Location: Horticultural Crops Disease and Pest Management Research Unit
Project Number: 2072-22000-046-022-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Sep 1, 2022
End Date: Oct 1, 2024
Objective:
1. Assessment of prevalence of potato cyst nematode rhabdovirus (PcRV) and potato cyst nematode picorna-like virus (PLV) in Globodera populations.
2. Assess PcRV and PLV transmissibility in Potato Cyst Nematode (PCN).
3. Use a metagenomic approach to identify viruses in other potato nematodes such as root-knot nematode (RKN).
Approach:
Objective 1: Assessment of prevalence of PcRV and PLV in Globodera populations. We will collect PCN cysts from infested potato fields that are being sampled by the APHIS personnel. Available PCN populations from South America will be also included in analyses. The nematodes will be examined for any morphological abnormalities and will be tested for the presence of PcRV and PLV using RT-PCR. Results from Objective 1 will be used to determine PcRV and PLV infection rates among field and laboratory populations of PCN. Transcriptome data from other Globodera spp. (G. pallida, G. rostochiensis, G. tabacum, G. mexicana) from various geographic locations that are publicly avaible on the NCBI SRA database, will be also screened for the presence of PcRV and PLV.
Objective 2: Assessment of PcRV and PLV transmissibility. To assess the mode of transmission, different PCN developmental stages will be assayed for PcRV and PLV using RT-PCR method. Viral sequences detected through the entire nematode lifecycle will determine whether the virus is transmitted vertically, from juvenile to egg. Virus-positive nematodes will be further subjected to fluorescent in situ hybridization (FISH) with sequence specific probes to identify which nematode tissues are impacted by PcRV and PLV. Knowledge gained from the proposed localization studies will help better understand the possible nature of viral transmission. Potential for a horizontal transmission will be examined by adding filtered lysates of infected nematodes to hatched uninfected PCN juveniles prior to inoculation on potato and PCN progeny will be tested for presence of viruses at the end of nematode reproduction cycle.
Objective 3: Identification of other new viruses potentially associated with plant-parasitic nematodes endangering potato production in Pacific Northwest. Using populations outlined above, nematodes will be subjected to the high-throughput sequencing (HTS) analysis. Total RNA will be extracted using a TRIzol reagent-based protocol. DNAse-treated, purified RNA will 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 NovaSeq platform, to produce at least 20M paired-end reads per sample. Bioinformatics analysis of the high-throughput sequencing data will be performed as described previously (Thompson et al. 2019, Dahan et al. 2020, 2022).
