Survey of Cacopsylla bidens, vector of 'Candidatus phytoplasma pyri', in Chilean pear orchards.

Pear decline, induced by the phytoplasma 'Candidatus Phytoplasma pyri', transmitted by pear psyllids, is one of the most devastating diseases on Pyrus communis in Europe and North America. Investigations of pear psyllids in 4 pear orchards in lower Austria showed the presence of Cacopsylla pyri, C. pyricola and C. pyrisuga at all locations. PCR analyses revealed overall phytoplasma infection rates for C. pyri of 5.4%, for C. pyricola, of 4.6%, for C. pyrisuga remigrants of 9.6% and for C. pyrisuga emigrants of 0%. The rates of PCR-positive C. pyri and C. pyricola individuals varied greatly in the course of the year, and the highest infection rates were observed in late summer, autumn and in late winter. In transmission experiments with healthy pear seedlings, winterform individuals of C. pyri and C. pyricola transmitted the pathogen to 19.2% (5 out of 26) and 4.8% (2 out of 41) of the test plants, respectively. The vectoring ability of C. pyrisuga was experimentally proven for the first time, and in transmission experiments with remigrants, 9.5% (2 out of 21) of the pear seedlings were infected. Our data indicate a significant risk of pathogen transmission in pear orchards during the greater part of the year, especially in late winter, early spring and autumn. Multilocus sequence analysis by aid of the genes aceF and imp allowed the discrimination between 15 phytoplasma types. Three so far undescribed aceF genotypes and four undescribed imp genotypes were identified.

'Candidatus Phytoplasma pyri', the pathogen associated with pear decline, affects pear trees across both the old and new worlds. However, research on this phytoplasma has been limited by the lack of genomic data. This study presents the first draft genome of 'Ca. P. pyri' using a strain from Chile, with its genomic features analyzed in comparison with the closely related 'Ca. Phytoplasma' species 'Ca. P. mali' and 'Ca. P. prunorum'. The draft genome spans 456,478 bp with a GC content of 20.4%. Key genes possibly associated with pathogenicity and potential pathogenic effectors were identified, and they are notably lacking orthologs of known effectors. A single potential mobile unit similar to that of 'Ca. P. mali' was identified. It is characterized by the absence of the transposase tra5 and the presence of the IS3 family transposase iSErh1. Multilocus sequence analysis of six genetic markers (16S rRNA gene, LSU36p, tuf, aceF, secA, and secY) from 10 Chilean and 10 Italian samples revealed high genetic uniformity among the Chilean strains, collected from five geographically distant orchards over a span of 13 months; by contrast, strains with greater diversity were detected among those from Italy, collected from a few localities over approximately 30 years. These findings suggest limited evolutionary divergence of this phytoplasma in Chile. This study provides a foundational framework for investigations into the pathogenic mechanisms and evolutionary dynamics of 'Ca. P. pyri'.

Simple SummaryPear psyllids are responsible for transmitting the pathogen ‘Candidatus Phytoplasma pyri’, causing the pear decline disease. Specific repellents are a potential method for controlling this pest and reducing the spreading of phytoplasma pear in orchards. Little is known about the chemical ecology of these insects. Based on recent literature, we tested the behavioral reactions of Cacopsylla pyri and Cacopsylla pyricola to the major synthetic compounds of clove essential oil in olfactometer experiments. In addition, we developed new nanofiber-based repellents dispensers and evaluated their repellent and host-odor masking activity in the laboratory and later in the field. Results demonstrate that only the synthetic mixture of the three major compounds of the clove essential was repellent to both psyllids species. When volatiles were formulated in nanofibers, they were not only repellent but could mask the odors of pear plants, disrupting the insects from finding the host. In the field, no differences in psyllids captures were observed in color-attractive sticky traps with repellent-loaded nanofibers or nanofibers without repellent. Our study also evaluated the release rates of volatiles from the nanoformulation. We discussed using nanofibers as volatile dispensers and the improvements necessary to use repellents as a management tool for pear psyllids in the field.Pear psyllids are the main vectors of the pathogen ‘Candidatus Phytoplasma pyri’ causing pear decline. Based on earlier reports, we tested the behavioral activity of the major synthetic compounds of clove essential oil (eugenol, eugenyl acetate, and β-caryophyllene) against Cacopsylla pyri and C. pyricola. Of six mixtures tested in olfactometer assays, a formulation consisting of three specific compounds (M6 mixture) demonstrated a repellent effect on both psyllid species. In addition, this formulation masked the odor of the host Pyrus communis cv. Williams Christ, disturbing the host finding ability of C. pyri. Electrospun fibers were produced with biocompatible polymers poly(ε-caprolactone), cellulose acetate, and solvents formic acid and acetic acid, loaded with the repellent mixture to test their efficacy as dispensers of repellents in laboratory and field. The fibers produced were repellent to C. pyri and effectively masked the odors of pear plants in olfactometer tests. In a pear orchard, we compared the captures of pear psyllids in green-colored attractive traps treated with nanofibers loaded with M6 mixture or unloaded nanofibers (blank). The result showed no differences in the captures of C. pyri between treatments. The release rates of volatiles from the fibers were evaluated weekly over 56 days. The fibers were able to entrap the major compound of the M6 mixture, eugenol, but the release rates were significantly reduced after 21 days. Our results suggest that biodegradable dispensers could be produced with electrospinning, but further improvements are necessary to use repellents as a management tool for pear psyllids in the field.

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'.

Pear decline (PD) is a serious disease of pear (Pyrus communis L.) caused by 'Candidatus Phytoplasma pyri', which belongs to the subgroup 16SrX-C of the apple proliferation (AP) group of phytoplasmas (3). Pear seedlings from the Agriculture and Agri-Food Canada (AAFC) pear breeding program, which have been selected for advanced test and grower trials, are routinely submitted to the Canadian Food Inspection Agency (CFIA) Sidney Laboratory (formerly, CFIA Centre for Plant Health, Saanichton, BC) for virus testing at the same time that propagation is initiated to produce trees for further evaluations. In early 2007, the CFIA reported that samples of two seedling selections submitted in 2005 tested positive for phytoplasmas by a nested PCR assay with phytoplasma universal primers P1/P7 (1), followed by phytoplasma universal primers fU5/rU3 (2) and real time PCR with universal phytoplasma primers developed by the CFIA-Sidney (personal communication). Phytoplasmas present in both selections were subsequently identified as 'Ca. P. pyri' strains by nested PCR with the P1/P7 primers followed by PD/peach yellow leaf roll (PYLR)-specific primers fPD/rPDS (2,4). These were the first PD-positive results from many samples submitted over the years for testing. Following PD-positive diagnoses for the seedling trees, others propagated from these seedling trees were removed from the nursery. When tested by PD-specific nested PCR (P1/P7 then fPD/rPDS), one selection had 39 of 79 nursery trees (49%) that were PD positive, while the other selection had 27 of 96 trees (28%) testing as PD positive. PCR amplification of DNA isolated from leaves of six of the propagated trees, with primer pair fPD/rPDS, yielded an ~1,400-bp product that was sequenced. A consensus sequence of 1,313 bp (GenBank Accession No. GU565959) was subjected to a nucleotide BLAST search of the NCBI database and showed 100% nt identity with sequences of phytoplasmas PD1 (AJ542543) and PYLR (Y16394). Subsequently, the PD-positive results from leaf, dormant shoot, and root tissues from the original seedling trees were confirmed by PD-specific nested PCR. On the original seedling trees, visible symptoms typical of PD, especially premature leaf coloration, were observed in late summer 2008 and samples taken of green and red leaves were subjected to PD-specific PCR. Red leaves were PD-positive, while green leaves were mostly PD-negative. Pear leaves, dormant shoots, and roots collected from research and commercial orchards in southern Ontario in 2007 and 2008 were subjected to PD-specific nested PCR (P1/P7 then fPD/rPDS), AP-specific nested PCR (P1/P7 then fO1/rO1) (2), as well as the universal phytoplasma nested PCR (P1/P7 then fU5/rU3), resulting in the identification of PD-positive trees of several cultivars. The sequence of the 1,057-bp amplicon from accession PYR0190 (selection HW615), with AP-specific primers fO1/rO1, was deposited in GenBank (GU475131). Although there have been no previous reports of PD in Ontario, Canada, it would appear that PD has been present for some time based on the number and distribution (both geographic and cultivar) of positive samples.

Aims: In this study, the best sampling time and tissues for phytoplasma detection in twenty pear trees (cv. Deveci) infected by ‘Candidatus Phytoplasma pyri’, causal agent of pear decline disease, in Bursa province of Turkey were investigated.Methods and Results: Sampling was done throughout the year in leaf midribs, shoot and root tissues, where as the flower tissues were tested once a year in March and fruit tissues in September. All samples were analyzed by nested-PCR using P1/P7 and fU5/rU3 universal primer pairs. Nested PCR products were digested with RsaI and SspI restriction enzymes. The results revealed that the detection rate of ‘Ca. P. pyri’ in different plant tissues was greatly depending on the sample collection period. The fruit tissues, which were only sampled in September due to the ripening time of Deveci pear cultivar in Bursa, showed the highest detection rate of ‘Ca. P. pyri’ (100%) followed by flower tissues (75%). The average detection rate in root, shoot tissues and leaf midribs was found as 43.75, 39.58 and 16.25%, respectively. The present results showed that the best plant tissues for detecting ‘Ca. P. pyri’ in pear trees were fruit columella and flowers. The highest detection rate of this phytoplasma in root tissues was found from November to March, whereas it could be detected whole year around except summer months in shoot samples in Turkey.Conclusions: For 'Candidatus Phytoplasma pyri', detection, if there is no seasonal limitation for testing, the most suitable tissues are fruits and flowers. When it comes to testing throughout the year, the most suitable tissues were determined as the root, the phloem and cambium layer of the shoots and the leaves, respectively.Significance and Impact of the Study: This study on seasonal variations of ‘Candidatus Phytoplasma pyri’ in different pear tissues has been first time investigated in Turkey. This preliminary data provides important knowledge on molecular detection of Ca. P. pyri, causal agent of pear decline disease for further studies and sertification-quarantine programmes of pear trees in Turkey.

“First report of ‘Candidatus Phytoplasma pyri’-related strain causing pear decline in Argentina"

A survey was made to determine the incidence of phytoplasmas in 39 sweet and sour cherry, peach, nectarine, apricot and plum commercial and experimental orchards in seven growing regions of Poland. Nested polymerase chain reaction (PCR) using the phytoplasma-universal primer pairs P1/P7 followed by R16F2n/ R16R2 showed the presence of phytoplasmas in 29 of 435 tested stone fruit trees. The random fragment length polymorphism (RFLP) patterns obtained after digestion of the nested PCR products separately with RsaI, AluI and SspI endonucleases indicated that selected Prunus spp. trees were infected by phytoplasmas belonging to three different subgroups of the apple proliferation group (16SrX-A, -B, -C). Nucleotide sequence analysis of 16S rDNA fragment amplified with primers R16F2n/R16R2 confirmed the PCR/Restriction Fragment Length Polymorphism (RFLP) results and revealed that phytoplasma infecting sweet cherry cv. Regina (Reg), sour cherry cv. Sokowka (Sok), apricots cv. Early Orange (EO) and AI/5, Japanese plum cv. Ozark Premier (OzPr) and peach cv. Redhaven (RedH) was closely related to isolate European stone fruit yellows-G1 of the 'Candidatus Phytoplasma prunorum' (16SrX-B). Sequence and phylogenetic analyses resulted in the highest similarity of the 16S rDNA fragment of phytoplasma from nectarine cv. Super Queen (SQ) with the parallel sequence of the strain AP 15 of the 'Candidatus Phytoplasma mali' (16SrX-A). The phytoplasma infecting sweet cherry cv. Kordia (Kord) was most similar to the PD1 strain of the 'Candidatus Phytoplasma pyri' (16SrX-C). This is the first report of the occurrence of 'Ca. P. prunorum', 'Ca. P. mali' and 'Ca. P. pyri' in naturally infected stone fruit trees in Poland.

Pear decline (PD) is one of the most devastating diseases of Pyrus communis in Europe and North America. It is caused by the pathogen 'Candidatus Phytoplasma pyri' and transmitted by pear psyllids (Cacopsylla pyri, C. pyricola, and C. pyrisuga). Identifying attractant and repellent volatile organic compounds (VOCs) could improve the development of alternative plant protection measurements like push-pull or attract-and-kill strategies against pear psyllids. Our objective was to investigate which chemical cues of the host plant could influence the host-seeking behavior of pear psyllids, and if cedarwood (CWO) and cinnamon bark (CBO) essential oils could serve as repellents. Based on the literature, the five most abundant VOCs from pear plants elicited EAG responses in both C. pyri and C. pyrisuga psyllid species. In Y-olfactometer trials, single compounds were not attractive to C. pyri. However, the main compound mixture was attractive to C. pyri and C. pyrisuga females. CWO and CBO were repellent against C. pyri, and when formulated into nanofibers (NF), both were repellent in olfactometer trials. However, CBO nanoformulation was ineffective in masking the odors of pear plants. In a field trial, attractive, repellent CWO and blank formulated NF were inserted in attractive green sticky traps. C. pyri captures in traps with CWO NF were statistically lower than in traps with the attractive mixture. Nevertheless, no statistical differences in the numbers of caught specimens were observed between CWO NF and those captured in green traps baited with blank NF. Transparent traps captured fewer psyllids than green ones. In a second field study with a completed different design (push-and-count design), dispensers filled with CBO were distributed within the plantation, and attractive green sticky traps were placed around the plantation. The numbers of trapped pear psyllids increased significantly in the border of the treated plantation, showing that psyllids were repelled by the EOs in the plantation. Although further field evaluation is needed to assess and improve their effectiveness, our results show that these aromatic compounds, repellent or attractive both in nanoformulations and marking pen dispensers, offer great potential as an environmentally sustainable alternative to currently applied methods for managing pear decline vectors.

In the last twenty years, three phytoplasma diseases were identified in Hungary, viz. European Stone Fruit Yellows (ESFY) (caused by Candidatus Phytoplasma prunorum), pear decline (caused by Candidatus Phytoplasma pyri), and apple proliferation (caused by Candidatus Phytoplasma mali). Candidatus Phytoplasma prunorum was isolated from apricot, peach, plum and japanese plum. Cacopsylla pruni the vector of ESFY was also isolated and identified. Infection of Candidatus Phytoplasma pyri was diagnosed from pear and Candidatus Phytoplasma mali was found on apple and pear. The three phytoplasmas cause different damages on their host plants. The most economically important phytoplasma disease is the ESFY. It seriously impairs apricot and japanase plum trees. After infection of apricots and japanese plums show yellowing and defoliation, and within a few years die in apoplexy-like symptoms. The disease on japanese plum is so severe that this fruit practically can not be cultivated in Hungary. Pear decline is the most serious problem especially in intensive pear plantations. The vector Cacopsylla pyri, C. pyrisuga and C. pyricola can be found in almost all pear orchards. Because of the regular presence of psyllids in intensive pear orchards the insecticide control is necessary. Apple proliferation is not an important disease in Hungary. All of our isolations of ’Candidatus Phytoplasma mali’ occured in organic orchards and record was not available in Hungary lately.

In einem faktoriellen Versuch wurde der Einfluss von drei nukleinsaure-basierten Nachweisverfahren und zwei Probenumfangen auf die Nachweisrate von ‘Candidatus Phytoplasma pyri’ (Birnenverfallserreger) bei drei Birnensorten/Unterlagenkombinationen im Jahresverlauf untersucht. Triebproben wurden in etwa monatlichem Abstand uber einen Zeitraum von 17 Monaten von naturlich infizierten Baumen entnommen. Als Nachweismethoden wurden die nested PCR (nPCR), real-time LAMP (rtLAMP) und in geringerem Umfang real-time PCR (rtPCR) verwendet. Die Probenumfange umfassten zwei Teilprobenmengen, bestehend aus drei bzw. zwei Triebstucken (insgesamt funf) je Baum. Das Nachweisniveau war stark von der getesteten Sorte abhangig. Es war hoch bei der Kreuzungsnummer ‘48–40–95’ (81,4%), aber niedrig bei ‘Conference’ (26,2%) und ‘Xenia’ (19,2%), in deren Wiederholungsbaumen teilweise fur langere Zeitabschnitte keine positiven Nachweise erhalten wurden, was zu insgesamt geringen Nachweisraten fuhrte. In der Summe aller Sorten gab es keine signifikanten Nachweisunterschiede zwischen der nPCR und rtLAMP, dagegen wurden mit der rtPCR signifikant hohere Nachweisraten im Vergleich mit den anderen Nachweisverfahren erzielt. Durch die Untersuchung von beiden Teilproben anstatt nur einer je Baum konnte die Nachweisrate in den meisten Fallen verbessert werden und ergab in der Summe aller Untersuchungen fur die Methoden und Sorten eine Steigerung um 29.4%. Die Nachweisrate variierte im Jahresverlauf in Abhangigkeit von der Methode und der Sorte, die meisten Baume konnten jedoch im Zeitraum Spatwinter bis Anfang Fruhjahr positiv getestet werden, auser bei ‘48–40–95’, in welcher der Erreger fast ganzjahrig nachgewiesen werden konnte. Ein Vergleich des Einflusses der untersuchten Faktoren auf die Nachweisrate ergab fur die Sorte/Unterlage den grosten Effekt, gefolgt von der Methode und dem Probenumfang mit etwa gleich grosen Auswirkungen.

The aim of this research was to investigate the relationship among leaf mineral nutrition, soil chemical fertility, presence of phytoplasma in fruit trees and susceptibility of the plant to become infected. In the experimental farm, pear and apricot orchards were cultivated, in separate areas far 30 metres each other and since 1997 they resulted respectively infected by 'Candidatus Phytoplasma pyri' and by 'Candidatus Phytoplasma prunorum'. In 1999 whole collections of these two species had been eradicated and substituted with new plants of the same species. Four pear plants had been selected in the old collection and had been covered by anti-aphid tissue. These pear plants (three infected by the phytoplasma and one healthy) and four healthy apricot plants were analysed for the presence of phytoplasma, for the nutritional status of leaves as well as for the soil chemical fertility. This study was carried out in two years, from May 2006 to September 2007. The results showed a Fe/Mn ratio unbalanced in the pear infected plants compared with the pear healthy ones. The pear leaves of the healthy plant didn't show any disequilibrium, while those of the infected plants showed the unbalanced ratio Fe/Mn either when the molecular test for phytoplasma detection was still negative. The apricot leaves, always resulted free from phytoplasmas, showed a K/Mg imbalance.

SUMMARY A survey was carried out in germplasm nurseries and commercial orchards from ten provinces in Turkey during 2003-2005. Samples were collected from trees showing European stone fruit yellows (ESFY) and Pear decline (PD) symptoms. A total of 270 stone fruit and six pear samples were tested by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP). Universal phytoplasma primers P1/P7 and R16F2n/R2 were used for one-step and nested PCR assays, respectively. Nested-PCR products were digested with RsaI, SspI and MseI restriction enzymes. Twentyeight of 270 samples including plum, apricot, peach and almond were infected by Candidatus Phytoplasma prunorum. Infection rate by the same phytoplasma species of germplasm nurseries containing mostly foreign cultivars was 54.8%, whereas it was only 3.2% in commercial orchards. Ca. Phytoplasma prunorum was detected for the first time in Antalya, Gaziantep, Icel, Isparta, and Yalova provinces in local apricot cultivars as well as Japanese plum, apricot, and almond cultivars imported from Japan. PCR and RFLP tests of pear samples collected from commercial orchards in the Bursa province showed that the local pear cultivar ‘Deveci’ was infected by Candidatus Phytoplasma pyri, which was also found in the pear psyllid Cacopsylla pyri L. To our knowledge, this is the first report of detection of Ca. Phytoplasma pyri in C. pyri in Turkey.

During the 2015 growing season, peach plants (Prunus persica) showing chlorotic, curling leaves and midrib thickening were detected in production fields in Mendoza province (Midwest region, Argentina). These symptoms resemble those described for Peach Yellow Leaf Roll disease, a ‘Candidatus Phytoplasma pyri’ related strain. By PCR of the 16S rDNA gene, a phytoplasma was detected in symptomatic samples. The RFLP profile, sequence identity and phylogenetic analyses showed that the Peach Yellows phytoplasma belongs to group 16SrX-C and is identical to those described as ‘Candidatus Phytoplasma pyri’. This is the first report of a phytoplasma from 16Sr group X infecting peach trees in Argentina and in South America.

A recent severe outbreak of pear decline in a four month-old orchard in Northern Italy is reported. The symptom was seen since July 2019 in about 30% of the plants which increased to 80% by the end of the summer season. Molecular analyses confirmed the presence of the pear decline phytoplasma (16SrX-C), ‘Candidatus Phytoplasma pyri’ in symptomatic and asymptomatic leaf materials and also in the roots of the symptomatic plants. The infected plants belong to the cultivar Abate Fetel grafted to quince MA; considering the frequency of the symptom appearance after transplantation and its spread in the field it is very likely that the rootstock was already phytoplasma infected and contributed to the quick dissemination of the pathogen in the field that was also emphasized by the presence of low numbers of the pear decline phytoplasma insect vector Cacopsylla pyri. The orchard is under process of uprooting and will be replanted with phytoplasma-free tested materials.