Inhibitory Effect Of Endophytic Bacteria Eb-28 From Tomato On Botrytis Cinerea Pers

Cucumber cotyledon method was chosed as basic method,and iprodione was used as primary fungicide matrial to develop a system for fungicide screening against cucumber gray mould.Different cultivars,different cotyledon age of cucumber,different cultivating time of B.cinerea mycilia,different testing condition for detached cucumber cotyledon and fungicide application methods were taken account of integrated screening system.An simple and rapid fungicide screening system was developed.In this system, changchunmici wasselected as a suitable cultivar for bioassay test.Eight days leaf age,1 day young hypha of B.cinerea,spraying,keeping wet by 0.5%water agar after inoculation,cultivating at 24℃with light and lesion diameter measuring 24～36 h after inoculation constituted the whole system.Four different fungicides,2%propamidine AS, 25%SYP-Z048 EC,50%procymidone WP and 40%pyrimethanil SC were tested for the stability of the system.One isolate Eb-28 that was isolated from tomato stem selected from 3 endophytic bacterium isolates strongly inhibited myceria grow of B.cinerea..Culturing conditions of strain Eb-28 showed that cultural time,cultural temperature, pH values of media and oxygen conditions could affect strain quantity.Result indicated that optimum cultural conditions of strain Eb-28 are cultural time 48 h,cultural temperature 30℃,6.0 pH values of media and oxygen conditions of 175 mL/250 mL.Eb-28 strain was identified by traditional and molecular biology method.Eb-28 strain was identified as Bacillus spp.according to the morphological characterization,culturing pattern,physiology and biochemical characteristics and Bergey's Manual of Determinative Bacteriology(Eighth Edition).Furthermore,it was finally identified as Bacillus subtilis by 16S rDNA sequence analysis.The inhibitive rates of Eb-28 reached to 51.68%in vivo against leision extension of tomato gray mould.Fermentation liquid of Eb-28 produced inhibitory zone with diameter over 20 mm against B.cinerea Autoclaved ferment had no inhibitory effects on B.cinerea Pers.It indicated that active substance of fermentation liquid lost its function after autoclaving.After treated by antibacterial products,the mycelia of phytopathogenic fungi appeared distortion with agglomerated protoplasm.The antifungal crude protein was extracted by the steps of ammonium sulfate precipitation,and the inhibitory zone against B. cinema was over 20 mm,however supernatants had no antifungal activity.This result suggested that antifungal substances in fermented filtrates of Eb-28 were precipitated by ammonium sulfate.The mycelia of B.cinerea appeared distortion and their protoplasm agglomerated after treated by fermentation liquid with antifungal proteins inside. Furthermore,the inhibition zone of antifungal crude protein against B.cinerea was over 20 mm at neutral and alkaline medium(pH range of 6.6～10.6),and antifungal crude protein displayed rather good stability at different temperature and ultraviolet radiation.