Pathogen Identification And The Establishment Of Detection Methods Of Heracleum Moellendorffii Hance Mottle Spot Disease

Heracleum moellendorffii Hance,a perennial root herb of the Umbelliferae.Mainly distributed in the northeast of China,it has rich nutritional value.In recent years,edible wild vegetables have become popular for their natural pollution-free quality and allowing people to feel the power of nature.Heracleum moellendorffii Hance has become one of the most popular edible wild vegetables because it is a kind of "medicine and food homologous".In order to meet market demand,many places in Heilongjiang Province began to cultivate Heracleum moellendorffii Hance artificially.Nowadays,the conventional cultivation industry of Heracleum moellendorffii Hance has been developed throughout the country.As the planting area expands year by year,it is vulnerable to diseases,which seriously affects its yield and quality.Cause huge economic losses to farmers.Although it has important economic value,little attention has been paid to its disease.In this study,when we conducted a plant virus survey on common crops,horticultural crops and other plants in Heilongjiang Province,we found that many Heracleum moellendorffii Hance exhibited typical virus symptoms.In order to study the cause of this disease,t he diseased Heracleum moellendorffii Hance was used as the research material,and the pathogen was identified through small RNA sequencing,RT-PCR amplification,gene cloning and other technologies,and two detection systems were established.The main research results as follows:(1)From July to September 2019,many Heracleum moellendorffii Hance exhibiting typical virus symptoms were found in Wangkui County,Heilongjiang Province.By using small RNA sequencing technology to identify the type of virus in the diseased samples,a new virus of the associatedsecoviridae family was found from the diseased Heracleum moellendorffii Hance.Then,using the strategy of segmented cloning,using RT-PCR and RACE technology for amplification,the whole genome sequence of the new virus was cloned.(2)Further analysis of the whole genome sequence of the new virus showed that the new virus includes two genomes RNA1 and RNA2(Gen Bank accession numbers are MW143070 and MW143071 respectively).RNA1 has a total length of 6616 nucleotides and encodes a polyprotein P1 with a predicted molecular weight of 222 k Da,and RNA2 has a total length of 5356 nucleotides and encodes a polyprotein P2 with a predicted molecular weight of 184 k Da.Among them,RNA1 contains 5 open reading frames,namely protease cofactor,helicase,viral-linked protein,protease and RNA dependent RNA polymerase;RNA2 contains 3 open reading frames,namely Movement protein),Large coat protein and Small coat protein.The nucleotide sequence homology with othersecoviridae viruses is 45.36～53.77%,and the genus has not yet been determined.And the virus is temporarily named as mountain celery yellow spot virus(MCYSV).(3)Two detection methods RT-PCR and RT-RAA have been established based on mountain celery yellow spot virus.Four pairs of primers were designed for screening according to their CP conservative position sequences.Among them,the primers RTSeco-CPI-F1 and RTSeco-CPI-R1 are used as primers for RT-PCR amplification,the optimal annealing temperature is 55℃,the c DNA dilution limit concentration for sensitivity detection is 100ng/μl ×10-3;RAASecocp S-F4 and RAASecocp S-R4 are used as primers for RT-RAA amplification,the optimal annealing temperature is 42℃,the RNA dilution limit concentration for sensitivity detection is 700ng/μl×10-5,it is 100 times higher than that of RT-PCR,and both detection methods have good specificity for mountain celery yellow spot virus.(4)In order to clarify whether the virus has other hosts,The diseased samples of MCYSV were inoculated on 10 different hosts by mechanical friction.After 15 days,the symptoms of disease were observed,and RNA from the leaves was extracted and RT-PCR was performed,the virus was not detected as a result.