Establishment And Application Of Multiplex PCR And Real-time PCR Molecular Detection System Of Soil–borne Fungal Pathogens In Wheat

Soil-borne diseases is one of the most important diseases in wheat. At present, the diseaseis spread quickly in wheat-growing areas in our country, and has a tendency to increase year by year,which makes it a serious threat to food production safety. The common soil-borne diseases in wheat aresharp eyespot, take-all, common root rot and crown rot. The symptoms among the soil bornepathogens are similar and the four diseases always occur mixed. At the same time, the traditionalmorphological identification of different fungi is time consuming and not suitable for rapiddiagnosis, so an effeicient and accurate method is significant and required.This research aims atestablishing a rapid, sensitive and simple detection technology for soil-borne disease by exporatorymolecular technologies research. The main discoveries of this study are the followings.（1）The establishment of Multiplex PCR detection systemTwo pairs of primers were designed according to the β-tubulin gene of Gaeumanomyces.graminis var.tritici and the ITS gene of B. sorokiniana, that is TAF/R、RbF/R. At the same time,take Fusarium graminearum specific primer Fg16NF/R and F.pseudograminearum specificprimer Fp1-1、Fp1-2as references. The multiplex PCR reaction solution was optimized. Theresults show that the amplification PCR products were consistent with the expected results and thefive pairs of primers were highly specific without non-specific bands. Consequently, the fiveprimers can distinguish the five pathogens effectively. The sensitivity assay showed that multiplexPCR detected as little as0.39ng of mycelium DNA. Infected wheat samples were collectedrandomLy from Fengqiu, Qinyang, Yuanyang and Taikang in Henan Province, and DNA wasextracted from the stem base of wheat and amplified using multiplex PCR. The pathogens thatoccurred in the four areas varied with R. cerealis and F. graminearum found in Fengqiu, R.cerealis,F. pseudograminearum and F. graminearum in Qinyang while both Fengqiu samples and Qinyangsamples were suspected WSE. All the tested pathogens except F.pseudograminearum are detectedin Zhoukou, and all the tested pathogens except G. graminis var. tritici are found in Yuanyang,while both Zhoukou samples and Yuanyang samples were suspected to be take-all. （2）The establishment of Real-time PCR detection systemSpecific primers for Real-time PCR are designed on the basis of β-tubulin gene of R.cerealisand G.graminis var.tritici and ITS gene of B.sorokiniana. With these primes、Fg16NF/R andFp1-1、Fp1-2, SYBR Green I Real-time PCR methods for each pathogen was established. Everydetection reaction reached a good amplification effect by optimizing the annealing temperature.For each primer, pathogen DNA standards and plasmid standards were prepared. According to the10times dilution gradient, DNA standard curves and plasmid standard curves were established,which all had a high linearity and good amplification efficiency.（3）Indoor potted experimentsIndoor potted experiments of five pathogens were carried out. The Real-time PCR detectionresults showed that, for R.cerealis and F.graminearum, when the inoculation amount was higherthan1g/100g soil, the pathogen can be detected after5days when sowing. When the inoculationamount was0.25g/100g soil, R.cerealis can be detected after10days when sowing, andF.graminearum can be detected after25days. For F. pseudograminearum, when the inoculationamount was8g/100g soil, it needed25days for detecting the pathogen, and when the inoculationamount was0.5g/100g soil, it needed60days. For G. graminis var. tritici and B.sorokiniana, theexperimented wheats didn’t have any symptoms and we did not detect any pathogen after60days.The Real-time PCR detection of the samples has a significant positive correlation with inoculationamount and disease index.（4）Detecting technology integrationA quick detecting technology for field samples is eatablished, that is, field samples are firstlydetected by multiple PCR for pathogenic species, and then quantitative analysis will be done byReal time PCR. This assay takes Xiping samples as an example for the qualitative and quantitativeanalysis of pathogenic fungi.