Pseudomonas Fluorescens R15 Induces Pepper Resistance Gene Analysis And Research On The Prevention And Control Methods Of Blight

Endophytic bacteria is a beneficial bacteria which elonized inside the plant,it could control plant diseases effectively.Now,endophytic bacteria had not been widely used in agriculture.One of the reasons was that the mechanisms against pathogens were unclear and the biocontrol effect could not control.An endophytic bacterium named Pseudomonas fluorescens R15 which had effective control on Phytophthora blight was selected.The results showed that the higher the colonization amount of P.fluorescens R15 in pepper roots,the better the biocontrol effect on Phytophthora blight was.When the colonization number of R15 was 6.3 ×105 cfu/g above,the control effect reached 100%and could maintain 14 days.Afterwards,the colonization amount of P.fluorescens R15 declined gradually,and the biocontrol effect of Phytophthora blight also went down.In order to explore the mechanisms of R15 against Phytophthora blight,cDNA-AFLP technique was used to analyse pepper leaves different genes expressed after challenge inoculation pathogen Phytophthora caaPsici that early inoculation R15,and definited the resistant genes against P.capsici induced by R15 when the amount in pepper roots was higher.Meanwhile,the study was to seek one or more mehods that kept the higher amount of R15 in pepper roots to stably control Phytophthora blight.The cDNA-AFLP technique was used to analyse the resistant genes against P.capsici induced by R15.The results showed that 30 selective amplification primers pairs obtained 137 differentially expressed transcripts derived fragments(TDFs).29 TDFs was successfully sequenced,of which 24 was up-regulated genes and 5 down-regulated.Blastx analyses in NCBI and functional annotations were then performed and the results revealed that 11 TDFs(37.9%of the sequenced total 29 TDFs)were highly homologous to unknown functional proteins genes and had no match to known genes.There were 3 TDFs products which belonged to metabolism,energy,protein destination and storage accounted for 10.3%respectively.Signal transduction,transcription and disease/defense protein accounted for 6.9%respectively.Finally,there was 1 TDF involved in cell structure,cell position and protein synthesis respectively,each accounted for 3.5%.We found that ethylene response element binding protein gene,heat shock protein 70 gene,cytochrome P450 protein gene,amine oxidase gene,calcium dependent protein kinase gene were involved in plant disease resistance according to previously reports.Four differential genes from TDFs were chosen for further qRT-PCR expression patterms to evaluate the validation of cDNA-AFLP.The results showed that these genes expression patterns confirmed the cDNA-AFLP profiles.In order to seek one or more mehods that kept the higher amount of R15 in pepper roots,using repeated cutting roots,repeated roots-cutting inoculation,repeated irrigating urea solution,repeated irrigating inoculation treated peppers that had been inoculated by endophytic bacterium R15 in the experiments.The results showed that the repeated cutting roots and repeated irrigating urea solution could not keep the colonization number of R15,and the control effect of Phytophthora blight was under the effect used by farm chemicals;The quantity of R15 significantly increased after each roots-cutting inoculation treatment.However,the control effect on phytophthora blight went down after cutting roots several times.The quantity of R15 maintained at 4.1 ×105 cfu/g above after repeated irrigating inoculation.What’s more,lhe biocontrol effect also could keep the leval at 90%～100%.Therefore,when the endophytic bacterium R15 was first inoculated in the peppers,the disease incidence would obviously decline after irrigating inoculation at every 15～20 d after inoculation.