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.     

Molecular tracing of the transmission routes of bois noir in Mediterranean vineyards of Montenegro and experimental evidence for the epidemiological role of Vitex agnus‐castus (Lamiaceae) and associated Hyalesthes obsoletus (Cixiidae)

Epidemiological aspects and transmission routes of bois noir (BN), a grapevine yellows disease induced by ‘Candidatus Phytoplasma solani’, have been exhaustively studied in the affected vineyards of continental Europe but not in the Mediterranean coastal zone. Because ‘Ca. Phytoplasma solani’ and its principal vector Hyalesthes obsoletus presumably originate from the Mediterranean, gaining knowledge of the epidemiological peculiarities of the disease in this area is essential for understanding its global spread and diversification, as well as for designing local management strategies. In this study, molecular epidemiology was applied to trace transmission pathways of ‘Ca. Phytoplasma solani’ in the Mediterranean vineyards of Montenegro, using multilocus sequence typing of tuf, vmp1 and stamp genes of the isolates associated with various hosts. Thus, ‘Ca. Phytoplasma solani’ was tracked from a tentative reservoir plant (inoculum source) through an associated vector population to the infected grapevine. Three pathways of transmission were documented, originating from Urtica dioica, Convolvulus arvensis and Vitex agnus‐castus; however, only the route originating from U. dioica was direct, whereas the latter two were overlapping and could be intermixed. Vitex agnus‐castus is a natural source of ‘Ca. Phytoplasma solani’, representing an important link in disease epidemiology in the Mediterranean and a possible origin of several genotypes occurring in central Europe. Experimental confirmation of the role of Vitex‐associated H. obsoletus in BN transmission in Montenegrin vineyards indicates its tentative role as a vector in the wide area of the Mediterranean, where some of the major wine‐producing regions are located.     

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.     

Leafhoppers Exitianus atratus and Amplicephalus funzaensis transmit phytoplasmas of groups 16SrI and 16SrVII in Colombia

The phytoplasmas of groups 16SrI (‘Candidatus Phytoplasma asteris’) and 16SrVII (‘Ca. Phytoplasma fraxini’) have been associated with phytoplasma diseases in several urban tree species in Bogotá, Colombia and surrounding areas. The insect vectors responsible for this phytoplasma transmission are unknown. The objectives of this study were to test for the presence of phytoplasmas in leafhopper species (Cicadellidae) collected in areas with diseased trees and to determine the phytoplasma transmission ability of two of these species. Leafhoppers of nine species were collected at two sampling sites and tested by nested or double nested PCR using primers for the 16S rRNA gene. The amplicons were subjected to RFLP and/or sequencing analysis. Phytoplasmas of group 16SrI were detected in morphospecies MF05 (Haldorus sp.), group 16SrVII in MF07 (Xestocephalus desertorum), MF08 (Empoasca sp.) and MF09 (Typhlocybinae), and both groups 16SrI and 16SrVII in MF01 (Empoasca sp.), MF02 (Typhlocybinae), MF03 (Scaphytopius sp.), MF04 (Amplicephalus funzaensis) and MF06 (Exitianus atratus). Transmission tests to uninfected bean plants (Phaseolus vulgaris) were performed using field collected A. funzaensis and E. atratus individuals in separate assays. After 5 weeks, the test plants exposed to individuals of both species of leafhoppers showed symptoms, suggesting phytoplasma infection. Phytoplasma groups 16SrI and 16SrVII were detected in the two groups of exposed plants, indicating that A. funzaensis and E. atratus were able to transmit both groups of phytoplasmas. This is the first report of insect vectors for phytoplasmas of group 16SrVII in the world and of 16SrI in South America.     

PM 7/62 (2) ‘Candidatus Phytoplasma mali’, ‘Ca. P. pyri’ and ‘Ca. P. prunorum’

Specific scope

This Standard describes a diagnostic protocol for ‘Candidatus Phytoplasma mali’, ‘Ca. P. pyri’ and ‘Ca. P. prunorum’. This Standard should be used in conjunction with PM 7/76 Use of EPPO diagnostic protocols

Specific approval and amendment

Approved as PM 7/62 Candidatus Phytoplasma mali and PM 7/63 Ca. P. pyri in 2006. Revised in 2017‐02 as a single Standard as PM 7/62 (2) with the addition of ‘Ca. P. prunorum’.     

Molecular analysis of ‘Candidatus Phytoplasma aurantifolia’ associated with phytoplasma diseases in Myanmar

During 2010 and 2011, typical phytoplasma disease symptoms such as little leaves, phyllody and witches’ brooms were observed on black gram, green gram, long bean, shaggy button weed and sesame plants from different regions of Myanmar. The symptomatic samples were analyzed by PCR using universal phytoplasma primers and characterized by sequencing 16S rRNA, ribosomal protein and translocase protein genes. Based on sequence and phylogenetic analysis of the three genes, the phytoplasmas associated with those plants belonged to members of ‘Candidatus Phytoplasma aurantifolia’. To our knowledge, black gram and shaggy button are new hosts for ‘Ca. P. aurantifolia’.     

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.     

The <Emphasis Type="Italic">Foc</Emphasis> gene governs resistance to race 3 of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">cucumerinum</Emphasis> in the cucumber cv. SMR-18

Bois noir (BN) is the most widespread European grapevine yellows disease caused by ‘Candidatus Phytoplasma solani’. Although our knowledge of the mechanisms of interactions of this pathogenic bacteria with host is largely unknown, the plant-pathogen system of BN is commonly used as a model system for studying grapevine yellows diseases. We applied here a conceptual model of general plant pathology – a disease triangle for describing interactions among the host plant, the pathogen and the environment. We generated a proof-of-concept statistical model for disease triangle using original experimental data and different statistical and data mining approaches for a selected system of ‘Ca. P. solani’ infection of cv. ‘Chardonnay’ grapevine plants. We monitored individual plants from a single vineyard over a period of six years. Phytoplasma content, the expression of 21 selected grapevine genes and environmental conditions were recorded and related to disease severity. Our model predicts that in described conditions BN is a function of the expression of grapevine gene VvDMR6, summer rainfall and abundance of ‘Ca. P. solani’. The greatest impact among elements of the disease triangle is attributed to the pathogen, and is independent of the pathogen titer. We showed that this first de facto representation of the disease triangle is useful for showing disease dynamics over several years and could be applied to other plant-pathogen systems. The overall results of this study will contribute to understanding of ‘Ca. P. solani’ biology and its interactions with grapevine host.     

Test performance study of isothermal amplification tests for the detection of Grapevine flavescence dorée phytoplasma and ‘Candidatus Phytoplasma solani’

This test performance study (TPS) was carried out on DNA samples from grapevine, clematis, fungi and bacteria to compare and validate loop‐mediated isothermal amplification (LAMP) tests for detection of Grapevine flavescence dorée phytoplasma and ‘Candidatus Phytoplasma solani’ (Grapevine Bois noir phytoplasma). Two LAMP tests, for Grapevine flavescence dorée phytoplasma and ‘Candidatus Phytoplasma solani’ (as developed by Kogov?ek and colleagues), with proven applicability for rapid laboratory or on‐site detection were included in this study. They were performed in 10 laboratories. In addition, the commercial Qualiplante/Hyris isothermal amplification test for Grapevine flavescence dorée phytoplasma was performed in three laboratories. The accuracy of the three tests was shown to be over 98%. Moreover, the high accuracy of these tests, which used different devices across different laboratories, confirmed their reproducibility.     

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 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.     

Genetic diversity of Czech ‘Candidatus Phytoplasma mali’ strains based on multilocus gene analyses

In several European countries apple trees are affected by apple proliferation disease, which is usually associated with the presence of ‘Candidatus Phytoplasma mali’. During 2010, samples from several apple trees displaying proliferation symptoms were collected throughout the Czech Republic to verify identity of phytoplasmas detected in association with the disease. The majority of the 74 apple trees examined using molecular tools were positive for ‘Ca. P. mali’ presence. The 16S–23S ribosomal genes, the ribosomal protein genes and the nitroreductase and rhodonase like genes were then studied to verify phytoplasma strain variability on multigenic bases. Two RFLP profiles and correspondingly two genetic lineages were found in the PCR-amplified fragments covering the 16S–23S rDNA spacer region. ‘Ca. P. mali’ strains belonging to rpX-A subgroup were identified in the majority of the apple tree sampled, whereas phytoplasmas belonging to the rpX-B subgroup were distributed sporadically. The apple proliferation subtypes AP-15 and AT-2 exhibited nearly equal occurrence; the AT-1 subtype and a mixture of the two or all three of the AP subtypes were infrequently found. The PCR/RFLP results were confirmed by nucleotide sequence analyses of selected ‘Ca. P. mali’ strains.     

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.     

First report of ‘Candidatus Phytoplasma asteris’ infecting hydrangea showing phyllody in Japan

Phytoplasma-induced floral malformations such as virescence, phyllody, and proliferation were observed on hydrangeas in Gunma Prefecture, Japan. Phylogenetic analyses based on 16S rRNA, secY, groEL, and amp gene sequences indicated that the affected hydrangea plants were associated with phytoplasmas belonging to ‘Candidatus Phytoplasma asteris’, but not to ‘Ca. P. japonicum’, which occurs in hydrangeas showing phyllody in Japan. This is the first molecular evidence of an association of ‘Ca. P. asteris’ with hydrangea plants in Japan.     

On the apple proliferation symptom display and the canopy colonization pattern of “<Emphasis Type="Italic">Candidatus</Emphasis> Phytoplasma mali” in apple trees

Notwithstanding the availability of several different real time PCR protocols for “Candidatus Phytoplasma mali”, it is still unclear how informative is the estimation of the concentration of phytoplasma cells in the leaves of apple proliferation infected trees, and how the reliability of the estimations may be affected by an erratic and uneven distribution of the pathogen in the host. Here we investigated these issues systematically and showed that phytoplasma concentration varies significantly among seasons, but not between two cultivars that appeared to have different degree of susceptibility on the basis of the symptoms displayed. In fully symptomatic trees sampled at the end of the season the phytoplasmas were detectable in most leaves, but in more than half of the leaves at low concentrations. Both the pattern of colonization of the canopy and the amount of phytoplasmas varied greatly in trees that show symptom remission, although a direct relation between symptom severity and colonization could not be established. The sampling of the apple canopy for the purpose of evaluation of concentration of “Candidatus Phytoplasma mali” should take into consideration the complex pattern of colonization and seasonal variation.     

First report of Almond witches’ broom phytoplasma1 (‘Candidatus Phytoplasma phoenicium’) causing a severe disease on nectarine and peach trees in Lebanon

Symptoms of shoot proliferation characteristic of phytoplasma diseases were observed on nectarine (Prunus persica var. nucipersica) and peach (P. persica) trees in the Sarada plain, south of Lebanon. The presence of phytoplasmas in the two orchards visited was confirmed by nested polymerase chain reaction using universal primers. The amplified DNA fragments were cloned and sequenced. Blast analysis of over 1000 nucleotides demonstrated the presence of ‘Candidatus Phytoplasma phoenicium’ which is considered to be the causal agent of Almond witches’ broom. This phytoplasma which belongs to the pigeon pea witches’ broom group (16SrIX) can be devastating since Almond witches’ broom has killed thousands of almond trees in Lebanon and Iran. Previous reports indicated that Almond witches’ broom may be transmitted by grafting to peach and nectarine under experimental conditions. This is the first report of a natural and epidemic spread of ‘Ca. Phytoplasma phoenicium’ in peach and nectarine. Farmers in the region were advised to eradicate the infected trees immediately. Further studies on the epidemiology of ‘Ca. Phytoplasma phoenicium’ and its vector(s) are recommended in order to develop successful eradication or disease management programmes.     

Survey and molecular detection of phytoplasmas associated with potato in Romania and southern Russia

In recent years, emerging phytoplasma diseases of potato (Solanum tuberosum L.) have increasingly become important in central and eastern Europe. Accurate identification of phytoplasmas and their insect vectors is essential to developing effective management strategies for diseases caused by these plant pathogens. Potato phytoplasma diseases in Europe were for a long time diagnosed only on the basis of visual symptoms. However, this approach is not very reliable and the use of modern molecular techniques such as polymerase chain reaction (PCR) is required in order to accurately determine the etiology of these phytoplasma diseases. A survey and identification of phytoplasmas associated with potato crops in Romania and southern Russia were conducted based on modern molecular techniques. Symptomatic potato plants were collected from several fields and tested for phytoplasmas by PCR. Also, selected crops and weeds in the vicinity of these potato fields were sampled and tested for phytoplasmas. Stolbur (“Candidatus Phytoplasma solani”; 16SrXII-A) was the only phytoplasma detected in potato and adjacent crops, including tomato (Solanum lycopersicum), pepper (Capsicum annuum), eggplant (Solanum melongena), and beet (Beta vulgaris). This phytoplasma was also detected in weeds, particularly Convolvulus arvensis, Cuscuta sp., and Euphorbia falcata. Genotyping of obtained stolbur isolates on tuf genes revealed that they all had the same RFLP profile corresponding to the tuf-type ‘b’ (VK Type II). Stolbur-affected potato plants produced a large number of spongy tubers that resulted in commercially unacceptable potato chips upon processing.