Effects Of Anti-TMV Active Substance FTDP On The Structure And Diversity Of The Rhizosphere Microbial Community Of Tobacco

Tobacco mosaic virus is a highly infectious and hostile plant virus that causes serious economic losses to agricultural production.At present,the prevention and treatment of TMV is still dominated by chemical drugs,but chemical drugs are not easy to degrade and affect the ecological environment.Therefore,the developing of green environmental protection antiviral biological drugs is of great significance.Protein TDP(TEER-decreasing protein)was extracted from Flammulina velutipes.Direct extraction and prokaryotic expression of recombinant protein f TDP can enhance the activity of defense enzymes and the expression of defense genes in tobacco plants,activate salicylic acid signaling pathway,and induce systemic acquired resistance(SAR)in plants.TDP,as novel anti-TMV active substances,has been well studied for their disease resistance mechanisms on the above-ground parts of tobacco plants,but has not been placed in the ecological context in which they are found.Rhizosphere microorganisms are one of the most complex ecosystems,which are pivotal to plant growth and immunity.The application of biopesticides may change the rhizosphere microbial community structure,affect the rhizosphere micro-ecological environment,and further affect plant growth.The changed rhizosphere microorganisms may also affect the antiviral efficacy of biopesticides through plants.Therefore,a comprehensive assessment of the effect of TDP proteins on the diversity of the rhizosphere microbial community of tobacco is an important guide for commercial production and field use.In this experiment,common tobacco K236 was used as the study target.Tobacco leaves were inoculated with TMV by friction after 24 h of protein f TDP spraying,and the TMV content was measured by quantitative enzyme-linked immunoassay.The results showed that the content of TMV in leaves was significantly decreased under the action of 200 μg / m L f TDP protein(P < 0.05).Therefore 200 μg/m L was determined to be the optimum concentration for use and this concentration was selected for testing in subsequent studies.During the tobacco harvest period in July 2019 and June 2020,different soil samples were collected from three regions(growing tobacco for many years)in Tanxi Township,Lichuan County,Fuzhou City,Jiangxi Province.The analysis of physical and chemical properties showed that the contents of nitrogen and phosphorus in soil A,B and C were 51.563 mg/kg and 5.429 mg/g,54.263 mg/kg and 5.670 mg/g,35.228mg/kg and 4.019 mg/g,respectively.Tobacco seedlings were transplanted after soil drying and sieving,and then three different treatment groups were set up: control group(CK)-spraying water for 24 h and friction inoculation of TMV;experimental group(TV)-sprayed f TDP protein(200 μg/m L)24 h after friction inoculation TMV;blank group(Blank)-healthy tobacco plants sprayed with clear water,with 9 potted plants in each treatment group,and 3 potted plants in each group.Due to the strong infectivity of TMV,the healthy group of tobacco was isolated from the treated group to grow in different spaces.The growth status(maximum leaf length,maximum leaf width,plant height and number of effective leaves)of the tobacco plants in the different treatment groups was measured from 1 to 20 d.In the soil of A area,the plant height of TV group was higher than that of CK group at 15 d and 20 d.In the soil of region B,the leaf width and leaf number of TV group were lower than those of CK group,and the plant height was significantly decreased(P <0.05).In the soil of area C,the leaf length of TV group reached the maximum at the 20 th day;leaf width was significantly higher than CK at15 d(P <0.05).The plant height of TV group was higher than that of CK group in 1-20 d.The results showed that the f TDP protein had no effect on the growth of diseased tobacco grown in soil in areas A and C,and to a lesser extent in area B.Using the above three treatment groups,as well as the fourth experimental group:the experimental group grew to 10 days after the application of f TDP protein.The rhizosphere soil was collected on the 20 th day,and the 16 S r RNA amplicon sequencing technology was used to analyze the microbial community structure diversity of tobacco rhizosphere under different treatments.α-diversity analysis showed that f TDP protein spraying reduced the abundance and diversity of the inter-root microbial community relative to the CK group.Comparing the number of genera annotated in the different treatment groups in the three regions,f TDP application and secondary spraying reduced the diversity of the inter-rhizosphere microbial community structure,but protein spraying increased the relative abundance of beneficial genera,e.g.Compared to the CK group,the spraying of the f TDP protein simultaneously increased the relative abundance of Sphingomonas,Streptomyces,Devosia,KF-JG30-C25 and Ramlibacter in regions A,B and C.The spraying of protein increased the relative abundance of Gemmatimonas,Ellin6067 and Burkholderia-Caballeronia-Paraburkholderia,Dyella in regions A,B and in regions A,C.Also in comparison to CK,protein spraying increased the population abundance of Actinospica,Nocardioides etc.,and significantly increased the populations of Gemmatimonas,Nocardioides etc.in region A.Increased relative abundance of Flavisolibacter,Ralstonia etc.,and significantly increased the number of Ramlibacter in region B.In contrast,the relative abundance of Muribaculaceae,Streptococcus and Acidibacter increased in the C region.Forty-one dominant strains were isolated from three regional TV groups using dilution plate coating,and the morphology of the strains was determined by Gram staining.The results showed that in group A,most of the bacteria were negative Gram stains and only two were Gram-positive.In groups B and C,most of the bacteria were Gram-positive and a few were Gram-negative.After 16 S r DNA sequencing and identification by gene matching,the isolates were assigned to the beneficial genera Arthrobacter 、 Brachybacterium 、 Microbacterium 、 Leifsonia 、 Bacillus 、Pseudarthrobacter、Bacterium、Stenotrophomonas.In summary,the optimum concentration of f TDP protein to be administered in this study was determined to be 200 μg/m L.Tobacco growth status differed between the three different soil environments,while f TDP use had no effect on tobacco growth.However,the application of TDP protein had an important impact on the diversity of virus-infected tobacco rhizosphere microorganisms,increasing the relative abundance of beneficial bacterial genera,providing new information for the analysis of complex antiviral pathways,and the isolation and identification of dominant strains laid the theoretical foundation for the subsequent excavation of TMV-resistant biocontrol bacteria.