Root nodules of Ceanothus caeruleus contain both the N2-fixing Frankia endophyte and a phylogenetically related Nod-/Fix- actinomycete

摘要

Attempts to isolate the N2-fixing endophyte of Ceanothus caeruleus (Rhamnaceae) root nodules, led to the isolation of nine actinomycete strains. Owing to their inability to fix nitrogen (Fix-) and nodulate (Nod-), they could not be regarded as the effective endophyte. Characterization was done based on morphological and physiological features and 16S rDNA sequence analysis. The effective Frankia endophyte was characterized without cultivation by amplification, cloning, and sequencing of nearly fulllength 16S rDNA and partial nifH genes. Phylogenetic analysis based on 16S rDNA revealed that both the effective endophyte and the isolated actinomycetes belong to two different but well-defined lineages within the family Frankiaceae. One lineage is formed mainly by uncultured endophytes that so far have resisted isolation, and the other includes only Fix-/Nod- isolates. Application of temperature gradient gel electrophoresis techniques to actinorhizal nodules allowed the detection and identification of 16S rDNA sequences from both the Fix+ and the Fix- nodule inhabitants. A note is made of the fact that these same two sequences 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.