Department of Genetics, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria 0002, South Africa1
ARC-Fruit, Vine and Wine Research Institute, Private Bag X5013, Stellenbosch 7599, South Africa2
Author for correspondence: Oliver Preisig. Fax +27 12 420 39 60. e-mail Oliver.Preisig{at}fabi.up.ac.za
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Various mycoviruses have been shown to mediate reduced virulence (hypovirulence) in their plant pathogenic hosts. A well-studied example is the hypovirus of the chestnut blight fungus, Cryphonectria parasitica (Shapira et al., 1991 ; Nuss, 1992
). The American chestnut (Castanea dentata) has virtually been eliminated from the landscape by C. parasitica, which was introduced into North America early in the 20th century (Anagnostakis & Waggoner, 1981
). In Europe, the presence of a hypovirulence-mediating dsRNA virus in the introduced population of C. parasitica reduced the devastating impact of this chestnut pathogen (Heiniger & Rigling, 1994
). The horizontal transmission of mycoviruses depends on hyphal fusion (anastomosis) between two compatible isolates (Ghabrial, 1998
). In Europe, a much smaller number of vegetative compatibility (VC) groups are found in the population of C. parasitica and this has facilitated spread of the hypovirus (Liu & Milgroom, 1996
).
Besides the hypovirulence-mediating hypovirus infecting C. parasitica, other mycoviruses conferring hypovirulence have been reported. These viruses include different mitoviruses infecting the mitochondria of Ophiostoma novo-ulmi (Hong et al., 1999 ), the totivirus Hv190SV of Helminthosporium victoriae (Huang & Ghabrial, 1996
) as well as the unclassified dsRNA elements in Leucostoma persoonii (Hammar et al., 1989
). In other cases, such as the totiviruses infecting Sphaeropsis sapinea, no significant effect of the virus on the host could be observed (Preisig et al., 1998
; Steenkamp et al., 1998
). In plant pathology, the interest in mycoviruses derives from their potential to actively apply their hypovirulence-mediating effect in the biological control of pathogens.
Ascomycetes belonging to the genus Diaporthe are important plant pathogens of numerous agronomic and tree crops worldwide. In South Africa, the filamentous fungus Diaporthe ambigua causes cankers on apple, pear and plum trees and their rootstocks. This disease can lead to the slow death of mature trees, while affected nursery rootstocks usually die rapidly. The pathogen thus has an immense effect on the productivity of orchards of pome and stone fruit trees (Smit et al., 1996a , 1998
).
Isolates of D. ambigua have been observed to differ in virulence and morphology. The hypovirulence of isolates is coincidental with the presence of a single dsRNA species of about 4 kb in the fungal mycelia (Smit et al., 1996b ). Fungal isolates containing these dsRNA elements are not only hypovirulent, but also show hypovirulence-associated traits. These traits included reduced phenol oxidase activity, reduced gallic acid oxidation, diminished oxalate accumulation and suppressed production of ascospores (Smit et al., 1996b
). The dsRNA was transmissible between dsRNA-containing and dsRNA-free isolates of D. ambigua of the same VC group through anastomosis. Through this approach, previously virulent, dsRNA-free isolates were converted to hypovirulence. This result supported the idea that the presence of dsRNA mediates hypovirulence in D. ambigua (Smit et al., 1996b
). However, only transformation or transfection of dsRNA-free fungal isolates with cDNA constructs or RNA from the genetic RNA element might present conclusive evidence for this effect. The putative hypovirulence-mediating dsRNA element in D. ambigua could then be an ideal agent to be developed for biological control of this serious canker pathogen of pome and stone fruit trees (Smit et al., 1998
).
For application of the D. ambigua dsRNA element in biological control, the dsRNA must be characterized at a molecular level. In this study, we confirm the viral origin of the dsRNA based on its complete cDNA sequence. Furthermore, this sequence analysis shows that D. ambigua is infected by a novel RNA mycovirus related to the plant virus family Tombusviridae.
![]() |
Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Extraction and purification of dsRNA.
Freeze-dried mycelium was ground and resuspended in 2x STE (0·1 M TrisHCl, 0·2 M NaCl and 2 mM EDTA, pH 6·8) with 1% SDS. The sample was incubated for 10 min at 60 °C and subsequently mixed with an equal volume of phenol. The mixture was shaken for 30 min and then centrifuged at 10000 r.p.m. in a Beckman JA25.50 rotor for 30 min at 4 °C. The aqueous phase was extracted with an equal volume of chloroform and separated by another centrifugation step. The aqueous phase was finally adjusted to 16% ethanol and centrifuged at 5000 r.p.m. for 5 min to pellet the precipitated genomic DNA. The supernatant was applied to a CF11 cellulose (Whatman) column (Valverde et al., 1990 ) prepared in a syringe without the plunger. The column was washed with 2x STE with 16% ethanol to separate the bound dsRNA from other nucleic acids. The dsRNA was eluted in 2x STE and subsequently precipitated with 0·6 vols isopropanol. The dsRNA pellet was washed with 70% ethanol. The dried sample was then resuspended in DEPC-treated ddH2O and separated by agarose gel electrophoresis. The dsRNA band was excised from the gel. An RNaid w/SPIN kit (BIO101) was used to isolate the dsRNA from the gel pieces. The purified dsRNA was stored in ddH2O at -20 °C.
Production of cDNA from viral dsRNA.
The cDNA synthesis was performed following the method of Gubler & Hoffmann (1983) using a cDNA synthesis kit (Roche Molecular Biochemicals). Mixed hexanucleotides were initially used to prime the first-strand cDNA synthesis of heat-denatured dsRNA (for 10 min at 99 °C) with AMV reverse transcriptase. The second strand was synthesized with E. coli DNA polymerase I from nicks introduced by RNase H in the RNA strand of the RNAcDNA hybrid. The random cDNA products were then digested with Sau3AI. The restriction digest was purified through a column of a High Pure PCR Product Purification kit (Roche Molecular Biochemicals) to remove products with a length under 100 bp. Products were then cloned into the BamHI site of pGEM-3Zf(+) vector and transformed into E. coli JM109 (Promega).
Sequence-derived 19- to 23-mer primers (MWG-Biotech) were used for RTPCR experiments (Titan One Tube RTPCR System; Roche Molecular Biochemicals) to amplify parts of the genome which were not cloned by the initial random cDNA synthesis. When necessary, the RTPCR products were cloned in the pGEM-T easy vector (Promega) prior to sequencing. The distal ends of the dsRNA were amplified by the 5' RACE (rapid amplification of cDNA ends) approach (Frohman, 1994 ; Preisig et al., 1998
) using a 5'/3' RACE kit (Roche Molecular Biochemicals) and sequence-derived, nested primers. Primer pair Oli64 (5' GTCGCATCTCACAGCCGAGCGC 3') and Oli80 (5' CTCACCAGCCTCCAACCG 3') was used to amplify the conserved coding region of the RDRP in RTPCRs.
Construction of a full-length cDNA of viral dsRNA.
The construction of a full-length cDNA clone of the viral dsRNA was based on two large overlapping partial RTPCR products (Fig. 1A). Both products were cloned in the pGEM T-Easy vector (Promega). The 2·8 kb 5' product was amplified using primer pair DaRV-5' (5' GGGAAATTTGTGAGATTATCGCC 3') and Oli65 (5' AACCTCGAGCACAGCGCAACG 3', including a XhoI restriction site) and cloned as pDV1. The 1·5 kb 3' product was amplified using primer pair Oli64 and DaRV-3' (5' GGGCCACAGGATCCGGAGAAC 3') and cloned as pDV2. The orientations of the clones were selected so that the 5' ends of viral cDNA were on the site of T7 promoter of the pGEM T-Easy vector. Plasmid pDV1 was linearized using the restriction enzymes XhoI and NsiI. While XhoI has one restriction site within the viral cDNA, NsiI cuts once in the multiple cloning site of pGEM T-Easy Vector. The 1·4 kb XhoINsiI fragment of pDV2 was excised from an agarose gel and then ligated into the linearized pDV1. The authenticity of the resulting, full-length cDNA clone pDV3 was confirmed by sequencing.
|
Sequences were analysed using the computer program Sequence Navigator (Perkin-Elmer) and the programs Translate, SIM/PRSS, PSORT, CLUSTALW and WU-BLAST listed at the ExPASY home page (http://www.expasy.ch/tools/). The GenBank/EMBL and SWISS-PROT/TrEMBL databases were used for homology searches.
Northern blot hybridization.
Total nucleic acids from 1 mg of mycelia of isolate CMW3407 and CMW5588 were separated in a 1% agarose gel in 1x Trisacetate/EDTA buffer. As positive controls, isolated dsRNA and in vitro-produced positive- and negative-stranded RNA from pDV3 were included. The latter were produced by the T7 or the SP6 RNA polymerase from pDV3, which was linearized with SpeI or NcoI (Fig. 1A), respectively. The agarose gel was denatured in 50 mM NaOH/150 mM NaCl for 30 min and subsequently neutralized in 1 M TrisHCl (pH 7·5)/1·5 M NaCl twice for 15 min. The nucleic acids were then transferred by a capillary blot onto positively charged nylon membrane (Roche Molecular Biochemicals). Hybridization with DIG-labelled, strand-specific RNA probes was carried out according to the manufacturers protocols in DIG Easy Hyb buffer at 68 °C (Roche Molecular Biochemicals). Colorimetric detection was done with NBT/BCIP.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Primers were designed from the short cDNA sequences. Different primer combinations were then randomly applied in RTPCR to amplify overlapping products to determine gaps between the sequenced parts of the putative genome. This worked in a few cases. In other cases RTPCR products were obtained from reactions in which only one specific primer amplified a product from its specific binding site. These products, which were very valuable for establishment of the sequence, were apparently due to nonspecific binding of the primer to a homologous region on the template close to its specific site. The second primers in such reactions were found to have been derived from a cDNA clone that was not of viral origin. Using the sequences of the RTPCR products, the cDNA sequence of the whole dsRNA element could be completed, with the exception of the terminal sequences. The distal 5' and 3' termini of the dsRNA were then determined by a 5' RACE approach (Frohman, 1994 ). For both ends, single PCR products were amplified with an oligo(dT) primer and a terminus-specific primer. The products were directly sequenced with nested primers. The termini did not show any variation in sequence or length.
The described sequencing strategy established the complete cDNA sequence of the dsRNA genetic element of D. ambigua. The ability to determine distinct ends of the dsRNA genetic element shows that the dsRNA is a linear molecule. The sequence consists of 4113 bp with a GC content of 53%. The accuracy of the sequence was confirmed through sequencing both strands of the full-length cDNA clone pDV3 (construction described in Methods; Fig. 1A). Only two sequence variations were observed, namely at positions 389 (G
A) and 1743 (G
A).
Sequence analysis of the complete cDNA of the dsRNA genetic element
Analysis of the complete cDNA sequence revealed the presence of two large open reading frames (ORFs). Translation of the first ORF might be initiated at the AUG start codon at position 576 and be terminated at the UAG stop codon at position 2085. Translation of the second ORF might be internally initiated at the AUG start codon at position 2202 to the stop codon UAG at position 3678. However, it is common in the translation of RNA viruses for ribosomes to readthrough UAG (amber) stop codons and to produce a fusion protein (Skuzeski et al., 1991 ). A readthrough of UAG at position 20852087 is supported by the fact that both ORFs are in the same frame, which is essential for a readthrough translation. Furthermore, the flanking region of this amber codon (2082UUU-UAG-GGA2090) is similar to the consensus sequence AAA-UAG-G (G/U)(G/A) around readthrough codons found in carmoviruses (Skuzeski et al., 1991
). Therefore, the two ORFs are most probably translated as a fusion protein from the AUG at position 576. Translation of this fusion protein might be extended over two more UAG codons at positions 3678 (the end of the second ORF) and 3846 and might stop at the UGA stop codon at position 3954. No other ORFs of significant length can be detected either upstream of position 576 or downstream of position 3954.
Considering the different possibilities for readthrough translations, proteins of 503, 1034, 1090 and 1126 amino acids might be translated from the genetic element of D. ambigua. Their predicted molecular masses would be 56·0, 114·9, 120·7 or 124·3 kDa, respectively. These predictions do not take the unknown substitutes for the amber codons into account. In the following characterization, the longest translation product, p125, which is 1126 amino acids long, will be considered. However, the shorter translation product p56 (N-terminal part of p125) might be the major translation product because readthrough events occur at a lower rate than translation termination (Skuzeski et al., 1991 ).
The PSORT program, employing the method of Klein et al. (1985) , predicts eight possible transmembrane helices for p125 (Fig. 2
). Six of these are predicted to occur at the N terminus, which is also part of p56, and two at the C terminus. Therefore, p56 and p125 could be associated with membranes of the host cell.
|
A reduced but still significant homology can be found outside the RDRP domain in the alignment of the three gene products (Fig. 2). However, the N-terminal part of p125 down to the potential readthrough codon, which might also be translated as p56, is N-terminally extended by 220 amino acids compared to the TCV and CarMV proteins (Fig. 2
). On its own, the protein p56 does not produce any significant homology to proteins in the SWISS-PROT and TrEMBL protein databases.
The potential translation product p125 shows clear homology to viral RDRPs. It can, therefore, be assumed that the dsRNA in D. ambigua is of viral origin. We have named the virus Diaporthe ambigua RNA virus (DaRV). Interestingly, the sequence of the DaRV genome does not include an ORF encoding a coat protein. In the cases of TCV and CarMV (Guilley et al., 1985 ; Carrington et al., 1989
), an ORF for a coat protein is located at the 3' end of their genomes, downstream of the coding region for the RDRP (Fig. 1B
).
Relative abundance of viral ssRNA versus dsRNA in D. ambigua
The dsRNA isolated from the infected D. ambigua isolate may represent the genomic or the replicative form of DaRV. In order to test if the DaRV genome is maintained in an ssRNA or in a dsRNA form, a Northern blot hybridization study was done. This also enabled determination of the relative abundance of viral ssRNA versus dsRNA.
Total nucleic acid preparations were obtained from the dsRNA-infected isolate CMW3407 and the dsRNA-free isolate CMW5588 of D. ambigua. These were separated by non-denaturing agarose gel electrophoresis. Isolated D. ambigua dsRNA as well as in vitro-produced positive-strand (400 bases shorter than the full-length) and negative-strand (full-length) RNA of DaRV were included as positive controls. Duplicate blots were probed with either a digoxigenin-labelled, positive-strand or negative-strand RNA probe of DaRV, respectively.
Hybridization with the probe for positive-strand RNA resulted in an intense signal in the lane in which total nucleic acid from the virus-infected isolate was separated. The signal at the position where the positive-strand RNA control migrated was very strong, while the slower migrating dsRNA could hardly be detected (Fig. 3A). A smear of RNA migrating faster than the full-length DaRV RNA was also observed. This band most probably represents shorter transcripts or different conformations of the positive-strand RNA of DaRV. With the negative-strand RNA probe, only a faint signal of dsRNA was observed in the total nucleic acid preparation from the virus-infected isolate (Fig. 3B
). No signal was observed with either probe in the lanes with nucleic acid from the virus-free isolate.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Mycoviruses other than DaRV have been reported to show distant sequence relationship to Tombusviridae. Koonin & Dolja (1993) studied the tentative phylogeny of positive-strand RNA viruses based on multiple alignments of the conserved RDRP domains. In their study, the Saccharomyces cerevisiae 20S and 23S narnaviruses grouped with tombusviruses among others in supergroup II. Recently, the fungal mitochondrial RNA replicons, the mitoviruses, were also reported to be part of this lineage (Hong et al., 1998
, 1999
). Because DaRV shows the highest sequence similarities to the Tombusviridae, we propose that DaRV is a member of supergroup II of positive-strand RNA viruses.
DaRV is among a number of mycoviruses that show distinct relatedness to plant viruses (Ghabrial, 1998 ). In addition to the narnaviruses and mitoviruses, it was previously shown that the hypoviruses of C. parasitica have common ancestry with plant potyviruses (Koonin et al., 1991
). Mushroom bacilliform virus, a barnavirus with an ssRNA genome, was also reported to share sequence similarity with plant viruses of the genus Polerovirus and to sobemoviruses (Revill et al., 1999
). However, there is no obvious relationship based on sequence similarity and genome organization between these viruses and DaRV, except around the consensus sequences in the RDRP domain.
The deviations from the conserved motifs observed in the DaRV RDRP are of particular interest with respect to the activity of the polymerase. These motifs are present in all classes of polymerases and certain polymerase functions are attributed to them (OReilly & Kao, 1998 ). Motif A is possibly involved in the coordination of divalent metal cofactors and the selection for NTP or dNTP. It is therefore interesting that the modified Motif A of DaRV (-D737-X-X-X-X-E742-; Fig. 2
), with a glutamate instead of an aspartate at position 742, resembles more the active site of DNA polymerases than that of an RDRP (OReilly & Kao, 1998
; Patel & Loeb, 2000
). The -G-D-D- consensus sequence of Motif C, which is also involved in the coordination of divalent metal ions, is highly conserved in RDRPs, especially the two aspartates (OReilly & Kao, 1998
). Therefore, it might be important for the function of the DaRV RDRP that the second aspartate in DaRV Motif C is replaced by an asparagine (Fig. 2
). The same, modified -G-D-N- sequence can only be found in the functional RDRP domain of the L proteins of nonsegmented negative-strand RNA viruses, like Rabies virus or Measles virus (Schnell & Conzelmann, 1995
).
These few but significant differences in the conserved motifs of the DaRV RDRP might cause an altered polymerase activity. However, the possibility that the viral sequence represents a defective RNA cannot be ruled out. This RNA could be in such a high abundance that the active viral genome could not be detected in the several, independent RTPCR amplifications that we did. Only a successful transfection or transformation of D. ambigua with in vitro-produced viral RNA or with a cDNA construct from DaRV, respectively, can definitely indicate that the described viral genome is functional.
The most significant difference in the genome organization of DaRV and the carmoviruses is that the DaRV genome encodes neither a coat protein (p38) nor small movement proteins (p7, p8 or p9) such as are found for TCV or CarMV (Fig. 1B). Movement proteins are essential for the intercellular spread in plants (Hacker et al., 1992
) and, therefore, might be dispensable in a mycovirus. In carmoviruses, coat proteins make up a large number of isometric virus particles of about 25 nm in diameter (Russo & Martelli, 1982
). A possibility remains that p56 or a cleaved part of it, for example the N-terminal extension, might function as a coat protein. However, no significant sequence similarity can be found between p56 and coat proteins of carmoviruses or other viruses. Without coat proteins, DaRV probably has no means to form virus particles. Experiments with a TCV mutant lacking the coding region for a coat protein showed that the mutant viral RNA was still able to replicate in the cells of inoculated plant leaves (Hacker et al., 1992
).
It might be assumed that the DaRV genome is either present as RNAp56/p125 nucleoprotein complexes in the cytoplasm or that the virus might be associated with membrane vesicles. While ribonucleoprotein complexes were reported for yeast narnaviruses (García-Cuéllar et al., 1997 ), the unencapsidated hypovirus of C. parasitica was found to be associated with fungal vesicles (Hansen et al., 1985
; Fahima et al., 1993
). The N-terminal, hydrophobic part of p56/p125 could favour a membrane association of DaRV. Furthermore, it has been shown that the replication of positive-stranded RNA virus genomes commonly takes place in close association with membranes (Schaad et al., 1997
). The N terminus of p56/p125 could ideally anchor the viral protein in a membrane through the six predicted transmembrane helices. Interestingly, the non-structural proteins of the tombusviruses Cymbidium ringspot virus and Carnation Italian ringspot virus target the peroxisomal or mitochondrial membranes based on their N-terminal sequences (Rubino & Russo, 1998
; Rubino et al., 2000
). The targeting is determined by the leader sequence as well as the transmembrane segments. Further studies will be needed to clarify the localization of DaRV in the cell.
The hypovirulence-associated mycovirus DaRV, with its relatively small genome size, is of great interest for the development of a biological control system for D. ambigua and other related plant pathogens. This study has characterized the virus at sequence level, which is an important step towards any advanced use of this virus in biological control. The development of protocols for transfection and transformation of the fungus with DaRV is under way. Such studies are essential to understand the mechanism involved in hypovirulence, as well as for a practical application of this virus in biological control.
![]() |
Acknowledgments |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Carrington, J. C., Heaton, L. A., Zuidema, D., Hillman, B. I. & Morris, T. J.(1989). The genome structure of turnip crinkle virus.Virology170, 219-226.[Medline]
Fahima, T., Kazmierczak, P., Hansen, D. R., Pfeiffer, P. & van Alfen, N. K.(1993). Membrane-associated replication of an unencapsidated double-strand RNA of the fungus, Cryphonectria parasitica.Virology195, 81-89.[Medline]
Frohman, M. A.(1994). On beyond classic RACE (rapid amplification of cDNA ends).PCR Methods and Applications4, S40-S58.[Medline]
García-Cuéllar, M. P., Esteban, R. & Fujimura, T.(1997). RNA-dependent RNA polymerase activity associated with the yeast viral p91/20S RNA ribonucleoprotein complex.RNA3, 27-36.
Ghabrial, S. A.(1998). Origin, adaptation and evolutionary pathways of fungal viruses.Virus Genes16, 119-131.[Medline]
Gubler, U. & Hoffmann, B. J.(1983). A simple and very efficient method for generating cDNA libraries. A simple and very efficient method for generating cDNA libraries.Gene25, 263-269.[Medline]
Guilley, H., Carrington, J. C., Balazs, E., Jonard, G., Richards, K. & Morris, T. J.(1985). Nucleotide sequence and genome organization of carnation mottle virus.Nucleic Acids Research13, 6663-6677.[Abstract]
Hacker, D. L., Petty, I. T. D., Wei, N. & Morris, T. J.(1992). Turnip crinkle virus genes required for RNA replication and virus movement.Virology186, 1-8.[Medline]
Hammar, S., Fulbright, D. W. & Adams, G. C.(1989). Association of double-stranded RNA with low virulence in an isolate of Leucostoma persoonii.Phytopathology79, 568-572.
Hansen, D. R., van Alfen, N. K., Gillies, K. & Powell, W. A.(1985). Naked dsRNA associated with hypovirulence of Endothia parasitica is packaged in fungal vesicles.Journal of General Virology66, 2605-2614.
Heiniger, U. & Rigling, D.(1994). Biological control of chestnut blight in Europe.Annual Review of Phytopathology32, 581-599.
Hong, Y., Cole, T. E., Brasier, C. M. & Buck, K. W.(1998). Evolutionary relationships among putative RNA-dependent RNA polymerases encoded by a mitochondrial virus-like RNA in the Dutch elm disease fungus, Ophiostoma novo-ulmi, by other viruses and virus-like RNAs and by the Arabidopsis mitochondrial genome.Virology246, 158-169.[Medline]
Hong, Y., Dover, S. L., Cole, T. E., Brasier, C. M. & Buck, K. W.(1999). Multiple mitochondrial viruses in an isolate of the Dutch elm disease fungus Ophiostoma novo-ulmi.Virology258, 118-127.[Medline]
Huang, S. & Ghabrial, S. A.(1996). Organization and expression of the double-stranded RNA genome of Helminthosporium victoriae 190S virus, a totivirus infecting a plant pathogenic filamentous fungus.Proceedings of the National Academy of Sciences, USA93, 12541-12546.
Klein, P., Kanehisa, M. & DeLisi, C.(1985). The detection and classification of membrane-spanning proteins.Biochimica et Biophysica Acta815, 468-476.[Medline]
Koonin, E. V. & Dolja, V. V.(1993). Evolution and taxonomy of positive-strand RNA viruses: Implications of comparative analysis of amino acid sequences.Critical Reviews in Biochemistry and Molecular Biology28, 375-430.[Abstract]
Koonin, E. V., Choi, G. H., Nuss, D. L., Shapira, R. & Carrington, J. C.(1991). Evidence for common ancestry of a chestnut blight hypovirulence-associated double-stranded RNA and a group of positive-strand RNA plant viruses. Proceedings of the National Academy of Sciences, USA88, 10647-10651.[Abstract]
Liu, Y.-C. & Milgroom, M. G.(1996). Correlation between hypovirus transmission and the number of vegetative incompatibility (vic) genes different among isolates from a natural population of Cryphonectria parasitica.Phytopathology86, 79-86.
Nuss, D. L.(1992). Biological control of chestnut blight: an example of virus-mediated attenuation of fungal pathogenesis.Microbiological Reviews56, 561-576.[Abstract]
OReilly, E. R. & Kao, C. C.(1998). Analysis of RNA-dependent RNA polymerase structure and function as guided by known polymerase structures and computer predictions of secondary structure. Virology252, 287-303.[Medline]
Patel, P. H. & Loeb, L. A.(2000). DNA polymerase active site is highly mutable: evolutionary consequences.Proceedings of the National Academy of Sciences, USA97, 5095-5100.
Preisig, O., Wingfield, B. D. & Wingfield, M. J.(1998). Coinfection of a fungal pathogen by two distinct double-stranded RNA viruses. Virology252, 399-406.[Medline]
Revill, P. A., Davidson, A. D. & Wright, P. J.(1999). Identification of a subgenomic mRNA encoding the capsid protein of mushroom bacilliform virus, a single-stranded RNA mycovirus.Virology260, 273-276.[Medline]
Rubino, L. & Russo, M.(1998). Membrane targeting sequences in tombusvirus infections.Virology252, 431-437.[Medline]
Rubino, L., Di Franco, A. & Russo, M.(2000). Expression of a plant virus non-structural protein in Saccharomyces cerevisiae causes membrane proliferation and altered mitochondrial morphology.Journal of General Virology81, 279-286.
Russo, M. & Martelli, G. P.(1982). Ultrastructure of turnip crinkle- and saguaro cactus virus-infected tissues. Virology118, 109-116.
Schaad, M. C., Jensen, P. E. & Carrington, J. C.(1997). Formation of plant RNA virus replication complexes on membranes: role of an endoplasmic reticulum-targeted viral protein.EMBO Journal16, 4049-4059.
Schnell, M. J. & Conzelmann, K.-K.(1995). Polymerase activity of in vitro mutated rabies virus L protein.Virology214, 522-530.[Medline]
Shapira, R., Choi, G. H. & Nuss, D. L.(1991). Virus-like genetic organization and expression strategy for a double-stranded RNA genetic element associated with biological control of chestnut blight. EMBO Journal10, 731-739.[Abstract]
Skuzeski, J. M., Nichols, L. M., Gesteland, R. F. & Atkins, J. F.(1991). The signal for a leaky UAG stop codon in several plant viruses includes the two downstream codons.Journal of Molecular Biology218, 365-373.[Medline]
Smit, W. A., Viljoen, C. D., Wingfield, B. D., Wingfield, M. J. & Calitz, F. J.(1996a). A new canker disease of apple, pear and plum rootstocks caused by Diaporthe ambigua in South Africa.Plant Disease80, 1331-1335.
Smit, W. A., Wingfield, B. D. & Wingfield, M. J.(1996b). Reduction of laccase activity and other hypovirulence-associated traits in dsRNA-containing strains of Diaporthe ambigua.Phytopathology86, 1311-1316.
Smit, W. A., Wingfield, B. D. & Wingfield, M. J.(1998). Integrated approach to controlling Diaporthe canker of deciduous fruit in South Africa.Recent Research Developments in Plant Pathology2, 43-62.
Steenkamp, E. T., Wingfield, B. D., Swart, W. J. & Wingfield, M. J.(1998). Double-stranded RNA and associated virulence in South African isolates of Sphaeropsis sapinea.Canadian Journal of Botany76, 1412-1417.
Valverde, R. A., Nameth, S. T. & Jordan, R. L.(1990). Analysis of double-stranded RNA for plant virus diagnostics.Plant Disease74, 255-258.
Received 29 June 2000;
accepted 31 August 2000.