Numerical taxonomy of Sarothamnus scoparius rhizobia.

Twenty nodule isolates from Sarothamnus scoparius (broom) growing in Poland and nine strains from plants growing in Japan were studied for phenotypic properties, plasmid presence, phage sensitivity, and host plant specificity. By numerical analysis of phenotypic properties, it was found that the studied nodule bacteria, originating from geographically different countries, constitute two separate groups affiliated to the bradyrhizobium cluster. The membership of S. scoparius rhizobia in the Bradyrhizobium genus was also supported by their long generation time, alkaline reaction in YEM medium with mannitol, lack of plasmids, and wide host plant range.

The root-nodule symbiosis between Sarothamnus scoparius L. and its microsymbionts.

When nitrogen fixing root nodules are formed, Sarothamnus scoparius (broom) is inoculated with its microsymbionts. Nodules studied under light and electron microscopy exhibited typical indeterminate nodule histology with apical, persistent meristem, age gradient of nodule tissues, and open vascular bundles, and also with some particular features such as: the presence of mitotic activity in the infected meristematic cells, lack of infection threads, distribution of bacteria by process of host cell division, and occurrence of a large bacteroid zone only with infected cells. The results of cross-inoculation tests have shown a broad host range for S. scoparius microsymbionts including not only the native host but also species such as: Lupinus luteus, Ornithopus sativa, Lotus corniculatus, Genista tinctoria, Chamaecitisus ratisbonensis, Macroptilium atropurpureum, and Phaseolus vulgaris. In addition, our data established a close symbiotic relationship of S. scoparius nodulators to Bradyrhizobium sp. (Lupinus) by comparison of the partial sequence of nodC gene of the strain CYT7, specific for the broom, to those from Bradyrhizobium sp. (Lupinus) strain D1 and others available in the public databases.

Symbiosis of Astragalus cicer with its microsymbionts: partial nodC gene sequence, host plant specificity, and root nodule structure.

Astragalus cicer (cicer milkvetch) nodule bacteria were investigated for host plant specificity and partial nodC gene sequences, whilst their native host was studied for the microscopic structure of root nodules. The strains under investigation formed nodules not only on the original host but also on Astragalus glycyphyllos, Astragalus sinicus, Lotus corniculatus, and Phaseolus vulgaris. The nodules induced on the cicer milkvetch were classified as indeterminate and characterized by apical, persistent meristem, a large bacteroid region with infected and uninfected cells, and elongated bacteroids singly located inside peribacteroid membranes. By comparison of the partial nodC gene sequences of a representative strain of astragali rhizobia to those contained in the GenBank database, a close symbiotic relationship of A. cicer microsymbionts to Rhizobium sp. (Oxytropis) was found.

Numerical analysis of Astragalus cicer microsymbionts.

Thirty-seven rhizobium strains, isolated from root nodules of Astragalus cicer (L.) (cicer milkvetch) deriving from different geographic regions, were compared with the representative strains of the known rhizobial species and genera by numerical analysis of phenotypic characteristics. Our results indicated that Astragalus cicer rhizobia were related to the bacteria of Mesorhizobium species and formed two major phena. One phenon, localized on Mesorhizobium loti branch, contained strains from Poland. Another cluster, placed in the vicinity of M. tianshanense, M. mediterraneum, M. ciceri, and M. huakuii, comprised cicer milkvetch nodule isolates from Canada, Ukraine, and one strain from Poland. The relationship of Astragalus cicer microsymbionts to bacteria of the Mesorhizobium species was also supported by phage typing.

Morphology and general characteristics of phages specific for Astragalus cicer rhizobia.

Three newly isolated phages, K1, K2, and C1, specific for A. cicer rhizobia were characterized by their morphology, host range, rate of adsorption, restriction endonuclease patterns, and DNA molecular weights. All three phages were classified to the morphological group B of Bradley's (Siphoviridae family) on the basis of presence of hexagonal in outline heads and long noncontractile tails. Phages K1, K2, and C1 are related by host range and restriction endonuclease patterns. The molecular weights of phage DNAs estimated from restriction enzyme digests were in the range from 64.6 kb to 68.5 kb.

Phylogenetic position of Mesorhizobium huakuii subsp. rengei, a symbiont of Astragalus sinicus cv. Japan.

The phylogenetic position of Rhizobium huakuii bv. renge, a symbiont of Astragalus sinicus cv. Japan (renge-sou), was studied. The following phylogenetic approaches were used: restriction fragment length polymorphism (RFLP)-polymerase chain reaction (PCR) analysis of a full-length 16S rDNA fragment, 16S rDNA analysis of the first 300-bp sequence, bacteriophage typing, and amplification of the genomic region by random primer. All the data suggest that strains of R. huakuii bv. renge should be classified into subspecies of the new genus Mesorhizobium (Jarvis et al., Inter. J. System. Bacteriol., 47, 895-898, 1997) and renamed M. huakuii subsp. rengei. All the strains fell into a tight cluster which included M. loti and M. huakuii. The strains isolated from root nodules on A. sinicus were divided into three groups as follows: group I, M. huakuii subsp. rengei B3, M. huakuii subsp. rengei My6, M. huakuii subsp. rengei My7, M. huakuii subsp. rengei My3, and M. huakuii subsp. rengei OUT30020; group II, M. huakuii subsp. huakuii CCBAU103(T), M. huakuii subsp. huakuii ACCC13005, M. huakuii subsp. huakuii 7653R, and Mesorhizobium sp. N-1; group III, Mesorhizobium sp. OUT30019. All the strains isolated in Japan except strains N-1 and OUT30019 were classified into group I. Strains in group I were sensitive to bacteriophage H1 which was isolated from rice-paddy soil in Japan. Strains in groups II and III except for M. huakuii subsp. huakuii 7653R were resistant to phage H1. Rhizobium sp. ACMP18, a native symbiont of Astragalus cicer that forms nodules on A. sinicus, showed close similarity to M. huakuii subsp. huakuii CCBAU103(T), and should thus be classified as a Mesorhizobium sp. Taken together, the results of the analyses indicate that M. huakuii subsp. rengei forms a subgroup which is distinct from M. huakuii subsp. huakuii strains isolated in China and that strain B3 is the type strain.

Induction of Rhizobium meliloti nodC gene by Alnus incana compounds.

An expression of nodC promoter of R. meliloti 1021, cloned in front of the Escherichia coli lacZ gene, was used to study the presence of R. meliloti nodC gene-inducing compound(s) in extracts and exudates of A. incana seeds and roots. The regulatory gene nodD was expressed at comparable level in bacterial culture of R. meliloti with and without the studied plant extracts and exudates, whereas the nodC-lacZ fusion expression was increased 4 times by seed exudates of grey alder and 2 times by seed extracts and root ingredients in comparison to the nodC-lacZ fusion expression in R. meliloti grown in a medium free of plant compounds. Induction of R. meliloti nodC gene expression by A. incana substances was also supported in plant test. Sterile filtrate of coculture of R. meliloti with seed exudates of A. incana induced root hair deformations and nodule-like structures on Medicago sativa.

Symbiotic properties of adenine requiring mutants of Rhizobium meliloti strain L5-30.

From the effective and prototrophic Rhizobium meliloti strain L5-30 two auxotrophic mutants were isolated: RM4 and RM221. These two mutants required adenine and adenine with thiamine for their growth, respectively. Both mutants nodulated lucerne plants ineffectively. Electron microscopic observations of the nodule tissue showed that its cells were not occupied by bacteria. Prototrophic revertants and transductants of these mutants showed high symbiotic effectiveness. It is assumed that adenine or adenine and thiamine requirements made impossible release of bacteria from the infection thread.

Localization of nodulation gene on the chromosome of Rhizobium meliloti.

Using N-methyl-N'-nitro-N-nitrosoguanidine mutant RM54 of Rhizobium meliloti L5-30 defective in the nodulation process (Nod-) and in the biosynthesis of adenine was obtained. Nod- phenotype of this mutant was not caused by the auxotrophic mutation. The nod gene is located on the chromosome. The wild type strain of R. meliloti and Nod- mutant RM54 harbour two indigenous plasmids having a molecular weight of 90 Mdal and about 300 Mdal.

Chromosomal gene controlling symbiotic nitrogen fixation in Rhizobium meliloti L5-30.

Auxotrophic Rhizobium meliloti strain RM 246 carries two independent mutations: in the biosynthesis of cysteine (cys) and symbiotic nitrogen fixation process (fix). These two mutations were mapped by transduction between his-240 and ade-4 markers. Cotransduction frequencies show the following order of genes: his-240 fix-1 cys-246 ade-4.

Starch contents in alfalfa nodules with effective and ineffective symbiotic responses.

Rhizobium meliloti auxotrophic and ineffective mutants induced nodules containing: 9.35 microg of starch per mg of the plant tissue (wet wt.) in the case of strain his-240 which did not occupy host cells and 2.7 to 3.5 microg of starch in strains: arg-55, cys-243, cys-244 and cys-246 which occupied part of the host cells (bacteroidal zone). Nodules induced by prototrophic and effective strains i.e. L5-30 (wilde type) and prototrophic transductants of arg-55 and his-240 mutants contained 0.13 to 0.06 microg of starch per mg of the plant tissue (wet wt.). These results indicate that iodometric estimation of starch contents in the nodules may by used as a simple test showing the quantity of plant cells infected by rhizobia.

Auxotrophic mutations related to symbiotic properties of Rhizobium meliloti strain L5-30.

Mutants isolated from effective R. meliloti strain L5-30 which required histidine (his-240), arginine+uracil (arg-55) and cysteine (cys-243, cys-244 and cys-246) showed also loss of effectiveness. Mutant requiring isoleucine+valine (ilv-74) was non-infective. Relation of the metabolic deficiency to the symbiotic properties of these mutants was tested comparing symbiotic response of their prototrophic revertants and transductants. It was found that all revertants and transductants of the strain his-240 were effective which suggests that histidine deficiency was the cause of their ineffectiveness. All revertants and transductants of the cysteine mutants were still ineffective. This result indicates two independent mutations which were not cotransductible. Prototrophic revertants of the mutant arg-55 were ineffective whereas 56.9 percent of transductants appeared effective suggesting close linkage of two mutations. i.e. auxotrophic and the other concerned with symbiotic effectiveness. Though one of 69 prototrophic transductants obtained from the non-nodulating mutant ilv-74 remained non-nodulating, it seems that changes in nodulating ability of the mutant are related to the auxotrophic requirements.

Structure of nodules induced by auxotrophic and ineffective mutants of Rhizobium meliloti strain L5-30 requiring cysteine, arginine+uracil and histidine.

Nodules produced by ineffective mutants of R. meliloti strain L5-30 requiring arginine+uracil (arg-55) and cysteine requiring mutants (cys-243, cys-244, cys-246) studied under light microscopy were found to be occupied by bacteria. This indicates on defect in transformation of these mutants into N2 fixing bacteroids. These defects were not associated with auxotrophy. In the nodules induced by histidine requiring mutant (his-240) only few host plant cells were occupied by bacteria. This indicate that his-240 mutant is defective in liberation from the infection thread and its multiplication since supplementation of the plant growth medium with 50 microgram/ml of L-histidine enabled establishment of fully effective association. Prototrophic transductants and revertants were fully effective.