phytoplasmal diseases of peach and associated phytoplasma ... - SIPaV

Journal of Plant Pathology (2014), 96 (1), 15-28

Edizioni ETS Pisa, 2014

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Offered Review

PHYTOPLASMAL DISEASES OF PEACH AND ASSOCIATED PHYTOPLASMA TAXA C. Marcone1, L.J. Guerra2 and J.K. Uyemoto3 1Department

of Pharmacy, University of Salerno, Via Giovanni Paolo II, 132, I-84084 Fisciano (Salerno), Italy State Department of Agriculture, North Bunn Road 24106, WA-99350 Prosser, Washington, USA 3USDA-ARS, Department of Plant Pathology, University of California, One Shields Avenue, CA-95616 Davis, California, USA 2Washington

Running title: Peach phytoplasmal diseases SUMMARY

INTRODUCTION

Phytoplasmal diseases occur wherever peach (Prunus persica) trees are grown. However, the causal agents differ considerably in taxonomy, insect vector relationships and geographic locations. X-disease of peach is widespread in North America, but does not occur elsewhere in the world. X-disease is induced by ‘Candidatus Phytoplasma pruni’, a member of the X-disease phytoplasma group (16SrIII group, subgroup 16SrIII-A). Peach rosette, peach red suture, and peach yellows, which occur in eastern United States and Canada are all caused by the X-disease phytoplasma. Another North America disease, peach yellow leaf roll (PYLR) is present in a limited area of northern California. Its causal agent is classified in the apple proliferation (AP) group, 16SrX group, as a subtype of the pear decline phytoplasma. In Europe, phytoplasmal diseases of peach are reported under the name European stone fruit yellows and incited by ‘Ca. P. prunorum’, a member of the AP group, subgroup 16SrX-B. ‘Ca. P. prunorum’ is closely related to the PYLR agent. In Lebanon and Iran, peach trees are affected by almond witches’ broom, a lethal disease incited by ‘Ca. P. phoenicium’, a member of the 16SrIX group, subgroup 16SrIX-B. Phytoplasmas of other phylogenetic groups, known to infect a wide range of plant hosts, have been identified in declining peach trees in several fruit-growing areas of the world. The pathological relevance of several ‘non-peach’ phytoplasmas requires further investigations as their presence was ascertained by nested PCR assays only.

In most of the peach (P. persica) production areas of the world, peach trees are severely affected by yellows and decline diseases of phytoplasmal etiology. Phytoplasmas are non-helical, wall-less bacterial plant pathogens of the class Mollicutes, that cause diseases collectively referred to as “yellows diseases”. Besides peach, phytoplasmas affect a wide range of plant species (Seemüller et al., 1998a; Lee et al., 2000; Bertaccini, 2007; Bertaccini and Duduk, 2009), many of which, especially those of temperate fruit trees, are of great economic importance. In diseased plants, phytoplasmas reside almost exclusively in sieve tube elements (Fig. 1A) and are transmitted to host plants by phloemfeeding homopteran insects, i.e., leafhoppers (Cicadellidae), planthoppers (Fulgoromorpha), or psyllids (Psyllidae) (Weintraub and Beanland, 2006). Even though phytoplasma DNAs were detected in embryos of lethal yellowingdiseased coconut palms and in seeds from phytoplasmainfected plants of lime, alfalfa, tomato, oilseed rape, maize and apricot, none gave rise to diseased seedlings (Nečas et al., 2008; Faghihi et al., 2011; Dickinson et al., 2013). Phytoplasmas are not sap-transmissible. However, they are commonly spread to new orchards via vegetative propagation and grafting of infected scions on healthy rootstocks (Lee et al., 2000; Dickinson et al., 2013). The introduction of DNA-based detection methods into phytoplasmology occurred two decades ago with the development of specific and sensitive PCR assays which proved useful for identifying phytoplasma infections in both plants and insect vectors. It also became possible to differentiate, characterize and classify phytoplasmas on a phylogenetic basis, using sequence and restriction fragment length polymorphism (RFLP) analyses of 16S ribosomal DNA (rDNA) (Seemüller et al., 1998a, 2002; Lee et al., 2000, 2007). In this way, approximately twenty major phylogenetic groups or subclades were identified within the phytoplasma clade. This number is generally in accordance with the phytoplasma groups or 16Sr groups established by RFLP analysis of PCR-amplified rDNA (Seemüller et al., 1998a, 2002; Lee et al., 2000, 2007). Each phytoplasma subclade (or corresponding 16Sr group) is considered to represent at least one distinct species under

Key words: phytoplasmal etiology, X-disease, peach yellow leaf roll, European stone fruit yellows, symptomatology, phytoplasma taxonomy.

Corresponding author: C. Marcone Fax: +39.089.969602 E-mail: [email protected]

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Peach phytoplasmal diseases

the provisional taxonomic status of ‘Candidatus’ (IRPCM, 2004). Within most of the phytoplasma groups, several distinct subgroups (16Sr subgroups) have been delineated on the basis of the RFLP analysis of 16S rDNA sequences (Lee et al., 2007). Recently, the number of 16Sr groups and subgroups was expanded to 32 and more than 100, respectively, via the use of a computer-simulated RFLP analysis method (Zhao et al., 2009, 2013; Nejat et al., 2013). To date, over 30 ‘Candidatus Phytoplasma’ species have been formally described (IRPCM, 2004; Davis et al., 2013). The aim of the present article is to summarize information on phytoplasmal diseases of peach trees, with emphasis on the molecular and taxonomic aspects of the associated phytoplasmas. PHYTOPLASMAL DISEASES OF PEACH

X-disease. X-disease is one of the most serious diseases of peach. It is widespread in North America and does not occur elsewhere in the world. The disease was first described in 1931 on sweet cherry (Prunus avium) in California as cherry buckskin. Shortly thereafter, epidemiological and graft-transmission studies showed that the causal agent of cherry buckskin also caused a lethal decline disease of peach, called leaf casting yellows. In 1933, a disorder of peach characterized by symptoms similar to those reported in California, was observed in Connecticut and called X-disease because of its unknown cause. In ensuing years, peach X-disease was reported throughout the United States and in Canada (for reviews see Gilmar and Blodgett, 1976; Kirkpatrick et al., 1995a; Uyemoto and Kirkpatrick, 2011). The disease is induced by a distinct phytoplasma, the X-disease agent ‘Candidatus Phytoplasma pruni’, a member of the X-disease phytoplasma group or 16SrIII group, subgroup 16SrIII-A (Davis et al., 2013). ‘Ca. P. pruni’, at the level of 16S rDNA sequences, is a homogeneous pathogen. Nucleotide sequence comparisons revealed that the 16S rDNA sequences of strains from eastern and western fruit-growing regions of the United States and Canada were identical or nearly so (Kirkpatrick et al., 1995a; Wang et al., 2012; Davis et al., 2013). Also, graft-inoculation studies have shown that geographically different strains inciting the typical X-disease symptoms were pathologically similar (Kirkpatrick et al., 1995a). In addition to peach, the X-disease phytoplasma infects several other cultivated and wild Prunus species including sweet cherry, sour cherry (P. cerasus), nectarine (P. persica var. nectarina), almond (P. dulcis), Japanese plum (P. salicina), chokecherry (P. virginiana) and bitter cherry (P. emarginata). Apricot (P. armeniaca) and European plum (P. domestica) are not known to be susceptible to X-disease phytoplasma. In one-year-old infections, symptoms are often limited to a single branch (due to feeding transmission by an insect vector). Evidence of internal spread, as shown by the appearance of symptoms in other parts of the tree,


becomes obvious in the growing seasons post inoculations. In peach and nectarine, symptoms are cultivar-dependent ranging from early defoliation (mild to moderate) without any obvious leaf markings to leaf yellowing by midsummer (Guerra, 1997). Other symptoms include leaves with irregularly shaped lesions that are red, chlorotic or necrotic. Necrotic tissues dehisce giving leaves a shot-hole appearance (Fig. 1B and C). Later, symptomatic leaves are prematurely shed and, depending on tree vigor, new terminal leaves may develop. Fruits borne on infected branches are small and lack flavor. Diseased trees exhibit branch dieback and trees succumb within five years. The X-disease agent is transmitted in nature by at least 13 species of polyphagous leafhoppers (Purcell, 1996). In the western United States, Colladonus montanus, C. geminatus, Fieberiella florii, Scaphytopius acutus and some Osbornellus species are the most important vectors, while Paraphlepsius irroratus appears to be the most important vector in Michigan and other eastern states (Fig. 1D and E). Although peach is highly susceptible to X-disease phytoplasma and readily infectible (via graft inoculation), it is a very poor host for acquisitions by leafhopper vectors. Thus, peach is regarded as a dead-end host, with no evidence of disease spread from tree to tree (Purcell, 1996). In the eastern United States, X-disease-infected chokecherry and bitter cherry trees, residing in close proximity to peach orchards, serve as the primary pathogen reservoirs from which leafhopper vectors readily acquire the X-disease phytoplasma (Fig. 1F). However, in the central valley of California, where wild Prunus hosts are absent, diseased sweet cherry trees are the most important pathogen source (Kirkpatrick et al., 1995a; Uyemoto and Kirkpatrick, 2011). In the foothills of California and in the cherry-growing areas of Washington state, chokecherries growing in the vicinity of orchards serve as a pathogen reservoir. We speculate that other ornamental trees of genus Prunus may be potential pathogen reservoirs. Species from other genera may also function as reservoirs, but their role on the epidemiology of the pathogen is not known. For example, common lilac (Syringa vulgaris) can be naturally infected with the X-disease phytoplasma but it is unknown whether it is reservoir of the pathogen (Guerra, 1997). Peach rosette, peach red suture, and peach yellows. Peach rosette (PR) was first observed in Georgia in 1881 (Smith, 1891). Subsequently the disease was reported primarily from the southeastern United States and as far west as Texas (KenKnight, 1976; Uyemoto and Scott, 1992; Kirkpatrick, 1995a; Scott and Zimmerman, 2001; Kirkpatrick et al., 2011). A severe outbreak occurred in Arkansas in 1977 (Kim and Slack, 1978). Although sporadic spread of the disease has been reported, PR is regarded as minor in importance (Scott and Zimmerman, 2001; Ragozzino, 2011). A characteristic disease symptom is the production of numerous multiple axillary buds and of excessive number


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Fig. 1. A, transmission electron micrograph showing phytoplasmas in sieve tubes. CW = cell wall; SP = sieve plate; Bar = 600 nm. B and C, typical foliar symptoms of X-disease on peach: irregularly shaped and necrotic lesions (B) and shot-hole appearance of the leaves (C). D and E, leafhopper vectors of the X-disease phytoplasma: Colladonus montanus (D) and Fieberiella florii (E). F, healthy-looking chokecherry (Prunus virginiana), a major pathogen reservoir of the X-disease agent. G and H, peach rosette symptoms: leaves of affected trees are appressed into distinct dense rosettes. Right, healthy tree (H). I, a disorder of peach resembling peach rosette, observed in southern Italy (Marcone et al., 1995). J, peach fruit with swollen and reddened suture, typical symptom of peach red suture. K and L, narrow and chlorotic leaves (K) and willowy growth (L), characteristic symptoms associated with peach yellows, called also little peach. (D and E courtesy J.K. Clark, University of California, Davis, CA, USA; G, H, J, K and L, courtesy S.W. Scott, Department of Entomology, Soils and Plant Sciences, Clemson University, SC, USA).

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of shoots with shortened internodes, due to death of terminal buds. As new leaves develop, they appear normal in size and appressed into distinct dense rosettes (Fig. 1G and H). At the base of these rosettes there are one or two abnormally long and straight leaves with inward rolled margins. Older leaves turn yellow and drop by early summer to leave tufts of younger leaves near the tips of otherwise bare shoots. Very few adventitious shoots develop in the interior of the tree canopy. The affected trees produce only few, small misshapened fruits that drop prematurely. Severely affected trees may succumb during the first year of symptom expression. Although an insect vector of PR has not been identified, natural spread into peach orchards is correlated with the close proximity to diseased wild plum (Prunus angustifolia) trees, in which the PR agent has been detected by PCR (Scott and Zimmerman, 2001). In southern Italy, a disorder of peach resembling PR (Fig. 1I) was reported in a small orchard near Salerno (Marcone et al., 1995). The etiology of this disorder was not elucidated, but two different phytoplasmas, based on RFLP analysis of PCR-amplified rDNA, were found, which were assigned to the aster yellows (AY) phytoplasma group or 16SrI group, and the X-disease group. Even though neither phytoplasma could be detected in diseased peach trees with the methods used (Marcone et al., 1995), both phytoplasmas were transmitted from diseased trees to Catharanthus roseus (periwinkle) via dodder (Cuscuta campestris) bridges. Before further studies could be completed, affected trees were destroyed and the disease was eradicated in southern Italy (Ragozzino, 2011). Peach red suture (PRS) has been reported from Michigan, Maryland, South Carolina and eastern Canada (for reviews see Larsen and Waterworth, 1995; Scott and Zimmerman, 2001). In recent years, it has not been economically important (Kirkpatrick et al., 2011). Leaves of infected trees turn yellowish-green to greenish-bronze a few weeks after petal fall and just before fruit maturity. Diseased trees also show premature autumn coloration, reduced terminal growth and proliferation of shoots with shortened internodes arising from vigorous branches. However, the most typical symptom occurs on the fruits as a premature ripening and softening in the suture area, while the fruit remains green and hard (Fig. 1J). The suture area is prominent, bumpy, swollen, with an intense dark red to purple color on red cultivars and an intense yellow color on yellow cultivars. Affected fruits are insipid. Peach yellows (PY), also called little peach, was first observed in Pennsylvania in 1791 (Kunkel, 1936). The disease spread gradually northward, through the New England states and into Canada and southward into Delaware, Maryland, Virginia, West Virginia, and North Carolina. PY has not been found in the far western or southern states or outside North America. European diseases called peach yellows are either caused by other phytoplasmas or have unknown etiologies. Epidemic outbreaks in the United States during the 19th and early 20th centuries caused


significant tree losses (Kirkpatrick, 1995b). Currently, disease incidence is low (Scott and Zimmerman, 2001; Kirkpatrick et al., 2011). Leaf buds on diseased peach trees, even those that should normally remain dormant, develop prematurely. Leaves produced from these buds are narrower and smaller than normal leaves, and as the season progresses, they become chlorotic and often develop red spots. Affected trees produce slender, branched, willowy shoots that grow upright from the main limbs, thus giving the tree a bushy appearance (Fig. 1K, L). Leaves borne on the abnormal shoots are dwarfed, severely chlorotic with the margins rolled upward and drop prematurely. As the disease progresses, diseased limbs die back and the trees succumb one to five years later. Fruits produced on diseased limbs ripen two to three weeks earlier than healthy fruits. They are of normal size but of low quality, usually with a bitter taste. In red-skinned cultivars, the fruit surface shows highly pigmented spots with red streaks in the flesh and a pronounced red color around the pit. The PY agent is transmitted by the plum leafhopper Macropsis trimaculata (Kirkpatrick, 1995b). Recent work, employing sequence and computer-simulated RFLP analyses of 16S rDNA, has shown that phytoplasmas associated with PR, PRS and PY diseases are to be regarded as ‘Ca. P. pruni’, and as members of subgroup 16SrIII-A. However, PR phytoplasma may represent a subgroup 16SrIII-A variant whose 16S rDNA differs by a single base substitution in a Sau3AI restriction enzyme site (Davis et al., 2013). Peach yellow leaf roll. An important phytoplasmal disease of peach, called peach yellow leaf roll (PYLR), is reported only from California. This disease was first observed in 1948 in the northern Sacramento Valley (Schlocker and Nyland, 1951). The incidence of PYLR remained relatively low until an epidemic outbreak occurred in the late 1970s and early 1980s when tens of thousands PYLR-infected peach trees were identified and removed (Purcell et al., 1981; Kirkpatrick and Uyemoto, 2011). Symptoms of PYLR are similar, but not identical, to those of X-disease in peach. In contrast to X-disease, PYLR causes chlorosis, downward curling of leaf tips and rolling of leaf margins in mid-summer due to a cork layer deposition (Fig. 2A, B and C). Leaves are normal in size and do not develop extensive shot-holing, typically associated with X-disease. Also, PYLR-infected trees are more severely damaged than those infected by X-disease phytoplasma and decline more rapidly. Yields of PYLR-affected peach trees are dramatically reduced by premature fruit drop. The causal agent is a member of the apple proliferation (AP) phytoplasma group or 16SrX group. Other members of this group include ‘Ca. P. mali’, ‘Ca. P. pyri’ and ‘Ca. P. prunorum’, the causative agents of AP, pear decline (PD) and European stone fruit yellows (ESFY), respectively (Seemüller and Schneider, 2004). Molecular and epidemiological evidence indicates that PYLR and


PD phytoplasmas are closely related. In various studies in which ribosomal and non-ribosomal DNA sequences were employed, the PYLR phytoplasma proved to be indistinguishable from the PD agent (Guerra, 1997; Kison et al., 1997; Blomquist and Kirkpatrick, 2002; Seemüller and Schneider, 2004). Significant differences between the PYLR phytoplasma and an European isolate of the PD agent were observed in the imp gene (Morton et al., 2003). However, the imp gene sequence of a PD isolate occurring in the same area where PYLR is present is not yet available. Work by Guerra (1997) showed that sequences of a region coding for putative transport proteins of PD phytoplasma isolates collected in California and Germany were identical to those of PYLR phytoplasma and shared 97.7% similarity with a PD isolate from Italy. Therefore, the PYLR phytoplasma was regarded as a subtype of the PD agent (Seemüller and Schneider, 2004) and was also designated as PD/PYLR phytoplasma (Guerra, 1997; Blomquist and Kirkpatrick, 2002). The PYLR phytoplasma is transmitted by the pear psyllid Cacopsylla pyricola (Fig. 2D) (Purcell et al., 1981; Blomquist and Kirkpatrick, 2002). Field transmissions to peach trees occurs when psyllids migrate in late autumn from pear orchards to neighboring peach orchards to overwinter there. In addition, the incidence of PYLR is highest in peach trees planted in the proximity of commercial pear orchards, and the incidence usually decreases with increasing distance from pear trees. Furthermore, since there is no evidence that PYLR spreads from peach to peach, pear trees are regarded as the primary pathogen reservoir (Purcell et al., 1981; Kirkpatrick and Uyemoto, 2011). PYLR phytoplasma is a pear pathogen and the only known phytoplasma infecting pear trees in the area where PYLR occurs (Sutter and Yuba counties, California) is the PD phytoplasma. Evidence based on diseased peach orchard surveys and vector transmission studies indicate that C. pyricola is, at best, an inefficient vector of PYLR phytoplasma. But, due in part to its abundance, the vector plays an important role in field spread of the pathogen (Purcell et al., 1981; Purcell, 1996). PYLR phytoplasma infections were also detected in Paraphlepsius species leafhoppers collected in pear orchards in northern California (Blomquist and Kirkpatrick, 2002). However, it remains to be demonstrated if Paraphlepsius species can transmit the pathogen. Although PYLR phytoplasma was experimentally transmitted through grafting to Japanese plum, it has not been reported to occur in nature on this host (Guerra, 1997). Uyemoto et al. (1992, 1999) detected PYLR phytoplasma in diseased almond trees causing brown line and decline of trees grafted on Marianna 2624 rootstock and kernel shrivel in trees grafted on peach rootstocks. The same authors graft-transmitted PYLR from peach to almond on both rootstocks, which developed symptoms similar to those of naturally infected almonds. Attempts to graft-transmit PYLR phytoplasma to apricot, European plum and sweet cherry have failed (Guerra, 1997).

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Outside northern California, pears affected by PD are cultivated in close proximity to peach orchards and, despite the consistent presence of the pear psyllid vector, there has not been a single case of PYLR-diseased peach trees. In the 1980’s, Kirkpatrick (1986) found that 10% of the PYLR symptomatic peach trees reacted positively with antibodies specific to X-disease phytoplasma (Kirkpatrick and Garrot, 1984) produced against an isolate that was transferred to celery from a peach tree with PYLR symptoms (Jensen, 1956). PD was recorded once in British Columbia in 1948 (McLarthy, 1948) and, a decade later, in California. However, peach trees with PYLR symptoms had been observed earlier than the PD phytoplasma or its vector were known to occur in California (Guerra, 1997). Perhaps the coexistence of X-disease and PD phytoplasmas has led to recombination of genetic material or to plasmid exchange, thereby giving rise to an unique subtype of PD agent, i.e., the PYLR phytoplasma, capable of infecting peach trees and other Prunus species, or, perhaps, a vector subpopulation arose which was able of transmitting the PD phytoplasma to peach trees. European stone fruit yellows. European stone fruit yellows (ESFY) is the common name of several economically important phytoplasma disorders of Prunus species in Europe. These disorders have been described under different names such as apricot chlorotic leaf roll, leptonecrosis of Japanese plum, yellows and decline diseases of peach and European plum, Molières disease of sweet cherry, and diseases affecting almond and flowering cherry (P. serrulata) (Lorenz et al., 1994). All of the above mentioned disorders are caused by the same pathogen, ‘Ca. P. prunorum’. This pathogen, taxonomically assigned to subgroup 16SrX-B within the AP group, is phylogenetically closely related to AP, PD, and PYLR phytoplasmas (Seemüller and Schneider, 2004; Lee et al., 2007). However, the ESFY phytoplasma is distinguished from other AP group fruit tree-infecting phytoplamas by RFLP analysis of PCRamplified 16S rDNA sequences employing SspI, BsaAI and RsaI restriction endonucleases (Marcone et al., 2010). The ESFY phytoplasma is a homogeneous taxon at the level of ribosomal DNA sequences. Sequence alignment revealed that the 16S rDNA sequences of several ‘Ca. P. prunorum’ strains from various locations in Europe are identical or nearly identical, showing similarity values between 99.8 and 100% (Seemüller and Schneider, 2004). Also, the pathogen exists as strains that greatly differ in aggressiveness. For example, some strains are avirulent or weakly virulent, inducing mild foliar symptoms and slightly reduced vigor but no tree death, while others are highly virulent and cause severe symptoms and a high mortality rate of affected trees. However, strains that show these pathological differences are indistinguishable at the genetic level with the methods thus far available (Kison and Seemüller, 2001; Marcone et al., 2010).

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Symptoms of ESFY on peach vary with the cultivar. In some white-fleshed cultivars, early fall and moderate rolling, curling, and premature casting of leaves are common (Fig. 2E, F and H). In certain yellow-fleshed cultivars, symptoms resemble those induced by X-disease and PYLR phytoplasmas. Leaves appear normal until mid-summer, then develop chlorotic and necrotic lesions giving rise to a shot-hole condition. As the season progresses, leaf margins roll longitudinally upward, leaf tips curl downward, and leaves turn hard and brittle, develop a reddish blush, and drop prematurely. More specific symptoms concern the enlargement of midribs and major lateral veins owing to corky depositions (Fig. 2G and H). These symptoms are most pronounced in late summer and autumn. Phloem discoloration has also been observed whereas off-season growth and premature break of leaf buds are typical symptoms of winter and early spring months (Fig. 2I, J, K). Vigor and productivity of infected trees are reduced, individual branches die-back, and trees decline within a few years. A disease incidence ranging from 5 to 25% has been recorded in peach orchards in Spain. The most affected were cvs Baby Gold and A-3 (Battle et al., 2012). Surveys in peach orchards in north-central Italy have shown 1-4% disease incidence. The highest infection rates were found in cvs Venus and Super Crimson Gold, grown on GF 677 (P. dulcis x P. persica) rootstocks (Poggi Pollini et al., 2001). In a previous work, in which many established and experimental Prunus rootstocks were examined by graftinoculation, PCR assays and DAPI fluorescence tests in an attempt to identify resistance in stone fruits, ESFY resistance could not be detected (Kison and Seemüller, 2001). Among the most susceptible genotypes were the peach rootstock Montclar and peach seedlings of Rutgers Red Leaf, and Rubira. Moderately to highly susceptible were peach rootstocks Higama and GF 305, hybrids GF 677 and Ishtara [(P. cerasifera x P. persica) x P. salicina] as well as several other peach rootstocks including Myrobalan (P. cerasifera), Marianna GF 8/1 (P. cerasifera x P. munsoniana) and P. insititia St Julien A, St Julien 2 and St Julien GF 655/2. Least susceptible were the P. domestica rootstocks: Achermann’s, Brompton, and P 2175, and P. cerasifera rootstock Myrabi (Kison and Seemüller, 2001). Work by Battle et al. (2012), who examined responses of five rootstocks to ESFY phytoplasma infections using graft-inoculation and nested-PCR assays, reported highest detection rates in Garner (P. persica x P. dulcis), followed by GF 677, Marianna GF 8/1, Cadaman (P. persica x P. davidiana) and Barrier (P. persica x P. davidiana). Similarly to other AP-grouped fruit tree phytoplasmas, ‘Ca. P. prunorum’ is spread by psyllid vectors. Cacopsylla pruni was identified as a vector in various European countries (Carraro et al., 1998, 2001; Jarausch et al., 2001, 2008; Thébaud et al., 2009; Battle et al., 2012). This psyllid is strictly oligophagous on Prunus species, completes one generation per year and overwinters as adult on shelter plants, usually conifers. At the end of winter/early spring,


overwintering adults migrate from shelter plants back to stone fruit trees (primary hosts) for oviposition. Both spring-time and overwintering adults transmit the ESFY agent to healthy Prunus plants. However, the transmission efficiency of spring-matured adults was lower than that of overwintering adults (Carraro et al., 2001; Thébaud et al., 2009). Wild Prunus species play an important role in the epidemiology of ‘Ca. P. prunorum’. P. spinosa (blackthorn), P. cerasifera and P. domestica may harbor the ESFY agent in latent fashion and are the preferred hosts of the vector C. pruni (Carraro et al., 2002; Jaraush et al., 2008). Almond witches’ broom. Almond witches’ broom (AlmWB) is a lethal, destructive disease of almond, widespread in Lebanon and Iran, which killed more than 100,000 almond trees in the past two decades (Choueiri et al., 2001; Abou-Jawdah et al., 2002; Verdin et al., 2003; Foissac et al., 2011). The disease is caused by ‘Ca. P. phoenicium’, which belongs to pigeon pea witches’ broom (PPWB) phytoplasma group or 16SrIX group, subgroup 16SrIX-B (Verdin et al., 2003; Lee et al., 2012). AlmWB phytoplasma is also known to occur in nature on peach, nectarine and rootstock GF 677 (Abou-Jawdah et al., 2002, 2009, 2012; Molino Lova et al., 2011; Salehi et al., 2011). Symptoms of diseased peach and nectarine trees include proliferation of slender suckers, arising mostly from roots or the base of trunks, shortened internodes, small chlorotic or light green, rolled leaves (Fig. 2L), and decline. Diseased trees do not set fruits. The most characteristic symptoms of affected rootstock GF 677 trees are shoot proliferation, witches’ brooms, stunting and dieback (Fig. 2M). Graftinoculation experiments have shown that inocula from AlmWB-affected almond trees induce in peach and nectarine symptoms similar to those observed in naturally infected trees (Abou-Jawdah et al., 2003, 2009; Verdin et al., 2003; Salehi et al., 2006). Although the insect vector is unknown, the rapid spread of the AlmWB phytoplasma in both well-managed and neglected almond orchards suggests involvement of an efficient vector (Abou-Jawdah et al., 2002, 2009). Recently, ‘Ca. P. phoenicium’-related strains were identified in declining almond, peach and nectarine trees in four stone fruit-growing areas of Lebanon. Based on computer-simulated RFLP analyses, these strains were assigned to subgroups 16SrIX-G, -F and -D (Molino Lova et al., 2011). However, it is unknown whether the newly identified strains of the AlmWB phytoplasma differ also in their epidemiological and pathological properties. Other phytoplasmal diseases of peach. European countries: in Italy a phytoplasma of the elm yellows (EY) phytoplasma group or 16SrV group, subgroup 16SrV-B was detected in young branches of a single declining peach tree in northern Italy (Paltrinieri et al., 2006). Again in Italy, Poland and Croatia, single, double or multiple infections with subgroup 16SrI-B, 16SrXII-A of the stolbur phytoplasma group or 16SrXII group, and 16SrX-A phytoplasmas


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Fig. 2. A-C, peach yellow leaf roll: symptoms of yellowing, enlargement of midribs and primary leaf veins and reduced fruit size. In A and C healthy leaves and fruits are on the right end site. D, pear psyllid Cacopsylla pyricola, natural vector of peach yellow leaf roll phytoplasma. E-K, symptoms on peach associated with European stone fruit yellows: leaf rolling and curling (E, F and H), enlargement of midribs and major lateral veins (G and H), phloem discoloration (I), off-season growth (J) and premature break of leaf buds (K). L and M, chlorotic leaves and dwarfed growth on peach (L) and shoot proliferation on rootstock GF 677 (M) associated with ‘Candidatus Phytoplasma phoenicium’ infections. (L courtesy M.G. Zamharir, Department of Plant Disease, Iranian Research Institute of Plant Protection, Tehran, Iran; M courtesy M. Salehi, Plant Protection Research Department, Shiraz, Iran).

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were found in peach, whereas mixed infections with peanut witches’ broom or 16SrII and 16SrXII group phytoplasmas were identified in one peach tree in Serbia. However, the presence of these phytoplasmas was very often not associated with pathological traits (Lee et al., 1995; Paltrinieri et al., 2001, 2006; Krizanac et al., 2010; Cieslinska and Morgas, 2011; Duduk et al., 2012). Jordan: peach trees with symptoms resembling those of X-disease were observed in the Al-Jubiha and Homret Al-Sahen areas. Diseased trees were infected by a phytoplasma assigned to the AY group on the basis of RFLP analysis of PCR-amplified 16S rDNA sequences (Anfoka and Fattash, 2004). Iran: genetically different phytoplasmas belonging to 16SrXII, 16SrII, and clover proliferation or 16SrVI groups were identified in peach trees with yellow canopy, little leaves, leaf rolling and rosetting in the central and northwestern regions of the country. These phytoplasmas are only distantly related to the AlmWB agent ‘Ca. P. phoenicium’, which is also known to infect peach in Iran (Zirak et al., 2010). Azerbaijan: a strain of ‘Ca. P. brasiliense’, a member of the hibiscus witches’ broom group or 16SrXV group, subgroup 16SrXV-A, was identified in one peach tree with yellow canopy in the Guba region. ‘Ca. P. brasiliense’ had not been previously reported to occur outside of Brazil and in hosts other than hibiscus (Balakishiyeva et al., 2011). India: an outbreak of peach tree decline was observed in the northwestern Himalayan region (Thakur et al., 1998). Primary symptoms resembled those of peach X-disease. Disease incidence of 70% occurred in some orchards. The phytoplasmal etiology of the disease was ascertained by graft-inoculation assays, fluorescence microscopy using the DNA-binding fluorochrome 4’-6-diamidino-2-phenylindole (DAPI), and symptoms remission following oxytetracycline injections. In the same country, a phytoplasma of the 16SrV group, subgroup 16SrV-B was reported as the causal agent of a peach yellows disease (PY-In) (Lee et al., 2004). This phytoplasma was closely related to subgroup 16SrV-B members, i.e., cherry lethal yellows (CLY) phytoplasma and the jujube witches’ broom (JWB) agent ‘Ca. P. ziziphi’, which are common in Asian countries. However, PY-In, CLY and JWB phytoplasmas were clearly distinguishable at the ribosomal protein (rpl22 and rps3) and at the secY gene sequence level (Lee et al., 2004). China: a peach yellows disease reported from the Sichuan province of China, has a causal agent showing a 99% 16S rDNA sequence similarity with the PY-In phytoplasma (Huang et al., 2009). More recently, Zhang et al. (2013) have described a red leaf condition of peach (PRL) in Shaanxi province, one of the most important peachgrowing areas of this country. The disease is characterized by reddening and rolling of the leaves, reduced growth, stunting and decline of the affected plants. The PRL agent is a member of the 16SrI group, subgroup 16SrI-C and is most closely related to the clover phyllody phytoplasma.


Argentina: peach trees in the Jujuy province, a recent peach-growing area in the country’s northwest, showed yellowing, reddening, leaf curling, shortened internodes and premature defoliation (Fernández et al., 2013). Diseased trees were infected by a subgroup 16SrIII-B phytoplasma which, by computer-simulated RFLP analyses, proved indistinguishable from the clover yellow edge (CYE) agent. Bolivia: a disease characterized by symptoms similar to those of PYLR was found in a peach orchard in the Santa Cruz province. A strain of the ‘Ca. P. australiense’, a member of the 16SrXII group, subgroup 16SrXII-B, was associated with the disease (Jones et al., 2005). Canada: two phylogenetically different phytoplasmas were identified in diseased peach accessions growing at the Canadian Clonal Genebank (Harrow, Ontario). Trees showing symptoms of decline, yellowing and reddening were infected by a ‘Ca. P. fraxini’-related strain, a member of the ash yellows phytoplasma group or 16SrVII group, subgroup 16SrVII-A. Trees affected by a peach rosette-like disease harbored a ‘Ca. P. asteris’-related strain which, on the basis of computer-simulated RFLP analysis, was assigned to a new subgroup (16SrI-W) within the AY group. Some trees were infected with both phytoplasmas (Zunnoon-Khan et al., 2010; Arocha-Rosete et al., 2011a, 2011b). Single or double infections with the two phytoplasmas were detected in specimens of the leafhopper Graminella nigrifrons collected in the Canadian Clonal Genebank orchards (Arocha-Rosete et al., 2011b). Therefore, G. nigrifrons was regarded as a potential vector of the subgroup 16SrI-W and 16SrVII-A phytoplasmas infecting peach in Canada. DETECTION

Prior to the introduction of DNA-based methods into phytoplasmology, mainly microscopic methods and, to a lesser extent, serological assays were employed for detecting phytoplasma infections in peach trees (MacBeath et al., 1972; Kirkpatrick et al., 1975, 1995a; Jones et al., 1974a, 1974b; Kim and Slack, 1978; Douglas, 1986; Kirkpatrick, 1991; Lederer and Seemüller, 1992; Poggi Pollini et al., 1993). Microscopic methods, which included transmission electron microscope (TEM) observations and DAPI fluorescence tests are unfit for epidemiological studies which require a quick identification of plant reservoirs and insect vectors for a given phytoplasma. In addition, they do not allow pathogen identification and are of little use when phytoplasma populations are very low and unevenly distributed in the plant host organs, as it is often true for peach. By contrast, DAPI fluorescence is simple, rapid, sensitive, less expensive than TEM, and enables microscopic screening of large numbers of samples. Using DAPI fluorescence tests, Douglas (1986) monitored the distribution of X-disease phytoplasma in diseased peach and


chokecherry trees during two growing seasons, finding a good correlation between symptom severity and extent of phytoplasma invasion. However, in chokecherry, pathogen invasion occurred earlier and was more extensively distributed than that observed in peach trees. Polyclonal antisera and monoclonal antibodies have been produced against the X-disease phytoplasma and were successfully used in ELISA, ISEM and immunofluorescence staining tests to detect and/or follow the multiplication of the pathogen in several plant hosts, including peach and chokecherry, as well as in the leafhopper vectors C. montanus and P. irroratus (Sinha and Chiykowski, 1984, 1986, 1990; Kirkpatrick and Garrot, 1984; Jiang et al., 1989; Guo et al., 1996, 1998). However, due to lack of specificity and sensitivity, serological methods are not widely employed for diagnostic purposes. Also, serological procedures are lacking for other major phytoplasmas infecting peach although an immunodominant membrane protein gene of ‘Ca. P. prunorum’ was isolated and expressed in Escherichia coli with the aim of raising highly specific monoclonal antibodies for diagnostic purposes (Mergenthaler et al., 2001). DNA-based methods were integrated in phytoplasma research when protocols for isolation and cloning phytoplasmal DNA were developed (for reviews see Kirkpatrick, 1991, 1997; Seemüller et al., 1998a). Cloned fragments of chromosomal DNA of X-disease phytoplasma and to, a lesser extent, of the PYLR agent, were used in dot and Southern blot hybridization assays as probes to detect phytoplasmal infections in field-collected diseased peach trees and in insect vectors. These assays were also employed in epidemiological studies to identify plant reservoirs and putative insect vectors of the above mentioned phytoplasmas (Kirkpatrick et al., 1987, 1990; Kirkpatrick and Purcell, 1987; Guerra, 1997; Uyemoto et al., 1998; Blomquist and Kirkpatrick, 2002). The major problem in the detection of phytoplasma infections by DNA hybridization assays especially in woody hosts, is the low sensitivity of the probes. Usually, no hybridization signals are obtained when phytoplasmas cannot be detected by DAPI fluorescence tests (Seemüller and Kirkpatrick, 1996). Currently, PCR technology is the most widely used method and has nearly completely replaced all other assays for phytoplasma detection. It offers several advantages including its versatility, relative simplicity, specificity and high sensitivity. Universal phytoplasma primers as well as group- and pathogen-specific primers have been developed, targeting ribosomal or non-ribosomal DNA sequences. Primers amplifying rDNA sequences are being used extensively. The sensitivity of detection can be increased by the use of nested PCR which is currently one of the most sensitive means, suitable for detecting extremely low-titer infections in woody plants. Information on primer sequences and primer combinations for detection of the various peach-infecting phytoplasmas can be found elsewhere (see reviews Seemüller et al., 1998b; Verdin et al.,

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2003; Hadidi et al., 2011). For successful detection, template DNA is extracted from leaf petioles, midribs or from phloem tissues prepared from stems or roots as described by Ahrens and Seemüller (1994). The amount of phytoplasmal DNA in total DNA extracted from infected plants or insects can be increased using phytoplasma-enrichment procedures (Ahrens and Seemüller, 1992; Kirkpatrick et al., 1995b). Real-time PCR assays using ribosomal or non-ribosomal primers have been employed for increased detection and quantification of ‘Ca. P. prunorum’ infections in various host plants and in the psyllid vector C. pruni (for review see Marcone et al., 2010). However, these assays have not yet been employed to examine ESFY-affected peach trees. A real-time PCR assay using ribosomal primers in combination with SYBR green stain was developed for sensitive detection of PYLR phytoplasma. This assays allowed detection of the PYLR agent in dormant buds of diseased peach trees (Sudarshana et al., 2011). Although the detection threshold of real-time PCR is largely similar to that of nested PCR, real-time PCR assays greatly reduce the risk of false positives arising from laboratory contamination with ‘amplicons’ from previous amplifications. In certification or quarantine programs, detection typically relies on biological indexing using sensitive woody indicators such as peach seedlings, GF 305 and/or cv. Elberta (Adams et al., 2001; Rowhani et al., 2005; Thompson et al., 2011; Polák et al., 2012). Although laboratory-based assays (serological and nucleic acid-based methods) have been widely adopted and integrated in certification and quarantine programs over the last years, they have not replaced biological assays. This is because biological indexing is still the method of choice for detection of unknown or uncharacterized graft-transmissible agents including phytoplasmas, and/or for comparing phytoplasma strains within a given taxon, which differ in aggressiveness. Since phytoplasmas differentially colonize plant tissue throughout the season, variations in transmission rates following graft-inoculations have been observed in many instances. This requires the determination of the best timing and tissues to be used for testing. Work by Uyemoto and Luhn (2006) has shown that for X-disease phytoplasma infecting sweet cherry, the transmission rate was 10% in spring (June) whereas it reached 64% in summer (August), irrespective of the age of the shoots used as inoculum. Besides cherry sources, graft-transmissions using scion buds from X-disease- and PYLR-affected peach trees, collected in June and August, respectively, yielded high rates of transmission (55% for both diseases). Similar results were obtained by Suslow and Purcell (1982) and Kirkpatrick et al. (1990), who demonstrated that the ability of the leafhopper vector C. montanus to acquire the X-disease phytoplasma from diseased sweet cherry trees correlated with the seasonal variation of the phytoplasma titer occurring in the leaves. X-disease phytoplasma in spring initially colonized and multiplied

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in the fruit peduncles rather than in the leaves reaching the highest concentration in peduncles when fruits were mature. Later, after ripening and senescence of the fruits, the pathogen persisted and continued to increase in the leaves. Therefore, the highest rates of infective C. montanus individuals were recorded in August-September. Rosenberger and Jones (1977) reported on the seasonal variations in transmission of X-disease phytoplasma in peach and chokecherry trees following graft-inoculations. Highest transmission rates were obtained in June-August (82-100%) and May-June (100%) for peach and chokecherry, respectively. The pathogen was also transmitted during winter, although at a low percentage in peach. Work by Seemüller et al. (1998c) showed that the ESFY phytoplasma can be efficiently transmitted by grafting of stem scions throughout the year including winter because this pathogen persists in the aerial parts of peach trees as well as of other Prunus species during the dormant season. It is also present at the root level throughout the year and the phytoplasma concentration is higher in the roots than in the stem of the same genotype. In France, Jarausch et al. (1999) monitored the colonization pattern of the ESFY phytoplasma in apricot, Japanese and European plum trees using DAPI fluorescence and PCR assays. Although the pathogen was detected throughout the year in the aerial parts, increased colonization (titers) in diseased trees occurred during summer (July-September). CONCLUSIONS

Phytoplasmal diseases are very frequently present where peach trees are grown. However, they differ considerably in the type of the associated phytoplasma(s), insect vector relationship and geographic distribution. X-disease, PR, PRS, PY, PYLR, ESFY and AlmWB are induced by specific phytoplasmas, known to infect in nature mainly Prunus spp. or only peach. The pathological relevance of these Prunus-specific pathogens, which belong to either the X-disease, AP or PPWB groups, has been demonstrated by countless diagnostic examinations and inoculation experiments. Phytoplasmas of other phylogenetic groups, which are known to infect a wide range of plant hosts, have been identified in declining peach trees in several fruit-growing areas of the world. However, the pathological relevance of these ‘non-peach’ phytoplasmas needs to be further investigated as their detection is mainly based on highly sensitive nested PCR assays but no information on pathogenicity is available. ACKNOWLEDGEMENTS

We are particularly indebted to A.H. Purcell, M. Salehi, S.W. Scott and M.G. Zamharir, each of whom gave us and/or offered many more of their photographs than we needed.

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