Further characterization of new symbiotic nitrogen fixing non-Frankia actinomycetes isolated from nodules of Alnus acuminata.

VALDÉS, D; HUSS-DANELL K; LAVIRE C; NORMAND P; WALL LG

Umea, Suecia

Simposio; The 14 th Meeting on Frankia and actinorhizal plants; 2006

We have continued the characterization of N2 fixing actinomycetes isolated from surface sterilized root nodules of Alnus acuminata. If nodules were never frozen we obtained Frankia like actinomycetes out of them. If nodules were frozen before isolation we could not get typical Frankia but other N-fix actinomycetes. As previously reported, all these particular isolates grew on N-free media and deformed root hairs of A. acuminata roots. Some of them were able to induce cell division in the cortex and scarce nodulation when inoculated at high doses, compare to control Frankia ArI3. Some isolates were able to stimulate nodulation when co-inoculated with low dose inoculum of ArI3 in ratio 10:1 and on the basis of fingerprint analysis of vesicles it seemed that could exist co-infection. More over, and surprisingly, when the isolate AaI4 was co-inoculated on A. acuminata with the non-infective heterologous Frankia BCU110501 (isolated from Discaria trinervis), good, effective and reproducible nodulation was obtained suggesting physiological complementation for nodulation.  On the basis of detailed morphological characterization of the isolates cultivated with or without N, and Scanning Electron Microscopy analysis, we conclude that at least two of these isolates, AaI4 and AaI5 were not Frankia. SEM revealed branched short chain sporangia containing bacillary spores, and the development of terminal spherical structures in nitrogen free medium. Transmission Electron Microscopy analysis of slides from nodules, induced by these isolates, revealed septated vesicles in infected cells. The analysis of 16s RNA gene sequences of AaI4 and AaI5 suggested that both strains were closely related to Nocardia brasiliensis, however sporangia morphology did not corroborate the relationship. It was not possible to amplified squalene hopene cyclase (shc) gene in AaI4 and AaI5 while it was positive for another isolate AaI15 obtained also from Alnus acuminata nodule that was Frankia, morphologically and because of its 16s rDNA gene sequence.