Study On Colonization Characteristics Of Paenibacillus Polymyxa In Tobacco Plants And Its Control Effect On Tobacco Bacterial Wilt

Ralstonia solanacearum is the pathogen that causes bacterial fusarium wilt of tobacco,and it poses a huge threat to the healthy cultivation of tobacco.The use of biological control agents is one of the important methods to prevent the infection of pathogenic bacteria.Paenibacillus polymyxa is a kind of peripheral flagellated rod-shaped bacteria that widely exists in soil,plant rhizosphere and tissues,and can produce a variety of Enzymes and secondary metabolites,coupled with the ability to form biofilms on plant roots,are also used as an effective biological control agent.The efficient colonization ability of biocontrol bacteria is the prerequisite for its stable biocontrol effect.Therefore,the colonization of Paenibacillus polymyxa in tobacco plants is very important for the control of tobacco bacterial wilt.In addition,the process of bacterial colonization in tobacco plants is affected by biological and non-biological factors,among which indigenous microorganisms are a factor that cannot be ignored in bacterial colonization.Based on this,this article designed an indoor pot experiment to observe and determine the colonization of Paenibacillus polymyxa in the roots,stems and leaves of tobacco plants by scanning electron microscopy and fluorescent quantitative PCR technology;secondly,analyze the Paenibacillus polymyxa by processing at different times Research on its colonization in tobacco roots and its relationship with bacterial wilt;finally,the colonization ability of indigenous bacteria on Paenibacillus polymyxa and its influence on tobacco bacterial wilt were investigated through indoor and field experiments.The main results of the study are as follows:1.The biofilm formation ability of Paenibacillus polymyxa and its colonization on the root surface of tobacco plants are clarified,and the colonization dynamics of Paenibacillus polymyxa in the roots,stems and leaves of tobacco plants are analyzed.The biofilm formation ability of Paenibacillus polymyxa was determined.The crystal violet method was used indoors and it was found that the absorbance value of Paenibacillus polymyxa at 600 nm measured at 24 h was 1.432,which was significantly higher than the 0.562 of the control Bacillus subtilis,indicating that Paenibacillus polymyxa had a strong The ability to form biofilms.Observe the colonization of Paenibacillus polymyxa on the root surface of tobacco plants,use confocal scanning electron microscopy to observe that Paenibacillus polymyxa can colonize the root tip,middle of the root,and the surface of the root base of the tobacco plant on the 3rd day after treatment.The quantity is Root tip>Root middle>Root base.The dynamic changes of the colonization of Paenibacillus polymyxa in the roots,stems and leaves of tobacco plants were analyzed,and the fluorescence quantitative PCR technology was used to determine that the colonization law of Paenibacillus polymyxa in the roots,stems and leaves of tobacco plants increased first and then decreased.Among them,a large number of colonizations were carried out at the roots of tobacco plants,and the highest number of colonizations in the stems of tobacco plants was 4.27×1011 copies/g stem tissue.2.Analyzed the competitive colonization of Paenibacillus polymyxa and Ralstonia solanacearum on the roots of tobacco plants,and evaluated the effect of early colonization of Paenibacillus polymyxa on the occurrence of tobacco bacterial wilt.The competitive colonization of Paenibacillus polymyxa and Ralstonia solanacearum in the roots of tobacco plants was explored,and the colonization amount of Paenibacillus polymyxa under the influence of Ralstonia solanacearum under the influence of Ralstonia solanacearum was 5.09×108copies/g.The root tissue was significantly lower than that Paenibacillus polymyxa was treated alone,and the colonization of Ralstonia solanacearum was also reduced 1-3 days before the colonization of Paenibacillus polymyxa.Analyzed the colonization of the tobacco plant roots of Paenibacillus polymyxa treated at different times and its relationship with the occurrence of tobacco bacterial wilt.Through indoor quantitative determination and pot experiment,it was found that the earlier the treatment time of Paenibacillus polymyxa,the stronger its colonization ability.,The more stable the colonization.At the same time,the earlier the treatment time of Paenibacillus polymyxa,the lower the incidence and disease index of tobacco bacterial wilt.3.The influence of indigenous bacteria on the biofilm formation and colonization ability of Paenibacillus polymyxa was explored,and the combined effect of indigenous bacteria and Paenibacillus polymyxa on the prevention and control of tobacco bacterial wilt was evaluated.Indigenous bacteria that are convenient to Paenibacillus polymyxa have been screened out.The laboratory research has selected 26 kinds of indigenous bacteria through single and mixed culture and quantitative analysis of Paenibacillus polymyxa to select the"convenient type"for the growth of Paenibacillus polymyxa.The 6species of bacteria are Pseudomonas fluorescens,Bacillus amyloliquefaciens,Bacillus pumilus,Bacillus altitudinis and Bacillus stratosphericus Among them,the fermentation broth of Bacillus stratosphere,Pseudomonas fluorescens and Bacillus pumilus can promote the formation of Paenibacillus polymyxa biofilm.To study the influence of indigenous bacteria on the colonization ability of Paenibacillus polymyxa,the laboratory analysis by fluorescence quantitative PCR method found that Pseudomonas fluorescens and Bacillus pumilus significantly promoted the colonization of Paenibacillus polymyxa on the first day after treatment.The colonies of 7.08×10¹²copies/g root tissue and 2.94×10¹²copies/g root tissue were significantly higher than the 3.32×10⁷copies/g root tissue of the control treatment,which promoted the colonization of Paenibacillus polymyxa.The effects of indigenous bacteria and Paenibacillus polymyxa on the occurrence of tobacco bacterial wilt were evaluated.Indoor pot experiment analysis found that the mixed treatment of Pseudomonas fluorescens and Paenibacillus polymyxa could reduce the occurrence of tobacco bacterial wilt,and the relative control effect Compared with the single treatment of Paenibacillus polymyxa,it increased by 1.64times.In summary,this thesis clarified that polymyxa spores first colonized the roots of tobacco plants in large numbers,and then colonized the stems of tobacco plants,and the colonization rule first increased and then decreased.Polymyxa spores were found.The simultaneous treatment of Pseudomonas fluorescens and Ralstonia solanacearum resulted in competitive colonization in the roots of tobacco plants;the early treatment of Pseudomonas fluorescens and the mixed treatment of indigenous bacterium Pseudomonas fluorescens were evaluated to promote its colonization ability on the roots of tobacco plants and improve the The prevention and control effect of tobacco bacterial wilt.The results of this study provide evidence for the application of early colonization techniques for biocontrol bacteria and lay a foundation for guiding the prevention and control of tobacco bacterial wilt.