A. A. Franco

Rhizobium tropici, a Novel Species Nodulating Phaseolus vulgaris L. Beans and Leucaena sp. Trees

A new Rhizobium species that nodulates Phaseolus vulgaris L. and Leucaena spp. is proposed on the basis of the results of multilocus enzyme electrophoresis, DNA-DNA hybridization, an analysis of ribosomal DNA organization, a sequence analysis of 16S rDNA, and an analysis of phenotypic characteristics. This taxon, Rhizobium tropici sp. nov., was previously named Rhizobium leguminosarum biovar phaseoli (type II strains) and was recognized by its host range (which includes Leucaena spp.) and nif gene organization. In contrast to R. leguminosarum biovar phaseoli, R. tropici strains tolerate high temperatures and high levels of acidity in culture and are symbiotically more stable. We identified two subgroups within R. tropici and describe them in this paper.

The contribution of N2-fixing tree legumes to land reclamation and sustainability in the tropics

Abstract Biodiversity, nutrient cycling and energy flux are the bases for sustainability of any system. Among the essential nutrients for plant growth, N is the most expensive and energy-consuming, and potentially is an environmental pollutant. Mixed crops with N2-fixing trees (NFTs) have been thought to maintain biodiversity and sustainability of systems in the tropics. However, most of the empirical knowledge and scientific research done with NFTs have taken for granted that all legume species nodulate and fix N under field conditions without inoculation or without supplementing phosphorus, the most common limiting essential nutrient for legumes in the tropics. In the last few years, surveys on nodulation of legume tree species and selection of highly efficient rhizobial strains for legume trees have received more attention. More than 600 species have been observed for their nodulation status in Brazil and from their nodules approximately 2400 rhizobial strains have been isolated. From 44 legume species with potential uses in the different agrosystems, efficient N2-fixing rhizobia have been selected and are available for inoculant production. New species of rhizobia or bradyrhizobia have been described and large collections of isolates are being developed. Useful legume tree species may contribute around 12 tons of dry litter and 190 kg of N ha−1 y−1 to renovate degraded soil. These studies have prompted use of nodulated and mycorrhizal legume trees to revegetate poor or depleted soils with the goal of restoring their fertility. Experiments with native and introduced legume tree species have been successful in revegetating exposed subsoil, open mining areas and acidic residues from bauxite mining without addition of organic soil. However, supplements of rock phosphate, gypsum, micronutrients and potassium are required.

Characterization of Rhizobia Isolated from Different Divergence Groups of Tropical Leguminosae by Comparative Polyacrylamide Gel Electrophoresis of their Total Proteins

Summary In an attempt to determine the taxonomic position of and the relationships between some 800 strains of bradyrhizobia and rhizobia isolated from nodules of tropical leguminous plants in the Amazon region and Atlantic forests of Brazil, 171 strains (of which 120 were slow or very slow growers) were selected for study by sodium dodecyl sulphate polyacrylamide gel electrophoresis of proteins (SDS-PAGE). The strains were chosen to represent culturally different isolates from different divergence groups of Leguminosae. Appropriate type and reference strains and also seven tropical strains isolated from Phaseolus vulgaris were included for comparison. At a mean correlation coefficient (r) of 0.86, 23 protein electrophoretic clusters were obtained. The majority of the isolates grouped in a large cluster that contained the type strain of Bradyrhizobium japonicum. This cluster could be divided in 5 subclusters that correlated only to some degree with previously determined DNA homology groups. A few of the isolates could be equated with the species Rhizobium fredii (syn. Sinorhizobium fredii), R. galegae and R. loti but many could not be allocated to currently described taxa. No correlation was noted between clusters obtained and the divergence groups of Leguminosae from which the strains were isolated.

New sources of high-temperature tolerant rhizobia for Phaseolus vulgaris L.

Common bean (Phaseolus vulgaris L.) represents an important crop in tropics, but previous screenings of Rhizobium leguminosarum bv. phaseoli did not show strains that could fix N2 in symbiosis with bean at temperatures higher than 35°C (Hungria and Franco, 1993). However, there are other rhizobia and bradyrhizobia species that nodulate some tropical leguminous trees and can fix N2 at high temperatures. In a trial of rhizobial strains isolated from leguminous trees, we found that 14 out of 21 isolates from Gliricidia, Lonchocarpus and Leucaena were also able to nodulate common beans at optimal temperatures (28/23°C, day/night). When we exposed beans inoculated with these strains to high temperature conditions, 40°C/8 h/day, some of them accumulated at flowering time as much or more N as bean plants receiving mineral N. These broad host-range sources of rhizobia capable of fixing nitrogen with bean at high temperature seem to have the potential to improve yields in tropical soils.

Effects of high temperature on nodulation and nitrogen fixation by Phaseolus vulgaris L.

Screening of Rhizobium leguminosarum bv. phaseoli strains showed some that were able to nodulate common beans (Phaseolus vulgaris L.) at high temperatures (35 and 38°C/8 h/day). The nodulation ability was not related to the capability to grow or produce melanin-like pigment in culture media at high temperatures. However, nodules formed at high temperatures were ineffective and plants did not accumulate N in shoots. Two thermal shocks of 40°C/8 h/day at flowering time drastically decreased nitrogenase activity and nodule relative efficiency of plants otherwise grown at 28°C. Recovery of nitrogenase activity began only after seven days, when new nodules formed; total incorporation of N in tops did not recover for 2 weeks. Non-inoculated beans receiving mineral N were not affected by the thermal shock, and when growing continuously at 35 or 38°C had total N accumulated in shoots reduced by only 18%.

Competition amongst rhizobial strains for the colonization and nodulation of two tropical legumes

SummaryPaired rhizobial strains supplied in several proportions were used to study inter-strain competition in association with Macroptilium atropurpureum (DC) URB (siratro) and Stylosanthes guianensis (Aubl.) Swartz (Stylo, line I R I 1022). Plants raised from surface sterilized seed, were grown on agar in large cotton-wool plugged tubes, and populations and inter-strain ratios determined in the inoculum and on the root at several times after inoculation. The nodules were mapped in order of appearance and the strains they contained identified at harvest.Related substrains and strains of similar growth habit competed more with each other in the colonization of the root surface than did a fast-growing strain in association with a typical slow grower. Capacity amongst slow-growing strains to dominate a paired competitor in the colonization of the root was a strain characteristic and was not affected by host. It was unrelated to effectiveness in the rhizobium-host association.In 5 of the 7 cases nodulation success could be related quantitatively to root-surface representation and a ‘competitive index’ calculated; in the remainder one of each pair overwhelmed the other over a wide range of inoculum ratios. It was not possible to relate competitive nodulating success to any single feature of the host: rhizobium symbiosis. In the two most striking cases relationship between competitiveness and N2-fixing effectiveness was reversed; in others competitiveness difference was as great between equally effective as between strains of differing effectiveness. In the case of Stylo there was a marked dominance of an ineffective over an effective competitor, which might be attributed to greater compatibility, as indicated by faster nodulaton by the ineffective strain. This last result argues against use of mixed inocula including any strain ineffective on any of the hosts for which the inoculum is recommended.

The response of field grownPhaseolus vulgaris to Rhizobium inoculation and the quantification of N2 fixation using15N

SummaryA field experiment was performed to assess the effects of Rhizobium inoculation and nitrogen fertilizer (100 kg N ha−1) on four cultivars of Phaseolus beans; Carioca, Negro Argel, Venezuela 350 and Rio Tibagi. In the inoculated treatment 2.5 kg N ha−1 of15N labelled fertilizer was added in order to apply the isotope dilution technique to quantify the contribution of N2 fixation to the nutrition of these cultivars.Nodulation of all cultivars in the uninoculated treatments was poor, but the cultivars Carioca and Negro Argel were well nodulated when inoculated. Even when inoculated, nodulation of the cultivars Venezuela 350 and Rio Tibagi was poor and these cultivars showed little response to inoculation in terms of nitrogen accumulation or grain yield. The estimates of the contribution of N2 fixation estimated using the isotope dilution technique, for the Carioca and Negro Argel cultivars, amounted to 31.7 and 18.4 kg N ha−1 respectively. These two cultivars produced 991 and 883 kg ha−1 of grain, respectively, when inoculated and 663 and 620 kg ha−1 with the addition of 100 kg N ha−1 of N fertilizer. The response to nitrogen was particularly poor due to high leaching losses in the very sandy soil at the experimental site.The Venezuela 350 and Rio Tibagi cultivars only responded to N fertilizer and not to inoculation with Rhizobium which stresses the great importance of selecting plant cultivars for nitrogen fixation in the field.

Nodulation of legume trees from South East Brazil

SummaryAn extended survey of nodulation of legume trees from South-East Brazilian forests was conducted. Six new species from the Caesalpinioideae, 23 from the Mimosoideae and 27 from the Papilionoideae are reported to have nodules. Nitrogenase activity (acetylene reduction) was tested for all nodules and rhizobia were isolated from the most active.

Stem and root nodules on the tropical wetland legume Aeschynomene fluminensis

SUMMARY Aeschynomene fluminensis Veil., originally obtained from flooded areas of the Pantanal Matogrossense region of Brazil, was grown under stem‐flooded or non‐flooded conditions for 70 d after inoculation with isolates of photosynthetic stem nodule rhizobia obtained from native A. fluminensis. Stem nodules formed only on submerged stems of flooded plants (mean of 25 per plant), and did not form on aerial parts, although they were capable of growing and fixing N2 after drainage of the stems. Root nodules formed on both non‐flooded and flooded plants but were usually decreased in number by flooding (from means of 124 to 51 per plant, respectively). Flooding (and stem‐nodulation) resulted in an increase in shoot (and a decrease in root) dry weight, regardless of rhizobial isolate. Stem nodules were attached by a wide collar of aerenchymatous tissue at the base of the nodule. There were large air spaces in the stem where nodules were subtended and these were continuous with nodule aerenchyma/outer cortex. In addition, aerenchyma and spongy tissue at the base of the nodule connected both flooded and non‐flooded root nodules to large intercellular spaces in the root cortex. The stem and root nodules were ovoid in shape, and essentially aeschynomenoid in type, i.e. the central infected tissue was without uninfected, interstitial cells. Root nodules had a similar structure to stem nodules (although stem nodules were generally larger), and flooded root nodules were approximately twice the size of non‐flooded nodules. The infected tissue of root and stem nodules consisted of spherical, bacteroid‐containing cells containing one or two rod‐shaped bacteroids per peribacteroid unit and prominent organelles. Infection threads were observed in root but not in stem nodules. The cortex of stem and root nodules had an apparent oxygen diffusion barrier, consisting of concentric layers of small cells with interlocking cell walls and few intercellular spaces. Cell layers external to these consisted of larger cells and intercellular spaces, with some spaces being occluded with an electron‐dense material that contained a glycoprotein recognized by the monoclonal antibodies MAC236 and MAC265. The amount of glycoprotein occlusions did not appear to differ between nodule types or treatments, although stem nodules contained intracellular glycoprotein vesicles adjacent to cell walls. The exterior of the nodules consisted of an epidermis of thin flattened cells with occasional lenticels. Amyloplasts were common in lower stem and hypocotyl nodules, but fewer in flooded or non‐flooded root nodules. Upper stem nodules (i.e. those within 6 cm of the water surface) differed from more profoundly submerged stem nodules by having chloroplasts throughout the cortex. Root nodules did not contain chloroplasts, and undifferentiated plastids were found mainly in lower stem nodules.

Atributos químicos e microbianos de solos sob diferentes coberturas vegetais no norte do Estado do Rio de Janeiro

The objectives of this study were to evaluate the influence of three soil cover species Acacia auriculiformis, Mimosa caesalpiniifolia and Corymbia citriodora compared to pasture and natural secondary forest on soil chemical and microbial attributes. The field survival rate of the species reached over 70% and the development in height and diameter (DAP) was adequate. The survival rate of Mimosa caesalpiniifolia was the highest among the legume species, while the development of Acacia auriculiformis was the best, mainly between 27 and 44 months. The forest species improved soil fertility adequately in the initial development stages, in spite of the reduction in the levels of organic matter. The potential of pasture to maintain the level of organic matter and improve soil fertility was shown. Microbial C and N were more discriminating, regarding changes in land cover than soil C content. The microbial quotients (qCO2, CBM/C, NBM/N and microbial C/N) detected differences among land covers more clearly than the chemical (C and N content) and microbial (CBM, NBM and RA) attributes.