| Potato(Solanum tuberosum)is one of the important food crops in China,especially in Guizhou Province.It is critical for strategic importancer to ensure the safety of potato production.However,Late Blight of Potato caused by the Phytophthora infestans has been a serious threat to potato yield,quality,and the overall development of its staple food strategy in China.As an excellent fungicide against this pathogen,mefenoxam is widely used in the control of the disease.In recent years,the resistance of the pathogenic mildew to mefenoxam has become increasingly evident.Researchers have analyzed its single nucleotide polymorphism(SNP)locus RPA190-T1145 A to correlate with the development of resistance,but it is still controversial.Based on this,this study aims to further clarify the relationship between the two.Currently,the analysis of SNPs generally relies on sequencing methods,which are complex and timeconsuming.Therefore,the development of a rapid and simple method with high specificity and sensitivity for SNPs detection is essential.T4 DNA ligase is often used for SNPs related detection because of its easy availability and low cost,but various methods still have serious defects of low specificity and need further improvement.In summary,this study aims to develop a high-fidelity single-stranded DNA cyclization system based on T4 DNA ligase,and combine it with the loop-mediated isothermal amplification(LAMP)detection technique to construct a simple SNPs detection method with high specificity and sensitivity,and apply it to the accurate detection of the P.infestans SNP RPA190-T1145 A,and then analyze the relationship between this locus and the development of mefenoxam resistance.Through the implementation of the project,the following research results were achieved.1.Isolation and identification of pathogens of Potato late blight in Guizhou province: samples of potato leaf disease were collected from 10 collection site in Guizhou province,and 135 strains were isolated,and all strains were P.infestans after morphological and ITS sequence identification analysis.2.Creation of a high-fidelity cyclization pattern: cyclizing the long bases at the 5’end of single-stranded DNA and asymmetric pairing of only 5 bases at the 3’ end,a high-fidelity cyclization pattern system was successfully constructed,which greatly improved the specificity of T4 DNA ligase ligation.3.Establishment of SNPs detection method: the constructed high-fidelity cyclization pattern was combined with LAMP detection to establish a highly specific and sensitive SNPs detection strategy,named as LR-LAMP.The visual result judgment based on color change or the result analysis of fluorescence signal can identify mutation rates as low as 0.1% or 0.01%,respectively.In addition,its detection sensitivity reached100 a M of target DNA.actual test samples were evaluated using P.infestans genomic DNA solution,and the results showed that the method can effectively distinguish the RPA190-T1145 A of P.infestans.4.Analysis of the resistance of P.infestans to mefenoxam and its correlation with the RPA190-T1145A: the measured isolates from Guizhou province were all sensitive to mefenoxam,and their RPA190-T1145 A were all intermediate T/A;the eight strains from outside the province showed sensitivity,and the other two strains showed resistance,and the RPA190-T1145 A were wild-type T or intermediate T/A,respectively.Therefore,this result suggests that the resistance of P.infestans to spermacetam and its RPA190-T1145 A locus do not uniquely correlate. |
