Burkholderia species are ancient symbionts of legumes

Burkholderia has only recently been recognized as a potential nitrogen-fixing symbiont of legumes, but we find that the origins of symbiosis in Burkholderia are much deeper than previously suspected. We sampled 143 symbionts from 47 native species of Mimosa across 1800 km in central Brazil and found that 98% were Burkholderia . Gene sequences defined seven distinct and divergent species complexes within the genus Burkholderia . The symbiosis-related genes formed deep Burkholderia -specific clades, each specific to a species complex, implying that these genes diverged over a long period within Burkholderia without substantial horizontal gene transfer between species complexes.     

西北部分地区苦马豆根瘤菌的表型多样性研究

将采集自甘肃、新疆、宁夏等地区的苦马豆根瘤,经分离、纯化获得48株未知菌株,并选取7株参比菌株,进行唯一碳、氮源利用、对抗生素及染料抗性、耐盐性、初始pH生长、生长温度范围及酶活性等共113项生理生化测定;采用数值分类方法对未知根瘤菌进行表型多样性分析。结果表明：供试菌株在碳氮源利用、抗生素敏感性、抗逆性等方面存在着差异。该地区苦马豆根瘤菌具有较强的耐盐、耐碱能力,所有菌株均能在初始pH9~12的YMA培养基上生长,35%菌株可耐受6%的NaCl。从数值分类树状图可见,未知供试菌株在56%的相似水平上聚在一起,在72%的相似水平上分为5个表观群。群Ⅰ有39株菌,在74.6%相似水平聚合,中心菌株为CCNWGS0215;群Ⅱ有5株菌,在76%的相似水平聚合,中心菌株为CCNWGS0228;群Ⅳ有2株菌,在81.5%的相似水平聚合,群Ⅲ和群Ⅴ分别只有1株菌,它们与模式株分离,可能为潜在的新种。     

OBPC Symposium: Maize 2004 &; beyond—Intracellular colonization of cereals and other crop plants by nitrogen-fixing bacteria for reduced inputs of synthetic nitrogen fertilizers

Summary The problem of environmental nitrogen enrichment is most likely to be solved by reducing the inputs of synthetic nitrogen fertilizers through the creation of cereals that, like legumes, are able to fix nitrogen. In legumes, rhizobia present intracellularly in vesicles in the cytoplasm of nodule cells fix nitrogen endosymbiotically. Rhizobia within these membrane-bounded compartments are supplied with energy from plant photosynthates and, in return, the bacteria provide the plant with biologically fixed nitrogen. Recently, we have demonstrated, using novel inoculation conditions with very low numbers of bacteria, that cells of the root meristems of maize, rice, wheat, and other major non-legume crops can be colonized intracellularly by the non-rhizobial, non-nodulating, nitrogen-fixing bacterium, Gluconacetobacter diazotrophicus, that occurs naturally in sugarcane. G. diazotrophicus expressing nitrogen-fixing genes is present in membrane-bounded compartments in the cytoplasm of cells of the root meristems of the target cereals and non-legume species, similar to the intracellular colonization of legume root nodule cells by rhizobia. In order to obtain an indication of the likelihood of adequate growth and yield of maize, for example, with reduced inputs of synthetic nitrogen fertilizers, we are determining the extent to which nitrogen fixation is correlated with systemic intracellular colonization by G. diazotrophicus, with minimal or zero inputs of synthetic nitrogen fertilizer.     

Phenotypic and genotypic characterization of rhizobia associated with Acacia saligna (Labill.) Wendl. in nurseries from Algeria

Twenty seven rhizobial strains associated with Acacia saligna grown in northern and southern Algeria were characterized, including generation time, host-range, the 16S rRNA gene and 16S–23S rRNA intergenic spacer restriction patterns, 16S rRNA gene sequence analysis and tolerance to salinity and drought. Cross inoculation tests indicated that 11 slow-growing isolates from northern nurseries were able to nodulate introduced Australian acacias exclusively, whereas 16 fast-growing isolates, mainly from southern nurseries, were capable of also nodulating native acacias. Restriction patterns and sequence analysis of the 16S rRNA gene showed that strains of the first group belonged to Bradyrhizobium while strains of the second group were related to Sinorhizobium meliloti and Rhizobium gallicum. Interestingly, five strains of the first group formed a distinct cluster phylogenetically close to Bradyrhizobium betae, a non-nodulating species causing tumour-like deformations in sugar beet roots. Bradyrhizobium strains were in general more sensitive to NaCl and PEG than the S. meliloti and R. gallicum representatives. Among the latter, strains S. meliloti BEC1 and R. gallicum DJA2 were able to tolerate up to 1 M NaCl and 20% PEG. This, together with their wide host-range among Acacia species, make them good candidates for developing inoculants for A. saligna and other acacia trees growing in arid areas.     

Host control over infection and proliferation of a cheater symbiont

Host control mechanisms are thought to be critical for selecting against cheater mutants in symbiont populations. Here, we provide the first experimental test of a legume host’s ability to constrain the infection and proliferation of a native‐occurring rhizobial cheater. Lotus strigosus hosts were experimentally inoculated with pairs of Bradyrhizobium strains that naturally vary in symbiotic benefit, including a cheater strain that proliferates in the roots of singly infected hosts, yet provides zero growth benefits. Within co‐infected hosts, the cheater exhibited lower infection rates than competing beneficial strains and grew to smaller population sizes within those nodules. In vitro assays revealed that infection‐rate differences among competing strains were not caused by variation in rhizobial growth rate or interstrain toxicity. These results can explain how a rapidly growing cheater symbiont – that exhibits a massive fitness advantage in single infections – can be prevented from sweeping through a beneficial population of symbionts.     

Mixed infections may promote diversification of mutualistic symbionts: why are there ineffective rhizobia?

While strategy variation is a key feature of symbiotic mutualisms, little work focuses on the origin of this diversity. Rhizobia strategies range from mutualistic nitrogen fixers to parasitic nonfixers that hoard plant resources to increase their own survival in soil. Host plants reward beneficial rhizobia with higher nodule growth rates, generating a trade‐off between reproduction in nodules and subsequent survival in soil. However, hosts might not discriminate between strains in mixed infections, allowing nonfixing strains to escape sanctions. We construct an adaptive dynamics model of symbiotic nitrogen‐fixation and find general situations where symbionts undergo adaptive diversification, but in most situations complete nonfixers do not evolve. Social conflict in mixed infections when symbionts face a survival–reproduction trade‐off can drive the origin of some coexisting symbiont strategies, where less mutualistic strains exploit benefits generated by better mutualists.     

Molecular diversity of rhizobia nodulating the invasive legume Cytisus scoparius in Australia

AIMS: To contribute to the understanding of Cytisus scoparius success at invading and establishing itself in Australia. METHODS AND RESULTS: Root-nodule bacteria isolated from C. scoparius, growing on five different sites and originally introduced to Australia, were compared with isolates from indigenous plants growing in France and isolates from native legumes growing on the same Australian sites as C. scoparius. Small-subunit rDNA from 251 isolates were analysed by PCR-RFLP and representatives from different genospecies were selected for sequencing. Phylogenetic analyses revealed a great diversity of lineages belonging to Bradyrhizobium, with one genospecies being specific for Cytisus both in Australia and in France, Rhizobium and Mesorhizobium and one falling outside the described genera of legume-nodulating bacteria. Principal component analysis showed that the Cytisus Australian rhizobial communities are more similar to each other than to their co-occurring native partners. CONCLUSIONS: Early established rhizobial symbionts may have an increased probability to contribute inoculum for the development of further nodules. SIGNIFICANCE AND IMPACT OF THE STUDY: This is a first report comparing rhizobia nodulating C. scoparius in its native and exotic environments. Cytisus scoparius symbionts were identified outside the Bradyrhizobium genus and a new lineage of legume-nodulating bacteria was identified.     

60Ma of legume nodulation. What's new? What's changing?

Current evidence suggests that legumes evolved about 60 million years ago. Genetic material for nodulation was recruited from existing DNA, often following gene duplication. The initial process of infection probably did not involve either root hairs or infection threads. From this initial event, two branched pathways of nodule developmental processes evolved, one involving and one not involving the development of infection threads to 'escort' bacteria to young nodule cells. Extant legumes have a wide range of nodule structures and at least 25% of them do not have infection threads. The latter have uniform infected tissue whereas those that have infection threads have infected cells interspersed with uninfected (interstitial) cells. Each type of nodule may develop indeterminately, with an apical meristem, or show determinate growth. These nodule structures are host determined and are largely congruent with taxonomic position. In addition to variation on the plant side, the last 10 years have seen the recognition of many new types of 'rhizobia', bacteria that can induce nodulation and fix nitrogen. It is not yet possible to fit these into the emerging pattern of nodule evolution.     

Coexistence of predominantly nonculturable rhizobia with diverse, endophytic bacterial taxa within nodules of wild legumes

A previous analysis showed that Gammaproteobacteria could be the sole recoverable bacteria from surface-sterilized nodules of three wild species of Hedysarum. In this study we extended the analysis to eight Mediterranean native, uninoculated legumes never previously investigated regarding their root-nodule microsymbionts. The structural organization of the nodules was studied by light and electron microscopy, and their bacterial occupants were assessed by combined cultural and molecular approaches. On examination of 100 field-collected nodules, culturable isolates of rhizobia were hardly ever found, whereas over 24 other bacterial taxa were isolated from nodules. None of these nonrhizobial isolates could nodulate the original host when reinoculated in gnotobiotic culture. Despite the inability to culture rhizobial endosymbionts from within the nodules using standard culture media, a direct 16S rRNA gene PCR analysis revealed that most of these nodules contained rhizobia as the predominant population. The presence of nodular endophytes colocalized with rhizobia was verified by immunofluorescence microscopy of nodule sections using an Enterobacter-specific antibody. Hypotheses to explain the nonculturability of rhizobia are presented, and pertinent literature on legume endophytes is discussed.