ROOT-NODULES OF CEANOTHUS-CAERULEUS CONTAIN BOTH THE N-2-FIXING FRANKIA ENDOPHYTE AND A PHYLOGENETICALLY RELATED NOD(-) FIX(-) ACTINOMYCETE/

Attempts to isolate the N-2-fixing endophyte of Ceanothus caeruleus (R hamnaceae) root nodules, led to the isolation of nine actinomycetous s trains. Owing to their inability to fix nitrogen (Fix(-)) and nodulate (Nod(-)), they could not be regarded as the effective endophyte. Char acterization was done based on morphological and physiological feature s and 16S rDNA sequence analysis. The effective Frankia endophyte was characterized without cultivation by amplification, cloning, and seque ncing of nearly full length 16S rDNA and partial nifH genes. Phylogene tic analysis based on 16S rDNA revealed that both the effective endoph yte and the isolated actinomycetes belong to two different but well-de fined lineages within the family Frankiaceae. One lineage is formed ma inly by uncultured endophytes that so far have resisted isolation, and the other includes only Fix(-)/Nod(-) isolates. Application of temper ature gradient gel electrophoresis techniques to actinorhizal nodules allowed us to detect and identify 16S rDNA sequences from both the Fix (+) and the Fix(-) nodule inhabitants. Interestingly, these same two s equences were detected on Hippophae rhamnoides nodules obtained after inoculation with Ceanothus caeruleus nodule suspensions. The isolates were located in the outer layers of the nodule.