Development of a loop-mediated isothermal amplification assay for rapid and sensitive detection of Globodera...

The potato cyst nematodes (PCNs), Globodera pallida and G. rostochiensis, cause substantial yield losses in potato worldwide. In the USA, Globodera ellingtonae, was described in 2008 after detection in Oregon and Idaho. Although G. ellingtonae reproduces on potato, the varieties assessed appear to be tolerant. Nonetheless, the reliable detection and discrimination of G. ellingtonae from G. pallida and G. rostochiensis is critical for regulatory decision-making, survey programs, and research, especially in regions with mixed infestations such as Idaho, Chile, Bolivia, and Argentina. Traditional morphological identification is labor-intensive, and requires specialized expertise, emphasizing the need for a rapid molecular diagnostic assay targeting G. ellingtonae so that it can be differentiated from G. pallida or G. rostochiensis. We developed a loop-mediated isothermal amplification (LAMP) assay for specific detection of G. ellingtonae based on the chorismate mutase gene, which exhibits species-specific sequence variation and alternative splicing patterns among Globodera spp. The assay showed high analytical specificity for G. ellingtonae, with no cross-reactivity observed with G. pallida or G. rostochiensis. It was completed within 30 min under isothermal conditions and demonstrated a 100-fold higher sensitivity for G. ellingtonae (100 fg) compared with conventional PCR (10 pg). This is the first report of a LAMP assay enabling rapid, sensitive, and species-specific detection of G. ellingtonae. The assay provides a simple and robust diagnostic tool suitable for integration into plant health regulatory and surveillance programs to support management of potential mixed PCN infestations.