Desenvolvimento de um método para o diagnóstico do complexo do Pineapple mealybug wilt-associated virus

Abstract

Pineapple mealybug wilt associated virus species (PMWaV) in association with Dysmicoccus brevipes cause mealybug wilt of pineapple. Virus dissemination occurs primarily through infected and asymptomatic propagative material. In this work, we validated a molecular methodology by RT-qPCR, to detect of the PMWaV-1, PMWaV-2 and PMWaV-3 virus in asymptomatic and symptomatic plants. Leaves and tissue culture plantlets from pineapple cultivars Smooth Cayenne, Pérola and Vitória were analysed by conventional RT-PCR and reverse transcription quantitative real-time (qRT-PCR). Mealybugs D. brevipes obtained from symptomatic wilt plants were analyzed. A specific genes for each virus of PMWaV was found in response to infection and was not expressed in healthy plant. In silico assessment four housekeeping were selected for accurate gene expression analysis in pineapple during biotic stress by RTqPCR. PMWaV-3 virus was prevalent in symptomatic and asymptomatic plants, and associated with PMWaV-2 virus in the symptomatic wilt plants. PMWaV-1 virus did not exhibited direct relation with the disease. The nutritional status of plants influenced the onset of symtoms and presence of PMWaV-2 virus. The PMWaV-1, PMWaV-2 and PMWaV-3 virus were detected in mealybugs colleted from diseased plants. Our results demonstrated transmission of the PMWaV-2 and PMWaV-3 by mealybugs. However, D. brevipes was not a good vector of PMWaV-1. This research provided a method for indexing plants for production or certification healthy propagative material. This prevent the spread of virus by propagation material to new crops areas and an important tool for epidemiological study of the mealybug wilt of pineapple. These is the first validation of an accurately method to detect three PMWaVs virus in asymptomatic and symptomatic plants.