Detection of Globodera pallida directly from soil sample using mt-COI region based LAMP assay

Abstract

Potato cyst nematodes (PCN), Globodera rostochiensis (Golden/yellow) and G. pallida (White), are economically important and relatively specialized pest of potato (Solanum tuberosum L.). Both the species are being identified based on cyst colour after 55-60 days after planting (DAP) however, after 65 DAP, we cannot differentiate based on cyst colour as both species turns brown. Moreover, the molecular techniques available to detect the PCN at species level is laborious, time consuming and costly. Therefore, development of rapid, accurate and economically cheap technique for detection of PCN at species level from the field is important to device effective management strategies for sustainable potato production. Accordingly, in the first instance, loop-mediated isothermal amplification (LAMP) assay was developed to detect G. pallida directly from soil by using the mitochondrial (mt-COI) gene specific primer. The LAMP assay was completed within 60 min at 60 °C isothermal conditions and the primer, efficiently detects the G. pallida without any cross reaction with G. rostochiensis, Meloidogyne incognita, M. javanica, Heterodera avenae, H. carotae, and Cactodera spp. In analytical sensitivity tests, the assay was able to detect G. pallida with 1000 times less DNA concentration (10 fg/µl) as compared to conventional PCR (10 pg/µl) and the LAMP product was visualized by using SYBR Gold nucleic acid dye and the assay can be highly useful in detection of G. pallida.