from soil isolates revealed that the strains nodulating the Cytisus plant in Urewera soil were Bradyrhizobium as expected. In the Nelson Lakes National Park soil however, the isolates from Clianthus were not Mesorhizobium as expected, but rather R.leguminosarum and a Paenibacillus strain. nodA gene sequences from these isolates indicated that both were bv.trifolii (type 4). No Paenibacillus spp.---or indeed any Firmicute---has previously been recorded nodulating any legume. Thus this strain may represent a novel rhizobial species. Additional experiments on this isolate will be conducted in future work.

It is possible that nodulation of native legumes with species other than Mesorhizobium occurs because their preferential symbiont, Mesorhizobium, is not present in the soil, or is present but lacks the appropriate symbiosis genes.

Further research s-Johns-work

Introduction Based on the results of this experiment, further experiments were performed using larger volumes of soil and from locations adjacent to legume populations, as well as pristine soils. Much of the experimental work was carried out by John Young (Landcare Research), and as such the work is not a formal part of this thesis.

Methods Soil from rhizospheres of native and introduced legumes and from pristine sites in the Tongariro and Urewera National Parks (a total of 30 sites) was baited with Clianthus and Cytisus seed into pots in a greenhouse, and nodulation was assessed after 14 weeks.

Results and Discussion Nodulation of Clianthus in native legume rhizosphere soil and of Cytisus in introduced legume rhizosphere soil occurred readily, as expected.

Nodulation of Clianthus did not occur in pristine soils, confirming the results of Table t-Soils for Mesorhizobium spp.in native and introduced legumes. The scarcity of nodulation of native legumes in pristine soils is consistent with that of earlier reports Greenwood78a,Sullivan96 that Mesorhizobium populations are present in soils but do not nodulate, perhaps because they lack an effective symbiosis island.

Nodulation of Cytisus did occur in pristine soils, supporting a concept of a ubiquitous, cosmopolitan existence of effective Bradyrhizobium in soils.

In the reciprocal pot treatments Clianthus was nodulated at high levels in the rhizosphere soil of introduced legumes, and Cytisus was nodulated in the rhizosphere soil of native legumes. This result suggests that the rhizosphere of legumes is a general reservoir of rhizobia. The conclusion is supported by the earlier isolation of R.leguminosarum from native legumes. Selected rhizobial strains isolated from nodules of bait native and introduced legume plants were identified as Bradyrhizobium, Mesorhizobium and Rhizobium. The presence of Bradyrhizobium strains in native legumes root nodules is surprising given the findings of this thesis to the contrary, although no experiments were exhaustive. These isolates have not yet been confirmed for host-range nodulating ability.