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PhD project - Introduction

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Rhizobia

In this thesis `rhizobia' are defined as bacteria capable of forming root nodules on legumes, mediated by nod genes. This term describes the phenotype (causing root nodules), but has no taxonomic relevance and should not be confused with the genus name Rhizobium (although `rhizobia' has been used by others for the plural form of Rhizobium). An equivalent term used by other researchers is `root nodule bacteria' (RNB) Zakhia04,Howieson05.

Rhizobia are soil-inhabiting bacteria with the potential for forming specific root structures called nodules. In effective nodules the bacteria fix nitrogen gas (N2) from the atmosphere into ammonia OGara76, which is assimilated by the plant and supports growth---particularly in nutrient deficient soils. In return the rhizobia are supplied with nutrients (predominantly dicarboxylic acids Lodwig03), and are protected inside the nodule structure VanRhijn95. In ineffective nodules no nitrogen is fixed, yet rhizobia are still supplied with nutrients, and in this situation the rhizobia could be considered parasitic Denison04b.

The nitrogen-fixing symbiotic relationship has been exploited in agriculture to enhance crop and pasture growth without the addition of nitrogen fertilisers. For this reason, the majority of research in this field has focused on herbaceous crop and forage legumes of agricultural significance. In contrast, few studies have been made of rhizobial associations among non-crop legumes, despite the fact that they may be ecologically important in the natural landscape Boring88.

Worldwide, there are an estimated 17000--19000 legume species Martinez96, although nodulating bacterial species have only been identified for a small proportion of these. To date (September 2006), 55 rhizobial species have been identified in twelve genera (Table t-Rhizobia-species). Most of the species are in the genera Rhizobium (from the Latin `root living'), Bradyrhizobium, Mesorhizobium and Ensifer (Sinorhizobium).

A detailed discussion of rhizobial systematics is presented in Section s-systematics, but important taxonomic distinctions are noted below. All currently known rhizobia are in the phylum Proteobacteria, most in the class Alphaproteobacteria, which contains six rhizobial families in a single order---Rhizobiales, as listed in the hierarchy below Bergeys-outline.

Rhizobiales
Rhizobiaceae
Rhizobium
Ensifer (Sinorhizobium)
Brucellaceae
Ochrobactrum
Phyllobacteriaceae
Phyllobacterium
Mesorhizobium
Bradyrhizobiaceae
Bradyrhizobium
Hyphomicrobiaceae
Azorhizobium
Devosia
Methylobacteriaceae
Methylobacterium

There are also three rhizobial species in two families in the
Betaproteobacteria, all of which are in the Burkholderiales order,
as listed below Bergeys-outline.

Burkholderiales
Burkholderiaceae
Burkholderia
Cupriavidus
Oxalobacteraceae
Herbaspirillum

Although it remains to be confirmed, it is possible that some
Gammaproteobacteria also nodulate legumes Benhizia04.

List of rhizobial species

Binomial Name & Authority
Rhizobium daejeonense & Quan 05
Rhizobium etli & Segovia 93
Rhizobium galegae & Lindstrom 89
Rhizobium gallicum & Amarger 97
Rhizobium giardinii & Amarger 97
Rhizobium hainanense & Chen 97
Rhizobium huautlense & Wang 98
Rhizobium indigoferae & Wei 02
Rhizobium leguminosarum & Frank 79 Fran k89
Rhizobium loessense & Wei 03
Rhizobium mongolense & vanBerkum 98
Rhizobium sullae & Squartini 02
Rhizobium tropici & Martinez 91
Rhizobium undicola & deLajudie 98a Young 01a
Rhizobium yanglingense & Tan 01a

Ensifer (Sinorhizobium) abri & Ogasawara 03
Ensifer adhaerens & Wang 02a Young 03b
Ensifer (Sinorhizobium) americanum & Toledo 03
Ensifer arboris & Nick 99 Young 03b
Ensifer fredii & Scholla 84 Young 03b
Ensifer (Sinorhizobium) indiaense & Ogasawara 03
Ensifer kostiensis & Nick 99 Young 03b
Ensifer kummerowiae & Wei 02 Young 03b
Ensifer medicae & Rome 96 Young 03b
Ensifer meliloti & Dangeard 26 Young 03b
Ensifer saheli & deLajudie 94 Young 03b
Ensifer terangae & deLajudie 94 Young 03b
Ensifer xinjiangense & Chen 88 Young 03b

Mesorhizobium amorphae & Wang 99a
Mesorhizobium chacoense & Velazquez 01a
Mesorhizobium ciceri & Nour 94a Jarvis 97
Mesorhizobium huakuii & Chen 91 Jarvis 97
Mesorhizobium loti & Jarvis 82 Jarvis 97
Mesorhizobium mediterraneum & Nour 95 Jarvis 97
Mesorhizobium plurifarium & deLajudie 98b
Mesorhizobium septentrionale & Gao 04b
Mesorhizobium temperatum & Gao 04b
Mesorhizobium tianshanense & Chen 95 Jarvis 97

Bradyrhizobium canariense & Vinuesa 05b
Bradyrhizobium elkanii & Kuykendall 93
Bradyrhizobium japonicum & Kirchner 96 Jordan 82
Bradyrhizobium liaoningense & Xu 95
Bradyrhizobium yuanmingense & Yao 02

Burkholderia caribensis & Vandamme 02
Burkholderia cepacia & Vandamme 02
Burkholderia phymatum & Vandamme 02
Burkholderia tuberum & Vandamme 02

Azorhizobium caulinodans & Dreyfus 88
Azorhizobium doebereinerae & Souza 06

Cupriavidus taiwanensis & Chen 01 x Vandamme 04

Devosia neptuniae & Rivas 03

Herbaspirillum lusitanum & Valverde 03

Phyllobacterium trifolii & Valverde 03

Methylobacterium nodulans & Jourand04

Ochrobactrum lupini & Trujillo 05

t-Rhizobia-species

Type species Parenthesis indicate original publication, following reference is subsequent reclassification

There are a number of species present in these rhizobial genera that have not been observed to form nodules, and therefore do not fit the functional definition of rhizobia. These include many of the species that were formerly known as Agrobacterium (e. g. R. larrymoorei, R. rubi, and R. vitis; Young01a,Young04a). However, there is recent evidence that other species formerly classified as Agrobacterium are capable of nodulation. For example R. radiobacterAs Agrobacterium tumefaciens in the publication. nodulates Phaseolus vulgaris, Campylotropis spp. , Cassia spp. Han05, and Wisteria sinensis Lui05. Both nodules and tumours were formed on Phaseolus vulgaris by R. rhizogenes strains containing a Sym plasmid Velazquez05.

There are also other species, although classified within genera commonly considered to be represented entirely by nodulating strains, in fact include strains apparently devoid of nodulation ability. For example Bradyrhizobium betae forms tumours on Beta vulgaris (Beetroot) but is not known to fix N Rivas04a. Mesorhizobium thiogangeticum is a sulfur-oxidising bacterium, and does not nodulate the tested legumes of Clitoria ternatea, Pisum sativum, and Cicer arietinum Gosh06. There are also non-symbiotic strains of Mesorhizobium (and other genera) that can become nodulating species by acquiring symbiosis genes Sullivan95.

The genus Sinorhizobium was recently reclassified to Ensifer on the basis of similarity of DNA sequences and priority of publication Willems03,Young03b. Ensifer adhaerens is a soil bacterium that attaches to other bacteria and may cause cell lysis Casida82. Although wild type E. adhaerens did not nodulate Phaseolus vulgaris nor Leucaena leucocephala, it did so when transformed with a symbiotic plasmid from Rhizobium tropici Rogel01, demonstrating its capacity to become a rhizobial species. Other E. adhaerens strains were subsequently isolated that nodulated legumes naturally. These form a single clade with Sinorhizobium in 16S rRNA and recA phylogenies leading Willems03 to suggest that these strains be reclassified as Sinorhizobium adhaerens. However, Ensifer Casida82 is the senior heterotypic synonym and thus takes priority Young03b. This means that all Sinorhizobium spp. must be renamed as Ensifer spp. according to the Bacteriological code Lapage90. In this thesis Ensifer is used exclusively.

Cupriavidus species have recently undergone several taxonomic revisions, being formerly known as both Wautersia and Ralstonia. This genus currently contains a single rhizobial species, and ten other non-symbiotic species Euzeby97.

Rhizobial systematics is rapidly changing, and recently many new species have been recognised. Novel species may also be associated with the native legumes of New Zealand.

 

 

New Zealand native legumes

Introduction

New Zealand has 33 species of legumes that are native. These are comprised of four genera: Sophora, Carmichaelia, Clianthus, and Montigena.

Sophora

figure [tb] [width=12cm]Sophora [Sophora chathamica]Sophora chathamica showing yellow bell-shaped flowers and mature seed pod (right of centre). p-Sophora figure

Sophora L. (1753) was named after sufayra, the arabic name for the tree. The Maori name (and the vernacular) for the endemic Sophora is `kowhai', from the word for yellow---which describes the colour of the flowers (Fig. p-Sophora).

There are eight species native to New Zealand: S. chathamica, S. fulvida, S. godley, S. longicarinata, S. microphylla, S. molloyi, S. prostrata and S. tetraptera Heenan01c. There are another 49 species in the genus Sophora that are not native to New Zealand. Species endemic to the Southern Hemisphere are in the Edwardsia sector of Sophora. Edwardsia members other than the New Zealand natives are from South America (S. macrocarpa), Lord Howe Island (S. howinsula), Hawaii (S. chrysophylla), La Reunion (S. denudata), Easter Island (S. toromiro), and Raivavae Island (S. raivavae). S. microphylla was considered to occur in Chile and on Gough Island in the south Atlantic Markham72, however Heenan01a considers these species to be Sophora cassioides, distinct from the New Zealand species. The type species of the genus is S. tomentosa which is closely related to sect. Edwardsia Heenan04,ILDIS.

Carmichaelia

figure [tb] [width=12cm]Carmichaelia [Carmichaelia australis]Carmichaelia australis showing the cladodes and mature seed pods. Insert: 2 magnification of flowers from the same plant. p-Carmichaelia figure

Carmichaelia R. Br. (1825) was named after Captain Dugald Carmichael, a Scottish army officer and botanist who collected plants in New Zealand Allen81. The English vernacular name is `New Zealand broom', and in Maori is variably known as tawao, makaka, maukoro, and tainoka NZPlant (illustrated in Fig. p-Carmichaelia).

The taxonomic history of this genus is complex, and has been confused by inadequate collections and intraspecific variation Heenan95b. The formerly recognised genera of Chordospartium, Corallospartium, Notospartium, and Huttonella are now included in Carmichaelia Heenan95a,Heenan98c,Heenan98a. In the most recent treatment Heenan95b,Heenan96b, there are 22 species of Carmichaelia native to New Zealand (C. appressa, C. arborea, C. astonii, C. australis, C. carmichaeliae, C. compacta, C. corrugata, C. crassicaule, C. curta, C. glabrescens, C. hollowayi, C. juncea, C. kirkii, C. monroi, C. muritai, C. nana, C. odorata, C. petriei, C. stevensonii, C. torulosa, C. uniflora, C. vexillata, and C. williamsii). An additional species, C. exsul, is found on Lord Howe Island in the Tasman Sea, 600 km east from Australia. The species exhibit remarkable diversity, from trees to prostrate forms a few centimetres high.

Carmichaelia is distributed throughout New Zealand, although most species are restricted to certain localities. Most of the diversity (15 species) is in the eastern South Island. They typically invade disturbed habitats on shallow poor soils, drought and frost prone areas, and alluvial soils Wagstaff99.

Clianthus

figure [tb] [width=12cm]Clianthus [Clianthus puniceus]Clianthus puniceus showing distinctive beak-shaped flowers. p-Clianthus figure

Clianthus Soland. ex Lindl. was named from the Greek kleos `glory' and anthos `flower' Allen81. The English vernacular name is `kakabeak' after its distinctive flowers shaped like a native parrot's (kaka) beak (Fig. p-Clianthus), it is known in Maori as `kowhai ngutukaka' Shaw97.

Once considered monotypic, in the most recent treatment Heenan95c,Heenan00, there are now two species (C. maximus and C. puniceus) native to New Zealand. It is found naturally only in isolated refuges in the eastern North Island. Formerly some Australian and Asian legumes were classified as Clianthus, these are now known as Swainsona and Sarcodum ILDIS.

Montigena

figure [tb] [width=12cm]Montigena [Montigena novae-zelandiae]Montigena novae-zelandiae, growing on a scree slope, with mature seed pods. Photo Peter Heenan. p-Montigena figure

Montigena (Hook. f. ) Heenan, is named from `mountain-born' referring to its habitat. Heenan98d. The English vernacular name is `scree pea' (Fig. p-Montigena).

Montigena novae-zelandiae is the only species in the Montigena genus. It was known as Swainsona novae-zelandiae until Heenan98d reclassified it based on morphological features. There are currently 55 Swainsona species, mostly in Australia ILDIS. Montigena has a distinctly different ITS sequence from other New Zealand legumes, but forms a clade with the Australian Swainsona Wagstaff99 (See Fig. p-legume-tree).

Montigena is endemic to the dry eastern mountains of the South Island of New Zealand, where it grows on partially stable scree slopes.

Evolution and history of New Zealand native legumes legume-history

Geology and palaeobotany

The archipelago of New Zealand began to split away from the larger landmass of Gondwana about 80 million years ago (mya) due to continental drift, although was still relatively close for another 10 to 20 million years Cooper93,Stevens95. The start of this separation coincides approximately with the date of the evolution of legumes Sprent94, although legumes were not abundant until 35--54 million years ago Doyle03. Hence all legumes must have arrived in New Zealand after its separation from Gondwana.

The historical presence of legumes in New Zealand is largely inferred from fossil pollen records. Fossils of Carmichaelia were detected in the ``late Pliocene Waipaoa series''of soils dating from less than 5 mya Oliver28, but Sophora is not common in fossil pollen records until the Pleistocene (1. 81 mya) Hurr99. Fossil pollen records also show that before the last Ice Age ended, 10000 years ago, New Zealand had an indigenous population of Acacia spp. Mildenhall72,Lee01.

table [tbp] Native legume taxonomic hierarchy tabularccccc Kingdom & 4cPlantae Division & 4cMagnoliophyta Class & 4cMagnoliopsida Order & 4cFabales Family & 4cFabaceae Subfamily & 4cFaboideae Tribe & Sophoreae & 2cCarmichaeliaeae & Galegeae SubTribe & & 3cCarmichaelinae Genus & Sophora & Carmichaelia & Montigena & Clianthus tabular

taxbox-Native 1cm After Pohill81-Carm. 1cm After Wagstaff99. table

The Carmichaelinae

The original classification of native legumes placed Carmichaelia and Montigena in the tribe Carmichaelieae, and Clianthus in the diverse tribe Galegeae Pohill81-Carm; however this classification is polyphyletic Wagstaff99, and recent evidence has suggested that Carmichaelia, Clianthus, Montigena, and the Australian genus Swainsona, form a single clade called Carmichaelinae at the subtribe rank Wagstaff99 (Tabletaxbox-Native).

Wagstaff99 used ITS sequences of 39 species of Carmichaelia, Clianthus, Montigena, Swainsona and related legumes, to determine the classification and origins of New Zealand legumes. Most species of Carmichaelia had nearly identical ITS sequences, indicating recent radiation. The results suggested that Carmichaelinae were derived from the Northern Hemisphere Astragalinae, and confirmed an earlier study of Heenan98a using 47 phenotypic characters.

figure [tbp] [width=12cm]legume-tree [Carmichaelinae phylogenetic tree]Phylogenetic tree of the New Zealand Carmichaelinae clade (in bold) and related genera (Galegeae tribe) from Australia and other countries, using ITS sequences. This penalised likelihood rate-smoothed Bayesian consensus phylogeny and the estimated ages were derived from the data and information provided in Wagstaff99. Carmichaelinae are mainly of Australia but with two independent lines in New Zealand. Figure modified from Lavin04, misspelling of `Swainsona' in original. Symbols: * -- Clianthus puniceus.
p-legume-tree figure

Lavin04 extended the work of Wagstaff99 by reanalysing the data using Bayesian methods to estimate the age of divergence of each clade (Fig. p-legume-tree). From these data it appears that the New Zealand Carmichaelinae, including all Carmichaelia species and Clianthus (marked on the tree by `*') diverged 5. 31. 1 mya, and all Carmichaelinae have a common origin 7. 50. 8 mya. Carmichaelia shares a common ancestor with Sutherlandia (found in Australia, Africa, and Mauritius ILDIS) 10. 42. 0 mya. These dates agree with those from fossil pollen.

In a large study of 235 genera using matK sequence data, Wojciechowski04 included Clianthus and Carmichaelia in a larger ``Astragalean clade'' including Swainsona, Colutea, Sutherlandia, Oxytropis, and Astragalus.

These publications show that the radiation of Carmichaelinae legume species into New Zealand was quite recent (compared to the diversification of legumes in the Northern Hemisphere). The ancestor of the Carmichaelinae derived from a Northern Hemisphere lineage and arrived (probably in Australia) between 10 and 7. 5 mya.

New Zealand Sophora

Sophora is distinct from the other legume genera of New Zealand, being a member of the Sophoreae tribe (Table taxbox-Native). Sophora is a diverse genus that has about 80 members spread throughout the world. Molecular analyses indicate that the genus is polyphyletic, and and comprises three distinct and unrelated lineages Kass95,Kass96,Crisp00,Pennington01.

New Zealand Sophora belong to a subset known as ``Sophora sect. Edwardsia'' Kass97,Heenan04. This sector is one of the largest groups in Sophora, and includes about 19 species whose distribution is centred on islands in the southern Pacific Ocean. Most species of sect. Edwardsia have identical ITS sequences, indicating a recent and rapid radiation Mitchell02.

There are competing theories on the origin of Sophora sect. Edwardsia. Some believe that they originated in Chile from a North American ancestor Sykes68,Pena00. Molecular genetics indicates the likely origin is from the North Western Pacific, from an Eurasian ancestor, in the last 2--5 million years, and dispersal occurred around the pacific via the buoyant saltwater-resistant seeds Hurr99,Mitchell02,Heenan04.

In summary it is proposed that New Zealand Sophora spp. derived from a separate legume lineage and geographical origin than the Carmichaelinae, and were dispersed to New Zealand perhaps a few million years later.

 

Exotic weed legumes in New Zealand

Introduction

The indigenous people of New Zealand---the Maori---arrived in the mid 13th century from Eastern Polynesia Irwin05. They brought with them new species, such as mammals (rats, dogs) and tuber plants (kumera, taro, yam), but there is no evidence that they brought any legumes Bellich96.

The first exotic legumes were introduced into New Zealand by settlers from Europe in the early 19th century. The settlers brought many plants and animals to establish familiar industries, and to remind them of their previous homelands. In their endemic habitats, these shrubs are in equilibrium with their natural flora, but in New Zealand, some have become serious invasive noxious weeds.

Legumes have several properties that make them successful invaders. They have a high seed production, often with many seeds per pod, and many pods per tree. Most legume seeds are able to survive long periods in soil banks due to their thick impervious testa Lee01. The mature plant generally lives for many years, and high-density seedling success allows rapid coverage of large areas. Possibly the success of legumes as invasive weeds is augmented by their ability to grow in nutrient deficient soils, in association with nitrogen-fixing rhizobia.

There are now over 100 naturalised legume species in New Zealand NZPlant. A small number of these have become common weeds and include: Chamaecytisus palmensis (tree lucerne), Cytisus scoparius (broom), Galega officinalis (goat's rue), Lathyrus latifolius (everlasting pea), Lotus pedunculatus (lotus), Lotus suaveolens (hairy birdsfoot trefoil), lupinus arboreus (tree lupin), Medicago lupulina (black medick), Medicago sativa (lucerne), Melilotus indicus (King Island melilot), Ornithopus perpusillus (wild serradella), various wattles (Acacia spp. ), Psoralea pinnata (dally pine), various Trifolium spp. (clover), Ulex europaeus (gorse), Vicia hirsuta (hairy vetch), Vicia sativa (vetch) Roy04. The woody species of Ulex, Cytisus, and Acacia are the most invasive, and are the three of this study.

table Weed legume taxonomic hierarchy tabularcccc Kingdom & 3cPlantae Division & 3cMagnoliophyta Class & 3cMagnoliopsida Order & 3cFabales Family & 3cFabaceae Subfamily & 2cFaboideae & Mimosoideae Tribe & 2cGenisteae & Acacieae Genus & Ulex & Cytisus & Acacia tabular

taxbox-Weed 3cm Note: Information from ILDIS table

Ulex and Cytisus are classified in the Genisteae tribe, and Acacia is in the Acacieae tribe (Tabletaxbox-Weed). These are distinct from the woody native New Zealand legumes, which belong to the tribes Sophoreae and Carmichaelinae.

 

Ulex europaeus

figure [tb] [width=12cm]Ulex [Ulex europaeus]Ulex europaeus showing spines and flowers beginning to open. p-Ulex figure

 

Ulex europaeus L. is known in the vernacular as whin, furze, or more commonly in New Zealand---gorse (Figp-Ulex).

There are some eleven Ulex species but only U. europaeus is important in New Zealand ILDIS. Its habitat is mostly disturbed and modified ecosystems, including river-beds, pasture, scrubland, forest margins and wasteland.

Gorse is native to Western Europe and was naturalised in New Zealand in 1867 Bellingham04, although Darwin09 recorded it at Waimate some thirty years earlier in December 1835. It was introduced as a `living fence', but outgrew its usefulness and was soon classified as a weed. It is now considered New Zealand's worst weed, and millions of dollars are spent annually in control Hill86. Gorse is also a problem in parts of Spain, Portugal, Chile, Hawaii, Ireland, coastal Oregon, and Southern Australia Roy04,Leary06

 

Cytisus scoparius

figure [tb] [width=12cm]Cytisus [Cytisus scoparius]Cytisus scoparius. Photo Jon J. Sullivan. p-Cytisus figure

Cytisus scoparius(L. )Link, is also classified as Sarothamnus scoparius (L. ) W. D. J. KochOccasionally incorrectly spelt as Sarathamnus. . It is commonly called `broom' or `scotch broom'. Cytisus has some 51 taxa ILDIS, but only C. scoparius is of importance in New Zealand (Fig. p-Cytisus).

Broom is common throughout New Zealand, particularly on the drier eastern side of the South Island, and the central North Island Fowler00. Its habitat is mostly river-beds, hedgerows, low-fertility hill country, scrubland, coastal areas, and waste land. It was originally from Europe, Asia, and Russia. In New Zealand it grows more vigorously than in its native range, with a greater maximum age and larger size. It was naturalised in New Zealand in 1872 Bellingham04.

Broom causes economic losses to agricultural and forestry operations, and occupies 0.92% of South Island farmable land Fowler00.

 

Acacia

Acacia (commonly called wattle) is a large genus with over 950 species ILDIS. Recent studies have shown that Acacia is polyphyletic and should be split into five genera Luckow05, although there are competing proposals for this. `Proposal 1584' would retypify Acacia: The type of the Australian taxon (A. penninervis) would be conserved over the current lectotype (A. scorpioides) of an African taxon Orchard05. Alternate proposals keep the lectotype, and reclassify some Acacia species as `Racosperma' Luckow05. `Acacia' will be used for the Australian species in this thesis. A summary of the events relating to the renaming of Acacia can be found at http://www. worldwidewattle. com/infogallery/nameissue/chronology. php.

figure [tb] [width=12cm]Acacia [Acacia longifolia]Acacia longifolia. Photo Brenda Foran. p-Acacia figure

Acacia longifolia (Andrews) Willd. (Sydney golden wattle) is investigated in this study. It is a serious invasive weed in Northland, where it was introduced to control sand dune erosion, but has now spread widely and invaded wetlands Hicks01. A. longifolia is also a significant problem in South Africa endangering the floristically unique Cape Floral Kingdom Dennill99,VanWilgen04.

 

Previous research on New Zealand rhizobia

Work on this project started in early 2002. At this time there were few reports of rhizobia nodulating native legumes apart from an Honours dissertation using a small number of strains McCallum96, and work in the 1960's--70's Greenwood69, Greenwood78a, Greenwood78b. Likewise, there were no investigations using molecular techniques of rhizobia nodulating gorse and broom in any country, although historical research lumped the strains into the inaccurately described `cowpea rhizobia' Pieters27,Wilson39a. It is not until recently that molecular techniques allowed affordable and accurate assessment of the phylogeny of bacterial strains.

In early studies, many host-range experiments were done Greenwood69, Jarvis77, Crow81, but interpretation of the data was difficult, as then the molecular mechanisms behind nodulation were not known, nor was it known that symbiosis genes were transmissible. A more comprehensive account of previous Rhizobium--legume research in New Zealand is presented in Section s-NZ-rhizobia-history.

This thesis will build on this previous work, assisted by modern techniques and knowledge.

Research objectives

This thesis aims to establish a better understanding of the nature (taxonomy, diversity, host-range, and distribution) of the associations between rhizobia and New Zealand's endemic and weed legume flora.

It is assumed that the native legume genera have co-evolved with nitrogen-fixing bacterial symbionts for millions of years, potentially leading to new species. In contrast the origin of rhizobia nodulating the recently introduced exotic legumes is unknown. Previous studies overseas [reviewed by][]Perret00 have shown that rhizobial strains differ in host-range specificity. Some (e. g. Ensifer fredii NGR234) are promiscuous, while others appear to be host specific (e. g. Rhizobium leguminosarum bv. trifolii). Based on this, there are three possibilities that could explain exotic legume nodulation: 1) Introduced legumes are promiscuous and use the same rhizobia as native legumes. 2) Introduced legumes use specific rhizobia that were recently introduced---perhaps in conjunction with exotic legumes. 3) Introduced legumes use specific rhizobia that were already present in New Zealand (possibly cosmopolitan).

Specific objectives for this thesis are:

 

  1. To establish the identity and diversity of the rhizobial species associated with New Zealand's endemic legume species.
  2. To establish the identity and presumptive origins of the rhizobial species associated with the woody legume weeds introduced into New Zealand.
  3. To determine the specificity and efficacy of the symbiotic associations of rhizobial species with both plant groups, endemic and woody weeds, by an investigation of their nodulation and nitrogen-fixing capacity.
  4. To investigate possible exchange of transmissible genetic elements between rhizobial species associated with endemic and introduced legumes.

 

Research strategy

To investigate the identity and diversity of rhizobia, a polyphasic strategy employing both phenotypic and phylogenetic characteristics was used Vandamme96. Phylogenetic analyses were based on the sequencing of three protein-coding conserved `housekeeping' genes (atpD, glnII, recA), and one ribosomal RNA gene (16S). Phenotypic characteristics included metabolic fingerprints based on substrate utilisation (Biolog), and whole cell fatty acid methyl ester profiles (FAME).

The symbiosis genes were investigated by sequencing a protein-coding gene (nodA) involved in Rhizobium--plant signalling, which is usually carried on a transmissible genetic element (plasmid or symbiosis island).

The efficacy of the symbiotic combinations was tested by inoculating legumes with rhizobial strains in host-range experiments. The potential to fix nitrogen was determined by acetylene reduction, and roots were visually examined for nodulation.