Screening, identification and optimization of fermentation conditions of an antagonistic endophytic bacterium from mulberry

Abstract:

[Objective] Application of antagonistic endophyte is a potential strategy for biological control of plant diseases. In this study, the antagonistic endophytic bacteria were isolated and screened from healthy mulberry, to provide candidate strains for the biological control of mulberry fruit sclerotiniose.[Methods] Tissue isolation method was used to isolate the endophytic bacteria from healthy mulberry tissues. The endophytic bacterium with strong and stable antagonistic activity on the pathogens of mulberry fruit sclerotiniose was screened by inhibition zone method. The antagonistic bacterium was identified through morphological features, cultural, physiological and biochemical characteristics and 16S rDNA phylogenetic analysis. Antimicrobial spectrum and thermal stability of cell-free fermentation supernatant were assayed by mycelial growth rate. The fermentation conditions and medium composition were optimized through single factor and orthogonal experiment.[Results] In total 55 endophytic bacteria were isolated from healthy mulberry. Among them, strain XP-27 was found to have strong and stable antagonistic activity against Sclerotinia sclerotiorum PZ-2 (the pathogen of mulberry fruit sclerotiniose). Morphological features, cultural, physiological and biochemical characteristics indicated that XP-27 belongs to genus of Bacillus. Based on 16S rDNA phylogenetic analysis, XP-27 was close to the Bacillus methylotrophicus and within the same clade with several strains of Bacillus methylotrophicus. Therefore, strain XP-27 was identified as Bacillus methylotrophicus, and named to be B. methylotrophicus XP-27. Antimicrobial spectrum assayed results showed that strain XP-27 had variously antagonistic activity on 10 plant pathogens and had excellent heat stability. The results of fermentation optimization showed that the optimal medium was composed of 1.00% beef extract, 1.50% starch, 0.05% K₂HPO₄, 0.10% MgSO₄·7H₂O and the optimal culture conditions were inoculation size of 7% for 120 h at 30℃ with initial pH 8.0.[Conclusion] The above results indicated that mulberry endophytic bacterium B. methylotrophicus XP-27 has strong antagonistic activity on S. sclerotiorum PZ-2, being a candidate strain for developing the biological control agent against mulberry fruit sclerotiniose.