Biochemical and epigenetic changes in phytoplasma‐recovered periwinkle after indole‐3‐butyric acid treatment

Aim: To elucidate the possible mechanism of phytoplasma elimination from periwinkle shoots caused by indole‐3‐butyric acid (IBA) treatment. Methods and Results: It has been shown that a transfer of in vitro‐grown phytoplasma‐infected Catharanthus roseus (periwinkle) plantlets from medium supplemented with 6‐benzylaminopurine (BA) to one supplemented with IBA can induce remission of symptoms and even permanent elimination of ‘Candidatus Phytoplasma asteris’ reference strain HYDB. Endogenous auxin levels and general methylation levels in noninfected periwinkles, periwinkles infected with two ‘Candidatus Phytoplasma’ species and phytoplasma‐recovered periwinkles were measured and compared. After the transfer from cytokinin‐ to auxin‐containing media, healthy shoots maintained their phenotype, methylation levels and hormone concentrations. Phytoplasma infection caused a change in the endogenous indole‐3‐acetic acid to IBA ratio in periwinkle shoots infected with two ‘Candidatus Phytoplasma’ species, but general methylation was significantly changed only in shoots infected with ‘Ca. P. asteris’, which resulted in the only phytoplasma species eliminated from shoots after transfer to IBA‐containing medium. Both phytoplasma infection and treatment with plant growth regulators influenced callose deposition in phloem tissue, concentrations of photosynthetic pigments and soluble proteins, H₂O₂ levels and activities of catalase (CAT) and ascorbate peroxidase (APX). Conclusion: Lower level of host genome methylation in ‘Ca. P. asteris’‐infected periwinkles on medium supplemented with BA was significantly elevated after IBA treatment, while IBA treatment had no effect on cytosine methylation in periwinkles infected with ‘Candidatus Phytoplasma ulmi’ strain EY‐C. Significance and Impact of the Study: Hormone‐dependent recovery is a distinct phenomenon from natural recovery. As opposed to spontaneously recovered plants in which elevated peroxide levels and differential expression of peroxide‐related enzymes were observed, in hormone‐dependent recovery changes in global host genome, methylation coincide with the presence/absence of phytoplasma.     

Niger Seed (Guizotia abyssinica), a New Host of ‘Candidatus Phytoplasma asteris’ in Iran

Shrubs of niger seed with phyllody and internode elongation symptoms suggestive of phytoplasma infections occurred in the central regions of Iran. Phytoplasma was detected by polymerase chain reaction (PCR) and nested PCR amplifications using phytoplasma universal primer pairs P1/P7 and R16F2n/R16R2. Using aster yellows group–specific primer pair rp(I)F1A/rp(I)R1A, a fragment of 1212 bp of the rp genes was amplified from DNA samples of infected plants. Random fragment length polymorphism (RFLP) analyses of R16F2n/R16R2‐amplified products using the CfoI restriction enzyme confirmed that Iranian niger seed phyllody phytoplasma is associated with aster yellows group phytoplasmas. Sequence analyses of the partial rp genes fragment indicated that the Iranian niger seed phyllody phytoplasma, which was collected from central regions of Iran, is related to ‘Candidatus Phytoplasma asteris’. This is the first report of a phytoplasma infecting the niger seed plant.     

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 AP15 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.     

Molecular Characterization of a Phytoplasma Associated with Potato Witches’‐broom Disease in Iran

Potato plants showing symptoms suggestive of potato witches’‐broom disease including witches’‐broom, little leaf, stunting, yellowing and swollen shoots formation in tubers were observed in the central Iran. For phytoplasma detection, Polymerase Chain Reaction (PCR) and nested PCR assays were performed using phytoplasma universal primer pair P1/P7, followed by primer pair R16F2n/R16R2. Random fragment length polymorphism analysis of potato phytoplasma isolates collected from different production areas using the CfoI restriction enzyme indicated that potato witches’‐broom phytoplasma isolate (PoWB) is genetically different from phytoplasmas associated with potato purple top disease in Iran. Sequence analysis of the partial 16S rRNA gene amplified by nested PCR indicated that ‘Candidatus Phytoplasma trifolii’ is associated with potato witches’‐broom disease in Iran. This is the first report of potato witches’‐broom disease in Iran.     

Molecular Characterization of Phytoplasmas Associated with Peach Diseases in Iran

Recently, peach trees showing leaf rolling, little leaf, rosetting, yellowing, bronzing of foliage and tattered and shot‐holed leaves symptoms were observed in peach growing areas in the central and north‐western regions of Iran. Polymerase chain reaction (PCR) and nested PCR using phytoplasma universal primer pairs P1/Tint, R16F2/R2, PA2F/R and NPA2F/R were employed to detect phytoplasmas. The nested PCR assays detected phytoplasma infections in 51% of symptomatic peach trees in the major peach production areas in East Azerbaijan, Isfahan, ChaharMahal‐O‐Bakhtiari and Tehran provinces. Restriction fragment length polymorphism (RFLP) analyses of 485 bp fragments amplified using primer pair NPA2F/R in nested PCR revealed that the phytoplasmas associated with infected peaches were genetically different and they were distinct from phytoplasmas that have been associated with peach and almond witches’‐broom diseases in the south of Iran. Sequence analyses of partial 16S rDNA and 16S–23S rDNA intergenic spacer regions demonstrated that ‘Candidatus Phytoplasma aurantifolia’, ‘Ca. Phytoplasma solani’ and ‘Ca. Phytoplasma trifolii’ are prevalent in peach growing areas in the central and north‐western regions of Iran.     

Auxin influences symptom expression and phytoplasma colonisation in periwinkle infected with periwinkle leaf yellowing phytoplasma

Auxin imbalance was suggested as a key factor in phytoplasma symptom development. Furthermore, remission of the symptoms of phytoplasma‐infected shoots can be promoted by culturing them in vitro in high‐auxin‐containing media. Therefore, effect of spraying 1‐naphthaleneacetic acid (NAA) on infected periwinkle (Catharanthus roseus) with periwinkle leaf yellowing (PLY) phytoplasma was examined. 1‐Naphthaleneacetic acid stimulated symptom development in phytoplasma‐inoculated shoots. Accelerated symptom development was associated with early accumulation of phytoplasmas. Two PATHOGENESIS‐RELATED (PR) genes, CrPR1a and CrPR1b, were induced by PLY phytoplasma infection, and the induction was suppressed by NAA. Therefore, the accelerated symptom development may be due to the suppression effect of NAA on PR‐related defence. However, while NAA promoted symptom development on shoots inoculated with phytoplasma, more non‐symptomatic shoots containing no phytoplasma were observed, suggesting that NAA prevents phytoplasma colonisation in non‐symptomatic shoots. The expression of two genes encoding jasmonic acid (JA) biosynthesis key enzymes, lipoxygenase and allene oxide cyclase, was downregulated in non‐symptomatic shoots of infected plants, and remained downregulated after auxin treatment. Therefore, the auxin‐promoted resistance should be JA independent. Because auxin may promote symptom development of PLY phytoplasma‐infected periwinkles, it may not link to plant resistance to phytoplasma infection.     

Occurrence of Phytoplasmas Related to Stolbur and to ‘Candidatus Phytoplasma japonicum’ in Woody Host Plants in China

During a survey in a limited area of the Shanxi province in China, phytoplasma symptoms were observed on woody plants such as Chinese scholar tree, apple, grapevine and apricot. The polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) analyses on the phytoplasma 16S ribosomal gene confirmed that symptomatic samples from all these species were infected by phytoplasmas. The molecular characterization of the pathogen, performed also with sequencing of polymerase chain reaction amplified 16S rDNA, showed that the phytoplasmas detected in all plant species tested are closely related with stolbur, but two samples from a Chinese scholar tree were infected with phytoplasmas related to ‘Candidatus Phytoplasma japonicum’. The presence of RFLP polymorphism was found in the 16S rDNA amplicons with three of the six enzymes employed in the majority of phytoplasma strains studied.     

Identification of two new phylogenetically distant phytoplasmas from Senna surattensis plants exhibiting stem fasciation and shoot proliferation symptoms

Sunshine trees (Senna surattensis) exhibiting unusual stem fasciation symptoms were observed in Yunnan, China. Morphological abnormalities of the affected plants included enlargement and flattening of stems and excessive proliferation of shoots. An electron microscopic investigation revealed presence of single membrane bound mycoplasma‐like bodies in sieve elements of symptomatic plants. With DNA templates extracted from diseased plants and phytoplasma universal primers P1/P7 and P1A/R16S‐SR, nested polymerase chain reactions produced amplicons of 1.5 kb. Subsequent restriction fragment polymorphism and nucleotide sequence analyses of the amplicons indicated that the diseased plants were infected by distinct phytoplasmas affiliated with two phylogenetically distant taxa classified in two 16Sr groups (16SrXII and 16SrV). This is the first report that sunshine tree is a natural host of two evolutionarily divergent phytoplasmas and the first report that a ‘Candidatus Phytoplasma australiense’‐related strain is present in China. The findings signal a significant expansion of both geographical distribution and host range of 16SrXII and 16SrV phytoplasmas.     

Asparagus officinalis: A New Host of ‘Candidatus Phytoplasma asteris’

Asparagus officinalis plants with severe fasciation of some spears were observed in southern Bohemia between 1998 and 2007. Nucleic acids extracted from these and asymptomatic plants were assayed with nested polymerase chain reaction (PCR) using the phytoplasma‐specific universal ribosomal primers P1/P7 and R16F2n/R2. The restriction profiles obtained from digestion of the PCR products with five endonucleases (AluI, HhaI, KpnI, MseI and RsaI) were identical in all phytoplasmas infecting asparagus in the Czech Republic and indistinguishable from those of phytoplasmas in the aster yellows group (subgroup 16SrI‐B). Sequence analysis of 1754 bp of the ribosomal operon indicated that the closest related phytoplasmas were those associated with epilobium phyllody and onion yellows. This is the first report of the natural occurrence of ‘Candidatus Phytoplasma asteris’ in A. officinalis.     

The genome of ‘Candidatus Phytoplasma solani’ strain SA-1 is highly dynamic and prone to adopting foreign sequences

Bacteria of the genus ‘Candidatus Phytoplasma’ are uncultivated intracellular plant pathogens transmitted by phloem-feeding insects. They have small genomes lacking genes for essential metabolites, which they acquire from either plant or insect hosts. Nonetheless, some phytoplasmas, such as ‘Ca. P. solani’, have broad plant host range and are transmitted by several polyphagous insect species. To understand better how these obligate symbionts can colonize such a wide range of hosts, the genome of ‘Ca. P. solani’ strain SA-1 was sequenced from infected periwinkle via a metagenomics approach. The de novo assembly generated a draft genome with 19 contigs totalling 821,322 bp, which corresponded to more than 80% of the estimated genome size. Further completion of the genome was challenging due to the high occurrence of repetitive sequences. The majority of repeats consisted of gene arrangements characteristic of phytoplasma potential mobile units (PMUs). These regions showed variation in gene orders intermixed with genes of unknown functions and lack of similarity to other phytoplasma genes, suggesting that they were prone to rearrangements and acquisition of new sequences via recombination. The availability of this high-quality draft genome also provided a foundation for genome-scale genotypic analysis (e.g., average nucleotide identity and average amino acid identity) and molecular phylogenetic analysis. Phylogenetic analyses provided evidence of horizontal transfer for PMU-like elements from various phytoplasmas, including distantly related ones. The ‘Ca. P. solani’ SA-1 genome also contained putative secreted protein/effector genes, including a homologue of SAP11, found in many other phytoplasma species.     

Detection and Identification of Aster Yellows Phytoplasma Associated with Lipstick Yellow Frond Disease in Malaysia

Yellowing symptoms similar to coconut yellow decline phytoplasma disease were observed on lipstick palms (Cyrtostachys renda) in Selangor state, Malaysia. Typical symptoms were yellowing, light green fronds, gradual collapse of older fronds and decline in growth. Polymerase chain reaction assay was employed to detect phytoplasma in symptomatic lipstick palms. Extracted DNA was amplified from symptomatic lipstick palms by PCR using phytoplasma‐universal primer pair P1/P7 followed by R16F2n/R16R2. Phytoplasma presence was confirmed, and the 1250 bp products were cloned and sequenced. Sequence analysis indicated that the phytoplasmas associated with lipstick yellow frond disease were isolates of ‘Candidatus Phytoplasma asteris’ belonging to the 16SrI group. Virtual RFLP analysis of the resulting profiles revealed that these palm‐infecting phytoplasmas belong to subgroup 16SrI‐B and a possibly new 16SrI‐subgroup. This is the first report of lipstick palm as a new host of aster yellows phytoplasma (16SrI) in Malaysia and worldwide.     

‘Candidatus Phytoplasma asteris’ and ‘Candidatus Phytoplasma aurantifolia’, New Phytoplasma Species Infecting Apple Trees in Iran

During several surveys in extensive areas in central Iran, apple trees showing phytoplasma diseases symptoms were observed. PCR tests using phytoplasma universal primer pairs P1A/P7A followed by R16F2n/R16R2 confirmed the association of phytoplasmas with symptomatic apple trees. Nested PCR using 16SrX group‐specific primer pair R16(X)F1/R1 and aster yellows group‐specific primer pairs rp(I)F1A/rp(I)R1A and fTufAy/rTufAy indicated that apple phytoplasmas in these regions did not belong to the apple proliferation group, whereas aster yellows group‐related phytoplasmas caused disease on some trees. Restriction fragment length polymorphism (RFLP) analyses using four restriction enzymes (HhaI, HpaII, HaeIII and RsaI) and sequence analyses of partial 16S rRNA and rp genes demonstrated that apple phytoplasma isolates in the centre of Iran are related to ‘Ca. Phytoplasma asteris’ and ‘Ca. Phytoplasma aurantifolia’. This is the first report of apples infected with ‘Ca. Phytoplasma asteris’ in Iran and the first record from association of ‘Ca. Phytoplasma aurantifolia’ with apples worldwide.     

Effects of ‘Candidatus Phytoplasma asteris’ on the Volatile Chemical Content and Composition of Grindelia robusta Nutt.

Grindelia robusta, a perennial herb, contains an essential oil that is used as an antitussive, sedative, and analgesic agent. During the spring of 2007, ‘Candidatus Phytoplasma asteris’‐related phytoplasmas were identified in plants showing virescence and phyllody symptoms. The qualitative and quantitative composition of the oil of healthy and infected plants was compared by gas chromatography/mass spectrometry. Samples from six symptomatic and five asymptomatic plants tested by nested PCR followed by RFLP analyses confirmed the presence of ‘Ca. P. asteris’ in all symptomatic samples. The oils from healthy and infected plants, obtained by steam distillation, contained 42 components; that of healthy plants contained a higher concentration of monoterpenes, especially limonene and bornyl acetate, which were nearly 50% higher.     

Aster Yellows Subgroup 16SrI‐C Phytoplasma in Rhododendron hybridum

Symptoms of unknown aetiology on Rhododendron hybridum cv. Cunningham's White were observed in the Czech Republic in 2010. The infected plant had malformed leaves, with irregular shaped edges, mosaic, leaf tip necrosis and multiple axillary shoots with smaller leaves. Transmission electron microscopy showed phytoplasma‐like bodies in phloem cells of the symptomatic plant. Phytoplasma presence was confirmed by polymerase chain reaction using phytoplasma‐specific, universal and group‐specific primer pairs. Restriction fragment length polymorphism analysis of 16S rDNA enabled classification of the detected phytoplasma into the aster yellows subgroup I‐C. Sequence analysis of the 16S‐23S ribosomal operon of the amplified phytoplasma genome from the infected rhododendron plant (1724 bp) confirmed the closest relationship with the Czech Echinacea purpurea phyllody phytoplasma. These data suggest Rhododendron hybridum is a new host for the aster yellows phytoplasma subgroup 16SrI‐C in the Czech Republic and worldwide.     

Molecular Characterization of Phytoplasmas Associated with Potato Purple Top Disease in Iran

Potato plants with symptoms suggestive of potato purple top disease (PPTD) occurred in the central, western and north‐western regions of Iran. Polymerase chain reaction (PCR) and nested PCR assays were performed using phytoplasma universal primer pair P1/P7 followed by primer pairs R16F2n/R16R2 and fU5/rU3 for phytoplasma detection. Using primer pairs R16F2n/R16R2 and fU5/rU3 in nested PCR, the expected fragments were amplified from 53% of symptomatic potatoes. Restriction fragment length polymorphism (RFLP) analysis using AluI, CfoI, EcoRI, KpnI, HindIII, MseI, RsaI and TaqI restriction enzymes confirmed that different phytoplasma isolates caused PPTD in several Iranian potato‐growing areas. Sequences analysis of partial 16S rRNA gene amplified by nested PCR indicated that ‘Candidatus Phytoplasma solani’, ‘Ca. Phytoplasma astris’ and ‘Ca. Phytoplasma trifolii’ are prevalent in potato plants showing PPTD symptoms in the production areas of central, western and north‐western regions of Iran, although ‘Ca. Phytoplasma solani’ is more prevalent than other phytoplasmas. This is the first report of phytoplasmas related to ‘Ca. Phytoplasma astris’, ‘Ca. Phytoplasma solani’ and ‘Ca. Phytoplasma trifolii’ causing PPTD in Iran.     

Dodder Transmission of Pear Decline, European Stone Fruit Yellows, Rubus Stunt, Picris echioides Yellows and Cotton Phyllody Phytoplasmas to Periwinkle

The pear decline, European stone fruit yellows and rubus stunt agents as well as the phytoplasmas causing Picris echioides (bristly oxtongue) yellows and cotton (Gossypium hirsutum) phyllody, respectively, were transmitted from naturally infected plants to the experimental host Catharanthus roseus (periwinkle) via dodder (Cuscuta spp.) bridges. The identities of the dodder-transmitted phytoplasmas were confirmed by restriction length fragment polymorphism analysis of polymerase chain reaction-amplified ribosomal DNA. On the basis of restriction profiles the cotton phyllody agent could be differentiated from the phytoplasma causing faba bean phyllody, a disease previously thought to be induced by the same organism as cotton phyllody.     

Survival,fecundity, and body mass of Amplicephalus curtulus influenced by ‘Candidatus Phytoplasma ulmi’ (16SrV‐A) infection

The leafhopper Amplicephalus curtulus Linnavuori & DeLong (Hemiptera: Cicadellidae) can transmit ‘Candidatus Phytoplasma ulmi’ (16SrV‐A) from a native Chilean shrub, Ugni molinae Turcz. (Myrtaceae), to ryegrasses. A recent study showed that this phytoplasma reduced the total protein content and the activity of detoxifying enzymes in A. curtulus, which could also affect its vector fitness. This study evaluated the effect of ‘Ca. Phytoplasma ulmi’ on the longevity, fecundity, and body mass of A. curtulus. Both females and males were exposed to ‘Ca. Phytoplasma ulmi’‐infected plants for 96 h, whereas a control group remained unexposed. Quartiles from adult emergence to 75% (t₇₅), 50% (t₅₀), and 25% (t₂₅) survival rates were determined for each leafhopper survival distribution. The dry weight was also established at the end of the experiment. The adult lifespan of phytoplasma‐infected males and females was significantly lower than that of the uninfected leafhoppers in quartile survival distributions t₅₀ and t₂₅. The phytoplasma‐infected males and females lived 3 and 4 weeks less than uninfected ones in the last quartile, respectively. Fecundity was established by number of nymphs per female (in four periods) in phytoplasma‐infected and uninfected assays. In general, the weekly pattern of the number of nymphs per phytoplasma‐infected female was lower than that of uninfected leafhoppers; it was 37% lower at the end of the experiment. Phytoplasma‐infected females weighed significantly less (11%) than uninfected individuals. Phytoplasma‐infected males weighed 8% less than uninfected ones, but this difference was not significant. Our data indicated that ‘Ca. Phytoplasma ulmi’ negatively affected the fitness of A. curtulus, and nymphs produced by phytoplasma‐infected females varied over time, which may influence the disease dynamics in nature or in field crops.     

Molecular Identification of a ‘Candidatus Phytoplasma ziziphi’‐related Strain Infecting Amaranth (Amaranthus retroflexus L.) in China

Amaranth (Amaranthus retroflexus L.) is a common weed that grows vigorously in orchards, roadside verges, fields, woods and scrubland in China. In 2009, phytoplasma disease surveys were made in orchards in Beijing, China, and stem/leaf tissues were collected from asymptomatic amaranths. Direct PCR using universal phytoplasma primers P1/P7 detected 16S rRNA gene sequences in every DNA sample extracted from the symptomless amaranths. Sequence alignment and phylogenetic analyses of the 16S rRNA gene determined that the amaranth phytoplasma strain was related to ‘Candidatus Phytoplasma ziziphi’. Furthermore, virtual RFLP pattern analysis showed that the amaranth phytoplasma belonged to the 16SrV‐B subgroup. This is the first report of symptomless plants containing a ‘Candidatus Phytoplasma ziziphi’‐related strain.     

Identification of phytoplasmas associated with sesame phyllody disease in southeastern Iran

Phyllody disease is a threat to sesame production in Kerman province, southeastern Iran. RFLP analysis of PCR products of phytoplasma-specific 16S rRNA gene (1.8 kb) and phylogenetic analyses of 16S-23S rDNA spacer region (SR) sequence indicated that the predominant agent associated with sesame phyllody in Kerman province is a phytoplasma with 100% similarity with eggplant big bud, and peanut witches’-broom phytoplasmas, members of “Candidatus Phytoplasma aurantifolia” from Iran and China, respectively. Among the samples tested, only one strain (SPhSr1), had a unique RFLP profile and its SR was 100% similar in nucleotide sequence with the phytoplasma carried by Orosius albicinctus and Helianthus annus witches’-broom phytoplasma from Iran, members of “Ca. Phytoplasma trifolii”. Virtual RFLP patterns of SPhJ2 (representative of the predominant PCR-RFLP profiles) SR sequence were identical to those of peanut witches’-broom phytoplasma (16SrII-A, JX871467). However, SPhSr1 SR sequence patterns resemble (99.7%) those of vinca virescence phytoplasma (16SrVI-A, AY500817).     

Cyclamen (Cyclamen persicum L.): a dead-end host species for 16Sr-IB and -IC subgroup phytoplasmas

Phytoplasmas belonging to the 16S rDNA subgroups IB and IC were found in five cyclamen (Cyclamen persicum L.) plants showing virescence and yellow stunted leaves and one plant showing phyllody, rolled and thickened leaves, respectively. Two cyclamens, representing the two syndromes, were chosen as source plants for transmission trials in which three leafhopper species, known as vectors of IB and IC subgroup phytoplasmas, were used to inoculate cyclamen and periwinkle [Catharanthus roseus (L.) G. Don] test plants. Out of 366 tested plants only one periwinkle exposed to Euscelis incisus was found harbouring a 16Sr‐IB phytoplasma. Out of 60 tested vector insects, only one adult of Macrosteles quadripunctulatus and two of E. incisus fed on 16Sr‐IB source cyclamen gave a positive amplification signal in nested PCR. This extremely low level of transmission to both cyclamen and the very susceptible periwinkle strongly suggests that cyclamen, commonly found infected in crops, is an unsuitable species for phytoplasma acquisition and can be regarded as a dead‐end host plant for phytoplasmas belonging to both IB and IC subgroups. Indications for glasshouse management are drawn from these findings. Among the leafhoppers investigated E. incisus falls most under suspicion since it feeds better than the others on cyclamen, was able to transmit the disease to one periwinkle plant, and IB phytoplasmas were detected in two individuals.