Transmission Characteristics Of Wheat Yellow Striate Virus By Its Leafhopper Vector Psammotettix Alienus

The leafhopper Psammotettix alienus is an agricultural pest,which can cause damage to gramineae crops.It can vector wheat dwarf virus(WDV)(genus Mastrevirus,family Geminiviridae)by feeding on plant sap,and make a great threat to crop production.In recent years,our lab has found that P.alienus can also transmit another virus-wheat yellow striate virus(WYSV)with a negative single-stranded RNA genome,which is a tentative species of the genus Nucleorhabdovirus of the family Rhabdoviridae.The interaction mechanism of the two viruses in leafhopper and its possible impact on disease epidemiology are not clear.Here,we firstly investigated the biological characteristics of WYSV transmitted by leafhopper P.alienus,and then further determine the propagation,distribution and dissemination route of WYSV in the vector leafhopper.On this basis,we investigated the ability of P.alienus to transmit WDV or WYSV when WYSV and WDV coexisted in the leafhopper.It laid the foundation for the analysis of the interaction mechanism between multiple viruses and insect vector and provided a scientific basis for the development of virus control strategies.Our results showed that,under the laboratory condition of 23 °C and 16 h daylight,the minimum period for acquisition access and inoculation access of WYSV was 5 min and 10 min,respectively.The minimum and the maximum latent period of WYSV were 10 days and 22 days,and majority(79.2%)was between 16-20 days.In addition,WYSV can be transovarially transmitted by leafhopper,and up to 45.1% of the progeny was WYSV positive.The relative expression of viral mRNA in P.alienus was continuously increased with time post acquisition from 2,4,8 to 16 days tested by RT-qPCR,indicating that WYSV can propagate in the vector P.alienus.Transmission electron microscopy revealed that typical bacilliform shaped WYSV particles with a size of about(200-270)nm×(35-40)nm located in the salivary glands and midgut of P.alienus and mainly distributed in the cytoplasm around the nucleus.Immunofluorescence assay combined with confocal microscopy suggested that WYSV spread to the filter chamber and the muscle layer of the anterior midgut of the leafhopper at 2 day post acquisition(DPA).The virus can spread to the midgut and hindgut muscle layer at 4 DPA.It was found that a small amount of virus can spread to salivary gland of some vector at 8 DPA.WYSV particle,typically distributed around the nucleus,were observed in salivary gland,filter chamber,and muscle layer of anterior midgut,mid midgut,posterior midgut and hind-gut at 14 DPA.Combined with establishing detection systems for WYSV and WDV with duplex RT-PCR and Nuclei acid dot-blot hybridization(NASH)and based on the identification of the biological characteristics of WYSV transmitted by leafhopper,we also carried out the study on the efficiency of P.alienus transmitting WYSV and WDV under different conditions of virus acqusition.From single infections,the leafhopper P.alienus had a higher ability to transmit WDV or WYSV,respectively.However,when leafhoppers acquired WYSV challenged with WDV after a 48-h delay,or WDV challenged with WYSV after a 48-h delay,our data suggested that both these sequential acquisition would significantly reduce the fraction of leafhoppers transmitting WDV.Also,WDV transmission was significantly delayed in leafhopper exposed to plants co-infected by both WYSV and WDV.In contrast,the exsit of WDV had no effect on the acquisition and transmission of WYSV.The above results laid the foundation for exploring the synergistic or antagonistic mechanism between virus and virus in the same vector.