A biocontrol bacterial strain against Zanthoxylum bungeanum root rot:screening, fermentation condition optimization, and preliminary identification of antimicrobial ingredients

Abstract:

[Background] Fusarium solani is the main pathogen of Zanthoxylum bungeanum root rot in Longnan, affecting the production of Z. bungeanum. [Objective] To screen out a strain for the biocontrol of F. solani and provide new bioresources for the development of microbial agents for the control of Z. bungeanum root rot. [Methods] In this study, we employed the plate confrontation method and 16S rRNA gene sequencing to screen and identify the bacterial strain with strong antimicrobial activity from the rhizosphere soil of Z. bungeanum. Box-Benhnken design was employed to optimize the fermentation conditions of the strain. Liquid chromatography-mass spectrometry (LC-MS) was used to study the changes of metabolites in the organic solvent phase of the bacterial fermentation broth. [Results] The antagonistic strain screened out was identified as Bacillus velezensis W-1, which showed the inhibition rates of 89% against F. solani and 57%–90% against Gibberella fujikuroi, Fusarium graminearum, Fusarium syphilis, Fusarium proliferatum, and Colletotrichum fioriniae. The fermentation conditions of W-1 were optimized as pH 6.5, 30℃, inoculum amount of 5%, and 180 r/min. The OD₆₀₀value of the fermentation broth obtained under the optimal conditions reached 1.93, which was 2.6 times higher than that of the LB basal medium. The antimicrobial activity of the sterile fermentation broth remained stable in the range of pH 2.0−9.0 and gradually decreased after the broth was irradiated with ultraviolet light (UVC 200−280 nm, 100 μW/cm²) for 10 min. The antimicrobial activity significantly decreased when the temperature exceeded 80℃. Ethyl acetate was the best organic solvent for the extraction of antimicrobial ingredients. The organic phase of the extract contained rich antagonistic substances, such as lipopeptides (surfactants), phosphate oligopeptides (antibiotics), butirosin, polyketone compounds, lysobactin, cyclic dipeptides, and gramicidin. [Conclusion] B. velezensis W-1 can secrete a variety of antimicrobial substances and has strong inhibitory effects on multiple common pathogenic fungi of plants. Moreover, its fermentation broth can tolerate acid, alkali, and high temperature, demonstrating great potential for application in the biocontrol of Z. bungeanum root rot.

Key words:Zanthoxylum bungeanum root rot;Fusarium solani;antagonistic bacteria;optimization of fermentation conditions