contained “conventional” virus-like particles, but all
the hypovirulent strains contained dsRNA whereas
virulent strains had no dsRNA. Electron micrographs
show that this dsRNA is contained in rounded or club-
shaped, membrane-bound vesicles in the cytoplasm
(Newhouse et al. 1983) and that, unlike many VLPs,
these can occur in significant amounts in the hyphal
apices of C. parasitica.
The spontaneous disease decline in Italy led French
workers to develop a highly successful biological
control program. Hypovirulent strains of C. parasitica
were cultured in the laboratory and inoculated at the
expanding margins of cankers in the field. Within a
short time the cankers stopped growing, and only
hypovirulent strains could be recovered where once the
virulent strain had been. This transmissible change of
phenotypewas always accompanied by the transmis-
sion of dsRNA.
Following the success of this program in Europe,
attempts were made to introduce hypovirulent strains
of Cryphonectria into the USA. But they gave only
partial and localized disease control, because the
pathogen population in the USA consists of numerous
vegetative compatibility groups (VCGs) that limit the
natural transfer of dsRNA, owing to cytoplasmic death
when the strains anastomose. In one early study about
0.5 hectare of natural chestnut forest in the USA was
found to contain at least 35 VCGs. More recently the
VCGs have been shown to be in a continuous state of
flux: samplings of identical trees over several years
showed that some of the predominant VCGs declined,
while new ones arose (Anagnostakis 1992). The Euro-
pean population of C. parasiticais much more uniform
in terms of VCGs, and this probably accounts for the
success of the biocontrol program in Europe.
Progress in understanding the role of dsRNA in
Cryphonectriawas significantly delayed by the lack of
a suitable transformation system for this fungus.
However, when a system was eventually developed
it led to rapid progress (Nuss 1992). The dsRNA of
C. parasiticawas found to be highly variable, with
lengths falling into three broad size ranges – S (small),
M (medium), and large (L, about 12.7 kb). As shown
in Fig. 9.15, all these types have the same terminal
regions – a poly-A tail at the 3′end and a 28-nucleotide
conserved sequence at the 5′end. They differ mainly
in the degree of internal deletion. The L-form seems to
be the full-length molecule, and the smaller forms are
defective (presumably functionless) derivatives which
accumulate in the hyphae. This high degree of variability
of RNA genomes is not unusual because there is no
effective proofreading system for RNA, to ensure that
it is copied faithfully.
With the development of a transformation system,
Nuss and his colleagues were able to produce a cDNA
from the full-length dsRNA and transform it into vir-
ulent strains. It caused the virulent strains to become
hypovirulent and to exhibit all the features typical of
hypovirulent strains – slow growth, pale coloration,
and reduced sporulation (the hypovirulence-associated
traits). Thus, dsRNA was shown unequivocally to be
the cause of hypovirulence. The cDNA could also be
used for molecular analysis of the dsRNA, which was
shown to consist of two open reading frames (ORFs) –
ORF A of 622 codons (nucleotide triplets) and ORF B
of 3165 codons. When a cDNA copy of ORF A was intro-
duced into Cryphonectria, it caused a loss of pigmen-
tation, a reduction of sporulation and a reduction
of laccase activity. But the transformed strain
remained virulent. By contrast, ORF B codes for a
polyprotein that was found to contain motifs charac-
teristic of RNA-dependent RNA polymerases and RNA
helicases. So, the primary function of ORF B could be
the replication and maintenance of the dsRNA, caus-
ing the loss of pathogenic virulence, whereas ORF A
encodes many of the hypovirulence-associated traitsFUNGAL GENETICS, MOLECULAR GENETICS, AND GENOMICS 175
Fig. 9.15Double-stranded RNA of hypovirulent strains of C. parasitica. Large (L) dsRNA is the full-length molecule com-
prising two conserved end regions and a central coding region of two open reading frames (ORFA and ORFB) which
confer hypovirulence. Medium (M) and small (S) dsRNAs are also commonly found in hyphae. They are internally deleted
copies of the L-dsRNA.