Phytoplasma Prunorum Research Articles

Spread of European fruit tree phytoplasma diseases

The genetically closely related ‘Candidatus Phytoplasma mali’, ‘Candidatus Phytoplasma prunorum’ and ‘Candidatus Phytoplasma pyri’ are associated with economically important diseases such as apple proliferation, European stone fruit yellows and pear decline in European fruit tree areas and are spread by psyllid vectors of the superfamily Psylloidea (Hemiptera, Sternorrhyncha). Although the relationship between these phytoplasmas, their hosts and the vectoring psyllids is almost highly specific, there are some interconnections and biological factors which influence disease epidemiology and vector ecology and which should be taken into consideration for risk assessment and vector control. For a reliable risk assessment of a putative vector is important to know its population dynamics, its transmission efficiency, its host plant preferences and its epidemiological cycle. Although psyllid vectors usually show a high host plant specificity, being either monophagous or oligophagous on closely related plant species, shelter plants may serve as sources of inoculum and hence interfere with the disease spread. Therefore, the knowledge of the epidemiological cycle of both, the phytoplasma and its main vector is crucial for the development of efficient control measures.

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.

First report of 'Candidatus Phytoplasma prunorum' infecting apricots in Tunisia.

‘Candidatus Phytoplasma prunorum’ was detected for the first time in Tunisia in apricot trees that showed early leaf reddening in autumn, off-season growth in winter followed by dieback, and bore small and tasteless fruits. Phytoplasma was detected by nested PCR using the universal phytoplasma primer pairs R16mF2/R1 and R16F2n/R2. An amplification product of the expected size (1.2 kbp) was obtained from samples collected from symptomatic but not from symptomless apricot trees. Amplicons subjected to RFLP analysis after digestion with endonucleases MseI and RsaI, gave patterns similar to those reported for the European stone fruit yellows phytoplasma (ESFY, 16SrX-B). Identification was further confirmed by PCR using ESFY-specific primer pairs (ECA1/ECA2).

First report of Candidatus 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.

Phytoplasma diseases of stone fruit trees in Turkey and their containment

Although fruit tree phytoplasmas were studied since 1999 in Turkey, there have been very limited studies and records on stone fruit tree phytoplasmas. The main symptoms reported on apricot, plum, peach and almond were chlorosis between veins, off season flowering and fruiting as a result of early bud breaking, longitudinal leaf rolling and quick die-back. More than 500 cultivated and wild Prunus plants in or nearby germplasm nurseries and commercial orchards during 2002–2009 were tested by using universal primers P1/P7 and fU5/rU3 in direct and nested PCR assays, respectively. Amplification products were digested with RsaI and SspI restriction enzymes. The average incidence of ‘Candidatus Phytoplasma prunorum’ was detected as 10.19%. The most infected stone fruit species were apricot and plum, followed by almond and peach. No phytoplasma was found in cherries and wild Prunus species.

European Stone Fruit Yellows Disease and Its Causal Agent ‘Candidatus Phytoplasma Prunorum’

The European stone fruit yellows (ESFY) caused by ‘Candidatus Phytoplasma prunorum’ (‘Ca. P. prunorum’), is a devastating, quarantine phytoplasma disease. This agent leads to serious damage in apricot and Japanese plum orchards, including decline of infected trees. The consequences are considerable economic losses in production. European plum and cherry are less susceptible to ESFY, although the ‘Ca. P. prunorum’ was also detected in these trees in several countries. ESFY seems to be limited to Europe and the neighboring regions (Asia Minor) as its incidence was reported from most of the southern and central European countries, Turkey, and Azerbaijan. ‘Ca. P. prunorum’ is transmitted by Cacopsylla pruni (Scopoli). The impact of ‘Ca. P. prunorum’ on stone fruit trees, as well as the methods of detection and identification and control management are reviewed.

Dieback of apricot plantations caused by 'Ca. Phytoplasma prunorum' in Borsod-Abaúj-Zemplén county (Northern-Hungary)

Plant diseases caused by phytoplasmas have increasing importance in all over the world for fruit growers. Lately, phytoplasma diseases occur on many fruit varieties and responsible for serious losses both in quality and quantity of fruit production. In the long-run these diseases cause destruction of fruit trees. The apricot phytoplasma disease (Ca. Phytoplasma prunorum) was first reported in Europe in 1924 from France. In 1992 the disease has also been identified in Hungary. On the base of growers' signals serious damages of "Candidatus Phytoplasma prunorum" Seemüller and Schneider, 2004 (formerly: European stone fruit yellows phytoplasma) could be observed in different stone fruit plantations in the famous apricot-growing area nearby Gönc town, Northern-Hungary. Field examinations have been begun in 2009 in several stone fruit plantations in Borsod-Abaúj-Zemplén County mainly in Gönc region which is one of the most important apricot growing regions in Hungary, named “Gönc Apricot Growing Area”. Our goals were to diagnose the occurrence of Ca. Phytoplasma prunorum on stone fruits (especially on apricot) in the North-Hungarian growing areas by visual diagnostics and confirm data by laboratory PCR-based examinations. All the 28 collected samples were tested in laboratory trials and at 13 samples from apricot, peach, sour cherry and wild plum were confirmed the presence of phytoplasma (ESFY). On the base of observations it seems evident that the notable losses caused by "Ca. Phytoplasma prunorum" is a new plant health problem to manage for fruit growers, especially apricot producers in Hungary.

Detection and Identification of ‘Candidatus Phytoplasma prunorum’, ‘Candidatus Phytoplasma mali’ and ‘Candidatus Phytoplasma pyri’ in Stone Fruit Trees in Poland

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

Preliminary results for obtaining nuclear-stock plum material in Romania.

A certification program focused on plum was established at Fruit Research & Development Station Bistrita, Romania started in 2008. The objective is to obtain in the first step the prebasic material and, subsequently, to establish plum mother plantations with basic material satisfying the EPPO recommended certification standards. Sixteen plum (Prunus domestica) cultivars (Iulia, Matilda, Geta, Zamfira, Ivan, Dani, Doina, Romaner, Elena, Jubileu 50, Delia, Flora, Renclod d’Althan, Stanley, Anna Spath and Carpatin) were subjected to this work. Selected plum trees from experimental orchards were monitored and tested. One shoot was collected from each candidate and tested before grafting onto virus-free Myrobolan 29C rootstock. Selected clones were also tested on woody indicators GF 305. Propagated material was individually tested one year later. All tests were done according to the EPPO recommendations. DAS-ELISA was used for Plum pox virus (PPV), Prune dwarf virus (PDV), Prunus necrotic ringspot virus (PNRSV), Apple chlorotic leaf spot virus (ACLSV), Apple mosaic virus (ApMV) and Myrobalan latent ringspot virus (MLRSV). To increase the sensitivity of PPV and PDV detection, IC-RT-PCR was performed. Spot-Real Time RT-PCR was also used to detect PPV. Nested-PCR method was made to detect Candidatus Phytoplasma prunorum. Delia cultivar was found infected by PPV and eliminated from propagation by grafting. This cultivar is subjected to pathogen elimination by using in vitro chemo-thermotherapy. The other fifteen cultivars were found virus-free both before and after propagation. The certification of this material as ‘Prebasic category’ is in progress.

Multilocus sequence analysis reveals the genetic diversity of European fruit tree phytoplasmas and supports the existence of inter-species recombination

The genetic diversity of three temperate fruit tree phytoplasmas 'Candidatus Phytoplasma prunorum', 'Ca. P. mali' and 'Ca. P. pyri' has been established by multilocus sequence analysis. Among the four genetic loci used, the genes imp and aceF distinguished 30 and 24 genotypes, respectively, and showed the highest variability. Percentage of substitution for imp ranged from 50 to 68 % according to species. Percentage of substitution varied between 9 and 12 % for aceF, whereas it was between 5 and 6 % for pnp and secY. In the case of 'Ca P. prunorum' the three most prevalent aceF genotypes were detected in both plants and insect vectors, confirming that the prevalent isolates are propagated by insects. The four isolates known to be hypo-virulent had the same aceF sequence, indicating a possible monophyletic origin. Haplotype network reconstructed by eBURST revealed that among the 34 haplotypes of 'Ca. P. prunorum', the four hypo-virulent isolates also grouped together in the same clade. Genotyping of some Spanish and Azerbaijanese 'Ca. P. pyri' isolates showed that they shared some alleles with 'Ca. P. prunorum', supporting for the first time to our knowledge, the existence of inter-species recombination between these two species.

MINERAL NUTRITION OF PEAR AND APRICOT TREES CULTIVATED IN SOUTHERN ITALY AREA DAMAGED BY PHYTOPLASMA MICROORGANISMS

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

RESEARCH ON THE VECTOR OF 'CANDIDATUS PHYTOPLASMA PRUNORUM' IN APRICOT TREES IN SOUTHERN ITALY

A research of vector(s) of the phytoplasmas responsible for apricot chlorotic leaf roll disease was carried out in a field plot where European Stone Fruit Yellows Phytoplasma (ESFYP), now named 'candidates Phytoplasma prunorum' ('Ca. P. prunorum'), had heavily infected an apricot collection in 1993. The plants were uprooted in May 1994 and, in 1997, the field was planted again with apricot trees. This report was elaborated with the data collected from February 2003 through July 2007. The only insect species found was Empoasca decedens Paoli with 5,085 insects collected. These insects were tested by direct and nested PCR to verify the presence of the phytoplasma. Individuals of E. decedens, present in the field each month in each year, resulted to be hosts of 'Ca. P. prunorum' in ten months out of twelve, but not of every year. The absence of phytoplasma in the two plants infected by 'Ca. P. prunorum' with E. decendens, four years after the acquisition of the pathogen, was also recorded.

CANDIDATUS PHYTOPLASMA PRUNORUM, THE CAUSAL AGENT OF EUROPEAN STONE FRUIT YELLOWS: AN OVERVIEW

‘Candidatus Phytoplasma prunorum’ is an important prokaryotic pathogen that infects stone fruits in Europe. It is known to cause several economically relevant disorders of Prunus spp. which are collectively referred to as European stone fruit yellows (ESFY). This organism is phylogenetically closely related to other important fruit trees pathogens such as apple proliferation (AP), pear decline (PD) and peach yellow leaf roll (PYLR) agents. Together they form a distinct phylogenetic cluster, the AP or 16SrX group. Like the other fruit tree phytoplasmas of the AP group, ‘Ca. P. prunorum’ exhibits a high host specificity. In nature, this pathogen has been reported to infect only plants in the genus Prunus and to be transmitted by one insect vector species, the psyllid (Psyllidae) Cacopsylla pruni. Also, ‘Ca. P. prunorum’ includes strains which greatly differ in virulence. This review summarizes the current knowledge of ‘Ca. P. prunorum’ with emphasis on advances that have been made during the last two decades in understanding molecular and epidemiological aspects. Prospects for disease management and future research which could provide insights into the largely unknown mechanisms involved in pathogenicity of ‘Ca. P. prunorum’, are also critically discussed.

'Candidatus Phytoplasma tamaricis', a novel taxon discovered in witches'-broom-diseased salt cedar (Tamarix chinensis Lour.)

Salt cedar trees with pronounced witches'-broom symptoms were observed in their natural habitat in China. 16S rRNA gene sequences unique to phytoplasmas were detected in every DNA sample extracted from stem and leaf tissues of the symptomatic trees, revealing a direct association between phytoplasma infection and the salt cedar witches'-broom (SCWB) disease. Phylogenetic analysis of the SCWB phytoplasma 16S rRNA gene sequence indicated that the SCWB phytoplasma belonged to a subclade consisting of several mutually distinct 'Candidatus Phytoplasma' taxa including 'Ca. Phytoplasma prunorum', 'Ca. Phytoplasma mali', 'Ca. Phytoplasma pyri' and 'Ca. Phytoplasma spartii'. Pairwise sequence similarity scores calculated from an alignment of near full-length 16S rRNA genes revealed that SCWB phytoplasma shared 96.6 % or less sequence similarity with each previously described or proposed 'Ca. Phytoplasma' taxon, justifying the recognition of SCWB phytoplasma as a novel taxon, 'Candidatus Phytoplasma tamaricis'. The distinct virtual RFLP pattern derived from the SCWB phytoplasma 16S rRNA gene sequence, together with its lower-than-threshold similarity coefficient values with RFLP patterns of any of the 29 previously established groups, supported the recognition of a new 16Sr group, designated 16SrXXX, salt cedar witches'-broom phytoplasma group.

Specific detection and quantification of the phytopathogenic agent ‘ Candidatus Phytoplasma prunorum’

‘ Candidatus Phytoplasma prunorum’ is a wall-less bacterium associated with European stone fruit yellows (ESFY), a severe disease of Prunus spp. (mainly apricot and Japanese plum trees). It can be spread by one insect vector, Cacopsylla pruni, and by the trade of infected material. The availability of PCR-based methods allowing a sensitive and specific detection of ‘ Ca. P. prunorum’ is crucial for this phytoplasma because, at present, it is uncultured and cannot be detected serologically. We developed a PCR test which, in contrast to the existing detection tools, provides a fast, specific and sensitive detection of ‘ Ca. P. prunorum’ in plants and insects. For studies requiring an absolute quantification of the phytoplasma titer, the same primers were used to develop a real-time PCR assay, including a standard for C. pruni. The sensitivity of these molecular tools was compared by serial dilutions and their specificity was assessed both in silico and experimentally for reference strains and field samples of the closely related phytoplasma ‘ Ca. P. prunorum’, ‘ Ca. P. pyri’ (pear decline agent) and ‘ Ca. P. mali’ (apple proliferation agent), as well as for representative strains of the ‘ Ca. Phytoplasma’ genus.

Efficient Transmission of ‘CandidatusPhytoplasma prunorum’ Is Delayed by Eight Months Due to a Long Latency in Its Host-Alternating Vector

Understanding at which spatiotemporal scale a disease causes significant secondary spread has both theoretical and practical implications. We investigated this issue in the case of European stone fruit yellows (ESFY), a quarantine vector-borne phytoplasma disease of Prunus trees. Our work was focused on the processes underlying disease spread: the interplay between the life cycles of the pathogen ('Candidatus Phytoplasma prunorum') and of the vector (Cacopsylla pruni). We demonstrated experimentally that C. pruni has only one generation per year and we showed that, at least in southeastern France, C. pruni migrates between conifers in mountainous regions (where it overwinters) and Prunus spp. at lower altitude (where it breeds). In acquisition-inoculation experiments performed with C. pruni over its period of presence on Prunus spp., both immature and mature C. pruni were hardly infectious (0.6%) despite effective phytoplasma acquisition and multiplication. We demonstrated that most immature vectors born on infected plants reach their maximum phytoplasma load (10(7) genomes per insect) only after migrating to conifers and that, after a life-long retention of the phytoplasma, their transmission efficiency was very high (60%) at the end of winter (when they migrate back to their Prunus host). Thus, most transmissions occur only after an effective latency of 8 months, following vector migrations and overwintering on conifers in mountainous regions. From this transmission cycle, we can infer that local secondary spread of ESFY in apricot orchards is marginal, and recommend that disease management strategies take more into account the processes occurring at a regional scale, including the role of wild Prunus spp. in ESFY epidemics.

Strain differentiation of Candidatus Phytoplasma mali by SSCP - and sequence analyses of the hflB gene.

Molecular analysis of the hflB gene ATP00464 of Candidatus Phytoplasma mali strains revealed sequence polymorphisms within this taxon. A 530 bp fragment from 42 accessions originating from apple proliferationinfected plants from Germany, Italy, and France was amplified with primers fHflB3-1/rHflB3. The variability of the amplimers was demonstrated by single-strand conformation polymorphism (SSCP) analysis that resulted in more than 20 different profiles. PCR fragments were sequenced to corroborate SSCP results. The nucleic acid homology within Ca. P. mali strains ranged from 94.2% to full identity, whereas the identity to the hflB gene of Candidatus Phytoplasma pyri and Candidatus Phytoplasma prunorum was at least 85.9% and 84.6% or higher, respectively. The homology on the protein level within Ca. P. mali was higher as a number of substitutions were silent mutations. The phylogenetic tree derived from the nucleic acid sequences revealed more than 20 different lineages and agreed with the SSCP typing. The molecular analysis of the hflB gene in combination with SSCP typing allowed high throughput analysis of samples and enabled differentiation of Ca. P. mali strains to a higher degree than published markers. The polymorphism of the hflB fragment predestine this gene as a molecular marker for tracking strains in epidemiological studies and characterizing, define and monitor inoculum strains used for resistance breeding projects.

Characterization of a Phytoplasma Associated with Pear Decline in Iran

AbstractSymptoms of pear decline (PD) were observed in several pear growing regions of Iran. Pear trees with typical symptoms of PD from Estahban (Fars Province) were examined for phytoplasma infection using polymerase chain reaction (PCR) assay. Graft inoculation of healthy pear trees with scions from diseased trees resulted in production of PD symptoms and transmission of phytoplasma as verified by PCR. Target DNA was amplified from symptomatic pear trees with fO1/rO1, an apple proliferation (AP) group‐specific primer pair. Physical and putative restriction fragment length polymorphism (RFLP) analyses of fO1/rO1 primed PCR products showed profiles corresponding to AP group, 16SrX‐C subgroup (Candidatus Phytoplasma pyri). Percent similarity values and phylogenetic analysis of fO1/rO1 primed sequences confirmed that, as a member of AP subclade, Estahban PD phytoplasma has a closer relationship to PD and peach yellow leaf roll phytoplasmas than to AP (Ca. Phytoplasma mali) and European stone fruit yellows (Ca. Phytoplasma prunorum) phytoplasmas. This is the first report of PD phytoplasma in the eastern Mediterranean.

HIGH INFECTION PRESSURE OF ESFY PHYTOPLASMA THREATENS THE CULTIVATION OF STONE FRUIT SPECIES

Stone fruit species are affected by severe disease caused by European stone fruit yellows phytoplasma (ESFY; ‘Candidatus Phytoplasma prunorum’). ESFY phytoplasma is transmitted to the host plants of Prunus spp. by the vector Cacopsylla pruni. The disease is graft-transmissible as well. The occurence of ESFY phytoplasma was monitored from 2004 to 2006 in a mother plant orchard of stone fruit species planted with virus free material in 2001 in the Primorska region of Slovenia. The total of 158 samples of mother plants were analysed in this period. The symptomatic and asymptomatic trees were analysed using molecular methods (PCR or nested-PCR). Among 15.7 % of sampled apricot trees (Prunus armeniaca) in the orchard, ESFY phytoplasma was detected in 70.0 % of samples. In the case of Japanese plum (Prunus salicina) samples were taken from one third of all Japanese plum trees and the presence of ESFY phytoplasma was confi rmed in all samples. In the European plum trees (Prunus domestica) the incidence of phytoplasma was determined in 51.0 % of sampled trees, where the plants in most cases did not show symptoms. ESFY phytoplasma was also detected in peaches and nectarines (Prunus persica) in 13.0 % of sampled trees while no detection of the phytoplasma was confi rmed in the samples of cherry trees (Prunus avium). With the survey performed in a mother plant orchard it was observed that especially young trees did not show typical symptoms and the infection was latent. In the year 2005, ESFY phytoplasma was detected in all tested samples of the vector Cacopsylla pruni captured in the vicinity of the mother plant orchard.

Development of a new probe for specific and sensitive detection of ‘Candidatus Phytoplasma mali’ in inoculated apple trees

AbstractA new TaqMan minor groove binding (MGB) probe and new PCR conditions were designed for quick, specific and sensitive detection of ‘Candidatus Phytoplasma mali’. The new probe can distinguish a single mismatch between ‘Ca. P. mali’ and ‘Candidatus Phytoplasma prunorum’, this constituting an improvement over a previously published method. In our study, the relative position of the mismatch in the MGB probe influenced greatly the specificity of detection. Our new real‐time PCR protocol was able to detect one plasmid copy in water and 100 copies in healthy plant DNA extracts. The sensitivity of this new real‐time PCR method, three other real‐time PCR protocols and a conventional PCR with fU5/rU3 primers was compared. Periwinkles and MM106 rootstocks were grafted with infected material and surveyed over time by symptom observation, conventional PCR and real‐time PCR. Phytoplasma infection was detected by symptom observation in all periwinkles within 4 months and in 75% of the MM106 rootstocks by the end of 7 months. Conventional PCR detected phytoplasma infection in all periwinkles within 4 months and in all MM106 rootstocks within 7 months. Best results were obtained by our real‐time PCR, which detected phytoplasma infection in all grafted plants within 3 months after inoculation.