Abstract:
ln recent years a lot of interest has focussed on the role of mycoviruses (fungal viruses)
that confer reduced pathogenicity to their fungal hosts. In several plant pathogenic fungi
viral double-stranded RNA (dsRNA) elements have been shown to be the agents
responsible for this attenuation of virulence.
In New Zealand and other temperate climates the plant pathogenic fungus, Botrytis
cinerea is an important pathogen of many horticultural crops. Control of this grey
mould disease still relies heavily on the use of fungicides to augment cultural practices-
Consequently, there is now increasing interest in the use of biological control as an
alternative means of control.
This study reports the first large-scale survey of dsRNA elements in B. cinerea.
Analysis of 200 New Zealand isolates from a range of hosts revealed 72% contained
dsRNA, with high variation in dsRNA profiles, both within and between groups of
fungal isolates from different hosts. DsRNA has been associated with isometric
particles in many different fungi. In this study virus-like particles (VLPs) were observed
in three of five dsRNA-containing isolates examined. Morphological types included
isometric particles of varying size classes (approx' 30, 35, 40 and 45 nm) and
bacilliform parricles (approx. 25 x 63 nm). In five isolates apparently lacking dsRNA no
isometric or bacilliform particles were found but flexuous rods with a modal length of
approx. 7ZO nm were detected in one of these isolates. Total RNA extraction of the
various partially purified VLP preparations gave similar profiles to those obtained from
mycelial dsRNA extractions of the same isolates, suggesting that the VLPs comprise
encapsidated dsRNA. Comparison of 12 dsRNA-containing and 12 dsRNA-free isolates
indicated slight differences between the two groups in radial growth rate, virulence on
bean leaves and sclerotial number, but the differences were minor. ln summary, while
dsRNA was found to be common among B. cinerea isolates in this study, the evidence
did not reveal any marked phenotypic effects of dsRNA presence.
The putative flexuous rod-shaped virus particles rn B. cinerea were studied further.
Similar particles have been observed in fungi before but none have been characterised at
the molecular level. This study reports the first complete nucleotide sequences of two
such mycoviruses, Botrytis Virus F (BVF) and Botrytis Virus X (BVX), and also the
first molecular characterisation of viruses infecting B. cinerea. The mycoviruses, which
were both present in the same B. cinerea isolate, were similar in size and morphology to
ssRNA plant viruses of the family Potexviridae. BVF and BVX contained ssRNA
genomes af 6827 and 6966 nucleotides respectively, excluding poly(A) tails. Computer
analysis of the genomic cDNA sequence of BVF revealed three potential open reading
frames (ORFs) encoding putative proteins of 20 kDa (ORFI),212 kDa (ORFZ) and 32
kDa (ORF3). ORFI, which overlapped ORF2 by 467 nt, did not reveal significant
homology with known protein sequences.O RF2, which showed significant sequence
identity to the replicase proteins of the plant virus families Tymoviridae and
potexviridae, contained a putative opal readthrough codon intermediate between the
helicase and RNA-dependent RNA polymerase (RDRP) regions. ORF3 shared amino
acid homology with coat proteins of the family Potexviridae. A putative defective RNA
(D RNA) of 829 nt was also detected which consisted of a 5' region of the parental
genome fused in-frame to the terminal 3' end, complete with poly(A) tail. BVX
revealed five potential ORFs encoding putative proteins of 158 kDa (ORFl), 30 kDa
(ORF2), 44y,Da(ORF3), 14 kDa (ORF4) and l4kDa (ORFs). ORFI showed significant
amino acid sequenceid entity to the replicase proteins and ORF3 to the coat proteins of
the potexviridae. The remaining ORFs did not reveal significant homology with known
protein sequences.
This is the first report of sequence similarities between mycoviruses and the plant virus
families Potexviridae and Tymoviridae. Comparisons of sequence and genome
organisation suggest that the mycoviruses BVF and BVX may each belong to a new as
yet unassigned genus in the plant virus family Potexviridae. An unexpected result from
this study was the high level of identity observed between the derived amino acid
sequences of the mycoviruses and plant viruses including a region of -7O% for the
RNA-dependent RNA polymerase (RDRP) region of BVX and similar regions in
several potex-like viruses of AIIium spp. This high level of interviral homology has not
previously been reported between the plant and fungal kingdoms and is possibly
indicative of a recent divergence from a common ancestor. These results suggest that
horizontal gene transfer between the plant and fungal kingdoms may occur more
frequently than has been appreciated to date. If so it may play a significant role in viral evolution.
In addition to providing novel phylogenetic information on the relationship between these
morphological types of viruses in fungi and in plants, characterisation of these mycoviral genomes provides a possible strategy for incorporating genes into Botrytis using viruses as vectors. In this context Botrytis strains infected with modified viruses could provide a new approach for the biocontrol of grey mould.