Screening Of Actinobacterial Resources In Sugarcane Rhizosphere And Study On Plant Growth Promoting Mechanism

Guangxi is the largest sugarcane producing area in China,and the sugar industry is also one of the pillar industries of Guangxi.The research and utilization of sugarcane microbial resources are increasingly important for the development of sugarcane industry.In this study,many actinobacterial stains were isolated with different media,further identification was carried out to understand the diversity of sugarcane-related actinobacteria,and different plant growthpromoting tests were done to screen out the potential dominant strains favorable for sugarcane growth.The nitrogen-free growth and ACC deaminase activity dominant strain WZS021 was obtained,and further experiments were done to detect the effect of actinobacteria on plant nitrogen fixation and drought resistance in sugarcane,and explain its physiological mechanism.The antifungal activity experiment in vitro found the antagonistic effect of strain BTU6 on sugarcane pineapple disease,the fermentation conditions and product stability were optimized,and the effective active ingredients were extracted.The main findings are as follows.1,The Gao's No.1 medium was found suitable for the isolation of actinobacteria from sugarcane rhizosphere.By using this medium,58 actinobacteria strains were isolated from sugarcane rhizosphere,and the 16 S r RNA identification found they belonged to: 52 strains(89%)of Streptomyces,1 strain(1%)of Leucobacter,2 strains(3%)of Nocardioides,1 strain(2%)of Leifsonia,1 strain of Amycolatopsis(2%),and 1 strain of Brevibacterium(2%).The genetic diversity and evolutionary relationships among the strains of Streptomyces were further analyzed by Rep-PCR.The PGPR characteristics showed the strains producing IAA capacity were the most(32,55%),followed by those with ACC deaminase activity(23,40%),and those with nitrogen-fixing potential(15,26%),with iron-producing ability(13,22%),and with dissolving inorganic phosphorus(12,21%).Among them,the strain WZS021 had the highest ACC deaminase activity(3.03 ?mol ?-ketobutyrate mg-1 protein h-1)and the best nitrogen fixation potential.2,Positive screening of secondary metabolite genes: PKSI 36(62%),PKSII 48(83%),NRPS 31(53%),phz E 31(53%),d TGD 28(48%),Halo 34(59%),CYP 26(45%).Pathogenic fungal antagonism: Ceratocystis paradoxa(de Seynes)Moreau 33(57%),Fusarium oxysporum f.sp.cubense 34(59%),Botrytis cinerea 42(72%),Rhizoctonia solani 35(60%),Fusarium oxysporun f.sp.niveum 23(40%),Alternaria brassicicola 34(59%),Colletotrichum acutatum 30(52%),and Cochliobolus heterostrophus 23(40%).Among them,the strain BTU6 was found antagonized to the pathogen of sugarcane pineapple disease.3,WZS021 was further identified as Streptomyces chartreusis by electron microscopy,carbon source utilization model,and gyr B gene amplification analysis.Nitrogen fixation experiments showed that the strain had high nitrogenase activity at 30 °C,p H 8,5% sucrose and 6 g L-1 KNO3.Using 15 N dilution method,the percentage of nitrogen fixation of WZS021 inoculated sugarcane seedlings was as high as 9.16% Ndfa compared to the control.Inoculation with WZS021 significantly increased the nitrogen metabolism-related enzyme activities(GS,NR,GOT,GPT)of sugarcane,which was beneficial to the accumulation of active substances(SP,NO3--N)related to nitrogen metabolism.After treatment with WZS021 and BTU6,the chemical properties of sugarcane rhizosphere soil changed significantly,soil fertility increased,urease,DHA and AKP activities increased.The structure diversity of microbial community was further highlighted by Hiseq high-throughput sequencing,which provided a better underground environment for plant growth.4,The strain WZS021 can effectively colonize the roots of sugarcane and promote root growth.Under drought stress,WZS021 can regulate antioxidant enzyme system(SOD,POD,CAT,MDA),proline and chlorophyll content,and plant growth hormone(IAA,ABA,ET),alleviating the harm of water loss in sugarcane.5,The genome of strain WZS021 was sequenced using Pac Bio RS II SMRT technology platform,and 22 contigs were obtained,totaling a genome sequence of about 9.92 Mb,and GC content was 71.34%.There were 17 s RNAs,21 r RNAs,72 t RNAs,9,561 protein-coding gene(CDS),and 35 secondary metabolic gene clusters.The genomic sequence was predicted using seven databases(GO,KEGG,COG,NR,TCDB,Swiss-Prot,Tr EMBL),and the potential genes for promoting sugarcane growth and improving drought resistance were listed.The genomic sequence was registered in NCBI with the accession number QVOF00000000.6,The optimum fermentation conditions were determined for strain BTU6: culturing at 28°C for 7 d with Gao's No.1 medium,5% original bacteria solution and 40% bottling rate.The antibacterial activity of the BTU6 antifungal material did not decrease significantly after storage for 30 d under room temperature,and it also had good acid and alkali resistance and thermal stability.The extraction of BTU6 secondary metabolites was the best using butanol,and the main antifungal active substances can be eluted with a solvent consisting of petroleum ether : ethyl acetate(7:1 or 5:1).Combined with thin layer chromatography and dextran column chromatography,the active components in BTU6 antagonizing the pathogen of sugarcane pineapple disease were extracted and purified.In summary,there are a large number of actinobacteria closely related to plant growth in sugarcane rhizosphere,and some actinobacteria play an important role in regulating the growth of sugarcane.In this study,the strain WZS021 had a positive effect on the nitrogen utilization and drought resistance of sugarcane,and the strain BTU6 produced antifungal active substances.Both strains have the potential to be developed into microbial fertilizers.