IDENTIFICATION OF N-2-FIXING PLANT-ASSOCIATED AND FUNGUS-ASSOCIATED AZOARCUS SPECIES BY PCR-BASED GENOMIC FINGERPRINTS

Most species of the diazotrophic Proteobacteria Azoarcus spp. occur in association with grass roots, while A. tolulyticus and A. evansii are soil bacteria not associated with a plant host, To facilitate species identification and strain comparison, we developed a protocol for PCR -generated genomic fingerprints, using an automated sequencer for frag ment analysis. Commonly used primers targeted to REP (repetitive extra genic palindromic) and ERIC (enterobacterial repetitive intergenic con sensus) sequence elements failed to amplify fragments from the two spe cies tested, In contrast, the BOX-PCR assay (targeted to repetitive in tergenic sequence elements of Streptococcus) yielded species-specific genomic fingerprints with some strain-specific differences. PCR profil es of an additional PCR assay using primers targeted to tRNA genes (tD NA-PCR, for tRNA(Ile)) were more discriminative, allowing differentiat ion at species-specific (for two species) or infraspecies-specific lev el. Our protocol of several consecutive PCR assays consisted of 16S ri bosomal DNA (rDNA)-targeted, genus-specific PCR followed by BOX-and tD NA-PCR; it enabled us to assign new diazotrophic isolates originating from fungal resting stages (sclerotia) to known species of Azoarcus. T he assignment was confirmed by phylogenetic analysis of 16S rDNA seque nces. Additionally, the phylogenetic distances and the lack of monophy ly suggested emendment of the genus Azoarcus: the unnamed species Azoa rcus groups C and D and a new group (E) of Azoarcus, which was detecte d in association,vith fungi, are likely to have the taxonomic rank of three different genera. According to its small subunit rRNA, the scler otium-forming basidiomycete,vas related to the Ustilagomycetes, facult atively biotrophic parasites of plants. Since they occurred in a field which was under cultivation with rice and wheat, these fungi might se rve as a niche for survival for Azoarcus in the soil and as a source f or reinfection of plants.