Establishment Of Real-time Fluorescence Quantitative PCR Detection Method For Tartary Buckwheat Leaf Spot And Indoor Screening Of Pesticides For Control

Nigrospora and Alternaria are important pathogens in agriculture and forestry,which endanger the quality and yield of various economic crops.In 2021,two pathogenic fungi were isolated from tartary buckwheat leaves infected by spot pathogens in our laboratory,and were identified as Nigrospora osmanthi and Alternaria alternata,respectively.The establishment of a sensitive,accurate and rapid detection methods is a powerful tool to study plant disease prevention and control measures,and Taq Man probe is one of the methods.As an important real-time fluorescent quantitative PCR technology,it has the characteristics of strong specificity and high sensitivity.In this paper,according to the translation elongation factor（TEF1-α）gene of N.osmanthi and its related species,and the Alternaria pathogen（Alt a1）gene of A.alternata and its related species,a pair of specific primers and a specific Taq Man probe were used to establish a q PCR detection method for tartary buckwheat igrospora leaf spot and Alternaria leaf spot respectively.These two q PCR detection methods are fast and sensitive.The whole reaction only takes 1 hour,and no subsequent processing of PCR products is required,The two methods provide an important reference for early and rapid detection of Nigrospora leaf spot and Alternaria leaf spot in tartary buckwheat.At the same time,seven common pesticides were selected for each of these two kinds of buckwheat leaf spot,and the indoor screening of pesticides was carried out,and the best pesticides that inhibit the growth of these two pathogens were found.The virulence regression equation and EC₅₀were obtained by SPSS26 for the screening of field pesticides against the two tartary buckwheat leaf spots.The results are briefly described as follows:1.A rapid detection system for N.Osmanthi was constructed.A pair of primers and a specific probe were screened from the translation elongation factor TEF1-αsequence of N.Osmanthi.The q PCR detection conditions were optimized as:the optimal annealing temperature was 57℃,the optimal final primer concentration was 0.6 tmol·L^-1,and the optimal final probe concentration was 0.4 tmol·L^-1.The sensitivity of this system is 2.6×10⁶copies·m L^-1（20μL system）,which is 100 times higher than the monitoring sensitivity under PCR conditions.The earliest pathogen detection time of q PCR fluorescence signal was 1 day after infection undergreen house conditions,which was 2 days earlier than diagnosis by naked eyes,and the detection rate was 81.8%.2.A rapid detection system for A.alternata was constructed.A pair of primers and a specific probe were screened according to the pathogenic gene Alt al of A.alternata.The q PCR detection conditions were optimized as:the optimal annealing temperature was 57℃,the optimal final primer concentration was 0.5 tmol·L^-1,and the optimal final probe concentration was 0.5 tmol·L^-1.The sensitivity of this system is 3.3×10⁷ copies·m L^-1（20μL system）,which is 10 times higher than the monitoring sensitivity under PCR conditions.The earliest pathogen detection time of q PCR fluorescence signal was 1 day after infection under green house conditions,which was 2 days earlier than diagnosis by naked eyes,and the detection rate was 66.7%.3.The indoor effects of 7 fungicides were verified,and the results showed that Sporgon had the best inhibitory effect on N.Osmanthi and A.alternata,with EC₅₀ values of 0.037mg·L^-1 and 0.035 mg·L^-1,respectively.Sporgon is suggested for further field screenings of pesticidesof pesticides for the control of the tartary buckwheat leaf spots.