CSIRO PUBLISHING www.publish.csiro.au/journals/app Australasian Plant Pathology, 2006, 35, 465–471 Characterisation of Pear blister canker viroid isolates from Australian pome fruit orchards P. A. Joyce A,B , F. E. Constable A,D , J. Crosslin C , K. Eastwell C , W. E. Howell C and B. C. Rodoni A A Department of Primary Industries, Knoxfield, 621 Burwood Highway, Victoria 3180, Australia. B Current address: Bureau of Sugar Experiment Stations Pty Ltd, 50 Meiers Road, Indooroopilly, Qld 4068, Australia. C 24106 North Bunn Road, Prosser, Washington State University–IAREC, WA 99350, USA. D Corresponding author. Email: fiona.constable@dpi.vic.gov.au Abstract. Pear blister canker viroid (PBCVd) was detected in pear (Pyrus sp.), nashi (Pyrus serotina) and quince (Cydonia oblonga) trees from various pome fruit growing regions of Australia using dot-blot hybridisation and RT-PCR techniques. Characteristic symptoms of PBCVd infection were not observed on the infected trees. This is the first report of PBCVd infecting nashi varieties. The 12 Australian PBCVd isolates showed 92–99% nucleotide sequence identity with previously published sequences of PBCVd from other countries. The Australian isolates fell into two distinct groups based on sequence similarity and secondary structures. The possible origin of PBCVd in Australia is discussed. Introduction Pear blister canker disease was first reported in England in 1959 on Williams, Comice and Laxton’s Superb cultivars of pears, and affected trees developed cankers on the bark (Cropley 1959). Pear blister canker viroid (PBCVd; genus Apscaviroid, family Pospiviroidae) was isolated and identified to be the causal agent of pear blister canker disease (Flores et al. 1991). PBCVd has a circular, single-stranded RNA genome that is 315 nucleotides in length and is most closely related to Grapevine yellow speckle viroid (Hern´ andez et al. 1992). PBCVd has been reported to occur in 10% of old French quince and pear varieties, in various pear cultivars from Italy, Tunisia and China and also in wild and cultivated pears from Greece (Loreti et al. 1997; Desvignes et al. 1999; Kyriakopoulou et al. 2001; Shamloul et al. 2002; Fekih Hassen et al. 2006). Grafting studies using PBCVd infected pear bark showed that the genera Pyrus, Pyronia, Cydonia and Sorbus are susceptible to the viroid, while nashi (Pyrus serotina) is resistant (Desvignes et al. 1999). As most pears infected with PBCVd are symptomless, diagnosis is most reliably conducted by double budding onto indicator hosts or using molecular detection tests. Double budding is generally onto Cultivar A20, which takes 2 to 3 years to show symptoms, or onto the highly susceptible Fieud lines 37 and 110, which show necrosis of the bark after 3–4 months and die within 6 months (Desvignes et al. 1999). Molecular methods that can be used to detect the viroid include hybridisation using a cRNA probe (Ambr´ os et al. 1995; Desvignes et al. 1999; Malfitano et al. 2004) and reverse transcription polymerase chain reaction (RT-PCR; Loreti et al. 1997; Faggioli et al. 2001; Faggioli and Ragozzino 2002; Shamloul et al. 2002; Ragozzino et al. 2004). The first Australian isolate of PBCVd was discovered during a routine quarantine check of bud wood of a promising pear cultivar developed in Australia and being exported overseas (Joyce et al. 2004). As this was the first time that PBCVd had been recorded in Australia, a small but targeted survey of pear trees from all of the major pome fruit growing regions in Australia was done during the winter of 2002. This was followed by two additional surveys of pome fruit trees from September 2003 to January 2005. This paper presents the results from these surveys. Methods Surveys and sampling The first survey for PBCVd was done in May 2002 and bud wood samples were collected from 63 pear trees (Pyrus sp.) in orchards from New South Wales, Queensland, South Australia, Tasmania, Western Australia and Victoria. One orchard in Victoria where PBCVd was detected in the first survey was revisited in 2003 and samples collected from 11 additional trees of different ages with different rootstock and scions. Additional surveys were done in New South Wales, Queensland, South Australia, Tasmania, Western Australia and Victoria during the period from September 2004 to January 2005 and 18 pear, seven nashi (P. serotina) trees and seven quince (Cydonia oblonga) trees were sampled. In this final round of surveys, older cultivars were selected © Australasian Plant Pathology Society 2006 10.1071/AP06050 0815-3191/06/040465