Agronomy for Sustainable Development, An International Journal in Agriculture and Environment

The species Phaseolus vulgaris is a promiscuous legume nodulated by several species of the family Rhizobiaceae. During a study of rhizobia nodulating this legume in Portugal, we isolated several strains that nodulate P. vulgaris effectively and also Macroptilium atropurpureum and Leucaena leucocephala, but they form ineffective nodules in Medicago sativa. According to phylogenetic analysis of the 16S rRNA gene sequence, the strains from this study belong to the genus Rhizobium, with Rhizobium rhizogenes and Rhizobium tropici as the closest related species, with 99.9 and 99.2 % similarity, respectively, between the type strains of these species and strain P1-7^T. The nodD and nifH genes carried by strain P1-7^T are phylogenetically related to those of other species nodulating Phaseolus. This strain does not carry virulence genes present in the type strain of R. rhizogenes, ATCC 11325^T. Analysis of the recA and atpD genes confirms this phylogenetic arrangement, showing low similarity with respect to those of R. rhizogenes ATCC 11325^T (91.9 and 94.1 % similarity, respectively) and R. tropici IIB CIAT 899^T (90.6 % and 91.8 % similarity, respectively). The intergenic spacer (ITS) of the strains from this study is phylogenetically divergent from those of R. rhizogenes ATCC 11235^T and R. tropici CIAT 899^T, with 85.9 and 82.8 % similarity, respectively, with respect to strain P1-7^T. The tRNA profile and two-primer random amplified polymorphic DNA pattern of strain P1-7^T are also different from those of R. rhizogenes ATCC 11235^T and R. tropici CIAT 899^T. The strains isolated in this study can be also differentiated from R. rhizogenes and R. tropici by several phenotypic characteristics. The results of DNA–DNA hybridization showed means of 28 and 25 % similarity between strain P1-7^T and R. rhizogenes ATCC 11235^T and R. tropici CIAT 899^T, respectively. All these data showed that the strains isolated in this study belong to a novel species of the genus Rhizobium, for which we propose the name Rhizobium lusitanum sp. nov.; the type strain is P1-7^T (=LMG 22705^T=CECT 7016^T).

Morphological variation in seed characters includes differences in seed size and shape. Seed shape is an important trait in plant identification and classification. In addition it has agronomic importance because it reflects genetic, physiological, and ecological components and affects yield, quality, and market price. The use of digital technologies, together with development of quantification and modeling methods, allows a better description of seed shape. Image processing systems are used in the automatic determination of seed size and shape, becoming a basic tool in the study of diversity. Seed shape is determined by a variety of indexes (circularity, roundness, andJindex). The comparison of the seed images to a geometrical figure (circle, cardioid, ellipse, ellipsoid, etc.) provides a precise quantification of shape. The methods of shape quantification based on these models are useful for an accurate description allowing to compare between genotypes or along developmental phases as well as to establish the level of variation in different sets of seeds.

Several bacterial strains were isolated from root nodules of Phaseolus vulgaris plants grown in a soil from Portugal. The strains were Gram-negative, aerobic, curved rod-shaped and motile. The isolates were catalase- and oxidase-positive. The TP-RAPD (two-primer randomly amplified polymorphic DNA) patterns of all strains were identical, suggesting that they belong to the same species. The complete 16S rDNA sequence of a representative strain was obtained and phylogenetic analysis based on the neighbour-joining method indicated that this bacterium belongs to the β-Proteobacteria and that the closest related genus is Herbaspirillum. The DNA G+C content ranged from 57·9 to 61·9 mol%. Growth was observed with many different carbohydrates and organic acids including caprate, malate, citrate and phenylacetate. No growth was observed with maltose, meso-inositol, meso-erythritol or adipate as sole carbon source. According to the phenotypic and genotypic data obtained in this work, the bacterium represents a novel species of the genus Herbaspirillum, and the name Herbaspirillum lusitanum sp. nov. is proposed. The type strain is P6-12^T (=LMG 21710^T=CECT 5661^T).

Rhizobium species strain NGR234 nodulates at least 35 diverse genera of legumes as well as the nonlegume Parasponia andersonii. Most nodulation genes are located on the 500-kilobase pair symbiotic plasmid, pNGR234a. Previously, three plasmid-borne host range determinants (HsnI, HsnII, and HsnIII) were identified by their ability to extend the nodulation capacity of heterologous rhizobia to include Vigna unguiculata. In this study, we show that HsnII contains two new nod-box linked hsn genes, nodS and nodU.nodS controls nodulation of the tropical tree Leucaena leucocephala, while the nodSU genes regulate nodulation of the pasture legume Desmodium intortum and the grain legume V. unguiculata. Regulation of the nod-box upstream of nodSU by the flavonoid naringenin was shown using a fusion with a promoterless lacZ gene. Determination of the nucleotide sequence of the nodS gene did not reveal homology with any gene in the EMBL library, although Bradyrhizobium japonicum USDA110 contains both nodS and nodU (M. Göttfert, S. Hitz, and H. Hennecke, Molecular Plant-Microbe Interactions 3:308-316, 1990). We suggest that broad host range in NGR234 is controlled in part by a nodD gene which interacts with a wide range of flavonoids, and in part by host-specific nod genes such as nodS.