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.     

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 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.     

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.     

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.     

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.     

Molecular typing of ‘<Emphasis Type="Italic">Candidatus</Emphasis> Phytoplasma mali’ and epidemic history tracing by a combined T-RFLP/VNTR analysis approach

Apple proliferation caused by ‘Candidatus Phytoplasma mali’ is a disease of apple trees gaining increasing importance in Europe. The present study describes a high-throughput method for simultaneous typing of ‘Ca. P. mali’ at two genetic loci. This novel approach combines terminal restriction fragment length polymorphism (T-RFLP) analysis of a putative rhodanese-like protein gene and the analysis of the variable number of tandem repeats (VNTR) of the ribosomal protein L22 gene. The typing approach was applied to analyse a collection of DNA isolates from 310 apple trees tested positive for ‘Ca. P. mali’. Samples were taken between 2002 and 2010 in South Tyrol (Northern Italy). In addition, 15 samples of Cacopsylla melanoneura and 19 of C. picta were typed. Seven combined genetic profiles were found in the samples of infected apple trees: AT-2/rpX-A (81.0%), AT-1/rpX-D (8.4%), AT-1/rpX-E (4.2%), AT-1/rpX-A (3.2%), AT-1/rpX-B (1.6%), AT-1/rpX-C (1.0%) and AP/rpX-A (0.3%), and one mixed infection AP + AT-1/rpX-A + rpX-D (0.3%). Subtype rpX-E was discovered for the first time. In C. melanoneura samples the most frequent subtype was AT-1/rpX-E, followed by AT-1/rpX-D and AT-1/rpX-C. All C. picta samples displayed subtype AT-2/rpX-A. Analysis of the temporal distribution of subtype frequencies in apple trees revealed that exclusively subtype AT-1 in combination with four rpX subtypes was present in South Tyrol in the period from 2002 to 2004. From 2006 onwards subtype AT-2/rpX-A became dominant with an average frequency of 90%. The data obtained suggest that there may be a co-adaptation of particular ‘Ca. P. mali’ subtypes with different insect vector species.     

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.     

Control of rubus stunt and stolbur diseases in Madagascar periwinkle with mycorrhizae and a synthetic antibacterial peptide

Three experimental treatments consisting of inoculation with an arbuscular mycorrhizal fungus, application of a synthetic antimicrobial peptide or application of a resistance inducer, were evaluated in Madagascar periwinkle as control methods for rubus stunt and stolbur diseases caused by ‘Candidatus Phytoplasma rubi’ and ‘Candidatus Phytoplasma solani’, respectively. Two experiments were conducted under controlled environment conditions. In the first experiment, 4 months after graft‐inoculating the phytoplasmas, the root colonization achieved by Rhizophagus irregularis significantly reduced both disease symptoms and the frequency of detection of the pathogens by real‐time PCR. In the second experiment, the antimicrobial peptide BP100 totally prevented disease symptoms, despite the molecular detection of the phytoplasmas in 75% and 50% of the plants inoculated with ‘Ca. Phytoplasma rubi’ and ‘Ca. Phytoplasma solani’, respectively, and was more effective than benzothiadiazole (BTH) at increasing resistance against the pathogenic infections. A potential combination of early mycorrhizal inoculation and BP100 antimicrobial peptide application is envisaged as a future control strategy for phytoplasma diseases.     

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.     

Different symptoms in carrots caused by male and female carrot psyllid feeding and infection by ‘Candidatus Liberibacter solanacearum’

Carrot psyllid Trioza apicalis was recently found to carry the plant pathogenic bacterium ‘Candidatus Liberibacter solanacearum’ (CLs). To confirm the transmission of bacteria by the psyllids and to dissect the symptoms caused in carrot plants by psyllid feeding and CLs infection, a greenhouse experiment with single psyllids feeding on separate plants was performed. A positive correlation was found between the amount of CLs bacteria in the psyllids and in the corresponding plants exposed to feeding, indicating CLs transmission. The female psyllid feeding caused more severe damage than male feeding, and resulted in a substantial decrease in the root weight. Female psyllid feeding also significantly reduced the carrot leaf weight and increased the number of curled leaves. The number of curled leaves was also increased by the nymphs when their number exceeded 10 per plant. A high titre of CLs bacteria significantly reduced root weight, while not affecting the weight or number of the leaves. However, the amount of CLs correlated with the number of leaves showing discolouration symptoms. Microscopy of infected carrot plants revealed that the phloem tubes throughout the whole plant, from leaf veins to the root tip, were colonized by bacteria. The bacterial cells appeared to be long and thin flexible rods with tapering ends and a transversally undulated surface. Microscopy also revealed collapsed phloem cells in the infected carrots. Damage in the phloem vessels is likely to reduce the sucrose transport from source leaves to the root, explaining the observed leaf discolouration and reduction in root weight.     

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.     

Differentially-regulated defence genes in Malus domestica during phytoplasma infection and recovery

To improve knowledge about plant/phytoplasma interactions and, in particular, about the ‘recovery’ phenomenon in previously-infected plants, we investigated and compared expression levels of several defence-related genes (four pathogenesis-related proteins and three jasmonate-pathway marker enzymes) in apple plants showing different states of health: vigorous (healthy), phytoplasma-infected, and recovered. Real Time-PCR analyses demonstrated that genes are differentially expressed in apple leaf tissue according to the plants’ state of health. Malus domestica Pathogenesis-Related protein (MdPR) 1, MdPR 2 and MdPR 5 were significantly induced in leaves of diseased and symptomatic plants compared to leaves of those plants that were healthy or recovered. On the other hand, levels of all the jasmonate (JA)-pathway marker genes that we selected for this study, were up-regulated in the leaves of recovered plants compared to the diseased ones. In conclusion, our study demonstrated that two different sets of defence genes are involved in the interactions between apple plants and ‘Candidatus Phytoplasma mali’ (‘Ca. P. mali’) and that these genes are differentially expressed during phytoplasma infection or recovery.     

Tomato can be a latent carrier of ‘Candidatus Liberibacter solanacearum’, the causal agent of potato zebra chip disease

‘Candidatus Liberibacter solanacearum’ was recently described as the causal agent of potato zebra chip disease. This pathogen occurs in North America, New Zealand, and Northern Europe on various crops, and may spread to other potato growing regions. Observation on ‘Ca. L. solanacearum’‐infected tomato and potato plants propagated in growth chambers over 5 years indicated that tomato plants (cvs Moneymaker and Roma) can be a latent carrier of ‘Ca. L. solanacearum’. Tomato plants graft‐inoculated with scions from latently infected tomato plants remained symptomless, but tested positive in a species specific PCR assay. ‘Ca. L. solanacearum’ was consistently detected in the top, middle and bottom portion of the symptomless tomato plants, including stem, petiole, midrib, vein, flowers and fruits. In tomato fruits, ‘Ca. L. solanacearum’ was evenly distributed in the tissues at the peduncle and style ends, as well as in the pericarp, and columella placenta tissues. This is the first report that ‘Ca. L. solanacearum’ is present in a plant reproductive organ. In contrast, potato plants (cvs. Jemseg, Atlantic, Shepody, Frontier Russet, Russet Burbank, Red Pontiac, and Russet Norkotah) grafted with scions from the same latently infected tomato plants resulted in typical symptoms of purple top, leaf scorch, and other disease symptoms in plants and brown discoloration in the vascular ring and medullary rays in tubers.     

In vitro screening for resistance to apple proliferation in Malus species

A screening system for apple proliferation resistance was developed, based on in vitro graft‐inoculation with the causal agent ‘Candidatus Phytoplasma mali’. For this, in vitro cultures of the field‐resistant apomictic genotypes Malus sieboldii, H0909, D2212 and the susceptible Malus × domestica genotypes Golden Delicious and rootstock M9 were established, as well as in vitro cultures of Rubinette and Golden Delicious infected with ‘Ca. P. mali’ strains PM4 and PM6, respectively. Healthy in vitro shoots were inoculated by micrografting with infected shoots used as graft tip. After 6 weeks graft contact no significant differences for graft quality were observed between healthy and infected grafts. Mortality of grafts and transmission rates were not significantly different among the different genotypes. The phytoplasma concentration in inoculated shoots was determined at different times post‐inoculation (p.i.) by quantitative real‐time PCR. Infected M. sieboldii and D2212 had lower phytoplasma concentration than the susceptible controls and showed no symptoms. H0909 showed an intermediate behaviour exhibiting lower phytoplasma concentrations with strain PM4 but growth was affected. The dynamics of phytoplasma concentration reached a maximum at 6–8 months p.i. for all genotypes but the values for 3–5 and 10–12 months p.i. were similar. The resistance of M. sieboldii and D2212 was confirmed in vitro. A significant difference in phytoplasma concentration was observed between strains PM4 and PM6.     

Tissue‐print and squash real‐time PCR for direct detection of ‘Candidatus Liberibacter’ species in citrus plants and psyllid vectors

Huanglongbing (HLB) disease is seriously threatening and/or damaging the citrus industry worldwide. Accurate detection of the three species associated with HLB disease, ‘Candidatus Liberibacter asiaticus’, ‘Candidatus Liberibacter africanus’ and ‘Candidatus Liberibacter americanus’, is essential for the preventive control of the disease. Real‐time PCR is a useful tool for bacterial detection. However, nucleic acid purification steps limit the number of samples that can be processed by PCR. Universal detection of ‘Ca. Liberibacter’ species was achieved by direct tissue‐printing and spotting of plant leaf petiole extracts or squashing of individual psyllids onto paper or nylon membranes. Primers were designed and used with TaqMan chemistry for accurate detection of the bacterium in immobilized targets (prints of 10 overlapping leaf pedicels per tree, or squashed single vectors), by extraction with water and direct use for real‐time PCR. This simplified method was validated and could detect HLB‐liberibacters in 100% of leaves with symptoms and 59% of symptomless leaves collected from HLB‐infected trees. The use of direct assays as template showed good agreement with use of purified DNA (κ = 0·76 ± 0·052). The squash assay allowed detection of the bacterium in 40% of mature Diaphorina citri that fed on leaves of HLB‐infected trees with or without symptoms. A commercial ready‐made kit based on this technology showed 96% accuracy in intra‐laboratory performance studies. The simplified direct methods of sample preparation presented herein can be effectively adopted for use in rapid screening of HLB agents in extensive surveys, certification schemes or for epidemiological and research studies.     

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.