Seed germination,seedling inoculation and establishment ofAlnus spp. in containers in greenhouse trials

Methods for production of containerized seedlings ofAlnus species were developed which permit nitrogen-fixing nodules to form on the root systems prior to outplanting, in order to provide an early nitrogen input during seedling establishment. The methods are based on procedures for inoculating root systems with suspensions ofFrankia (Actinomycetales), applied either directly in the container cell as a soil drench at the time of seeding, or as a root dip for seedlings transplanted into the containers. Germination of dried, stored seed was enhanced by light and by presoaking for 16 h in water. Pretreatments to overcome seed dormancy or to eliminate fungal pathogens did not further enhance germination. Some loss of seedlings was recorded in the early stages of growth shortly after germination, which is a factor in calculating projected seedling yield. Nodulation and seedling growth were evaluated in terms of growth media characteristics. Seedlings performed well in peat-vermiculite, at soil pH between 5.5 and 8.0.     

Identification of 3-deoxy-lyxo-2-heptulosaric acid in the core region of lipopolysaccharides from Rhizobiaceae

A 3-deoxy-2-heptulosaric acid (DHA), very probably with the lyxo-configuration, was identified in the R-core region of lipopolysaccharides from nodulating strains of Rhizobium leguminosarum, Rhizobium meliloti and from all three biovars of the phytopathogenic Agrobacterium tumefaciens. Its structure could be deduced from the fragmentation pattern of the corresponding alditol acetates obtained after reduction of the 2-keto and the 1.7-carboxy groups by sodium borohydride or sodium borodeuteride. DHA in lipopolysaccharide was not destroyed by periodate and is therefore not in a terminal position. Two DHA-containing oligosaccharides, namely glucosyl (1----4)-3-deoxy-2-heptulosaric acid and rhamnosyl-rhamnosyl-(1----5)-3-deoxy-2-heptulosaric acid could be tentatively identified by mass spectrometric methods amongst the products of mild acidic hydrolysis of lipopolysaccharides of Rhizobium leguminosarum strain 24. The two types of non-nodulating mutants of Rhizobium leguminosarum included in this study did not contain 3-deoxy-2-heptulosaric acid.     

Characterization of Bradyrhizobium strains indigenous to Western Australia and South Africa indicates remarkable genetic diversity and reveals putative new species

Bradyrhizobium are N₂-fixing microsymbionts of legumes with relevant applications in agricultural sustainability, and we investigated the phylogenetic relationships of conserved and symbiotic genes of 21 bradyrhizobial strains. The study included strains from Western Australia (WA), isolated from nodules of Glycine spp. the country is one genetic center for the genus and from nodules of other indigenous legumes grown in WA, and strains isolated from forage Glycine sp. grown in South Africa. The 16S rRNA phylogeny divided the strains in two superclades, of B. japonicum and B. elkanii, but with low discrimination among the species. The multilocus sequence analysis (MLSA) with four protein-coding housekeeping genes (dnaK, glnII, gyrB and recA) pointed out seven groups as putative new species, two within the B. japonicum, and five within the B. elkanii superclades. The remaining eleven strains showed higher similarity with six species, B. lupini, B. liaoningense, B. yuanmingense, B. subterraneum, B. brasilense and B. retamae. Phylogenetic analysis of the nodC symbiotic gene clustered 13 strains in three different symbiovars (sv. vignae, sv. genistearum and sv. retamae), while seven others might compose new symbiovars. The genetic profiles of the strains evaluated by BOX-PCR revealed high intra- and interspecific diversity. The results point out the high level of diversity still to be explored within the Bradyrhizobium genus, and further studies might confirm new species and symbiovars.     

A study of nodulation and nitrogen fixation of alder on the purplish soils in China

Summary The alder has a perennial nodule cluster. The nodule amount on the roots increases with tree age. The N₂-fixing activity of nodules decreases with nodule age. Purple coloured soils with various soil pHs and CaCO₃ contents are, in the main, the ones which influence nodulation and N₂-fixing. Higher N₂-fixing capacity existed in the neutral and low calcium soils. High calcium soils and acid soils can restrain nodulation and the N₂-fixing rate significantly. On the slope, where calcarous light loams are found, the annual nitrogen fixation capacity of alder and cypress mixed plantations, less than 10 years old, is 16 or 17 kg/ha yr, but in the valley, a pure alder plantation can reach 40 kg/ha yr.     

Mutational analysis of the Bradyrhizobium japonicum common nod genes and further nod box-linked genomic DNA regions

Summary By insertional and deletional marker replacement mutagenesis the common nod region of Bradyrhizobium japonicum was examined for the presence of additional, essential nodulation genes. An open reading frame located in the 800 bp large intergenic region between nodD1 and nodA did not appear to be essential for nodulation of soybean. Furthermore, a strain with a deletion of the nodI- and nodJ-like genes downstream of nodC had a Nod⁺ phenotype. A mutant with a 1.7 kb deletion immediately downstream of nodD1 considerably delayed the onset of nodulation. This region carried a second copy of nodD (nodD2). A nodD1-nodD2 double mutant had a similar phenotype to the nodD2 mutant. Using a 22-mer oligonucleotide probe partially identical to the nod box sequence, a total of six hybridizing regions were identified in B. japonicum genomic DNA and isolated from a cosmid library. Sequencing of the hybridizing regions revealed that at least three of them represented true nod box sequences whereas the others showed considerable deviations from the consensus sequence. One of the three nod box sequences was the one known to be associated with nodA, whereas the other two were located 60 to 70 kb away from nif cluster I. A deletion of one of these two sequences plus adjacent DNA material mmutant 308) led to a reduced nodulation on Vigna radiata but not on soybean. Thus, this region is probably involved in the determination of host specificity.Dedicated to Prof. Giorgio Semenza on the occasion of his 60th birthday     

TheRhizobium-legume symbiosis Two methods to discriminate between nodules and other root-derived structures

Summary Two methods have been developed in order to discriminate between lateral roots, nodules and root-derived structures which exhibit both root and nodule histological features and which can develop on legumes inoculated with certainRhizobium mutants. The first method, known as the clearing method, allows the observation by light microscopy of cleared undissected root-structures. The second, known as the slicing method, is a complementary technique which provides a greater degree of structural information concerning such structures. The two methods have proved invaluable in defining unequivocally the nature of the interaction between a rhizobial strain and a legume host.     

Codon usage and G+C content in Bradyrhizobium japonicum genes are not uniform

To date, the sequences of 45 Bradyrhizobium japonicum genes are known. This provides sufficient information to determine their codon usage and G+C content. Surprisingly, B. japonicum nodulation and NifA-regulated genes were found to have a less biased codon usage and a lower G+C content than genes not belonging to these two groups. Thus, the coding regions of nodulation genes and NifA-regulated genes could hardly be identified in codon preference plots whereas this was not difficult with other genes. The codon frequency table of the highly biased genes was used in a codon preference plot to analyze the RSRj9 sequence which is an insertion sequence (IS)-like element. The plot helped identify a new open reading frame (ORF355) that escaped previous detection because of two sequencing errors. These were now corrected. The deduced gene product of ORF355 in RSRj9 showed extensive similarity to a putative protein encoded by an ORF in the T-DNA of Agrobacterium rhizogenes. The DNA sequences bordering both ORFs showed inverted repeats and potential target site duplications which supported the assumption that they were IS-like elements.     

Identification of host range determinants in the Rhizobium species MPIK3030

Summary R. meliloti primarily nodulates Medicago sativa but cannot nodulate Macroptilium atropurpureum. By introducing an 11.4 kb region into R. meliloti from the Symplasmid of Rhizobium strain MPIK3030, the host range of the R. meliloti transconjugants were shown to be extended to M. atropurpureum, one of the hosts of MPIK3030 but not normally nodulated by R. meliloti. The region responsible for host range extension was isolated by mass conjugating a clone bank from MPIK3030 into the R. meliloti wild type, and subsequent screening for nodulation on M. atropurpureum. Using deleted derivatives of a plasmid reisolated from endosymbiotic bacteria, the host range region was further narrowed down to three EcoRI fragments. Tn5 mutagenesis allowed the isolation of three discrete regions on an 11.4 kb section, which are involved in the extension of host range to M. atropurpureum. Finally, complementation experiments performed with R. meliloti common nod and hsn mutants indicated that none of the genes involved in the early steps of nodulation, including host-range functions, can be complemented by genes carried on the 11.4 kb fragment derived from MPIK3030.     

Effect of some carbamate pesticides on nodulation,plant yield and nitrogen fixation byPisum sativum andVigna sinensis in the presence of their respective rhizobia

Summary Six carbamate pesticides namely 1-naphthol, sevin, dimetilan, trematan, NaDDC and dymid were studied to see their effect on nodulation and nitrogen fixation inPisum sativum andVigna sinensis. Low concentrations of the pesticides have little effect on nodulation and nitrogen fixation, whereas higher concentrations adversely effect these processes. The results also indicate that then sensitivity depends upon the species of the Rhizobium and also the type of the pesticide. Pesticides belonging to the carbamate group differ in their capacity to affect nodulation and nitroge fixation.     

Numerical taxonomic analysis of cross-inoculation patterns of legumes and Rhizobium

Summary A survey was made of published results of tests of the capacity of Rhizobium derived from one legume genus to nodulate plants from other genera. The data were derived from more than 14,000 separate cross-inoculation trials involving species from 165 genera of legumes. Numerical taxonomic techniques were applied to 113 of the genera for which results of substantial cross-infection tests were available. The data were examined using mean character difference coefficients re-expressed as total and positive-only similarity coefficients. The resulting similarity matrices were clustered by the unweighted pair-group method using arithmetic averages. Eighteen affinity groups were defined at the 70% similarity level. With few exceptions, the physiological and cultural behavior of the rhizobia was consistent within the defined groups. Two broad categories were suggested in the numerical taxonomic analysis, and their validity is discussed in regard to the geographic, physiological and cultural characteristics of the legumes and their Rhizobium microsymbionts. The taxonomic and agronomic value of this approach and the new groupings are discussed.