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Cacopsylla pruniisa psyllid that has been known since 1998 as the vector of the bacterium ‘Candidatus Phytoplasma prunorum’, responsible for the European stone fruit yellows (ESFY), a disease that affects species of Prunus. This disease is one of the major limiting factors for the production of stone fruits, most notably apricot (Prunus armeniaca) and Japanese plum (P. salicina), in all EU stone fruit-growing areas. The psyllid vector is widespread in the Western Palearctic, and evidence for the presence of the phytoplasma that it transmits to species of Prunus has been found in 15 of the 27 EU countries. Recent studies showed that C. pruni is actually composed of two cryptic species, which can be differentiated bymolecular markers. A literature review on the distribution of C. pruni was published in 2012, but it only provided presence or absence information at the country level and without distinction between the two cryptic species. Since 2012, numerous new records of the vector in several European countries have been published. We ourselves have acquired a large amountof data from sampling in France and other European countries. We have also carried out a thorough systematic literature review to find additional records, including all the original sources mentioningC. pruni (or its synonyms) since the first description by Scopoli in 1763. Our aim was to create an exhaustive georeferenced occurrence catalog, in particular in countries that are occasionnaly mentioned in the literature with little detail. Finally, for countries that seem suitable for the proliferation of C. pruni (USA, Canada, Japan, China, etc.), we digged deeper into the literature and reliablesources (e.g. checklist)to better subtanciateits current absence from those regions. Information on the distribution ranges of these vector psyllids is of crucial interest in order to best predict the vulnerability of stone fruit producing countries to the ESFY threat in the foreseeable future. We give free access to a unique file of 1975 records of all occurrence data in our possession concerning C. pruni, which we have gathered through more than twenty years of sampling efforts in Europe or through intensive text mining. We have made every effort to retrieve the source information for the records extracted from litterature (1201 records). Thus, we always give the title of the original reference, together with the page(s) citing C. pruni and, if possible, the year of sampling. To makethe results of this survey publicly available, we give a URL to access the literature sources. In most cases, this link allows to freely download a PDFfile. We also give access to information extracted from GBIF (162 exploitable data points on 245 occurrences found in the database), which we thoroughly checked and often supplemented to make the information more easily exploitable. We give access to our own unpublished georeferenced and genotyped record from 612samples taken over the last 20 years in several European countries (Switzerland, Belgium, Netherlands, Spain, etc.). Theseinclude two countries (Portugal and North Macedonia) for which the presence of C. pruni had notbeen reported before.Asour specimens have been genotyped (74 sites with species A solely, 202 with species B solely, and 310 with species A+B), our new data enable a better view of the geographical distribution of the two species at the Palaearctic scale.

Phytoplasmas are specialized small bacteria restricted to the phloem tissue and spread by hemipterans feeding on plant sieve tube elements. As for many other plant pathogens, it is known that phytoplasmas alter the chemistry of their hosts. Most research on phytoplasma-plant interactions focused on the induction of plant volatiles and phytohormones. Little is known about the influence of phytoplasma infections on the nutritional composition of phloem and consequences on vector behavior and development. The plum psyllidCacopsylla prunitransmits ‘CandidatusPhytoplasma prunorum’, the causing agent of European Stone Fruit Yellows (ESFY). While severalPrunusspecies are susceptible for psyllid feeding, they show different responses to the pathogen. We studied the possible modulation of plant-insect interactions by bacteria-induced changes in phloem sap chemistry. Therefore, we sampled phloem sap from phytoplasma-infected and non-infectedPrunus persicaandPrunus insititiaplants, which differ in their susceptibility to ESFY and psyllid feeding. Furthermore, the feeding behavior and development ofC. pruninymphs was compared on infected and non-infectedP. persicaandP. insititiaplants. Phytoplasma infection did not affect phloem consumption byC. pruninymphs nor their development time. In contrast, the study revealed significant differences betweenP. insititiaandP. persicain terms of both phloem chemistry and feeding behavior ofC. pruninymphs.Phloem feeding phases were four times longer onP. insititiathan onP. persica, resulting in a decreased development time and higher mortality of vector insects onP. persicaplants. These findings explain the low infestation rates of peach cultivars with plum psyllids commonly found in field surveys.

BGPI : équipe 6; International audience; ‘Candidatus Phytoplasma’ species are mostly transmitted from plant to plant by phloem feeding hemipterans, primarily leafhoppers (Cicadellidae) and planthoppers (Fulgoroidea) (Hemiptera, Auchenorrhyncha). However, there is one group of phytoplasmas, the 16SrX or apple proliferation group, whose members are transmitted by psyllid vectors of the superfamily Psylloidea (Hemiptera, Sternorrhyncha). These psyllid-transmitted phytoplasmas are genetically closely related and are associated with economically important diseases of fruit trees such as pear decline, apple proliferation and European stone fruit yellows. The psyllid vector species of these phytoplasmas are also closely related and all belong to the genus Cacopsylla. Both, phytoplasmas and psyllid vectors, are geographically limited to the Palaearctic region, mainly Europe. Only pear decline and peach yellow leaf roll phytoplasmas have probably been introduced to America along with their vectors. As phytoplasma-infected trees cannot be cured and resistant plant material is not available to the growers, preventive control measures such as vector control are of paramount importance to limit the spread of these diseases. Thus, detailed knowledge about the biology and ecology of the vector species, their host plants as well as knowledge about the transmission parameters is crucial.

Peach (Prunus persica) is in many countries severely affected by yellows and decline diseases of phytoplasmal etiology. These diseases are caused by phylogenetically different phytoplasmas. X-disease is one of the most serious diseases of peach. It is widespread in North America and has not been observed elsewhere. This disease is induced by a distinct phytoplasma, the X-disease agent ‘Candidatus Phytoplasma pruni’, which is a member of the X-disease phytoplasma group or 16SrIII group, subgroup 16SrIII-A. ‘Ca. P. pruni’ is, at the level of 16S rDNA sequences, a homogeneous pathogen. Peach rosette, peach red suture, and peach yellows, also called little peach, are three other diseases which are mainly known from eastern North America. These diseases are currently of minor importance. They are also caused by the X-disease phytoplasma. Peach yellow leaf roll (PYLR) is a major disease of peach that occurs in California. The causative agent, the PYLR phytoplasma, is a member of the apple proliferation (AP) group and is most closely related to the pear decline phytoplasma. In Europe, peach is affected by an economically important disorder known as European stone fruit yellows. This disorder is induced by ‘Ca. P. prunorum’ which is a member of the AP group, subgroup 16SrX-B. ‘Ca. P. prunorum’ is closely related to other important temperate fruit tree phytoplasmas including the PYLR agent. In Lebanon and Iran, peach is affected by almond witches’-broom, a lethal disease that is induced by ‘Ca. P. phoenicium’. This phytoplasma is a member of the 16SrIX group, subgroup 16SrIX-B. 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 worldwide. However, the pathological relevance of these non-peach phytoplasmas needs to be investigated in many cases.

The molecular identification and characterization of phytoplasmas infecting grapevine in four locations of different grape-growing regions of Serbia is reported. Samples from symptomatic and asymptomatic grapevine plants were collected during 2002 and 2003. Detection and identification of phytoplasmas were done by the restriction fragment length polymorphism analysis (RFLP) of polymerase chain reaction (PCR) amplified 16S rDNA. Grapevine yellows were associated with three molecularly distinguishable phytoplasmas. Flavescence dorée phytoplasmas (elm yellows group - 16SrV-C subgroup) were present only in the Župa Aleksandrovac region; in other surveyed regions Bois noir phytoplasmas (stolbur group - 16SrXII-A subgroup) were detected; a mixed infection of European stone fruit yellows (apple proliferation group - 16SrX-B subgroup) and Bois noir phytoplasmas was identified in one sample. A finer molecular characterization by the RFLP analysis on rpS3 and SecY genes of Flavescence dorée phytoplasmas from Župa Aleksandrovac allowed to verify that the Serbian genotype is indistinguishable from a strain from the Veneto region, Italy. Characterization performed on tuf gene of Bois noir phytoplasmas showed lack of amplification of samples from Erdevik. The identity of HpaII profiles on the tuf gene PCR products was observed between samples from Palić and Radmilovac, this pattern appears indistinguishable from one of the two profiles obtained in samples from the Italian grapevines used as reference strains.

BGPI : équipe 6; Diseases and insect vectors

BGPI : équipe 6; International audience; 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.

Phytoplasmas are microorganisms associated with a number of severe plant diseases affecting many areas of diverse agricultural cultivations worldwide. The discovery of a new group of plant pathogens related to bacteria led to the finding of polymorphic, wall-less prokaryotes, located in the phloem of many plant species affected by yellows-type diseases believed to be caused by viruses, considering their infectious nature, and transmission by insects. Molecular data have provided considerable insights into their molecular diversity and genetic interrelationships and significant taxonomic progress has been achieved by the study of the 16S ribosomal gene with full sequencing of four strains. However there is a gap between taxonomy and diseases since it is not uncommon that the same disease is associated with molecularly differentiable phytoplasmas and this is quite common with diseases described in different areas of the world (Lee et al., 1998). During the last ten years, the use of molecular methods has enabled the detection and characterization of phytoplasmas associated with major fruit tree and grapevine diseases in Europe

Recovery phenomena, have been intensively studied for apple proliferation and grapevine yellows. Several recent findings in apricot orchards in Friuli-Venezia Giulia indicate the importance of this strategy also for the control of European Stone Fruit Yellows (ESFY) infections, caused by “Candidatus Phytoplasma prunorum”. The aim of this work was to investigate the occurrence of recovery in 2 experimental orchards of apricot cv. Bergeron on “Wavit”, located in the province of Trento, where the disease has been constantly spreading since 2000. This included visual inspections for typical symptoms and a diagnostic method based on Spot Real-Time Reverse Transcription PCR assay. The results obtained showed that three different groups of plants infected by ESFY were present: many plants with typical symptoms, some recovered and some symptomless infected plants. To date no interesting plant genotypes showing resistance to ESFY are available, moreover most of the insecticide applications have no efficacy in controlling the vector than the disease. Both use of pathogen-free propagation material and resort to natural recovery could be adopted as an integrated approach for the control of this phytoplasma disease. For those reasons the research will continue with the aim to quantify the concentration of the pathogen in the recovered plants.

L'ECA est une maladie importante qui, en France, affecte principalement les abricotiers et les pruniers japonais.Il est causé par la bactérie Candidatus Phytoplasma prunorum. La forte variabilité d'expression des symptômes peut rendre difficile son diagnostic visuel, mais aujourd'hui des tests moléculaires permettent une caractérisation formelle.Dans le milieu naturel, le phytoplasme est hébergé dans les Prunus sauvage (prunelliers, myrobalans).Les deux espèces sœurs du psylles (groupées sous le nom de Cacopsylla pruni) capables de disséminer cet agent pathogène ont une seule génération par an. Les adultes migrent des Prunus aux conifères avant l'été puis des conifères aux Prunus en fin d'hiver. La transmission se fait sur le mode persistant et ce sont essentiellement les psylles qui se sont infestés avant leur départ des Prunus qui sont susceptibles de transmettre le phytoplasme dans les vergers 8 mois plus tard.La pertinence des stratégies de lutte possibles (notamment la gestion des Prunus sauvages) est discutée sur la base de ces connaissances.

UMR BGPI Equipe 6 ; UMR DAP Equipe DGBPublication Inra prise en compte dans l'analyse bibliométrique des publications scientifiques mondiales sur les Fruits, les Légumes et la Pomme de terre. Période 2000-2012. http://prodinra.inra.fr/record/256699; Understanding at which spatiotemporal scale a disease causes significant secondary spread has both theoretical and practical implications. We investigated this issue in the case of European stone fruit yellows (ESFY), a quarantine vector-borne phytoplasma disease of Prunus trees. Our work was focused on the processes underlying disease spread: the interplay between the life cycles of the pathogen ('Candidatus Phytoplasma prunorum') and of the vector (Cacopsylla pruni). We demonstrated experimentally that C. pruni has only one generation per year and we showed that, at least in southeastern France, C. pruni migrates between conifers in mountainous regions (where it overwinters) and Prunus spp. at lower altitude (where it breeds). In acquisition-inoculation experiments performed with C. pruni over its period of presence on Prunus spp., both immature and mature C. pruni were hardly infectious (0.6%) despite effective phytoplasma acquisition and multiplication. We demonstrated that most immature vectors born on infected plants reach their maximum phytoplasma load (107 genomes per insect) only after migrating to conifers and that, after a life-long retention of the phytoplasma, their transmission efficiency was very high (60%) at the end of winter (when they migrate back to their Prunus host). Thus, most transmissions occur only after an effective latency of 8 months, following vector migrations and overwintering on conifers in mountainous regions. From this transmission cycle, we can infer that local secondary spread of ESFY in apricot orchards is marginal, and recommend that disease management strategies take more into account the processes occurring at a regional scale, including the role of wild Prunus spp. in ESFY epidemics.

Since the beginning of the twentieth century symptoms of apricot tree decline were observed in France and Italy: Morvan in 1977 named the disease associated with leptonecrosis (Goidanich, 1934) or with new sprouting in winter "apricot chlorotic leaf rolling" (ACLR). Only since the late 1970ies these symptoms were associated with phytoplasma infections, since electron or fluorescence microscopy (by DAPI-staining) allowed to detect phytoplasmas as single cells in sieve tubes (Fig. 1) and transmission experiments to other stone fruit and indicator plants were successfully carried out (Morvan, 1977; Goidanich et al., 1980; Giunchedi et al., 1982, Pastore et al., 1995). European stone fruit yellows (acronym: ESFY) has been proposed as the common name for phytoplasma-related diseases in European stone fruits (Kison et al., 1997). Among others it includes the French ECA or ACLR, which is a quarantine organism of EPPO (OEPP/EPPO, 1986), included in the EPPO certification scheme for virus tested fruit trees (OEPP/EPPO, 1991/1992).The presence of ESFY disease has been reported in France, Spain, Italy, Greece, and in Hungary, Romania, Switzerland, Germany, former Yugoslavia, the UK and Austria (Nemeth, 1986; Morvan, 1977, Davies and Adams 2000, Laimer da Câmara Machado et al. 2001a) causing decline and death to apricot, Japanese plum, more rarely to peach (Llacer and Medina, 1988) and to almond, flowering cherry and European plum (Seemüller et al. 1998) http://www.boku.ac.at/iam/pbiotech/phytopath/v_esfy.html). An increasing presence of phytoplasma associated diseases such as leptonecrosis on Japanese plum (Prunus salicina) and chlorotic leaf roll on apricot (Prunus armeniaca) has been observed in commercial orchards in several European regions in the last twenty-five years (Giunchedi et al., 1978; Desvignes and Cornaggia, 1982; Dosba et al., 1991; Bertaccini et al., 1993; Laimer et al., 2001; Torres et al., 2004). Prunus rootstocks are also affected by this disease (Dosba et al. 1991, Jarausch et al. 1998). ESFY phytoplasmas have also been detected in wild Prunus species, e.g. Prunus spinosa and P. cerasifera (Carraro et al. 2002) and cherry (Prunus avium) (Paltrinieri et al., 2001). In recent years ESFY phytoplasma has been detected in other wild plants such as Rosa canina, Celtis australis and Fraxinus excelsior (Jarausch et al., 2001) as well as in grapevine in Hungary (Varga et al., 2000) and in Serbia (Duduk et al., 2004).

To investigate the genetic variability of fruit tree phytoplasmas belonging to the 16SrX phylogenetic group, we used a Multilocus Sequence Typing strategy (MLST). Sequences of four non-ribosomal genetic loci (aceF, pnp, imp and secY) were determined among a collection of Candidatus Phytoplasma prunorum, Ca. P. mali and Ca. P. pyri isolates. Sequences alignment and phy-logenetic analyses confirmed the classification based on rDNA16S phylogeny. The four genetic loci displayed specific signatures clearly correlated with the recent definition of the corresponding Candidatus species. However, a total congruence was not ob-served inside species, between phylogenetic trees constructed with the different loci. A divergent Ca. P. prunorum variant from Azerbaijan, recently characterized through aceF sequencing, again presented a divergent imp gene. The previously characterized Ca. P. prunorum hypovirulent strains had the same aceF sequence but were discriminated with the imp marker. Ca.P. pyri strains detected in Lebanese Pear Decline isolates also form a distinct cluster according to aceF and imp markers. Imp and secY phyloge-netic analyses pointed out a new Ca. P. prunorum cluster comprising strains never detected in psyllid vectors (non circulative strains ?). On the opposite, the circulative Ca.P. prunorum strains were distributed among all phylogenetic group identified. For Ca. P.mali, discrimination of apple proliferation isolates was in agreement with previous studies.

National audience

National audience

Publication Inra prise en compte dans l'analyse bibliométrique des publications scientifiques mondiales sur les Fruits, les Légumes et la Pomme de terre. Période 2000-2012. http://prodinra.inra.fr/record/256699; 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.

Hydrogen peroxide (H2O2) localization and roles of peroxidases, malondialdehyde and reduced glutathione were compared in leaves of apricot (Prunus armeniaca) plants: asymptomatic, European Stone Fruits Yellows (ESFY)-symptomatic and recovered. Nested PCR analysis revealed that ‘Candidatus Phytoplasma prunorum’, is present in asymptomatic, symptomatic and recovered apricot trees, confirming previous observations on this species, in which recovery does not seem to be related to the disappearance of phytoplasma from the plant. H2O2 was detected cytochemically by its reaction with cerium chloride, which produces electron-dense deposits of cerium perhydroxides. H2O2 was present in the plasmalemma of the phloem cells of recovered apricot plant leaves, but not in the asymptomatic or symptomatic material. Furthermore, by labelling apricot leaf tissues with diaminobenzidine DAB, no differences were found in the localization of peroxidases. Protein content in asymptomatic, symptomatic and recovered leaves was not significantly different from one another. In contrast, guaiacol peroxidase activity had the following trend: symptomatic > recovered > asymptomatic, whereas reduced glutathione content followed the opposite trend: asymptomatic > recovered > symptomatic. Moreover, no differences were observed in malondialdehyde concentrations between asymptomatic, symptomatic and recovered leaves. The overall results suggest that H2O2 and related metabolites and enzymes appear to be involved in lessening both pathogen virulence and disease symptom expression in ESFY-infected apricot plants.

UMR BGPI Equipe 6; International audience; A network was created in France to obtain data on the seasonal activity of the psyllid Cacopsylla pruni, vector of European stone fruit yellows phytoplasma. C. pruni was collected on Prunus plants by beating trays. The insect was found in every prospected area. The reimmigrants (adults that have overwintered) were found on Prunus from February to May and the new generation from May to June. Not any C. pruni was found on Prunus after July. Prunus spinosa appeared the preferred host plant compared to the other monitored Prunus. The data are discussed in relation to the control of the phytoplasma disease.

A 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.