Prunus necrotic ringspot and prune dwarf viruses in New Zealand

New Zealand Journal of Agricultural Research ISSN: 0028-8233 (Print) 1175-8775 (Online) Journal homepage: http://www.tandfonline.com/loi/tnza20 Prunus necrotic ringspot and prune dwarf viruses in New Zealand G. A. Wood To cite this article: G. A. Wood (1971) Prunus necrotic ringspot and prune dwarf viruses in New Zealand, New Zealand Journal of Agricultural Research, 14:2, 515-525, DOI: 10.1080/00288233.1971.10427114 To link to this article: https://doi.org/10.1080/00288233.1971.10427114 Published online: 24 Jan 2012. Submit your article to this journal Article views: 120 View related articles Citing articles: 3 View citing articles Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalinformation?journalcode=tnza20

515 PRUNUS NECROTIC RINGSPOT AND PRUNE DWARF VIRUSES IN NEW ZEALAND By P. R. FRY* AND G. A. WOOD* (Received 17 September 1970) ABSTRACT In New Zealand Prunus necrotic rings pot virus (NRSV) is carried without symptoms on peach, nectarine, apricot, and Japanese plum. Tatter leaf symptoms develop on the rootstock clone FI2/I and some sweet cherry varieties. Prune dwarf virus (PDV) has been found only in mixed infections. NRSV is widespread in some peach varieties, cherries, and plums, but incidence is low in apricots and nectarines. PDV is widespread only in cherries. NRSV was seed transmitted in 0.75% of seed from infected Golden Queen and in 12% of seed from Muir peach. Two of 16 trees pollinated with pollen from infected trees became infected. Spread of NRSV was detected in all three orchards indexed in three successive seasons. NRSV was eliminated by hot-water treatment of budwood for 10 min at 55 c or 20 min at 50, and PDV was eliminated after 10 min at 55. Hot-air treatment followed by tip grafting eliminated both NRSV and PDV from most cherry tips after heating for 13 to 24 days. NRSV was eliminated from peach tips after 17 and 24 days' heating, and from plum after 21 days. The NRSV particle was "spherical", 20 to 25 mj' in diameter. Serological relationship to a Californian almond calico strain of NRSV was demonstrated. INTRODUCTION Prunus necrotic ringspot (NRSV) and prune dwarf (PDV) were recorded in New Zealand by Chamberlain (1961), who had obtained preliminary results suggesting that both viruses were widespread in sweet cherry (Prunus avium L.). NRSV is thought to have been recorded first from plum (P. salicina Lindl.) in Kentucky, U.S.A., in 1932, and was described in California in cherry in 1939 and peach (P. persica (L.) Batsch) in 1941 (Richards and Cochran 1954). PDV was described first from plum in New York State by Thomas and Hildebrand (1936). Both viruses are now known to be of world-wide distribution. Until these viruses were transmitted to herbaceous hosts, allowing partial purification and studies of.. Plant Diseases Division, Department of Scientific and Industrial Research, P.B., Auckland, New Zealand. N.Z. Journal of Agricultural Research (1971), 14: 515-25

516 Prunus viruses Fig. l-"tatter leaf" symptoms of NRSV on leaves of F12/1 cherry rootstock. [Photo 1. W. Endt serological reactions, their relationship to each other remained uncertain. From evidence reviewed by Fulton (1968), however, it is now clear that NRSV and PDV are oorelated viruses. NRSV FIELD SYMPTOMS NRSV was originally named in North America for its symptoms on peach. Some varieties and seedlings were severely affected after inoculation, producing ringspots on leaves, shot-hole, bud killing, tip dieback, and sometimes death (Cochran et al. 1951). In New Zealand it is carried without symptoms by apricot (P. armeniaca L.), peach, nectarine (P. persica (L.) Batsch), and Japanese plum (P. salicina). On the cherry rootstock clone Fl2fl and on some sweet cherry varieties

P. R. FRY AND G. A. WOOD 517 NRSV causes a "tatter leaf" condition. Symptoms appear, in spring, as irregular, brown necrotic spots sometimes forming wavy lines across the leaf. Later, in summer, these necrotic spots fall out, and severely affected leaves become torn and ragged (Fig. 1). PDV Where PDV has been found in peach, plum, apricot, and cherry in New Zealand orchards, one or more other viruses have always been present in the infected trees, so no symptoms can be described for PDV alone. Mixed infections NRSV and PDV usually occur together in peach, nectarine, and apricot trees showing a rosette symptom with shortened internodes and small, upward-rolled leaves. Their contributions to the symptoms of this disorder are not yet clear (Fry and Wood 1969). In North America and England it has been suggested that strains of NRSV in combination with strains of PDV may be responsible for sour cherry yellows symptoms in sour cherry (P. cerasus L.) (Cropley et al. 1964). Very few sour cherries are grown in New Zealand, and the condition is unknown here. Attempts to induce sour cherry yellows symptoms on Montmorency sour cherry by inoculating with naturally occurring mixtures of NRSV and PDV have been unsuccessful. This may be due to climatic influences or to the strain mixtures used. METHODS OF DIAGNOSIS NRSV and PDV infections have been diagnosed by the use of the following indicators: 1. Shirofugen oriental flowering cherry (P. serrulata Lindl.) When buds from trees infected with either NRSV or PDV are budded on Shirofugen cherry a local necrotic reaction develops around the inserted buds a few weeks after inoculation (Milbrath and Zeller 1945). Infected wood turns brown, then black, and usually oozes quantities of gum (Fig. 2). Inspection for necrosis 3 to 4 weeks after inoculation can determine whether the inoculum buds contain virus. The disadvantage of this method is that the Shirofugen reaction does not differentiate between NRSV and PDV (Hampton et al. 1966). 2. Italian prune (P. domestica L.) When buds from a PDV -infected tree are inserted below healthy Italian prune buds on plum or peach seedling stock, development of the Italian prune shoot is restricted, and shoots develop leathery, strap-like leaves (Thomas and Hildebrand 1936) (Fig. 3). Onset and severity of symptoms are variable. Symptoms usually appear in the season after budding, but may be delayed until the second or third season. NRSV is symptomless in Italian prune.

518 Prunus viruses 3. Cucumber (Cucumis sativus L., c.v. Short Prickly) and squash (Cucurbita pepo L., c.v. Buttercup) Mechanical transmission of both NRSV and PDV to cucumber and squash occurs readily if tip leaves from infected Prunus trees are crushed in 2.5% nicotine and inoculated to the cotyledons using a suitable abrasive (Cadman 1959). On cucumber, NRSV produces chlorotic local lesions followed by chlorotic mottling of the first-formed leaf and necrosis of the growing point. Lateral shoots that develop chlorotic mottle with or without further necrosis may be produced. PDV does not cause local lesions, but after 2 to 3 weeks a mild mottle develops on young leaves. On Buttercup squash, NRSV causes necrotic lesions on inoculated cotyledons, and there is no systemic spread of infection. Two to three weeks after inoculation with PDV a bright yellow vein banding develops on young leaves, which may become completely yellowed. Transmission of both viruses to cucumber and Buttercup squash occurs readily in spring and early summer, but is not usually successful in mid or late summer, probably because the viruses occur in low concentration during the warmer summer months. INCIDENCE Information on the occurrence of NRSV and PDV has been obtained by indexing blocks of trees in peach orchards, and by testing selected bud wood trees of stone fruit varieties that are used for propagation by the nursery trade. Further records have come from test trees chosen at random in orchards. Peach:- NRSV was present in all 5 of the orchard blocks examined. These were Golden Queen in Nelson (18 out of 20 trees in a mature block); Golden Queen in Hawke's Bay (48 out of 198 and 16 out of 100 trees in two 7-year-old blocks); Fortuna in Hawke's Bay (all 17 trees tested in a 4-year-old block); and Muir in Central Otago (all 51 trees in a 20-year-old block). Budwood trees of Culemborg, Paragon, and Mary's Choice were infected with NRSV, but those of Dixired. Fairhaven, Golden Queen, Halehaven, Redskin, and Southland were free from both viruses. POV was found only in trees of Culemborg, Golden Queen, Ideal, and Mary's Choice, which were also infected with NRSV and showing rosette symptoms (Fry and Wood 1969). Nectarine:- Although budwood trees of the varieties W. C. Fripp, Goldmine, and John Rivers were free from both viruses, orchard trees of Goldmine, Murray, and New Yorker showing rosette symptoms were infected with both. Apricot: - Budwood trees of the varieties Bolton, Dundonald, Moorpark, Newcastle, Oullin's Early, Roxburgh Red, and Trevatt were free from both viruses, but single orchard trees of Early Royal, Moorpark, and Roxburgh Red were infected with NRSV. A further Moorpark showing rosette symptoms was infected with both NRSV and PDV.

P. R. FRY AND G. A. WOOD 519 Fig. 2-Wood of Shirofugen cherry budded with healthy (left) and NRSV-infected (right) buds of Golden Queen peach. [Photo I. W. Endt Fig. 3--8hoot growth of Italian prune showing healthy (left) and PDV-affected (right) growth. [Photo I. W. Endt Cherry: - Budwood trees of the varieties Bigarreau Pelissier, Dawson (Noir de Guben), Early Rivers, and Florence were infected with NRSV but not PDV, and those of Bedford Prolific, Chapman, Early Cluster, St Margaret, and Werder's Early Black were infected with both viruses. Randomly sampled orchard trees of Bing, Black Tartarian, Ramon Oliva, White Heart, and William's Favourite were infected with NRSV, but results of indexing for PDV are not yet available. No cherry has been found free from both viruses. Plum: - Budwood trees of the varieties Billington, Black Doris, Omega. Purple King, Santa Rosa. and Satsuma were infected with NRSV. and Sultan was infected with both viruses. Neither virus was found in a budwood tree of Duff's Early Jewel.

520 Prunus viruses The above results show that NRSV is widespread in plums and cherries and in some peach varieties. Most apricots and nectarines tested were not infected. PDV is not common in peaches, nectarines, apricots, or plums, but is widespread among cherries. 1. Budding and grafting METHODS OF TRANSMISSION Both viruses are readily transmitted by budding or grafting, diseased trees being produced if either stock or scion is infected. Transmission occurs even when the inoculum bud dies, provided it remained alive long enough for some temporary tissue union to have occurred (Fridlund 1967). 2. Seed transmission Seed transmission of NRSV in cherry seed was demonstrated by Cochran (1946), who later also found infection in peach seedlings grown from seed saved from infected trees (Cochran 1950). Millikan (1959) showed that in peach varieties transmission may be up to 16% in seed from infected plants, and Wagnon et ai. (1960) obtained transmission rates of 1.1 % to 1l.7% in peach varieties. Fridlund (1966) found 2% and 4% infection in lines of commercial peach seed. PDV is transmitted in seed of cherry (Cation 1949), but transmission in peach seed has not been reported. Evidence for seed transmission of NRSV in peach in New Zealand has been obtained from experiments with two varieties. Golden Queen seed was saved from an orchard tree infected with both NRSV and PDV. Muir seed was taken at random in a block in which all trees were infected with NRSV. One hundred and fifty seedlings of each variety were indexed, and one Golden Queen seedling (0.75%) and 18 Muir seedlings (12 % ) were infected with NRSV. This confirms reports by other workers that transmission rates differ in different varieties. Whether this is a varietal difference in capability or due to difference in virus strains is not known. No PDV was detected in any of the 150 Golden Queen seedlings raised from seed from the tree carrying both NRSV and PDV. 3. Pollen transmission Transmission of NRSV by pollen to seeds produced on virusfree sour cherries was demonstrated by Gilmer and Way (1960). Plantto-plant transmission by pollen has been demonstrated for both viruses in sour cherry (Gilmer and Way 1963; Gilmer 1965) and forpdv in Buttercup squash (Das and Milbrath 1961). During the past 4 years we have attempted pollen transmission to a total of 16 virus-free trees, including 6 peach varieties, growing in nursery rows. Flowers to be pollinated on each tree were emasculated at the balloon stage and brush pollinated with pollen from NRSVinfected Golden Queen peach. The pollinated flowers were covered with

P. R. FRY AND G. A. WOOD 521 plastic bags until fruitlets had formed. All flowers not pollinated were removed before opening. All flowers were also removed from a comparable number of adjacent trees which acted as un inoculated controls. On each tree 20 to 30 flowers were used, and the fruit set varied from 2 to 24. Trees were indexed for NRSV during the autumn by taking 3 or 4 buds from twigs near fruit which had set on brush-pollinated plants or by sampling at random from the controls. and inserting them in Shirofugen. Transmission occurred only in 1967, when two Tatura Sunset trees became infected. While the possibility of spread by other agency must be admitted. this is considered unlikely, as adjacent control plants in the same nursery row remained uninfected. It is therefore concluded that the two trees were infected by pollen transmission. 4. Mechanical transmission Sap expressed from leaves of rosaceous plants contains phenolic compounds which rapidly inactivate viruses (Bawden and Kleczkowski 1945). Transmission to herbaceous hosts and from herbaceous hosts to Prunus spp. is assisted by the use of nicotine or high ph buffers (Cadman 1959) to prevent this inactivation. Fridlund (1967) has shown that when plants are budded some tissue union is necessary for transmission to occur. Thus there is no spread of infection by contamination of implements with infected sap during budding or pruning operations. SPREAD OF INFECTION IN NURSERY AND ORCHARD (a) Nursery Infection in commercial nurseries may come from either rootstock or scion. Seedling rootstocks may be infected by seed transmission if seed is saved from infected trees. Clonally propagated rootstocks become infected if cuttings are taken from plants that have been worked with infected material. Budding or grafting with infected scions gives rise to infected trees. (b) Orchard Though spread may occur by natural root grafting. the main method of spread in orchards appears to be by infected pollen. Davidson and George (1964) found that in sour cherry in the Niagara Peninsula, Canada, NRSV spread slowly in orchards less than 4 years old but rapidly in older orchards where the greater amount of blossom facilitated pollen transmission. PDV inducing sour cherry yellows symptoms did not spread rapidly until trees were more than 10 years old. Under New Zealand conditions NRSV has been found to spread in peach orchards. Indexing of a 5-year-old block of Golden Queen peach trees in a Hawke's Bay orchard in the 1967-68 season showed that 7 of the 102 trees tested were infected with NRSV. In the following years further trees in the orchard were included in the indexing programme. In the 1968-69 season 28 out of 195 (14%) and in the 1969-70 season 48 out of 198 (24%) trees were infected.

522 Prunus viruses Two small blocks of Golden Queen at the Oratia Research Orchard were used in another experiment. In block A planted in 1962, 20 of the 37 trees were infected with NRSV by the 1966-67 season when indexing commenced. No increase in infection was detected in 1967-68, but one further tree was infected in each of the next two seasons. Block B, of 35 trees, was planted in 1963 and was free from NRSV infection when indexed during the 1966-67 and 1967-68 seasons. In 1968-69 one tree was infected, and in 1969-70 four trees were infected. No information is available on the field spread of PDV under local conditions. PARTIAL PURIFICATION AND SEROLOGY Partial purification was attempted only with NRSV using an isolate, H.242, from Golden Queen peach, which had been maintained by serial passage in cucumber for several months. The source of virus was cucumber cotyledons harvested 5 days after inoculation. These were crushed in 1.5 times their weight of 0.02M Na2HP04 plus 0.02M sodium thioglycollate plus O.OIM sodium DIECA, adjusted to ph 8.0 with NaH 2 P04. The extract was clarified using the hydrated calcium phosphate method of Fulton (1959) and the virus was separated from other components by two cycles of low- and high-speed centrifugation followed by density gradient centrifugation using a 10 to 40% sucrose gradient for 90 min at 35,000 rpm in the Spinco SW39 rotor. Gradients were sampled using an ISCO Density Gradient Fractionator which gave good separation between virus and normal plant protein. Electron microscope observations confirmed the presence of viruslike particles in infectious preparations. When negatively stained with 2% phosphotungstic acid (adjusted to ph 6.0) and printed at a magnification of 125,000, the particles appeared spherical with a diameter of from 20 to 25 mp.. This agrees with the measurements of 20.6 to 23.5 mp' obtained by Fulton (1959). Serological tests were made by the Octerlony double-diffusion method using 0.75% agar in 0.14M NaCl. An antiserum was prepared against the NRSV, H.242 isolate, in a rabbit, by giving three injections of approximately 1 mg of virus at weekly intervals and bleeding 12 days after the last injectior. This antiserum had a titre of to. Three months later two intramuscular injections of approximately 1 mg of virus were given 14 days apart using Freund's adjuvant. Bleeding 1 month later gave an anti-serum with a titre of too. These antisera reacted specifically with the homologous antigen, there being no reaction to normal plant proteins in either purified preparations or cucumber sap. The H.242 virus preparation gave a single line of precipitation with an antiserum to an almond calico strain of NRSV supplied by Prof. G. Nyland, University of California, Davis. When H.242 antigen and a NRSV isolate from Muir peach were placed in adjacent wells to react with H.242 antiserum, spur formation occurred, showing that different strains or strain mixtures had been isolated from Golden Queen and Muir peach trees.

P. R. FRY AND G. A. WOOD 523 HEAT THERAPY Heat treatment of infected plants to obtain virus-free material was used by Ehlers (1957), who cured sour cherry of PDV by dipping budwood in hot water at 35 c for 30 hr or 50 for 22 min, and by Nyland (1960), who grew trees at 38 to eliminate a number of viruses. including NRSV and PDV, from cherries after 2 weeks and peaches after 3 weeks. In our experiments we have used both hot-water treatment of budwood and hot-air treatment of plants. For hot-water treatment, budsticks of NRSV -infected Myrobalan plum (P. cerasifera Ehrh.) and NRSV plus PDV-infected Italian prune were immersed in water at 50 c for 10, 20, or 30 min or at 55 for 10 min. After treatment the sticks were cooled by plunging into water at room temperature. Three buds from each stick were then budded into Shirofugen flowering cherry to test for the presence of NRSV or PDV. Buds that were still infected produced a necrotic reaction and soon died, but those that were free from infection were still alive when inspected 6 weeks later. NRSV was eliminated from all 3 Myrobalan plum buds heated at 55 for 10 min or 50 for 20 or 30 min, but not from those heated at 50 for 10 min. Both NRSV and PDV were eliminated from all 3 Italian prune buds heated at 55 for 10 min, but those heated at 50 for 10, 20, or 30 min gave a positive reaction on Shirofugen, showing that one or both viruses were still present. Potted plants given hot-air treatment were placed in a temperaturecontrolled chamber at 32 c + 2 for 7 to 10 days to stimulate lateral growth. The temperature was then raised to 38 + 2 and shoot tips removed when they had developed sufficiently, 13 to 24 days later. Shoot tips. approximately 1 cm long, were grafted on to virus-free seedlings. Where tip propagation was successful buds from subsequent growth were later indexed on Shirofugen. With cherries both NRSV and PDV were eliminated from one Chapman tip heated at 38 for 13 days and Early Cluster heated for 16 (2 tips), 21 (2 tips). and 24 (1 tip) days. One Bedford Prolific tip taken after 15 days' heating still gave a positive reaction on Shirofugen. Plums and peaches that were heat treated contained only NRSV. It was eliminated from single tips of Satsuma and Billington plums heated for 21 days and from two Paragon peach tips after 17 days' heating. from two Mary's Choice peach tips heated for 21 days. but not from one Halford peach tip heated for 14 days. CONTROL Transmission of infection between and within orchards occurs with the transfer of pollen from infected to healthy trees. Indexing of orchard trees and removal of any found to be infected would be uneconomic because of the high incidence of infection. Control measures must therefore be directed at the production of virus-free trees for the establishment of new orchards that should be planted a sufficient distance from existing orchards to prevent, or at least minimise. the transfer of infected pollen.

524 Prunus viruses Davidson and George (1964) found that NRSV could spread over a distance of at least 800 yards and PDV about 100 yards, but most infections od: both occur within 50 ft of a known source. The production of virus-free trees by nurseries depends on a supply of un infected scion and rootstock material. Because infection is widespread, a heat treatment programme has been initiated with the object of producing virus-free scion material. Elimination of NRSV and PDV has been successful to date with two peach, three plum, and two cherry varieties. It is intended to establish a collection of virus-free stone fruit varieties and stocks in an area isolated from sources of infection. From this area scion wood will become available for propagation. This scheme will be similar to those established in many other countries for the maintenance and distribution of virus-free propagating material. Seed for use as rootstocks should be saved from trees that are free from both viruses. If clonal rootstocks are used, cuttings should be taken only from trees known to be virus free. Acknowledgments Dr S. Bullivant, Cell Biology Department, Auckland University, for the electron microscope examinations; Mr F. M. Mitchell for purification of virus and preparation of antisera; and officers of the Horticulture Division, Department of Agriculture, for collection of samples from orchards. REFERENCES BAWDEN, F. c.; KLECZKOWSKI, A 1945: Journal of Pomological and Horticultural Science 21: 2. CADMAN, C. H. 1959: Journal of General Microbiology 20: 113. CATION, D. 1949: Phytopathology 39: 37. CHAMBERLAIN, E. E. 1961: Orchardist of N.Z. Supplement Nov. 1%1. CocHRAN, L. C. 1946: Science 104: 269. -----. 1950: Phytopathology 40: 964 (abs). CocHRAN, L. C.; HUTCHINS, L. M.; MILBRATH, J. A; STOUT, G. L.; ZELLER, S. M. 1951: u.s. Department of Agriculture Handbook 10: 71. CROPLEY, R.; GILMER, R. M.; POSNETTE, A F. 1964: Annals of Applied Biology 53: 325. DAS, C. R.; MILBRATH, J. A 1961: Phytopathology 51: 489. DAVIDSON, T. R.; GEORGE, J. A 1964: Canadian Journal of Plant Science 44: 471. EHLERS, C. G. 1957: Dissertation Abstracts 17: 1879. FRIDLUND, P. R. 1966: Plant Disease Reporter 50: 740. 1967: Phytopathology 57: 1296. FRY, P. R.; WOOD, G. A 1969: Orchardist of N.Z. 42: 397.

P. R. FRY AND G. A. WOOD 525 FULlON, R. W. 1959: Virology 9: 522. 1968: Vll Europiiisches Symposium "ber Viruskrankheiten der Obstbiiume: 123. GILMER, R. M. 1965: Phytopathology 55: 482. GILMER, R. M.; WAY, R. D. 1960: Ibid. 50: 624. ---- 1963: Plant Disease Reporter 47: 1051. HAMPlON, R. 0.; AICHELE, M. D.; BLODGETT, E. C. 1966: Phytopathology 56: 65. MILBRATH, J. A.; ZELLER, S. M. 1945: Science 101: 114. MILLIKAN, D. F. 1959: Plant Disease Reporter 43: 82. NYLAND, G. 1960: Phytopathology 50: 380. RICHARDS, B. L.; COCHRAN, L. C. 1954: Bulletin of Utah Agricultural College 384: 40. THOMAS, H. E.; HILDEBRAND, E. M. 1936: Phytopathology 26: 1145. WAGNON, H. K.; TRAYLOR, 1. A.; WILLIAMS, H. E.; WEINBERGER, 1. H. 1960: Plant Disease Reporter 44: 117.