Effects Of Plant Viral Inhibitor FTDP On Fungal Community Structure And Diversity In Tobacco Rhizosphere Soil And Screening Of TMV Biocontrol Bacteria

Tobacco mosaic virus（TMV）belongs to the typical positive single stranded RNA model virus of the Tobamovirus.TMV has a wide host range,causes severe damage and casualties,and is difficult to prevent and treat.The research,development and application of antiviral biological drugs are of great significance for the prevention and control of TMV.Rhizosphere soil microorganisms play an important role in rhizosphere environment,which can directly or indirectly affect the growth state of plants,and may also affect the efficacy of biological pesticides.It is of great significance for ecological security assessment and sustainable development of biopesticides to study the response of microbial changes in tobacco rhizosphere soil to biopesticides and to mine new microbial resources to control TMV from the dominant bacteria enriched by biopesticides.Previous laboratory studies have shown that fTDP,a prokaryotic expression product,can induce resistance to TMV in common tobacco and significantly affect the abundance and structure of tobacco rhizosphere bacteria during its use,but its effect on tobacco rhizosphere fungi has not been studied.In this experiment,three sites were selected in the tobacco planting base of Tanxi Township,Lichuan County,Fuzhou City,Jiangxi Province during the tobacco harvest period in July 2020 and June 2021.Site A（116.96°E;27.26°N）,Site B（116.96°E;27.27°N）,Site C（117.00°E;27.27°N）to collect soil for growing common tobacco K326.Treatment is as follows:Blank:spray tobacco leaves with distilled water;CK:TMV was inoculated by friction 24 hours after spraying tobacco leaves with distilled water.;TV:TMV was inoculated 24 h after fTDP protein spray.After 20 days,the rhizosphere soil was collected and ITS amplicon sequencing was used to determine and analyze the fungal community diversity of tobacco rhizosphere soil.At the same time,culturable biocontrol bacteria were isolated and screened from tobacco rhizosphere soil microorganisms enriched with fTDP and the resistance of fermentation broth to TMV was tested.The results are as follows:（1）Ascomycetes,Chytromycetes and Basidiomycetes were the dominant fungal phyla in the three treatment groups in the three regions A,B and C,and the horizontal community structure of the phyla in the three regions was similar but the abundance was different.Ascomycota had the highest abundance in region A,which were 35.21%,35.77%and 44.88%（Blank A,TVA,CKA）,9.44%,32.63%and 18.72%（Blank B,TVB,CKB）,and 20.59%and 23.49%in region C,respectively.10.46%（Blank C,TVC,CKC）.The abundance of Chytridomycta was the highest in region B and C,with 15.90%,13.61%and 22.11%in region A（Blank A,TVA,CKA）,39.63%,49.03%and 62.83%in region B（Blank B,TVB,CKB）and 47.70%,45.34%,52.10%（Blank C,TVC,CKC）in region C.Basidiomycetes were the third dominant fungal phylum（Blank A:2.88%,TVA:5.07%,CKA:2.11%;Blank B:6.33%,TVB:3.00%,CKB:9.67%;Blank C:9.14%,TVC:5.59%,CKC:3.56%）.（2）Eurotiomycetes was the endemic fungal class with the highest abundance in region A,12.50%,17.40%,26.27%（Blank A,TVA,CKA）in the three treatments,respectively.Sordariomycetes was the second dominant class,and the abundance in Region A was 20.77%,5.61%,16.20%（Blank A,TVA,CKA）.In region B,7.23%,27.55%,15.58%（Blank B,TVB,CKB）,and in region C,Blank C was 15.84%,respectively.TVC:17.96%;CKC:7.93%.Chytridomycetes were the fungi class with the highest abundance in region B and C,showing in region A:15.80%,13.42%,22.00%（Blank A,TVA,CKA）,in Region B:37.80%,48.32%,61.88%（Blank B,TVB,CKB）,in region C:46.51%,45.00%,51.35%（Blank C,TVC,CKC）.Agaricomycetes was the third dominant class in region B and C,with 6.30%,2.90%,9.42%（Blank B,TVB,CKB）in region B and 8.79%,3.79%,3.29%（Blank C,TVC,CKC）in region C,respectively.（3）Compared with CKA,the abundance of Humicola（1.82%）,Mortierella（1.55%）,Fusarium（1.29%）,Pluteus（1.07%）and Zopfiella（0.03%）,increased in TVA.In region B,compared with CKB,TVB increased Humicola（13.10%）,Phlyctochytrium（4.41%）,Penicillium（0.90%）,Chaetomium（0.79%）,Talaromyces（0.71%）,Aspergillus（0.22%）,Mortierella（0.10%）and Pluteus（0.03%）.In the C region,Compared with CKC,the contents of TVC increased by Penicillium（1.58%）,Fusarium（1.23%）,Saitozyma（1.23%）,Zopfiella（0.97%）,Heliocybe（0.93%）,Talaromyces（0.84%）,Funneliformis（0.81%）,Mortierella（0.36%）,Idriella（0.09%）,Coprinus（0.07%）and Clitopilus（0.01%）.In regions A,B and C,the same dominant bacteria genus in the rhizosphere of tobacco treated with fTDP protein was Talaromyces.In addition to Talaromyces,there were other dominant bacteria genera in regions A and B,including Mortierella,Chaetomium and Claviceps.The dominant bacteria in C region were different from the other two places.The changes of rhizosphere soil microbial communities in different regions may be related to the physical and chemical properties of NH₃,TN,TP,NO₂^-and p H in soil,and soil fungi have different responses to the changes of different physical and chemical properties.（4）Culturable microorganisms were identified and the anti-TMV effect of fermentation broth was screened.The tobacco rhizosphere soil of TV group was collected to prepare soil bacterial suspension.The bacterial solution was coated on LB nutrient agar by dilution plate coating method.Single colonies were selected for isolation and purification,and ten strains with different colony characteristics were co-cultured.After 16S rDNA was determined,BLAST comparison was conducted on NCBI,they are Rhodococcus sp.,Microbacillus sp.,Pseudomonas sp.And Streptomyces sp..Among them,three strains had good resistance to TMV:HXC1and Rhodococcus sp.strain HBUM200062 had the highest similarity rate of 99.77%,which was preliminarily identified as Rhodococcus.The similarity rate between XZN1 and Pseudomonas sp.P7 was the highest of 99.08%,which was preliminarily determined as Pseudomona.And the similarity rate of XZX1 to Microbacterium sp.strain FT211 was 99.00%,which showed that it was Microbacterium.After 24 hours of applying fermented broth to tobacco plants,followed by inoculation with TMV,leaf samples were collected at 1,3,5,7,and 9 days to evaluate their resistance against TMV.Tobacco plants treated only with TMV served as the control group（CK）.The quantification of TMV-CP using real-time quantitative PCR revealed that HXC1,XZX1,and XZN1 could suppress TMV proliferation.Among the HXC1 and XZX1 groups,the TMV-CP content was lowest on the seventh day,while for the XZN1 group,it was lowest on the fifth day.Enzymatic assays related to plant defense showed that compared to the CK group,the POD,PAL,and SOD activities in the tobacco plants treated with fermented broth were enhanced.Analysis of POD activity indicated that the HCX1 group reached its peak on the ninth day,with a 364.29%increase,while the XZX1 and XZN1 groups reached their peaks on the third day,with 400%increases for both.PAL activity measurements showed that the HCX1,XZX1,and XZN1 groups reached their peaks on the ninth day,with increases of 17.95%,94.87%,and192.31%,respectively.SOD activity measurements revealed that the HCX1 and XZN1 groups reached their peaks on the ninth day,with increases of 229.27%and278.50%,respectively.The strains HXC1,XZX1,and XZN1 may possess potential biocontrol properties against TMV.The fungal community structure of the tobacco rhizosphere soil treated with fTDP protein was similar in phylum and class level,but different in content in region A,B and C.The enrichment of different beneficial bacteria genera at the genus level changed the structure of fungal community and enriched the community diversity.And three strains with good resistance to TMV were screened to provide resources for green biopesticide.