Lamp and Real-Time PCR for Detection of Passion Fruit Woodiness Disease (PWD) Causal Viruses

Abstract

Passion fruit (Passiflora edulis [Sims]) is an important economic crop grown for both domestic and export market world-wide. In Kenya, passion fruit productivity is low due to both biotic and abiotic constraints. Passion fruit woodiness disease (PWD) is the most important viral disease and the most limiting factor for passion fruit production worldwide. Cowpea Aphid Borne Mosaic Virus (CABMV) reported to be present in Brazil, South Africa, Uganda and Rwanda is the major causal agent of woodiness disease in Kenya. This and other pests contribute largely to non-availability of clean planting material. This study aimed at improving the diagnostics of passion fruit woodiness disease through development of molecular-based diagnostic assays. SYBR Green-based real-time PCR and loop-mediated isothermal amplification (LAMP) were optimized and evaluated for detection of the causal agents of woodiness disease in passion fruit. The effect of different sample storage conditions for passion fruit leaf samples on RNA quality and suitability for passion fruit virus diagnostics were also evaluated. This was to determine the most reliable method for handling sample materials from field to the laboratory with the aim of achieving rapid detection of the viruses. To evaluate the different sample storage methods, passion fruit leaf samples were collected from the field and stored for 1 and 2 weeks using FTA® cards, RNAlater, cold ice followed by transfer to -80˚C freezer, drying on silica gel and drying in between the sheets of newsprints (as herbarium) before RNA extraction and subsequent downstream assays. Good RNA yield and quality were obtained from samples stored in silica gel for 1 and 2 weeks after collection. Further analysis confirmed that RNA extracted from samples stored in silica gel was suitable for RT-PCR amplification. The assay conditions of LAMP and SYBR Green real-time PCR assay were optimized followed by specificity and sensitivity determination. The lowest limit of detection for the LAMP assay and real-time PCR was determined using tenfold dilution series were found to be 10-7 and 10-6, respectively. No cross-reaction was observed when other viruses were included in the assay indicating that the assays were 100% specific. There is need for adoption of these molecular diagnostic methods in Passion fruit nursery certification programs for early stage screening of planting materials.