Studies On Diversity Of Nitrogen-fixing Actinomycetes From Root-nodules Of 12 Plants

In this study, we used root-nodule homogenate method and isolated 60 nitrogen-fixing actinomycetes from root nodules of 12 plants, which were collected from Yunnan, Jilin,Inner Mongolia and Hainan of China. The nitrogen-free media of BAP, S, JA, ISP4 and FTW were used for isolation of strains. The results show that the efficiency of BAP medium and S medium with relatively simple components were much better than other media. We also found that the bacteriostatic effect of the combination of nalidixic acid and cycloheximide was better than the same dose combination of streptomycin and nystatin, streptomycin and cycloheximide and nalidixic acid and nystatin.The morphological observation of the isolated strains show that strain 49615 produced a single spore on substrate mycelium; substrate mycelium of strain 49640 were abundant and aeril mycelium were short; strain 49641 had rich substrate mycelium and short spore chains; Strain 49601 and other 14 strains could produce brown, yellow, brown, or other soluble pigments.The results of physiological and biochemical showed that all of the isolated strains could utilized glucose and arabinose as the sole carbon source. In addition to strain 49616,all of the isolated strains could use xylose as the sole carbon source. All of the strains could not grow in the medium with 10% NaCl; All of the strains could not hydrolyze cellulose. All of the strains could use rescin glycosides.Most of strains could reducte nitrate and hydrolyze starch. All of the strains could not utilize sodium acetate, only strain 49611, strain 49612, strain 49616 were able to use sodium citrate,only strain 49601 could liquefact gelatin and only strain 49606 could use sodium oxalate.The 16S rDNA sequence analysis of the isolated 60 strains showed that the 60 strains were located into 4 genus: strain 49601 and other 37 strains belonged to Micromonospora, strain 49639 and others 19 strains belonged to Streptomyces, strain 49659 belonged to Nonomuraea, Strain 49660 belonged to Pseudonocardia.The results of nodulation experiments showed that the host plants Alnus nepalensis, Coriaria sinica and Elaeagnus angustifolia growed better than the control group and the Coriaria sinica grewed nodules after the infection of strain 49616 and strain 49625,which belonged to Micromonospora. The mycelium of actinomycetes and other structures in root nodule were found by observing the ultramicrocut, and also amplified specific sequences in the total DNA of nodules by using specific primers of Frankia , Micromonospora, Streptomyces, and Pseudonocardia , as a result,there were different specific bands were getten , which showed that there were different kinds of actinomycetes strains and species diversity in 12 root nodules.