2.2.46
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
Double-stranded RNA (dsRNA) has been detected in all major fungal taxonomic groups. DsRNA elements associated with virus-like particles (VLPs) are usually found encapsidated in isometric particles, whereas those not associated with VLPs are unencapsidated and located in vesicles of the cytoplasm. In most cases, dsRNA has asymptomatic effects on the host, although changes in colony growth, sporulation or pigmentation have been noted. Of particular interest to agriculture is the reduction of pathogenicity (hypovirulence) caused by the presence of dsRNA. For example, in Cryphonectria parasitica the transmission of dsRNA and hypovirulence between isolates has been utilized successfully for biocontrol of chestnut blight in Europe. We have been working on Fusarium oxysporum f. ;sp. phaseoli (Fop), the pathogen of Phaseolus sp. that causes bean yellows or wilt disease in all bean growing regions of the world. Extraneous bands were noted in total nucleic acid preparations of some Fop isolates during RFLP and RAPD characterization. The objectives of this study were to determine if in Fop and other Fusaria these bands were: (1) dsRNA; (2) useful taxonomic characters; (3) influential on host development; 4) associated with VLPs; and (5) correlated with pathogenicity.

Materials and methods
Extractions of total nucleic acids by standard protocols and dsRNA by cellulose chromatography were conducted on isolates from 32 putative F. oxysporum f. ;sp. phaseoli, seven F. ;oxysporum formae speciales and four other Fusarium spp. Enzyme digests were conducted on samples with various combinations of RNase A, NaCl, and/or DNase I. Radial growth rate was measured over a 14-day period for all isolates grown on PDA, and conidia counts were obtained. Partially purified VLPs were isolated from dsRNA containing and not containing isolates, UV absorbance was measured at 260 ;nm, and RNA was extracted from the VLPs. In addition, untreated mycelia and VLPs were negatively stained; and mycelia were fixed, resin embedded and sectioned for examination by TEM.

Results and conclusions

Two 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.