ISOLATION AND ANALYSIS OF DOUBLE-STRANDED RNA AND VIRUS-LIKE PARTICLES IN FUSARIUM OXYSPORUM F. SP. PHASEOLI
SL WOO1, S MUCCIFORA2, M LORITO1, F SCALA1, A ZOINA1 and C. NOVIELLO1
1Dipartimento di Arboricoltura, Botanica e Patologia Vegetale - sezione Patologia Vegetale, UniversitÓ degli Studi di Napoli "Federico II", 80055 Portici (NA), Italy; 2Dipartimento di Biologia Evolutiva, UniversitÓ di Siena, Italy.
Background and Objectives
Materials and methods
Results and conclusionsTwo isolates pathogenic and one no longer pathogenic to bean were found to contain extraneous bands below undigested DNA in gel electrophoresis of nucleic acid samples. Extracts obtained by a specific procedure from these isolates plus another three F. ;oxysporum isolates contained dsRNA elements which varied in number, from 1 to 7 bands, and in size, from 0.9 to 7.0. ;kb. When samples were digested with DNase or RNase in high salt buffers there was no effect on the bands, whereas DNA and single-stranded RNA were digested by the respective enzyme treatments. This differential digestion indicated that the extraneous bands were dsRNA, which has never been reported for F. oxysporum f.. ;sp. phaseoli. The analysis of numerous Fusaria showed that since dsRNA is infrequent, it was not suitable for general isolate characterization. However, when present, dsRNA was an important character to identify specific isolates. No apparent physiological effects on growth were observed in isolates containing these elements.
Isometric VLPs averaging 30-50 ;nm were observed by TEM examinations of samples obtained from VLP extractions and in fresh mycelia of five dsRNA containing isolates, but not in the dsRNA non-containing isolates. No VLPs were detected in sections of resin embedded mycelia. Significant differences in 260 ;nm absorbance of RNA extracts from VLPs between the dsRNA containing and non-containing isolates suggest an association of dsRNA with VLPs and a viral origin. Gel electrophoresis of RNA from VLP samples did not produce banding profiles similar to those of the dsRNA, which may imply that the dsRNA elements were not encapsidated or the amount of RNA extracted from the VLPs was insufficient. The relationship of dsRNA to pathogenicity in F. ;oxysporum is being investigated since different elements were found in both pathogenic and non-pathogenic isolates.