Marc Neyra

Characterization of tropical tree rhizobia and description of Mesorhizobium plurifarium sp. nov.

A collection of strains isolated from root nodules of Acacia species in Senegal was analysed previously by electrophoresis of total cell protein, auxanographic tests, rRNA-DNA hydridization, 16S rRNA gene sequencing, DNA base composition and DNA-DNA hybridization [de Lajudie, P., Willems, A., Pot, B. & 7 other authors (1994). Int J Syst Bacteriol 44, 715-733]. Strains from Acacia were shown to belong to two groups, Sinorhizobium terangae, and a so-called gel electrophoretic cluster U, which also included some reference strains from Brazil. Further taxonomic characterization of this group using the same techniques plus repetitive extragenic palindromic-PCR and nodulation tests is presented in this paper. Reference strains from Sudan and a number of new rhizobia isolated from nodules of Acacia senegal, Acacia tortilis subsp. raddiana and Prosopis juliflora in Senegal were included. As a result of this polyphasic approach, the creation of a new species, Mesorhizobium plurifarium, is proposed for a genotypically and phenotypically distinct group corresponding to the former cluster U and containing strains isolated from Acacia, Leucaena, Prosopis and Chamaecrista in West Africa (Senegal), East Africa (Sudan) and South America (Brazil). The type strain of Mesorhizobium plurifarium ORS 1032 has been deposited in the LMG collection as LMG 11892.

Polyphasic characterization of rhizobia that nodulate Phaseolus vulgaris in West Africa (Senegal and Gambia).

Fifty-eight new isolates were obtained from root nodules of common bean (Phaseolus vulgaris) cultivated in soils originating from different agroecological areas in Senegal and Gambia (West Africa). A polyphasic approach including both phenotypic and genotypic techniques was used to study the diversity of the 58 Rhizobium isolates and to determine their taxonomic relationships with reference strains. All the techniques performed, analysis of multilocus enzyme electrophoretic patterns, SDS-PAGE profiles of total cell proteins, PCR-RFLP analysis of the genes encoding 16S rRNA and of the 16S-23S RNA intergenic spacer region (ITS-PCR-RFLP), auxanographic tests using API galleries and nodulation tests lead to the consensus conclusion that the new rhizobial isolates formed two main distinct groups, I and II, belonging to Rhizobium tropici type B and Rhizobium etli, respectively. By MLEE R. etli and group II strains showed several related electrophoretic types, evidencing some extent of internal heterogeneity among them. This heterogeneity was confirmed by other techniques (ITS-PCR-RFLP, SDS-PAGE and host-plant-specificity) with the same nine distinct strains of group II showing some differences from the core of group II (54 strains).

Symbiotic and Taxonomic Diversity of Rhizobia Isolated from Acacia tortilis subsp. raddiana in Africa

A collection of rhizobia isolated from Acacia tortilis subsp. raddiana from various sites in the North and South of Sahara was analyzed for their diversity at both taxonomic and symbiotic levels. On the basis of whole cell protein (SDS-PAGE) and 16S rDNA sequence analysis, most of the strains were found to belong to the Sinorhizobium and Mesorhizobium genera where they may represent several different genospecies. Despite their chromosomal diversity, most A. tortilis Mesorhizobium and Sinorhizobium symbionts exhibited very similar symbiotic characters. Nodulation tests showed that the strains belong to the Acacia-Leucaena-Prosopis nodulation group, although mainly forming non-fixing nodules on species other than A. tortilis. Most of the strains tested responded similarly to flavonoid nod gene inducers, as estimated by using heterologous nodA-lacZ fusions. Thin layer chromatography analysis of the Nod factors synthesized by overproducing strains showed that most of the strains exhibited similar profiles. The structures of Nod factors produced by four different Sinorhizobium sp. strains were determined and found to be similar to other Acacia-Prosopis-Leucaena nodulating rhizobia of the Sinorhizobium-Mesorhizobium-Rhizobium branch. They are chitopentamers, N-methylated and N-acylated by common fatty acids at the terminal non reducing sugar. The molecules can also be 6-O sulfated at the reducing end and carbamoylated at the non reducing end. The phylogenetic analysis of available NodA sequences, including new sequences from A. tortilis strains, confirmed the clustering of the NodA sequences of members of the Acacia-Prosopis-Leucaena nodulation group.

Diversity of indigeneous bradyrhizobia associated with three cowpea cultivars (Vigna unguiculata (L.) Walp.) grown under limited and favorable water conditions in Senegal (West Africa)

The diversity of Bradyrhizobium strains nodulating three cowpea (Vigna unguiculata L. Walp.) cultivars in favorable and water-limited conditions occuring at flowering was analysed. PCR- Restriction Fragment Length Polymorphism (PCR-RFLP) analysis of 16S-23S rDNA intergenic spacer region (IGS) directly applied on 85 crushed nodules distinguished four genetic profiles, IGS types I, II, III and IV. The distribution of these IGS types according to water conditions and cowpea cultivars (B-21, TN 88-63 and Mouride) showed that nodulating strains appeared more diverse in water-limited condition. More than three quarters of prospected nodules presented the IGS type I. They were formed on all three cultivars and in both water conditions. Only a small part of nodules was distributed between the IGS type II, III and IV. Nodules showing the IGS types II and III were found mainly in limited conditions on TN 88-63 and Mouride cultivars, whereas nodules presenting the IGS type IV were collected only from cultivars B-21 and Mouride, in both water conditions. Strains corresponding to the different profiles were isolated. The phylogenetic analysis of 16S rRNA gene sequences showed that they belong to the genus, Bradyrhizobium. The sequence analysis of 16S-23S rDNA IGS revealed that the strains exhibiting IGS types II, III and IV were closely related to some Faidherbia albida isolates from Senegal. IGS type II can be assigned with at least 98% similarity to Bradyrhizobium genospecies IV. IGS types III and IV showed more than 96% similarity with genospecies VII and could belong to the same genospecies. IGS type I, the most frequent, exhibits low IGS similarity with reported sequences in the databases, and could represent a new genospecies. (African Journal of Biotechnology: 2003 2(1): 13-23)

Genetic Diversity of Acacia seyal Del. Rhizobial Populations Indigenous to Senegalese Soils in Relation to Salinity and pH of the Sampling Sites

The occurrence and the distribution of rhizobial populations naturally associated to Acacia seyal Del. were characterized in 42 soils from Senegal. The diversity of rhizobial genotypes, as characterized by polymerase chain reaction restriction fragment length polymorphism (RFLP) analysis of 16S–23S rDNA, performed on DNA extracted from 138 nodules resulted in 15 clusters. Results indicated the widespread occurrence of compatible rhizobia associated to A. seyal in various ecogeographic areas. However, the clustering of rhizobial populations based on intergenic spacer (IGS) RFLP profiles did not reflect their geographic origin. Four genera were discriminated on the basis of 16S rRNA gene sequences of the strains representative for the IGS-RFLP profiles. The majority of rhizobia associated to A. seyal were affiliated to Mesorhizobium and Sinorhizobium 64 and 29%, respectively, of the different IGS-RFLP profiles. Our results demonstrate the coexistence inside the nodule of plant-pathogenic non-N2-fixing Agrobacterium and Burkholderia strains, which induced the formation of ineffective nodules, with symbiotic rhizobia. Nodulation was recorded in saline soils and/or at low pH values or in alkaline soils, suggesting adaptability of natural rhizobial populations to major ecological environmental stress and their ability to establish symbiotic associations within these soil environments. These results contribute to the progressing research efforts to uncover the biodiversity of rhizobia and to improve nitrogen fixation in agroforestry systems in sub-Saharan Africa.

Phenotypic characteristics of root-nodulating bacteria isolated from Acacia spp. grown in Libya.

Thirty isolates of root-nodulating bacteria obtained from Acacia cyanophylla, A. karroo, A. cyclops, A. tortilis (subsp.raddiana), Faidherbia albida and Acacia sp., grown in different regions of Libya, were studied by performing numerical analysis of 104 characteristics. Three fast- and one slow-growing reference strains from herbaceous and woody legumes were included. Five distinct clusters were formed. The fast-growing reference strains were separated from the isolates whereas the slow-growing was included in cluster 4. With the exception of one cluster, the majority of clusters were formed regardless of the host plant or site of origin. Based on plant tests, generation times, acid production and carbon utilization the isolates were diverse (fast and slow-growing isolates). Like slow-growing isolates, most of the fast-growing isolates appeared to be non-specific, nodulated many species from the same genus notably F. albida, known to nodulate only with slow-growing strains. Most clusters grew at temperatures 35 °C and 37 °C; some grew at temperatures above 40 °C. The majority of isolates grew at acid and alkaline pH and only one isolate grew below pH 4. Most isolates were able to utilize many amino acids as nitrogen sources and to reduce nitrate. Urea was hydrolysed by all clusters. Monosaccharides and polyols were used by slow and fast-growing isolates as the only carbon sources whereas assimilation of disaccharides varied: Some isolates, like slow-growing isolates, failed to utilize these carbon sources. Most isolates were unable to utilize polysaccharides. Regarding tolerance to NaCl on agar medium, the majority of isolates were unable to grow at a concentration of 2% NaCl, but some were highly resistant and there was one isolate which grew at 8% NaCl. Most isolates were resistant to heavy metals and to antibiotics.

Distribution of roots, arbuscular mycorrhizal colonisation and spores around fast-growing tree species in Senegal

Abstract Roots and soil were sampled from around four leguminous tree species in 10-year-old plots at Bandia, Senegal. Assessments of root concentration (cm per 100 cm3 soil) and mycorrhizal colonisation (% of root length) were made and related to the abundance of spores in the soil and the above ground growth (stem diameter at 30 cm height) of the trees. Root concentrations in Acacia nilotica and Acacia tortilis plots were greater than those found in Prosopis juliflora and Acacia aneura plots at all three depths examined (0–10, 10–25, 25–50 cm). Root concentration decreased with soil depth in all plots and was greatest nearest the tree in the Acacia nilotica and Acacia tortilis plots. Mycorrhizal colonisation was highest on Prosopis juliflora roots and lowest on Acacia tortilis roots. Colonisation was not affected by distance from the tree and decreased with depth only in the Acacia aneura plot. Numbers of spores recovered from soils were generally low (27 per 100 g dry wt. soil) and were concentrated in the upper 10 cm of soil. Spores were most numerous in the Acacia aneura plot and least numerous in the Prosopis juliflora plot. Positive relationships were found between spore numbers and root concentration in the Prosopis juliflora plot and between spore numbers and mycorrhizal colonisation in the Acacia aneura plot. Of the four tree species examined, root and mycorrhizal distributions of Prosopis juliflora and Acacia nilotica showed most promise for use in agroforestry systems.

Effect of rhizobial inoculation methods and host plant provenances on nodulation and growth of Acacia senegal and Acacia nilotica

AbstractThe purpose of this work was to determine the most efficient methods of inoculation to significantly improve nodulation and growth of Acacia senegal and Acacia nilotica, grown under greenhouse conditions. Our results showed that inoculation using dissolved alginate beads containing rhizobia significantly improved the growth of both acacia species better than the growth of plants in other treatments. The experiment with A. nilotica was conducted in two unsterilised soils from different areas. Plants grown in soil from Bel Air were well-nodulated and showed better growth than plants grown in soil from Sangalkam. However, no difference between these soils was shown between the several methods of inoculation and their effect on the nodulation and growth of plants. An interaction between A. senegal and A. nilotica provenances and the effect of inoculation with rhizobia was also demonstrated. Bel Air provenance of A. senegal, Dahra and RIM provenances of A. nilotica grew best of several provenances tested. These results suggest that (1) it may be possible to improve growth and yield of A. senegal and A. nilotica by careful selection of each symbiotic partner; and (2) nursery-grown seedlings of A. senegal and A. nilotica should be inoculated, just after sowing, with dissolved alginate beads containing a mixture of selected rhizobia. Résumé. L’object de notre travail était de déterminer la méthodologie d’inoculation la plus efficace pour améliorer significativement la nodulation et la croissance d’Acacia senegal et d’Acacia nilotica poussant dans des conditions de serre. Nos résultats ont montré que l’inoculation utilisant des billes d’alginate dissoutes améliorait significativement la croissance des deux espèces d’Acacia par rapport à celle des plantes des autres traitements. L’expérience avec A. nilotica a été faite dans deux types de sol non stériles. Nous avons observé que les plants poussant dans le sol de Bel Air étaient plus nodulés et mieux développés que ceux poussant dans le sol de Sangalkam. Cependant, aucune différence significative entre les deux sols n’a été mise en évidence en termes d’effet des différentes méthodologies d’inoculation sur la nodulation et la croissance des plantes. Il a aussi été démontré l’existence d’une interaction entre les provenances d’A. senegal et d’A. nilotica et l’effet de l’inoculation avec rhizobium. C’est ainsi que les provenances Bel Air d’A. senegal, Dahra et RIM d’A. nilotica ont le mieux poussé parmi les différentes provenances testées. L’ensemble de ces résultats suggèrent: (1) il devrait être possible d’améliorer la croissance et la production d’A. senegal et d’A. nilotica par une sélection des deux partenaires symbiotiques; (2) Les jeunes plantules d’A. senegal et d’A. nilotica produites en pépinière devraient être inoculées juste après le semis avec des billes d’alginate dissoutes contenant un cocktail de souches sélectionnées.

Nitrogen in interstitial waters in the Sahel : natural baseline, pollutant or resource?

Nitrate in the unsaturated zone between the soil surface and the water table was studied in agroforestry Parklands in north western Senegal by examination of samples obtained by hand auger. Depending on location, water tables existed at depths between 10 and 35m below ground. Previous studies of groundwater in this region had found that large concentrations of nitrate were unconnected with anthropogenic activity. The objective of this study was to determine whether nitrogen fixing vegetation had a role in groundwater nitrate accumulation and whether roots of trees were located deeply enough to access the nitrate. Accordingly, sample profiles were augered close to stems of nitrogen fixing trees, non-nitrogen fixing trees and also in adjacent areas that were unaffected by tree presence. These adjacent areas were typically open pasture or cultivated fields. Tree fine roots were quantified in the samples and examined for the presence of mycorrhizas. Similarly, sand/soil samples were examined and tested for the presence of nitrogen fixing rhizobia that were capable of forming functional nodules on appropriate host plants. Concentrations of nitrate were greatest in soils beneath nitrogen fixing trees and nitrate was more plentiful in profiles augered beneath nitrogen fixing crops than it was elsewhere suggesting that N-fixation was the source of the nitrate. The concentrrations of nitrate that were found in the unsaturated zone were greatly in excess of the WHO recommended limit for nitrate in drinking water. High NO3-N/Cl ratios confirm insitu production of nitrate, and indicate that this is a natural baseline occurrence related to N-fixation. The nitrate is moving down the profile and impacts the groundwater unless it can be intercepted by plant roots. NO3-N amounts in solution in the soil profile varied between 75 and 1000kg ha−1 beneath trees and between 120 and 400kg ha−1 in areas outwith tree crowns. Although these quantities of N occupy the lower end of the range of N values obtained in north American deserts, they comprise a considerable dryland resource where amounts of organic fertilizer are limited and where cost prohibits the use of commercial fertilizers. Roots of both nitrogen fixing and non-nitrogen fixing trees were deep enough to access the nitrate but the small amounts of available water at intermediate depths suggest that large scale uptake of nitrate will only be possible in the wetter zones located close to the water table. Shallow roots tended to be more heavily colonized by mycorrhizas than deeper roots but mycorrhizas were recovered from roots located 22m below ground. Tree roots and rhizobia had similar patterns of distribution. They were commonest close to the soil surface, less frequent at intermediate depths and tended to increase in frequency close to the water table.

Ectomycorrhizal symbiosis enhanced the efficiency of inoculation with two Bradyrhizobium strains and Acacia holosericea growth

A bstractTwo strains of Bradyrhizobium sp., Aust 13C and Aust 11C, were dually or singly inoculated with an ectomycorrhizal fungus, Pisolithus albus to assess the interactions between ectomycorrhizal symbiosis and the nodulation process in glasshouse conditions. Sequencing of strains Aust 13C and Aust 11C confirmed their previous placement in the genus Bradyrhizobium. After 4 months’ culture, the ectomycorrhizal symbiosis promoted plant growth and the nodulation process of both Bradyrhizobium strains, singly or dually inoculated. PCR/RFLP analysis of the nodules randomly collected in each treatment with Aust 13C and/or Aust 11C: (1) showed that all the nodules exhibited the same patterns as those of the Bradyrhizobium strains, and (2) did not detect contaminant rhizobia. When both Bradyrhizobium isolates were inoculated together, but without P. albus IR100, Aust 11C was recorded in 13% of the treated nodules compared to 87% for Aust 13C, whereas Aust 11C and Aust 13C were represented in 20 and 80% of the treated nodules, respectively, in the ectomycorrhizal treatment. Therefore Aust 13C had a high competitive ability and a great persistence in soil. The presence of the fungus did not significantly influence the frequencies of each Bradyrhizobium sp. root nodules. Although the mechanisms remain unknown, these results showed that the ectomycorrhizal and biological nitrogen-fixing symbioses were very dependent on each other. From a practical point of view, the role of ectomycorrhizal symbiosis is of great importance to N2 fixation and, consequently, these kinds of symbiosis must be associated in any controlled inoculation.