Random compound mutagenesis breeding of Bacillus amyloliquefaciens X030 with high resistance to pathogenic bacteria on grass carp

Abstract Background: Bacillus amyloliquefaciens X030 (BaX030) was obtained by screening from peanut fields in Henan Province, China. It had broad-spectrum antibacterial activity against fish pathogens Aeromonas hydrophila and Aeromonas veronii. In order to improve its antibacterial effect, BaX030 was carried out the compound mutagenesis that atmospheric and room temperature plasma (ARTP) combined with nitrosoguanidine (NTG). Results: The result showed the yield of macrolactin A and oxydifficidin of the mutant N-11 were increased by 2.01 times and 3.68 times, respectively. Re-sequencing found that the corresponding 9th and 15th gene clusters had 4 and 6 SNP mutations, respectively, the 15th gene cluster also had 7 InDel mutations. Scanning electron microscopy observed that the N-11 became thin and long. The results of qRT-PCR indicated that feeding the N-11 can increase the expression of immune factors in the liver or kidney tissue of grass carp. It can also significantly reduce the mortality and the surface symptoms of grass carp that was infected by two pathogens through HE staining and protection experiments.Conclusion: This is the first report that after ARTP-NTG compound mutagenesis, a high-yielding strain of macrolactin A and oxydifficidin were increased by 2.01 times and 3.68 times compared with the original strain, respectively, which laid the foundation for elucidating its biological regulation, they had antibacterial effects on Aeromonas hydrophila and Aeromonas veronii. We combined re-sequencing to find the mutation sites of gene clusters and. The probiotic strain N-11 can quickly activate the immune protection mechanism of grass carp to resist pathogenic bacteria.