Isolation,identification and biocontrol effect of endogenous antagonistic bacteria against Alternaria alternata

【Objective】The biocontrol strains with good antagonistic effect against the pathogens of honeysuckle leaf spot were selected from the leaves of honeysuckle,in order to provide suitable strain resources for the biological control of honeysuckle leaf spot and lay a foundation for the high-quality development of honeysuckle industry. 【Method】In this study,honeysuckle leaves were used as materials to isolate and purify endophytic Bacillus strains using the dilution separation method. The strains with antagonistic effects on Alternaria alternata were screened using the Oxford cup plate confrontation method. The effects of antagonistic bacteria on the morphology of A. alternata were evaluated by a centrifuge tube co-culture test. The inhibitory effect of the lipopeptide crude extracts of antagonistic bacteria on the mycelial growth of A. alternata were detected using Oxford cup plate confrontation test. The biocontrol effect of antagonistic bacteria on honeysuckle leaf spot in pot assay were evaluated under greenhouse conditions. Antagonistic bacteria were identified based on morphological,physiological,biochemical,and molecular biological characteristics. Moreover,the antimicrobial spectrum of antagonistic bacteria were determined by plate confrontation method. 【Result】A total of 120 endophytic Bacillus strains were isolated from honeysuckle leaves. Through the plate confrontation method,four strains were selected that exhibited effective antagonistic properties against the leaf spot pathogen of honeysuckle. The antagonistic effects of these four strains on the leaf spot pathogen were not significantly different(P>0.05). Strain HC-14 was randomly chosen for further study. The results of the plate confrontation test showed that strain HC-14 had a good antagonistic effect against the leaf spot pathogen with an inhibition rate of 49.38%. The results of the centrifuge tube temperature bath showed that the supernatant of strain HC-14 had a strong inhibitory effect on A. alternate. The results of the Oxford cup plate test showed that the crude extract of lipopeptide antibiotics of strain HC-14 had a certain inhibitory effect on A. alternate,and the inhibitory rate was 11.44%. Pot experiment results showed that the disease indexes of the prevention group,therapeutic group and protection group were only 6.94,6.35 and 4.21 on the 21st day after inoculation with strain HC-14 fermentation solution by spraying,and the control effects were 76.62%,78.59%,and 85.79% respectively. Combined with the results of colony morphology,physiology,biochemistry and molecular identification,the strain HC-14 was identified as B. subtilis.Antibactrial spectrum result of strain HC-14 indicated that it had inhibition effects on ten common pathogens at various degrees except Ralstonia solanacearum. 【Conclusion】B. subtilis strain HC-14 has wide antibacterial spectrum,thus has the potential for biocontrol against leaf spot disease in honeysuckle.