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Twenty-two isolates of Anabaena azol1ae derived from seven Azolla species from various geographic and ecological sources were characterized by DNA-DNA hybridization. Cloned DNA fragments derived from the genomic sequences of three different A. azollae isolates were used to detect restriction fragment length polymorphism among all symbiotic anabaenas. DNA clones were radiolabeled and hybridized against southern blot transfers of genomic DNAs of different isolates of A. azollae digested with restriction endonucleases. Eight DNA probes were selected to identify the Anabaena strains tested. Two were strain specific and hybridized only to A. azollae strains isolated from Azolla microphylla or Azolla caroliniana. One DNA probe was section specific (hybridized only to anabaenas isolated from Azolla ferns representing the section Euazolla), and five other probes gave finer discrimination among anabaenas representing various ecotypes of AzoUa species. These cloned genomic DNA probes identified 11 different genotypes of A. azoUae isolates. These included three endosymbiotic genotypes within Azolla filiculoides species and two genotypes within both A. caroliniana and Azolla pinnata endosymbionts. Although we were not able to discriminate among anabaenas extracted from different ecotypes of Azolla nilotica, Azolla mexicina, Azolla rubra and Azolla microphyUa species, each of the endosymbionts was easily identified as a unique genotype. When total DNA isolated from free-living Anabaena sp. strain PCC7120 was screened, none of the genomic DNA probes gave detectable positive hybridization. Total DNA of Nostoc cycas PCC7422 hybridized with six of eight genomic DNA fragments. These data imply that the dominant symbiotic organism in association with Azolla spp. is more closely related to Nostoc spp. than to free-living Anabaena spp. The aquatic fern Azolla sp. Lam. (27) and its cyanobacterial symbiont Anabaena azollae Strasburger (26) make substantial contributions to fixed nitrogen supplies for rice crops. The agronomic importance of Azolla sp. is related to its ability to grow very successfully in habitats where little or no combined nitrogen is available. In the symbiotic association, the Azolla fern provides nutrients, including fixed carbon, and a protective cavity in each leaf for Anabaena colonies and in exchange receives fixed atmospheric nitrogen and possibly growth-promoting substances (13, 17, 19, 20). Most present taxonomic treatments recognize seven distinct Azolla species on the basis of reproductive and morphological features (14). They are grouped into two sections. Section Euazolla includes Azolla caroliniana Willdenow, A. filiculoides Lamarck, A. mexicana Presl, A. microphylla Kalfuss, and A. rubra Brown, and section Rhizosperma includes A. pinnata Brown and A. nilotica De Caisne. A. azollae Strasburger is the only species mentioned in symbiotic association with Azolla sp. (14, 26). Taxonomists place A. azollae among the cyanobacteria, order Nostocales, family Nostocaceae (24). The evidence that there are, in fac...

The method of horizontal agarose gel electrophoresis was used to demonstrate the presence of indigenous plasmid DNAs in different isolates of the symbiotic cyanobacterium Anabaena azollae. All isolates extracted from seven distinct species of the host fern Azolla were found to possess one to three cryptic plasmids ranging in sizes from 35 to 100 MDa (million daltons). Anabaenas isolated from Azolla caroliniana, Az. nilotica, and Az. pinnata species contained a single plasmid band of molecular mass approximately 60 MDa, whereas other endosymbiotic cyanobacteria extracted from Azolla filiculoides, Az. rubra, Az. mexicana, and Az. microphylla were shown to possess two or three covalently closed circular (CCC) DNAs. Cloned DNA fragments derived from the plasmid sequences of two different An. azollae isolates were used as hybridization probes. Hybridization data indicated that these symbiotic cyanobacteria possess different but related plasmid species and that it is possible to construct specific plasmid DNA probes capable of distinguishing among several strains of the symbiotic anabaenas. Several heterologous DNA probes, including Rhizobium symbiotic genes, were used to seek homologous sequences on the An. azollae plasmids. DNA sequences homologous to the nod box and nodMN genes were present on the Anabaena plasmids. Moreover, homology of a key Rhizobium exopolysaccharide (exoY) gene to the An. azollae CCC DNAs was detected. In addition, the introduction of the An. azollae plasmid clone into Rhizobium Exo− mutant (exoY) resulted in the Exo+ transconjugants. Those findings suggest that some of the An. azollae plasmids may play a role in symbiotic interactions with Azolla fem. Key words: Anabaena azollae, Azolla, symbiosis, cyanobacterium, plasmids.

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