Comparative analyses of molecular detection protocols for grapevine fanleaf virus (GFLV) in Xiphinema index

Abstract This study evaluated different approaches for detecting grapevine fanleaf virus (GFLV) in its nematode vector, Xiphinema index , using reverse transcription polymerase chain reaction (RT-PCR). Four sample preparation strategies were compared, each based on a distinct approach and tested under identical conditions. Two approaches involved RNA extraction prior to RT-PCR: column-based RNA extraction (using the RNeasy Plant Mini Kit) and magnetic bead-based RNA extraction (using the MagMAX RNA Isolation Kit). The other two methods were tested without RNA extraction prior to RT-PCR: direct RT-PCR and brief heat treatment. Samples containing one, two or five nematodes from three different vineyards were analysed to assess the effect of sample size on the efficiency of GFLV detection in X. index . Transmission electron microscopy (TEM) confirmed the presence of GFLV-like particles in the anterior region of individual adult females of X. index , supporting the molecular detection results. Among the tested methods, column-based RNA extraction using the RNeasy Plant Mini Kit showed the highest detection probability and most consistent performance under the conditions evaluated, including samples containing one or two adult females of X. index . Detection success increased with higher nematode numbers, with a more pronounced effect observed for methods without prior RNA extraction. These results demonstrate that both sample preparation strategy and nematode number influence GFLV detection efficiency in vector populations and highlight the importance of optimised sample preparation and sampling strategies for reliable GFLV detection, with potential applications in vineyard monitoring, replanting and phytosanitary management.