M. E. Aouani

Genotypic diversity and symbiotic effectiveness of rhizobia isolated from root nodules of Phaseolus vulgaris L. grown in Tunisian soils

Abstract One hundred and sixty isolates of rhizobia were sampled from the root nodules of common bean (Phaseolus vulgaris L.) cultivated in Tunisian soil samples originating from three geographically distinct fields. Plasmid profiling was used as a primary method to rapidly screen the isolates, and then 38 plasmid types were recorded among the 160 isolates. A sample representing the majority of plasmid types was chosen for further characterization by restriction fragment length polymorphism (RFLP) analysis of genomic DNA using chromosomal and symbiotic gene probes, and by their ability to nodulate a potential alternative host, Leucaena leucocephala. One third of the isolates showed a high similarity to Rhizobium gallicum isolated from common bean in France, another third showed the same characteristics as the R. etli-R. leguminosarum group, while the remaining isolates could not be related to any of the five species nodulating bean. When reexamined for nodulation, some of these isolates, showing similarities to R. tropici and Agrobacterium with respect to colony morphology and growth in different media, failed to nodulate their original host. The R. gallicum-like isolates, R. etli-like isolates, and R. leguminosarum-like isolates were recovered from regions where bean is frequently grown, while in fields which had not been cultivated with beans for at least the 10 previous years, solely unrecognized taxa of ineffective isolates were recovered. We detected variations in the symbiotic regions, but certain pSym RFLP patterns for nifH were conserved between Tunisian, French, and Austrian populations of bean rhizobia. Evaluation of symbiotic effectiveness showed that R. gallicum-like isolates and R. etli-like isolates were effective, whereas some R. leguminosarum-like isolates were ineffective. Furthermore, effective isolates were also found among the unrecognized taxa.

Development of real-time PCR assay for detection and quantification of Sinorhizobium meliloti in soil and plant tissue.

Aims:  Sinorhizobium meliloti is a nitrogen‐fixing alpha‐proteobacterium present in soil and symbiotically associated with root nodules of leguminous plants. To date, estimation of bacterial titres in soil is achieved by most‐probable‐number assays based on the number of nodules on the roots of test plants. Here, we report the development of two real‐time PCR (qPCR) assays to detect the presence of S. meliloti in soil and plant tissues by targeting, in a species‐specific fashion, the chromosomal gene rpoE1 and the pSymA gene nodC.

Genetic diversity and salt tolerance of bacterial communities from two Tunisian soils

Microbial ecology studies on arid soils are particularly important for the analysis of biological functions during desertification. Although much is known about the arid saline flora, few researches have directly compared the bacterial communities of saline arid soils with cultivated soils in Northern Africa. Bacterial communities present in two soils from Soliman (north of Tunisia), one salty and neglected, and the other cultivated, were investigated by using both cultivation dependent and independent approaches. The first approach was used to assess the presence of salt tolerant bacteria and the relationships among salt (NaCl) resistance phenotype, soil characteristics and phylogenetic assignment of strains. Total community analysis, performed by T-RFLP on total DNA, was carried out to investigate the relationships between total community fingerprinting with cultivated isolates diversity. The cultivated isolates from salty soil were more genetically diverse, harbouring strains that can grow at high salt concentration. Moreover, the salt resistance of isolates was found not to be related to any particular phylogenetic group, being widespread among isolates belonging to different bacterial subdivisions. Ribotype richness, evaluated as number of different T-RFLP bands (TRFs), was shown to be higher in the agricultural soil than in the salty soil and several agricultural soil-specific TRFs were detected.

Development of a cultivation-independent approach for the study of genetic diversity of Sinorhizobium meliloti populations.

The development of a species‐specific marker for the analysis of the genetic polymorphism of the nitrogen‐fixing symbiotic bacterium Sinorhizobium meliloti directly from environmental DNA is reported. The marker is based on terminal‐restriction fragment length polymorphism (T‐RFLP) methodology targeting specifically the 16S‐23S Ribosomal Intergenic Spacer of S. meliloti. Species‐specificity and polymorphism of the marker were tested on DNA extracted from soil samples and from a collection of 130 S. meliloti bacterial isolates. These primers and the T‐RFLP approach proved useful for the detection and analysis of polymorphism of S. meliloti populations.

Use of Plasmid Profile and RFLP Marker to Evaluate Genetic Diversity of a Local Collection of Common Bean Rhizobia

Rhizobia which are able to form root nodules on Phaseolis vulgaris are highly diverse. They are currently classified in three species: R. leguminosarum bv. phaseoli (Jordan, 1984), R. etli bv. phaseoli (Segovia et al., 1993) and R. tropici (Martinez-Romero et al., 1991). Two other groups have been Characterized in France as new genomic species: Rhizobium sp. H. 152 and R. sp. R602 (Laguerre et al., 1993, Geniaux et al., 1993). All the strains cited so far are of american or european origin, very little information is available about rhizobia nodulating beans in Africa.

Competitiveness Between Imported Strains and Local Isolates of Common Bean Rhizobia

Lack of efficient modulation for common bean in tunisian soils has been observed for a long time. Then, inoculation could be used to solve this problem. However, it is well known that inoculated rhizobia often fail to nodulate in foreign soils, because of the adverse effect of environmental factors and highly competitive indigenous adapted rhizobia (Wolff et al., 1991; Graham, 1992; Thies et al., 1992). Thus, in selection process, it is necessary to predict the tolerance of inoculated strains to chemical factors, and their ability to compete with native rhizobia.