Research On The Role Of Secondary Symbiont In The Transmission Of Barley Yellow Dwarf Virus By Sitobion Miscanthi

Sitobion miscanthi（Takahashi）（it was mistakenly identified as Sitobion avenae（Fabricius）in China,and is still commonly used）is an important pest on many economic crops（especially wheat）.In our country,the annual reduction in wheat production caused by direct damage（such as feeding on leaves or ears）or indirect damage（such as excretion of honeydew or transmission of viruses）causes up to 30%reduction in wheat production,which seriously threatens our country’s wheat production and food security.The aphid is also vector of Barley yellow dwarf virus（BYDV）,causing the disease to spread.BYDV is extremely harmful to wheat.It is one of the important virus diseases in our country’s wheat producing areas.Preliminary research has shown that:The secondary symbionts and the virus share the same place in the body of aphid,and the interaction relationship is complicated.Therefore,by exploring the interaction mechanism of secondary symbionts-mediator-plant virus,it can provide a theoretical basis for preventing the epidemic of plant viruses by regulating the secondary symbionts,thereby contributing to the prevention and control of diseases.In this study,we tested the infection of secondary symbionts in the monoclonal system of different S.miscanthi,and selected 2 monoclonal strains non-infected and 2monoclonal strains only infected with one secondary symbiont to add or eliminate secondary symbionts by adding secondary symbionts enrichment solution or antibiotics to artificial diet.Then we obtained the natural non-infected and artificially-added clones of S.miscanthi with the same genetic background,as well as the natural-infected and artificially eliminated clones of S.miscanthi with the same genetic background.Based on this,we studied the difference in the acquisition,retention and transmission efficiency of BYDV-PAV between aphid-infected and non-infected symbionts under the same genetic background,so as to explore and answer the role of secondary symbionts in the host aphids to transmit barley yellow dwarf virus.The results are as follows:1.Secondary symbionts detection:collecting adult S.miscanthi from Yangling wheat field,and using single head to raise 34 monoclonal strains.The 16S r DNA specific primers were used to detect seven secondary symbionts:H.defensa,R.insecticola,Rickettsia,Wolbachia,S.symbiotica,Spiroplama and Arsenophonus.The results showed that the monoclonal strains 27(N27^S-)and 28(N28^S-)were not infected with any secondary symbionts,the monoclonal strain 15(N15^Ri+)was only infected with Rickettsia,the monoclonal strains 21(N21^Re+)were only infected with R.insecticola.The other monoclonal strains infect two or more secondary symbionts,shows that co-infection is more common in S.miscanthi.2.Target secondary symbiont addition:The 2-day-old aphids of N27^S-and N28^S-infect with no secondary symbionts was reared on artificial diet containing the target secondary symbiont（Rickettsia）enrichment solution for 7 days,and then single aphid was transfered to a single wheat plant and reared continuously for 3 generations,forming a physiologically stable artificial monoclonal line A27^Ri+containing Rickettsia.3.Target secondary symbiont elimination:The 2-day-old aphids of N15^Ri+（infect with secondary symbionts Rickettsia）and N21^Re+（infect with Secondary symbionts R.insecticola）was reared on artificial diet containing specific antibiotic combinations（ampicillin,cefotaxime and gentamycin）for 7 days,and then single aphid was transfered to a single wheat plant and reared continuously for 3 generations,forming a physiologically stable artificial monoclonal line A15^Ri-and A21^Re-both infect with no secondary symbionts.4.The impact of target secondary symbiont on the transmission of BYDV-PAV:（1）Compared with N27^S-,the amount of BYDV-PAV obtained by A27^Ri+S.miscanthi was significantly reduced,the amount of virus retained in the body at the early stage was significantly increased,and the amount of virus in the host wheat in the early stage after transmission was significantly reduced;（2）Compared with A15^Ri-,N15^Ri+S.miscanthi acquires the virus in quantity and efficiency significantly increased,and the amount and efficiency of virus storage in the early stage of the body were significantly increased,and the amount of virus in the host wheat in the early stage after transmission was significantly reduced.The above results indicate that Rickettsia could promote the ability of host aphids to store virus and inhibit their ability to transmit virus,but the ability to capture the virus shows promotion and inhibition due to differences in host aphids populations.In summary,under artificial feed conditions,we have successfully obtained the artificial infected or non-infected single clones that are consistent with the genetic background of the natural S.miscanthi,through the target symbiont enrichment solution（addition process）or specific antibiotic formulation method（elimination process）;based on this,we further compared the differences in the ability of acquiring,maintaining and transmitting BYDV-PAV between the infected and non-infected S.miscanthi aphid.It was found that the secondary symbiont Rickettsia could increase the retention of BYDV-PAV and reduce its transmission ability,indicates that the secondary symbionts play an important role in the transmission of barley yellow dwarf virus by the host S.miscanthi.This research provides a favorable support for further in-depth exploration of the secondary symbionts-mediator-plant virus interaction mechanism,and at the same time provides a theoretical basis for preventing the epidemic of plant viruses by regulating the secondary symbionts.