Restriction fragment length polymorphism of Azospirillum strains

Abstract: Total DNA of various Azospirillum strains representing different species was digested with restriction enzymes, Southern blotted and hybridized with four A. brasilense probes. Pairwise comparison of the conserved hybridization fragments was applied to calculate sequence divergence and to group the strains using the unweighted pair group method. The resulting dendrogram grouped the strains according to the known species indicating that the analysis of the restriction fragment length polymorphism is an useful tool for characterizing Azospirillum isolates.     

Diversity of Plasmid Profiles and Conservation of Symbiotic Nitrogen Fixation Genes in Newly Isolated Rhizobium Strains Nodulating Sulla (Hedysarum coronarium L.)

Forty-five Rhizobium strains nodulating sulla (Hedysarum coronarium L.), isolated from plants grown in different sites in Menorca Island and southern Spain, were examined for plasmid content and the location and organization of nif (nitrogen fixation) and nod (nodulation) sequences. A great diversity in both number and size of the plasmids was observed in this native population of strains, which could be distributed among 19 different groups according to their plasmid profiles. No correlation was found between plasmid profile and geographical origin of the strains. In each strain a single plasmid ranging from 187 to 349 megadaltons hybridized to Rhizobium meliloti nifHD and nodD DNA, and in three strains the spontaneous loss of this plasmid resulted in the loss of the nodulation capacity. In addition to the symbiotic plasmid, 18 different cryptic plasmids were identified. A characteristic cryptic plasmid of >1,000 megadaltons was present in all strains. Total DNA hybridization experiments, with nifHD and portions of nodC and nodD genes (coding for common nodulation functions) from R. meliloti as probes, demonstrated that both the sequence and organization of nif and common nod genes were highly conserved within rhizobia nodulating sulla. Evidence for reiteration of nodD sequences and for linkage of nodC to at least one copy of nodD was obtained for all the strains examined. From these results we conclude that Rhizobium strains nodulating sulla are a homogeneous group of symbiotic bacteria that are closely related to the classical fast-growing group of rhizobia.     

Characterization of the formae speciales of Fusarium oxysporum causing wilts of cucurbits by DNA fingerprinting with nuclear repetitive DNA sequences.

The genetic relatedness of five formae speciales of Fusarium oxysporum causing wilts of cucurbit plants was determined by DNA fingerprinting with the moderately repetitive DNA sequences FOLR1 to FOLR4. The four FOLR clones were chosen from a genomic library made from F. oxysporum f. sp. lagenariae 03-05118. Total DNAs from 50 strains representing five cucurbit-infecting formae speciales, cucumerinum, melonis, lagenariae, niveum, and momordicae, and 6 strains of formae speciales pathogenic to other plants were digested with EcoRV and hybridized with 32P-labeled FOLR probes. The strains were clearly distinguishable at the formae specialis level on the basis of FOLR DNA fingerprints. Fifty-two fingerprint types were detected among the 56 strains by using all FOLR probes. These probes were used to infer phylogenetic relationships among the DNA fingerprint types by the unweighted pair group method using averages and parsimony analysis. The fingerprint types detected in each of the formae speciales cucumerinum, lagenariae, niveum, and momordicae were grouped into a single cluster. However, two different genetic groups occurred in the formae specialis melonis. The two groups also differed in pathogenicity: one group caused wilts of muskmelon and oriental melon, while the second was pathogenic only to muskmelon. The fingerprint types of different formae speciales pathogenic to plants other than cucurbits were distinguishable from one another and from the fingerprints of the cucurbit-infecting strains. These results suggest that the cucurbit-infecting formae speciales are intraspecific variants distinguishable at the DNA level and in their host range.     

The Occurrence of the Glucose-inducible Transport Systems for Glucose,Proline, and Arginine in Different Species of Chlorella

Incubation of the green alga Chlorella vulgaris (strain K, Tanner and Kandler, 1967) with glucose leads to the induction of a glucose transport system and of two amino acid transport systems. Because it was not clear whether the regulation of 3 different transport systems by glucose is specific to our strain of Chlorella or whether it is a general property of the genus Chlorella, 11 other free living and symbiotic Chlorella species and strains were tested for glucose-inducible glucose, arginine and proline transport. It was found that nearly all Chlorella species possess glucose and amino acid uptake systems. Often they were constitutive, although in some species they were induced or stimulated by glucose. According to the transport activities of the different Chlorella species and strains, a physiological classification of Chlorella was constructed, resulting in 3 groups: the C. fusca vacuolata, the C. vulgaris and the symbiotic Chlorella group. Our Chlorella (strain K) obviously belongs to the C. vulgaris group and forms a link to symbiotic Chlorella strains. This suggests that the possession of the glucose-regulated transport systems is of advantage for Chlorella in symbiotic situations, whereas the constitutive systems are useful for free living Chlorella.     

Description of two biovars in the Rhizobium galegae species: biovar orientalis and biovar officinalis

Twenty-six Rhizobium galegae strains, representing the center of origin of the host plants Galega orientalis and G. officinalis as well as other geographic regions, were used in a polyphasic analysis of the relationships of R. galegae strains. Phage typing, lipopolysaccharide (LPS) profiling, pulsed field gel electrophoresis (PFGE) profiling and rep-PCR (use of repetitive sequences as PCR primers for genomic fingerprinting) with REP and ERIC primers investigated nonsymbiotic properties, whereas plasmid profiling and hybridisation with a nif gene probe, and with nodB, nodD, nod box and an IS sequence from the symbiotic region as probes, were used to reveal the relationships of symbiotic genes. The results were used in pairwise calculations of distances between the strains, and the distances were visualised as a dendrogram. Indexes of association were compared for all tests pooled, and for chromosomal tests and symbiotic markers separately, to display the input of the different categories of tests on the grouping of the strains. Our study shows that symbiosis related genetic traits in R. galegae divide strains belonging to the species into two groups, which correspond to strains forming an effective symbioses with G. orientalis and G. officinalis respectively. We therefore propose that Rhizobium galegae strains forming an effective symbiosis with Galega orientalis are called R. galegae bv. orientalis and strains forming an effective symbiosis with Galega officinalis are called R. galegae bv. officinalis.     

Monophyly of nodA and nifH genes across Texan and Costa Rican populations of Cupriavidus nodule symbionts

nodA and nifH phylogenies for Cupriavidus nodule bacteria from native legumes in Texas and Costa Rica grouped all strains into a single clade nested among neotropical Burkholderia strains. Thus, Cupriavidus symbiotic genes were not acquired independently in different regions and are derived from other Betaproteobacteria rather than from alpha-rhizobial donors.     

Cytisus villosus from Northeastern Algeria is nodulated by genetically diverse Bradyrhizobium strains

Fifty-one rhizobial strains isolated from root nodules of Cytisus villosus growing in Northeastern Algeria were characterized by genomic and phenotypic analyses. Isolates were grouped into sixteen different patterns by PCR-RAPD. The phylogenetic status of one representative isolate from each pattern was examined by multilocus sequence analyses of four housekeeping genes (16S rRNA, glnII, recA, and atpD) and one symbiotic gene (nodC). Analysis of 16S rRNA gene sequences showed that all the isolates belonged to the genus Bradyrhizobium. Phylogenetic analyses based on individual or concatenated genes glnII, recA, and atpD indicated that strains cluster in three distinct groups. Ten out of the sixteen strains grouped together with Bradyrhizobium japonicum, while a second group of four clustered with Bradyrhizobium canariense. The third group, represented by isolates CTS8 and CTS57, differed significantly from all other bradyrhizobia known to nodulate members of the Genisteae tribe. In contrast with core genes, sequences of the nodC symbiotic gene from all the examined strains form a homogeneous group within the genistearum symbiovar of Bradyrhizobium. All strains tested nodulated Lupinus angustifolius, Lupinus luteus, and Spartium junceum but not Glycine max. From these results, it is concluded that C. villosus CTS8 and CTS57 strains represent a new lineage within the Bradyrhizobium genus.     

Phylogenetic diversity and symbiotic functioning in mungbean (Vigna radiata L. Wilczek) bradyrhizobia from contrast agro-ecological regions of Nepal

Nepal consists wide range of climatic and topographical variations. Here, we explored the phylogeny of native mungbean bradyrhizobia isolated from different agro-ecological regions of Nepal and accessed their nodulation and nitrogen fixation characteristics. Soil samples were collected from three agro-ecological regions with contrasting climate and topography. A local mungbean cultivar, Kalyan, was used as a trap plant. We characterized isolates based on the full nucleotide sequence of the 16S rRNA, ITS region, and nodA genes; and partial sequences of nodD1 and nifD genes. We found 50% of isolates phylogenetically related to B. yuanmingense, 13% to B. japonicum, 8% to B. elkanii, and 29% to novel phylogenetic origin. Results of the inoculation test suggested that expression of different symbiotic genes in isolates resulted in different degrees of symbiotic functioning. Our results indicate B. yuanmingense and novel strains are more efficient symbiotic partners than B. elkanii for the local mungbean cv. Kalyan. We also found most mungbean rhizobial genotypes were conserved across agro-ecological regions. All the strains from tropical Terai region belonged to B. yuanmingense or a novel lineage of B. yuanmingense, and dominance of B. japonicum related strains was observed in the Hill region. Higher genetic diversity of Bradyrhizobium strains was observed in temperate and sub-tropical region than in the tropical region.     

Isolation of unique nucleic acid sequences from rhizobia by genomic subtraction: Applications in microbial ecology and symbiotic gene analysis

A subtraction hybridization and PCR amplification procedure was used to isolate two Rhizobium DNA probes which exhibited high degrees of specificity at different levels of taxonomic organization and which could be used as tools for detection of rhizobia in ecological studies. First, a probe was isolated from Rhizobium leguminosarum bv. trifolii strain P3 by removing those Sau3A restriction fragments from a P3 DNA digest which cross hybridized with pooled DNA from seven other strains of the same biovar. The remaining restriction fragments hybridized to DNA from strain P3 but not to DNA from any of the seven other strains. In a similar experiment another DNA probe, specific for the Rhizobium leguminosarum bv. phaseoli and Rhizobium tropici group, was generated by removing sequences from R. leguminosarum bv phaseoli strain Kim 5s with pooled subtracter DNA from eight other Rhizobium, Bradyrhizobium and Agrobacterium species. The same subtraction hybridization technique was also used to isolate symbiotic genes from a Rhizobium species. Results from a 1:1 subtractive DNA hybridization of the broad host range Rhizobium sp NGR234 against highly homologous S. fredii USDA257, combined with those from competitive RNA hybridizations to cosmid digests of the NGR234 symbiotic plasmid, allowed the identification of several NGR234 loci which were flavonoid-inducible and not present in S. fredii USDA257. One of these, ORF-1, was highly homologous to the leucine responsive regulatory protein of E. coli.     

Characterization by genotypic methods of symbiotic Nostoc strains isolated from five species of Gunnera

The genetic diversity of ten symbiotic Nostoc strains isolated from different Gunnera species was investigated. The strains were analyzed using molecular methods with different taxonomic resolutions, including restriction fragment length polymorphisms (RFLP) of the PCR-amplified 16S ribosomal gene and the 16S-23S internal transcribed spacer (ITS) region combined with computer-assisted analyses. The functional gene hetR, assigned to heterocyst differentiation, was used for denaturing gradient gel electrophoresis. A high genetic diversity was observed among the isolates even in the conserved gene coding for the small ribosomal unit. No correlation was observed between clustering of cyanobacteria and the host species of Gunnera.     

Genetic diversity and phylogeny analysis of Anabaena azollae based on RFLPs detected in Azolla-Anabaena azollae DNA complexes using nif gene probes

The cyanobacterium Anabaena has both symbiotic and free-living forms. The genetic diversity of Anabaena strains symbiotically associated with the aquatic fern Azolla and the evolutionary relationships among these symbionts were evaluated by means of RFLP (restriction fragment length polymorphism) experiments. Three DNA fragments corresponding to nif genes were cloned from the free-living cyanobacterium Anabaena PCC 7120 and used as probes. A mixture of Azolla, Anabaena and bacterial DNA was extracted from Azolla fronds and digested with two restriction enzymes. Single-copy RFLP signals were detected with two of the probes in all Azolla Anabaena examined. Multiple-copy RFLP signals were obtained from the third probe which corresponded to a part of the nif N gene. A total of 46 probe/enzyme combinations were scored as present or absent and used to calculate pairwise Nei's genetic distances among symbiotic Anaebaena strains. Phylogenetic trees summarizing phenetic and cladistic relationships among strains were generated according to three different evolutionary scenarios: parsimony, UPGMA and neighbour joining. All trees revealed identical phylogenetic relationships. Principal component analysis was also used to evaluate genetic similarities and revealed three groups: group one contains the cyanobacteria associated with plants from the Azolla section, group two contains those associated with plants from the pinnata species and group three contains those associated with plants from the nilotica species. The same groups had already been identified earlier in a random amplified polymorphic DNA (RAPD) analysis of Azolla-Anbaena DNA complexes, suggesting that the present Azolla taxonomy should be revised. We now suggest a taxonomy of Anabaena azollae that is parallel to such a revised Azolla taxonomy. An Azolla chloroplast DNA sequence derived from Oryza sativa was also used as an RFLP probe on Azolla DNA to confirm the presence of plant DNA in the total genomic DNA extracted from ferns with or without the symbiont. Our results also suggest that total DNA extracted from the Azolla-Anabaena complexes includes both plant and symbiont DNA and can be used equally well for RFLP analysis of host plant or symbiotic cyanobacteria.     

Diversity and compatibility of peanut (Arachis hypogaea L.) bradyrhizobia and their host plants

A high degree of genetic diversity among 125 peanut bradyrhizobial strains and among 32 peanut cultivars collected from different regions of China was revealed by using the amplified fragment length polymorphism (AFLP) technique. Eighteen different peanut bradyrhizobial genotypes and six peanut cultivars were selected for symbiotic cross-inoculation experiments. The genomic diversity was reflected in the symbiotic diversity. The peanut cultivars varied in their ability to nodulate with the strains used. Some cultivars had a more restricted host range than the others. Also the strains displayed a range of nodulation patterns. In yield formation there were clear differences between the plant cultivar/bradyrhizobium combinations. There was good compatibility between some peanut bradyrhizobial strains and selected cultivars, with inoculation resulting in well-nodulated, high-yielding symbiotic combinations, but no plant cultivar was compatible with all strains used. The strains displayed a varying degree of effectiveness, with some strains being fairly effective with all cultivars and others with selected ones. The AFLP genotypes of the strains did not explain the symbiotic behavior, whereas the yield formation of the plant cultivars was more related to the genotype. It is concluded that to obtain optimal nitrogen fixation efficiency of peanut in the field, compatible plant cultivar-bradyrhizobium combinations should be selected either by finding inoculant strains compatible with the plant cultivars used, or plant cultivars compatible with the indigenous bradyrhizobia.     

PCR use of highly conserved DNA regions for identification of Sinorhizobium meliloti

A PCR identification method in which four primers that recognize homologous conserved regions in the Sinorhizobium meliloti genome are used was developed and tested. The regions used for identification were the nodbox 4 locus, which is located in one of the symbiotic megaplasmids, and the mucR gene, which is located in the chromosome. The new method was used to establish a collection of S. meliloti strains from polluted soils.     

Identification of Different Agrobacterium Strains Isolated from the Same Forest Nursery

Several Agrobacterium strains isolated from the same forest nursery from 1982 to 1988 were compared by serological, biochemical, and DNA-DNA hybridization methods. Similarities among strains belonging to biovar 2 were observed by indirect immunofluorescence, whereas biovar 1 strains showed serological heterogeneity. Electrophoretic analysis of bacterial envelope-associated proteins showed that few bands appeared in the strains belonging to biovar 1, whereas many proteins appeared in the case of biovar 2 strains. Chromosomal DNA was analyzed with six random C58 chromosomal fragments. None of the six probes hybridized to the DNA of the two biovar 2 strains. One of the probes gave the same hybridization pattern with all biovar 1 strains, whereas the other probes yielded different patterns. The vir regions were closely related in the different pathogenic strains. The T-DNA and replication regions were less conserved and showed some variations among the strains.     

Four unnamed species of nonsymbiotic rhizobia isolated from the rhizosphere of Lotus corniculatus.

Previously, we found that genetically diverse rhizobia nodulating Lotus corniculatus at a field site devoid of naturalized rhizobia had symbiotic DNA regions identical to those of ICMP3153, the inoculant strain used at the site (J. T. Sullivan, H. N. Patrick, W. L. Lowther, D. B. Scott, and C. W. Ronson, Proc. Natl. Acad. Sci. USA 92:8985-8989, 1995). In this study, we characterized seven nonsymbiotic rhizobial isolates from the rhizosphere of L. corniculatus. These included two from plants at the field site sampled by Sullivan et al. and five from plants at a new field plot adjacent to that site. The isolates did not nodulate Lotus species or hybridize to symbiotic gene probes but did hybridize to genomic DNA probes from Rhizobium loti. Their genetic relationships with symbiotic isolates obtained from the same sites, with inoculant strain ICMP3153, and with R. loti NZP2213T were determined by three methods. Genetic distance estimates based on genomic DNA-DNA hybridization and multilocus enzyme electrophoresis were correlated but were not consistently reflected by 16S rRNA nucleotide sequence divergence. The nonsymbiotic isolates represented four genomic species that were related to R. loti; the diverse symbiotic isolates from the site belonged to one of these species. The inoculant strain ICMP3153 belonged to a fifth genomic species that was more closely related to Rhizobium huakuii. These results support the proposal that nonsymbiotic rhizobia persist in soils in the absence of legumes and acquire symbiotic genes from inoculant strains upon introduction of host legumes.     

Phenotypic and Genotypic Comparison of Symbiotic and Free-Living Cyanobacteria from a Single Field Site

PCR amplification techniques were used to compare cyanobacterial symbionts from a cyanobacterium-bryophyte symbiosis and free-living cyanobacteria from the same field site. Thirty-one symbiotic cyanobacteria were isolated from the hornwort Phaeoceros sp. at several closely spaced locations, and 40 free-living cyanobacteria were isolated from the immediate vicinity of the same plants. One of the symbiotic isolates was a species of Calothrix, a genus not previously known to form bryophyte symbioses, and the remainder were Nostoc spp. Of the free-living strains, two were Calothrix spp., three were Chlorogloeopsis spp. and the rest were Nostoc spp. All of the symbiotic and all but one of the free-living strains were able to reconstitute the symbiosis with axenic cultures of both Phaeoceros and the liverwort Blasia sp. Axenic cyanobacterial strains were compared by DNA amplification using PCR with either short arbitrary primers or primers specific for the regions flanking the 16S-23S rRNA internal transcribed spacer. With one exception, the two techniques produced complementary results and confirmed for the first time that a diversity of symbiotic cyanobacteria infect Phaeoceros in the field. Symbionts from adjacent colonies were different as often as they were the same, showing that the same thallus could be infected with many different cyanobacterial strains. Strains found to be identical by the techniques employed here were often found as symbionts in different thalli at the same locale but were never found free-living. Only one of the free-living strains, and none of the symbiotic strains, was found at more than one sample site, implying a highly localized distribution of strains.     

Genomic instability in Rhizobium phaseoli.

Experience from different laboratories indicates that Rhizobium strains can generate variability in regard to some phenotypic characteristics such as colony morphology or symbiotic properties. On the other hand, several reports suggest that under certain stress conditions or genetic manipulations Rhizobium cells can present genomic rearrangements. In search of frequent genomic rearrangements, we analyzed three Rhizobium strains under laboratory conditions that are not considered to cause stress in bacterial populations. DNAs from direct descendants of a single cell were analyzed in regard to the hybridization patterns obtained, using as probes different recombinant plasmids or cosmids; while most of the probes utilized did not show differences in the hybridization patterns, some of them revealed the occurrence of frequent genomic rearrangements. The implications and possible biological significance of these observations are discussed.     

Three Phylogenetic Groups of nodA and nifH Genes in Sinorhizobium and Mesorhizobium Isolates from Leguminous Trees Growing in Africa and Latin America

The diversity and phylogeny of nodA and nifH genes were studied by using 52 rhizobial isolates from Acacia senegal, Prosopis chilensis, and related leguminous trees growing in Africa and Latin America. All of the strains had similar host ranges and belonged to the genera Sinorhizobium and Mesorhizobium, as previously determined by 16S rRNA gene sequence analysis. The restriction patterns and a sequence analysis of the nodA and nifH genes divided the strains into the following three distinct groups: sinorhizobia from Africa, sinorhizobia from Latin America, and mesorhizobia from both regions. In a phylogenetic tree also containing previously published sequences, the nodA genes of our rhizobia formed a branch of their own, but within the branch no correlation between symbiotic genes and host trees was apparent. Within the large group of African sinorhizobia, similar symbiotic gene types were found in different chromosomal backgrounds, suggesting that transfer of symbiotic genes has occurred across species boundaries. Most strains had plasmids, and the presence of plasmid-borne nifH was demonstrated by hybridization for some examples. The nodA and nifH genes of Sinorhizobium teranga ORS1009^T grouped with the nodA and nifH genes of the other African sinorhizobia, but Sinorhizobium saheli ORS609^T had a totally different nodA sequence, although it was closely related based on the 16S rRNA gene and nifH data. This might be because this S. saheli strain was originally isolated from Sesbania sp., which belongs to a different cross-nodulation group than Acacia and Prosopis spp. The factors that appear to have influenced the evolution of rhizobial symbiotic genes vary in importance at different taxonomic levels.     

Phenotypic and molecular characterization of indigenous rhizobia nodulating chickpea in India

In a combined approach of phenotypic and genotypic characterization, 28 indigenous rhizobial isolates obtained from different chickpea growing regions in peninsular and northern India were analyzed for diversity. The field isolates were compared to two reference strains TAL620 and UPM-Ca142 representing M. ciceri and M. mediterraneum respectively. Phenotypic markers such as resistance to antibiotics, tolerance to salinity, temperature, pH, phosphate solubilization ability, growth rate and also symbiotic efficiency showed considerable diversity among rhizobial isolates. Their phenotypic patterns showed adaptations of rhizobial isolates to abiotic stresses such as heat and salinity. Two salt tolerant strains (1.5% NaCl by T1 and T4) with relatively high symbiotic efficiency and two P-solubilising strains (66.7 and 71 microg/ml by T2 and T5) were identified as potential bioinoculants. Molecular profiling by 16S ribosomal DNA Restriction Fragment Length Polymorphism (RFLP) revealed three clusters at 67% similarity level. Further, the isolates were differentiated at intraspecific level by 16S rRNA gene phylogeny. Results assigned all the chickpea rhizobial field isolates to belong to three different species of Mesorhizobium genus. 46% of the isolates grouped with Mesorhizobium loti and the rest were identified as M. ciceri and M. mediterraneum, the two species which have been formerly described as specific chickpea symbionts. This is the first report on characterization of chickpea nodulating rhizobia covering soils of both northern and peninsular India. The collection of isolates, diverse in terms of species and symbiotic effectiveness holds a vast pool of genetic material which can be effectively used to yield superior inoculant strains.