European Stone Fruit Yellows Phytoplasma Research Articles

Evaluation of susceptibility to Plum pox virus and European stone fruit yellows phytoplasma in Japanese plum and pluot cultivars

Evaluation of susceptibility to Plum pox virus and European stone fruit yellows phytoplasma in Japanese plum and pluot cultivars

Development of three duplex real-time RT-PCR assays for the sensitive and rapid detection of a phytoplasma and five viral pathogens affecting stone fruit trees

Three duplex real-time reverse-transcription polymerase chain reaction (real-time RT-PCR) assays based on TaqMan chemistry, were developed for the simultaneous detection and specific quantification of apple chlorotic leafspot virus (ACLSV), plum pox virus (PPV), prunus necrotic ringspot virus (PNRSV), prune dwarf virus (PDV), peach latent mosaic viroid (PLMVd) and the European stone fruit yellows (ESFY) phytoplasma, which are considered among the most important pathogens affecting stone fruit trees. The quantitative RT-PCR (RT-qPCR) assays were optimized using RNA transcripts (linearized plasmid was used for the assay optimization of the ESFY phytoplasma) of known concentrations. No differences in sensitivity were recorded between the duplex and singleplex RT-qPCR assays. The amplification efficiency of the duplex assays reached 91.1–95.8%, while the linear range of quantification was from 20 to 2 × 107 RNA/linearized plasmid transcripts for PLMVd and ESFY phytoplasma, 40 to 4 × 107 RNA transcripts for ACLSV, PPV and PDV, and 102 to 108 RNA transcripts for PNRSV, respectively. The duplex RT-qPCR assays, which were validated using both characterized isolates from all pathogens and field samples from Prunus species in Northern Greece, exhibited a broad detection range. Overall, the developed methods comprise useful tools that could be applied for the simultaneous and reliable detection of graft-transmissible pathogens in certification programs of Prunus spp.

Phytoplasma infection status survey in plum psyllid (Cacopsylla pruni) population

European Stone Fruit Yellows (ESFY) phytoplasma disease causes an increasing amount of damage. This is especially true to the Gönci growing region. The insect vector of the disease, which has been shown to have a vector role during transmission experiments, is the plum psyllid (Cacopsylla pruni). In 2018, during the swarming period of plum psyllid, in 3 different settlements (Boldogkőváralja, Nagyvárad and Bekecs) 265 psyllidswere collected, from which 165 were plum psyllid. Molecular biology studies showed 106 individuals infected with phytoplasma, of which 20 were males and 86 were females.

First report of Candidatus Phytoplasma prunorum, the European stone fruit yellows phytoplasma on peach trees on the territory of Canton of Geneva, Switzerland

In recent years, ‘Ca. P. prunorum’, the agent of ESFY was reported from several apricot orchards of Canton of Wallis, the main apricot production region in Switzerland (Genini and Ramel, 2004). The psyllid vector Cacopsylla pruni was also found in several locations in the Lake Geneva area (Ackermann et al., 2006). The presence of the disease and of its proven vector C. pruni at the eastern part of the Lake Geneva area, as well as the existing risk of dissemination of ESFY to other stone fruit orchards along the Lake Geneva are the reasons for which ESFY needs to be studied further. ESFY and its possible dissemination through C. pruni on the territory of Canton of Geneva, bordering France, has never been yet studied. In 2016, visual observations were conducted in several stone fruit orchards near Geneva. Plant material was obtained from peach trees, displaying some of the typical symptoms such premature leaf colouration, leaf-roll, tree decline (Sabaté et al., 2015), in the autumn, when the concentration of the phytoplasma in the upper parts of the trees is the highest. Phloem was prepared from branches and was extracted with a CTAB-based adapted protocol (Lefort and Douglas, 1999). PCR amplification of phytoplasma DNA was achieved with the universal primers: fP1/rP7 (Deng and Hiruki, 1991; Schneider et al., 1995). All positive samples were tested with the ESFY-specific non-ribosomal primers ECA1/ECA2 (Jarausch et al., 1998). Typical symptoms of ESFY, like premature leaf colouration, leaf yellowing with reddish edges, leaf-roll, severe chlorosis, die-back of top branches and partial or, complete decline of the trees (Figure 1) were found in a peach orchard (GPS coordinates: 46°15'17.4"N 6°12'40.7"E) located in the area of Collonge-Bellerive, close to Geneva city. Ten trees were sampled in this orchard. The presence of ‘Ca. P. prunorum’ was confirmed in two of them (Figure 2 and Figure 3). At the beginning of spring 2017, the monitoring of the ESFY symptoms in the infested orchard was resumed. Early bud break was found on the two infected trees and the infection was confirmed again by PCR (Figure 4 and Figure 5). The same symptoms were observed in many trees of this orchard, which correlated with the previous autumn observations. Trapping confirmed the presence of the insect vector Cacopsylla pruni (Bodnár et al., 2018) in the infected area. In order to understand more about the origin and the diversity of ESFY phytoplasma in this area, additional plant and insect samples will be analyzed. The correlation between symptoms and infected plants will be studied further. We report here for the first time on the occurrence of ‘Candidatus Phytoplasma prunorum’ (‘Ca. P. prunorum’) the agent of ESFY on the territory of Canton of Geneva.

Examination of the plum psyllid (Cacopsylla pruni Scopoli), a vector of European Stone Fruit Yellows (ESFY) phytoplasma in the countryside of Boldogkőváralja (Hungary)

European Stone Fruit Yellows(ESFY) phytoplasma disease on apricot was identified by molecular methods for the first time in 1992 in Hungary. Currently, the pathogen is known as’ Ca. Phytoplasma prunorum’. Since, the pathogen become wide-spreaded and manyof the apricot-plantations had to be eliminated. In 2009 and 2010 in the countryside of Boldogkőváralja the infection of the apricot-plantations was about 77%. As the disease spreading and causing large damages, we studied the roleo fpossible vector,the plum psyllid (Cacopsylla pruni Scopoli) in the areas of Boldogkőváralja, North-East-Hungary. Studies were done in the year of 2016, monitoring the behaviour of the psyllids in four different areas, and caught 41 them for further examination. We collected plant samples from those apricot trees, on which the plum psyllids were caught.

Review of the biology of plant psyllid (Cacopsylla pruni, Scopoli 1763), and its role in the spreading of European stone fruit yellows, ESFY-phytoplasma with Hungarian data

The European stone fruit yellows (ESFY) phytoplasma disease caused by pathogen ’Ca. Phytoplasma prunorum’ induces serious damages in cherry, sour cherry, peach, and apricot orchards mostly in Europe. Its known vector is the plum psyllid (Cacopsylla pruni). Many articles report on the biology (morphology, taxonomy, life cycle etc.) and the method of transmission of the pathogen by the vector, and the possibilities of their control. This paper reviews our knowledge about the vector, and summarises the results of an inland research carried out in a northeastern Hungarian apricot orchards. Our goal was to show some important data for the farmers or anyone who is interested in this disease and its vector. And give some known method that we can protect our orchards against them to prevent the appearance of the disease. As the psyllid that became infected with the pathogen can hold its infectionous capacity during their lifetime, it is very important to have enough knowledge about their lifecycle, that we can determine the right time and method to control them. We also have to know how to identify them; therefore, this paper lists several important data which can be helpful. The most important keys of identification are their wing color, which dark borwn in the apex and brown is in the remaining part of the forewing. The length of the antennae is also an important factor, since other genuse’s species have longer antennae than twice the width of the head. C. pruni has as long antennae as twice the width of the head. They return to Prunus species in early spring and we have to protect our orhards in this period against them. We have to use preparations with a knock down effect on them to prevent the inoculation of the pathogen into the trees in our orchards.

General phytoplasma detection by a q-PCR method using mycoplasma primers

Phytoplasmas and mycoplasmas are bacteria belonging to the class Mollicutes. In this study, a fine tuning of quantitative polymerase chain reaction (qPCR) with a universal mycoplasma primer pair (GPO3F/MGSO) targeting the 16S rRNA gene was carried out on phytoplasmas. The dissociation curves of DNAs from Catharanthus roseus phytoplasma-infected micropropagated shoots and from phytoplasma field-infected plant samples showed a single peak at 82.5 °C (±0.5) specifically detecting phytoplasmas belonging to several ribosomal groups. Assay specificity was determined with DNA of selected bacteria: ‘Candidatus Liberibacter solanacearum’, Xylella fastidiosa, Ralstonia solanacearum and Clavibacter michiganensis. No amplification curves were observed with any of these tested bacteria except ‘Ca. L. solanacearum’ that was amplified with a melting temperature at 85 °C. Absolute quantification of phytoplasma titer was calculated using standard curves prepared from serial dilutions of plasmids containing the cloned fragment GPO3F/MGSO from European stone fruit yellows phytoplasma. Phytoplasma copy number ranged from 106 to 103 according with the sample. The sensitivity evaluated comparing plasmid serial dilutions resulted 10−6 for conventional PCR and 10−7 for qPCR. The latter method resulted therefore able to detect very low concentrations of phytoplasma in plant material.

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.

Molecular Test to Assign Individuals within the Cacopsylla pruni Complex

Crop protection requires the accurate identification of disease vectors, a task that can be made difficult when these vectors encompass cryptic species. Here we developed a rapid molecular diagnostic test to identify individuals of Cacopsylla pruni (Scopoli, 1763) (Hemiptera: Psyllidae), the main vector of the European stone fruit yellows phytoplasma. This psyllid encompasses two highly divergent genetic groups that are morphologically similar and that are characterized by genotyping several microsatellite markers, a costly and time-consuming protocol. With the aim of developing species-specific PCR primers, we sequenced the Internal Transcribed Spacer 2 (ITS2) on a collection of C . pruni samples from France and other European countries. ITS2 sequences showed that the two genetic groups represent two highly divergent clades. This enabled us to develop specific primers for the assignment of individuals to either genetic group in a single PCR, based on ITS2 amplicon size. All previously assigned individuals yielded bands of expected sizes, and the PCR proved efficient on a larger sample of 799 individuals. Because none appeared heterozygous at the ITS2 locus (i.e., none produced two bands), we inferred that the genetic groups of C . pruni , whose distribution is partly sympatric, constitute biological species that have not exchanged genes for an extended period of time. Other psyllid species (Cacopsylla, Psylla, Triozidae and Aphalaridae) failed to yield any amplicon. These primers are therefore unlikely to produce false positives and allow rapid assignment of C . pruni individuals to either cryptic species.

First report of 'Candidatus Phytoplasma prunorum' infecting apricots in Tunisia.

‘Candidatus Phytoplasma prunorum’ was detected for the first time in Tunisia in apricot trees that showed early leaf reddening in autumn, off-season growth in winter followed by dieback, and bore small and tasteless fruits. Phytoplasma was detected by nested PCR using the universal phytoplasma primer pairs R16mF2/R1 and R16F2n/R2. An amplification product of the expected size (1.2 kbp) was obtained from samples collected from symptomatic but not from symptomless apricot trees. Amplicons subjected to RFLP analysis after digestion with endonucleases MseI and RsaI, gave patterns similar to those reported for the European stone fruit yellows phytoplasma (ESFY, 16SrX-B). Identification was further confirmed by PCR using ESFY-specific primer pairs (ECA1/ECA2).

Dieback of apricot plantations caused by 'Ca. Phytoplasma prunorum' in Borsod-Abaúj-Zemplén county (Northern-Hungary)

Plant diseases caused by phytoplasmas have increasing importance in all over the world for fruit growers. Lately, phytoplasma diseases occur on many fruit varieties and responsible for serious losses both in quality and quantity of fruit production. In the long-run these diseases cause destruction of fruit trees. The apricot phytoplasma disease (Ca. Phytoplasma prunorum) was first reported in Europe in 1924 from France. In 1992 the disease has also been identified in Hungary. On the base of growers' signals serious damages of "Candidatus Phytoplasma prunorum" Seemüller and Schneider, 2004 (formerly: European stone fruit yellows phytoplasma) could be observed in different stone fruit plantations in the famous apricot-growing area nearby Gönc town, Northern-Hungary. Field examinations have been begun in 2009 in several stone fruit plantations in Borsod-Abaúj-Zemplén County mainly in Gönc region which is one of the most important apricot growing regions in Hungary, named “Gönc Apricot Growing Area”. Our goals were to diagnose the occurrence of Ca. Phytoplasma prunorum on stone fruits (especially on apricot) in the North-Hungarian growing areas by visual diagnostics and confirm data by laboratory PCR-based examinations. All the 28 collected samples were tested in laboratory trials and at 13 samples from apricot, peach, sour cherry and wild plum were confirmed the presence of phytoplasma (ESFY). On the base of observations it seems evident that the notable losses caused by "Ca. Phytoplasma prunorum" is a new plant health problem to manage for fruit growers, especially apricot producers in Hungary.

RESEARCH ON THE VECTOR OF 'CANDIDATUS PHYTOPLASMA PRUNORUM' IN APRICOT TREES IN SOUTHERN ITALY

A research of vector(s) of the phytoplasmas responsible for apricot chlorotic leaf roll disease was carried out in a field plot where European Stone Fruit Yellows Phytoplasma (ESFYP), now named 'candidates Phytoplasma prunorum' ('Ca. P. prunorum'), had heavily infected an apricot collection in 1993. The plants were uprooted in May 1994 and, in 1997, the field was planted again with apricot trees. This report was elaborated with the data collected from February 2003 through July 2007. The only insect species found was Empoasca decedens Paoli with 5,085 insects collected. These insects were tested by direct and nested PCR to verify the presence of the phytoplasma. Individuals of E. decedens, present in the field each month in each year, resulted to be hosts of 'Ca. P. prunorum' in ten months out of twelve, but not of every year. The absence of phytoplasma in the two plants infected by 'Ca. P. prunorum' with E. decendens, four years after the acquisition of the pathogen, was also recorded.

HIGH INFECTION PRESSURE OF ESFY PHYTOPLASMA THREATENS THE CULTIVATION OF STONE FRUIT SPECIES

Stone fruit species are affected by severe disease caused by European stone fruit yellows phytoplasma (ESFY; ‘Candidatus Phytoplasma prunorum’). ESFY phytoplasma is transmitted to the host plants of Prunus spp. by the vector Cacopsylla pruni. The disease is graft-transmissible as well. The occurence of ESFY phytoplasma was monitored from 2004 to 2006 in a mother plant orchard of stone fruit species planted with virus free material in 2001 in the Primorska region of Slovenia. The total of 158 samples of mother plants were analysed in this period. The symptomatic and asymptomatic trees were analysed using molecular methods (PCR or nested-PCR). Among 15.7 % of sampled apricot trees (Prunus armeniaca) in the orchard, ESFY phytoplasma was detected in 70.0 % of samples. In the case of Japanese plum (Prunus salicina) samples were taken from one third of all Japanese plum trees and the presence of ESFY phytoplasma was confi rmed in all samples. In the European plum trees (Prunus domestica) the incidence of phytoplasma was determined in 51.0 % of sampled trees, where the plants in most cases did not show symptoms. ESFY phytoplasma was also detected in peaches and nectarines (Prunus persica) in 13.0 % of sampled trees while no detection of the phytoplasma was confi rmed in the samples of cherry trees (Prunus avium). With the survey performed in a mother plant orchard it was observed that especially young trees did not show typical symptoms and the infection was latent. In the year 2005, ESFY phytoplasma was detected in all tested samples of the vector Cacopsylla pruni captured in the vicinity of the mother plant orchard.

Detection of phytoplasma ESFY in apricot trees using phloem and petioles

ESFY phytoplasma (European stone fruit yellows phytoplasma) is nowadays one of the most important plant diseases, especially on apricots and peaches, and it belongs to the list of organisms for which quarantine is required in the Czech Republic. The aim of this study was to determine the best period for tissue extraction and the best technique for ESFY detection. It was also to investigate the possibility of isolating DNA for use in ESFY detection from the leaf-stalks of randomly chosen symptomatic and asymptomatic apricot trees. Results of the amplification of DNA extracted from leaf-stalk and phloem sampled from 2-year old woody shoots during the years 2003 and 2004 were statistically analysed and compared, and visible disease symptoms were simultaneously evaluated and compared to the results of molecular detection. DNA isolation from leaf-stalks can be considered as less significant and reliable than isolation from phloem sampled from 2-year old woody shoots.

Spreading of ESFY Phytoplasmas in Stone Fruit in Catalonia (Spain)

AbstractA survey was carried out in nine stone fruit commercial orchards located in Barcelona province where plum and apricot trees of different cultivars showing European stone fruit yellows (ESFY) symptoms were present. A 4‐year survey with visual inspection of symptoms in one apricot orchard showed a rather high ESFY disease spread, also in a Japanese plum plantation newly infected plants were detected every year in a similar rate (about 2%). All the inspected symptomatic trees were polymerase chain reaction (PCR) tested and ESFY phytoplasma identity was confirmed by restriction fragment length polymorphism analyses and sequencing of ribosomal DNA amplification products. In apricot plantation the detection of ESFY phytoplasma was also tested on 69 asymptomatic trees sampled in summer 2002. The nested PCR with 16SrX group‐specific primers allowed detection of ESFY phytoplasmas in 50% of the trees that indeed showed symptoms by the next winter (2003). The molecular detection of ESFY phytoplasma in asymptomatic apricot trees indicates the risk of maintaining phytoplasma foci in the fields where eradication is based only on visual inspection.

'Candidatus Phytoplasma mali', 'Candidatus Phytoplasma pyri' and 'Candidatus Phytoplasma prunorum', the causal agents of apple proliferation, pear decline and European stone fruit yellows, respectively.

Apple proliferation (AP), pear decline (PD) and European stone fruit yellows (ESFY) are among the most economically important plant diseases that are caused by phytoplasmas. Phylogenetic analyses revealed that the 16S rDNA sequences of strains of each of these pathogens were identical or nearly identical. Differences between the three phytoplasmas ranged from 1.0 to 1.5% of nucleotide positions and were thus below the recommended threshold of 2.5% for assigning species rank to phytoplasmas under the provisional status 'Candidatus'. However, supporting data for distinguishing the AP, PD and ESFY agents at the species level were obtained by examining other molecular markers, including the 16S-23S rDNA spacer region, protein-encoding genes and randomly cloned DNA fragments. The three phytoplasmas also differed in serological comparisons and showed clear differences in vector transmission and host-range specificity. From these results, it can be concluded that the AP, PD and ESFY phytoplasmas are coherent but discrete taxa that can be distinguished at the putative species level, for which the names 'Candidatus Phytoplasma mali', 'Candidatus Phytoplasma pyri' and 'Candidatus Phytoplasma prunorum', respectively, are proposed. Strains AP15R, PD1R and ESFY-G1R were selected as reference strains. Examination of available data on the peach yellow leaf roll (PYLR) phytoplasma, which clusters with the AP, PD and ESFY agents, confirmed previous results showing that it is related most closely to the PD pathogen. The two phytoplasmas share 99.6% 16S rDNA sequence similarity. Significant differences were only observed in the sequence of a gene that encodes an immunodominant membrane protein. Until more information on this phytoplasma is available, it is proposed that the PYLR phytoplasma should be regarded as a subtype of 'Candidatus Phytoplasma pyri'.

Identification of genes expressed in response to phytoplasma infection in leaves of Prunus armeniaca by messenger RNA differential display

The messenger RNA (mRNA) differential display technique was applied to the identification and isolation of genes whose transcription was altered in leaves of Prunus armeniaca infected by European stone fruit yellows (ESFY) phytoplasma belonging to ribosomal subgroup 16SrX-B. Four genes whose steady-state levels of expression significantly changed in response to phytoplasma infection were isolated and identified. The results obtained show that two group of genes are affected by phytoplasma infection in apricot leaves. The first group comprises genes that are up-regulated by phytoplasma presence: in particular, a gene encoding the heat-shock protein HSP-70, a gene encoding a metallothionein (MT) and another homologous to the EST 673 cDNA clone of P. armeniaca, whose function was unknown. The other gene identified in our analysis is down-regulated by phytoplasma presence. It encodes a protein having homology to an amino acid transporter of Arabidopsis thaliana. Our findings demonstrate the usefulness of mRNA differential display approach for the detection of plant metabolic pathways affected by phytoplasma infection.

Seasonal infectivity of Cacopsylla pruni, vector of European stone fruit yellows phytoplasma

SummaryEpidemiology of European stone fruit yellows was studied by focussing on the life cycle and transmission characteristics of the vector Cacopsyllapruni. The proportion of both phytoplasma positive and inoculative insects was determined for the first C. pruni adults back colonising the stone fruit trees in spring and for the new generations of the vector, hatched at the beginning of summer. We showed that in spring, as soon as the insects moved to stone fruit trees from shelter plants, they were infective. After the vector fed on infected stone fruit trees, the proportion of phytoplasma positive insects increased. The new generation colonising Prunus species also acquired the phytoplasma from their hosts although several of these insects completed the latency period on secondary hosts. Results showed that the risk of natural transmission of European stone fruit yellows‐phytoplasma by C. pruni within orchards is high when the vector is present. These results have implications for the control of European stone fruit yellows.

Detection of the phytosanitary status of stone fruit cultivars under in vitro conditions in Hungary

Prunus species are prone to infections by a range of pathogens (LAIMER 2002). Stone fruit plantations are mainly affected by Plum pox virus (PPV), Prune dwarf virus (PDV), Prunus necrotic ringspot virus (PNRSV) and European Stone Fruit Yellows phytoplasma (ESFY). The production of elite plants of pathogen-free stone fruit cultivars includes the application of effective methods for the detection and elimination of the major stone fruit tree viruses and phytoplasmas. The method of micropropagation offers a quick possibility for breeders to introduce new cultivars into the production. Virus testing by molecular techniques of in vitro plants requires only tiny samples and reduces the danger of false negatives. First results of the virus testing originated from in vitro peach cultivars are presented.

Role of wild Prunus species in the epidemiology of European stone fruit yellows

Several uncultivated trees of the species Prunus spinosa, P. cerasifera and P. domestica, sampled both adjacent to European stone fruit yellows (ESFY)‐infected orchards and in isolation from cultivated stone fruit plants, were found to be infected by ESFY phytoplasma. These species were also colonized by Cacopsylla pruni, vector of the ESFY agent. In contrast, uncultivated species of Prunus avium, P. cerasus and P. mahaleb hosted neither the pathogen nor the vector. Insect‐ and graft‐transmission trials of ESFY phytoplasma conducted under controlled conditions confirmed the data obtained in the field. The role played by the wild Prunus species is discussed and appears to be fundamental in the epidemic cycle of the disease.