Aplicação e validação de metodologia para identificação e caracterização molecular de vírus associado à Atrofia da Coroa do Coqueiro.

Abstract

Coconut is a crop of great economic importance in Brazil. The country is the world's leading producer of coconut water and the world's fifth largest producer of coconut fruit. As with other crops, coconut is also susceptible to pests and phytosanitary problems that can affect the production. Coconut crown atrophy (CCA) was first reported in 2012 in Brazil. The disease name is attributed to the symptoms induced the in plants, which can cause fruit loss, necrosis, and leaf atrophy. CCA etiology and its possible vectors are hitherto unknown. High throughput sequencing studies of coconut palms showing disease symptoms identified a DNA-Rep sequence similar to DNA-Rep associated with viruses belonging to the Nanoviridae family. In the present study, we describe and validate methods for the identification of coconut crown atrophy virus – CCAV, a possible multipartite virus associated with CCA. We also standardize a nanopore sequencing method to access CCAV diversity. Palm samples from five Brazilian states were collected from symptomatic and asymptomatic coconut plants. The sequence of the largest common region (CR-M) of previously cloned and sequenced CCAV components was used to design primer pairs to amplify all genomic components of CCAV in a multiplex inverse PCR reaction. Then, the amplicons were sequenced in a MinION Mk1B sequencer (Oxford Nanopore Technologies). The CR1-Mix primers used in multiplex PCR proved to be efficient in accessing the genetic diversity of the coconut crown atrophy virus (CCAV) genomes of the studied isolates, especially from Pará state . Multiplex PCR associated with nanopore sequencing proved to be a fast, effective and assertive tool for molecular characterization of CCAV when compared to other cloning and sequencing techniques. Finally, phylogenetic studies suggest that CCAV is a member of a new taxon that could represent a new genus within the Nanoviridae family or a member of a new family of plant ssDNA genome viruses.