Phaseolus vulgaris is nodulated in northern Spain by Rhizobium leguminosarum strains harboring two nodC alleles present in American Rhizobium etli strains: biogeographical and evolutionary implications.(Report)

Introduction

Phaseolus vulgaris is a legume indigenous to Central and South American countries that was introduced in Europe after the 15th century. This legume establishes symbiosis with different fast-growing rhizobia in American countries, but the predominant endosymbiont is Rhizobium etli, whose American origin has been proposed (Aguilar et al. 1998, 2004; Bernal and Graham 2001; Segovia et al. 1993; Silva et al. 2003). Taxonomically, R. etli originated as a consequence of the reclassification of Rhizobium leguminosarum bv. phaseoli type I (Segovia et al. 1993) and is closely related to Rhizobium phaseoli ATCC 14482T (Ramirez-Bahena et al. 2008). The seeds of P. vulgaris were introduced from America to different continents, namely Europe, through Spain and Portugal, and Africa, through the Canary Islands. These seeds could have delivered rhizobia nodulating P. vulgaris to these continents, since at least R. etli has been reported to be carried by bean grains, on their testa (Perez-Ramirez et al. 1998). The presence of this species has been detected in Africa (Mhamdi et al. 1999; Beyene et al. 2004) and Europe (southern Spain), as it is the most common endosymbiont of Phaseolus in some of these soils (Herrera-Cervera et al. 1999; Rodriguez-Navarre et al. 2000). In southern Spain, 2 nodC alleles ([alpha] and [gamma]), commonly harbored in R. etli strains, have been found (Aguilar et al. 2004). However, in Africa and the Canary Islands, P. vulgaris may be nodulated by Sinorhizobium meliloti strains that carry a different nodC gene (Mnasri et al. 2007; ZurdoPineiro et al. 2009). The dominance of R. etli (73% of strains) in southern Spain and the presence of the 2 nodC alleles found in this species in America supports the hypothesis that this species was introduced in Spain by associating with bean seeds. Nevertheless, the data obtained in southern Spain correspond to soils without a cultivation history of the common bean, as northern Spain is the main producer region with the longest history of cultivation of this legume. Since only 6 strains isolated in a single soil of this region had been previously analysed (Velazquez et al. 2001), and none of them were identified as R. etli, we performed a wider study of strains nodulating common bean in different soils from northern Spain. The phylogenetic relationships of these strains were analysed on the basis of several chromosomal loci (rrs, atpD, and recA genes and 16S-23S intergenic spacer) and a symbiotic locus (nodC), and a comparative study was carried out with respect to strains that have been isolated from other geographical locations and whose genes are available in databases.

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Material and methods

Isolation of rhizobial strains and nodulation tests

A total of 43 rhizobial isolates (Table 1) able to form effective nodules on P. vulgaris were isolated according to Vincent (1970) on medium containing yeast extract--mannitol--agar, from common bean plants growing in 4 different locations in northern Spain, in which different varieties of this legume are commonly cultivated in rotation with maize, potatoes, or sugar beet. The locations Riego de la Vega (42[degrees]23'N, 5[degrees]58'W), Sueros de Cepeda (42[degrees]36'N, 6[degrees]02'W), and Bercianos del Paramo (42[degrees]22'N, 5[degrees]42'W) belong to Leon province, and Barco de Avila to Avila province (40[degrees]21'N, 5[degrees]31'W). The nodulation experiments were carried out on Phaseolus vulgaris 'Rinon', Pisum sativum 'Frisson', Trifolium repens 'Huia', and a local variety of Vicia ervilia, as previously described (Ramirez-Bahena et al. 2009).

Two primer randomly amplified polymorphic DNA (TPRAPD) pattern analysis

DNA extraction and TP-RAPD analysis were performed as reported in Rivas et al. (2002), using the primers pair 879F-1522R at 2 mmol/L final concentration and an AmpliTaq Gold reagent kit (Applied Biosystems Inc., Foster City, Calif., USA). PCR conditions were as follows: preheating at 95[degrees]C for 9 min; 35 cycles of denaturing at 95 [degrees]C for 1 min; annealing at 50[degrees]C for 1 min and extension at 72 [degrees]C for 2 min; and a final extension at 72[degrees]C for 7 min. An 8 [micro]L aliquot of loading solution (40% sucrose and 0.25% bromophenol blue) was added to 25 [micro]L of PCR product. The electrophoresis was carried out on a 1.5% agarose gel in TBE buffer (100 mmol/L Tris, 83 mmol/L boric acid, 1 mmol/L EDTA, pH 8.5) at 6 V/cm. The gels were stained in a solution containing 0.5 [micro]g/mL ethidium bromide and photographed under ultraviolet light.

Sequence analysis of 16S rRNA, recA, atpD, and nodC genes and the 16S-23S intergenic spacer

The amplification and sequencing of 16S rRNA gene was carried out according to Rivas et al. (2007a), internal transcribed spacer (ITS) according to Kwon et al. (2005), recA and atpD according to Gaunt et al. (2001), and nodC as described by Laguerre et al. (2001). PCR amplification and gene sequencing were performed as was recently described by Ramirez-Bahena et al. (2009). The sequences obtained were compared with those from GenBank using the BLASTN program (Altschul et al. 1990) and aligned using the Clustal W …