| Tobacco bacterial wilt is a typical soil-borne disease with a wide range of occurrence,heavy harm and difficult control,Researchers or agricultural practitioners have made a lot of attempts to control tobacco bacterial wilt,including the implementation of fallow crop rotation,screening and breeding of resistant varieties of tobacco bacterial wilt,the use of chemical agents,etc.The compre-hensive use of these means has alleviated the occurrence of tobacco bacterial wilt to some extent,but in practical application,the effective prevention and control of tobacco bacterial wilt still faces many limitations,and biological control itself has the advantages of green,non-pollution,safety and low cost.In this study,biocontrol strains with antagonistic effect on tobacco bacterial blight and good control effect on tobacco bacterial wilt were screened by collecting rhizosphere soil of non-pathogenic tobacco strains,then isolate and identify the bacteria.1.Twenty-four strains of bacteria with antagonistic effect on tobacco bacterial wilt were screened out from the 184 strains,and LSW-4 was screened out through subsequent pot experiment and field experiment,which was identified as Pseudomonas fluorescens with effective prevention and control of tobacco bacterial wilt.A total of 24 antagonistic bacteria were screened out from 184 strains of tobacco rhizosphere soil by plate confrontation method.Subsequently,LSW-4,an antagonistic bacterium with better control effect on tobacco bacterial wilt,was screened out through pot experiment and field experiment.Morphological and transmission electron microscopy observation,16 S r DNA sequencing analysis,growth curve determination,whole cell fatty acid methyl ester determination,Phylogenetic analysis of gyr B(Gyrase B subunit),rpo B(B subunit of RNA polymerase)and rpo D(D subunit of RNA polymerase)gene sequences indicated that LSW-4 was Pseudomonas fluorescens.2.The pot experiment results showed that root irrigation treatment with antagonistic bacteria LSW-4 could promote the root growth of tobacco plant.It was recommended to use 1×108 cfu /m L and inoculation with antagonistic bacteria LSW-4 3 days before inoculating Ralstonia solanacearum had the best control effect on tobacco bacterial wilt,which could reach more than 80%.The pot experiment showed that although there were no significant differences in average plant height,maximum leaf length and maximum leaf width,there were significant differences in root length between the treatment group and the clear water control group after 20 days.The average root length after LSW-4 treatment was 22.38 cm,while the average root length of the clear water control group was only 16.12 cm.The results showed that when the disease incidence of tobacco bacterial wilt was 66.66% in the blank control group,The disease incidence of the antagonistic bacteria LSW-4 concentration 1×108 cfu /m L,1× 107 cfu /m L and 1×106 cfu /m L were 11.11%,23.61% and 44.45%,respectively,and the relative control efficacy were 83.33%,64.58% and 33.32%,respectively.Evaluation of control effect of LSW-4 on tobacco bacterial wilt with different inoculation time.The results showed that inoculation of antagonistic bacteria LSW-4 in advance for 3 days could effectively prevent and control tobacco bacterial wilt,control efficacy was 69.23%.The effect of early or late inoculation of antagonistic bacteria LSW-4 on the control of tobacco bacterial wilt was not ideal.3.The field experiment results showed that the antagonistic bacteria LSW-4 could effectively prevent and control tobacco bacterial wilt,and the combination of LSW-4 and cow manure fermentation organic fertilizer could improve the control effect of tobacco bacterial wilt.The evaluation of field control effect of different microbial agents on tobacco bacterial wilt showed that antagonistic bacteria LSW-4 treatment could effectively reduce tobacco bacterial wilt,and the final disease incidence of tobacco bacterial wilt in the treatment group was only 10.00%,while the disease incidence of tobacco bacterial wilt in the blank control group was 61.67%.The disease index was 5.78 after treatment with antagonistic bacteria LSW-4,and 28.85 after treatment with blank control.The relative control effect was 79.97%.The results of the evaluation of the prevention and control effect of LSW-4 combined with different organic fertilizers on tobacco bacterial wilt showed that the single application of antagonistic bacteria LSW-4 or the combination of LSW-4 and organic fertilizer had a good control effect on tobacco bacterial wilt.The disease incidence of tobacco bacterial wilt in blank control group was 38.89%,and the disease index was 13.95.When LSW-4 was combined with cow manure fermentation organic fertilizer,the incidence rate was 11.67%,the disease index was 5.12,and the control effect was 63.30%.Antagonistic bacteria LSW-4 had the second highest control effect against tobacco bacterial wilt,with control effect of 63.73%.After single use of antagonistic bacteria LSW-4,the disease incidence was 14.44%,the disease index was 5.06,and the control effect was 63.73%.4.The analysis of microbial community structure in rhizosphere soil of tobacco showed that there were significant differences in the composition of microbial community between the healthy soil and the disease soil at the OTU level.At the genus level,Ralstonia and Pseudomonas were significantly enriched in the affected soil.PCOA analysis was performed on the community structure of bacteria and fungi in the healthy soil(LSH)and the disease soil(LSU)at the OTU level,and the results showed that there were significant differences in the community structure of bacteria and fungi in the unaffected soil and the affected soil,as the sample distance between the groups was greater than the sample distance within the group.LEf Se discriminant analysis was conducted to screen the soil microbial groups significantly enriched in the uninfected or infected soil with significant differences between groups.The results showed that,at the genus level,both Ralstonia and Pseudomonas were significantly enriched in the disease soil.5.LSW-4 root irrigation can significantly change the soil bacterial community.Statistical analysis of β-diversity showed that at the OTU level,on June 23,there was no significant difference between the soil treated with LSW-4 and the blank control,whether it was bacteria or fungi.on July 18,after LSW-4 treatment,the effect on bacterial community was great,and there was a significant difference.Through LEf Se discriminant analysis of soil samples treated with LSW-4,soil samples collected on June 23 showed that Rhodanobacter was significantly enriched after treatment with LSW-4 at the level of bacterial community genus.The fungi with significant enrichment after LSW-4 treatment were Pseudeurotium,Setophoma,Rickenella and Cotylidia.In the soil sample of July 18,Obscuribacterales were significantly enriched after LSW-4 treatment,while at the level of fungi genus,Codinaea and Scytalidium were significantly enriched after LSW-4 treatment. |
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