Tree Protection News :: November 2001

Abstracts - 6th International Symposium on Positive Strand RNA viruses

STUDIES OF THE EFFECTS OF DIAPORTHE AMBIGUA RNA VIRUS (DaRV) ON NATURALLY INFECTED AND TRANSFECTED ISOLATES OF DIAPORTHE AMBIGUA

The fungus Diaporthe ambigua is an important canker pathogen of stone fruits and apples in South Africa. The symptoms associated with Diaporthe canker include sunken, pointed lesions with longitudinal cracks on infected trees. Different isolates of D. ambigua differ in morphology and display varying degrees of pathogenicity. Hypovirulent isolates were found to be infected by a 4113 bp positive-strand RNA virus that has been named Diaporthe ambigua RNA virus (DaRV). This mycovirus has significant potential as a biocontrol agent of Diaporthe and other plant pathogenic fungi. The aim of this study was, therefore, to consider whether the hypovirulence in naturally infected isolates of D. ambigua is solely the result of the resident DaRV, or whether other factors are necessary to confer hypovirulence on the host. DaRV was thus cloned in a pGEM T-Easy vector using RT-PCR. The T7 polymerase was used to produce, in vitro, RNA from DaRV cDNA clone. The RNA was used to transfect by electroporation, virus-free isolates of D. ambigua. The transfected D. ambigua isolates did not display any morphological changes. Further studies are underway to consider the effects on pathogenicity and other traits found in naturally infected isolates of the fungus.

A NOVEL RNA VIRUS IN THE FUNGUS DIAPORTHE AMBIGUA IS RELATED TO PLANT TOMBUSVIRIDAE

RNA viruses infect a wide range of fungi. They are often detected as dsRNA elements in nucleic acid isolations or as virus-like particles by electron microscopy. Positive-stranded RNA genomes have been found for a few mycoviruses. The effects of mycoviruses on their hosts vary from lytic to cryptic. In some fungi, for example the chestnut blight pathogen Cryphonectria parasitica, virus infection drastically reduces the virulence. Viruses mediating hypovirulence have potential as biological control agents. In this study, a dsRNA element was isolated and characterized from a hypovirulent isolate of Diaporthe ambigua. This fungus is a serious canker pathogen of pome and stone fruit trees. The dsRNA represents the replicative stage of a positive-stranded RNA virus, which we have named D. ambigua RNA virus (DaRV). The genome sequence contains 4113 bases and has a GC content of 53%. Two large ORFs are present in the same reading frame. The second ORF is most probably translated by readthrough of an UAG stop codon of the first ORF. This results in a protein with a predicted molecular mass of 125 kDa. Significant homology can be found to the non-structural proteins of carmoviruses of the positive-strand RNA virus family Tombusviridae, which also contain the RDRP domain. The coding regions for coat proteins of Tombusviridae are usually situated at the 3' end of their genomes, but such an ORF is not present in the case of DaRV. Therefore, DaRV is most probably not encapsidated but might occur as RNA-RDRP complexes. DaRV is a mycovirus with a unique genome organization and is distantly related to the plant virus family Tombusviridae.

CHARACTERIZATION OF MITOVIRUSES ASSOCIATED WITH THE FUNGAL PATHOGEN, CRYPHONECTRIA CUBENSIS

The fungal pathogen, Cryphonectria cubensis causes considerable damage to Eucalyptus plantations worldwide. It is, therefore, essential to develop an effective control strategy against Cryphonectria canker. Planting of disease tolerant clones is one approach. The potential to reduce the virulence of the pathogen through virus-mediated hypovirulence is also an attractive option. In this study slow growing isolates from a South African C. cubensis population were screened for the presence of dsRNA using CF11 cellulose column chromatography. Some of the isolates were found to contain dsRNA elements with a size of ca. 2.5 kb. Sequence analysis of cDNA produced randomly from the dsRNA revealed that these elements represent at least three genomes related to mitochondrial mitoviruses (Narnaviridae). These are similar to those found in C. parasitica and the dutch elm disease fungus, Ophiostoma novo-ulmi. Northern blot hybridization was performed to determine whether the virus genome predominantly occurs in the + or - single stranded RNA or ds RNA form. Results indicate that the mitoviruses associated with C. cubensis are positive ssRNA genomes. A greenhouse inoculation trial was conducted to determine whether the isolates containing the mitovirus display reduced virulence. No reduction in virulence was observed for these isolates. It is, therefore, unlikely that these viruses will be useful as biological control agents although further studies will be necessary to better understand their role.