Phytoplasma Infection Research Articles

Detection, Identification, and Characterization of Phytoplasmas Infecting Apple and Pear Trees in Belarus.

In Samochvalovichi, Belarus, apple and pear tree root samples were examined for the presence of phytoplasmas using a universal 16S rDNA-based PCR assay. Out of 27 tested apple trees, 23 were found to be infected by 'Candidatus Phytoplasma mali' and 46 out of 58 pear trees were positive for the presence of 'Ca. P. pyri.' Species were identified by sequence analysis of the 16S rDNA amplicons. The molecular diversity of the phytoplasma isolates was examined by analysis of an hflB gene using single-strand conformation polymorphism (SSCP) and sequence analysis. Therefore hflB gene amplicons from 'Ca. P. mali' and 'Ca. P. pyri' accessions were cloned after amplification. Screening of 640 cloned hflB fragments by SSCP analysis revealed the presence of eight different profiles for 'Ca. P. mali' and 12 different profiles for 'Ca. P. pyri.' The variants were sequenced and compared in multiple alignments. The nucleic acid homology among the hflB gene fragments ranged between 95.4 and 100.0% and 81.3 to 100.0% for 'Ca. P. mali' and 'Ca. P. pyri,' respectively, indicating a high genetic variability within the species. This is the first report on the occurrence of 'Ca. P. mali' and 'Ca. P. pyri' in Belarusian apple and pear trees and their molecular diversity.

Impact of insecticides treatment on phytoplasma infection risk in apple orchards

During 2013-2015, a monitoring study was carried out on the migration, abundance, and infectivity of Cacopsylla picta and C. melanoneura in apple orchards that were under different types of management. The presence of symptomatic and non-symptomatic apple trees infected by 'Candidatus Phytoplasma mali' was studied. It was demonstrated that the infectivity of psyllid vectors is the same without regard to the growth management applied in the orchard. The potential risk of phytoplasma spreading in the orchards under an integrated management was lower due to the side-effects of insecticides on psyllids. Their shorter occurrence, lower abundance and the absence of a new vector generation were observed and compared to the orchard grown under organic management.

Molecular Identification and Diversity of ‘Candidatus Phytoplasma solani’ Associated with Red-leaf Disease ofSalvia miltiorrhizain China

Reddening disease has recently been threatening Salvia miltiorrhiza in China, ranging from 30 to 50%. The main symptoms observed, such as plant stunting, inflorescence malformation, leaf reddening, fibrous roots browning, skin blackening and eventually root rot, are typically associated with phytoplasma infection. The presence of phytoplasmas was demonstrated through phytoplasma-specific PCR, with the expected amplification (1.8 kb) from symptomatic S. miltiorrhiza plants from Shangluo, Shangzhou and Luonan fields in Shaanxi Province of China. The sequences of 16S rRNA, tuf, secY and vmp1 genes amplified from LN-1 phytoplasma shared the closest homologies of 99%, 100%, 99% and 98% with those of the reference strain Candidatus Phytoplasma solani (subgroup 16SrXII-A), respectively. The phylogenetic trees showed that LN-1 phytoplasma clustered with the members of 16SrXII-A group, including Ca. P. solani. Computer-simulated restriction fragment length polymorphism analysis further supported this classification. Diversity analysis showed that all ‘Ca. P. solani’ strains identified from the three different regions examined shared 100% identical 16S rRNA, tuf, secY and vmp1 nucleotide sequences. To the best of our knowledge, this is the first report of phytoplasma infecting the medicinal plant of S. miltiorrhiza. The results demonstrate that ‘Ca. P. solani’ is the presumptive aetiological agent of S. miltiorrhiza reddening disease in China.

Evidence for the role of an invasive weed in widespread occurrence of phytoplasma diseases in diverse vegetable crops: Implications from lineage-specific molecular markers

During the period from 2011 to 2013, several plant diseases repeatedly occurred in vegetable crops grown in Yuanmou County, Yunnan Province, China. Affected plants included cowpea, sword bean, string bean, tomato, lettuce, and water spinach. The diseased plants exhibited symptoms of witches'-broom growth and floral deformations, linking each disease to phytoplasmal infection. Phylogenetic and virtual RFLP analyses of the phytoplasmal 16S rRNA gene sequences amplified from DNA of diseased plants revealed that all of the individual strains present in the diverse vegetable plants were affiliated with a single ‘Candidatus Phytoplasma’ species (‘Ca. Phytoplasma aurantifolia’) and a single ribosomal subgroup (16SrII-A). While presence of subgroup 16SrII-A phytoplasma in this geographic region was reported previously, such widespread infections in diverse plant hosts are unveiled for the first time in this study. In pursuing the source of the infections, we found that areas surrounding the affected vegetable fields were extensively invaded by parthenium weeds (Parthenium hysterophorus); and many of the weed plants exhibited abnormal morphologies that were suspicious of, and later diagnosed with, phytoplasmal infections. Results from genotyping of 16S rRNA and lineage-specific immunodominant membrane protein genes revealed that the vegetable-infecting phytoplasmas and the parthenium weed phytoplasma belong to the same genetic lineage. The findings indicate that parthenium weed poses a substantial risk as a reservoir of phytoplasmal infection of nearby agricultural crops in the geographic region since the ecosystems of Yuanmou are insect-rich, and parthenium weed is known to attract diverse leafhoppers. Further studies are warranted to assess the impact of farmland invasions by the noxious weed and to devise practical measures for improved weed control.

Looking inside phytoplasma-infected sieve elements: A combined microscopy approach using Arabidopsis thaliana as a model plant

Phytoplasmas are phloem-inhabiting plant pathogens that affect over one thousand plant species, representing a severe threat to agriculture. The absence of an effective curative strategy and the economic importance of many affected crops make a priority of studying how plants respond to phytoplasma infection. Nevertheless, the study of phytoplasmas has been hindered by the extreme difficulty of culturing them in vitro and by impediments to natural host plant surveys such as low phytoplasma titre, long plant life cycle and poor knowledge of natural host-plant biology. Stating correspondence between macroscopic symptoms of phytoplasma infected Arabidopsis thaliana and those observed in natural host plants, over the last decade some authors have started to use this plant as a model for studying phytoplasma-plant interactions. Nevertheless, the morphological and ultrastructural modifications occurring in A. thaliana tissues following phytoplasma infection have never been described in detail. In this work, we adopted a combined-microscopy approach to verify if A. thaliana can be considered a reliable model for the study of phytoplasma-plant interactions at the microscopical level.The consistent presence of phytoplasma in infected phloem allowed detailed study of the infection process and the relationship established by phytoplasmas with different components of the sieve elements. In infected A. thaliana, phytoplasmas induced strong disturbances of host plant development that were mainly due to phloem disorganization and impairment. Light microscopy showed collapse, necrosis and hyperplasia of phloem cells. TEM observations of sieve elements identified two common plant-responses to phytoplasma infection: phloem protein agglutination and callose deposition.

First finding of a ‘Candidatus Phytoplasma fraxini‘‐related strain associated with disease of olive in Iran

Iran is becoming a globally significant olive producer. During a survey in 2015, olive trees with symptoms of shoot proliferation, stunting, yellowed and little leaves (Fig. 1) typical of phytoplasma infection were observed in Roudbar region, Gilan province, Iran. Approximately 35% of the samples collected showed symptoms. To determine if a phytoplasma was associated with the disease, total DNA was extracted using a CTAB-based method (Doyle & Doyle, 2) from leaves of five healthy and 20 diseased trees. DNA was analysed by PCR using phytoplasma universal primers P1/P7 (Ahrens & Seemuller, 1) in first round PCR, and R16F2n/R16R2 (Gundersen & Lee, 3) in nested PCR. This resulted in fragments of the expected size in all symptomatic plants. All of the healthy plants tested negative for phytoplasmas. The nested PCR product of one sample was sequenced directly (using primers R16F2n/R16R2) on both strands by Macrogen Co. (Korea). Sequences were assembled using SeqMan (Lasergene), manually adjusted when necessary, and deposited in GenBank (Accession No. KX298840). BLAST analysis of the 16Sr DNA showed the highest identity (99%) with members of the 16SrVII phytoplasma group. Phylogenetic analysis with other related phytoplasmas using ClustalX and MEGA6 identified the phytoplasma, designated olive little leaf phytoplasma, as a ‘Candidatus Phytoplasma fraxini‘-related strain (Fig. 2), with 99% sequence identity to the reference strain of the species (Ashy4, JQ868445). These results extend the geographical distribution and host range of ‘Ca. P. fraxini’. To our knowledge this is the first report of a phytoplasma related to ‘Ca. P. fraxini’ infecting olive, and the study of natural reservoirs and vectors will be important to understand this disease.

Periwinkle proliferation disease associated with 16SrI-B phytoplasma in Mexico

Catharantus roseus, known as periwinkle, is highly susceptible to phytoplasma infection. Periwinkle plants showing proliferation symptoms were detected during 2013–2014 in four geographically distant states in Mexico. The presence of phytoplasmas was confirmed through the amplification of 16S F2nR2 and cpn60 UT sequences from symptomatic plants. Sequencing, phylogenetic analysis and in vitro RFLP revealed that the isolates were ‘Candidatus Phytoplasma asteris’-related strains and members of the 16SrI-B subgroup, confirming the association of this phytoplasma group with periwinkle proliferation disease in Mexico. We also demonstrated that the use of the approximately 550 pb cpn60 universal target sequences allow the differentiation of two 16SrI-B strains, designated here as MePP-Centre, and MePP-South.

Identification of microRNAs and their targets in Paulownia fortunei plants free from phytoplasma pathogen after methyl methane sulfonate treatment.

MicroRNAs (miRNAs) play major roles in plant responses to various biotic and abiotic stresses by regulating gene expression at the transcriptional and post-transcriptional levels. Paulownia witches' broom (PaWB) disease caused by phytoplasmas reduces Paulownia production worldwide. In this study, we investigated the miRNA-mediated plant response to PaWB phytoplasma by Illumina sequencing and degradome analysis of Paulownia fortunei small RNAs (sRNAs). The sRNA and degradome libraries were constructed from healthy and diseased P.fortunei plants and the plants free from phytoplasma pathogen after 60mgL(-1) methyl methane sulfonate treatment. A total of 96 P.fortunei-conserved miRNAs and 83 putative novel miRNAs were identified. Among them, 37 miRNAs (17 conserved, 20 novel) were found to be differentially expressed in response to PaWB phytoplasma infection. In addition, 114 target genes for 18 of the conserved miRNA families and 33 target genes for 15 of the novel miRNAs in P.fortunei were detected. The expression patterns of 14 of the PaWB phytoplasma-responsive miRNAs and 12 target genes were determined by quantitative real-time polymerase chain reaction (qPCR) experiments. A functional analysis of the miRNA targets indicated that these targeted genes may regulate transcription, stress response, nitrogen metabolism, and various other activities. Our results will help identify the potential roles of miRNAs involved in protecting P.fortunei from diseases.

An effector of apple proliferation phytoplasma targets TCP transcription factors-a generalized virulence strategy of phytoplasma?

SummaryThe plant pathogen Candidatus Phytoplasma mali (P. mali) is the causative agent of apple proliferation, a disease of increasing importance in apple‐growing areas within Europe. Despite its economic importance, little is known about the molecular mechanisms of disease manifestation within apple trees. In this study, we identified two TCP (TEOSINTE BRANCHED/CYCLOIDEA/PROLIFERATING CELL FACTOR) transcription factors of Malus x domestica as binding partners of the P. mali SAP11‐like effector ATP_00189. Phytohormone analyses revealed an effect of P. mali infection on jasmonates, salicylic acid and abscisic acid levels, showing that P. mali affects phytohormonal levels in apple trees, which is in line with the functions of the effector assumed from its binding to TCP transcription factors. To our knowledge, this is the first characterization of the molecular targets of a P. mali effector and thus provides the basis to better understand symptom development and disease progress during apple proliferation. As SAP11 homologues are found in several Phytoplasma species infecting a broad range of different plants, SAP11‐like proteins seem to be key players in phytoplasmal infection.

Molecular identification of a 16SrII-D phytoplasma associated with sunflower phyllody in India

Symptoms of phyllody on sunflower in India have previously been reported however, lately, the frequency of phyllody observed has increased. Transmission electron microscope (TEM) examination and PCR including nested PCR analyses using universal primers (P1/P7 and R16F2n/R2) specific to phytoplasma confirmed that the floral abnormalities are due to phytoplasma infection. Nucleotide BLAST analysis of the PCR sequences showed highest identity with sequences of phytoplasma members of the 16SrII group (‘Candidatus Phytoplasma aurantifolia’). Further, phylogenetic analysis of the 16S rRNA sequences clustered the sequences of both sunflower (HAP1) and sesame (SIP1) with other members of 16SrII. ‘Candidatus Phytoplasma’ species assignment and 16Sr group/subgroup classification using iPhyClassifier confirmed that the phytoplasma causing sunflower phyllody is a ‘Candidatus Phytoplasma aurantifolia’ related strain and belongs to the 16SrII-D group. The molecular characters of sunflower phytoplasma were compared with that of the phytoplasma associated with sesame phyllody (used as reference) which showed that the R16F2n/R2 sequences of both the phytoplasmas are identical indicating that the 16SrII-D phytoplasma that is associated with sesame phyllody can infect sunflower as well.

Identification of a novel subgroup 16SrII-U phytoplasma associated with papaya little leaf disease

Papaya is an important fruit crop cultivated in tropical and subtropical regions. Papaya little leaf (PLL) disease was observed in China. The phytoplasma 16S rRNA gene was detected from symptomatic papaya trees via PCR using phytoplasma universal primers P1/P7 followed by R16F2n/R16R2. No amplification products were obtained from templates of asymptomatic papaya trees. These results indicated a direct association between phytoplasma infection and PLL disease. Comparative and phylogenetic analyses of 16S rRNA gene sequences indicated that the papaya-infecting phytoplasmas under study belonged to the peanut witches' broom phytoplasma group (16SrII). Genotyping through use of computer-simulated RFLP analysis of 16S rRNA genes and coefficients of RFLP pattern similarities (0.97) reveal that the PLL phytoplasma was placed in a new subgroup. In this article, we describe the molecular characterization of a new phytoplasma associated with PLL disease and propose that the PLL phytoplasma be considered as a novel subgroup, 16SrII-U.

An 1-Aminocyclopropane-1-carboxylate (ACC) deaminase-expressing endophyte increases plant resistance to flavescence dorée phytoplasma infection

Flavescence dorée is an epidemic yellows disease of grapevine, caused by a phytoplasma (FDP), for which there is currently no cure. We assessed whether the endophyte Pseudomonas migulae 8R6, able to synthesize 1-aminocyclopropane-1-carboxylate (ACC) deaminase, can limit the phytoplasma-induced damages in periwinkle, a model plant hosting FDP. Plant protection induced by 8R6 and its mutant, impaired in ACC deaminase synthesis, was compared. Fifteen plants per treatment were used; FD infection was transmitted by grafting. Evaluation of symptoms was performed every 4 days for 40 days. The presence and the amount of FDP were assessed by nested PCR and qPCR, respectively. Images of phytoplasma inside the infected plants were obtained by transmission electron microscopy. The strain 8R6 significantly reduced the number of symptomatic plants (53% vs 93%). While the density of FDP inside the leaves was unaffected by the bacterial strains, the FDP titre was under the quantification threshold in 38% of the plants inoculated with strain 8R6. Microscopical observations showed damaged FDP cells in plants inoculated with strain 8R6. The ACC deaminase activity of the endophytic bacteria P. migulae 8R6 helps the plant to regulate the level of the stress-related hormone ethylene, leading to significantly improved resistance to phytoplasma infection.

Combined microscopy and molecular analyses show phloem occlusions and cell wall modifications in tomato leaves in response to 'Candidatus Phytoplasma solani'.

Callose deposition, phloem-protein conformational changes and cell wall thickening are calcium-mediated occlusions occurring in the plant sieve elements in response to different biotic and abiotic stresses. However, the significance of these structures in plant-phytoplasma interactions requires in-depth investigations. We adopted a novel integrated approach, based on the combined use of microscopic and molecular analyses, to investigate the structural modifications induced in tomato leaf tissues in presence of phytoplasmas, focusing on vascular bundles and on the occlusion structures. Phloem hyperplasia and string-like arrangement of xylem vessels were found in infected vascular tissue. The diverse occlusion structures were differentially modulated in the phloem in response to phytoplasma infection. Callose amount was higher in midribs from infected plants than in healthy ones. Callose was observed at sieve plates but not at pore-plasmodesma units. A putative callose synthase gene encoding a protein with high similarity to Arabidopsis CalS7, responsible for callose deposition at sieve plates, was upregulated in symptomatic leaves, indicating a modulation in the response to stolbur infection. P-proteins showed configuration changes in infected sieve elements, exhibiting condensation of the filaments. The transcripts for a putative P-protein 2 and a sieve element occlusion-related protein were localized in the phloem but only the first one was modulated in the infected tissues.

Multiplex Real-Time qPCR Assay for Simultaneous and Sensitive Detection of Phytoplasmas in Sesame Plants and Insect Vectors.

Phyllody, a destructive and economically important disease worldwide caused by phytoplasma infections, is characterized by the abnormal development of floral structures into stunted leafy parts and contributes to serious losses in crop plants, including sesame (Sesamum indicum L.). Accurate identification, differentiation, and quantification of phyllody-causing phytoplasmas are essential for effective management of this plant disease and for selection of resistant sesame varieties. In this study, a diagnostic multiplex qPCR assay was developed using TaqMan® chemistry based on detection of the 16S ribosomal RNA gene of phytoplasmas and the 18S ribosomal gene of sesame. Phytoplasma and sesame specific primers and probes labeled with different fluorescent dyes were used for simultaneous amplification of 16SrII and 16SrIX phytoplasmas in a single tube. The multiplex real-time qPCR assay allowed accurate detection, differentiation, and quantification of 16SrII and 16SrIX groups in 109 sesame plant and 92 insect vector samples tested. The assay was found to have a detection sensitivity of 1.8 x 102 and 1.6 x 102 DNA copies for absolute quantification of 16SrII and 16SrIX group phytoplasmas, respectively. Relative quantification was effective and reliable for determination of phyllody phytoplasma DNA amounts normalized to sesame DNA in infected plant tissues. The development of this qPCR assay provides a method for the rapid measurement of infection loads to identify resistance levels of sesame genotypes against phyllody phytoplasma disease.

Association of 16SrII-C phytoplasma with lentil phyllody disease in Pakistan

In April 2011, lentil (Lens culinaris) plants were found with symptoms reminiscent of phytoplasma infection for the first time in Pakistan. Floral malformation, chlorosis, little leaf and extensive proliferation of branches were the most common symptoms. For the confirmation of phytoplasma, total DNA was extracted separately from eight symptomatic plants and was amplified by nested PCR using universal 16Sr DNA phytoplasma primers P1/P7. One representative PCR product was sequenced in both directions using primers R16F2n/R16R2 and was shown to have 100 % identity to phytoplasmas of the 16SrII-C subgroup. The infectious agent was successfully transmitted both by grafting and by the leafhopper Orosius albicinctus, while transmission via seed failed to produce disease symptoms under glasshouse conditions. Infection of lentils with phytoplasmas has not previously been reported in the literature, so to our knowledge, this is the first report of phytoplasma from the 16SrII-C group naturally infecting lentil in Pakistan.

Expression Stabilities of Candidate Reference Genes for RT-qPCR in Chinese Jujube (Ziziphus jujuba Mill.) under a Variety of Conditions.

Reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) is a powerful method for evaluating patterns of gene expression. Jujube whole-genome sequencing has been completed, and analysis of gene function, an important part of any follow-up study, requires the appropriate selection of reference genes. Indeed, suitable reference gene selection for RT-qPCR is critical for accurate normalization of target gene expression. In this study, the software packages geNorm and NormFinder were employed to examine the expression stabilities of nine candidate reference genes under a variety of conditions. Actin-depolymerizing factor 1 (ACT1), Histone-H3 (His3), and Polyadenylate-binding protein-interacting protein (PAIP) were determined to be the most stably expressed genes during five stages of fruit development and ACT1, SiR-Fd, BTF3, and Tubulin alpha chain (TUA) across different tissues/organs. Whereas ACT1, Basic Transcription factor 3 (BTF3), Glyceraldehyde-3-phosphate dehydrogenase (GADPH), and PAIP were the most stable under dark conditions. ACT1, PAIP, BTF3, and Elongation factor 1- gamma (EF1γ) were the most stably expressed genes under phytoplasma infection. Among these genes, SiR-Fd and PAIP are here first reported as stable reference genes. When normalized using these most stable reference genes, the expression patterns of four target genes were found to be in accordance with physiological data, indicating that the reference genes selected in our study are suitable for use in such analyses. This study provides appropriate reference genes and corresponding primers for further RT-qPCR studies in Chinese jujube and emphasizes the importance of validating reference genes for gene expression analysis under variable experimental conditions.

Photosynthetic responses to phytoplasma infection in Chinese jujube

Phytoplasma is one of the most devastating plant pathogens. Jujube witches' broom (JWB) is a typical and highly fatal phytoplasma disease of Chinese jujube (Ziziphus jujuba Mill.), which is widely cultivated in Asia. To further elucidate the mechanism of plant-phytoplasma interaction, we first compared the effects of phytoplasma infection on photosynthetic pigments and activities between a JWB-resistant cultivar (Xingguang) and a susceptible cultivar (Pozao). Total chlorophyll and carotenoid levels were significantly decreased in the susceptible cultivar at later stages of infection, but were remarkably increased in the resistant cultivar at the earlier stages. Compared to uninfected plant, a significant decrease in the main photochemical parameters (Fv/Fm, ΦPSII and qP) was recorded at the initial stages of infection in the resistant cultivar, but occurred at later stages in the susceptible cultivar. Meanwhile, the qRT-PCR results of four key photosynthesis-related genes (ZjGluTR, ZjCBP, ZjRubisco and ZjRCA2) demonstrated that the expression patterns were similar in uninfected cultivars, but up-regulated in resistant cultivar and down-regulated in the susceptible one at 12 wks after grafting inoculation. Collectively, our data indicated that the resistant cultivar ‘Xingguang’ undergoes a decrease in initial stage (inhibiting phytoplasma multiplication) and then a rapid enhancement of photosynthetic activity (helping jujube recovery) in response to phytoplasma infection, while the susceptible cultivar ‘Pozao’ displays a later decrease in photosynthetic activity. The novel photosynthetic response pattern of the resistant cultivar may contribute to its stronger immunity to phytoplasma infection, which provides new insights into plant-phytoplasma interactions.

FIRST REPORT OF ‘CANDIDATUS PHYTOPLASMA SOLANI’ IN PEPPER AND CELERY IN BOSNIA AND HERZEGOVINA

In August 2015, in Bijeljina (Semberija province of Bosnia and Herzegovina) pepper and celery plants were surveyed for phytoplasma infections. During the survey pepper plants (Capsicum annuum ) of cvs Fortesa, Niska Sipka and Amanda showing stunting and leaf yellowing were observed, while celery plants (Apium graveolens L.) variety ‘Giant Prague’, expressed leaf whitening and stunting; the percentages of symptomatic plants ranged from 20 to 30%, respectively. Leaf samples from symptomatic and asymptomatic plants were tested to verify phytoplasma presence using nested-PCR/RFLP analyses. Results of 16S rDNA RFLP analyses with Tru1I and tuf typing using HpaII (Langer and Maixner, 2004) indicated the presence of a ‘Candidatus Phytoplasma solani’-related strain in all the symptomatic samples tested. There was no variability in the tuf gene and only tuf-type b was detected. Four symptomatic pepper and two celery samples were selected for further characterization amplifying vmp1 and stamp genes (Fialova et al., 2009; Fabre et al., 2011). The nucleotide sequences of the obtained amplicons were submitted to GenBank (accession Nos. KU340846-51 and KU295501-06 for vmp1 and stamp sequences, respectively). Homology with ‘Ca. P. solani’ sequences from database was 96- 100% for the vmp1 and 99-100% for the stamp gene. Combined RFLP analyses using Tru1I and Hpy188I on stamp and RsaI on vmp1 genes distinguished four ‘Ca. P. solani’ lineages indicating the presence of genetic variability among the phytoplasmas studied. This is the first report of ‘Ca. P. solani’ presence in symptomatic pepper and celery in Bosnia and Herzegovina.

Multilocus genotyping of a ‘CandidatusPhytoplasma aurantifolia’‐related strain associated with cauliflower phyllody disease in China

AbstractA new cauliflower disease characterised by the formation of leaf‐like inflorescences and malformed flowers occurred in a seed production field located in Yunnan, a southwest province of China. Detection of phytoplasma‐characteristic 16S rRNA gene sequences in DNA samples from diseased plants linked the cauliflower disease to phytoplasmal infection. Results from phylogenetic and virtual restriction fragment length polymorphism analyses of the 16S rRNA gene sequence indicated that the cauliflower‐infecting agent is a ‘Candidatus Phytoplasma aurantifolia’‐related strain and is a new member of the peanut witches'‐broom phytoplasma group, subgroup A (16SrII‐A). Multilocus genotyping showed close genetic relationship between this cauliflower phytoplasma and a broad host range phytoplasma lineage found only in East Asia thus far. Molecular markers present in the secY and rp loci distinguished this phytoplasma from other members of the subgroup 16SrII‐A.

'Candidatus Phytoplasma cirsii', a novel taxon from creeping thistle [Cirsium arvense (L.) Scop

Creeping thistle [Cirsium arvense (L.) Scop.] and dahlia (Dahlia sp.) plants showing typical symptoms of phytoplasma infection including yellowing, stunting, inflorescence and proliferation, were sampled; the presence of phytoplasma was confirmed by standard PCR using universal primers. RFLP analysis allowed classification of the detected phytoplasma strains CirYS, CirYS1 and DahlP within the 16SrXI group, the unique restriction profile F2nR2 fragment obtained in silico by iPhyClassifier indicated that they belong to the new 16SrXI-E subgroup. Genetic analysis of the 16S rRNA gene revealed that the studied strains shared less than 97.5% similarity with all of the previously described 'Candidatus Phytoplasma' species. The closest relatives are 'Candidatus Phytoplasma cynodontis' and 'Candidatus Phytoplasma oryzae' with 96.8% and 96.6% similarity. All strains studied bear three specific regions in the 16S rRNA gene, discriminating them from the other phytoplasma species. Phylogenetic analysis of the 16S rRNA and secA genes confirmed this specificity, as the creeping thistle and dahlia phytoplasma strains clustered in a distinguishable lineage group. The uniqueness of the genetic analysis agrees with the biological characterization of the studied phytoplasma strains, their host range, and geographical distribution. The strains only infect dicotyledonous plants in Europe, contrary to their closest relatives. Based on their unique properties, it could be concluded that the studied phytoplasma strains represent a discrete group that is proposed as a novel taxon 'Candidatus Phytoplasma cirsii', with strain CirYS as a reference strain.