Detection Of Wheat Blue Dwarf Phytoplasma In Its Insect Vector By Immunofluorescence

Wheat blue dwarf is one of the most important diseases of wheat in northwest China,which seriously affects the wheat production in northwest China.The pathogen of Wheat Blue dwarf is Wheat Blue dwarf phytoplasma,which is transmitted by the leafhopper Psammotettix striatus L.At present,electron microscopy is widely used to detect the Wheat Blue dwarf Phytoplasma in the leafhopper.For the electron microscopy method,there are some disadvantages,such as: the detection equipment is expensive;the time required for detection is longer;the localization of the wheat blue dwarf phytoplasma cannot be observed at the whole level of the organization.In this study,we detect of Wheat Blue Dwarf phytoplasma in its insect vector by Immunofluorescence successfully.The method can be used to locate the distribution of wheat blue dwarf phytoplasma in the leafhopper.By this study,we laid a solid foundation for exploring the pathway of wheat blue dwarf phytoplasma in the leafhopper.Capturing leafhopper from Shaanxi HanCheng health wheat field,we raise the leafhopper and transfer nymph on the healthy wheat,then get healthy leafhopper.Transfer the healthy leafhopper on the infected WBD wheat,at different time after feeding,the nested PCR amplification method is used to detect these leafhoppers.The results showed that after feeding 7 d,about 80% leafhoppers infected with WBD,and the leafhopper could be used for the subsequent immunofluorescence experiment.Extract the total protein of the leafhopper infected with WBD,then use western blot method to detect the specificity of antibody.The result show that there is a specific band at 21 Kda,consistent with the IMP protein size.It proves that the IMP polyclonal antibody has good specificity and can be used in subsequent experiments.Based on the above experimental results,leafhopper vector was dissected out and then processed for the confocal laser scanning microscopy.A clear green dot-like fluorescence was detected in the digestive of the leafhopper.The results showed that the wheat blue dwarf phytoplasma in the leafhopper was successfully located.