Location: Emerging Pests and Pathogens ResearchTitle: Development of a species-specific PCR for detection and quantification of Meloidogyne hapla in soil using the 16D10 Root-Knot Nematode Effector Gene
|GORNY, ADRIENNE - Cornell University - New York|
|HAY, FRANK - Cornell University - New York|
|PETHYBRIDGE, SARAH - Cornell University - New York|
Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/16/2019
Publication Date: 2/19/2019
Citation: Gorny, A., Wang, X., Hay, F., Pethybridge, S. 2019. Development of a species-specific PCR for detection and quantification of Meloidogyne hapla in soil using the 16D10 Root-Knot Nematode Effector Gene. Plant Disease. https://doi.org/10.1094/PDIS-09-18-1539-RE.
Interpretive Summary: The Northern root-knot nematode (Meloidogyne hapla) is an important pest of numerous agricultural crops. Molecular methods for rapid detection and quantification of nematode pests are needed to support management decisions. In this study, a qPCR assay using a nematode effector gene as a diagnostic marker was developed for identifying M. hapla. This qPCR assay was shown to be a reliable and highly sensitive method for detecting and quantifying M. hapla in soil, adding a new tool for molecular diagnostics of root-knot nematode species.
Technical Abstract: The Northern root-knot nematode (Meloidogyne hapla) is an important soilborne pathogen of numerous agricultural crops in temperate regions. Accurate detection and quantification is vital to supporting informed pest management decisions. However, traditional methods of manual nematode extraction and morphology-based identification are time consuming and prone to error from misidentification of species. Furthermore, molecular assays targeting the internal transcribed spacer region may lead to inaccurate results due to intraspecific variability. The Meloidogyne spp. effector gene 16D10 was assessed as a target for a SYBR Green-I quantitative PCR (qPCR) assay for detection and quantification of M. hapla. M. hapla-specific qPCR primers were developed and evaluated for specificity against five M. hapla isolates and 14 other plant-parasitic nematodes. A standard curve was generated by relating quantification cycle to the log of M. hapla population densities artificially introduced into soil. The influence of soil inhibitors on quantitative amplification was assessed by generating a dilution series from DNA extracted from pure cultures and inoculated soil. Extracts from soil produced significantly later Cq values than those produced from pure culture extracts. The utility of the qPCR was evaluated using soil samples collected from naturally infested potato fields, resulting in a significant positive relationship between populations estimated using qPCR and populations derived from manual counting. The qPCR developed in this study provides a useful method for detecting and quantifying M. hapla in soil, and demonstrates the utility of effector genes in plant-parasitic nematode diagnostics. The ability to use effector genes as targets for qPCR and other molecular detection and quantification methods may also open additional avenues of novel research and support development of improved species level diagnostics.