Study On The Activity And Mechanism Of Trichoderma Harzianum TMN-1 Inducing Tobacco Bacterial Wilt Resistance

Tobacco bacterial wilt is a typical soil-borne rhizome disease in tobacco planting.This disease has a wide range of occurrence and is difficult to control.Once it occurs in a large area,it will have a huge impact on tobacco production.It is one of the main factors that restrict tobacco production and hinder the healthy and sustainable development of the tobacco industry.In the process of modern agricultural tobacco production,methods for controlling tobacco bacterial wilt include rotation and selection of resistant varieties,but the main control methods still rely on chemical control.Biological control is a control method that has been widely used in the prevention and treatment of rhizome diseases in recent years.Biocontrol microorganisms are increasingly favored by researchers because of their wide distribution and easy availability in nature.Compared with chemical control,biological control The prevention itself has the advantages of being green,pollution-free,safe,and low-cost.In this study,a rhizosphere soil was collected from healthy tobacco plants in healthy plots of tobacco bacterial wilt disease area,and a biocontrol fungus Trichoderma harzianum TMN-1 was isolated and purified by dilution coating.Growth and biological activity of tobacco bacterial wilt,combined with indoor and field prevention and treatment of bacterial wilt,through software statistics,to clarify the mechanism and effect of isolated strains on the induction of tobacco bacterial wilt resistance.It provides theoretical basis and practical methods for the application of the biocontrol strain Trichoderma harzianum TMN-1 in the control of tobacco rhizome diseases.1.Isolate,purify and identify a Trichoderma harzianum strain,clarifying the biological characteristics of the strainThe experimental strains were isolated by dilution coating plate method and selective medium,and the isolated strains were biologically identified by optical microscope;the isolated strains were molecularly identified by ITS sequence.The results showed that the isolated strains grew vigorously on the PDA medium,the colony was white at the beginning,the mycelium was flocculent or filamentous,and grew radially from the center,and the lag phase was that the green spore cluster was densely surrounded by the ring or Concentric distribution;the main axis of the conidiophore and the end of each branch of the peduncle 3-5 rounded arrangement;the peduncle ampoule or flask type,the bottom is constricted and narrowed to a thin neck,the top spores;the conidia are spherical or Oval,light green,smooth edges without protrusions.The morphological characteristics are the same as Trichoderma harzianum;ITS sequence alignment results show that the isolated strain is 100%homologous to Hypocrea lixii.Combined with the morphological characteristics,the isolated strain was identified as Trichoderma harzianum and named by Trichoderma harzianum TMN-1.Based on the purified strains,this study continued to develop the effect of Trichoderma harzianum TMN-1 strain on tobacco growth.The effect of different concentrations of Trichoderma spore suspension（1×10⁸ spores/mL,1×10⁷ spores/mL,1×10⁶ spores/mL）on the germination of tobacco seeds was detected on the plate by seed soaking method,and different concentrations were determined The biological effect of the spore suspension on tobacco seeds,and then on the basis of the above concentration using root irrigation to explore the effect of this concentration on the growth of tobacco seedlings.The results showed that after soaked with different concentrations of Trichoderma spore suspension,the tobacco seeds could significantly increase the germination potential of tobacco seeds（P<0.05）.Tobacco seeds reached the peak germination period after 60 h.Compared with the control group,The potential and germination index increased by 22.03%,22.92%,20.86%and 19.33%,36.31%and 23.81%,respectively.After 96 h,the germination rate of tobacco seeds was calculated,and there was no significant difference in germination rate among the treatments.In the greenhouse,the roots of tobacco plants were irrigated with biocontrol strain spore suspension,which can significantly promote the growth of flue-cured tobacco average plant height,average leaf length,average leaf width and maximum root length.The traits were 1.57,1.22,1.16,2.48 times higher than the control group;the aboveground fresh weight,aboveground dry weight,belowground fresh weight,and belowground dry weight treatment groups were 1.79,2.17,3.73,2.94 times higher than the control group.The antibacterial activity of biocontrol strains against Ralstonia solanacearum was initially evaluated by the plate antagonism experiment,and the effect of ethyl acetate extract of the liquid sterile fermentation broth of biocontrol strains on the growth of Ralstonia solanacearum was also evaluated.The results showed that the isolated strains did not show bacteriostatic effects on Ralstonia solanacearum on PDA plates.At the same time,subsequent experiments also showed that the sterile fermentation broth extracts of the isolated strains did not show significant effects on Ralstonia solanacearum in B medium.Of inhibition.Through root irrigation,the effect of different concentrations of spore suspension on tobacco bacterial wilt was investigated,and the applicable concentration of Trichoderma harzianum spore suspension was determined to be 1×0⁸ spores/mL.On the basis of the applicable concentration,Continue to study the effect of different use time on the occurrence of tobacco bacterial wilt.The spore suspension was used through root irrigation at different times.The results showed that root irrigation 3 days in advance can significantly improve the control of Trichoderma harzianum TMN-1 strain on tobacco bacterial wilt.Through the SMSA test,the effect of Trichoderma harzianum TMN-1 on root content of Ralstonia solanacearum in tobacco roots at different time periods was investigated.The results showed that the use of Trichoderma harzianum TMN-1 strain spore suspension to inoculate Ralstonia solanacearum in roots could significantly reduce the content of R.solanacearum in tobacco roots within 1-5 days.2.Clarified the physiological,biochemical and molecular mechanism of the induction of tobacco resistance by Trichoderma harzianum TMN-1 strainOn the basis of clarifying the indoor biocontrol effect of biocontrol bacteria on tobacco bacterial wilt,further determination of peroxidase（POD）,superoxide dismutase（SOD）,phenylalanine ammonia lyase in tobacco leaves（PAL）specific activity,the results show that Trichoderma can significantly induce the activity of peroxidase and phenylalanine ammonia-lyase in tobacco.On the basis of exploring the effect of Trichoderma harzianum TMN-1 strain on the specific activity of defense enzymes in tobacco plants,continue to explore genes related to salicylic acid signaling pathway,jasmonic acid/ethylene signaling pathway PR1a/c,PR2,EFE-26,ACC Oxidase and PDF1.2 relative expression.The results showed that Trichoderma can significantly stimulate the upregulation of the two genes PR1a/c and PR2 in the salicylic acid pathway,and the upregulated expression of the ACC Oxidase gene in the jasmonic acid/ethylene pathway.3.Analyzed the effect of Trichoderma harzianum TMN-1 strain on the microbial community composition of tobacco plant rhizosphereUnder field conditions,biocontrol agents fermented by Trichoderma harzianum TMN-1 strain were applied during the transplanting period of tobacco seedlings,and the rhizosphere soil of tobacco plants was collected during the tobacco harvest period,and analyzed by high-throughput sequencing technology.The community structure of fungal microorganisms and bacterial microorganisms in the soil.The results of alpha analysis showed that the use of Trichoderma can reduce the diversity of soil microbial communities,and the impact on fungi was greater than that on bacteria.The analysis results of the abundance composition of bacterial microbial groups showed that Pseudomonas,Chryseobacterium,Klebsiella,Allorhizobium,Paenarthrobacter,Sphingobium,Variovorax,Pedobacter are highly abundant In the control group.Among them,the abundance of Chrysobacterium,Rhizobium,Paenarthrobacter and Rhabdobacteria was significantly higher than that of the control group.The result of the level analysis of the fungal group shows that the fungi are mainly Fusarium fungi and their abundance accounts for more than 80%of the fungal group.The abundance of Campylospora and Mortierella in the control group was higher than that in the treatment group,and the abundance of Campylospora was significantly higher than that in the treatment group.Screening of the key microbial factors affecting the occurrence of bacterial wilt in the treatment of soil rhizosphere by LEfSe analysis resulted in 7 bacterial groups:Alternaria,Chitinophaga,Chthoniobacter,Filimonas,Parafilimonas,and 7 fungal groups:Cyberlindnera,Apiotrichum,etc.can be used as potential indicator bacterial strains to inhibit bacterial wilt.4.The fermentation conditions of Trichoderma harzianum TMN-1 strain biocide were explored,and its indoor relative control effect against bacterial wilt could reach 61.56%,and the field relative control effect could reach 56.8%.Under laboratory conditions,the effects of different solid fermentation substrates,different inoculation amounts,different water contents,different inoculation concentrations,and different fermentation temperatures on solid fermentation products were explored.At the same time,the best fermentation conditions obtained in the laboratory were explored Next,the impact on the fermentation cycle of biocontrol strains.The results show that under sterile conditions,rice husk powder is the best solid fermentation substrate.The optimal conditions for solid fermentation of Trichoderma harzianum TMN-1 strain are:solid fermentation at 28℃,while maintaining the initial moisture content of the fermentation substrate at 30%-50%,and the inoculation amount is not less than 4%The best fermentation effect of Trichoderma can be achieved.The fermentation period is 7-8 days.The optimal concentration of Trichoderma spores in the fermentation product can reach about 1×10¹⁰ spores/g.The indoor pot experiment results of the fermentation agent showed that the solid fermented Trichoderma agent can effectively prevent and control the occurrence of tobacco bacterial wilt,and the relative control effect against bacterial wilt could reach 61.56%;Trichoderma can effectively promote the growth of tobacco,and also has a good control effect on tobacco bacterial wilt,and the relative control effect can reach 56.8%.