Grapevines escaping trunk diseases in New Zealand vineyards have a distinct microbiome structure.

Grapevine trunk diseases (GTDs) are a substantial challenge to viticulture, especially with a lack of available control measures. The lack of approved fungicides necessitates the exploration of alternative controls. One promising approach is the investigation of disease escape plants, which remain healthy under high disease pressure, likely due to their microbiome function. This study explored the microbiome of grapevines with the disease escape phenotype. DNA metabarcoding of the ribosomal internal transcribed spacer 1 (ITS1) and 16S ribosomal RNA gene was applied to trunk tissues of GTD escape and adjacent diseased vines. Our findings showed that the GTD escape vines had a significantly different microbiome compared with diseased vines. The GTD escape vines consistently harbored a higher relative abundance of the bacterial taxa Pseudomonas and Hymenobacter. Among fungi, Aureobasidium and Rhodotorula were differentially associated with GTD escape vines, while the GTD pathogen, Eutypa, was associated with the diseased vines. This is the first report of the link between the GTD escape phenotype and the grapevine microbiome.

Root diameter, host specificity and arbuscular mycorrhizal fungal community composition among native and exotic plant species.

Plant root systems rely on a functionally diverse range of arbuscular mycorrhizal fungi to, among other benefits, extend their nutrient foraging. Extended nutrient foraging is likely of greatest importance to coarse-rooted plants, yet few studies have examined the link between root traits and arbuscular mycorrhizal fungal community composition. Here, we examine the relationship between root diameter and the composition of arbuscular mycorrhizal fungal communities in a range of native and exotic plant species. We characterized the arbuscular mycorrhizal fungal communities of 30 co-occurring native and exotic montane grassland/shrubland plant species in New Zealand. We found that plant root diameter and native/exotic status both strongly correlated with arbuscular mycorrhizal fungal community composition. Coarse-rooted plants had a lower diversity of mycorrhizal fungi compared with fine-rooted plants and associated less with generalist fungal partners. Exotic plants had a lower diversity of fungi and fewer associations with nondominant families of arbuscular mycorrhizal fungi compared with native plants. These observational patterns suggest that plants may differentially associate with fungal partners based on their root traits, with coarse-rooted plants being more specific in their associations. Furthermore, exotic plants may associate with dominant arbuscular mycorrhizal fungal taxa as a strategy in invasion.

Soil Arbuscular Mycorrhizal Fungal Communities Differentially Affect Growth and Nutrient Uptake by Grapevine Rootstocks.

Arbuscular mycorrhizal fungi (AMF) deliver potentially significant services in sustainable agricultural ecosystems, yet we still lack evidence showing how AMF abundance and/or community composition can benefit crops. In this study, we manipulated AMF communities in grapevine rootstock and measured plant growth and physiological responses. Glasshouse experiments were set up to determine the interaction between rootstock variety and different AMF communities, using AMF communities originating under their own (i.e., "home") soil and other rootstocks' (i.e., "away") soil. The results revealed that specific AMF communities had differential effects on grapevine rootstock growth and nutrient uptake. It was demonstrated that a rootstock generally performed better in the presence of its own AMF community. This study also showed that AMF spore diversity and the relative abundance of certain species is an important factor as, when present in equal abundance, competition between species was indicated to occur, resulting in a reduction in the positive growth outcomes. Moreover, there was a significant difference between the communities with some AMF communities increasing plant growth and nutrient uptake compared with others. The outcomes also demonstrated that some AMF communities indirectly influenced the chlorophyll content in grapevine leaves through the increase of specific nutrients such as K, Mn, and Zn. The findings also indicated that some AMF species may deliver particular benefits to grapevine plants. This work has provided an improved understanding of community level AMF-grapevine interaction and delivered an increased knowledge of the ecosystem services they provide which will benefit the wine growers and the viticulture industry.

Physiological stage drives fungal community dynamics and diversity in Leptospermum scoparium (manuka) flowers.

Flowers are an important niche for microbes, and microbes in turn influence plant fitness. As flower morphology and biology change rapidly over time, dynamic niches for microbes are formed and lost. Floral physiology at each life stage can therefore influence arrival, persistence and loss of microbial species; however, this remains little understood despite its potential consequences for host reproductive success. Through internal transcribed spacer 1 (ITS1) community profiling, we characterized the effect of transitioning through five floral stages of manuka (Leptospermum scoparium), from immature bud to spent flower, and subsequent allocation to seed, on the flower-inhabiting fungal community. We found nectar-consuming yeasts from Aureobasidium and Vishniacozyma genera and functionally diverse filamentous fungi from the Cladosporium genus dominated the anthosphere. The candidate core microbiota persisted across this dynamic niche despite high microbial turnover, as observed in shifts in community composition and diversity as flowers matured and senesced. The results demonstrated that floral stages are strong drivers of anthosphere fungal community assembly and dynamics. This study represents the first detailed exploration of fungi through floral development, building on fundamental knowledge in microbial ecology of healthy flowers.

Diversity and Bioactivity of Endophytic Actinobacteria Associated with Grapevines.

Grapevine trunk diseases (GTDs) are a significant problem for New Zealand viticulture. Endophytic actinobacteria are of interest as potential biocontrol agents due to their ability to inhibit plant pathogens and improve plant growth. However, no studies have investigated the diversity of actinobacteria associated with grapevines in New Zealand vineyards and their bioactivity. Actinobacteria diversity in different 'Sauvignon blanc' vine tissues from three vineyards (conventional and organic management, and different vine ages) was assessed using different methods and media. Forty-six endophytic actinobacteria were isolated, with more isolates recovered from roots (n = 45) than leaves (n = 1) and shoot internodes (n = 0). More isolates were recovered from the organic (n = 21) than conventional (n = 8) vineyard, mature (25-year old; n = 21) than young (2-year old; n = 2) vines and using a tissue maceration technique (n = 40). Actinomycete Isolation Agar, International Streptomyces Project 2, and Starch Casein media were effective for actinobacteria isolation. Most of the isolates recovered belonged to Streptomyces, with one isolate identified as Mycolicibacterium. Forty isolates were assessed for antifungal activity and plant growth-promoting (PGP) characteristics. Of these, 13 isolates had antifungal activity against test GTD pathogens (Dactylonectria macrodidyma, Eutypa lata, Ilyonectria liriodendri, Neofusicoccum parvum, and N. luteum). Eighteen isolates exhibited more than one PGP trait; 25siderophore production (n = 25), phosphate solubilization (n = 6), and indole acetic acid production (n = 16). Two strains, Streptomyces sp. LUVPK-22 and Streptomyces sp. LUVPK-30, exhibited the best antifungal and PGP properties. This study revealed the diversity of culturable endophytic actinobacteria from grapevines in New Zealand vineyards and their biocontrol potential against GTD pathogens.

DNA metabarcoding reveals high relative abundance of trunk disease fungi in grapevines from Marlborough, New Zealand.

Grapevine trunk diseases (GTDs) are a threat to grape production worldwide, with a diverse collection of fungal species implicated in disease onset. Due to the long-term and complex nature of GTDs, simultaneous detection of multiple microbial species can enhance understanding of disease development. We used DNA metabarcoding of ribosomal internal transcribed spacer 1 (ITS1) sequences, supported by specific PCR and microbial isolation, to establish the presence of trunk pathogens across 11 vineyards (11-26 years old) over three years in Marlborough, the largest wine producing region in New Zealand. Using a reference database of trunk pathogen sequences, species previously associated with GTD, such as Cadophora luteo-olivacea, Diplodia seriata, Diplodia mutila, Neofusicoccum australe, and Seimatosporium vitis, were identified as highly represented across the vineyard region. The well-known pathogens Phaeomoniella chlamydospora and Eutypa lata had especially high relative abundance across the dataset, with P. chlamydospora reads present between 22 and 84% (average 52%) across the vineyards. Screening of sequences against broader, publicly available databases revealed further fungal species within families and orders known to contain pathogens, many of which appeared to be endemic to New Zealand. The presence of several wood-rotting basidiomycetes (mostly Hymenochaetales) was detected for the first time in the Marlborough vineyard region, notably, the native Inonotus nothofagii which was present at 1-2% relative abundance in two vineyards.

High throughput pH bioassay demonstrates pH adaptation of Rhizobium strains isolated from the nodules of Trifolium subterraneum and T. repens.

The purpose of developing this high throughput assay was to determine whether there was evidence of pH adaptation in strains of rhizobia which nodulate subterranean clover (SC) and white clover (WC), and whether this was related to the pH of the soil of origin. pH is a first-order factor influencing the niche preferences of soil microorganisms and has been convincingly shown to be a key driver of soil bacterial communities. Naturalised strains of Rhizobium spp. that are pH-adapted may have the potential to better compete and/or persist in acidic or alkaline soils compared with introduced commercial strains. Three pilot studies were conducted to design the optimised bioassay. This bioassay tested the effect of pH-amended yeast mannitol broth (seven pH values from pH 4.5-9.0), across three time points, on the in vitro growth of 299 Rhizobium strains isolated from the nodules of SC and WC. The media pH where strains demonstrated fastest growth was related to the pH of the soil that strains were isolated from. However, the correlation between media pH and soil pH was strongly influenced by the growth of strains from alkaline soils (alkaline adaptation), especially in strains isolated from SC nodules.

AMF Community Diversity Promotes Grapevine Growth Parameters under High Black Foot Disease Pressure.

Black foot disease is one of the main grapevine root diseases observed worldwide and is especially problematic in New Zealand. Arbuscular mycorrhizal fungi (AMF) have been shown to reduce infection and mitigate the effect of black foot disease on grapevine rootstocks. In contrast to prior studies, which have limited their focus to the effect of one, two or a combination of only a small number of AMF species, this study used whole AMF communities identified from 101-14, 5C and Schwarzmann rootstocks sampled from New Zealand vineyards. The effect of AMF on black foot disease was investigated in a 'home' and 'away' experiment using three commercial grapevine rootstocks. The study produced some evidence that AMF treatments lowered disease incidence at 5 cm and disease severity in vines by 40% to 50% compared to the vines inoculated with the pathogen only. This work also showed that the presence of high disease incidence may have limited the potential disease protective effect of AMF community. However, despite the high disease incidence and severity, AMF inoculation increased vine growth parameters by 60% to 80% compared to the vines inoculated with the pathogen only. This study is the first to provide an understanding on how young grapevine rootstocks inoculated with their 'home' and 'away' AMF communities would respond to challenge with a black foot pathogen species mixture. Further research is required to understand the mechanistic effect of AMF colonization on the increase of grapevine growth parameters under high black foot disease pressure.

Influence of blueberry tissue type, wounding and cultivar on susceptibility to infection by Neofusicoccum species.

AIM: Botryosphaeriaceae causing stem blight and dieback of blueberry are important pathogens limiting economic production worldwide. This study investigated the pathogenicity and relative virulence of isolates from the Neofusicoccum species commonly associated with blueberries in New Zealand on different tissues and cultivars of blueberries. METHODS AND RESULTS: Both wounded and non-wounded fruit and flower buds and wounded attached soft green and hard green shoots were susceptible to infection by conidia of Neofusicoccum australe, Neofusicoccum parvum and Neofusicoccum ribis. N. ribis was generally most virulent, followed by N. parvum and then N. australe. Inoculation of potting mixture with N. australe or N. ribis conidia showed that potting mixtures were not a source of inoculum for infection of blueberry roots. Wounded and non-wounded leaf buds, fruit and wounded soft green shoots and hard green shoots of the different cultivars tested were susceptible to infection by N. parvum and N. ribis. Whilst the fruit of all cultivars were similarly infected, infection incidence in inoculated leaf buds was lowest in "Blue Bayou" and "Ocean Blue". Cultivar susceptibility differed when tested on soft green shoots compared with hard green shoots, with shortest lesions developed on "Maru" on soft green shoots, and "Centra Blue" and "Ocean Blue" on hard green shoots. CONCLUSIONS: All tested above-ground blueberry tissues, including non-wounded tissue, were susceptible to Neofusicoccum spp. All the cultivars assessed were susceptible to infection, although they varied in their relative susceptibility depending on the tissue assessed. SIGNIFICANCE AND IMPACT OF THE STUDY: The potential for non-wounded tissue to become infected indicate that fungicides may need to be applied to protect all tissue, not just wounds.

Fungal Communities in the Native New Zealand Medicinal Plant Pseudowintera colorata (Horopito) Are Determined by Plant Organ Type and Host Maturity with Key Members Promoting Plant Growth.

The plant Pseudowintera colorata is well known for its antimicrobial and medicinal properties and is endemic to New Zealand. Using PCR-Denaturing gradient gel electrophoresis (DGGE), we investigated the factors influencing the composition of endophytic fungal communities in P. colorata from ten distinct sites across New Zealand. Our results showed that plant organs of P. colorata influenced the diversity and richness of endophytic fungi (PERMANOVA, p < 0.05). In addition, plant maturity and its interactions revealed that endophytic fungal communities formed discrete clusters in leaves, stems, and roots of mature and immature P. colorata plants (PERMANOVA; p = 0.002, p = 0.001 and p = 0.039, respectively). For identifying isolates with biocontrol potential, dual culture tests were set up against four different phytopathogenic fungi. Isolates with high activity (zone of inhibition > 10 mm) were sequenced and identified as Trichoderma harzianum, Pezicula neosporulosa, Fusariumtricinctum, Metarhizium sp., and Chaetomium sp. Applying selected endophytic fungi (n = 7) as soil drenchers significantly increased the growth of P. colorata seedlings and produced more internodes. Seedling shoots treated with Trichoderma sp. PRY2BA21 were 2.2 × longer (8.36 cm) than the untreated controls (3.72 cm). Our results elucidate the main plant factors influencing fungal community composition and demonstrate a role for endophytic fungi in P. colorata growth and further demonstrate that medicinal plants are a rich source of endophytes with potential as biocontrol agents.

Community Structure, Diversity and Potential of Endophytic Bacteria in the Primitive New Zealand Medicinal Plant Pseudowintera colorata.

Although the importance of the plant microbiome in commercial plant health has been well established, there are limited studies in native medicinal plants. Pseudowintera colorata (horopito) is a native New Zealand medicinal plant recognized for its antimicrobial properties. Denaturing gradient gel electrophoresis (DGGE) and Illumina MiSeq analysis of P. colorata plants from ten sites across New Zealand showed that tissue type strongly influenced the diversity and richness of endophytic bacteria (PERMANOVA, P < 0.05). In addition, two OTUs belonging to the genus Pseudomonas (Greengenes ID: 646549 and 138914) were found to be present in >75% of all P. colorata leaf, stem and root samples and were identified as the members of the P. colorata "core endomicrobiome". Culture-independent analysis was complemented by the recovery of 405 endophytic bacteria from the tissues of P. colorata. Some of these cultured endophytic bacteria (n = 10) showed high antagonism against four different phytopathogenic fungi tested. The influence of endophytic bacteria on plant growth was assessed by inoculating P. colorata seedlings. The mean shoot height of seedlings treated with Bacillus sp. TP1LA1B were longer (1.83×), had higher shoot dry weight (1.8×) and produced more internodes (1.8×) compared to the control.

Non-host larvae negatively impact persistence of the entomopathogen Beauveria bassiana in soil.

A better understanding of the ecology of the insect pathogenic fungus, Beauveria bassiana, in soil is needed to identify reasons behind the variable efficacy often seen after field application. A transformed strain of a candidate commercial strain of B. bassiana (F418 gfp tr3), expressing the green fluorescent protein and the hygromycin B resistance gene, was used to assess the effects of the larvae of a host insect, Tenebrio molitor L. (Coleoptera: Tenebrionidae), a non-host, Costelytra zealandica (Coleoptera: Scarabaeidae) and the absence of larvae on the persistence of F418 gfp tr3 in pasteurised and non-sterile soil over 4 months. In the presence of a T. molitor larvae, F418 gfp tr3 populations increased significantly in pasteurised and non-sterile soil; however, populations increased less in non-sterile soil than in pasteurised soil. Lower populations of F418 gfp tr3 were recovered in pasteurised soil in the presence of C. zealandica larvae than in pasteurised soil without larvae. No difference was observed between F418 gfp tr3 populations in non-sterile soil with a non-host larvae or without larvae. Accompanying studies showed that F418 gfp tr3 conidia germinated and produced appressoria on live and excised cuticle of non-host (C. zealandica) larvae but infection did not occur, leading to a net loss of viable conidia in the soil. Conidia administrated orally to C. zealandica larvae were viable on recovery from faecal samples, suggesting that ingestion of the fungus by the larvae had little impact on the viable fungal population. Soil bacterial and fungal community patterns were analysed using Single-Strand Conformation Polymorphism (SSCP) and showed a correlation between changes in F418 gfp tr3 persistence in pasteurised and non-sterile soil and changes in soil communities in the presence of a host insect, non-host insect or in the absence of insect. In pasteurised soil, non-specific germination of F418 gfp tr3 conidia on the non-host larval cuticle and the presence of antagonistic bacteria introduced with the field-collected larvae are most likely responsible for the differences observed. The more complex microbial community structures in non-sterile soil could lead to fungistasis, preventing potentially antagonistic bacteria degrading conidia or inhibiting attachment and germination on the non-host larval cuticle, resulting in the observed lack of difference between non-host and no larval treatments.

Detection of the Entomopathogenic Fungus Beauveria bassiana in the Rhizosphere of Wound-Stressed Zea mays Plants.

Entomopathogenic fungi from the genus Beauveria (Vuillemin) play an important role in controlling insect populations and have been increasingly utilized for the biological control of insect pests. Various studies have reported that Beauveria bassiana (Bals.), Vuill. also has the ability to colonize a broad range of plant hosts as endophytes without causing disease but while still maintaining the capacity to infect insects. Beauveria is often applied as an inundative spore application, but little research has considered how plant colonization may alter the ability to persist in the environment. The aim of this study was to investigate potential interactions between B. bassiana and Zea mays L. (maize) in the rhizosphere following inoculation, in order to understand the factors that may affect environmental persistence of the fungi. The hypothesis was that different isolates of B. bassiana have the ability to colonize maize roots and/or rhizosphere soil, resulting in effects to the plant microbiome. To test this hypothesis, a two-step nested PCR protocol was developed to find and amplify Beauveria in planta or in soil; based on the translation elongation factor 1-alpha (ef1α) gene. The nested protocol was also designed to enable Beauveria species differentiation by sequence analysis. The impact of three selected B. bassiana isolates applied topically to roots on the rhizosphere soil community structure and function were consequently assessed using denaturing gradient gel electrophoresis (DGGE) and MicroRespTM techniques. The microbial community structure and function were not significantly affected by the presence of the isolates, however, retention of the inocula in the rhizosphere at 30 days after inoculation was enhanced when plants were subjected to intensive wounding of foliage to crudely simulate herbivory. The plant defense response likely changed under wound stress resulting in the apparent recruitment of Beauveria in the rhizosphere, which may be an indirect defensive strategy against herbivory and/or the result of induced systemic susceptibility in maize enabling plant colonization.

High spatial variation in population size and symbiotic performance of Rhizobium leguminosarum bv. trifolii with white clover in New Zealand pasture soils.

Biological nitrogen fixation through the legume-rhizobia symbiosis is important for sustainable pastoral production. In New Zealand, the most widespread and valuable symbiosis occurs between white clover (Trifolium repens L.) and Rhizobium leguminosarum bv. trifolii (Rlt). As variation in the population size (determined by most probable number assays; MPN) and effectiveness of N-fixation (symbiotic potential; SP) of Rlt in soils may affect white clover performance, the extent in variation in these properties was examined at three different spatial scales: (1) From 26 sites across New Zealand, (2) at farm-wide scale, and (3) within single fields. Overall, Rlt populations ranged from 95 to >1 x 108 per g soil, with variation similar at the three spatial scales assessed. For almost all samples, there was no relationship between rhizobia population size and ability of the population to fix N during legume symbiosis (SP). When compared with the commercial inoculant strain, the SP of soils ranged between 14 to 143% efficacy. The N-fixing ability of rhizobia populations varied more between samples collected from within a single hill country field (0.8 ha) than between 26 samples collected from diverse locations across New Zealand. Correlations between SP and calcium and aluminium content were found in all sites, except within a dairy farm field. Given the general lack of association between SP and MPN, and high spatial variability of SP at single field scale, provision of advice for treating legume seed with rhizobia based on field-average MPN counts needs to be carefully considered.

Community Structure of Endophytic Actinobacteria in a New Zealand Native Medicinal Plant Pseudowintera colorata (Horopito) and Their Influence on Plant Growth.

The role of plant endophytic Actinobacteria remains poorly understood with no reports of these communities in New Zealand native plants. This first investigation of endophytic Actinobacteria in New Zealand targeted the culturally significant medicinal shrub Pseudowintera colorata (horopito) as a model plant. Community analysis in plant tissues collected from ten geographically distinct sites showed that tissue type had the strongest influence on diversity and richness of endophytic Actinobacteria. More denaturing gradient gel electrophoresis (DGGE) bands were obtained from stems (n = 18) compared to roots (n = 13). Sequencing analysis of the major bands (n = 20) identified them as uncultured bacteria, Streptomyces sp. and Angustibacter peucedani. Using two Actinobacteria-specific media, nine isolates were recovered from surface-sterilised P. colorata tissues. This was approximately 12% of the total taxa and correlated well with culturable numbers in international studies. In vitro analysis of the functionality of these strains showed that Streptomyces sp. PRY2RB2 inhibited all the tested phytopathogenic fungi (n = 4), Streptomyces sp. UKCW/B and Nocardia sp. TP1BA1B solubilised phosphate and produced siderophores. The functionality of the phosphate solubilising strains (n = 2) in vivo was investigated by inoculation of P. colorata seedlings. After 4 months, the mean shoot height of seedlings treated with Nocardia sp. TP1BA1B was 1.65× longer, had higher shoot dry weight (1.6×) and number of internodes (1.67×) compared to control. This study identified for the first time a key group of endophytic Actinobacteria that are likely to be important in the ecology of New Zealand flora.

The Bacterial Signature of Leptospermum scoparium (Manuka) Reveals Core and Accessory Communities with Bioactive Properties.

Leptospermum scoparium or manuka is a New Zealand native medicinal plant that produces an essential oil with antimicrobial properties. This is the first study to investigate the structure and bioactivity of endophytic bacteria in manuka by using a combination of cultivation-independent (DGGE) and dependent approaches. A total of 23 plants were sampled across three sites. Plants were considered either immature (3-8 years) or mature (>20 years). The endophyte community structure and richness was affected by plant tissue and bacterial communities became more stable and uniform as plant maturity increased. A total of 192 culturable bacteria were recovered from leaves, stems and roots. Some bacterial isolates showed in vitro biocontrol activity against two fungal pathogens, Ilyonectria liriodendri and Neofusicoccum luteum and a bacterial pathogen, Pseudomonas syringae pv. actinidiae. A high proportion of bacterial endophytes could produce siderophores and solubilise phosphate in vitro. Gammaproteobacteria was the most variable class, representing the majority of cultivated bacteria with bioactivity.

Genome sequence of the clover symbiont Rhizobium leguminosarum bv. trifolii strain CC275e.

Rhizobium leguminosarum bv. trifolii strain CC275e is a highly effective, N2-fixing microsymbiont of white clover (Trifolium repens L.). The bacterium has been widely used in both Australia and New Zealand as a clover seed inoculant and, as such, has delivered the equivalent of millions of dollars of nitrogen into these pastoral systems. R. leguminosarum strain CC275e is a rod-shaped, motile, Gram-negative, non-spore forming bacterium. The genome was sequenced on an Illumina MiSeq instrument using a 2 × 150 bp paired end library and assembled into 29 scaffolds. The genome size is 7,077,367 nucleotides, with a GC content of 60.9 %. The final, high-quality draft genome contains 6693 protein coding genes, close to 85 % of which were assigned to COG categories. This Whole Genome Shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession JRXL00000000. The sequencing of this genome will enable identification of genetic traits associated with host compatibility and high N2 fixation characteristics in Rhizobium leguminosarum. The sequence will also be useful for development of strain-specific markers to assess factors associated with environmental fitness, competiveness for host nodule occupancy, and survival on legume seeds (New Zealand Ministry of Business, Innovation and Employment program, 'Improving forage legume-rhizobia performance' contract C10X1308 and DairyNZ Ltd.).

Phylogenetic congruence of lichenised fungi and algae is affected by spatial scale and taxonomic diversity.

The role of species' interactions in structuring biological communities remains unclear. Mutualistic symbioses, involving close positive interactions between two distinct organismal lineages, provide an excellent means to explore the roles of both evolutionary and ecological processes in determining how positive interactions affect community structure. In this study, we investigate patterns of co-diversification between fungi and algae for a range of New Zealand lichens at the community, genus, and species levels and explore explanations for possible patterns related to spatial scale and pattern, taxonomic diversity of the lichens considered, and the level sampling replication. We assembled six independent datasets to compare patterns in phylogenetic congruence with varied spatial extent of sampling, taxonomic diversity and level of specimen replication. For each dataset, we used the DNA sequences from the ITS regions of both the fungal and algal genomes from lichen specimens to produce genetic distance matrices. Phylogenetic congruence between fungi and algae was quantified using distance-based redundancy analysis and we used geographic distance matrices in Moran's eigenvector mapping and variance partitioning to evaluate the effects of spatial variation on the quantification of phylogenetic congruence. Phylogenetic congruence was highly significant for all datasets and a large proportion of variance in both algal and fungal genetic distances was explained by partner genetic variation. Spatial variables, primarily at large and intermediate scales, were also important for explaining genetic diversity patterns in all datasets. Interestingly, spatial structuring was stronger for fungal than algal genetic variation. As the spatial extent of the samples increased, so too did the proportion of explained variation that was shared between the spatial variables and the partners' genetic variation. Different lichen taxa showed some variation in their phylogenetic congruence and spatial genetic patterns and where greater sample replication was used, the amount of variation explained by partner genetic variation increased. Our results suggest that the phylogenetic congruence pattern, at least at small spatial scales, is likely due to reciprocal co-adaptation or co-dispersal. However, the detection of these patterns varies among different lichen taxa, across spatial scales and with different levels of sample replication. This work provides insight into the complexities faced in determining how evolutionary and ecological processes may interact to generate diversity in symbiotic association patterns at the population and community levels. Further, it highlights the critical importance of considering sample replication, taxonomic diversity and spatial scale in designing studies of co-diversification.

Burkholderia sp. induces functional nodules on the South African invasive legume Dipogon lignosus (Phaseoleae) in New Zealand soils.

The South African invasive legume Dipogon lignosus (Phaseoleae) produces nodules with both determinate and indeterminate characteristics in New Zealand (NZ) soils. Ten bacterial isolates produced functional nodules on D. lignosus. The 16S ribosomal RNA (rRNA) gene sequences identified one isolate as Bradyrhizobium sp., one isolate as Rhizobium sp. and eight isolates as Burkholderia sp. The Bradyrhizobium sp. and Rhizobium sp. 16S rRNA sequences were identical to those of strains previously isolated from crop plants and may have originated from inocula used on crops. Both 16S rRNA and DNA recombinase A (recA) gene sequences placed the eight Burkholderia isolates separate from previously described Burkholderia rhizobial species. However, the isolates showed a very close relationship to Burkholderia rhizobial strains isolated from South African plants with respect to their nitrogenase iron protein (nifH), N-acyltransferase nodulation protein A (nodA) and N-acetylglucosaminyl transferase nodulation protein C (nodC) gene sequences. Gene sequences and enterobacterial repetitive intergenic consensus (ERIC) PCR and repetitive element palindromic PCR (rep-PCR) banding patterns indicated that the eight Burkholderia isolates separated into five clones of one strain and three of another. One strain was tested and shown to produce functional nodules on a range of South African plants previously reported to be nodulated by Burkholderia tuberum STM678(T) which was isolated from the Cape Region. Thus, evidence is strong that the Burkholderia strains isolated here originated in South Africa and were somehow transported with the plants from their native habitat to NZ. It is possible that the strains are of a new species capable of nodulating legumes.

Rhizobia with 16S rRNA and nifH similar to Mesorhizobium huakuii but Novel recA, glnII, nodA and nodC genes are symbionts of New Zealand Carmichaelinae.

New Zealand became geographically isolated about 80 million years ago and this separation gave rise to a unique native flora including four genera of legume, Carmichaelia, Clianthus and Montigena in the Carmichaelinae clade, tribe Galegeae, and Sophora, tribe Sophoreae, sub-family Papilionoideae. Ten bacterial strains isolated from NZ Carmichaelinae growing in natural ecosystems grouped close to the Mesorhizobium huakuii type strain in relation to their 16S rRNA and nifH gene sequences. However, the ten strains separated into four groups on the basis of their recA and glnII sequences: all groups were clearly distinct from all Mesorhizobium type strains. The ten strains separated into two groups on the basis of their nodA sequences but grouped closely together in relation to nodC sequences; all nodA and nodC sequences were novel. Seven strains selected and the M. huakuii type strain (isolated from Astragalus sinicus) produced functional nodules on Carmichaelia spp., Clianthus puniceus and A. sinicus but did not nodulate two Sophora species. We conclude that rhizobia closely related to M. huakuii on the basis of 16S rRNA and nifH gene sequences, but with variable recA and glnII genes and novel nodA and nodC genes, are common symbionts of NZ Carmichaelinae.