Screening Of Compound Agents For Control Of Tobacco Target Spot Pathogen And Study On Biocontrol Bacterium And Fungal Virus

Rhizoctonia solani Kühn is a plant soil-borne pathogenic fungus with a wide host range,infecting at least 260 species of plants worldwide and causing serious damage to many crops.Tobacco target spot is a foliar disease of tobacco caused by the asexual stage of R.solani fusion group AG-3,which has broken out in many areas of China in recent years and has the tendency to spread nationwide.Currently,tobacco target spot disease is mainly controlled by chemical agents,combined with agricultural measures.However,the disease mainly occurs during the peak and maturity of tobacco,with short incubation period and strong epidemic,and once it occurs,the loss is serious.Therefore,it is of great theoretical and practical significance to find efficient and safe chemical and biological control methods to reduce chemical application and scientifically control tobacco target spot disease.In this study,we investigated the effective control pathways of tobacco target spot disease by screening effective chemical and biological compound formulations,screening of biocontrol bacteria and fungal viruses,and evaluation of control efficacy.The specific results of the study are as follows:1.Screening of effective compounding formulations of chemical and biological agents and field efficacy research.（1）The strain CZ-56 was isolated from tobacco rhizosphere soil in tobacco fields affected by tobacco target spot disease in Chenzhou,Hunan province by dilution coating method.The average inhibition rate of cz-56 was 83.33±0.68%,and it affected the growth of mycelium.The strain CZ-56 was identified as Pseudomonas mosselii by morphological characteristics,physiological and biochemical analysis and multi-gene phylogenetic analysis.（2）The optimal formula was selected from 45 groups of compound formulations and its control effect was determined in the field.The six agents were combined according to five mass ratios to obtain the optimal formula:when the complex ratio of flononil and butocurphenol was 1:1,the synergistic effect was the best,EC₅₀value was 0.032 mg/L,synergistic coefficient was 3.06,showing synergistic effect.The relative control effect of field test was 77.44%,which was better than that of single agent.2.Screening and identification of antagonistic bacteria against tobacco target spot disease,determination of prevention efficacy and growth promotion potential.（1）Using the dilution coating method,711 strains were isolated from the inter-root soil of healthy tobacco in Chenzhou,Hunan China,and 13 strains were found to have more than70%inhibition of tobacco target spot by the plate standoff test,among which the average inhibition rate of strain CZ-56 was 83.33%.The strain CZ-56 was identified as Pseudomonas mosselii by combining morphological characteristics,physiological and biochemical and multi-gene phylogenetic methods.（2）The prevention and treatment effects of the strain were analyzed by spraying in vitro leaves and living pot.The results of in vitro leaf inoculation showed that fermentation liquor treatment of tobacco could effectively prevent and treat the expansion of target spot disease.In the experiments of potted inoculation prevention and treatment,the fermentation broth treatment had better inhibition effect on tobacco target spot pathogen.（3）The growth promoting potential of CZ-56 on tobacco was analyzed by root irrigation.It was found that the fermentation broth and 6.4×10⁸CFU/m L bacterial suspension could promote the growth of tobacco seedlings to varying degrees,indicating that CZ-56 could promote the growth of tobacco seedlings and the accumulation of nutrients.（4）CZ-56 increased Polyphenol oxidase（PPO）,Peroxidase（POD）,Superoxide dismutase（Superoxide dismutase）in tobacco seedlings.SOD,L-phenylalanine ammoniate-lyase（PAL）enzyme activity.The activity of PPO,SOD,PAL and POD resistant enzymes in leaves of tobacco seedlings treated with fermentation broth was significantly increased.The results showed that the fermentation broth of strain CZ-56 could promote the increase of resistant enzyme activity of tobacco.3.Screening,molecular identification and biological functions of fungal viruses of tobacco target spot pathogens on host fungi.（1）Twenty-eight strains of tobacco target spot disease isolated from Hunan and Yunnan were subjected to ds RNA extraction,and five strains with different ds RNA band types were obtained,and the comparison of pathogenicity revealed that strains zlcy-4 and zlcy-2 were less pathogenic.Strain zlcy-2 was selected for the next step of molecular biology and biological characterization.（2）At least 11 ds RNA sequences were found in strain zlcy-2 by ds RNA high-throughput sequencing and conventional sequencing,including 7 RNA coding-dependent RNA polymerase（Rd Rp）.Based on sequence homology alignment,Two viruses belonging to Mitoviridae were identified as Rhizoctonia solani mitovirus 106（Rs MV106）and Rhizoctonia solani mitovirus 107（Rs MV107）.One belonged to Botourmiaviridae and was named Rhizoctonia solani botourmiavirus 9（Rs BV9）;Four belonged to the Partitiviridae family,they have been named Rhizoctonia solani partitivirus 16（Rs PV16）,Rhizoctonia solani partitivirus 17（Rs PV17）and Rhizoctonia solani partitivirus 18（Rs PV18）.All viruses in zlcy-2 were transferred horizontally to non-toxic strain Y-11 to obtain derivative strains Y-2-1,Y-2-2 and Y-2-3 by mycelial standoff culture and RT-PCR assay,and it was found that the virulent strains showed a decrease in the number of nuclei and pathogenicity.Further,strain Y-2-2 was confronted with non-virulent strain Y-11,and derivative strains Y-2-2-1 and Y-2-2-2 containing all viruses and Y-2-2-3 lacking Rs MV107 and Rs PV8 viruses were obtained,and comparison of pathogenicity revealed that strains Y-2-2-1,Y-2-2-2 and Y-2-2-3were also less pathogenic,but strain Y-2-2-3 was less pathogenic compared to non-toxic strain Y-11,the pathogenicity of strain Y-2-2-3 was reduced to a lesser extent compared with that of virus-free strain Y-11.Since Rs PV8 was reported to have no significant effect on host virulence,it was speculated that Rs MV107 or Rs MV107 combined with Rs PV8 affect the virulence of the strain zlcy-2,which was related to hypovirulence.Furthermore,in vitro inoculation and prevention test with tobacco leaves was conducted.It was found that after pretreatment with virus-containing strain zlcy-2,the disease degree of the virulence strain was significantly reduced,suggesting that zlcy-2 and its associated virus had biocontrol potential.In summary,the present study screened effective compound formulas from pharmaceuticals,antagonistic bacteria with growth-promoting effect and host resistance induction,and viruses with weak virulence characteristics to the pathogenic bacteria,which provided effective practical materials and theoretical basis for further control of tobacco target spot disease,and also provided reference for the control of other diseases caused by other R.solani.