Rapid Diagnosis Of Soybean Root Diseases Based On Loop-mediated Isotheral Amplication(LAMP)

Soybean root diseases caused by a dozen of species of pathogenic fungi and oomycete are the main causes of yield loss in soybean production.Often these diseases have similar symptoms,not easy to distinguish,and difficult to accurately diagnose.Based on the loop-mediated isothermal amplification(LAMP),we established eight LAMP detection systems for eight major soybean root diseases respectively,and could specificity detect the pathogens from disease tissues,and then rapidly diagnosed the diseases.It is important to guide disease control in time.Based on the above mentiumed results,we imoroved the operation of LAMP and the instruments,developed a system to diagnose soybean diseases in the fileds.Furthermore,we combined LAMP with 96-well plates,and developed a high-throughput detection system that could diagnose nine soybean root diseases at once.The results of this study have important value to enhancing the technical standard of plant disease diagnosis.Fusarium root rot of soybean is a worldwide disease that causes serious damage to soybean production in China.In this study,we established five LAMP technology systems to rapid diagnose Fusarium root rot in soybean in the fields;they were CYP51C-Fo-LAMP,CYP51C-Fe-LAMP,CYP51C-Fg-LAMP,TEF-1α-Fs-LAMP and TEF-1α-Fp-LAMP,could specifically detect Fusarium oxysporum,F.equiseti,F.graminearum,F.solani and F.proliferatum.All the LAMP assays could detect the pathogen in diseased soybean tissues in the field,and efficiently amplified the target genes over 60 min at 62℃,a yellow-green color(visible to the naked eye)or intense green fluorescence(visible under ultraviolet light)was only observed in the presence of Fusarium spp.After addition of SYBR Green I.Using the five assays described here,we successfully and rapidly diagnosed suspective diseased soybean samples collected in the field from Jiangsu,Anhui,Wuhan and Yunnan provinces.We thus describe a new method for the rapid diagnosis of Fusarium root rot of soybean.We report a new method of direct detection of pathogenic fungi in infected soybean tissues.The method rapidly diagnosis soybean seedling blight caused by Rhizoctonia solani and soybean charcoal rot caused by Macrophomina phaseolina,and features LAMP.The primers were designed and screened using internal transcribed spacers(ITS)as target DNAs of both pathogens.We developed an ITS-Rs-LAMP assay for R.solani and an ITS-Mp-LAMP assay for M.phaseolina that could detect the pathogen in diseased soybean tissues in the field.Both LAMP assays efficiently amplified the target genes over 60 min at62℃.A yellow-green color(visible to the naked eye)or intense green fluorescence(visible under ultraviolet light)was only observed in the presence of R.solani or M.phaseolina after addition of SYBR GreenⅠ.The detection limit of the ITS-Rs-LAMP assay was 10pg/μL genomic DNA;and that of the ITS-Mp-LAMP assay 100 pg/μL genomic DNA.Using the two assays described here,we successfully and rapidly diagnosed suspective diseased soybean samples collected in the field from Jiangsu and Anhui provinces.We thus describe a new method for the rapid diagnosis of soybean seedling blight by R.solani and soybean charcoal rot by M.phaseolina.In this study,we main to establish a LAMP system to rapid diagnose red crown rot of soybean in the fields.Red crown rot(RCR)of soybean caused by Calonectria ilicircola is an important disease in soybean.Typical symptoms of RCR are as follows:Chlorotic,yellowish and blighted leaves,and wilting of the plants.Taproots were blackened and rotted.Nuntous of reddish spherical perithelia appeared on basal stems and roots of soybean.But in the early and middle stage,its symptoms are relatively similar to the root rot caused by Fusarium spp.In order to make more accurate diagnoses,we report a new method of direct detection of pathogenic fungi in infected soybean tissues.The method rapidly diagnosis RCR of soybean caused by C.ilicircola and features Loop-mediated isothermal amplification(LAMP).The primers were designed and screened usingβ-tublin gene as target DNAs of the pathogen.We developed a TUB-Ci-LAMP assay for C.ilicircola that could detect the pathogen in diseased soybean tissues in the field.And this assay needs only a vacuum cup,reaction temperature of 62℃for 65 min,then add SYBR Green I as indicator could be directly observed.The detection limit was 10 pg/μL genomic DNA.In order to make the diagnosis of our system simpler,and more suitable for operating in the fileds,we based on the original system,according to the disease tissue DNA extraction,operation of LAMP and experimental instruments,made some improvement.Then the disease soybean tissues DNA extraction does not relied on centrifuge,and a vacuum used to replace PCR amplifier to supply isothermal heat resource(nearly isothermal heat resource)that LAMP assay needed.LAMP reagent mixed in advance to simplify the process of operation.Eventually we developed a LAMP system that could be carried out directly into the fields to diagnose soybean root diseases independent laboratory conditions.In this study,a molecular detection system with LAMP was developed and applied for the detection of 9 pathogens that causing soybean root diseases,simultaneous detection of three samples of high-throughput detection technology.This research combined CYP51C-Fo-LAMP,CYP51C-Fe-LAMP,CYP51 C-Fg-LAMP,TEF-1α-Fs-LAMP,TEF-1α-Fp-LAMP,A3apro-Ps-LAMP,ITS-Rs-LAMP,ITS-Mp-LAMP,TUB-Ci-LAMP those nine LAMP assays those detected Fusarium oxysporum,F.equiseti,F.graminearum,F.solani,F.proliferation,Phytophthora sojae,Rhizoctonia solani,Macrophomina phaseolina,Calonectria ilicicola respectively.The detection system had the advantages of rapidity,without needing special equipment and judgment by the naked eye,and providing reference for molecular detection and disease diagnosis for laboratory of grass-roots units and inspection and quarantine agencies.