Real-time PCR And RPA Were Used To Detect The Overwintering Bacterial Source Of Wheat Stripe Rust From Qinghai Province

Qinghai province is an important source base for stripe rust in summer in China.Wheat is planted and distributed throughout the year without interruption.Winter wheat,late spring wheat and self-growing wheat seedlings provide a large number of hosts for the continuous infection and epidemic of wheat stripe rust.The frequent variation of stripe rust toxicity resulted in the loss of resistance and the frequent outbreak of stripe rust caused serious economic losses.In recent years,due to the mass of the source area of the country of core management,longnan bacteria sources,gradually reduce the summer area,with the rising in winter wheat planting area of qinghai province,qinghai has become China’s wheat stripe rust was the summer area and the bacteria source quantity biggest bacteria source base,area can complete the winter at the same time,form the anniversary of the cycle,wintering bacteria source to epidemics of diseases need to clear and evaluation.In this study,q-PCR and RPA were used to analyze the overwintering of wheat stripe rust in Qinghai Province.The main results are as follows:(1)In order to study the bacterial source quantity of wheat stripe rust in leaves of gley stage in Qinghai Province,this study established a detection system of bacterial source quantity of wheat stripe rust in gley stage by using real-time fluorescence quantitative PCR with the wheat stripe rust extension factor EF1 as the primer.The results showed that the EF1 primer could amplify 243 bp specific target fragment from the g DNA of wheat stripe rust leaf.The sensitivity of Quantitivate PCR(q PCR)was 100 times that of conventional PCR.Stripe rust could be detected in the leaves of Mingxian 169 after inoculation from day 1 to day 8,and the amount of stripe rust increased exponentially with the change of days.The detection system established in this study can quickly and accurately detect the bacterial quantity of wheat stripe rust in gly stage leaves.(2)In this study,primers PS-RPA-F,PS-RPA-R and p S-LF-probe for recombinant enzyme polymerase technology were designed with the effective gene PS4593 of wheat stripe rust as the target gene.Combined with flow chromatography paper,the detection method for recombinant enzyme polymerase of wheat stripe rust was established.Stripe rust could be detected within 10 min,which was consistent with the result of common PCR.The lower limit of wheat stripe rust detected by this technique was 10fg/μL,and there was no difference between the detection result and conventional PCR.In addition,the RPA rapid detection method can detect wheat stripe rust from wheat leaves in the field gleying stage,which has strong practicability.Therefore,the RPA detection method of wheat stripe rust established in this study has the characteristics of high efficiency,rapid,universal practicality and simple operation,etc.,providing a theoretical basis for the early detection and prevention of wheat stripe rust.(3)Six overwintering monitoring sites will be set up in Qinghai province from 2020 to 2021.The monitoring results show that dashijia village,Xiadoba Village,Sheren village and Ahetan village have formed the disease center,and dashijia village and Ahetan village have the highest rate of diseased leaves.The overall overwintering situation of labeled wheat in the field after greenhouse transplanting was as follows: Ahetan Village > Dashijia Village > Sheren Village >Xiaduba village > Shangduba village and Helga village.The incidence in flowering and heading stage was as follows: 70% of marked plants were infected in Dashijia Village,Guide County;The incidence rate in Ahetan village was 46.15%.The incidence of other markers was mild.By analyzing the average daily temperature in this year,the average daily temperature of Dashijia village and Helga Village ranged from-5℃ to 0℃ from late December to late January of the next year,and the lowest average daily temperature of Shangduoba Village,Xiaduoba village and Ahetan village in this period reached-8℃.By analyzing the correlation between temperature and humidity and bacteria source quantity,it was found that the bacteria source quantity of wheat stripe rust was significantly correlated with the average temperature in December and January,and with the humidity in November,January and February.On the whole,the temperature and humidity in January had a great correlation with the amount of stripe rust bacteria.The comprehensive analysis results show that;In 2020-2021,wheat stripe rust could successfully overwinter in Dashijia Village,Guide County,Shangduoba Village,Sheren Village and Ahetan village,Hualong County,and the incidence was the most serious in Dashijia village and Ahetan village.