JounNlr or Frnl,mNrlrroN AND BrorxcrxrpnrNc Yol. 77, No. 6, 630-635. 1994 Enterobacter cloacae 4105, Isolated from the Surface of Root Nodules of Astragqlus sinicus cv. Japan, Stimulates Nodulation by Rhizobium huakuii bv. renge YONG XU,' AKIRA YOKOTA,2 HIROKAZU SANADA,I MAKOTO HISAMATSU,3 MINEO ARAKI,I HYEON-JE CHO,I TSUTOMU MORINAGA, r .lNo YOSHIKATSU MUROOKAT * Department of Fermentation Technology, Faculty of Engineering, Hiroshima University, I-Kagamiyama, HigashtHiroshima 724,1 Institutefor Fermentation, Osaka, Jusohonmachi, Yodogawa-ku, Osaka 532,2 and Department of Biological Resources, Faculty of Agriculture, Mie University, Tsu, Mie,3 lapan Received 24 November l993/Accepted 2 March 1994 Several bacterial strains \ilere isolated from the surface of root nodules ol Astragalus sinicus cv. Japan (known as renge-sou in Japanese), a green manure legume that grows in winter and which is used in rice fields to fertilize the soil in both Japan and China. These bacterial strains stimulated the nodulation on renge-sou induced by strains of Rhizobium huakuii bv. renge. From a taxonomic characterization of the isolates, the strains were found to belong to the species Enterobacter cloacae. It was found that strains of E. cloacae in- creased the number and weight of nodules and the yield of the host plant when these strains were inoculated with a strain ol R. huakuii bv. renge both in a test-tube nodulation assay and in soil from a rice field. E cloacae inflitenced nodulation at an appropriate ratio of cells of two bacterial strains. The timing of the inocula- tion oJ the two strains onto the host plant was also important. The effect of E. cloacae on the nodulation of renge-sou may be due to bacterial products such as exopolysaccharides, Chinese milk vetch (Astrogalus sinicus) or renge-sou in Japanese (A. sinicus cv. Japan) is a green manure le- gume that grows in the winter. Strains of this species are widely used in rice fields to fertilize the soil in Japan and in the southern part of China. The plant forms a symbi- otic relationship with soil bacteria, forming root nod- ules, in which nitrogen fixation occurs. The bacterium isolated from nodules of ,4. sinicus cv. China has been identified as a new species, Rhizobium huakuii (l). Recently, we isolated and identified a new biovariety of bacterium, R. huqkuii bv. renge that infects and forms nodules on A. sinicus cv. Japan (2). A test-tube system for assaying nodulation on roots of renge-sou has been developed using this bacterium. During the course of isolation of bacteria from renge- sou nodules, it was found that fast-growing bacteria were attached to the surface of the root nodules of renge-sou. Characterization of these bacterial strains and the effect of the bacteria on the formation of nodules that occurs in the presence of R. huakuii bv. renge were studied. MATERIALS AND METHODS Bacterial strains The bacterial strains used in this study are listed in Table l. Media Enterobacter cloacqe strains were main- tained on LB medium (3) while R. huakuii strains were maintained on TY medium (4). NA (5), PGA (2), KYM (2), and M9 minimal medium (6) were used to examine the taxonomical characteristics of the bacteria. For solid media, agar was added at 15 g/1. Strains of E. cloacae and R. huqkuii were grown in YEM medium (7). For the nodulation assays, nitrogen-free modified medium (NFR) (2) was used. Isolation of bacteria from the surface of renge-sou nodules Nodules were obtained from roots of renge- sou (.4. sinicus cultivar Japan) which had been cultivat- ed in a rice field near the campus of Hiroshima Univer- sity, Higashi-Hiroshima. All of the nodules from a sin- gle plant were detached from the roots, washed 5 times with distilled water and placed together in an Eppendorf tube. The nodules were treated with sodium hypochlorite (6%, v/v) for 5min and then washed three times with sterile distilled water. The nodules were mixed thorough- ly with I ml of 0.8% saline, after which 0.1ml of the mixture was put into 5.0m1 of YEM medium and cul- tured overnight at 28oC. Serial dilutions of these cul- tures were used for the isolation of single colonies on solid LB medium. Isolation of R. huakuii bv. renge from crushed nod- ules Strains of R. huakuii bv. renge were isolated from the crushed nodules that had been surface-steri- lized by treatment with ethanol (70%, v,/v), and then with sodium hypochlorite (6%, v/v) as described previ- ously (2). Plant nodulation test Nodulatiop was tested by using a test-tube assay system as described previously (2). Three-day-old seedlings were transferred to sterile test tubes (25 mm x 120 mm), and an appropriate dilu- tion of bacteria in 03% saline was added. The inoculat- ed plants were grown on nitrogen-free NFR medium in a controlled-environment cabinet at 20"C with 16-h of light per 24-h cycle at a light intensity of approximately 350 microeinsteins per mm2 per s at the top of the plants. Nodulation tests were performed in triplicate. Seedlings were examined periodically for up to 20 or 60 d for the number of nodules and the weight of both the nodules and host plants (without nodules). For the nodulation test with rice field soil, the seeds * Corresponding author. 630