| Crown gall of cherry blossom is a soil-borne bacterial disease that occurs in various parts of our country.Because of its special way of causing disease,chemical control can not have a good effect,and chemical control is not environmental friendly.Therefore,it is particularly important to select effective biocontrol strains to control the disease.In preliminary research in our laboratory,the strain B.velezensis JK-XZ8 was screened from the rhizosphere of flowering cherry trees in Xuzhou,Jiangsu Province,which has control effect on cherry crown gall.This strain was initially identified as Bacillus subtilis.Since many strains in the genus Bacillus are difficult to distinguish from each other,this study identified the biocontrol strain JK-XZ8 more accurately,and made a preliminary study on the colonization of the strain in the root system of cherry blossoms and the mechanism of inducing resistance to cherry blossoms.In addition,the strain was mixed with organic fertilizer to form biological fertilizer and applied in the field..The main findings are as follows:The physiological and biochemical characteristics of biocontrol strain JK-XZ8 showed that its starch hydrolysis,lactose utilization,inulin fermentation acid production,egg yolk phospholipase and horse chestnut hydrolysis were positive,and its starch hydrolysis ability was stronger than that of the standard control strain Bacillus subtilis 3610.Three conserved genes gyr A,gyr B and pur H were cloned and the phylogenetic tree was constructed.As a result,the biocontrol strain JK-XZ8 was identified as Bacillus velezensis.By labeling the biocontrol strain JK-XZ8 with antibiotics,the labeled strain JK-XZ8Rif had no significant difference in growth ability and bacteriostatic ability with the wild strain.When the labeled strain was applied to potted saplings,it was found that biocontrol strains were detected in the rhizosphere and root surface of cherry blossoms.The colonization density of this strain in the rhizosphere was 27.78×10~3 cfu/g on the fifth day,and then gradually decreased,while the colonization density of the root surface on the fifth day was 14.77×10~3 cfu/g,and then tended to stabilize between 2.77×10~3 cfu/g and 3.22×10~3 cfu/g.The greenhouse experiment was used to test the effect of JK-XZ8 on the prevention and therapy of crown gall.The results showed that compared with 87.5%of the incidence of pathogenic bacteria alone,the incidence of the prevention group was 48.15%,and the therapy group was 66.67%.Sole application of strain JK-XZ8 could increase the activities of PPO and PAL enzyme without affecting the activities of SOD.The activities of SOD and PPO increased in the prevention group.However,the activity of PAL increased in both the prevention and therapy group.In terms of defense-related genes,the expression of the salicylic acid pathway defense-related genes PAL and PR-1 in cherry roots increased in the prevention and therapy group.The application of the biocontrol bacterium JK-XZ8 alone increased the expression of MYC2,a key component of jasmonic acid signaling,which was 6 times higher than that of CK treatment.On the third day,both the COI and MYC2 genes of the jasmonic acid pathway were significantly increased in the prevention and therapy groups.This study showed that the application of JK-XZ8 reduced the incidence of crown gall in cherry saplings before or after inoculation with the pathogen,but the prevention treatment was better.Both the enzyme activities and the expression of defense-related genes showed that the biocontrol bacterium induced plant resistance via not only ISR but also SAR in cherry saplings,thereby preventing crown gall.By testing the growth of strain JK-XZ8 in four different organic fertilizers,it was found that it grew well in chicken manure.The chicken manure was combined with the biocontrol bacteria JK-XZ8 for field experiment.One month after the treatment,it was found that the number of A.tumefaciens in the rhizosphere of cherry blossoms was significantly lower than that of CK and K84 in the treatment of applying biocontrol bacteria alone or in combination with organic fertilizer.At the same time,the germination rate of cherry tree saplings in saline-alkali soil can be improved to a certain extent.The biocontrol bacteria JK-XZ8 can prevent and cure cherry blossom crown gall in the field by up to 50%,which is better than the K84 biological fertilizer(24%)used in the market.At the same time,the control effect of the combination of biocontrol bacteria and organic fertilizer was 37.5%.They are also better than K84.By measuring the plant height and diameter of cherry blossoms under different treatments,it was found that there was no significant difference in net plant height growth and diameter between different treatments.Using high-throughput sequencing technology to further analyze the changes of the microbial community in the rhizosphere soil of the cherry blossoms in the wild,the results showed that after one month of treatment,each treatment would reduce its unique OTU number,and the application of biocontrol bacteria JK-XZ8 alone reduced the most.The unique OUT number of JK-XZ8 and organic fertilizer compound is second only to CK treatment;after seven months of bacterial application,different treatments have increased its unique OUT number,and the compound treatment of biocontrol bacteria JK-XZ8 and organic fertilizer increases the most.However,no matter at what stage,the treatment with application of biocontrol bacteria JK-XZ8 alone had a higher abundance than other treatments.At the same time,the Tax4Fun function prediction showed that the functional performance of each treatment was the same under different treatments and time,but the functional intensity was different.In the short term,the application of biocontrol bacteria JK-XZ8 alone can improve the metabolic function of the soil;single application of chicken manure or K84 bacterial fertilizer treatment can improve the function of cellular processes.In the longer term,the effect of single application of chicken manure on the rhizosphere soil function of cherry blossoms was basically the same as that of CK,while the single application of antibacterial treatment improved its cellular process and environmental information processing function.Throughα-diversity analysis,it was found that there was no significant difference in species richness and diversity in the rhizosphere soil community under different treatments in the twoperiods.These results indicated that biocontrol bacteria JK-XZ8 or its combination with organic fertilizer will not affect the stability of the overall community structure of the cherry rhizosphere,and was beneficial to the soil microbial community to some extent.Crude extraction of the biocontrol strain JK-XZ8 and its antagonism with cherry crown gall disease pathogen PX-1 showed that there were substances in the crude extract which had antagonistic effects against A.tumefaciens PX-1.The bacteriostatic substances were resistant in high temperature,acid and alkali,and could not be degraded by proteinase K and ultraviolet rays.The strain contains lipopeptide genes bmy B,itu D,and fen D,and polyketide genes PK1,PK2,and PK3,which were detected by PCR. |
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