Etiology and Host Range of a Closterovirus Associated with Plum Bark Necrosis-Stem Pitting Disease Diana B. Marini, Former Graduate Student, Y.-P. Zhang, Former Postdoctorate Associate, A. Rowhani, Specialist, and J. K. Uyemoto, Research Plant Pathologist, USDA-ARS, all in the Department of Plant Pathology, University of California, Davis 95616 domestica L. plum cultivars—Angeleno, ABSTRACT Black Amber, Black Beaut, Casselman, Marini, D. B., Zhang, Y.-P., Rowhani, A., and Uyemoto, J. K. 2002. Etiology and host range of a French Improved, Friar, Shiro, Stanley, and Closterovirus associated with plum bark necrosis-stem pitting disease. Plant Dis. 86:415-417. Laroda—were propagated (by a commer- cial propagator) on Citation rootstock Diseased plum (Prunus salicina) cv. Black Beaut trees developed stem gumming, severe bark (peach × plum hybrid). In spring 1997, the necrosis, and stem pitting symptoms on the woody cylinder of the main trunk and scaffold trees were transplanted to the field at UCD branches. The sucker shoots of the peach (Prunus persica) cv. Nemaguard understocks exhib- and spaced 45 cm apart and 90 cm between ited oak-leaf patterns, but lacked the wood or bark markings. Other susceptible hosts included sets of five trees per cultivar. Choice of almond (Prunus dulcis), sweet (Prunus avium) and Japanese flowering (Prunus serrulata) cher- ries, and several plum (Prunus salicina) and prune (Prunus domestica) varieties. A purified plum cultivar was based, in part, on the preparation containing high molecular size dsRNAs was obtained initially from diseased cherry relative popularity and acreage planted in (P. av i um × Prunus pseudocerasus) cv. Colt tissues. Healthy preparations were devoid of simi- California. Later that fall, two or three lar sized dsRNAs. Reverse transcriptase-polymerase chain reaction (RT-PCR) assays with de- trees per cultivar were graft-inoculated, generate oligonucleotide Closterovirus primers, designed from the HSP70 gene, were used to each with three peach budchips. Non- amplify two DNA fragments of 0.67 and 0.56 kbp. The larger cDNA product was cloned, se- grafted trees served as healthy controls. In quenced (AF195501), and compared with other viral sequences. Depending on the number of September 2000, all plum trees were re- nucleotides used in the comparisons, identities ranged from 77% for Grapevine leafroll associ- moved, trunks autoclaved, and bark tissue ated virus to 3 to 44% for Little cherry virus-1. Specific primers from the 0.67 kbp cDNA se- removed to expose the woody cylinder. quence were designed and used in subsequent RT-PCR assays. The associated 0.67 kbp HSP70 In the greenhouse, dormant seedlings of amplicon of Plum bark necrosis-stem pitting associated virus was detected in all graft- cherry (Prunus tomentosa Thumb.) cv. inoculated Prunus species and varieties except prune (P. domestica var. French Improved). Nanking, cherry (P. avi um (L.) L.) cv. Mazzard, and peach cv. GF 305 were pot- ted during January 1998. Later in March, Plum bark necrosis-stem pitting (PBNSP) ated virus, and the putative causal agent, three diseased peach budchips or plum disease was first observed in 1986 affect- was identified as a Closterovirus and des- bark patches per tree were graft-inoculated ing plum (Prunus salicina Lindl.) cv. ignated as Plum bark necrosis stem-pitting on three to five plants per species. All pot- Black Beaut trees in two orchards in Di- associated virus (PBNSPaV). The host ted plants were transplanted in the field at nuba, CA (8). All of the Black Beaut plum range, symptoms induced, and develop- UCD in April 1998. One to three trees per trees had been whip-grafted with a single ment of a reverse transcriptase-polymerase species were used as healthy controls. cultivar Wickson plum scion per tree to chain reaction (RT-PCR) assay for the In the orchard, two or three mature trees serve as a pollenizer, and all Wickson associated Closterovirus are described. each of peach cv. Fay Elberta, cherry cv. Bing, grafts failed. A year later, the same trees almond (P. dulcis (Mill.) D.A. Webb) cv. were grafted a second time with another MATERIALS AND METHODS Mission, and cherry (P. avi um × P. pseudo- source of Wickson scion wood, which Inoculum source. The virus source was cerasus) cv. Colt supporting scions of flower- successfully callused and grew. Mean- a Black Beaut plum tree maintained at ing cherry cv. Shirofugen were inoculated while, scaffold branches with the failed UCD that was graft-inoculated with tissue with 15 peach budchips each. One healthy grafts began to exude copious amounts of from diseased Black Beaut plum trees tree per species was included as a control. dark-colored gum and developed necrotic taken in a commercial orchard (8). In the cDNA cloning and sequencing. Puri- bark and stem pitting symptoms. Disease second year postinoculation, the trunks and fied dsRNAs were prepared as described incidence approached 100%, and both sites scaffold branches of grafted trees devel- (9) from leaves removed from inoculated- were eventually replanted. We hypothe- oped gummosis, bark necrosis, and stem diseased and noninoculated-healthy trees sized the first collection of Wickson scions pitting symptoms. In addition, sucker of Colt cherry, Shirofugen flowering was diseased and the second healthy. growth arising from peach (Prunus persica cherry, and peach suckers and bark tissues Subsequent graft-transmission tests per- (L.) Batsch) cv. Nemaguard understocks of Black Beaut plum trees. These prepara- formed at University of California at Davis showed chlorotic oak-leaf patterns. No wood tions were used as templates in RT-PCR (UCD) proved the infectious nature of marks were observed on the main peach where cDNAs were amplified using the PBNSP (8). In the present study, an associ- trunks. Plum bark and peach bud tissues Closterovirus HSP70 degenerate primers (i.e., root sucker growth) were used as in- (7). The products were resolved by elec- ocula for graft-inoculations and as positive trophoresis in 1.5% agarose gel and size Corresponding author: J. K. Uyemoto controls in RT-PCR assays. The inoculum estimates determined with a 1-kb DNA E-mail: [email protected] source tree had previously tested negative Ladder standard (Life Biotechnologies Accepted for publication 12 December 2001. for Prunus necrotic ringspot virus and Prune Inc., Rockville, MD). The PCR products dwarf virus by enzyme-linked immunosor- were extracted and purified from the aga- bent assay and flowering cherry (Prunus rose gels using a commercial Bio-Spin Publication no. D-2002-0214-02R serrulata Lindl.) cv. Shirofugen indexings, DNA purification column (BIORAD Inc., This article is in the public domain and not copy- viruses commonly found in Prunus species. rightable. It may be freely reprinted with custom- Hercules, CA) and cloned with TOPO TA ary crediting of the source. The American Phyto- Plum cultivars and Prunus species. Cloning kit (Invitrogen Inc., Carlsbad, pathological Society, 2002. Five trees each of nine P. salicina and P. CA). The cloning plasmid was pCR 2.1- Plant Disease / April 2002 415 Fig. 1. Bark necrosis symptoms on Plum bark necrosis-stem pitting associated virus–infected Black Beaut plum tree. TOPO, and the resulting recombinant DNA plasmids were purified with the QIAprep spin miniprep kit (Qiagen Inc., Valencia, CA). Recombinant plasmids were selected, and those containing inserts were identified by restriction enzyme analysis (4). The clones with the 0.67 fragments of PCR-amplified DNAs were sequenced at the DNA Sequencing Facility, UCD. Spe- cific primers for the associated Closterovi- rus were then designed from the sequence Fig. 2. Profile of dsRNAs purified from symptomatic leaves of Colt cherry trees infected with Plum information. The RT-PCR products were bark necrosis-stem pitting associated virus (PBNSPaV). Lane 1, molecular size marker of Cucumber mosaic virus dsRNAs; lane 2, dsRNA profile of healthy Colt cherry leaves; lane 3, dsRNA profile of separated by electrophoresis through 1.5% PBNSPaV-infected Colt leaves; lane 4, dsRNA profile of PBNSPaV-infected Colt roots; and lane 5, agarose gels using TAE buffer as per 1-kb DNA marker (Life Biotechnologies). Electrophoresis was done in 6% polyacrylamide gel. Zhang et al. (10). Sample preparation and RT-PCR as- says. A rapid assay method developed for After budbreak and incubation for 6 mosaic virus (CMV) dsRNAs and the 1-kb grapevine viruses (3) was used to prepare months, both inoculated Colt cherry trees DNA ladder standard. petiole and bark tissues of diseased and developed leaves with chlorotic ring spots. Due to conserved sequences among healthy Black Beaut plum trees. On GF 305 peach, Casselman, Laroda, and Closteroviruses, use of the HSP70 degen- In May and in September 1999 (2 years Shiro plum cultivars, leaf symptoms of erate primers resulted in amplification of postinoculation), all graft-inoculated and chlorotic ring spots, oak-leaf and line- two fragments (0.67 and 0.56 kbp). Only healthy tree species were assayed by RT- pattern were observed 12 months post- the larger cDNA fragment was cloned and PCR. Approximately 0.5 g of bark tissues inoculation. Similarly, during the second sequenced (GenBank accession number: (located immediately beneath the bud- full growing season postinoculation, bark AF195501) and compared with sequences chip/bark patch inocula) and leaf petioles gummosis, necrosis, and stem pitting in the GenBank. The percentages of arising nearest the inoculation sites were symptoms began to develop on trunks of HSP70 nucleotide sequence identities were sampled. On some trees, leaves with virus- Angeleno, Black Beaut, and Friar trees. In 44% with Little cherry virus-1 (LChV-1); like symptoms along with bark samples the third year postinoculations, bark and 50 to 51% with Grapevine leafroll associ- were also assayed. All extracts were made stem symptoms intensified (Fig. 1). Con- ated virus-4 and -5 (GLRaV-4, -5); 59% at a tissue-to-grinding buffer ratio of 0.2 comitantly, leaf symptoms were absent on with Citrus tristeza virus; and 76 to 77% g:5.0 ml.