Characterization of a new causal agent associated with chlorotic spots in citrus

Abstract

Brazil is responsible for 56% of the world orange juice production; however, its productivity is frequently affected by several diseases such as citrus leprosis (CL), caused by Brevipalpus mites transmitted viruses (BTVs). BTVs induce chlorotic and/or necrotic localized lesions in fruits, leaves and branches; under natural conditions, they do not systemically infect plants. BTVs belong to the genera Dichorhavirus and Cilevirus. During the years of 2017-2019, sweet orange and mandarin samples collected in the states of Espírito Santo, Minas Gerais, and Bahia, exhibiting symptoms similar to those caused by CL and citrus zonate chlorosis (ZC), were sent to our lab. ZC is a disease of putative viral etiology, uncharacterized molecularly and possibly transmitted by brevipalpus mites. Although the samples tested negative in transmission electron microscopy (MET) and RT-PCR assays using primers that specifically amplify several BTVs, they were amplified with degenerated primers that detect members of family Kitaviridae. The present work aims to identify and characterize the causal agent of the described symptoms through high-throughput sequencing (HTS). In parallel, at least one citrus sample with ZC from the herbarium of the Instituto Biológico, collected from the period of 1933-1969, will be sequenced using HTS. The sequences of the new causal agent will be compared with those of other kitaviruses and the original ZC sample, allowing to establish the proper phylogenetic relationships, evolutionary parameters to propose a taxonomic classification for this virus. In addition, specific primers will be designed to detect the virus(es) involved in the symptoms, and its(their) biological transmission will be attempted with viruliferous brevipalpus mites to Arabidopsis thaliana plants. The molecular tools developed in this work shall support the studies on the diversity of viruses affecting citrus orchards in Brazil. Moreover, it is expected an increased relevance of emergent (or reemergent) citrus diseases in the future, due to climate changes and drastic restrictions in the use of available agrochemicals for the control of the mite vector in the management of diseases. This study is part of one of the objectives of the project "Understanding the molecular biology and ecology of plant-virus-vector relationships: Towards Sustainable, Integrated Virus Management Strategies" (Fapesp-NWO, proc. 2017/50222-0), coordinated by the advisor of this MS dissertation and to which this proposal is associated. (AU)