Study On Detection Techniques Of Three Important Pathogenic Of Sunflowers

With the intensification of globalization,the communication and cooperation among countries are getting closer and closer.Timely detection of significant harmful pathogens is of great significance for the import and export of sunflower agricultural products.In domestic and international trade,these three sunflower pathogens can be spread over long distances with seeds or disease residues.The sunflower yellow wilt fungus is Verticillium dahliae,which has a very wide host range and can survive in the soil in the form of microscopic nuclei for more than 10 years.Sunflower black stem disease(Leptosphaeria lindquistii Frezzi)and sunflower white rust(Albugo tragopogi(Persoon)Schr?ter var.helianthi Novotelnova)are important imported quarantine pests in China,posing a serious threat to the sunflower industry.serious threat to the sunflower industry.In order to stop the spread of the disease,it is important to establish a technical system for accurate,sensitive and rapid detection of the target pathogens.The current rapid detection of three important sunflower pathogens contains polymerase chain reaction(PCR),real-time fluorescence quantitative PCR technology(q PCR),loop-mediated isothermal amplification(LAMP),recombinant enzyme polymerase amplification(RPA),etc.In recent years,with the development of CRISPR/Cas12 precision detection technology,the technology in the detection of human pathogens and some plant pathogens In recent years,with the development of CRISPR/Cas12 precision detection technology,this technology has gained new progress in the detection of human pathogens and some plant pathogens,but there is still a gap in the detection of soilborne pathogens.The aim of this study was to establish and optimize the RPA-CRISPR/Cas12 a rapid detection and identification system for Verticillium dahliae,Leptosphaeria lindquistii Frezzi and Albugo tragopogi(Persoon)Schr?ter var.helianthi Novotelnova.Based on GAPDH gene of Verticillium dahliae,Actin gene of Leptosphaeria lindquistii Frezzi and DQ007498.1 gene of Albugo tragopogi(Persoon)Schr?ter var.helianthi Novotelnova,efficient and specific CRISPR RNA(cr RNA)and RPA amplification primers were designed and screened.The detection system has two visualization modes,the lateral flow chromatography strip(PLFS)mode and the fluorescence(FRB)mode,respectively.In this study,the RPA reaction time,Cas12 a cleavage time,cr RNA efficiency,pathogen specificity and sensitivity of the rapid detection system were optimized,respectively.The technique can complete the detection of target pathogens within 40 min under the reaction condition of constant temperature of 37°C,and the lower limit of detection is 10 genomic copies;the whole detection technology system does not require large instrumentation and is suitable for field and on-site detection,with the advantages of high accuracy,sensitivity and specificity;the technique broadens the application of CRISPR/Cas12 a nucleic acid detection in soil-borne diseases.It also provides an important reference for the rapid detection and identification of other quarantine pathogens.