Screening Of Bacillus Velezensis K-9 And Its Biocontrol Mechanism Against Potato Scab Disease

The development of potato industry is related to food security,and potato scab is the main soil-borne disease threatening the potato industry.Potato scab has occurred in Yunnan Province,Hebei Province,Xinjiang Uygur Autonomous Region,Gansu,Shanxi,Heilongjiang and Shandong provinces.Scab affects not only potatoes but also other root crops(radishes,sweet potatoes and beets),greatly reducing the economic value of tubers.The pathogen of scab disease has a strong stress effect on crops,and the pathogen can live in soil or plant residues for up to 10 years,which is difficult to control.In order to develop biocontrol agents for scab disease,an antagonistic bacterium with obvious inhibition on pathogens was obtained from potato tubers.Plate antagonism,indoor and field indirect seed tests were conducted to evaluate the biocontrol effects of scabies antagonistic strains on potato scab disease.The antibacterial substances were identified by genomics and mass spectrometry,and the inhibitory mechanism of the biocontrol bacterium against potato scab was revealed from the aspects of cytology,physiology,biochemistry and molecular biology.The following main results were obtained:1.In potato fields,243 bacterial strains of scabies were isolated from lightly diseased tubers at the point of contact between diseased spots and healthy epidermis.We screened 45 strains of antagonistic endophytic bacteria by the plate confrontation method.Among them,the bacteriostatic rate of strain K-9 was 44.90±1.99%,and it had growth-promoting characteristics,so it was used as the dominant strain for further study.By morphological observation,physiological and biochemical analysis,16 S r RNA gene sequence,gyr B gene sequence and DNA-DNA hybridization analysis,the strain K-9 was identified as Bacillus velezensis.2.The genome of Bacillus velezensis K-9 was 3891530 bp in size and 46.45% in GC content.The results of secondary metabolite prediction and gene function annotation showed that the K-9genome contained 11 secondary metabolite synthesis gene clusters,and LCI protein-producing lci gene was found.These genes were related to biosynthesis of biocontrol metabolites such as surfactant,lysobacterin,ferritic carrier and bacteriostatin.Fifty-eight genes related to colonization processes(motility,biofilm formation and polysaccharide degradation)were also found in K-9 genome.3.Bacillus velezensis K-9 showed broad-spectrum antibacterial effect on crop pathogens.Plate antagonism tests showed that strain K-9 had in dish antagonism against 8 species of crop pathogenic fungi,and the inhibition rate against 6 species of pathogen was more than 50%.According to the inhibition rate from large to small: Rhizoctonia solani,Colletotrichum gloeosporioides,Curvularia lunata,Bipolaris zeicola,Alternaria,Alternaria solani.The results indicated that strain K-9 had broad-spectrum bacteriostatic effect on crop pathogens.4.Different components of Bacillus velezensis K-9 had certain inhibitory effects on the growth of potato scab,and the best inhibitory effect was found in fermentation broth,with the bacteriostasis rate of 53.92%.Response surface method was used to optimize the optimal combination of soluble starch 0.2%,ammonium sulfate 3%,manganese chloride 0.01% in the fermentation medium of strain K-9.The optimal bacteriostatic activity of the active substance was obtained by fermentation culture with 2% inoculum,20-40% liquid loading and p H of 7,which was cultured at 180 rpm and 34 ℃ for 24 h.5.The prevention and growth promotion effects of Bacillus velezensis on potato were determined by field experiment.The strain K-9 increased the dry matter quality,plant height and stem diameter,leaf number and leaf area index of potatoes from seedling stage to maturity stage.These results indicated that strain K-9 could accelerate dry matter accumulation and promote potato growth and development.In 2021 and 2022,strain K-9 increased the potato yield by12.44% and 12.65%,improved tuber quality,and significantly improved soluble protein quality.Strain K-9 not only reduced the area of potato scab disease,but also reduced the depth of potato scab disease.In 2021,the disease index of the experimental field was 68.98±0.01%,the scab was serious,and the control effect was 33.01±0.96%.In 2022,the disease index of the experimental field was 66.72±2.13%,and the prevention and treatment efficiency was 45.42±4.38% when the symptoms of scab were still not optimistic.The effects of Bacillus velezensis K-9 on increasing production and preventing disease are complementary.6.Studies on the stability of bacteriostatic active substances of Bacillus velezensis K-9showed that strain K-9 had good storage stability,acid-base stability,light stability,genetic stability and UV stability,but could not withstand 80 ℃ high temperature.The antibacterial activity of the active substance decreased significantly after being treated with protease,and then ammonium sulfate precipitation method was used to obtain crude protein extract,which had antagonistic activity against pathogenic bacteria.Qualitative analysis was conducted by LS-MS/MS mass spectrometry.It was found that the extracellular protein substances contained bacteriostatic active substances,such as biosynthetic protein,subtilin,volgenin synthetase,flagellin and chitosan enzyme,as well as their synthetic catalytic substances,which played a role in bacteriostatic effect.The bacterial strain K-9 could stabilize in soil and potato roots,which was conducive to niche competition,which was the direct bacteriostatic mechanism of the bacterial strain.7.Bacillus velezensis K-9 increased POD and PAL enzyme activities,decreased CAT enzyme activities and MDA content in potato roots,and induced plant resistance to scab disease.The nutrient content and enzyme activity of potato rhizosphere soil were increased.The number of potato scab pathogens in rhizosphere soil was decreased.The bacterial and fungal community structure of rhizosphere soil was changed,and the relative abundance of Luteimonas,Rhodanobacter and Trichoderma were significantly increased during the whole growth period of potato.And significantly reduced the relative abundance of Fusarium.By inducing resistance of potato plants and regulating the potato rhizosphere microflora,the pathogen can be indirectly inhibited,which is the indirect bacteriostatic mechanism of the strain.