Nutricion

Castillo et al. Virology Journal 2011, 8:38 http://www.virologyj.com/content/8/1/38

METHODOLOGY

Open Access

Rapid isolation of mycoviral double-stranded RNA from Botrytis cinerea and Saccharomyces cerevisiae
Antonio Castillo*, Luis Cottet, Miguel Castro, Felipe Sepúlveda
Abstract
Background: In most of the infected fungi, the mycoviruses are latent or cryptic, the infected fungus doesnot show disease symptoms, and it is phenotypically identical to a non-infected strain of the same species. Because of these properties, the initial stage in the search for fungi infected with mycoviruses is the detection of their viral genome, which in most of the described cases corresponds to double-stranded RNA (dsRNA). So to analyze a large number of fungal isolates it is necessary to have asimple and rapid method to detect dsRNA. Results: A rapid method to isolate dsRNA from a virus-infected filamentous fungus, Botrytis cinerea, and from a killer strain of Saccharomyces cerevisiae using commercial minicolumns packed with CF11 cellulose was developed. In addition to being a rapid method, it allows to use small quantities of yeasts or mycelium as starting material, being obtainedsufficient dsRNA quantity that can later be analyzed by agarose gel electrophoresis, treated with enzymes for its partial characterization, amplified by RT-PCR and cloned in appropriate vectors for further sequencing. Conclusions: The method yields high quality dsRNA, free from DNA and ssRNA. The use of nucleases to degrade the DNA or the ssRNA is not required, and it can be used to isolate dsRNAfrom any type of fungi or any biological sample that contains dsRNA.

Background Mycoviruses or fungal viruses have properties that differentiate them from viruses that infect animals, plants and bacteria [1-4]; they do not infect intact cells and are transmitted vertically by intracellular routes (meiosis and mitosis) and horizontally by anastomosis of compatible hyphae or through sexual mating ofyeast cells. Mycoviruses may also be latent and/or cryptic, since in most cases the infected fungus does not show disease symptoms and is phenotypically identical to a non-infected strain of the same species. Due to these peculiarities, the initial stage in the search for infected fungi with mycoviruses is the detection of their viral genome, which in most of the described cases corresponds todsRNA [1-4]. Although the number of ssRNA viruses described so far, such as the F and X viruses of Botrytis cinerea [4-6], has increased considerably, dsRNA continues to be the more predominant mycoviral genome. Therefore, to analyze a
* Correspondence: antonio.castillo@usach.cl Laboratorio de Virología de Hongos, Departamento de Biología, Facultad de Química y Biología, Universidad de Santiago deChile. Avenida Libertador Bernardo O’Higgins 3363, Estación Central, Santiago, Chile

large number of fungal isolates it is necessary to have a rapid method that allows the isolation and partial characterization of viral dsRNA using small amounts of mycelia or yeast cells as starting material. Some of the main and general methods described until now to isolate dsRNA molecules are: total nucleicacid isolation and further enzymatic digestion of the DNA and ssRNA [7]; phenol acid extraction (pH 4.5) in the presence of ammonium sulphate [8]; boiling of the fungal sample in the presence of a high salt concentration buffer [9], and use of CF11 cellulose, a chromatographic resin that allows the selective separation of dsRNA from DNA and ssRNA, using 16% ethanol in the elution buffer [10-13].All of the former methods require a considerable quantity of initial sample to obtain sufficient dsRNA for its later characterization, so it is very difficult to analyze a large number of fungal isolates with these techniques. Of the previous methodologies, the most widely used one is chromatographic separation on CF11-cellulose, since it allows getting dsRNA free of ssRNA, rRNA or tRNA, without...