European stone fruit yellows phytoplasmas associated with a decline disease of apricot in southern England

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1 Plant Pathology (2000) 49, 635±639 European stone fruit yellows phytoplasmas associated with a decline disease of apricot in southern England D. L. Davies*² and A. N. Adams Horticulture Research International, East Malling, West Malling, Kent, ME19 6BJ, UK Phytoplasmas detected by fluorescence microscopy and polymerase chain reaction (PCR) have been discovered infecting Prunus trees at a site in south-east England. The pathogens were detected in tissue samples taken in autumn and also in spring. The symptoms in infected trees varied from severe decline to absence. PCR experiments using group-specific primers to amplify regions of the 16S RNA gene indicated that the phytoplasmas are similar to European stone fruit yellows isolates occurring in southern and eastern Europe. This is the first record of phytoplasmas in Prunus species in the UK. The origin of the infection is unknown. The implications of this new disease for the fruit industry are discussed. Keywords: apricot, England, fruit, phytoplasmas, stone, yellows Introduction Phytoplasmas cause a range of persistent diseases in fruit trees throughout the world. In Europe, apple proliferation (AP), pear decline (PD), and a range of diseases in Prunus species associated with European stone fruit yellows (ESFY) phytoplasmas are the most common. They are taxonomically related and belong to the apple proliferation group, but each one is considered to be specific to its respective host genus. Primers have been developed (Lorenz et al., 1995; Smart et al., 1996; Kison et al., 1997; Jarausch et al., 1998) that allow PD, AP and ESFY to be distinguished by polymerase chain reaction (PCR). Alternatively, broader-spectrum primers may be used and the PCR product digested with restriction endonucleases. In the UK, pear decline is the only fruit tree phytoplasma known to be widespread (Davies et al., 1992), although an isolated occurrence of apple proliferation has also been recorded (Davies et al., 1986). The phytoplasmas collectively termed ESFY (Lorenz et al., 1994) are associated with a wide range of diseases in southern Europe, including apricot chlorotic leafroll (Morvan, 1977, 1988), plum leptonecrosis (Giunchedi et al., 1982) and decline diseases in plum, peach and almond (Lederer & SeemuÈ ller, 1992; Poggi Pollini et al., 1993, 1995). The psyllid species Cacopsylla pruni has been reported to be a vector of the pathogen (Carraro et al., 1998a). There has been no *To whom correspondence should be addressed. ² david-l.davies@hri.ac.uk Accepted 28 April record to date of any phytoplasma disease occurring in Prunus species in the UK. This paper describes the occurrence of a severe decline disease in several apricot trees at a site in south-east England, and the subsequent discovery and identification of phytoplasmas associated with the diseased trees and with a number of other Prunus trees from the same site. Materials and methods Sources of plants The apricot and plum cultivars examined were growing at the National Fruit Collection in Kent and are listed in Table 1. The majority of trees were over 30 years old and had been grafted to St Julien rootstocks. They had all been propagated in the UK, but the source of most of the original graftwood was obscure. Malus explants infected with AP, and Prunus explants infected with ESFY (a gift from Dr Wolgang Jarausch and Micheline Lansac) were maintained by micropropagation as described by Jarausch et al. (1996). Pyrus explants infected with the PD phytoplasma were also maintained by micropropagation as described by Davies & Clark (1994). A German isolate of ESFY was propagated in Catharanthus roseus, and was supplied by Dr E SeemuÈ ller. St. Julien rootstocks were whip- and tongue-grafted in March with infected scions that were approximately 6 cm long with two buds. This material was kept in a gauze tunnel. Q 2000 BSPP 635

2 636 D. L. Davies and A. N. Adams Table1 Phytoplasmas detected by PCR amplification using the universal primers U5/U3 in a collection of apricot (Prunus armeniaca) and plum (P. domestica) cultivars Cultivar Species Symptoms a Result Alfred P. armeniaca ± Catherina P. armeniaca 1 Kember P. domestica ± Kea P. domestica ± Early Moorpark P. armeniaca ± Rode R Claude d'oultins P. domestica ± Farmingdale P. armeniaca Decline, leaf curl 1 Buhler Fruhtzwetsche P. domestica ± Prune Paschal P. domestica ± Kecskemeti P. armeniaca 1 Old Cape P. armeniaca Sparse, pale foliage 1 Veeblue P. domestica ± Monsieur Jaune P. domestica ± Riland P. armeniaca 1 Washington P. domestica ± Stanley P. domestica ± Sunglo P. armeniaca ± Uhinks Reine-Claude P. domestica ± George Calver P. domestica ± Victoria Late Sport P. domestica ± Wilson's Delicious P. armeniaca 1 Oneida P. domestica 1 Royale P. armeniaca ± President P. domestica ± a All trees were symptomless except where indicated. Detection of phytoplasmas Fluorescence microscopy was carried out as described by SeemuÈ ller (1976). DNA for PCR amplification was prepared from the petioles or bark of samples collected from the field or gauze tunnel, and from the stems of micropropagated samples, as described by Ahrens & SeemuÈ ller (1992). PCR experiments were performed in 20 ml reaction volumes containing 1±5 ng DNA, 0 4 mm of each primer (listed in Table 2), 0 25 mm of each dntp, 1 5 mm MgCl 2 and 0 5 units of Taq polymerase (Gibco BRL, Paisley, UK) with the manufacturer's supplied buffer. Thermocycling was performed using a Hybaid Table 2 Primers used for the PCR detection of pear decline (PD), apple proliferation (AP) and European stone fruit yellows (ESFY) phytoplasma isolates Primer pair Target Specificity Reference 01F/01R 16S rdna Fruit tree Lorenz et al., 1995 phytoplasma PDF/01R 16S rdna PD/AP Lorenz et al., 1995 U5/U3 16S rdna Universal Lorenz et al., 1995 phytoplasma ATF/ASR 16S rdna/its PD/AP Smart et al., 1996 PDF/PDR 16SrDNA/ITS PD Lorenz et al ATF/PruS 16SrDNA ESFY Smart et al. 1996; Kison et al., ECA1/ECA2 Genomic ESFY Jarausch et al., 1998 OmniGene thermocycler (Hybaid, Teddington, UK) for 35 cycles with 1 min each for denaturation, annealing and extension. In the final cycle the extension step was extended to 10 min. PCR products were separated by electrophoresis through 15 g L 21 agarose gels containing 0 5 mg ml 21 ethidium bromide and viewed with a UV transilluminator. Results Bark samples were examined from a 20-year-old apricot tree cv. Farmingdale showing severe symptoms of decline with pale, curled leaves (Fig. 1). Longitudinal sections through the sieve tubes showed the characteristic structures of phytoplasmas. DNA samples prepared from the same tree were tested by PCR using the universal primers U5/U3. A product of the expected size was obtained confirming a phytoplasma infection. This prompted a further investigation of the tree together with a number of other trees nearby. A second collection of samples made in October was examined by PCR also using U5/U3 primers. A product of the expected size was obtained from samples taken from cvs Farmingdale, Catherina, Kecskemeti, Old Cape, Riland and Wilson's Delicious, together with the plum cultivar Oneida (Table 1). Only cvs Farmingdale and Old Cape were showing disease symptoms. Samples from the rootstock suckers from the infected trees were also examined 1 month later, but the only ones to give a product by PCR were Farmingdale and Catherina. Also examined, from an adjacent site, were two trees each of Catherina, Farmingdale, Kecskemeti, Old Cape, Wilson's Delicious, and the plum cultivar Oneida. These trees were approximately 5 years old and had been propagated from the trees now found to be infected in the original collection. All of the younger trees appeared to be free of infection when examined by PCR. Two further sets of samples were taken from the Prunus collection the following March and April. Two trees each of Catherina, Farmingdale and Kecskemeti, and one tree each of Old Cape, Riland, Wilson's Delicious and Oneida, were examined. In the first set of samples, positive results were again obtained from Catherina (2/2), Farmingdale (1/2), Kecskemeti (1/2), Old Cape and Riland (Fig. 2), but there was only a very faint product from the Wilson's Delicious sample. In the second collection, a clear product was also obtained from the Wilson's Delicious sample (not shown), but in both collections there was no product detectable in samples prepared from the Oneida tree that had yielded a product the previous year. The samples taken in March were also used to graft St Julien rootstocks. Most of the grafts were unsuccessful, with the exceptions of the rootstocks grafted with Kecskemeti, Old Cape, Wilson's Delicious and Oneida. PCR amplification of DNA samples prepared from the grafted trees in October yielded a product from cvs Kecskemeti and Old Cape, confirming that the phytoplasmas detected in the graftwood in early spring had been viable. The Old

3 Stone fruit yellows in southern England 637 Figure 1 Apricot tree cv. Farmingdale showing symptoms of yellowing, leaf curl and decline. A symptomless shoot is shown in the foreground. Cape tree was also showing symptoms consistent with the symptoms on the mother tree. A range of primers for the detection of fruit tree phytoplasmas were used in PCR experiments with DNA samples prepared from the six infected apricot trees (Table 2). The phytoplasma from the plum cultivar could not be included in this comparison. The six apricot samples were indistinguishable on this basis. The results were compared with samples prepared from ESFY isolates of German and French origin, together with apple proliferation- and pear decline-infected samples (Table 3). The primer pairs U5/U3, 01F/01R and PDF/01R yielded a product with all the samples examined. The English apricot isolates and the French and German ESFY isolates also yielded a product with the primers ATF/PruS and ECA1/ECA2, which were designed for the detection of phytoplasmas from Prunus species. The ESFY isolate from France appeared to be slightly different from the German and English isolates in also giving a product with the primers ATF/ASR. On this basis, the phytoplasmas occurring in the UK apricot trees appear to be similar to the German ESFY isolate, and are clearly distinguishable from the phytoplasmas associated with pear decline that are widespread in the UK. Discussion This paper describes a severe decline disease in apricot trees at a site in south-east England, and the detection of phytoplasmas associated with this disease and with a number of other Prunus trees growing at the same site. Apricot chlorotic leafroll is a notifiable disease in the UK, and this is the first report of a phytoplasma Figure 2 Agarose gel electrophoresis of PCR products from apricot samples obtained using primers U5/U3. Tracks: 1, MW markers; 2,3, Catherina; 4,5, Farmingdale; 6,7, Kecskemeti; 8, Old Cape; 9, Riland; 10, Wilson's Delicious; 11, Oneida (plum); 12, positive control; 13, negative control.

4 638 D. L. Davies and A. N. Adams Table 3 PCR results using a range of fruit tree primers to differentiate between pear decline (PD), apple proliferation (AP) and European stone fruit yellows (ESFY) phytoplasmas from the UK, Germany (DE) and France (FR) PD AP ESFY (UK) ESFY (DE) ESFY (FR) 01F/01R PDF/01R U5/U ATF/ASR 1 1 ± ± 1 PDF/PDR 1 ± ± ± ± ATF/PruS 1 ± ECA1/ECA2 ± ± , Product of the correct size detected; ±, no product detected. infection in Prunus species in the UK. The apricot cultivar originally tested was severely affected, but the plum and many of the other apricot cultivars that were also infected were showing no obvious symptoms. The source of the infection is unknown. The trees were all relatively old (.30 years) and were propagated in the UK from sources in the UK, USA and South Africa. The severity of the disease in the worst-affected cultivars could imply a recent infection rather than one acquired through propagation. PCR comparisons using a range of primer combinations to distinguish pear decline in the UK from apple proliferation and ESFY isolates in France and Germany indicate that the phytoplasmas associated with the UK Prunus samples were similar to ESFY. The use of primers designed to amplify 16S rdna, as well as the primers (ECA1/ECA2) designed from a cloned genomic fragment specifically to detect ESFY isolates, yielded a similar conclusion. On this basis, the phytoplasmas in this study were clearly distinguishable from those associated with the pear decline widespread in the UK. Therefore this paper reports a new phytoplasma disease in the UK. Unlike the phytoplasmas associated with pear decline and apple proliferation, the phytoplasmas associated with Prunus species appear to be detectable during early spring as well as later in the growing season. This is consistent with a similar finding for German isolates of ESFY (SeemuÈ ller et al., 1998), and suggests that there is no `low-risk' time of year to propagate from potentially infected trees. This was confirmed in this study by the transmission of phytoplasmas by grafting infected material to experimental rootstocks in early spring. However, there was no evidence of infection in younger trees at a neighbouring site to the original Prunus collection that had been propagated 5 years previously from the trees examined in this study. This supports the suggestion that the infection in the older trees was a recent phenomenon. The phytoplasmas collectively termed ESFY (Lorenz et al., 1994) are associated with a wide range of diseases in southern Europe, including plum leptonecrosis (in the Japanese plum, P. salicina), apricot chlorotic leafroll, and peach and almond decline. The psyllid species C. pruni has been reported to be a vector of the disease (Carraro et al., 1998a) in northern Italy. This psyllid species is also resident in the UK (Hodkinson & White, 1979), although little is known about its distribution and behaviour, as it is not regarded as a pest. There is an increasing interest in planting apricot cultivars in the UK as an alternative to some of the more traditional fruit crops. It is unknown at present whether the phytoplasmas associated with the Prunus species in this paper represent an isolated occurrence, or are part of a more widespread infection which is apparent only when relatively sensitive apricot cultivars become infected. Some European plum cultivars (Prunus domestica) are reported to be tolerant to ESFY, the symptomless infection becoming apparent only when grafted with the indicator cultivar Ozark Premier (Carraro et al., 1998b). Future work is necessary to establish the prevalence of phytoplasmas in Prunus species in the UK, their occurrence in nurseries, and their epidemiology. Acknowledgements This work was funded by the Ministry of Agriculture, Fisheries and Food, project number HH1754STF, and was conducted within the terms of Plant Health Licence PHL 5C/2982. The authors wish to thank the staff at the National Fruit Collection for assistance with sample collection. References Ahrens U, SeemuÈ ller E, Detection of DNA of plant pathogenic mycoplasma-like organisms by a polymerase chain reaction that amplifies the sequence of the 16S rrna gene. Phytopathology 82, 828±32. Carraro L, Osler R, Loi N, Ermacora P, Refatti E, 1998a. Transmission of European stone fruit yellows phytoplasma by Cacopsylla pruni. Journal of Plant Pathology 80, 233±9. Carraro L, Loi N, Ermacora P, Osler R, 1998b. High tolerance of European plum varieties to plum leptonecrosis. European Journal of Plant Pathology 104, 141±5. 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