Marion F. Bateson

On the evolution and molecular epidemiology of the potyvirus Papaya ringspot virus

The potyvirus Papaya ringspot virus (PRSV) is found throughout the tropics and subtropics. Its P biotype is a devastating pathogen of papaya crops and its W biotype of cucurbits. PRSV-P is thought to arise by mutation from PRSV-W. However, the relative impact of mutation and movement on the structure of PRSV populations is not well characterized. To investigate this, we have determined the coat protein sequences of isolates of both biotypes of PRSV from Vietnam (50), Thailand (13), India (1) and the Philippines (1), and analysed them together with 28 PRSV sequences already published, so that we can better understand the molecular epidemiology and evolution of PRSV. In Thailand, variation was greater among PRSV-W isolates (mean nucleotide divergence 7.6%) than PRSV-P isolates (mean 2.6%), but in Vietnamese populations the P and W biotypes were more but similarly diverse. Phylogenetic analyses of PRSV also involving its closest known relative, Moroccan watermelon mosaic virus, indicate that PRSV may have originated in Asia, particularly in the Indian subcontinent, as PRSV populations there are most diverse and hence have probably been present longest. Our analyses show that mutation, together with local and long-distance movement, contributes to population variation, and also confirms an earlier conclusion that populations of the PRSV-P biotype have evolved on several occasions from PRSV-W populations.

PAPAYA RINGSPOT POTYVIRUS : ISOLATE VARIABILITY AND THE ORIGIN OF PRSV TYPE P (AUSTRALIA)

We have sequenced the coat protein gene of nine isolates of papaya ringspot virus (PRSV) including six Australian and three Asian isolates and compared these with four previously reported sequences of PRSV. There was up to 12% sequence variation between isolates at the nucleotide level. However, there was no significant difference between the sequences obtained from Australian isolates irrespective of whether they were PRSV type P (cucurbit or papaya infecting) or PRSV type W (cucurbit infecting) and these isolates were more closely related to one another than to any other isolate. These results imply that PRSV-P, first recorded in Australia in 1991, arose locally from PRSV-W (first recorded in Australia in 1978) rather than being introduced. Further, there was no consistent sequence difference between PRSV-P and PRSV-W isolates that would obviously account for their host range difference.

Construction of bacterial artificial chromosome libraries and their application in developing PCR-based markers closely linked to a major locus conditioning bruchid resistance in mungbean (Vigna radiata L. Wilczek)

Bacterial artificial chromosome (BAC) libraries have been widely used in different aspects of genome research. In this paper we report the construction of the first mungbean (Vigna radiata L. Wilczek) BAC libraries. These BAC clones were obtained from two ligations and represent an estimated 3.5 genome equivalents. This correlated well with the screening of nine random single-copy restriction fragment length polymorphism probes, which detected on average three BACs each. These mungbean clones were successfully used in the development of two PCR-based markers linked closely with a major locus conditioning bruchid (Callosobruchus chinesis) resistance. These markers will be invaluable in facilitating the introgression of bruchid resistance into breeding programmes as well as the further characterisation of the resistance locus.

Genetically engineered immunity to Papaya ringspot virus in Australian papaya cultivars

Papaya ringspot virus (PRSV), which has recently been identified inAustralia, is the major limiting factor in papaya production worldwide. In thispaper we report the development of two Australian papaya cultivars that areimmune to infection with PRSV. Papaya somatic embryos were transformed viamicroprojectile bombardment using a construct containing an untranslatable PRSVcoat protein coding region. Immunity was demonstrated by repeated inoculationinthe glasshouse and eighteen months in the field. The immune lines were shown tohave up to five copies of the transgene by Southern hybridisation. Northernhybridisation showed that the coat protein transcript in the immune linesappeared to be degraded; therefore, the mechanism of resistance appears to bepost transcriptional gene silencing via RNA degradation. We have taken aproactive approach to control of PRSV in Australia by developing geneticallyengineered resistance before the Australian papaya industry has been decimatedby the inevitable spread of PRSV.

Banana bract mosaic virus: characterisation using potyvirus specific degenerate PCR primers

SummaryBanana bract mosaic (BBMV) is a relatively new, non-persistently aphid transmitted disease of bananas in the Philippines. Partially purified preparations from infected plants contained low numbers of flexuous virions, 660 to 760 nm in length, and a 38kDa protein, possibly the coat protein, which reacted with a general potyvirus antiserum in western blots. There were insufficient virions for conventional antiserum production or cDNA synthesis. Therefore, DNA was amplified using potyvirus-specific degenerate primers and reverse transcriptase PCR. The PCR products were cloned, sequenced and analysed and contained a 5′ open reading frame of up to 150 amino acids and a 3′ untranslated region of up to 190 nucleotides which were clearly related to the C-terminal half of the coat proteins and the 3′ untranslated regions, respectively of potyviruses. The BBMV open reading frame amino acid sequence was most similar to the C-terminal half of the maize dwarf mosaic potyvirus coat protein (71.3% similarity) and the BBMV 3′ untranslated region was most similar to that of ornithogalum mosaic potyvirus (39.6% similarity). Our results show that BBMV is a distinct potyvirus and also demonstrate the application of virus group specific primers in the characterisation of previously undescribed viruses.

Detection and characterization of pangola stunt Fijivirus from Australia using cloned cDNA probes

SummaryFour cDNA clones were generated from the genomic dsRNA of an Australian isolate of pangola stunt Fijivirus (PaSV). Each clone hybridized with nucleic acid extracts from PaSV infected plants but not healthy plants. Further, each clone hybridized with more than one segment of the PaSV dsRNA genome. One clone was used to demonstrate that homology existed between the Australian isolate of PaSV and a South American isolate of PaSV although the isolates differed in the sizes of the genomic dsRNAs and in the vector species. The clone also hybridized with some segments of the maize rough dwarf Fijivirus genome.