Transmission of ‘Candidatus Phytoplasma pyri’ by naturally infected Cacopsylla pyri to peach,an approach to the epidemiology of peach yellow leaf roll (PYLR) in Spain

Peach orchards in the northeast of Spain were severely affected in 2012 by a previously unreported disease in this area. The symptoms included early reddening, leaf curling, decline, abnormal fruits, and in some cases death of the peach trees. All the infected peach samples were positive for ‘Candidatus Phytoplasma pyri’, but none were infected by the ‘Ca. Phytoplasma prunorum’. In this work, potential vectors able to transmit ‘Ca. Phytoplasma pyri’ from pear to peach and between peach trees were studied and their infective potential was analysed at different times of the year. Transmission trials of the phytoplasma with potential vectors to an artificial feeding medium for insects and to healthy peach trees were conducted. Additionally, isolated phytoplasmas were genetically characterized to determine which isolates were able to infect peach trees. Results showed that the only insect species captured inside peach plots that was a carrier of the ‘Ca. Phytoplasma pyri’ phytoplasma was Cacopsylla pyri. Other insect species captured and known to be phytoplasma transmitters were present in very low numbers, and were not infected with ‘Ca. Phytoplasma pyri’ phytoplasma. A total of 1928 individuals of C. pyri were captured in the peach orchards, of which around 49% were phytoplasma carriers. All the peach trees exposed to C. pyri in 2014, and 65% in 2015, were infected by ‘Ca. Phytoplasma pyri’ 1 year after exposure, showing that this species is able to transmit the phytoplasma to peach. Molecular characterization showed that some genotypes are preferentially determined in peach.     

Frequency and seasonal distribution of pear psylla infected with the pear decline phytoplasma in california pear orchards

ABSTRACT Pear decline (PD) is an important disease of Pyrus communis fruiting cultivars in Europe, Asia, and the Americas. PD is caused by a phloem-limited phytoplasma that, in California, is transmitted from diseased to healthy trees by pear psylla, Cacopsylla pyricola. The percentage of phytoplasma-infected pear psylla has never been assessed in the United States in field-collected insects. Pear psylla were collected monthly from PD-infected trees from three orchards in northern California. Individual psylla were tested for the presence of PD phytoplasma, using both a quantitative DNA hybridization and PD phytoplasma-specific polymerase chain reaction (PCR) assays. The percentage of infected psylla ranged from 0 to 45% depending on the orchard, the month and year collected, and the method of detection. The PD phytoplasma was detected in both the winterform and summerform pear psylla. Significantly more infected psylla were detected with PCR than with DNA hybridization analysis in two of the three orchards. The number of PD phytoplasma per pear psylla was estimated to range from 1 x 10(6) to 8.2 x 10(7). The percentage of PD-infected pear psylla found in the three northern California pear orchards suggests that both winterform and summerform pear psylla could be important in the transmission of PD.     

Detection and identification of the phytoplasma associated with pear decline in Taiwan

Pear decline (PD) is an important phytoplasmal disease that occurs mainly in Europe and North America. In 1994, pear trees exhibiting symptoms typical of PD disease were observed in orchards of central Taiwan. The sequence of 16S rDNA and 16S–23S rDNA intergenic spacer region (ISR) of the causative agent of pear decline in Taiwan (PDTW) were amplified with polymerase chain reaction (PCR) using a DNA template prepared from the diseased leaves. Sequence analysis of 16S rDNA revealed that the PDTW agent was closely related to the phytoplasmas of the apple proliferation group that cause diseases in stone fruits, pear and apple. Consistent with the result of 16S rDNA sequence analysis, sequence analysis of the 16S–23S rDNA ISR and putative restriction site analyses of 16S rDNA and 16S–23S rDNA ISR sequences provided further support for the view that the PDTW phytoplasma causing pear decline in Taiwan may represent a new subgroup of the apple proliferation group. According to the rDNA sequence of PDTW phytoplasma, two specific PCR primer pairs, APf2/L1n and fPD1/rPDS1, were designed in this study for the detection of the etiological agent in pear trees and insect vectors. Based on the sequence analyses of the PCR-amplified fragments, two species of pear psyllas, Cacopsylla qianli and Cacopsylla chinensis, were found to carry PDTW phytoplasma.     

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.     

Pear decline in Spain

Pear decline in Spain, associated with the presence of phytoplasmas in sieve tubes, was studied. Samples of healthy and diseased pear trees were tested to confirm the presence of the pathogen. The polymerase chain reaction (PCR) technique was used, with universal and specific primers. Specimens of Cacopsylla pyri were also analysed by PCR. Phytoplasmas were detected in 79% of trees with premature reddening, in 67% of trees with weakness and necrotic spots (cv. Limonera) and in 20% of trees without symptoms. The pathogen was also detected in the psyllids, indicating that C. pyri could be the vector of the disease in Spain.     

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.     

Natural enemies of the pear psylla<Emphasis Type="Italic">Cacopsylla pyri</Emphasis> in treated<Emphasis Type="Italic">vs</Emphasis> untreated pear orchards in Antalya,Turkey

Populations of pear psylla,Cacopsylla pyri (L.) (Rhynchota: Psyllidae), and its natural enemies were sampled on pears in Antalya province during the years 2000–2002. Thirty-two species of predators and three species of parasitoids were detected to be associated withC. pyri in Antalya province. The heteropterans (especially anthocorid species) were the most abundant predator group. Among the anthocorids,Anthocoris nemoralis (Fabricius) (Heteroptera: Anthocoridae), whose population was closely related to the dynamics of the pear psylla population, was generally the principal antagonist ofC. pyri. Other species recorded during the study were the miridDeraeocoris spp., the chrysopidChrysoperla carnea L., and many coccinellid species. Although these predators are polyphagous, as are theOrius species, their populations were related to the psylla fluctuations. Among the parasitoid complex belonging to the order Hymenoptera, the encyrtidTrechnites psyllae (Ruschka) was the only primary parasitoid. Two hyperparasites,viz., Syrphophagus mamitus (Walker) andPachyneuron aphidis (Bouché) (Encyrtidae and Pteromalidae, respectively), were detected in the surveys. Also, this study revealed new information concerning the pear psylla parasitoid complex in Turkey. WhileT. psyllae andS. mamitus are new records to Turkish parasitoid fauna,C. pyri is a new host record forP. aphidis. The population development and abundance of pear psylla and its natural enemies, as well as the parasitization rates in treated and untreated pear orchards, are presented here. http://www.phytoparasitica.org posting May 6, 2004.     

人工培养液在植原体昆虫介体筛选中的适用性

研究了一种人工培养液对各种常见的昆虫(主要是叶蝉)的亲和性和适用性.结果表明,该人工培养液适于本试验中大多数昆虫的人工饲养.用此方法,悬钩子广头叶蝉Macropsis.ftscula Zetterstedt和桤树广头叶蝉Oncopsis alniSchrank分别被再次确认为悬钩子矮化植原体和桤树黄化植原体的传播介体;田旋花麦蜡蝉Hyalesthes obsoletus Signoret再次被确认为葡萄黄化(stolbur)植原体的传播介体.此前,上述三种叶蝉已被传统的人工接种方法鉴定为相应植原体的传播介体.危害桤树的河谷树叶蝉Allygus modestus Scott尽管虫体DNA检测结果经常为阳性,但迄今其人工培养液的检测结果都是阴性,因此,我们认为河谷树叶蝉不是桤树黄化植原体的传播介体.Eppendorf管人工培养液饲养法不仅适用于潜在的植原体介体昆虫的筛选鉴定,而且可用于介体昆虫的生物防治研究.此外,本研究首次发现自然感染了葡萄上的一种被德国人称为"Vergi-lungskrankheit"植原体(AY组)的草地脊冠叶蝉Aprodes makarovi Zachvatkin.     

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.     

Inoculum availability and seasonal survival of Potebniamyces pyri in pear orchards

Potebniamyces pyri (anamorph Phacidiopycnis piri) is the causal agent of Phacidiopycnis rot, a postharvest disease of pear fruit (Pyrus communis). Infections of pear fruit by P. pyri occur in the orchard, and symptoms develop after harvest during storage or in the market. P. pyri also is the cause of a canker and twig dieback disease of pear trees. To determine inoculum availability of P. pyri, dead bark and dead fruit spurs were periodically collected in two commercial ‘d’Anjou’ pear orchards and examined for the presence and viability of fruiting bodies of P. pyri. To determine seasonal survival of P. pyri, 2-year-old twigs of ‘d’Anjou’ pear in a research orchard were inoculated approximately monthly over 2 years with P. pyri and monitored for canker development. Inoculated twigs were removed from the trees 6 months post inoculation and examined for formation, viability of pycnidia of P. pyri, and reisolation of the pathogen. In both commercial orchards, all sampled trees were infected by P. pyri; viable pycnidia of P. pyri were observed on 42–78 % of the sampled bark and 5–32 % of the sampled fruit spurs; and viable conidia were observed at all sampling times during the fruit growing season. Apothecia of P. pyri also were observed on sampled dead bark and fruit spurs, but at a frequency ranging from 0 % to 19 %. P. pyri was recovered from inoculated twigs 6 months after inoculation at all sampling times during the 2-year study, but recovery frequency varied. P. pyri formed pycnidia on most cold-injured and inoculated twigs. The results suggest that: i) the conidial state of P. pyri is the main type of inoculum in pear orchards in the region; ii) viable inoculum of P. pyri for potential fruit infections is available during the pear fruit-growing season; iii) P. pyri can form pycnidia on cankers of twigs infected by the fungus at different seasons during the year; and iv) P. pyri can survive as mycelium in diseased pear twigs year round in the orchard.     

Possible phytoplasma transovarial transmission in the psyllids Cacopsylla melanoneura and Cacopsylla pruni

The possible transovarial transmission of two phytoplasmas, ' Candidatus Phytoplasma mali' and ' Candidatus Phytoplasma prunorum', through their respective psyllid vectors Cacopsylla melanoneura and Cacopsylla pruni , was investigated. Different life stages of the progeny of infected female psyllids were analysed by PCR detection of phytoplasma DNA. While ' Ca. Phytoplasma mali' could not be detected in any of the C. melanoneura life stages tested, ' Ca. Phytoplasma prunorum' could be detected in eggs, nymphs and newly emerged adults of C. pruni . Infectivity tests using both nymphs and newly emerged adults of C. pruni showed that ' Ca. Phytoplasma prunorum' inherited from infected females can be transmitted to healthy plum plants. Although further validations are required, these findings open up new perspectives on the study of the epidemiology of diseases associated with European stone fruit yellows.     

梨衰退植原体Cycleave 实时荧光PCR检测方法的建立

 根据植原体16S rDNA 保守区设计Cycling 探针LST2probe及引物,建立了梨衰退植原体Cycleave实时荧光PCR检测方法。结果表明,探针LST2probe 能特异的检测梨衰退植原体,供试同一组内不同亚组植原体及参试病原细菌均为阴性,检测灵敏度可达0.5 pg/μL。该Cycleave实时荧光PCR检测方法可用于梨衰退植原体的快速检测,并为其他有害生物鉴定提供借鉴依据。     

Oncopsis alni (Schrank) (Auchenorrhyncha: Cicadellidae) as a Vector of the Alder Yellows Phytoplasma of Alnus glutinosa (L.) Gaertn.

Alder yellows phytoplasma was detected by PCR in Alnus glutinosa trees in the Palatine and Mosel areas of Germany. The restriction profiles obtained by TaqI and AluI digestion of a PCR amplified ribosomal DNA fragment from this phytoplasma and a periwinkle isolate of alder yellows from Italy (ALY) could not be distinguished while elm yellows isolates from Europe and North America led to different fragment patterns. Different restriction profiles for ALY and the German alder phytoplasma were obtained by TruI digestion of a non-ribosomal DNA fragment. Phloem feeding insects were collected from infected alder trees. Phytoplasmas of the elm-yellows group were detected by PCR in psyllids and the leafhopper Oncopsis alni. These pathogens were indistinguishable from the phytoplasma found in alder. Only O. alni was able to transmit the pathogen to healthy alder seedlings. Thus, it is the first insect known to transmit this phytoplasma. This leafhopper could be responsible for the ubiquitous infection of Alnus glutinosa due to its close association with alder and its wide distribution in Europe.     

A New Natural Planthopper Vector of Stolbur Phytoplasma in the Genus Pentastiridius (Hemiptera: Cixiidae)

A new disease of sugar beet called Syndrome des Basses Richesses, which appeared in Burgundy and Franche-Comté, France, in 1991, is of uncertain aetiology. However, evidence for aerial transmission of the disease, symptom similarity with yellow wilt and preliminary results of phytoplasma detection, support the hypothesis of a phytoplasma being associated to the disease. A search for a natural phytoplasma vector, was conducted in Franche-Comté in 1997 and 1998, in an area where sugar beet crops had been affected since 1996. A cixiid, tentatively identified as Pentastiridius beieri, not described in the preceding years and not formerly reported as a phytoplasma vector, was present in sugar beet plots in high populations from June to August in 1997 and 1998. Individuals were captured and used for transmission experiments to periwinkle and sugar beet seedlings. They were further tested for the presence of a phytoplasma in their body, using PCR amplification of 16S rDNA of phytoplasmas. In 1997 and 1998, from 2% to 13.3% of the individuals carried a stolbur phytoplasma and insects which tested positive, appeared to have transmitted, through feeding, a stolbur phytoplasma to periwinkles and to sugar beets. This cixiid, whose vectoring capacity of stolbur phytoplasma to plants, is now clearly demonstrated, is available for experimental inoculations, in order to examine the role of phytoplasmas in the Syndrome des Basses Richesses, through the observation of symptom expression in phytoplasma-inoculated plants.     

Laboratory evaluation of a botanical natural product (<Emphasis Type="Italic">AkseBio2</Emphasis>) against the pear psylla<Emphasis Type="Italic">Cacopsylla pyri</Emphasis>

A botanical natural product,AkseBio2, was evaluated under laboratory conditions for its oviposition deterrent, ovicidal and larvicidal (nymphicidal) effects against the pear psyllaCacopsylla pyri (L.) (Hemiptera: Psyllidae). The product exhibited a strong oviposition deterrent effect for winterform and summerform females and caused a reduction in the total number of eggs laid in both choice and no-choice assays. Significant mortalities in freshly laid eggs (0–48 h) and various nymphal stages of the pest were recorded in toxicity assays. At a concentration of 0.1% (formulation), the highest biological activity of the product was recorded against the young (1st and 2nd) nymphal stages (up to 87.4% mortality) in comparison with the other biological stages of the pest. It was less active against the older (3rd-5th) nymphs, causing 62.1% mortality at the same concentration. In assays with non-target organisms, a significant negative effect was not observed. There were no significant changes on treated plants up to 7 days after treatment in any trial, nor was there any phytotoxicity on plant tissue as a result ofAkseBio2 treatments. The results suggest that the product can be used in psylla control instead of synthetic insecticides and may serve as an integrated pest management (IPM) component in pear orchards. http://www.phytoparasitica.org posting July 14, 2004.     

The insect vector Cacopsylla picta vertically transmits the bacterium ‘Candidatus Phytoplasma mali’ to its progeny

The phloem‐sucking psyllid Cacopsylla picta plays an important role in transmitting the bacterium ‘Candidatus Phytoplasma mali’, the agent associated with apple proliferation disease. The psyllid can ingest ‘Ca. Phytoplasma mali’ from infected apple trees and spread the bacterium by subsequently feeding on uninfected trees. Until now, this has been the most important method of ‘Ca. Phytoplasma mali’ transmission. The aim of this study was to investigate whether infected C. picta are able to transmit ‘Ca. Phytoplasma mali’ directly to their progeny. This method of transmission would allow the bacteria to bypass a time‐consuming reproductive cycle in the host plant. Furthermore, this would cause a high number of infected F₁ individuals in the vector population. To address this question, eggs, nymphs and adults derived from infected overwintering adults of C. picta were reared on non‐infected apple saplings and subsequently tested for the presence of ‘Ca. Phytoplasma mali’. In this study it was shown for the first time that infected C. picta individuals transmit ‘Ca. Phytoplasma mali’ to their eggs, nymphs and F₁ adults, thus providing the basis for a more detailed understanding of ‘Ca. Phytoplasma mali’ transmission by C. picta.     

Incidence of ‘Candidatus Liberibacter europaeus’ and phytoplasmas in Cacopsylla species (Hemiptera: Psyllidae) and their host/shelter plants

Psyllids, as vectors of phloem-restricted plant pathogens, are serious agricultural pests. Fruit tree phytoplasmas are transmitted by different Cacopsylla spp., while other psyllids are known vectors of liberibacters. Recently, the bacterium ??Candidatus Liberibacter europaeus?? was found in pear trees and in Cacopsylla pyri (Linnaeus), the vector of ??Ca. Phytoplasma pyri??. This new species does not cause symptoms in plants and is probably a symbiont rather than a pathogen. Based on these findings and the assumption that ??Ca. Liberibacter europaeus?? is widespread, we studied its distribution in the genus Cacopsylla and in the respective host and shelter plants (where psyllids aestivate and overwinter), as well as its possible co-presence with ??Ca. Phytoplasma?? spp. We tested 14 Cacopsylla species and 11 plant species from northwestern Italy, Hungary and Israel, characterized by warm oceanic, temperate continental and warm Mediterranean climatic conditions, respectively. ??Ca. Liberibacter europaeus?? was common within the Cacopsylla genus, being present in nine of the 14 species screened as well as in most host plants, whereas none of the shelter plants tested positive for this bacterium. Altogether, these findings indicate the presence of ??Ca. Liberibacter europaeus?? in continental zones, whereas it does not seem to be widespread in the Mediterranean region. Furthermore, lack of specific symptoms in all infected plants confirms an endophytic relationship with this bacterium, while its abundance in insects suggests a beneficial role for the host. Co-infections with phytoplasmas, observed in insects and plants, require further study to evaluate the possible interactions between them.     

Composition,abundance and phytoplasma infection in the hawthorn psyllid fauna of northwestern Italy

Hawthorn (Crataegus monogyna) is one of the natural hosts of Cacopsylla melanoneura, the acknowledged vector of ‘Candidatus Phytoplasma mali’, the causal agent of Apple Proliferation disease, a serious and growing problem for apple production in Europe, particularly in northern Italy. Wild plants could be important sources of both insects and phytoplasmas, but their role in the epidemiology of phytoplasma diseases and their insect vectors has never been thoroughly examined. Cacopsylla melanoneura’s primary host is hawthorn, a plant closely related to apple which often grows wild near orchards. Other psyllid species feed on hawthorn, but no data are available on their possible role as phytoplasma vectors. We investigated the hawthorn’s psyllid fauna in northwestern Italy using yellow sticky traps, beat trays, and molecular analyses from 2003–2005, to study the relationship between hawthorn, the phytoplasma and the insect vector. Population dynamics were monitored, and insects and hawthorn samples were analysed by polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP), and DNA sequencing for the presence of phytoplasmas. Cacopsylla melanoneura was the dominant psyllid species, followed by C. peregrina, C. affinis and C. crataegi. PCR and RFLP analyses revealed the presence of different fruit tree phytoplasmas in hawthorn plants, and in all four psyllid species.     

Expression of phytoplasma‐induced witches’ broom disease symptoms in acid lime (Citrus aurantifolia) trees is affected by climatic conditions

Witches’ broom disease (WBD), caused by ‘Candidatus Phytoplasma aurantifolia’, is a serious disease of acid lime (Citrus aurantifolia) in Oman and the UAE. However, little is known about the distribution of phytoplasma and the expression of WBD symptoms in different geographical locations. A survey was carried out in 18 districts in Oman and the UAE covering 143 orchards and 5823 acid lime trees. ‘Candidatus Phytoplasma aurantifolia’ was detected in acid lime in all the 18 surveyed districts. However, the development of typical symptoms of WBD was only observed in 12 districts. Districts in which the phytoplasma was present but symptoms were not expressed were located either in desert areas or in areas characterized by semitropical conditions. Phylogenetic analysis of 16 phytoplasma isolates from trees developing WBD symptoms and six phytoplasma isolates from trees with no WBD symptoms showed that all isolates share an identical 16S rRNA sequence, belonging to subgroup II‐B. Quantitative PCR analysis showed that the concentration of phytoplasma is significantly higher (8800–801 000 copies) in leaves developing WBD symptoms compared to 2–268 copies in symptomless leaves from the same trees and 8–874 copies in acid lime trees from areas where disease symptoms were not expressed. The lack of expression of WBD symptoms under certain environmental conditions may suggest that symptom development and phytoplasma are affected by certain unfavourable environmental conditions. These findings could provide a basis for managing WBD through encouraging lime cultivation under climatic conditions less conducive to WBD symptom expression.     

Oviposition deterrency and deterrent stability of some oily substances against the pear psylla<Emphasis Type="Italic">Cacopsylla pyri</Emphasis>

Oviposition deterrents have the potential to prevent insect infestations in agroecosystems. In the present study, the level and duration of this behavioral effect was evaluated against pear psylla,Cacopsylla pyri (L.) (Hemiptera: Psyllidae), a serious pest of pears in Turkey. The activity of four oily substances — cotton seed oil, fish-liver oil, neem oil and summer oil — was tested against winterform and summerform females of the pest in laboratory and field experiments, in order to determine their oviposition deterrency and stability as deterrents. In the laboratory assays, the initial (3 days after treatments) deterrency of all test materials was 100%. While decreases in initial deterrency started after 1 week for neem oil and after 2 weeks for cotton seed oil, fish-liver oil and summer oil exhibited 100% deterrent activity against winterforms, but not summerforms during the 3-week test period. The last two also exhibited strong oviposition deterrent activity for both forms ofC. pyri females in field trials. A significant reduction in the total number of eggs laid was observed in the field plots. Data showed that summer oil and fish-liver oil are the most promising oviposition deterrents against pear psylla females. http://www.phytoparasitica.org posting Sept. 28, 2004.