Isolating And Characteirzation Of ACC Deaminase-contaning Rhizobacteira And Endophytic Bacteira From Eucommia Ulmoides And Gynostemma Pentaphyllum

A total of33bacterial isolates were screened from the rhizospheres and the rootsof Eucommia ulmoides and Gynostemma pentaphyllum for their ACC-degradingability, and these isolates were identified as species by biochemical test and16SrDNA sequence analysis. Five strains from the roots of Eucommia ulmoides wereidentified as Pseudomonas koreensis, Klebsiella (2strains) and Enterobacter (2strans), and four strains from the rhizospheres of Eucommia ulmoides were identifiedas Bacillus (2strains), Arthrobacter nicotianae and Pseudomonas taiwanensis. Ninestrains from the roots of Gynostemma pentaphyllum were identified as Pseudomonas(4strains), Paenibacillus xylanilyticus (2strains), Bacillus (2strains), Staphylococcusxylosus, and fifteen strains from the rhizospheres of Gynostemma pentaphyllum wereidentified as Bacillus (5strains), Acinetobacter (4strains), Enterobacter (2strains),Stenotrophomonas, Enterococcus casseliflavus, Chryseobacterium jejuense andPaenibacillus xylanexedens. The ACC deaminase activity assay demonstrated that the11strains of fourteen endophytic strains isolated from the roots of Eucommiaulmoides and Gynostemma pentaphyllum were expressed the ACC deaminse activity,while the13strains of nineteen rhizobacteria isolated from the rhizospheres ofEucommia ulmoides and Gynostemma pentaphyllum expressed the ACC deaminseactivity. These results demonstrated that the ACC deaminase activity of Pseudomonasand Acinetobacter strains was significantly higher than other genus of bacteria, andsuggesting the numbers and bacterial species of the ACC deaminase-containingstrains was significant differences between the roots and rhizosphere of two plants.The ACC deaminase structural (acdS) genes of14strains from the roots and therhizospheres were amplified by PCR using the degenerate primers. PCR showed thatthe1kp in size were amplified from the genomic DNA of7strains, and a1000bpDNA fragments were amplified from the plasmid DNA of2strains. While the acdSgene isolated from Pseudomonas koreensis JDM-2through PCR amplification wascloned and expressed in Escherichia coli M15. The sequence analysis showed that theacdS gene was1017bp in length, and DNA homology of the acdS genes between thePseudomonas koreensis JDM-2and the previously reported different bacterial strainsin GenBank was86%to99%. SDS-PAGE showed a single protein band with amolecular weight of37.18kDa expressed in Escherichia coli M15, and its enzymeactivity was0.214U/mg. To further study the growth promoting activity of above-mentioned ACCdeaminase-containing strains on rice was determined the antibacterial property andthe production of siderophore of these strains. The results showed that: the4strainsisolated from the roots of Eucommia ulmoides displayed antibacterial activity againstEscherichia coli and Bacillus subtilis, and the8strains isolated from the roots ofGynostemma pentaphyllum displayed antibacterial activity against Escherichia coli,Sarcina lutea and Candida albicans. However, all of the strains isolated from therhizospheres of Eucommia ulmoides and Gynostemma pentaphyllum did not showantibacterial activity to any tested microorganisms. And the7strains of them wereproduced the high level of siderophores, it suggesting that no correlation between thesiderophores production and antibacterial activity of these strains.The ability of these strains to promote the growth of rice in sterile growthpouches was studied. Results of growth promoting assays showed that there was nosignificant effect on seed germination and shoot growth of rice, while the8strainsincluding the JDG-127, JDM-2, JDM-235, JDG-6, JDG-7, JDG-14, JDG-122andrecombinant M15/pQE30ACDS were significantly increased root length compared tothe control. However, the JDG-32strain significantly inhibited the developing andgrowth of roots. This results indicated that no significant differences of growthpromoting activity between the rhizobacteria and the endophytic bacteria on rice.