Preferential occurrence of diazotrophic endophytes, Azoarcus spp., in wildrice species and land races of Oryza sativa in comparison with modern races

Several diazotrophic species of Azoarcus spp. occur as endophytes in the pi oneer plant Kallar grass. The purpose of this study was to screen Asian wil d rice and cultivated Oryza sativa varieties for natural association with t hese endophytes. Populations of culturable diazotrophs in surface-sterilize d roots were characterized by 16S rDNA sequence analysis, and Azoarcus spec ies were identified by genomic fingerprints. A. indigens and Azoarcus sp. g roup C were detected only rarely, whereas Azoarcus sp. group D occurred fre quently in samples of flooded plants: in 75% of wild rice, 80% of land race s of O. sativa from Nepal and 33% of modern cultivars from Nepal and Italy. The putatively endophytic populations of diazotrophs differed with the ric e genotype, The diversity of cultured diazotrophs was significantly lower i n wild rice species than in modern cultivars. In Oryza officinalis (from Ne pal) and O, minuta (from the Philippines), Azoarcus sp, group D were the pr edominant diazotrophic putative endophytes in roots. In contrast, their num ber was significantly lower in modern cultivars of O. sativa, whereas numbe rs and diversity of other diazotrophs, such as Azospirillum spp., Klebsiell a sp., Sphingomonas paucimobilis, Burkholderia sp. and Azorhizobium caulino dans were increased. In land races of O, sativa, the diazotrophic diversity was equally high; however, Azoarcus sp, was found in high apparent numbers . Similar differences in populations were also observed in a culture-indepe ndent approach comparing a wild rice (O. officinalis) and a modern-type O. sativa plant: in clone libraries of root-associated nitrogenase (nifH) gene fragments, the diazotrophic diversity was lower in the wild rice species. New lineages of nifH genes were detected, e,g, one deeply branching cluster within the anf (iron) nitrogenases, Our studies demonstrate that the natur al host range of Azoarcus spp, extends to rice, wild rice species and old v arieties being preferred over modern cultivars.