Selection of Stable Real-Time Quantitative PCR Reference Genes for the Beech Leaf Disease Nematode Litylenchus crenatae

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 etiological 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 analyse its gene expression profiles. Accurate quantification of gene expression using reverse transcription quantitative PCR (RT-qPCR) requires stable internal reference genes for normalisation. 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. Utilising these reference genes, we further characterised the expression profiles of four parasitism-related genes. These target genes were assessed across different nematode developmental stages collected from infected buds and leaves. Overall, our results provide suitable reference genes for accurate gene expression studies in L. crenatae, contributing to a better understanding of the molecular interaction between this nematode and its beech hosts.