Isolation and identification of an antagonistic endophytic bacterium from mulberry for biocontrol against Boeremia exigua

Abstract:

[Objective] Antagonistic endophytic bacteria were isolated and screened from the healthy stems of mulberry using Boeremia exigua (the pathogen of mulberry snags rotten leaves disease) as target, for the biological control of mulberry snags rotten leaves disease.[Methods] Endophytic bacteria were isolated from mulberry by tissue isolation method, and the antagonistic bacteria with stable antifungal activity were screened out by the combination of inhibition zone method and confrontation culture method. The antagonistic bacterium was identified through morphological features, cultural, physiological and biochemical characteristics and phylogenetic analysis based on 16S rDNA, gyrA and gyrB sequence. The thermal stability and antimicrobial spectrum of cell-free fermentation supernatant were assayed by inhibition zone method and mycelial growth rate method, respectively. Finally, the antimicrobial mechanism was explored through observation the affection of the antagonistic bacteria on B. exigua GXH1, amplification the key genes that were involved in the biosynthesis of antimicrobial active substances, and the lipopeptide compounds produced by antagonistic bacteria were extracted by acid precipitation and analyzed by LC-MS.[Results] In total 17 endophytic bacteria were isolated from healthy mulberry. Among them, strain NPJ13 exhibited strong and stable antagonistic activity against B. exigua GXH1. Morphological features, cultural, physiological and biochemical characteristics analysis results indicated that NPJ13 belongs to genus of Bacillus. Phylogenetic analysis results based on 16S rDNA, gyrA and gyrB genes revealed that NPJ13 strain and several strains of Bacillus velezensis were in the same minimal clade. Therefore, strain NPJ13 was identified as Bacillus velezensis and named B. velezensis NPJ13. The results of thermal stability test showed that strain NPJ13 had superior heat stability. Antimicrobial spectrum showed that strain NPJ13 had different degrees of antagonism against 12 kinds of pathogenic fungi, such as Botrytis cinerea SWU5, Scleromitrula shiraiana SXSG-5, Sclerotinia sclerotiorum PZ-2, Phytophthora nicotianae SWU20 and so on. The hyphaes of B. exigua GXH1 treated by B. velezensis NPJ13 would become distorted, intumescent, broken and more transparent. The results of functional genes detection showed that the genome of B. velezensis NPJ13 contains 5 antimicrobial metabolite synthesis related genes, which are PKSI, NRPS, Sfp, ItuD, Srfc. Surfactins and iturins were detected from the lipopeptide compounds of strain NPJ13 by LC-MS.[Conclusion] The above results indicated that mulberry endophytic bacterium B. velezensis NPJ13 has strong antagonistic activity on B. exigua GXH1, being a candidate strain for developing the biological control agent against mulberry snags rotten leaves disease.

Key words:mulberry endophytic bacterium;antagonism;mulberry snags rotten leaves disease;antagonistic mechanism