Symbiotic diversity of Pisum sativum L. ecotype humile and the associated Rhizobium leguminosarum strains

Within the species Pisum sativum L. several groups (ecotypes) can be distinguished. In a previous paper (Lie, 1981), the great variability with regard to symbiotic association with Rhizobium, within the pea ecotype fulvum* was described. Although based on only a limited number of pea lines, variation was also observed within the ecotypes P. s. abyssinicum and P. s. elatius (3). In this paper a symbiotic variation within pea lines, belonging to the ecotype P. s. humile, will be reported. In addition a preliminary study was made of the biodiversity of the associated Rhizobium strains, based on the nodX gene and ribosomal genes.

Large-seeded peas were collected in Turkey, Central Anatolia near the airport of Ankara (cv. Ankara), in the Cukurova plain near Adana (cv. Adana) and in the Taurus mountains on the road from Adana to Ankara (cv. Pozanti). An additional line was kindly supplied by Dr. Papastilianou from Cyprus (cv. Cyprus).

A standard group of Rhizobium leguminosarum bv viceae strains PF2, Tom and F13 was used. In addition new R. leguminosarum strains from different regions, Turkey, Syria, Nepal and N.E. China, were isolated. The bacteria were cultivated in a yeast-mannitol broth, and pDNA was isolated using the alkaline lysis method (6). Primers according to (1) were used for PCR amplification of putative nodX homologue sequences, and preliminary detection was based the presence and size of the PCR product, after electrophoresis in a 1.5% agarose gel. Further detection was performed by hybridization using a radio-labeled nodX probe. In a limited number of cases, a PCR-based sequencing technique was performed to determine the sequence of the nodX gene homologues.

To study the biodiversity of rhizobial strains a fingerprint was made of the intergenic space (IGS) between the 16S and 23S ribosomal RNA genes, after PCR amplification followed by RFLP analysis using three restriction enzymes (5).

The large symbiotic variation among the large-seeded humile group of pea lines is shown in Table 1. However, pea cv. Adana from S. Turkey seems to be even more specific, because it only can be nodulated by bacteria coming from its own region, and not by other bacterial strains from Turkey like strain Tom (4). All Rhizobium strains capable of nodulating these humile lines were found to contain gene nodX. An interesting case was observed in Rhizobium strain 310a, presumably originating from Europe. This strain is unable to nodulate pea cv. Afghanistan, and as shown above also not the humile pea lines. However, using PCR amplification a nodX homologue could be detected.

Table 1. Symbiotic specialization between Pisum sativum ecotype humile with R. leguminosarum strains from Europe, Anatolia (C. Turkey), Cukorova plain (S. Turkey) and Israel.

- no nodules, + low number, +++ high number of nodules,
I no, E/I low and E high nitrogen fixation.
*occasionally a few plants with some nodules.

Very slight differences in the sequence of the nodX homologues in the Rhizobium strains studied could be detected. Only one base difference was detected in the sequence of the nodX homologues of Rhizobium strains 310a and F13, when compared with that of the standard strain Tom. In nodX from Rhizobium strain Adana, which is more specialized, three base differences were observed.

To know if all the nodX⁺ bacteria belong to one specific group, or to different groups, a study was also made of the chromosomal background of the Rhizobium strains originating from geographically widely separated regions. Fingerprints of the intergenic spacer of the ribosomal RNA genes revealed considerable divergence among these bacterial strains. Therefore, we conclude that little or no relationship exists among these nodX containing Rhizobium strains coming from widely different regions.