Graduate Thesis Or Dissertation
 

Development of Quantitative PCR Assays to Aid in Root-knot Nematode (Meloidogyne spp.) Diagnostics and Resistance Breeding Efforts in the Pacific Northwest

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  • In the Pacific Northwest (PNW), the two most common root-knot nematodes are Meloidogyne hapla and Meloidogyne chitwoodi. These nematodes can infect a wide variety of crops and can cause significant losses. Currently, it is common for the field of nematology to use labor-intensive microscopy to identify plant-parasitic nematodes based on morphology. There is a need for molecular techniques such as quantitative PCR (qPCR) for faster, more reliable Meloidogyne spp. identification and quantification. Still, there is difficulty with designing molecular tools for the Meloidogyne spp. present in the PNW due to the limited amount of sequence data for PNW populations in the NCBI Genbank repository. The gene Hsp90 was chosen as the assay target because it is conserved among most living organisms but has highly variable functionality, conferring enough sequence variation for primer and probe design. One set of primers with two species-specific probes were designed for use in hydrolysis probe singlepex or multiplex qPCR to detect and quantify M. chitwoodi and M. hapla in the PNW. Chapter 2 of this thesis describes the development of the Hsp90 multiplex qPCR for M. hapla and M. chitwoodi and its use in field samples from a diagnostic laboratory to compare morphological and molecular diagnostics. This molecular assay is not able to distinguish between M. fallax and M. chitwoodi, but M. fallax is not found in the PNW. No cross reaction was observed among plant-parasitic nematodes commonly found in the PNW and the assay was able to detect populations of M. hapla and M. chitwoodi from areas of North America outside of the PNW. High DNA concentrations of M. hapla or M. chitwoodi affected the proficiency with which the assay could detect low DNA concentrations of the opposite target nematode in the sample. The reliability of testing 1 M. hapla or 1 M. chitwoodi in a sample was 50% and 80%, respectively. A test of three soils from the PNW did not indicate that soil type had an effect on Ct value. In the comparison between morphological and molecular field samples obtained from a diagnostic laboratory, the standard curve was unreliable. In determining presence or absence of M. hapla and M. chitwoodi, the multiplex qPCR and morphological diagnostics were in agreement 68% of the time. A subset of 25 samples where morphological and molecular diagnostics disagreed on the presence of M. chitwoodi were run in singleplex with the probe specific to M. chitwoodi and 17 of those 25 samples replicated the result of the multiplex assay. In Chapter 3, a high-throughput screening method termed “the canister assay” was optimized for evaluating the reproduction of M. chitwoodi on potato. In the canister assay, soil is added to the canister and planted with potato. The canister is then inoculated with M. chitwoodi and incubated at a constant temperature for the duration of the experiment. The canisters are then harvested and eggs are extracted and enumerated to calculate reproduction factor (RF = final population density/initial population density). Among the factors investigated, inoculating at time of planting and an incubation period of at least 6 weeks caused the greatest increase in RF values. The canister assay was also applied to a potato breeding population, for which enumeration of extracted eggs at the end of the experiment by microscopy was compared to the Hsp90 M. chitwoodi singleplex qPCR. There was no significant difference in calculated RF values between molecular and microscope enumeration. However, when samples with a low nematode density are considered (less than 200 eggs) there is a significant difference in egg density estimations, with the qPCR producing much more sensitive results. Broadly, the research in this thesis aims to contribute to the high-throughput methods that will advance nematology research and diagnostic efforts through molecular biology techniques.
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  • Northwest Potato Consortium
  • USDA-ARS CRIS project #2072-12220-004-00D
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