Establishment And Application Of LAMP Detection Methods For Phytophthora Parasitica And Thielaviopsis Basicolai

Black shank and root black rot caused by Phytophthora nicotianae and Thielaviopsis basicola are two soil-borne diseases that seriously threaten the production of tobacco.Accurate identification of pathogenic is of great significance for disease diagnosis and timely development of targeted control measures.In this experiment,the pathogenic ITS was used as the target sequence,and loop-mediated isothermal amplification(LAMP)of P.nicotianae and T.basicola was established.Not only is the test result visible,specific,and sensitive,And the reaction conditions are simple.Below are key research findings.Using Hydroxynaphthol blue(HNB)as the dye and ITS as the target sequence of the ribosomal transcript spacer region of P.nicotianae,using LAMP design software to design four primers,and by optimizing the LAMP reaction system,the P.nicotianae LAMP Rapid detection system(Phy-LAMP).The detection result of this system can be directly judged by the color change of the reaction solution,and the detection result is verified by 2 % agarose gel electrophoresis.This study verified the specificity and sensitivity of the established system,and tested the suspected diseased tobacco and their rhizosphere soil collected in the field.The results show that: in the optimized Phy-LAMP reaction system,in the specific detection,only the P.nicotianae LAMP detection reaction solution is blue(positive reaction),and the color of other control fungus LAMP detection reaction solution is purple(negative reaction);in the sensitivity detection,LAMP can detect 100 fg/μL,which is 1000 times higher than that of PCR.Detection of DNA extracted from 5 samples of suspected diseased tobacco,3samples were detected as positive,and P.nicotianae were isolated from conventional tissue samples of positive diseased samples;DNA test results of diseased rhizosphere soil samples and diseased tobacco test results are consistent.Inoculation of P.nicotianae on the tobacco seedlings cultivated indoors for 2 days can detect the target fungus by LAMP method.Using SYBR Green I as the dye and the ITS sequence of T.basicola.As the target gene sequence,four primers were designed using LAMP design software.By optimizing the LAMP reaction system,a rapid detection system(Tb-LAMP)of T.basicola was established.The detection result of this system can be directly judged by the color change of the reaction solution,and the detection result is verified by 2%agarose gel electrophoresis.This study verified the specificity and sensitivity of the established system,and tested the suspected diseased tobacco and their rhizosphere soil collected in the field.The results show that: in the optimized Tb-LAMP reaction system,in the specific detection,only the T.basicola LAMP detection reaction solution is yellow-green(positive reaction),and the color of other control fungus LAMP detection reaction solutions are orange(negative reaction);in the sensitivity detection,LAMP can detect 1 pg/μL,which is 10 times higher than that of PCR.Detection of DNA extracted from 10 samples of suspected diseased tobacco,of which2 samples were positive,Positive samples can be separated into T.basicola by carrot dish method The detection result of DNA extracted from the rhizosphere soil of the diseased tobacco and the detection of the diseased tobacco The results are consistent.The LAMP detection system for P.nicotianae and T.basicola established in this experiment has good specificity and sensitivity,can effectively detect the target fungus in diseased tobacco tissues and rhizosphere soil,provides a reliable diagnosis for disease diagnosis and a positive significance for the early prevention of diseases.