Application Of Loop-mediated Isotheral Amplication (LAMP) In Rapid Detection Of Three Soybean Stem Pathogens

Diaporthe phaseolorum var.meridionalis,Diaporthe phaseolorum var.caulivora and Phomopsis longicolla are three significant soybean stem pathogens.What's more,Diaporthe phaseolorum var.meridionalis and Diaporthe phaseolorum var.caulivora are plant quarantine fungus,Phomopsis longicolla causes a serious economic damage in China.It is urgent for us,on the one hand,to strength the quarantine work of Diaporthe phaseolorum var.meridionalis and Diaporthe phaseolorum var.caulivora,on the other hand,to improve the early detection technology of Phomopsis longicolla.rDNA sequences between closely related species are highly conserved,limiting the development of species-specific detection primers.Bioinformatical analysis indicated that translation elongation factor la(tefla)sequence was suitable for species-specific detection.The loop-mediated isothermal amplification(LAMP)assay targeting the translation elongation factor la(tefla)was used to establish methods for visual detection of Diaporthe phaseolorum var.meridionalis,Diaporthe phaseolorum var.caulivora and Phomopsis longicolla.We proceeded the specificity,sensitivity,plant inoculation assay and actual samples detection.The DPM-LAMP and DPC-LAMP assays efficiently amplified the target elements in 60 min at 64°C.The specificity was evaluated against D.phaseolorum var.meridionalis,Diaporthe phaseolortum var.caulivora,Phonmopsis longicolla and other isolates.Adding SYBR Green I after reaction,a positive yellow-green color by the naked eye was observed only in the presence of the corresponding fungi,whereas other isolates showed orange color.Meanwhile,the target fungi DNA products were visualized as a ladder-like banding pattern on 2%gel electrophoresis.The detection limit of the DPM-LAMP and DPC-LAMP assays was 10 pg·?L-1 and 1 pg·?L-1 of genomic DNA per reaction respectively.The assays also detected target fungus from inoculated soybean tissues and the DPM-LAMP and DPC-LAMP assays can only detect 50 and 10 ascospores respetively in 10 g soybean residues which are carried by the soybean trading between China and other countries.PL-LAMP was also proceeded.The specificity of the method was tested from 54 P.longicolla isolates,13 oomycetes isolates and 28 other fungal isolates.A positive color(sky blue)was only observed in the presence of P.longicolla with the addition of hydroxynaphthol blue(HNB)prior to amplification,whereas other isolates showed no color change.The sensitivity of this method can detect as little as 100 pg/uL fungal DNA.In addition,the assay also detected P.longicolla from diseased soybean tissues and residues from different origins.The results demonstrated that the LAMP assay provides a rapid and sensitive tool for detecting P.longicolla in plants and in production fields.High-throughput molecular detection technology which could greatly improve detection efficiency is a top priority.In our study,Combing with the 8 united PCR reaction tube,a novel molecular detection system with loop-mediated isothermal amplification(LAMP)was developed and applied for the detection of the dangerous soybean quarantine pathogens including Diaporthe phaseolorum var.meridionalis(DPM),Diaporthe phaseolorum var.caulivora(DPC)and Phialophora gregata f.sp.sojae(PGS).Based on the target genes translation elongation factor la(tefla)and internal transcription spacer(ITS),specific LAMP primers were designed.Each of species was amplified in a water bath at 640C for 70 min.what's more,the result can be observed with naked eyes by observing the color change of the reaction mixture,the color was sky-blue only in the exist of the target pathogens,whereas others showed violet.The detection limit for the each LAMP assay(DPM,DPC,PGS)was 100 pg·?L-1,100 pg·?L-1 and 10 pg·?L-1.We also detected the soybean residues which were carried by the soybean trading between China and other countries.The detection system had the advantages of rapidity,without needing special instruments,and judgment with the color change of reaction mixture.Establishment of LAMP method adapted to a 8 united PCR reaction tube provides a new alternative method for the rapid detection of soybean quarantine diseases diagnostic.In conclusion,this is the first report of the application of the LAMP assay technique for the rapid and specific detection of Diaporthe phaseolorum var.meridionalis,Diaporthe phaseolorum var.caulivora and Phomopsis longicolla.The LAMP assay developed in this study show great potential for the rapid diagnosis of soybean pathogens in production fields.Moreover,establishment of LAMP method adapted to a 8 united PCR reaction tube provides a new alternative method for the rapid detection of soybean quarantine pathogens diagnostics.