Mapping the spread of apricot chlorotic leaf roll (ACLR) in southern France and implication of Cacopsylla pruni as a vector of European stone fruit yellows (ESFY) phytoplasmas

An epidemiological study on European stone fruit yellows (ESFY) phytoplasmas infecting Prunus fruit trees was carried out from 1994 to 2000 in Languedoc-Roussillon (southern France). The spread of the disease was monitored for 7 years by visual observation of symptoms and by PCR detection of the phytoplasma in an experimental orchard planted with apricot hybrid seedlings. This indicated that aerial vectors were responsible for disease spread, and that transmission rates were low at the beginning of the spread. Seventy thousand homopteran insects were captured within and in the surroundings of highly ESFY-infected apricot orchards, of which about 10 000 were used in PCR and nested-PCR assays with universal ribosomal and ESFY-specific nonribosomal primers to detect ESFY phytoplasmas. The other insects were confined in cages for trials of transmission to test plants. ESFY phytoplasmas could not be detected by PCR in any of the leafhopper species captured but could be detected in the psyllid Cacopsylla pruni caught on Prunus domestica and Prunus cerasifera rootstock suckers of apricot trees and on Prunus spinosa . Nested PCR revealed ESFY phytoplasmas in one individual of the deltocephalid Synophropsis lauri captured on an apricot tree. Transmission trials confirmed the role of Cacopsylla pruni as the ESFY phytoplasma vector in France. When apricot seedlings were used as bait plants from April to November during two consecutive years, no natural transmission could be demonstrated. However, one out of 50 apricot seedlings left for the whole year in the orchard became infected. An early spring ESFY infection is in agreement with both the natural transmission results and the life cycle of Cacopsylla pruni .     

Phytoplasma transmission by in vitro graft inoculation as a basis for a preliminary screening method for resistance in fruit trees

In vitro grafting was tested as a technique for inoculating Prunus rootstock Prunus marianna GF 8-1 with European stone fruit yellows (ESFY) phytoplasmas and apple rootstock Malus pumila MM106 with apple proliferation (AP) phytoplasmas. In vitro shoot cultures of ESFY-infected Prunus marianna GF 8-1 and AP-infected Malus pumila MM106 were used as graft inoculum to transmit the phytoplasmas to the respective healthy rootstock. Phytoplasma transmission was assessed after a graft contact of 1, 2 or 3 months. Healthy autografts were used as controls to monitor parameters of in vitro grafting. Successful graft union formation ranged from 58 to 79% irrespective of the plant species and the sanitary state of the graft. Pathogen-specific polymerase chain reaction (PCR) was used to test the inoculated rootstocks for the presence of ESFY and AP phytoplasmas, respectively. The rate of ESFY phytoplasma transmission in successful Prunus -grafts increased from 69 to 94% with the time of contact. AP phytoplasma transmission to Malus occurred in 80 to 97% of successful grafts. To our knowledge this is the first report of phytoplasma transmission by grafting in vitro . The results provide a good basis for the establishment of a preliminary in vitro screening method for phytoplasma resistance in Prunus and Malus .     

Distribution of ‘Candidatus Phytoplasma prunorum’ and its vector Cacopsylla pruni in European fruit‐growing areas: a review

European stone fruit yellows (ESFY) is an EU‐listed I/AII disease affecting Prunus spp. caused by ‘Candidatus Phytoplasma prunorum’. This paper reports the results from a systematic literature review approach that sought to determine the geographic distribution of ‘Ca. Phytoplasma prunorum’ in European fruit‐growing areas. Evidence for the presence of the phytoplasma was found for 15 of the 27 EU countries. It is prevalent in the most important stone fruit production areas of Central and Southern Europe, where it causes substantial impact in apricots (Prunus armeniaca), Japanese plums (P. salicina) and peaches (P. persica). In Northern European areas where these hosts are not produced, it is occasionally found on tolerant species (P. domestica). However, because surveys of the disease status of tolerant hosts are not performed, it remains unclear whether the pathogen is absent in Northern Europe or survives in tolerant cultivated or wild hosts. No reports of ESFY were found from the southernmost part of Europe: Portugal, Spain (Andalucia, Castile–La Mancha), Italy (Sicily, Puglia), Greece (Crete), Cyprus and Malta. This may be explained by the absence of the favoured wild hosts of the vector. Moreover, it remains unclear if the vector finds suitable conditions for aestivation and overwintering in these regions.     

Detection and characterization of phytoplasmas in diseased stone fruits and pear by PCR-RFLP analysis in Turkey

During the late summer-early autumn of 2002, surveys were carried out in Turkey to determine the presence of phytoplasma diseases in fruit trees. Phytoplasmas were detected and characterized by PCR-RFLP analysis and TEM technique in stone fruit and pear trees in the eastern Mediterranean region of the country. Six out of 24 samples, including almond, apricot, peach, pear and plum, gave positive results in PCR assays. RFLP analysis usingSspI andBsaAI enzymes of PCR products obtained with primer pair f01/r01 enabled identification of the phytoplasmas involved in the diseases. Stone fruit trees, including a local apricot variety (‘Sakıt’) and a pear sample, were found to be infected with European stone fruit yellows (ESFY, 16SrX-B) and pear decline (PD, 16SrX-C) phytoplasmas, respectively. This is the first report in Turkey of PD phytoplasma infecting pear and of ESFY phytoplasma infecting almond, apricot, myrobalan plum and peach; ESFY phytoplasma infecting Japanese plum was previously reported. http://www.phytoparasitica.org posting July 21, 2005.     

Detection and Indentification of European Stone Fruit Yellows and Other Phytoplasmas in Wild Plants in the Surroundings of Apricot Chlorotic Leaf Roll-affected Orchards in Southern France

Between 1994 and 1998 a field study was conducted to identify plant hosts of the European stone fruit yellows (ESFY) phytoplasma in two apricot growing regions in southern and southwestern France where the incidence of apricot chlorotic leaf roll was high. A total of 431 samples from 51 different plant species were tested for the presence of phytoplasmas by PCR using universal and ESFY-specific primers. ESFY phytoplasma was detected in six different wild growing Prunus species exhibiting typical ESFY symptoms as well as in symptomless dog rose bushes (Rosa canina), ash trees (Fraxinus excelsior) and a declining hackberry (Celtis australis). The possible role of these plant species in the spread of ESFY phytoplasma is discussed. PCR-RFLP analysis of ribosomal DNA amplified with the universal primers was carried out to characterize the other phytoplasmas found. Thus, elm yellows phytoplasma, alder yellows phytoplasma and rubus stunt phytoplasma were detected in declining European field elm trees (Ulmus carpinifolia Gled), in declining European alder trees (Alnus glutinosa) and in proliferating Rubus spp. respectively. The presence of rubus stunt phytoplasma in great mallow (Malva sylvestris) and dog rose was demonstrated for the first time. Furthermore, the stolbur phytoplasma was detected in proliferating field bindweed (Convolvulus arvensis) and a previously undescribed phytoplasma type was detected in red dogwood (Cornus sanguinea). According to the 16S rDNA-RFLP pattern this new phytoplasma belongs to the stolbur phytoplasmas group.     

First report of <Emphasis Type="Italic">Candidatus</Emphasis> Phytoplasma prunorum infecting almonds in Tunisia

Candidatus Phytoplasma prunorum was detected for the first time in almond (Prunus dulcis Mill.) cv. ‘Abiod’ in Tunisia. Infected trees showed emergence of new growth during dormancy and leafed out before flowers opened in addition to early defoliation in summer. Phytoplasma was detected by nested polymerase chain reaction (PCR) using universal phytoplasma primer pairs P1/P7 and F2n/R2. A band with expected size was observed in samples collected from five symptomatic, but not symptomless almond trees. PCR products (1.2 kbp) were used for restriction fragment length polymorphism (RFLP) analysis after digestion with endonucleases RsaI and SspI. RFLP patterns obtained were similar to those reported previously for the European stone fruit yellows (ESFY, 16SrX-B). Identification has been further confirmed by PCR using ESFY specific primer pairs (ECA1/ECA2). This is the first report of Ca. Phytoplasma prunorum infecting almonds in Tunisia.     

Untersuchungen zur Europäischen Steinobstvergilbung (ESFY) in Deutschland

From 2003 to 2007 surveys have been conducted in different stone fruit growing regions in southwest Germany to detect European stone fruit yellows (ESFY) disease in Germany. Samplings have been done regularly in selected reference orchards in the regions Neuwieder Becken, Rheinhessen, Vorderpfalz and Südpfalz in summer on trees showing ESFY typical symptoms as well as on branches of trees with unspecific symptoms. All samples have been analysed by PCR for infection with Candidatus Phytoplasma prunorum. The phytoplasma could be detected in all investigated regions on the cultivated Prunus species P. armeniaca, P. persica and P. domestica. No infection was found in wild Prunus species. The main spread of the disease appeared on apricot while peach and European plum were less affected. A good correlation between symptoms and molecular detection of the pathogen could be shown for the typical symptoms in summer and winter for apricot as well as for peach. During regular psyllid captures in the reference orchards the population dynamics of Cacopsylla pruni could be described in southwest Germany for several years. By PCR-testing all collected insects individually a yearly natural infection rate of about 1–2% of all individuals of C. pruni could be calculated.     

Fast and sensitive on-site isothermal assay (LAMP) for diagnosis and detection of three fruit tree phytoplasmas

Over the years, real-time PCR outflanked endpoint PCR in phytopathogen diagnostics, mainly because of the increase in sensitivity and timesaving aspects of the technique. However, a time consuming 16S rRNA-based nested PCR method is still the gold standard for phytoplasma diagnosis. This is also the case for phytoplasma detection in Malus, Pyrus and Prunus, the three main host plants of apple proliferation (AP), pear decline (PD) and European stone fruit yellows (ESFY) phytoplasma, respectively. The last decade, loop-mediated isothermal amplification (LAMP) (Notomi et al. 2000) is gaining a lot in significance and is also for phytoplasmas expected to become a widely used reliable diagnostic tool. High specificity and sensitivity which also requires a less stringent need for DNA purification, and the short analysis time and the limited equipment requirements makes the LAMP method a fast and affordable alternative with great point-of-care diagnostic potential. In this paper, we present a LAMP primer set for the ribosomal group 16SrX, containing the important fruit tree phytoplasmas AP, PD and ESFY. The primers were developed and validated for fast and sensitive detection and general use for diagnosis. We foresee that the LAMP technique will also have its application in on-site diagnosis of the fruit tree phytoplasmas during inspections and surveys.     

Incidence and distribution of ‘Candidatus Phytoplasma prunorum’ and its vector Cacopsylla pruni in Spain: an approach to the epidemiology of the disease and the role of wild Prunus

‘Candidatus Phytoplasma prunorum’ is the causal agent of the European stone fruit yellows (ESFY) disease. This phytoplasma affects wild and cultivated species of Prunus to different degrees, depending on their susceptibility. ‘Candidatus Phytoplasma prunorum’ is present in the four regions of Spain surveyed in this study (Aragon, Catalonia, Extremadura and Valencia) with a variable incidence. Results showed that ‘Ca. Phytoplasma prunorum’ was detected in all of the cultivated Prunus species studied, except P. avium and P. dulcis, and was widespread in Spain. The most affected species was P. salicina, with symptoms including early bud break and blooming, leaf curling and yellowing, collapse, and a major decrease in production. In some plots in the Baix Llobregat area of Barcelona province (Catalonia), the incidence of ESFY on P. salicina was as high as 80%. The insect vector, Cacopsylla pruni, was present in all four of the regions studied, with the highest captures in yellow sticky traps in Catalonia on P. mahaleb and in Extremadura in peach orchards. In Baix Llobregat, large populations of C. pruni were present on infected P. mahaleb bushes, and with high infection rates. This was a key factor in the local pathogenic cycle that caused a major ESFY outbreak in the nearby P. salicina orchards. In the Ebro valley (Lleida and Aragon) and Valencia, the surveys showed very low incidences of the disease and low C. pruni populations.     

Identifying risk factors for European stone fruit yellows from a survey

ABSTRACT European stone fruit yellows (ESFY) is becoming a major economic problem for Prunus growers in Europe. The causal agent ("Candidatus Phytoplasma prunorum") and its vector (Cacopsylla pruni) have been identified, but the present knowledge of the risk factors for this disease relies, at best, on specific experiments. To assess the relative significance of several factors correlated with ESFY incidence in the field, an exhaustive survey was performed on apricot and Japanese plum orchards in the Crau plain (France). After a preliminary multivariate exploration of the data, we used a logistic regression model to analyze and predict the cumulative number of diseased trees on the basis of a set of quantitative (age, planting density, and area of the orchard) and categorical variables (species, cultivar, and rootstock). Because of the nature of the data, we used an overdispersed binomial model and we developed a parametric bootstrap procedure based on the beta-binomial distribution to obtain confidence intervals. Our results indicated that the age, species, and cultivar of the scion were the major factors explaining the observed number of diseased trees. The planting density and the rootstocks used in the zone under study were less significant, and the area of the orchard had no effect. The residuals of the model showed that some explanatory variables had not been taken into account, because part of the remaining variability could be explained by a grower effect. The spatial distribution of the residuals suggested that one of the reasons for this grower effect was the correlation between orchards closer than 100 m, possibly caused by the flight behavior of infectious vectors.     

Transmission Characteristics of the European Stone Fruit Yellows Phytoplasma and its Vector Cacopsylla Pruni

A study was carried out on the transmission parameters of the European stone fruit yellows phytoplasma by the vector Cacopsylla pruni. In the greenhouse, using groups of psyllids, the minimum acquisition period was 2–4 days, the minimum latent period 2–3 weeks and the minimum inoculation period 1–2 days. The vectors retained infectivity until their death. Under natural conditions retention of infectivity in C. pruni lasts through the winter and the following spring, when the overwintering insects reach the stone fruit trees, they are already infected and infective. The research shows that the vector C. pruni transmits the European stone fruit yellows phytoplasma in a persistent manner.     

Detection ofplum pox virus in ornamentalPrunus cerasifera

Plum pox virus (PPV) is the causal agent of sharka disease. It is a serious threat for temperate fruit, mainly apricots, plums and peaches. In order to study the ability ofPPV to infect wild and ornamentalPrunus species, several wild, native ornamental stone fruits and weeds were analyzed as possible reservoirs ofPPV. Five species of ornamental stone fruits and 24 species of weeds were evaluated between 2000 and 2004. The virus was not found in the weeds but was detected in one species of ornamental stone fruit, purple cherry-plums (Prunus cerasifera Pissardii). ThePPV strain M was identified by DASI-ELISA and confirmed by IC-RT-PCR. Additionally, mealy plum aphid (Hyalopterus pruni) was determined as a vector ofPPV inP. cerasifera. This is the first report on the reservoir potential of ornamental stone fruit trees and weeds forPPV in Turkey. http://www.phytoparasitica.org posting July 19, 2006;     

Genetic comparison of the peach yellow leaf roll agent with European fruit tree phytoplasmas of the apple proliferation group

Restriction fragment length polymorphism (RFLP) analysis of PCR-amplified ribosomal DNA and Southern blot hybridization using cloned chromosomal DNA fragments from the apple proliferation (AP) phytoplasma as probes were used to investigate the genetic relationship of the California peach yellow leaf roll (PYLR) agent with phytoplasmas causing fruit tree diseases in Europe. This comparison showed that the California PYLR phytoplasma is closely related to apple proliferation (AP), pear decline, and European stone fruit yellows phytoplasmas and that it is a member of the phylogenetic AP group. The PYLR agent could clearly be distinguished from the AP and European stone fruit yellows phytoplasmas by Southern blot hybridization with DNA fragments from the AP phytoplasma and by RFLP analysis of ribosomal DNA employing Ssp I, Bsa AI, and Rsa I restriction endonucleases. However, the PYLR phytoplasma was indistinguishable from the pear decline agent by RFLP analysis of PCR-amplified ribosomal DNA.     

Characterisation of a 16SrII phytoplasma strain associated with bushy stunt of hawkweed oxtongue (Picris hieracioides) in south-eastern Serbia and the role of the leafhopper Neoaliturus fenestratus (Deltocephalinae) as a natural vector

A 2-year study of host association, molecular characterisation and vector transmission of a phytoplasma related to the 16SrII group in a vineyard of south-eastern Serbia was conducted. Grapevine, eight common weeds and 31 Auchenorrhyncha species were collected and analysed for phytoplasma presence. PCR-RFLP analyses of the 16S rRNA gene identified the presence of a new strain of phytoplasma related to the 16SrII group in P. hieracioides with symptoms of stunting or bushy stunting. Grapevine samples, all without symptoms, were negative for phytoplasma presence. Plants of Erigeron annuus, Cynodon dactylon, Daucus carota and P. hieracioides, either exhibiting symptoms of yellowing or without symptoms, were positive for the presence of stolbur phytoplasma. Among the tested cicada species, seven were infected with phytoplasmas from the aster yellows group, two with stolbur phytoplasma, two with 16SrII phytoplasma, and one with the 16SrV-C phytoplasma subgroup. The phytoplasma strain of the 16SrII group was recorded in approximately 50?% of the collected leafhopper species Neoaliturus fenestratus and in a few specimens of the planthopper Dictyophara europaea. The vector status of N. fenestratus was tested using the second generation of the planthopper in two separate transmission trials with P. hieracioides and periwinkle seedlings. In both tests, the leafhopper successfully transmitted 16SrII phytoplasma to exposed plants, proving its role as a natural vector of this phytoplasma in Europe. A finer molecular characterisation and phylogenetic relatedness of the 16SrII phytoplasma strain by sequence analyses of the 16S rRNA and ribosomal protein genes rpl22-rps3 indicated that it was most closely related to the 16SrII-E subgroup.     

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

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.     

A phytoplasma closely related to the pigeon pea witches'-broom phytoplasma (16Sr IX) is associated with citrus huanglongbing symptoms in the state of São Paulo, Brazil

In February 2007, sweet orange trees with characteristic symptoms of huanglongbing (HLB) were encountered in a region of S?o Paulo state (SPs) hitherto free of HLB. These trees tested negative for the three liberibacter species associated with HLB. A polymerase chain reaction (PCR) product from symptomatic fruit columella DNA amplifications with universal primers fD1/rP1 was cloned and sequenced. The corresponding agent was found to have highest 16S rDNA sequence identity (99%) with the pigeon pea witches'-broom phytoplasma of group 16Sr IX. Sequences of PCR products obtained with phytoplasma 16S rDNA primer pairs fU5/rU3, fU5/P7 confirm these results. With two primers D7f2/D7r2 designed based on the 16S rDNA sequence of the cloned DNA fragment, positive amplifications were obtained from more than one hundred samples including symptomatic fruits and blotchy mottle leaves. Samples positive for phytoplasmas were negative for liberibacters, except for four samples, which were positive for both the phytoplasma and 'Candidatus Liberibacter asiaticus'. The phytoplasma was detected by electron microscopy in the sieve tubes of midribs from symptomatic leaves. These results show that a phytoplasma of group IX is associated with citrus HLB symptoms in northern, central, and southern SPs. This phytoplasma has very probably been transmitted to citrus from an external source of inoculum, but the putative insect vector is not yet known.     

Production of Monoclonal Antibodies against Apple Proliferation Phytoplasma and their Use in Serological Detection

Two monoclonal antibodies were obtained against the apple proliferation phytoplasma that provide easy, rapid, specific and sensitive serological detection. They reacted specifically by using ELISA and immunofluorescence techniques with apple proliferation-infected periwinkles and apple trees from different regions in northern Italy and Slovenia, but not with several other phytoplasma isolates. We did not observe any monoclonal antibody reaction even using phytoplasmas belonging to the same phylogenetic group such as European stone fruit yellows and pear decline. Two serological techniques, immunofluorescence and ELISA, were compared with DAPI staining and PCR. From July until leaf fall ELISA was as sensitive as PCR but was more rapid and convenient than PCR; immunofluorescence was useful for specific detection of apple proliferation phytoplasma on roots throughout the year. Serological techniques could be conveniently applied in the roots, stems and leaves of apple trees depending on specific phenological stages of the plants.     

Detection and molecular characterization of a phytoplasma affecting Prunus persica L. in Jujuy, Argentina

Peach (Prunus persica L.) plants with symptoms of yellowing, reddening, curling and leaf necrosis, premature defoliation and internode shortening were observed in production fields in Jujuy province (Argentina). A phytoplasma was detected by PCR using the universal primer pairs P1/P7 and R16F2n/R16R2 in all the symptomatic samples analysed. The RFLP profile of PCR products, amplified with R16F2n/R16R2 primers, shows that this phytoplasma, named Argentinean Peach Yellows (ArPY), belongs to subgroup 16Sr III-B. The phylogenetic analysis of the 1244 bp 16S rDNA cloned sequence, grouped the ArPY phytoplasma into the X-disease group with a closer relationship with CFSD, PssWB and ChTDIII phytoplasmas. This is the first report of a phytoplasma infecting peach trees in Argentina.     

Development of a multiplex TaqMan real-time RT-PCR assay for simultaneous detection of Asian prunus viruses, plum bark necrosis stem pitting associated virus, and peach latent mosaic viroid

Asian prunus viruses (APV 1, APV 2 and APV 3), Plum bark necrosis stem pitting associated virus (PBNSPaV) and Peach latent mosaic viroid (PLMVd) are pathogens that infect Prunus species. A single-tube multiplex, TaqMan real-time RT-PCR assay was developed for the simultaneous detection and identification of these pathogens. The protocol includes amplification and detection of a fluorogenic cytochrome oxidase gene (COX) as an internal control. The results of the multiplex TaqMan RT-PCR assay correlated with those from conventional RT-PCR, with a 10-fold increase in sensitivity in the multiplex real-time format. The efficiency and accuracy of the assay was evaluated by testing stone fruit trees from positive control collections and several orchard locations. Several mixed infections of target pathogens were detected in peach orchard samples. This assay is simple, rapid and cost-effective and can be used by quarantine and certification programs where numerous stone fruit trees need to be tested for these pathogens.     

Detection of phytoplasma by polymerase chain reaction of insect feeding medium and its use in determining vectoring ability

A polymerase chain reaction (PCR)-based method was developed for the detection of phytoplasma in insect feeding medium (sucrose). A correlation was established between the transmissibility of Flavescence dorée phytoplasma in the experimental leafhopper vector Euscelidius variegatus and its detection by PCR in the insect feeding medium. However, phytoplasma were detected in the insects' bodies 3 weeks before they began to transmit. Hence, PCR assays of the sucrose medium reflected phytoplasma vectoring ability probably by detecting it in the insect saliva, whereas detection of phytoplasma in the insect's body did not identify it as a vector. The assay was applied to two field-collected leafhoppers suspected of being phytoplasma vectors in Israel (Orosius albicinctus and Anaceratagallia laevis). The presence of phytoplasma in the body of specimens of the latter species was assayed by PCR in 1999. Phytoplasmas were detected in insects' bodies throughout the year, with no specific seasonal pattern. In the saliva, however, no phytoplasma could be detected in the autumn. This seasonal pattern supported the validity of the feeding-medium tests and their correlation to the insect's ability to transmit phytoplasma. Transmission assays indicated, to our knowledge for the first time, that O. albicinctus and A. laevis are vectors of phytoplasma in Israel. A simple PCR-based assay is thus provided, circumventing the need for tedious biological assays and enabling epidemiological studies of phytoplasma transmissibility on a large scale.