Project Number: 8062-22000-021-24
Start Date: Sep 01, 2013
End Date: Aug 31, 2016
Mixed infections will be created either by allowing a single aphid sequential acquisition access periods on two different source plants, each infected with a different PVY strain, followed by an inoculation access period on a healthy potato seedling. Alternatively, two individual aphids will be allowed acquisition access periods on two different source plants, each infected with a different PVY strain. Then, both aphids will be placed on the same healthy plant. We will vary the time and place of how the two aphids are allowed to inoculate the healthy plant; i.e. both aphids put on the same leaf at the same time, placed on the same leaf in sequential inoculation access periods, or placed on different leaf positions either at the same or sequential time periods. This will allow us to determine if virus infection by two different strains is influenced by the time and location of inoculation as will happen in the field. Plants that are found to be infected with pairs of PVY strains will be used as source plants for virus acquisition and transmission experiments. Initial experiments will use one clone of M. persicae. Subsequent experiments will investigate other populations of M. persicae and multiple populations of and non-colonizing species. Alate (winged) aphids will be allowed optimal acquisition access periods on leaves found to contain mixtures of PVY strains. Single aphid will be moved to healthy recipient potato plants for a 24 hr inoculation access period before the plants are fumigated to kill the aphid and moved into a greenhouse. Ten-15 single aphids will be fed on each source leaf and three-five source leaves will be used for each treatment. Transmission efficiency will be measured as the number of plants that become infected over the total number of plants infested with single aphids fed on a single source leaf. The virus source leaves will be taken from various positions on the plant and the relative levels of the two virus strains will be determined by Real Time RT-PCR. Source tissue will be frozen immediately after use and re-evaluated by RT-PCR to determine if the relative titers have changed between the initial analyses to identify the source tissue and the time the leaf was used in the transmission experiment. Tissue will also be used for analysis of all virus genomes present by deep sequencing. This will provide information not only on relative levels of the strains replicating in the source tissue, but also the genetic composition of the virus population so we can determine if specific genotypes of the virus are preferentially acquired and transmitted to the new host plants. Recipient plants will be analyzed by ELISA 10-14 days post infestation to determine which are infected. Subsequently, multiplex RT-PCR of the infected plants will determine which virus strains were transmitted and deep sequencing and subsequent analysis of virus specific sequences will allow comparison of the virus genotypes in the source tissue and the recipient tissue.