| Mulberry is an important raw material for silkworm breeding,and it is also an economical forest crop for both medicine and food.Mulberry has been widely used in food and health care,livestock and poultry feed,and ecological restoration,and has received increasing attention from people.Mulberry mosaic virus,as a mulberry virus disease,will lead to the deterioration of the quality of mulberry leaves,the decline of the yield,the dwarf plant and the decline of the resistance of mulberry,which seriously restricts the healthy and stable development of sericulture industry.With the development of mulberry industry,the occurrence rule,detection technology and prevention and control means of mulberry mosaic disease need to be solved in the new situation.In this study,Collect 182 samples of mulberry leaves with wrinkled and mosaic symptoms from 16 counties in four provinces and regions in Guangdong,Guangxi,Hainan and Chongqing,also collect 3 samples of thrips,leafhoppers and mulberry branch from mulberry garden.Separate and purify virus in the leaves of mulberry disease by densit gradient centrifugation.Observe the morphological characteristics of the virus by electron microscope,and prove the pathogenicity of virus was by the Coch rule.PCR and high throughput sequencing were used to detect the virus species in the leaves of mulberry disease.Genome sequencing and bioinformatics analysis of the viruses in four provinces were carried out.According to the virus conservative sequence,design specific detection primers and establish the PCR and qPCR detection methods.182 samples of mulberry leaves,3 samples insects and mulberry tissues were tested by PCR,and the relationship between the virus and disease,season,geography and other factors and the spread of the virus were analyzed.The primary results are as follows:?1?Isolate two conjoined virus particles from the leaves of the mulberry disease.The average size of the virus was 27.84±1.71nm and the width was 17.05±1.13nm,which was the same as the morphological characteristics of the virus in the geminivirus.?2?Two viruses were found in the mulberry leaf material that was detected by high throughput sequencing and PCR,and the mulberry mosaic dwarf associated virus?MMDaV?of the Geminiviridae and the Mulberry vein banding associated virus?MVBaV?of the Orthotospovirus.?3?The whole genome sequence of 29 copies of MMDaV in four silkworm regions was obtained.Through bioinformatics analysis,there were obvious geographical differences among the viruses in each region,which were divided into two major strains,HNZX and ZSZX.There were two strains of MMDaV in Guangdong,Guangxi,and Hainan.?4?According to MMDaV coat protein gene and MVBaV nucleocapsid protein gene nucleic acid sequence,designed the specific PCR and qPCR primers and constructed the positive detection plasmid.The minimum plasmid concentration detected by MMDaV PCR specific primers was 1×10-6ng/?L,and the minimum effective plasmid detection concentration of qPCR was 1 x 10-6ng/?L.The minimum plasmid concentration detected by PCR specific primers of MVBaV was 1×10-5ng/?L.?5?Samples of mulberry leaves collected from different regions and at different seasons,thrips and leafhoppers from mulberry garden were tested by PCR.It was found that there were two kinds of mixed infection of MMDaV and MVBaV,in which MMDaV is common in the mulberry leaf with wrinkles and mosaic.MVBaV is common in mulberry leaf with mosaic.MVBaV is obviously affected by seasonal temperature,and there is obvious latent phenomenon under high temperature,And the thrips and leafhoppers carry all two viruses.The virus can survive the winter in the roots of mulberry trees,and will grow with the growth of mulberry trees next year.The innovation of the full text:The study found that there were two viruses of MMDaV and MVBaV in the disease samples of mulberry with mosaic and wrinkles.Establish the detection techniques and methods of MMDaV?Patent application number:201711281273.8?and the detection techniques and methods of MVBaV?Patent application number:201810552526.9?. |
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