Construction Of Engineering Agent For Biocontrol Of Radopholus Similis And Study Of Its Biocontrol Effectiveness And Fermentation Systems

Radopholus similis(Cobb,1893)Thorne,1949 is an important plant parasitic nematode,widely distributed in tropical and subtropical regions.It also has been listed as a quarantine plant pest in China.In addition to doing seriously damage on baana(Musa spp.)growth as the main cause of banana yield loss in the world,it also endangers food crops,fruit trees,vegetables and various ornamental plants.R.similis is still a worldwide problem,pesticide is commonly used as a more effective approach to control R.similis.However,chemical pesticides using has great harm to human and animal,plant and other beneficial organisms,results in increasing restrictions in many countries and regions.Therefore,it has extremely urgent to exploring new strategies and new approaches to control R.similis.biological control because of its characteristics of environmental protection,economical and sustainable,is attracted more and more attention and research.Nematophgous microorganism's extracellular protease was proved to be an important virulence factors in the infection against nematodes.In this study,we constructed a microbial metagenomics fosmid library of banana rhizosphere soil that occured R.similis,and screened a nematicides active protease gene(pase4)from the metagenomics library.In addition,we screened an advantage associated bacteria strain “Pseudomonas fluorescens(pf36)” from ten different R.similis populations.On this base,we transfered the nematicides active gene pase4 into pf36 cell by vector transformation and triparental mating transduction and made gene pase4 express correctly.Then used mediator pf36 strain,which could expressed nematicides active gene pase4,to suppress the growth development and infection of R.similis,so as to achieve the purpose of prevention and control of R.similis.In the end,we determined and evaluated the biological characteristics,protease production,genetic stability,soil colonization ability,nematicides activity in vitro,biocontrol effect and crop safety of the genetic reconstruction strain.The main results of study are listed as follows.1.We carried out the high-throughput sequencing and species diversity analysis of bacteria,archaea 16 S rDNA and fungal 18 S rDNA of banana rhizosphere soil,it found that the richness and diversity from high to low were followed by bacteria,fungi,archaea,and the dominant groups were Acidobacteria GP1,Penidiella and Fervidicoccus,respectively.There were a certain percentage of unclassified sequences in each group.2.We constructed a rhizosphere soil microbial metagenomics fosmid library.This library contained about 35000 clones with the average insert fragment of 30 kb,including 1.05 Gbp microbial genetic information.At the same time,six fosmid clones(Pro1~Pro6)with protease activity were obtained by function-driven screening method.The subclone library wsa constructed and subclone Pro1',Por4' and Pro6' which had protease activity were screened.The fermentation supernatants of the three subclones(Pro1',Por4' and Pro6')all had varying degrees of nematocidal activity,and Por4' was the best treatment.3.The insertion sequence analysis of subclones Pro1 ',Pro4' and Pro6 'showed that each insert sequence contained a complete open reading frame(ORF),each ORF encoded the corresponding protease(PASE1,PASE4 and PASE6),respectively.All three proteases were secreted hydrophilic proteins.4.We obtained the mature protease PASE4(about 32 kDa)by prokaryotic expression.It was confirmed that PASE4 within significant nematocidal activity could effectively degrade the tissue of R.similis.In addition,we first established a method for breeding aseptic R.similis by using pure carrot callus.5.We obtained fifty-three associated bacteria strains that were isolated from ten different R.similis populations,and confirmed that Burkholderia cepacia,Pseudomonas fluorescens and Bacillus cereus were the dvantage associated bacteria strains of the tested nematode population.We selected Pseudomonas fluorescens pf36 as a target strain for being to modify.6.We transfered the nematicides active gene pase4 into pf36 cell by vector transformation and triparental mating transduction,and obtained genetic modified strains p4MCS-pf36 and p4Tn5-pf36.The p4MCS-pf36 had higher protease production and nematocidal activity than p4Tn5-pf36,but p4Tn5-pf36's genetic stability,soil colonization capacity and potting control effect is better than the p4MCS-pf36.7.We determined the optimized fermentation system for p4Tn5-pf36 producing protease: culture components were glycerol 10 mL/L,yeast extract 15 g/L and KCl 1 g/L;culture conditions were temperature 34.5?,initial pH 7.66,inoculation quantity 8.0%,rotational speed 274 r/min,protease activity after optimization reach up to 109.928 U/mL,1.58 fold increased.In conclusion,this study screened a protease gene pase4 with strong nematocidal activity from the microbial metagenomicslibrary of banana rhizosphere soil,and obtained an advantage associated bacteria strain “Pseudomonas fluorescens(pf36)” isolated from different R.similis populations.The gene pase4 was successfully transfered into pf36 strain by molecular biology method,and expressed correctly.Obtained a genetic stable strain with a certain biocontrol effect on R.similis.Preliminary achieved the purpose to suppress the growth development and infection of R.similis mediated by associated bacteria expressing the biocontrol factor.The result provide an new efficient and safe method for prevention and cotrol of R.similis and set up a new efficient,environmental protection and sustinability strategy and way to control the plant parasitic nematodes.