Chrysanthemum stunt viroid in Brazil: survey, identification, biological and molecular characterization and detection methods

Abstract

In Brazil, the ornamental flowers and plants market moves in the wholesale and retail more than two billion US dollars annually, and chrysanthemum stands out as one of the most valuable commercial species. The stunting disease induced by Chrysanthemum stunt viroid (CSVd) has become a serious problem in chrysanthemum production systems worldwide. CSVd incites also colour breaking and retards flowering, but in many situations it does not induce visible symptoms, facilitating its spread in the field and passing international borders unnoticed. In Brazil there are few studies on this pathogen, with a single report of its possible occurrence in chrysanthemum in the State of São Paulo. In this work we aimed at surveying, identifying and characterizyng viroids in chrysanthemum crops in the State of São Paulo; challenging chrysanthemum varieties with a Brazilian CSVd isolate; and establishing diagnostic strategies to strengthen quarantine and indexing programs. Our survey showed that CSVd is widely disseminated in chrysanthemum crops in the State of São Paulo. All evaluated chrysanthemum varieties were susceptible of infection, although infection was sometimes symptomless. The complete genome sequencing of seven CSVd isolates showed high nucleotide identity when compared with other isolates from databases. Specific oligonucleotides were designed for conventional RT-PCR and RT-qPCR CSVd analysis. Results show that CSVd can be identified with a sensitivity about 100,000 times higher than PAGE. Dotblot proved also to be highly sensitive and, consequently, most useful for large scale diagnosis. This is the first biological and molecular identification and characterization of CSVd isolates in Brazil.