| Mulberry diseases and insect pests are important factors that affect the growth and development of mulberry trees and hence hindering the development of the sericultural industry.Mulberry virus disease has become the most serious disease infecting mulberry trees in recent years.Mulberry mosaic leaf roll-associated virus(MMLRaV)is an isometric virus identified from mulberry trees exhibiting symptoms such as mosaic,leaf curling,and stunted growth.MMLRaV is a novel member of the genus Nepovirus subgroup A.it has been reported to occur in mulberry fields in the Jiangsu,Zhejiang and Yunnan provinces of China with an infection rate ranging between 5%-95%.The leaves from aging or old mulberry trees as well as those from mulberry trees infected with MMLRaV consequently have a decreased yield and leaf quality which also affects the yield and quality of silkworm cocoons.There has not been any report of chemical agent that can effectively control plant viral diseases.However,prevention and control measures such as of early diagnosis,early detection,and early treatment when practiced can effectively control the spread of plant viruses and limit the damages they cause.Although the detection method of MMLRaV using antiserum has been established,the detection accuracy rate is only 84%,and detection can easily be missed as a result.Therefore,it is necessary to establish a more reliable detection method that is fast,simple,more sensitive,and suitable for large-scale detection of MMLRaV.1.RT-PCR was used to detect MMLRaV in field samples.The total RNA of mulberry was reverse transcribed by random primers,and the field detection of mulberry samples was realized by PCR amplification of detection primers.The results showed that MMLRaV positive signals were detected in 27 of 62 mulberry samples,and the detection rate of MMLRaV was 43.5%.2.Established the Nucleic Acid Spot Hybridization(NASH)detection method of MMLRaV.Using the non-radioactive substance digoxin as the label,specific DNA probes were prepared by random primer labeling,and then a series of analyses were performed on the specificity and sensitivity of the probes.The NASH detection method was optimized as follows:(1)The prepared probe is used to hybridize the positive and the negative samples respectively.The result showed that the positive sample had hybridization signal while the negative sample had no hybridization signal,indicating that the probe had good specificity.(2)For the positive plasmid infected with MMLRaV(with initial concentration of 336.2ng/μL)it was diluted to a 10~0-10~7 dilution gradient,and hybridized with the prepared specific DNA probe.The results showed that the hybridization signal can still be detected when the plasmid is diluted to 10~6,and the detection sensitivity of the probe reaches the picogram level.(3)The positive samples loaded with 1μL,1+1μL,and 2μL were hybridized with specific DNA probes,and the results showed that the positive samples with three loading amounts showed similar hybridization signals.(4)Total mulberry RNA and a denaturant were mixed.The samples were then loaded and hybridized with the prepared specific DNA probes.The results showed that the positive samples with a denaturant and without denaturant had similar hybridization signals.(5)The positive samples were hybridized with the probes for varying periods of 2 h,4 h,6 h,8 h,and 16 h.The results showed that a hybridization time less than 6 h will have weak hybridization signal or low sensitivity,whereas a hybridization time greater than 6 h tends to have a stable hybridization signal.Therefore,the NASH detection method for MMLRaV in the field constituted 1μL sample loading,no denaturant addition,a hybridization time of 50℃and a hybridization time of at least 6 h.This optimized NASH detection method was used to detect the total RNA of 62 suspected infected mulberry trees in the four mulberry fields(A,B,C,D)in Zhenjiang.25 samples out of the total were detected using the MMLRaV positive hybridization signals,while the remaining 27 samples were detected to be MMLRaV positive by using the RT-PCR detection method.Comparing the results of both methods,the accuracy of the NASH detection method to the RT-PCR detection was 92.6%.By comparing the NASH detection accuracy rate to the MMLRaV antiserum detection method accuracy of 84%,it indicates that,the NASH detection method,which is fast,simple,more sensitive,and suitable for large-scale detection.It also has an obvious advantage in reducing the chances of missing the detection of mulberry diseased plants for MMLRaV,making it a more preferable method.The establishment of the NASH detection method for MMLRaV has important application value for the early prevention and control strategy of MMLRaV in mulberry fields.This would also serve as the gateway to the development of strategies for the prevention and control of other mulberry viruses.The prepared specific probes would provide raw materials for the subsequent in situ hybridization analysis to explore the distribution of MMLRaV in mulberry tissues,which is useful to the research on the pathogenicity of MMLRaV in mulberry and promotes the prevention and control of mulberry diseases. |
