Screening And Identification Of Actinomycetes And The Biocontrol Efficacy Against Fusarium Oxysporum F.sp.vasinfectum

The Fusarium wilt of cotton,caused by Fusarium oxysporum f.sp.vasinfectum（Atk.）W.C.Snyder&H.N.Hans（FOV）,is one of the most devastating diseases of cotton.The current recommendations for managing the disease include using the resistant varieties,rotation and chemical control.Chemical fungicide treatment can take effect at the beginning of application.However,excessive use of chemical fungicides in agriculture has led to environmental pollution,deteriorating human health,and development of pathogen resistance to fungicide.Therefore,a serious search is needed to identify alternative methods for plant protection,which is less dependent on chemicals and is more environmentally friendly.In this study,actinomycetes with high inhibitory activity against FOV were selected by comprehensive screening utilizing plate confrontation,greenhouse experiments and secondary metabolites detection.In particular,XN-04 was chosen for subsequent research.The optimization of fermentation medium and the preliminary study on the antifungal metabolite of the strain XN-04 were carried out.The results obtained are listed as follows:（1）Isolation,screening and identification of biocontrol actinomycetes.A total of 32culturable actinomycete isolates（numbered GS-01 to GS-10 and XN-01 to XN-22）were obtained from the rhizosphere soil collected in Gansu and Xining and examined for antagonistic activity to the FOV in vitro using plate confrontation method.Among them,GS-01,GS-03,XN-01 and XN-04 demonstrated strong inhibitory effect against FOV,and exhibited broad-spectrum antifungal activity.Based on the results of morphological observation,cultural characteristics,physiological and biochemical characteristics,ecological condition tolerance,and 16s r DNA sequence analysis,strain GS-01,GS-03,XN-01 and XN-04 were tentatively identified as Amycolatopsis speibonae,Streptomyces spectabilis,S.atroolivaceus and S.alfalfae,respectively.（2）The effect of actinomyces on cotton seedlings and its control effect on Fusarium wilt of cotton.In greenhouse experiments,the results showed that the actinomyces treatment showed obvious promoting effect on biomass accumulation of cotton as compared to CK.The results also showed that the control efficiency for GS-01,GS-03,XN-01 and XN-04reached 40.50%,51.72%,49.49%and 56.58 respectively.Particularly,the control effect of the strain XN-04 was better than that of carbendazim（52.40%）.（3）Detection of secondary metabolites produced by actinomycetes.The synthesis ability of fungal cell wall hydrolase（chitinase,cellulase,etc.）and the growth-promoting factors（siderophore,IAA,etc.）were detected in four actinomycetes.The results showed that all four actinomycetes had the potential to control plant diseases and promote plant growth.（4）Optimization of medium components for strain XN-04.Under the shaking culture,the antifungal metabolite of XN-04 was secreted inside the cell.First,the medium components were optimized by single factor analysis.Then,seven factors were selected for further optimization and their best concentrations were determined.Soluble starch,soybean cake powder and K₂HPO₄were considered to be the most significant variables using Plackett-Burman design（PBD）.The steepest climbing experiment and RSM based on the three selected factors were utilized to determine the interactions among these three variables,and fine-tune the concentrations in order to maximize biomass.A verification experiment was then carried out under these optimized conditions,a 7.5-fold increase of the biomass was achieved by RSM.（5）Preliminary separation of antifungal metabolite of the strain XN-04.The results showed that the antifungal metabolite produced by XN-04 solubled in organic solvent.The Rf spectrum of this metabolite belonged to the fifth class antibiotic using Doskochilova eight solvent system.（6）The stability analysis of antifungal metabolite of the strain XN-04.The results showed that the antifungal metabolite produced by XN-04 was stable when the temperature is lower than 100℃,the p H range 2-12,within 96 h of light treatment and storage at 4°C for8 months.The stability decreased slightly within 240 min of ultraviolet irradiation treatment.This antifungal metabolite was also stable to proteinase K.（7）The genome of strain XN-04 was sequenced.The whole genome size were 8.28Mb,which contains a chromosome（8.25 Mb）and a plasmid（24.56 Kb）.The content of G+C%in genome was 72.06%.A total of 33 secondary metabolites biosynthesis gene clusters were predicted based on anti SMASH5.0.These results provided theory basis and feasible measure for exploiting the strain XN-04 to control plant fungal diseases.Meanwhile,the genome analyse will be useful for carrying out genetic manipulation of the strain at the molecular level.