Abundance of Frankia from Gymnostoma spp. in the rhizosphere of Alphitonia neocaledonica,a non-nodulated Rhamnaceae endemicto New Caledonia

The capacity of different soils of New Caledonia to induce nodulation in Gymnostoma poissonianum was studied. The soils were sampled under five Gymnostoma species, Alphitonia neocaledonica (a non-nodulated endemic Rhamnaceae) and Pinus caribea (an introduced species) growing in various ecological conditions. Using G. poissonianum as trap-host, we observed a higher abundance of Frankia from Gymnostoma spp. in the rhizosphere of A. neocaledonica as compared with bare soils and P. caribea rhizosphere. The nodulating capacity of A. neocaledonica rhizosphere was almost similar to that of the five Gymnostoma species (symbiotic host) studied in the same stations. In comparison, bare soils or rhizosphere of P. caribea had poor nodulating capacities. We isolated fourteen Frankia strains from nodules of G. poissonianum after baiting with the rhizospheric soils of five Gymnostoma and A. neocaledonica. Using the PCR/RFLP method, we confirmed the similarity with those already described. Frankia was abundant in the rhizosphere of A. neocaledonica in all the sites studied. One explanation could be a positive tropism of Frankia towards species belonging to families having nodulated species, which is the case of A. neocaledonica endemic in New Caledonia. We can suppose that the non-nodulated plants belonging to these families can excrete some chemical substances able to attract Frankia and to induce its proliferation.     

Abundance of Frankia from Gymnostoma spp. in the rhizosphere of Alphitonia neocaledonica, a non-nodulated Rhamnaceae endemicto New Caledonia

The capacity of different soils of New Caledonia to induce nodulation in Gymnostoma poissonianum was studied. The soils were sampled under five Gymnostoma species, Alphitonia neocaledonica (a non-nodulated endemic Rhamnaceae) and Pinus caribea (an introduced species) growing in various ecological conditions. Using G. poissonianum as trap-host, we observed a higher abundance of Frankia from Gymnostoma spp. in the rhizosphere of A. neocaledonica as compared with bare soils and P. caribea rhizosphere. The nodulating capacity of A. neocaledonica rhizosphere was almost similar to that of the five Gymnostoma species (symbiotic host) studied in the same stations. In comparison, bare soils or rhizosphere of P. caribea had poor nodulating capacities. We isolated fourteen Frankia strains from nodules of G. poissonianum after baiting with the rhizospheric soils of five Gymnostoma and A. neocaledonica. Using the PCR/RFLP method, we confirmed the similarity with those already described. Frankia was abundant in the rhizosphere of A. neocaledonica in all the sites studied. One explanation could be a positive tropism of Frankia towards species belonging to families having nodulated species, which is the case of A. neocaledonica endemic in New Caledonia. We can suppose that the non-nodulated plants belonging to these families can excrete some chemical substances able to attract Frankia and to induce its proliferation.     

Population size and diversity of Frankia in soils of Ceanothus velutinus and Douglas-fir stands

The influence of host plants on Frankia populations was investigated using soils from Ceanothus velutinus (Dougl.) and Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) stands. Population sizes of Ceanothus-infective Frankia in the soils were measured using plant bioassays with C. velutinus, C. sanguineus (Pursh), and C. integerrimus (H. & A.) as trap plants. The Frankia population in soil from the C. velutinus stand soil was about 10 times higher than that from the Douglas-fir stand. This result supports previous reports that, although the presence of host plants increases Frankia populations, Frankia persist without host plants. Nodulation capacities of the three trap plants were not significantly different. All nodules showed N₂ fixation activity using the acetylene reduction assay. The diversity of Frankia that nodulated trap plants was examined using repetitive intergenic DNA and the polymerase chain reaction (rep-PCR). A newly designed, direct repeat sequence and a BOX sequence were used as rep-PCR primers. The results showed that infective Frankia in the two soils contained a common group of Frankia as well as some Frankia strains unique to each soil. The level of host specificity of the infective Frankia was low; however, one group of Frankia nodulated only C. integerrimus seedlings. Taken together, the results suggest that the higher populations in the soil from the C. velutinus stand may be due to preferential increases in particular groups of Frankia.     

Genetic diversity among Elaeagnus compatible Frankia strains and sympatric-related nitrogen-fixing actinobacteria revealed by nifH sequence analysis

Elaeagnus compatible Frankia isolates from Tunisian soil have been previously clustered with Frankia, colonizing Elaeagnaceae and Rhamnaceae in two different phylogenetic subgroups, while strain BMG5.6 was described as a new lineage closely related to Frankia and Micromonospora genera. In this study we further assess the diversity of captured Frankia and the relationship with BMG5.6-like actinobacteria, by using nifH gene sequences. Using PCR-RFLP screening on DNA extracted from lobe nodules, additional microsymbionts sharing BMG5.6 features have been detected proving a widespread occurrence of these actinobacteria in Elaeagnus root nodules. Neighbour-Joining trees of Frankia nifH sequences were consistent with previously published 16S rRNA and GlnII phylogenetic trees. Although four main clades could be discerned, actinobacterial strain BMG5.6 was clustered with Frankia strains isolated from Elaeagnus. The present study underscored the emanation of new diazotrophic taxon isolated from actinorhizal nodules occupying intermediate taxonomic position between Frankia and Micromonospora. Moreover, its aberrant position in nifH phylogeny should open network investigations on the natural history of nitrogen-fixing gene among actinobacteria.     

Effect of temperature and soil moisture on the survival and symbiotic effectiveness of Frankia spp.

Comparison of the effects of temperature on the growth in culture (increase in protein) of Frankia showed that three strains isolated from Casuarina were more tolerant of high temperature (45°C) than a strain from Alnus rubra. Optimal temperatures for growth of the Casuarina strains were in the range 25–30°C. Growth of the Alnus strain was good at 25°C but poor at 37°C. High temperatures (35–40°C) during storage for 7 months of these Frankia strains in sand, inoculated initially with liquid culture or with Frankia incorporated into alginate beads and permitted to dry, resulted in substantial loss of infectivity for the host plant species. Loss in infectivity was greater with an Alnus Frankia strain than strains from Casuarina cunninghamiana, C. equisetifolia and C. junghuniana. Three Frankia strains from C. equisetifolia were incorporated into a sand/perlite mixture with three different moisture regimes (field moisture capacity – wet: watered and maintained at field capacity; watered to field capacity but then allowed to dry – moderately wet; or watered to half field capacity and then permitted to dry – dry) and then stored for 12 weeks at 25°C and 35°C. Assessment by the most probable number (MPN) technique of the infectivity of the sand mixture for nodulation of C. equisetifolia showed significant interactions between Frankia strain, temperature and soil moisture content. The infectivity of Frankia strains ORS020607 and UGL020602q was not affected by incubation in wet sand at 25°C but fell by more than half after 12 weeks in moderate and dry conditions. Changes in infectivity were similar when incubation was at 35°C. By contrast, the infectivity of UGL020603q fell substantially under all moisture conditions and at both temperatures. The data show the importance of screening for tolerance of both temperature and moisture content when selecting strains for preparation of inoculum for use in hot climates. Received: 25 January 1996     

In vitro growth of five Frankia isolates in the presence of four phenolic acids and juglone

The effects of four phenolic acids and juglone on the growth of five Frankia isolates were determined using a total protein assay and light microscopy. Juglone and eaffeic acid, at 100 μM and 1 mM concentrations respectively, were most inhibitory to growth of the Frankia isolates, although an isolate from Alnus crispa [Alnus viridis ssp. crispa (Ait.) Turril] nodules exhibited significantly greater growth compared to the other isolates at identical treatment levels. Gentisic, o-hydroxyphenylacetic and vanillic acids were less inhibitory to growth of the five Frankia isolates than juglone and caffeic acid. Juglone inhibited vesicle production of two isolates that produced vesicles in controls. Juglone, caffeic acid and vanillic acid caused increased hyphal ramification, while vanillic acid also induced Frankia to form numerous spherical structures unlike vesicles or sporangia in appearance. None of the isolates tested had the ability to utilize the phenolic acids as sole carbon and energy sources at 1 mM concentrations.     

Lotononis angolensis forms nitrogen fixing, lupinoid nodules with phylogenetically unique, fast-growing, pink-pigmented bacteria, which do not nodulate L. bainesii or L. listii

Root-nodule bacteria that nodulate the legume genus Lotononis are being investigated to develop new forage species for agriculture. Bacteria isolated from nodules of Lotononis angolensis were fast-growing, highly mucoid and pink-pigmented, and on the basis of 16S rRNA phylogeny <94% related to other genera in the Alphaproteobacteria. Root-nodule bacteria isolated from other Lotononis species (L. bainesii, L. solitudinis and L. listii) resembled the more common dry, slow-growing, pink-pigmented rhizobia previously described for L. bainesii. These isolates could be attributed to the Methylobacterium genus, although not to the type species Methylobacterium nodulans. Further differences were uncovered with nodulation studies revealing that nodule isolates from L. angolensis were effective at nitrogen fixation on their host plant, but could nodulate neither L. bainesii nor L. listii. Reciprocal tests showed isolates from L. bainesii, L. listii and L. solitudinis were incapable of nodulating L. angolensis effectively. Nodule morphology for L. bainesii, L. angolensis and L. listii was characteristically lupinoid, with little structural divergence between the species, and with nodules eventually enclosing the entire root.     

Genetic diversity of rhizobia associated with indigenous legumes in different regions of Flanders (Belgium)

We investigated the diversity of rhizobia isolated from different indigenous legumes in Flanders (Belgium). A total of 3810 bacterial strains were analysed originating from 43 plant species. Based on rep-PCR clustering, 16S rRNA gene and recA gene sequence analysis, these isolates belonged to Bradyrhizobium, Ensifer (Sinorhizobium), Mesorhizobium and Rhizobium. Of the genera encountered, Rhizobium was the most abundant (62%) and especially the species Rhizobium leguminosarum, followed by Ensifer (19%), Bradyrhizobium (14%) and finally Mesorhizobium (5%). For two rep-clusters only low similarity values with other genera were found for both the 16S rRNA and recA genes, suggesting that these may represent a new genus with close relationship to Rhodopseudomonas and Bradyrhizobium. Primers for the symbiotic genes nodC and nifH were optimized and a phylogenetic sequence analysis revealed the presence of different symbiovars including genistearum, glycinearum, loti, meliloti, officinalis, trifolii and viciae. Moreover, three new nodC types were assigned to strains originating from Ononis, Robinia and Wisteria, respectively. Discriminant and MANOVA analysis confirmed the correlation of symbiosis genes with certain bacterial genera and less with the host plant. Multiple symbiovars can be present within the same host plant, suggesting the promiscuity of these plants. Moreover, the ecoregion did not contribute to the separation of the bacterial endosymbionts. Our results reveal a large diversity of rhizobia associated with indigenous legumes in Flanders. Most of the legumes harboured more than one rhizobial endosymbiont in their root nodules indicating the importance of including sufficient isolates per plant in diversity studies.     

Conservation genetics and ecology of Angiopteris chauliodonta Copel. (Marattiaceae), a critically endangered fern from Pitcairn Island, South Central Pacific Ocean

Angiopteris chauliodonta, endemic to remote Pitcairn Island, was until recently thought to occur in only two small populations. Survey work carried out on the island in 1997 increased the number of populations to six, but as the total number of plants found was 774 (of which only 147 were mature adults) the species should be regarded as critically endangered. The species occurred in native fern-rich Homalium taypau and Metrosideros collina forest that was in many areas heavily invaded by Syzygium jambos and Lantana camara. RAPD analysis identified related populations but there was no correlation between genetic and geographical distance. The highest levels of genetic diversity was partitioned within populations (H_S=0.154; D_ST=0.116), although the larger populations were not necessarily the most diverse. Threats are primarily due to forest clearance, invasive species and erosion. Conservation management for this species will be through reinforcement of existing populations to maximise their genetic diversity and translocation of new populations to suitable habitats.     

Diversity and phylogeny of rhizobial strains isolated from Lotus uliginosus grown in Uruguayan soils

Lotus uliginosus is generally nodulated by rhizobia of the genus Bradyrhizobium when used for improvement of Uruguayan pastures. The genetic diversity and phylogenetic relationships of 111 isolates from nodules of L. uliginosus collected from four fields with or without prior inoculation history were analyzed in this study. Genetic diversity estimated by ERIC-PCR revealed 75 different genomic fingerprints, and showed a relatively greater value compared with other methods and varied by soil type. 16S ribosomal RNA gene RFLP analysis revealed three different ribogroups, A, B and C, with 71 isolates in ribogroup A, three isolates in ribogroup B and only one in ribogroup C. Phylogenetic analyses based on 16S RNA gene sequences, ITS, as well as atpD, recA and glnII gene sequences indicated that ribogroup A strains were affiliated with B. japonicum bv. genistearum strains. The three isolates in ribogroup B did not clearly associate with any Bradyrhizobium species described previously and could represent a novel species within this genus. Unlike B. japonicum strains these isolates were able to nodulate and fix nitrogen with other Lotus species as well as with Spartium, a leguminous shrub. The unique isolate in ribogroup C clustered with Mesorhizobium and appeared genetically and phenotypically related to broad host-range Mesorhizobium sp. NZP2037. Our data suggest that Uruguayan soils contain native or naturalized bradyrhizobia that are able to nodulate L. uliginosus as efficiently as the commercial strain NZP2309 but could have adaptive advantages making them more suitable for inoculant purposes.     

Nuclear and chloroplast microsatellite markers to assess genetic diversity and evolution in hazelnut species, hybrids and cultivars

The US Department of Agriculture, Agricultural Research Service, National Clonal Germplasm Repository in Corvallis, Oregon, preserves more than 800 accessions of hazelnut (Corylus), including C. avellana cultivars and representatives of 10 other recognized shrub and tree species. Characterization and study of genetic diversity in this collection require cross-transferable markers, such as trinucleotide microsatellite or simple sequence repeat (SSR) markers and universal chloroplast SSR markers. We developed new SSR markers and evaluated 114 Corylus accessions representing 11 species and 44 interspecific hybrids. Eight of 23 SSRs generated easy-to-score alleles in all species and seven were highly polymorphic. For those seven, the average heterozygosity was moderate at 0.49, and mean allele number, genetic diversity and polymorphism information index were high at 11.71, 0.79 and 0.76, respectively. The three most polymorphic SSRs were CaC-C008, CaC-C040 and CaC-C118. Neighbor-joining (NJ) clustering and structure analysis agreed with classical taxonomic analysis and supported inclusion of C. maxima within the large polymorphic species, C. avellana. Analysis also indicated that C. californica is a distinct species rather than a botanical variety of C. cornuta. Six universal cpSSRs were polymorphic in Corylus and generated 21 distinct chlorotypes with an average of 3 alleles per locus. Diversity at these cpSSRs was high and ranged from 0.33 to 0.64, with an average of 0.54. Incongruence in NJ topologies between the nuclear and chloroplast markers could be attributed to chloroplast capture related to hybridization during the ancestral diversification of the genus, or to homoplasy. The phylogeographical relationships among the 21 chlorotypes in the 11 Corylus species support Asia as a refugium where several hazelnut lineages survived during glaciation and from which they continued to evolve after dispersal from Asia through the Mediterranean to Europe, and across the Atlantic and/or the Bering land bridge to North America.     

Genetic diversity of indigenous common bean (Phaseolus vulgaris) rhizobia in two Brazilian ecosystems

Although rhizobia for common bean (Phaseolus vulgaris L.) are established in most Brazilian soils, understanding of their genetic diversity is very poor. This study characterized bean strains from two contrasting ecosystems in Brazil, the Northeast Region, with a semi-arid climate and neutral soils and the South Region, with a humid subtropical climate and acid soils. Seedlings of the cultivars Negro Argel and Aporé were used to trap 243 rhizobial isolates from 12 out of 14 sites. An analysis of ERIC-PCR products revealed enormous variability, with 81% of the isolates representing unique strains considering a level of 70% of similarity. In general, there was no effect of either the bean cultivar, or the ecosystem on rhizobial diversity. One-hundred and one strains showing genetic relatedness (ERIC-PCR) less than 70% were further analyzed using restriction fragment length polymorphism (RFLP) of the 16 S rDNA cleaved with five restriction enzymes. Twenty-five different profile combinations were obtained. Rhizobium etli was the predominant species, with 73 strains showing similar RFLP profiles, while 12 other strains differed only by the profile with one restriction enzyme. Fifty strains were submitted to sequencing of a 16 S rDNA fragment, and 34 clustered with R. etli, including strains with RFLP-PCR profiles similar to those species or differing by one restriction enzyme. However, other strains differing by one or two enzymes were genetically distant from R. etli and two strains with identical profiles showed higher similarity to Sinorhizobium fredii. Other strains showed higher similarity of bases with R. tropici, R. leguminosarum and Mesorhizobium plurifarium, but some strains were quite dissimilar and may represent new species. Great variability was also verified among the sequenced strains in relation to the ability to grow in YMA at 40 °C, in LB, to synthesize melanin in vitro, as well as in symbiotic performance, including differences in relation to the described species, e.g. many R. etli strains were able to grow in LB and in YMA at 40 °C, and not all R. tropici were able to nodulate Leucaena.     

Diversity of rhizobia nodulating Lotus corniculatus grown in northern and southern regions of Uruguay

Diversity of rhizobia nodulating Lotus corniculatus grown in geographical regions with different rainfall regimes in northern and southern Uruguay, was estimated using 168 root nodule isolates. ERIC-PCR analysis revealed no correlation between observed fingerprints and the geographical origin of isolates. Despite the commercial strain U510 has been used for decades to inoculate L. corniculatus, none of the isolates corresponded to this strain. Phylogenetic analyses using 16S rRNA and atpD genes, and ITS sequences clustered all the isolates within genus Mesorhizobium. A great majority of the isolates likely belong to the species M. huakuii, as does the commercial strain U510. The remaining isolates were closely related to either M. septentrionale or M. caraganae. Although no M. loti-like bacteria were identified, all isolates carried symbiotic genes closely related to M. loti and other narrow host range Lotus rhizobia. A significant portion of the Uruguayan isolates were as efficient as the reference strain U510 in symbiosis with L. corniculatus. A few of the isolates were also capable of nitrogen fixation in symbiosis with L. uliginosus, albeit with lower efficiency than reference strains. Our results indicate that rhizobia nodulating L. corniculatus in Uruguay are genetically and phenotypically diverse, and that the commercial strain U510 is probably not adapted to survive the Uruguayan edaphoclimatic conditions.     

Non-rhizobial peanut nodule bacteria promote maize (Zea mays L.) and peanut (Arachis hypogaea L.) growth in a simulated crop rotation system

The term “Plant Growth Promoting Bacteria” or PGPB designates a diverse group of prokaryotic microorganisms that can increase plant growth by diverse mechanisms. Some PGPB are capable of colonizing root inner tissues and constitute endophytic populations. Incorporation of these microorganisms into agricultural practices may constitute a valid alternative to increase crop productivity in a sustainable and environmentally friendly production scheme, reducing the application of agrochemicals. In a previous work, we described the characterization of bacteria belonging to Pseudomonas, Enterobacter and Klebsiella obtained from surface sterilized peanut nodules. In addition, we showed that some of these isolates were able to promote several peanut growth and symbiotic parameters. Bounded to the results from this particular study, and considering their potential ability to interact with different plant species, in this work we assessed the effects of their inoculation in maize (Zea mays L.) under controlled conditions. Furthermore, we analyzed growth promotion in a simulated peanut–maize crop rotation system. Finally, we determined the plant growth promoting (PGP) properties present in the isolates. Results indicated that all bacteria are able to significantly promote maize and peanut growth, and that they also displayed plant growth promotion activity in maize growing in a peanut–maize crop rotation sequence.     

Isolation and symbiotic characteristics of Mexican Frankia strains associated with Casuarina

In the absence of available symbiotic nitrogen-fixing Frankia strains associated with Casuarina trees in Mexico for reforestation purposes, isolation was undertaken using root nodules from trees growing in different habitats in Mexico, from the coast of the Gulf of Mexico up to 2550 m above the sea level. A total of 24 strains were isolated and clonal cultures were obtained from one filament of each strain. The use of acetate as the sole carbon source was essential for the isolation of the endosymbiont from the nodules due to the fact that other contaminant actinomycetes utilize propionate. Clonal cultures were obtained, and cultural and symbiotic characteristics of pure cultures were assessed. All strains grew well in stirred DPM (defined propionate medium) with no mineral nitrogen. Isolates showed hyphae, multilocular sporangia and characteristic vesicles. The presence of the gene nifH was also demonstrated, with all strains being able to nodulate Casuarina equisetifolia. Nitrogenase activity (acetylene reduction) of the formed root nodules varied among the different associations depending on the isolate used to inoculate the plants. Several of the isolates can be used as inoculants for the propagation of Casuarina trees.     

Presence and dispersal of infective Frankia in peat and meadow soils in Sweden

Summary Use of the N₂-fixing grey alder, Alnus incana (L.) Moench, as a short-rotation crop for energy production is currently being explored. To evaluate the need for inoculation of alders, the distribution of infective propagules of Frankia in the soil at potential sites for alder plantations was examined. Uninoculated grey alder seedlings were grown in three types of soil. Frequent nodulation was found in a meadow soil which had been free from actinorhizal plants for nearly 60 years, but the alder seedlings failed to nodulate in peat soil from two different bog sites. One of these bogs had been exploited for peat and the surface layer of the peat had been removed, so that the soil samples were taken from deep layers of the peat. At the other site, an area of cultivated peat, there were no infective propagules of Frankia in plots without alders; the infective Frankia was present in plots only where it had been introduced by inoculated alders. There was no detectable air-borne dispersal of Frankia. Instead, water movement might account for the dispersal of Frankia in peat. Although the apparent absence of Frankia in these peat soils necessitates inoculation of alder seedlings before planting out, this makes it possible to introduce and maintain Frankia strains with selected beneficial characteristics, since there is no competition from an indigenous Frankia flora.     

Mapping pathways of possible phage-mediated genetic interchange among soil bacilli

Bacillus pumilus (Strain W43) has been shown to sustain the growth of an unusually large number of different phages. From 22 isolates 16 distinguishable phages have been obtained. Phage BPPX which is similar to the defective particle PBSX of B. subtilis is induced by four of the phages. When tested against a variety of Bacillus spp 12 of the non-defective phages had host ranges crossing at least one species line: Phage K13 infected 14 of 25 strains, distributed among six species some of which are considered to be taxonomically distant. It is suggested that the relatively restricted host ranges noted for most Bacillus phages may result from the use. as test organisms, of bacteria isolated from soils ecologically distinct from the source(s) of phage. A genetic “circuit diagram” constructed from the host range table, maps possible genetic connexions between various soil bacilli made possible by the phages. These data are set in the context of recent theories which postulate that viruses are agents of accelerated cell evolution.     

Phytophthora species producing toxin active on tobacco

Extracts of oat cultures of 53 isolates of Pythium and Phytophthora and an isolate of Achlya were tested for their ability to cause watersoaking, laminar collapse, and dehydration of excised tobacco leaves. Extracts of oat cultures of 10 of 11 isolates of P. cryptogea Pethy. & Laff., 4 of 5 isolates of P. megasperma Drechs., all 5 isolates of P. drechsleri Tucker, and 3 of 4 isolates of P. erythroseptica Pethy, were toxic to tobacco laminae. Mycelial extracts of three P. cryptogea isolates obtained from three diverse sources had properties similar to a previously studied toxin from P. cryptogea. Extracts of oat cultures of three P. cryptogea isolates and an isolate of P. megasperma caused foliar toxicity and growth reduction of tobacco transplants. Mycelial extracts of isolates of four species of Phytophthora active on excised leaves were also active in inhibiting plant growth. Most isolates of these four species of Phytophthora appear to produce identical or similar toxins. Although not parasitic to tobacco, those species are potentially detrimental to tobacco.     

Susceptibility of Pythium spp and other fungi to antagonism by the mycorparasite Phytium Oligandrum

Phytium spp and isolates within species differed in susceptibility to the mycoparasite Pythium oligandrum Drechs., as evidenced by their degree of inhibition by it on cellulose and ability to support its growth across their colonies on agar. Yet no Phythium sp. was highly susceptible to it, and P. graminicola Subramanian was highly resistant.No evidence was found that P. oligandrum produces toxins active against other fungi. In liquid culture, P. oligandrum grew on undisturbed colonies of Phialophora sp. (highly susceptible) but not P. ultimum Trow or Fusarium culmorum (W. G. Sm.) Sacc. (moderately resistant) and not Rhizoctonia solani Kuhn (highly resistant). It grew in culture filtrates of Phialophora sp., P. ultimum and F. culmorum, utilizing organic nitrogen and thiamine released by these fungi, but not in culture filtrates of R. solani. It grew on mycelial macerates of all these fungi, though poorly on those of R. solani. Resistance to parasitism by P. oligandrum seems to reside, at least partly, in low levels of nutrient release from host hyphae.     

Genetic diversity of rhizobia associated with indigenous legumes in different regions of Flanders (Belgium)

We investigated the diversity of rhizobia isolated from different indigenous legumes in Flanders (Belgium). A total of 3810 bacterial strains were analysed originating from 43 plant species. Based on rep-PCR clustering, 16S rRNA gene and recA gene sequence analysis, these isolates belonged to Bradyrhizobium, Ensifer (Sinorhizobium), Mesorhizobium and Rhizobium. Of the genera encountered, Rhizobium was the most abundant (62%) and especially the species Rhizobiumleguminosarum, followed by Ensifer (19%), Bradyrhizobium (14%) and finally Mesorhizobium (5%). For two rep-clusters only low similarity values with other genera were found for both the 16S rRNA and recA genes, suggesting that these may represent a new genus with close relationship to Rhodopseudomonas and Bradyrhizobium. Primers for the symbiotic genes nodC and nifH were optimized and a phylogenetic sequence analysis revealed the presence of different symbiovars including genistearum, glycinearum, loti, meliloti, officinalis, trifolii and viciae. Moreover, three new nodC types were assigned to strains originating from Ononis, Robinia and Wisteria, respectively. Discriminant and MANOVA analysis confirmed the correlation of symbiosis genes with certain bacterial genera and less with the host plant. Multiple symbiovars can be present within the same host plant, suggesting the promiscuity of these plants. Moreover, the ecoregion did not contribute to the separation of the bacterial endosymbionts. Our results reveal a large diversity of rhizobia associated with indigenous legumes in Flanders. Most of the legumes harboured more than one rhizobial endosymbiont in their root nodules indicating the importance of including sufficient isolates per plant in diversity studies.