A Rapid Detection Method For The Plant Pathogen Phialophora Gregata F.sp.sojae Based On Loop-mediated Isothermal Amplification

Brown stem rot of Soybean(Phialophora gregata f.sp.sojae,PGS)infection of soybean plants caused by brown rot of soybean.The disease was first discovered in 1942 in the US state of Illinois,after repeatedly been reported in soybean growing areas of North America and Canada,the US soybean production suffered a great loss,yield losses of up to 30%,up 66%.PGS is listed as the European and Mediterranean Plant Protection Organization(EPPO)A1 class quarantine pest,it is one of China’s plant quarantine harmful fungi.At present,PGS mainly discover in Brazil,the Midwest and southeastern US states,Canada,Argentina,Egypt,Mexico,the former Yugoslavia,Japan and other places,which takes place particularly severe in north-central United States and Canada,resulting in the local soybean production a great threat.But in our country,soybean stem brown rot caused by the PGS in our country has not reported occurrence for our port quarantine diseases.Notomi and so on developed a loop-mediated isothermal amplification(loop-mediated isothermal amplification,LAMP)in 2000.We report the development of loop-mediated isothermal amplification(LAMP)assays targeting ITS element for visual detection of Phialophora gregata f.sp.sojae(PGS).Via utilizing the technology of LAMP,and setting the ITS as target sequence,we designed four special LAMP primers and two loop primers and therefore based on this we set up the LAMP system of testing Phialophora gregata f.sp.sojae(PGS).The ITS-LAMP assay was evaluated for specificity,sensitivity,detections of artificial incubation and diseased soybean tissue.The ITS-LAMP assay efficiently amplified the target element in less than 60 min at 64 ℃ and was evaluated for specificity and sensitivity.The specificity was evaluated against PGS and other fungi isolates.Since the entry of the soybean plant residue sample containing pods,bean stalks,chopped leaves,a variety of microorganisms and field of mud and other ingredients,then we do not know whether the use of LAMP technology in the case of the presence of these factors,the accurate detection of PGS in the material.For this reason,we use of different concentrations of spores PGS soy added to plant residue samples not contain a certain amount of PGS provide by Jiangsu Entry-Exit Inspection and Quarantine Bureau to simulate the actual of LAMP detection sensitivity of the detection of PGS in the port.Detection sensitivity of PGS-LAMP,the higher the better it will improve the detection efficiency quarantine these two bacteria in the port.And ultimately determine the sensitivity of the application of the LAMP technology to detect PGS is 50 spores/lOg soybean plant residues under laboratory conditions.After determining the LAMP rapid molecular detection of PGS can be applied to detect soybean plants debris in the port under laboratory conditions,we actually test nine soybean plant residues provided by Jiangsu CIQ with PGS-LAMP.The results showed that the nine samples of soybean plant residues have one carrying PGS.In this study,the actual test results of loop-mediated isothermal amplification technology for detecting soybean stem fructicola in the port showed that:LAMP detected on soybean stem fructicola port is a new simple and efficient testing which have feasible and higher detection sensitivity.we successfully established a LAMP-based technology system for rapid detection of PGS for inspection and quarantine of imported soybeans.