Field Investigation And Multiplex PCR Detection Of Viruses Disease From Wheatmelon Plants

China is one of the major producers of watermelon and northwest is the most important high quality production. However, watermelon plants have been infected by many diseases. Resulting in great losses in watermelon production, when it is serious, it can lead to no production for the whole field. It has seriously affected the enthusiasm of Watermelon planting. It was reported that 5 major virus have infected watermelon: they are Watermelon mosaic virus(WMV), Squash mosaic virus(SqMV),Zucchini yellow mosaic virus(ZYMV), Tobacco mosaic virus(TMV) and Cucumber mosaic virus(CMV),but they will also occur complex infection..We have investigate the virus watermelon virus disease for the disease occurrence, damage extent and occurrence rule in six watermelon growing regions of Shaanxi province from 2007 to 2008.The results can be listed as follows:(1) It is very common to find mosaic symptoms on watermelon leaves in every regions of Shaanxi province. It can be observed in a early time. With the development of disease and plant, the top leaves will be turn to dark green, light green and white mottle mosaic. Sometimes it will cause infection of the whole field.(2) In addition to Fufeng, other watermelon leaves are occurred blister-like protrusions. There is evident difference among different watermelon cultivar.(3) In Yangling and Zhouzhi, there are different degrees of shrinkage malformation in leaves, late leaf necrosis.(4) Symptoms of small plants and leaves have occurred in Hancheng, Fufeng and Zhouzhi, when it is serious, the leaves showed fern-like or chicken feet.(5) Leafroll has a low incidence and only occurred in Fufeng sporadicallyIn order to secure watermelon yield and quality, it is imperative to develop a sensitive, accurate and rapid method for identifying watermelon t diseases. Reaction components and reaction cycling parameters were optimized for establishing a multiplex PCR system to simultaneous detection of ZYMV,WMV,TMV,SqMV and CMV on the basis of the present situation of watermelon disease in shaanxi province. Specific primer pairs were designed according to the respective pathogen isolate sequence data available from the Gen Bank and were used for reliable detection of the different pathogens by simplex- and mRT-PCR. The mRT-PCR system was optimized in terms of the concentrations of the main ingredients such as Taq DNA polymerase, dNTPs, primers, Mg2+and PCR conditions including annealing temperature and amplification cycles. With this system were ZYMV,WMV,TMV,SqMV and CMV in mixed infection samples successfully amplified to obtain five specific fragments of 542 bp (ZYMV), 485 bp (WMV), 410 bp (TMV), 354 bp (SqMV)and 293 bp (CMV), which were the same as the designed ones in size. Sequence homology analysis and the result pof PCR stability determination have confirmed the reliability of the established mRT-PCR system. Thus an mRT-PCR system for simultaneous detection of the five pathogens was developed. The mRT-PCR system is able to detect five diseases simultaneously when it is Applied to practical thus greatly increasing detection efficiency and reducing detection cost. So this mRT-PCR provides a reliable, sensitive, rapid method for detecting watermelon pathogens, embodying the advantages of mRT-PCR.