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A rapid and simple PCR method for identifying isolates of the genus Azospirillum within populations of rhizosphere bacteria

Shime–Hattori, A., Kobayashi, S., Ikeda, S., Asano, R., Shime, H., Shinano, T.
Journal of applied microbiology 2011 v.111 no.4 pp. 915-924
Azospirillum, DNA primers, Rhodocista centenaria, cell suspension culture, genes, nucleotide sequences, plant growth-promoting rhizobacteria, polymerase chain reaction, rhizosphere, rhizosphere bacteria, ribosomal RNA, roots, soil
Aims: To develop a rapid and simple genus‐specific polymerase chain reaction (PCR) method for detecting and identifying isolates of the genus Azospirillum which is well‐recognized as plant growth‐promoting rhizobacterium. Methods and Results: Nine pairs of PCR primers were designed based on the Azospirillum 16S rRNA, ipdC, nifA and nifH genes to assess their genus specificity by testing against 12 Azospirillum (from seven species) and 15 non‐Azospirillum reference strains, as compared with the fAZO/rAZO pair reported by Baudoin et al. (J Appl Microbiol, 108, 2010, 25). Among the primer pairs assessed, the Az16S–A pair designed on the 16S rRNA gene sequence showed the highest genus specificity: it successfully yielded a single amplicon of the expected size in all the 12 Azospirillum strains and for a close relative, Rhodocista centenaria. The PCR with the Az16S–A primers generated a detectable amount of the amplicon from ≥103 CFU ml−1 of Azospirillum cell suspensions even in the presence of contaminants and accurately discriminated Azospirillum and non‐Azospirillum species in both 35 Azospirillum‐like and 70 unknown isolates from plant roots and rhizosphere soils. Conclusions: We developed a rapid and simple PCR method for detecting and identifying Azospirillum isolates within populations of rhizosphere bacteria. Significance and Impact of the Study: The method developed would serve as a useful tool for isolating a variety of indigenous Azospirillum bacteria from agricultural samples.