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.     

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.     

PM 9/25 (1) Bactericera cockerelli and ‘Candidatus Liberibacter solanacearum’

Specific scope

This Standard describes a national regulatory control system for Bactericera cockerelli and the bacterial pathogen ‘Candidatus Liberibacter solanacearum’ the cause of zebra chip disease in potato. The scope is as follows:

• Exclusion from the EPPO region of B. cockerelli an efficient vector of ‘Ca. L. solanacearum’ within solanaceous crops (e.g. potato, tomato)
• Eradication of incursions of B. cockerelli
• Exclusion from the EPPO region of ‘Ca. L. solanacearum’ haplotypes A and B. Although reference will only be made to haplotypes A and B, the Standard would also apply to new non‐European haplotypes of ‘Ca. L. solanacearum’ which may have different host ranges, or which may be vectored more efficiently by psyllids which are widespread in the region.

The reduction of the risk of spreading ‘Ca. L. solanacearum’ haplotypes C, D and E to potato production systems and potatoes being moved within the EPPO region may be recommended in future when more information is available but is not covered in this Standard.

Specific approval

First approved in 2017‐09.     

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

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

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

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

New natural host plants of ‘Candidatus Phytoplasma pini’ in Poland and the Czech Republic

The presence of phytoplasmas in seven coniferous plant species (Abies procera, Pinus banksiana, P. mugo, P. nigra, P. sylvestris, P. tabuliformis and Tsuga canadensis) was demonstrated using nested PCR with the primer pairs P1/P7 followed by R16F2n/R16R2. The phytoplasmas were detected in pine trees with witches’ broom symptoms growing in natural forest ecosystems and also in plants propagated from witches’ brooms. Identification of phytoplasmas was done using restriction fragment length polymorphism analysis (RFLP) of the 16S rDNA gene fragment with AluI, MseI and RsaI endonucleases. All samples showed RFLP patterns similar to the theoretical pattern of ‘Candidatus Phytoplasma pini’, based on the sequence of the reference isolate Pin127S. Nested PCR‐amplified products, obtained with primers R16F2n/R16R2, were sequenced. Comparison of the 16S rDNAs obtained revealed high (99·8–100%) nucleotide sequence identity between the phytoplasma isolates. The isolates were also closely related to four other phytoplasma isolates found in pine trees previously. Based on the results of RFLP and sequence analyses, the phytoplasma isolates tested were classified as members of the ‘Candidatus Phytoplasma pini’, group 16SrXXI.     

Transmission of ‘Candidatus Liberibacter solanacearum’ in carrot seeds

A protocol for the specific detection and quantification of ‘Candidatus Liberibacter solanacearum’ in carrot seeds using real‐time PCR was developed. The bacterium was detected in 23 out of 54 carrot seed lots from 2010 to 2014, including seeds collected from diseased mother plants. The average total number of ‘Ca. L. solanacearum’ cells in individual seeds ranged from 4·8 ± 3·3 to 210 ± 6·7 cells per seed from three seed lots, but using propidium monoazide to target live cells, 95% of the cells in one seed lot were found to be dead. Liberibacter‐like cells were observed in the phloem sieve tubes of the seed coat and in the phloem of carrot leaf midrib from seedlings. The bacterium was detected as early as 30 days post‐germination, but more consistently after 90 days, in seedlings grown from PCR positive seed lots in an insect‐proof P2 level containment greenhouse. Between 12% and 42% of the seedlings from positive seed lots tested positive for ‘Ca. L. solanacearum’. After 150 days, symptoms of proliferation were observed in 12% of seedlings of cv. Maestro. ‘Candidatus Liberibacter solanacearum’ haplotype E was identified in the seeds and seedlings of cv. Maestro. No phytoplasmas were detected in seedlings with symptoms using a real‐time assay for universal detection of phytoplasmas. The results show that to prevent the entry and establishment of the bacterium in new areas and its potential spread to other crops, control of ‘Ca. L. solanacearum’ in seed lots is required.     

PM 7/128 (1) Xanthomonas axonopodis pv. allii

Specific scope

This Standard describes a diagnostic protocol for Xanthomonas axonopodis pv. allii. 1 This Standard should be used in conjunction with PM 7/76 Use of EPPO diagnostic protocols.

Specific approval and amendment

First approved in 2016‐09.     

PM 7/28 (2) Synchytrium endobioticum

Specific scope

This Standard describes a diagnostic protocol for Synchytrium endobioticum. 1 This Standard should be used in conjunction with PM 7/76 Use of EPPO diagnostic protocols.

Specific approval and amendment

Approved in 2003‐09. Revision approved in 2017‐06.     

PM 7/21 (2) Ralstonia solanacearum,R. pseudosolanacearum and R. syzygii (Ralstonia solanacearum species complex)

Specific scope

This Standard describes a diagnostic protocol for Ralstonia solanacearum, Ralstonia pseudosolanacearum and Ralstonia syzygii, i.e. phylotype/sequevar strain in the Ralstonia solanacearum Species Complex (RSSC). 1 It should be used in conjunction with PM 7/76 Use of EPPO diagnostic protocols.

Specific approval and amendment

Approved in 2003‐09. First revised in 2018‐02.     

Molecular diversity of ‘Candidatus Phytoplasma pyri’ isolates in Slovenia

A European quarantine organism ‘Candidatus Phytoplasma pyri’ causing devastating pear decline disease has been reported to affect pear trees in several European countries. In this study a multilocus sequence analysis was successfully used to gain detailed insight into the molecular diversity of thirty closely related ‘Candidatus Phytoplasma pyri’ isolates from different orchards in Slovenia. Among three genomic regions analyzed, the 16S/23S rRNA intergenic spacer region was the most conserved among Slovenian isolates with 99.7 % sequence identity, yielding only three distinct genotypes. On the other hand, five different genotypes were detected when analyzing secY and aceF genomic regions that shared sequence identity of 94.8 and 97.2 %, respectively. Six of the detected genotypes, specifically four in the secY region and one in each of the two other analyzed genomic regions, were unique for Slovenia. At least eight different haplotypes were found with multilocus sequence analysis, indicating high molecular diversity among Slovenian ‘Ca. P. pyri’ isolates. Haplotypes were clustered into two major clusters, separated by at least 45 mutations. No connection was established between haplotype occurrence and cultivar type.     

PM 7/36 (2) Diabrotica virgifera virgifera

Specific scope

This Standard describes a diagnostic protocol for Diabrotica virgifera virgifera. This Standard should be used in conjunction with PM 7/76 Use of EPPO diagnostic protocols.

Specific approval and amendment

Approved in 2003‐09. Revised in 2017‐02.     

PM 7/2 (2) Tobacco ringspot virus

Specific scope

This Standard describes a diagnostic protocol for Tobacco ringspot virus 1 This Standard should be used in conjunction with PM 7/76 Use of EPPO diagnostic protocols.

Specific approval and amendment

First approved in 2000‐09. Revision approved in 2017‐03     

Prima phacie1: a new European Food Safety Authority funded research project taking a comparative approach to pest risk assessment and methods to evaluate pest risk management options

In late 2009, a European Food Safety Authority (EFSA)‐funded project (Prima phacie) began work to review and test methodologies for conducting pest risk assessment by means of case studies on three phytoplasmas (Candidatus Phytoplasma mali, Ca. P. prunorum, Ca. P. pyri); two bacteria (Acidovorax avenae subsp. citrulli, Xanthomonas citri [=X. axonopodis] pv. citri); two fungi (Guignardia citricarpa, Mycosphaerella dearnessii); two nematodes (Meloidogyne chitwoodi, M. fallax); and an insect (Anoplophora glabripennis). Multiple risk assessment schemes and methods will be applied to each of the case study pests, allowing for a comparative assessment of methods. Methods to assess the effectiveness of possible risk management options for each pest will also be evaluated. The project will further develop the scientific basis for pest risk assessment within the European Community and identify methodologies most suitable for conducting risk assessments and for evaluating the effectiveness of possible risk management options by the EFSA Panel on Plant Health in order to support European decision making. The project lasts 29 months, and is being conducted by an international consortium of 11 partners consisting of phytosanitary organizations, research institutes and a university. Results will be disseminated via conventional publications and at a workshop in March 2012.     

High diversity,expanding populations and purifying selection in phytoplasmas causing coconut lethal yellowing in Mozambique

In this study, the putative phytoplasma species causing coconut lethal yellowing disease in Mozambique and Tanzania were characterized. The 16S rRNA and secA genes were sequenced. Phylogenetic analysis revealed that Mozambican coconut phytoplasmas belong to three different types: ‘Candidatus Phytoplasma palmicola’ 16SrXXII‐A, a second strain that was previously isolated in Tanzania and Kenya (16SrIV‐C), and a third strain that was different from all known lethal yellowing phytoplasma species. The third strain potentially represents a novel species and is closely related to pine phytoplasma. Co‐infection with ‘Ca. Phytoplasma pini’‐related and ‘Ca. Phytoplasma palmicola’ 16SrXXII‐A strains was observed. Furthermore, sequence variation in ‘Ca. Phytoplasma palmicola’ at the population level was consistent with purifying selection and population expansion.     

PM 7/89 (2) Heterodera glycines

Specific scope

This Standard describes a diagnostic protocol for Heterodera glycines. 1 This Standard should be used in conjunction with PM 7/76 Use of EPPO diagnostic protocols. Terms used are those in the EPPO Pictorial Glossary of Morphological Terms in Nematology. 2

Specific approval and amendment

Approved in 2008–09. Revision approved in 2017–11.     

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

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