Isolation And Characterization Of Pseudomonase Species From Sugarcane Rhizopheric Soil And Their Influence On The Growth Of Sugarcane

Sugarcane is the world's major sugar and energy crop,and Guangxi is the main sugarcane producing area in China and produces about 70%cane sugar in China.However,farmers apply rather high doses of fertilizers to increase cane yields in Guangxi sugarcane area.High dose of fertilization not only increases the production cost but also causes serious environmental pollution.Therefore,it is necessary to isolate and develop functional microbes that can be used as targeted fertilizers as an alternative to traditional fertilizer applications.In this study,Pseudomonas strains were isolated from the rhizosphere of sugarcane growning fields in Guangxi.PCR technique was used for detecting the nifH and antibiotic related genes,and their genetic diversity assessed through Rep-PCR fingerprinting.Only stains CY4 and CN11 were selected for further studies based on their plant growth promoting traits and antagonistic ability against sugarcane pathogens in vitro conditions.BIOLOG phenotypic profiling revealed the metabolic versatility of the selected strains.The colonization ability of the selected strains was evaluated by GFP-tagged technique.Both the selected strains could promote the growth of sugarcane after innoculated to two sugarcane cultivars,and 15N isotope dilution assays confirmed these bacterias could fix nitrogen associated sugarcane.The main results are as follows.1.Total 250 strains were isolated from the rhizosphere soil of sugarcane growing fields,and 100 isolates were selected on the basis of preliminary screening.Based on the 16S rDNA sequencing,30 isolates were identified as Pseudomonas species.2.All the 30 Pseudomonas isolates showed various different PGP traits,26 were positive for phosphate solubilization,20 displayed siderophore production ability,12 were able to produce ammonia,and 13 were capable of producing HCN.Fungal antagonistic test showed that 14 strains were resistant to sugarcane smut pathogen(U.Scitaminea),and 11 resistant to sugarcane pineapple disease pathogens(C.paradoxa).All the selected bacterial strains showed ACC deaminase activity ranging from 15.13 to 77.0 ?mol mg-1 h-1,all bacterial strains were able to produce IAA in the range of 13.12 to 312.07 ?g mL-1.Their nitrogen-fixation efficiency rangeg from 6.16 to 108.30 ?mol C2H2 h-1 mL-1.3.Molecular approach used for analyzing nitrogen-fixing genes and antibiotic related genes,the nifH gene was amplified only in 10 strains,4 showed the PCA-related amplified band size of 1.15 kb,5 showed PRN-related gene amplification at around 786 bp,and 9 showed positive amplification at 587 bp of the HCN-related gene.4.GFP-tagged technique showed that both the selected strains Pseudomonas CY4 and CN11 colonized the whole sugarcane plantlets as for roots,stems and leaves.The growth promoting effects of the strains CY4 and CN11 on sugarcane plant were investigated with pot experiment under greenhouse conditions.Results indicated that both strains could promote growth of sugarcane plant,increased the plant height,fresh weight,endogenous honrmone contents and nitrogen fixation efficiency.The strains CY4(P.koreensis)and CN11(P.entomophila)turned out to be very efficient in terms of enhancing the growth and development of sugarcan plants.Therefore,these two isolates may be used as inoculums or in biofertilizer production for enhancing growth and nutrients,as well as for improving nitrogen levels,in sugarcane and other crops.