RNA viruses of Sphaeropsis sapinea and Diaporthe ambigua and their possible use as biological control agents

Abstract

Sphaeropsis sapinea and Diaporthe ambigua are important pathogens of forest and orchard tree species, respectively. Some isolates of S. sapinea are co-infected with two dsRNA viruses, SsRVl and SsRV2. Isolates of D. perjuncta (formerly thought to be D. ambigua) are infected with a positive-stranded RNA virus known as DaRV. While S. sapinea is. infected with a heterogeneous mixture of dsRNA elements of different sizes, D. perjuncta is infected with a single virus. This presents excellent opportunity for biocontrol of Diaporthe. The aim of this study was to assess these three viruses for possible application as biological control agents of S. sapinea and D. ambigua. This was' done by transfecting these with in vitro-produced RNA from the cloned viral genomes and assessing the pathogenicity of the transfected isolates on apples and apple trees. Attempts to transfect S. sapinea spheroplasts with SsRVl and SsRV2 failed. Co¬transfection of S. sapinea spheroplasts with both viruses also failed. Three isolates of D. ambigua and a single isolate of a Phomopsis sp. were successfully transfected with DaRV. Attempts to transfect the same fungi with a mutant of DaRV, bearing six codons for histidine immediately downsteam of an AUG thought to be a start codon for the translation of ORFl, failed. DaRV was originally thought to be isolated from D. ambigua. The fungal isolates transfected with DaRV were thought to be D. ambigua. The transfectants did not resemble the naturally-infected isolate. The ITS regions from the ribosomal DNA operon of these isolates were amplified using ITS 1 and ITS4 primer pair. The blast search revealed that the ITS sequence of the naturally-infected isolates are identical to D. perjuncta. One virus-free isolate was identified as a Phomopsis sp. while three other virus-free isolates were identified as D. ambigua. A PCR-based RFLP was developed to differentiate the naturally-infected D. perjuncta isolates from the virus¬free Phomopsis sp. and D. ambigua isolates. In the growth and pathogenicity studies, a DaRV-transfected, wild-type and negative control isolate of one Phomopsis and three D. ambigua isolates, were used. The DaR V -transfected Phomopsis sp. had a higher growth rate than the wild-type isolate. This DaRV-transfected Phomopsis sp. was more virulent on apples than the wild-type isolate. The wild-type isolate was slightly more virulent than the DaR V -transfected Phomopsis sp. on apple trees. There were no significant differences in growth rates between the DaRV-transfected and wild-type isolates of D. ambigua CMW5587 and D. ambigua CMW5287. There were no significant differences in virulence on apples between the DaRV-transfected and wild-type isolates of these fungi. The DaRV-transfected D. ambigua CMW5287 was more virulent than the wild-type isolate on apple trees. The DaRV-transfected D. ambigua CMW5587 had the same virulence as the wild-type isolate on both apples and apple trees. The DaRV-transfected D. ambigua CMW5288 had a slower growth rate than the wild-type isolate. There were no significant differences in virulence on apples between these isolates. The wild-type isolate of this isolate was significantly more virulent on apple trees than the DaRV-infected isolate. Although transfection was successfully done, the effects of DaRV on the Phomopsis sp. and D. ambigua isolates are not conclusive. In order to obtain conclusive results, virus-free isolates of D. perjuncta must be transfected. During the course of this study, there were no available virus-free isolates of this fungus.