Identification and fermentation condition optimization of antagonistic bacteria against Lasiodiplodia theobromae

【Objective】To screen antagonistic biocontrol strains against Lasiodiplodia theobromae，so as to provide bacterial resources and guidance for areca root rot caused by L.theobromae.【Method】 Healthy areca root and rhizosphere soil in the areca orchard where root rot disease was detected in Hainan were taken as test materials.Tissue homogenization method and plate dilution gradient culture method were used to isolate root endophytic bacteria and rhizosphere soil bacteria，and flat-stand method was used to screen antagonistic bacterial strains.Inhibitory effect of the antagonistic strains on 3 kinds of pathogenic fungi of areca root diseases，Fusarium oxysporum，Thielaviopsis paradoxa and Lasiodiplodia theobromae，was determined through the flat-stand method.The strains were identified through morphological observation，physiological and biochemical determination and 16S rDNA sequence analysis.The pot experiment was conducted to verify the efficacy of the antagonistic strains against by areca root rot caused by L.theobromae.Single factor change experiment was used to screen the best fermentation conditions for antagonistic strains.【Result】 Five strains with strong antagonistic effect were screened through flat-stand method，in which strain wrj-2-5 had the highest average inhibitory rate of 74.07%.Strain wrj-2-5 had a strong inhibitory effect on the mycelial growth of F.oxysporum，T.paradoxa and L.theobromae，and the inhibition rate was above 70.00%.Taking results of morphological observation，physiological and biochemical determination and 16S rDNA analysis into account，strain wrj-2-5 was identified as Pseudomonas aeruginosa.The indoor pot test showed that the strain wrj-2-5 had a good preventive effect on areca root rot caused by L.theobromae.Disease incidence rate of the control group was 60.00%，and in the experimental group，plants grew well and no disease was detected.It was found that the optimized medium formula of antagonistic strain wrj-2-5 was as follows:1.5 g yeast extract，1.5 g glucose，0.2 g MgSO₄·7H₂O and 100 mL water；the optimized fermentation condition was as follows： rotating speed 240 r/min，temperature 28℃，culture time 36 h，initial pH 6 and inoculation amount of 8%.【Conclusion】 Strain wrj-2-5 is P.aeruginosa，which has a good inhibitory effect on the F.oxysporum，T.paradoxa and L.theobromae，and it has a good control effect on areca root rot caused by L.theobromae，which can be taken as resources for making areca root rot disease biocontrol bacterial agent.