Location: Temperate Tree Fruit and Vegetable Research
Project Number: 2092-22000-022-024-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Jun 1, 2021
End Date: May 31, 2024
Objective:
The goal of our study is to identify viral pathogens that could be developed or propagated as biological control agents for potato psyllid. Objective 1: 1A: Examine the transmissibility of the novel BcPLV between infected and uninfected potato psyllids. 1B: Determine whether observed morphological abnormalities or colony crashes are associated with high rates of virus infection with colony populations. Objective 2: Screen wild and laboratory populations of potato psyllids for the presence of Bactericera cockerelli Picorna-like virus (BcPLV). Objective 3: Use a metagenomic approach to identify other viral pathogens potentially infecting potato psyllids.
Approach:
Objective 1: We will examine how readily BcPLV is transmitted among psyllids and explore the possible effects of virus infection on potato psyllid. Objective 1A: To examine the transmissibility of BcPLV, we will confine virus-infected and uninfected psyllids to potato leaves using sleeve cages. Initial experiments will include cLso-uninfected psyllids only. Treatment pairs will test whether the virus is transmitted by contact or close proximity, sexually, or via plant conduits. Objective 1B: We suspect that the viral load will fluctuate within colony cages. Twenty psyllids from each colony cage will be collected from colony cages every three weeks (approx. 1 generation). The adults will be examined for the presence of morphological abnormalities, which will be photo documented. The psyllids will then be tested for the presence or absence of BcPLV using diagnostic RT-PCR. In addition to collecting psyllids, population fluctuations within cages will also be monitored to determine if colony “crashes” are associated with high rates of virus infection. Objective 2: We will collect potato psyllid adults from potato fields and from the uncultivated host plant, matrimony vine, using a vacuum sampler. The psyllids will be examined for presence of morphological abnormalities and will be tested for the presence of BcPLV using RT-PCR. We will also screen potato psyllids captured in 3D printed traps and sticky card traps deployed in potato fields for another study involving beet leafhopper. Results from Objective 2 will be used to 1) determine BcPLV infection rates among field populations of potato psyllid, and 2) determine whether BcPLV can be readily detected in psyllids captured on sticky card traps, or if preservation of virus particles by use of the 3D printed traps is necessary for accurate detection of the virus. Objective 3. The three colonies of psyllids belonging to the central, northwestern, and western haplotypes will be subjected to the HTS analysis, using 10-20 pooled psyllids per colony. Total RNA will be extracted using a TRIzol reagent-based protocol. DNAse-treated, purified RNA will then be depleted of ribosomal RNA using the RiboMinus kit for RNA-Seq. Before proceeding further, quality of resulting ribo-depleted RNA will be checked by fragment analysis. The ribo-depleted RNA will then be submitted to the Genomics Resources Core at the University of Idaho for library preparation, multiplexing, and sequencing on the Illumina MiSeq platform. Bioinformatics analysis of the high-throughput sequencing data will be performed as described previously. Any matches to the custom-prepared virus sequence database entries will be confirmed through the RT-PCR or PCR verification using specific primers, with subsequent Sanger-based sequencing of amplified PCR fragments.
