Novel Molecular Approaches to Identify and Control Plant Parasitic Nematodes

Scott Anderson

doi:10.7273/000006349

Among the many types of plant parasitic nematodes (PPNs), the root-knot nematodes (RKN), genus Meloidogyne, cause the largest amount of crop loss world-wide. These nematodes are obligate biotrophs, meaning that they rely solely on plants for their food, restricting the nutrients available to their hosts, and decreasing crop yield and production.Traditionally, PPNs have been dealt with by applying nematicides, but over the last few decades these chemicals have been phased out or banned due to their effects on humans and the environment. To avoid overreliance on expensive and dangerous nematicides, and to find a long-term robust solution to RKNs, a new form of control is badly needed. Another problem in controlling RKNs is knowing which species are present in a field via objective and reliable methods to prescribe appropriate management strategies in a timely fashion. Currently, nematology heavily relies on microscopy to identify and quantify nematodes based on morphology; this is a low throughput, labor intensive, and technical skill which takes years to master. Thus, plant pathologists have been developing molecular techniques for faster, easier nematode identification. In this research, we endeavored to 1) develop a time saving and reliable molecular assay for identifying three RKN species: M. chitwoodi, M. fallax, and M. minor; 2) investigate the potential for using ferroptosis, an evolutionarily conserved form of programmed cell death triggered by omega-6 polyunsaturated fatty acids (PUFAs) as an alternate form of RKN control; and 3) identify key fatty acid synthesis genes in order to evaluate their impact in the RKN lifecycle when silenced via host-induced gene silencing (HIGS). First, a molecular beacon qPCR assay for M. chitwoodi, M. fallax, and M. minor was developed that could detect the three species in a single multiplexed reaction. This assay was shown to reliably distinguish between these three RKN species. It was also sensitive enough to determine the species of RKN from a single juvenile and had no cross reaction with other economically destructive RKN species (M. incognita, M. javanica, M. arenaria, or M. hapla). In addition to nematode identification, developing nematode control tools was a major component of my research. To test the applicability of ferroptosis as a means of controlling RKNs, we created transgenic tomato plants that produced gamma-linolenic (GLA) and dihomo-gamma-linolenic acid (DGLA) in their roots and challenged them with M. incognita. Because no reproducible reduction in RKN hatching was observed, it was concluded that roots producing GLA/DGLA had no measurable effects on M. incognita reproduction. Finally, two putative acetyl-CoA carboxylase (ACC) orthologs were characterized in silico in M. incognita, MiACC1 and MiACC2. In Caenorhabditis elegans, these enzymes are necessary early on in fatty acid synthesis, and their absence causes disruptions in lipid biosynthesis and development. Additionally, previous research showed that knocking-down these genes in a closely related cyst nematode, Heterodera schachtii, led to a delayed development phenotype. We attempted to knock these genes down via HIGS by creating Arabidopsis thaliana lines that produced dsRNA targeting both MiACCs. RKNs feeding on these transgenic roots showed a delayed development phenotype. These results are similar to the observations in previous studies that used pesticides or exogenously supplied dsRNA to reduce ACC activity in PPNs and C. elegans. Overall, my research has produced a high throughput, reliable, and technically simple assay for identifying different RKN species, furthered our understanding of the fatty acid pathways in RKNs, and probed the effectiveness of feeding RKNs omega-6 PUFAs as an alternative control method to pesticides.

Novel Molecular Approaches to Identify and Control Plant Parasitic Nematodes

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