Potential and genome-wide analysis of Bacillus velezensis Mr12 in preventing apple ring rot and other diseases

Abstract:【Objective】Malus sieversii is not only an important wild life resource in China, but also a good germplasm resource for resistances to disease, insect, and drought. The endophytic bacteria in M. sieversii have high potential application value for biological control of apple diseases. To explore bio- control potential of endophytic Bacillus velezensis Mr12 isolated from M. sieversii, the antibacterial broad-spectrum, antibacterial mechanism and the stability of antibacterial active substances of strain Mr12 under high temperature, ultraviolet radiation and other conditions were determined and analyzed. The molecular mechanism underlying the biocontrol processes were studied.【Methods】The antagonis- tic effect of strain Mr12 against six pathogenic fungi (Fusarium graminearum, Gaeumannomyces graminis, Rizoctonia cerealis, Fusarium proliferatum, Botrytis cinerea and Colletotrichum karstii) was evaluated by confrontation culture method. The stability of antifungal metabolite of its sterile superna- tant against B. dothidea was determined. After the sterile filtrate of Mr12 heat treatment, acid-base treat- ment, proteinase K treatment and ultraviolet treatment, the antibacterial rates of the sterile filtrate were tested. The effect of the sterile filtrate on the spore germination and mycelial growth of B. dothidea was studied. Potential biocontrol molecular foundation of strain Mr12 was analyzed by the complete ge- nome sequencing.【Results】The strain Mr12 exhibited antagonistic abilities to the six plant pathogens, and the inhibition rate was between 55% and 91%. In addition, it still maintained the antifungal activity after treatment of acidic, alkaline, heat (< 80 °C), proteinase K and ultraviolet radiation. It could cause the abnormal growth of mycelium and inhabit spore germination. It was sequenced and assembled. The genome length was 4 001 152 bp, including a complete circular chromosome and a plasmid; the chro- mosome length was 3 920 120 bp (GenBank accession number is CP066337), and the plasmid length was 81 032 bp ( The accession number of GenBank is CP066338). The GC content of the genome was 46.50%. It was predicted to encode 3953 genes with an average length of 872 bp. The coding sequence accountes for 86.12% of the entire genome sequence. Complete genome sequence analysis showed that the strain Mr12 contained gene clusters related to the biosynthesis of a variety of polypeptide and polyketide compounds, such as difficidin, fengycin, bacillibactin and surfactin. And the genes encoding enzymes like β-1,3-glucase and chitinase could hydrolyze the pathogen cell wall. The results of bio- chemical analysis further proved the ability of this strain to produce casein and cellulase.【Conclusion】The strain Mr12 had an antagonistic effect on B. dothidea, Phomopsis mali and Colletotrichum gloeo- sporioides. This study further clarified that the strain had an effect on F. graminearum, G. graminis, R. cerealis, F. proliferatum, B. cinerea and C. karstii. It had strong inhibitory activity against fungal patho- gens in fruit trees, wheat, vegetable, and so on. The inhibitory activity was stable. The antibacterial rate of the fermentation filtrate of the strain Mr12 was still high even with ultraviolet irradiation and acidbase treatments. Genome analysis revealed that the disease prevention effect of the strain Mr12 could be achieved by producing lipopeptides, hydrolase enzymes and inducing plant disease resistance, etc. In addition, the whole genome analysis results also showed that the strain Mr12 contained lipopolysaccha- ride, salicylic acid, acetolactate decarboxylase and acetolactate synthase and other genes related to the induction of plant resistance. It would be valuable to further evaluate the strain Mr12 as potential bio- controlling strains to plant diseases.