| Cucumber is one of most important vegetatable in China. However, cucumber plants have suffered from soil-borne diseases due to the changes of soil environment. Vascular wilt of cucumber, caused by Fusarium oxysporum f. sp. cucumerinum J. H. Owen (FOC) is a severe disease, leading to serious losses of cucumber production all over the world. Thus control of the disease is an urgent need worldwide. However, there is not an effective measure to control the disease. The routine chemical control of Fusarium wilt results in pesticide residues. Biocontrol, which has been proving economical and environmentally friendly, is getting more and more attention.This paper presents the screening and identification of antagonistic bacterial strains against Fusarium oxysporum f. sp. Cucumerinum. The spectrum of the antagonist to different pathogens and the optimum fermentation conditions of the strain was studied. Lipopeptides synthesis-related genes were detected by PCR. Fermentation conditions for antagonistic substance production and their characteristics were also tested. Then the bioorganic fertilizer (BIO) was made by inoculating the strain into organic fertilizer and its biocontrol efficiency of cucumber Fusarium wilt was studied in pot experiments.The results obtained were as follows:(1) Totally35strains were isolated could inhibit Fusarium oxysporum f. sp. Cucumerinum. and among them the strain WQJ-100showed the best inhibition to Fusarium oxysporum f. sp. Cucumerinum with inhibition rate63.6%. Based on morphological, physiological and biochemical characteristics, and16S rRNA gene sequence analysis the strain WQJ-100was primarily identified as Bacillus vallismortis.(2) The strain WQJ-100also had antagonistic effects against several pathogenic fungi, such as V F. osyporunm f. sp melonis, F. osyporunm f. sp cubense, Phytophthora capsici, and the pathogen inhibition rate were more than60.0%. The strain WQJ-100showed the maximum inhibition activity to V. dahliae Kleb with inhibition rate75.8%.(3) Genes fenB, bam and ituA, ituB, ituC and ituD, responsible for synthesis of antibiotics Fengycin, Bacillomycin and Iturin were found in strain WQJ-100acillus vallismortis WQJ-100(4) the strain WQJ-100could get the maximum growth under conditions as following:2%of inoculum, sucrose as carbon source and pepton as nitrogen source respectively,10mL LB medium without NaCl, initial pH7.0, incubation at40℃for36h, with180r·min-1. The optimal conditions for producing antimicrobial substance by the strain WQJ-100were as following:1%of inoculum, incubation at35℃with180r·min-1for72h,50mL LB medium with initial pH7in250mL flask.(5) Crude lipopeptides were extracted from the strain WQJ-100culture. The crude extract could inhibit the growth of Fusarium oxysporum f. sp. Cucumerinum observably. The lipopeptides crude could endure acidity and it was very stable after treatment at pH from3to9. The crude extract was also stable to UV after different time treatment. Antagonistic substance was heat-stable, which still maintained strong inhibition against Fusarium oxysporum f. sp. Cucumerinum after80℃treatment for30min. The crude extract was stable to pepsin and proteinase K, but it was sensitive to trypsin to some extent.(6) Pot experiments were performed to investigate the effects of bio-organic fertilizers (BIO) made from the organic fertilizer and the strain WQJ-100. The results showed that BIO could decrease the incidence of fusarium wilt disease and increase cucumber yield. The disease incidence of cucumber was15.0%when the BIO was applied both in the nursery and the pot soil, which was much lower than that of control (83.3%), and the control effect reach80.7%.(7) Compared with the control treatment, BIO treatment could promote the photosynthesis and chlorophyll contents of leaves and increase the shoot and root biomass. BIO application also could raise the length and activities of roots compared with control.(8) Application of BIO strongly reduced the number of pathogen in rhizosphere soil. Pathogen populations were5.51×104CFU g-1DW (dry weight) in BIO-applied rhizosphere soil, while it was7.77×104CFU g-1DW in control rhizosphere soil. Soil microbial community structure was greatly changed by application of BIO. The number of actinomycota and bacteria including Bacillus vallismortis in rhizosphere soil were increased markedly in BIO treatment compared with those of control. In contrast, pathogen and fungal numbers in the rhizosphere soil was dramatically higher in control than in BIO treatment.(9) The activities of superoxidase (SOD), peroxidase (POD), catalase (CAT) of cucumber leaves in BIO treatment were much higher than that in control. The leaf MDA contents in cucumber treated in organic fertilizer and bio-organic fertilizer were significantly lower than that in control after55d of transplantation. |
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