NodC-based evaluation of the occurrence of bacteria in nodules of Pisum sativum isolated on YEM agar

The bacterial nodulation (nod) genes are essential in the formation process of root nodules. This study was aimed to verify the occurrence of nodule-associated bacteria in two pea varieties (“Tarchalska” and “Klif ”) inoculated with Rhizobium inoculants – Nitragine™ and a noncommercial one produced by the Polish Institute of Soil Science and Plant Cultivation (IUNG). The number of colonies isolated on yeast extract mannitol (YEM) agar from the nodules of “Klif ” inoculated with IUNG inoculants was significantly higher than the number of colonies isolated from other variants. Species identification was based on sequencing of 16S rDNA, which revealed that despite careful sterilization of nodules, sequences of other bacterial species were detected. Among them, one sequence belonged to Rhizobium leguminosarum (isolated from IUNG inoculant). To assess the presence of nodulation-capable Rhizobium, amplification of the nodC gene was performed, which revealed that of 29 samples, 19 were positive. The remaining isolates, including reference strain and bacteria isolated from Nitragine™, lacked this gene. The results show that pea nodules harbor a very diverse community of bacteria. The lack of nodC gene in some strains isolated from plants inoculated with Nitragine™ and with IUNG inoculant proves that even if R. leguminosarum are abundant, they may not be efficient in nodulation.