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ARS Home » Northeast Area » Beltsville, Maryland (BARC) » Beltsville Agricultural Research Center » Mycology and Nematology Genetic Diversity and Biology Laboratory » Research » Publications at this Location » Publication #426724
Research Project: Plant-associated Nematode Management and Systematics and USDA Nematode Collection Curation

Location: Mycology and Nematology Genetic Diversity and Biology Laboratory

Title: Selection of stable real-time quantitative PCR reference genes for the beech leaf disease nematode Litylenchus crenatae

Author
item WOLF, EMILY - Oak Ridge Institute For Science And Education (ORISE)
item MARRA, ROBERT - Connecticut Agricultural Experiment Station
item Reis Vieira, Paulo

Submitted to: Forest Pathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/1/2025
Publication Date: 9/23/2025
Citation: Wolf, E., Marra, R., Reis Vieira, P.C. 2025. Selection of stable real-time quantitative PCR reference genes for the beech leaf disease nematode Litylenchus crenatae. Forest Pathology. 55(5). Article e70039. https://doi.org/10.1111/efp.70039.
DOI: https://doi.org/10.1111/efp.70039

Interpretive Summary: Beech leaf disease (BLD) is spreading quickly through beech forests in the northeastern United States and Canada. This emerging disease is associated with an invasive leaf-infecting nematode (a microscopic roundworm) introduced from Asia. To understand the molecular mechanisms underlying this nematode parasitism, it is essential to analyze its gene expression profiles. In this study, we validate a set of reference nematode genes and select the most stable genes for the BLD nematode. This represents the first step towards identifying the nematode genes essential for the development of BLD. This information will be critical for plant pathologists and other engaged in the detection and monitoring this disease in both forest and beech nursery settings.

Technical Abstract: Beech leaf disease (BLD) is rapidly spreading throughout beech forests in the northeastern regions of North America, posing a significant ecological threat to these ecosystems. The biological agent in this disease is the foliar nematode Litylenchus crenatae. To unravel the molecular mechanisms governing L. crenatae parasitism and pathogenicity, it is essential to analyze its gene expression profiles. Accurate quantification of gene expression using reverse transcription quantitative PCR (RT-qPCR) requires stable internal reference genes for normalization. To date, no comprehensive studies have identified or validated suitable internal reference genes for L. crenatae across relevant stages of BLD. In this study, we evaluated nine candidate reference genes from L. crenatae and systematically evaluated their expression stability across various developmental stages and plant-interaction conditions. Our analysis identified two genes coding an EF-hand domain-containing protein (EF-hand) and a Ubiquitin-conjugating enzyme 2 (UBQ2), that exhibited the most stable expression profiles, indicating their suitability as internal controls for RT-qPCR assays in this nematode. Utilizing these validated reference genes, we further characterized the expression profiles of four parasitism-related genes. These target genes were assessed across different nematode developmental stages, as well as during key phases of host invasion and tissue interaction. Overall, our results provide suitable reference gene candidates for accurate gene expression studies in L. crenatae, contributing to a better understanding of the molecular interaction between this nematode and its beech hosts.