Aster Yellows Phytoplasma Research Articles

Molecular detection of aster yellows phytoplasma and "Candidatus Liberibacter solanacearum" in carrots affected by the psyllid Trioza apicalis (Hemiptera: Triozidae) in Finland.

Molecular detection of aster yellows phytoplasma and "Candidatus Liberibacter solanacearum" in carrots affected by the psyllid Trioza apicalis (Hemiptera: Triozidae) in Finland.

Detection of Elm Yellows Phytoplasma in Elms and Insects Using Real-Time PCR.

A rapid and accurate method to detect the common strain of elm yellows (EY) phytoplasma in elm and insect samples was developed using a real-time polymerase chain reaction (PCR) procedure based on the TaqMan minor-groove-binder probe. Primers and probe were designed based on the EY phytoplasma-specific translocation protein secY gene DNA sequence. Success of the DNA extraction procedure was evaluated by amplifying the chloroplast trnL gene of Ulmus americana. The real-time PCR assay reacted positively with EY and EY phytoplasma strain ULW DNA, an isolate which occurs in Europe. It did not cross-react with Illinois EY or aster yellows phytoplasma DNA, both of which are known to occur in elm trees in the United States, nor did it amplify several other phytoplasmas belonging to the 16SrV and other phylogenetic groups. The real-time PCR protocol was used to identify 30 EY-positive elm trees on The Pennsylvania State University, University Park campus. Threshold cycle (CT) values obtained from the EY phytoplasma-infected elm trees ranged from 15 to 37. EY phytoplasma was detected in several leafhopper taxa. This real-time PCR method can be used for the diagnostic screening of elm trees and for the identification of possible insect vectors of EY phytoplasma.

First Report of Spiroplasma citri in Carrot in Europe.

In 2008 and 2009, symptoms of curling, yellow and purple discoloration of leaves, stunting of shoots and tap roots, and formation of bunchy, fibrous secondary roots were observed in commercial carrot (Daucus carota L.) fields located in several production areas of Spain (Alicante, Albacete, Segovia, and Valladolid). Incidence of this disease was almost 100% in individual affected fields. Similar symptoms were reported from 1997 to 1998 in various carrot production areas of Spain (the Canary Islands, Segovia, and Madrid) and were associated with infection of stolbur and aster yellows phytoplasmas (2). Moreover, the observed symptoms resembled those caused by Spiroplasma citri in carrots affected by the carrot purple leaf disease recently reported in the United States (4). Studies were conducted to investigate whether S. citri and phytoplasmas were associated with the observed carrot symptoms. Total DNA was extracted from 0.5 g of phloem tissue of 13 symptomatic and 3 asymptomatic plants with DNeasy Plant Mini Kit (Qiagen, Valencia, CA). DNA samples were analyzed by nested-PCR assays using primers pair P1/P7 (1) and R16F2n/R16R2n (3) for phytoplasmas and ScR16F1/ScR16R1 followed by ScR16F1A/ScR16R2 (4) for S. citri detection. DNA of a known strain of S. citri (Sediag, Longvic, France) was used as a positive control of the assay. Analyses revealed that 8 of the 13 symptomatic plants tested positive for S. citri; the plants were collected from three different provinces of Spain, namely, Alicante, Valladolid, and Segovia. Two symptomatic plants were double infected by S. citri and a phytoplasma strain belonging to the Aster yellows group (16SrI), subgroup 16SrI-A. However, none of the symptomatic plants presented single infection with phytoplasmas. S. citri identity was determined by sequencing two nested PCR products (1.1 kb) that yielded identical sequences deposited in the GenBank database (Accession Nos. HM124555 and HM124556). BLAST analysis showed 100% nt identity with a sequence of S. citri from carrot (Accession No. DQ112019) associated with the new carrot disease referred to as 'carrot purple leaf reported in Washington State (4). To our knowledge, this is the first report of S. citri associated with carrot in Europe.

Detection of aster yellows phytoplasma DNA in seed and seedlings of canola (Brassica napus and B. rapa) and AY strain identification

A study was conducted to identify the strains of aster yellows (AY) disease present in crops of Brassica napus and B. rapa grown near Medstead, Saskatchewan. AY phytoplasma DNA was detected in midrib, stem and root tissues of several symptomless plants as well as plants exhibiting typical AY disease symptoms. Most symptomatic and symptomless, AY-infected plants produced normal-looking and misshapen seeds. However, for both Brassica species, symptomatic plants produced significantly more seeds containing phytoplasma DNA than symptomless, AY-infected plants. Also, significantly more misshapen seeds contained phytoplasma DNA than normal seeds. Phytoplasma DNA belonging to subgroups 16SrI-A and 16SrI-B was detected in symptomatic and symptomless, AY-infected plants and in seed of these plants. The new AY strain sequences were registered in Genbank. The study reports for the first time the detection of AY strains in seedling tissues of both Brassica species. The research also showed that spiral cleaning has the potential to remove seeds that contain phytoplasma DNA in B. napus.

Phytoplasma PMU1 exists as linear chromosomal and circular extrachromosomal elements and has enhanced expression in insect vectors compared with plant hosts

Phytoplasmas replicate intracellularly in plants and insects and are dependent on both hosts for dissemination in nature. Phytoplasmas have small genomes lacking genes for major metabolic pathways. Nevertheless, their genomes harbour multicopy gene clusters that were named potential mobile units (PMUs). PMU1 is the largest most complete repeat among the PMUs in the genome of Aster Yellows phytoplasma strain Witches' Broom (AY-WB). PMU1 is c. 20 kb in size and contains 21 genes encoding DNA replication and predicted membrane-targeted proteins. Here we show that AY-WB has a chromosomal linear PMU1 (L-PMU1) and an extrachromosomal circular PMU1 (C-PMU1). The C-PMU1 copy number was consistently higher by in average approximately fivefold in insects compared with plants and PMU1 gene expression levels were also considerably higher in insects indicating that C-PMU1 synthesis and expression are regulated. We found that the majority of AY-WB virulence genes lie on chromosomal PMU regions that have similar gene content and organization as PMU1 providing evidence that PMUs contribute to phytoplasma host adaptation and have integrated into the AY-WB chromosome.

Molecular characterization of a new isolate of phytoplasma associated with malformation and twisting of floral spikes of Gladiolus in India

In 2007–2009, severe virescence, malformation and twisting of flower spikes and yellowing of entire plants were observed in various Gladiolus cultivars growing in the gardens of the National Botanical Research Institute, Lucknow, India. The disease symptoms were very similar to symptoms in Gladiolus caused by the Aster yellows phytoplasma identified from Poland. Disease incidence was low (1.1–3.4%), but the severity of symptoms was high. A phytoplasma infection was detected in nine of 13 cultivars by PCR followed by nested PCR using universal phytoplasma primers P1/P6 or R16F2n/R16R2, respectively. An amplicon of ~1.2 kb obtained from the nested PCR was cloned and sequenced. Sequence analysis of the PCR amplicon revealed high (94–98%) identities and the closest phylogenic relationships with several isolates of Aster yellows phytoplasma of ‘Candidatus Phytoplasma asteris’ (16SrI group). Thus, the phytoplasma isolate of Gladiolus was identified as a new isolate of ‘Ca. P. asteris’ (16SrI group). In silico analysis of the phytoplasma isolate clearly indicated that the isolate was distinct from other Indian isolates of this phytoplasma.

Mobile units of DNA in phytoplasma genomes

Phytoplasmas are obligate symbionts of plants and insects that are responsible for significant yield losses in diverse crops. Genome sequencing has revealed that many phytoplasma genomes appear to contain repeated genes organized in units of approximately 20 kb. These 'potential mobile units' (PMUs) resemble composite replicative transposons. PMUs contain several genes for recombination and some also contain putative 'virulence genes'. Genome alignments suggest that PMUs are involved in phytoplasma genome instability and recombination. In this edition of Molecular Microbiology, Hogenhout and colleagues report that one PMU from the aster yellows phytoplasma strain Witches' Broom (AY-WB) can exist as both a linear PMU within the chromosome and as an extrachromosomal circular form. The copy number of the circular form is much higher in the insect vector compared with the plant, and expression levels of genes present on the PMU are also higher in the insect. These observations suggest not only that this PMU could be a mobile element, but that it could also be involved in a phase-variation mechanism that allows the phytoplasma to adapt to its different hosts.

Effect of arbuscular mycorrhizal fungi inoculation on aster yellows phytoplasma-infected tobacco plants

The objective of the research was to asses if arbuscular mycorrhizal fungi (AM) can modify the effect of two aster yellows phytoplasma strains infection in tobacco plants. Tobacco plants experimentally inoculated with aster yellows phytoplasma strains did not develop visible disease symptoms. However, PCR examination indicated that the inoculated plants were phytoplasma infected. Mycorrhiza inoculation had a positive effect on the shoot height of healthy plants, but did not influence shoot growth and weight of phytoplasma-infected plants. The roots of all mycorrhiza-inoculated plants were slightly reduced but significant differences were found in the total root length of plants infected with the phytoplasma strain AY1. AM inoculation had a positive effect on photosynthetic activity of tobacco plants infected with the phytoplasma strain AYSim, but net photosynthesis of tobacco infected with the phytoplasma strain AY1 was decreased. Transpiration rate and calcium content of AM and phytoplasma-infected plants were not affected. The mechanisms underlying these interactions are discussed and a direct action of the AM fungus is hypothesized.

First Report of Aster Yellows Phytoplasma in Grapevines in South Africa.

For many years phytoplasma diseases have caused serious losses in most of the major grape-growing regions of the world, except South Africa, where a mixed phytoplasma infection was first reported in 2006 (1). During the early growing season of 2006, symptoms consistent with phytoplasma disease were observed in vineyards in the Olifants River Valley. Symptoms included yellowing of leaves, incomplete lignification of shoots, shortening of internodes, and the abortion of growth tips and immature bunches. Symptomatic shoots and leaves from grapevine cultivars (Merlot, Shiraz, Cabernet Sauvignon, Ruby Cabernet, Pinotage, Corinth, Chardonnay, Columbar, Chenin blanc, Sauvignon blanc, Sultana, and Regal) were collected during the early growing season (November) of 2006, 2007, and 2008. Total DNA was extracted from 32 of these samples (from single plants in the same vineyards over the 3 years) with the Invisorb Spin Plant Mini Kit (Invitek, Berlin, Germany) and tested by nested PCR using two universal primer pairs, P1/P7 and R16F2n/R16R2 (3). The first round of PCR of the 2006 samples yielded 1.8-kb fragments for 17 of the samples, while the nested PCR yielded an additional seven positive samples, confirming the necessity of nested PCR for reliable diagnosis. A similar trend was observed in the 2007 and 2008 PCR test results. All asymptomatic plants, which were included as negative controls, and water controls were negative by nested PCR. Twenty-four 1,245-bp amplicons, generated by nested PCR, were excised from gels, purified with a NucleoSpin Extract II Kit (Macherey-Nagel, Düren, Germany) and directly sequenced. Sequence data was compiled with the BioEdit Version 7.0.4.1 sequence alignment editor software (2), aligned using ClustalW Version 1.4 (4), and a consensus sequence was generated (GenBank Accession No. GQ365729). A BLAST search of the NCBI GenBank database using the individual sequences revealed high sequence identities (≥99%) with the aster yellows phytoplasma group (16SrI) and specifically with the subgroup 16SrI-B. In a comparison of the sequences of the 1.2-kb PCR fragments of 24 local samples with each other, sequence identities of ~99% were observed. These results clearly illustrate that all vines screened were infected with the same phytoplasma. Single nucleotide differences observed between different isolates may indicate the presence of closely related sequence variants of this phytoplasma. Aster yellows occurs worldwide and has been reported to infect grapevine-South Africa can now be added to this list. During the three seasons of our study, the area in which symptomatic vineyards were observed increased significantly, indicating spread by a biological vector. Moreover, infected vineyards were noticed in two other South African grape-growing regions. In contrast to the previous report, which reported a mixed infection of phytoplasmas of groups 16SrXII-A and 16SrII-B (1), PCR screening and sequencing of more than 40 individual samples from these areas confirmed these all to be infected with aster yellows phytoplasma only. To our knowledge, this is the first report of the detection and identification of an aster yellows phytoplasma causing grapevine yellows disease in South Africa.

Response of mycorrhizal periwinkle plants to aster yellows phytoplasma infection

The objective of our research was to assess if arbuscular mycorrhizal (AM) fungal colonization can modify the effect of infection by two aster yellows phytoplasma strains (AY1, AYSim) in Catharanthus roseus plants. Both phytoplasma strains had a negative effect on the root fresh weight, but they differed in symptoms appearance and in their influence on photosynthetic and transpiration rates of the periwinkle plants. AM plants showed significantly reduced shoot fresh weight, while the transpiration rate was significantly increased. AM fungal colonization significantly affected shoot height and fresh weight of the plants infected by each phytoplasma strains as well as the root system of plants infected with the more aggressive AYSim phytoplasma strain. Double inoculation did not reduce the negative effects induced with phytoplasma alone on the photosynthetic activity of phytoplasma-infected plants.

Elucidation of the Roles of Blackcurrant reversion virus and Phytoplasma in the Etiology of Full Blossom Disease in Currants.

To determine the roles of phytoplasmas and Blackcurrant reversion virus (BRV) in the etiology of full blossom disease (FBD), we conducted graft and dodder transmission experiments. Scions from FBD-affected Ribes rubrum were grafted onto red currants, white currants, and black currants. Red and white cultivars revealed symptoms of FBD, whereas blackcurrant displayed symptoms of BRV. No differences in symptoms were observed between plants infected with BRV only and those infected with BRV and phytoplasma. Aster yellows phytoplasma subgroup 16SrI-C was transferred from FBD-infected red currants to periwinkle, where symptoms of green and yellow petal were observed. Back-transmission of phytoplasma to currant seedlings of red and black currant was not successful. Scions of periwinkle infected with aster yellows phytoplasmas of subgroup 16SrI-C and 16SrI-B, which were bottle-, bark-, and approach-grafted onto seedlings of red and black currant, resulted in positive but symptomless transmission of phytoplasma to red currant. We conclude that FBD symptoms are induced by BRV rather than by phytoplasma, which was originally described as the causal agent of FBD.

Detection and molecular characterization of an aster yellows phytoplasma in rhodiola in Alberta, Canada

Rhodiola plants exhibiting symptoms typical of phytoplasma infection were observed at an experimental farm near Edmonton, Alberta, Canada, in 2007. Phytoplasma infection was confirmed with a polymerase chain reaction (PCR)-assay using 16S ribosomal RNA primers developed specifically for phytoplasmas; the primers could amplify a band from tissue samples derived from infected, but not from asymptomatic, plants. Comparison between the sequence of the PCR product and the 16S ribosomal RNA gene sequences of other phytoplasmas available in GenBank indicated that the phytoplasma identified in rhodiola is a member of the subgroup 16SrI-B (aster yellows subgroup B).

First Report of Aster Yellows Phytoplasma in Goldenrain Tree (Koelreuteria paniculata) in Korea

Aster yellows phytoplasma was detected for the first time in goldenrain tree (Koelreuteria paniculata) growing in Sinpyeong-myeon, Jeollabuk-do, South Korea. DNA was extracted from the infected leaf samples and part of the 16S rDNA, rp operon and tuf gene were amplified using R16F2n/R2 and gene-specific primers. The sequence analysis showed that the phytoplasma was closely related (99%) to members of the Aster Yellows (AY) group, and belonging to 16Sr I, subgroup B. Moreover, the 16S rDNA sequences of the isolate showed 88–96% identity with members of other 16Sr and undesignated groups. Based on the sequence identity and phylogenetic studies, it was confirmed that phytoplasma infecting goldenrain tree in South Korea belongs to the AY group.

First Report of Aster Yellow Phytoplasmas (‘Candidatus Phytoplasma asteris’) in Canadian Grapevines

In North America, elm yellows, aster yellows (AY), and X-disease phytoplasmas have been detected in American grapevines (1), and recently, Bois noir was detected in Canadian vineyards from British Columbia (BC) and Ontario (ON) (2). Typical symptoms of grapevine yellows (GY) include leaf rolling and chlorosis, uneven or total lack of lignification of canes, flower abortion or berry withering, and stunting. In 2006 and 2007, independent surveys were conducted by the Canadian Food Inspection Agency (CFIA) and Agriculture and Agri-Food Canada (AAFC) to detect phytoplasmas in Canadian vineyards containing different cultivars in BC, ON, Québec (QC), Nova Scotia, New Brunswick, and Prince Edward Island. The CFIA collected and tested 651 fresh leaf samples from recently imported grapevines and older grapevines in the same or neighboring blocks displaying symptoms typical of those associated with disease caused by phytoplasmas. Many vineyards were surveyed only once. AAFC collected and tested 3,485 samples from symptomatic and asymptomatic grapevines from established vineyards in ON, BC, and QC. The same vineyards were sampled in ON and BC both years; QC vineyards were only sampled in 2007. AAFC-collected leaf samples were freeze dried and stored at -20°C before processing. CFIA samples were tested by a modified real-time PCR assay and TaqMan probe targeting the 16S ribosomal RNA gene that detects a wide range of known phytoplasmas (2). Positive samples were confirmed by conventional PCR using the phytoplasma-specific primers P1/P7 (3) and the resulting ~1,800-bp fragment was cloned and sequenced as previously described (2). DNA extracted by AAFC was amplified by nested PCR technology with universal phytoplasma specific primer pairs P1/P6 and R16R2/R16F2 (3) and the resulting 1,200-bp fragment was cloned and sequenced. Two plants, one located in ON in 2006 and the other in BC in 2007, were found to be infected with an AY-like phytoplasma by the CFIA. The phytoplasmas detected in both infected plants had a 99.9% nt sequence identity with AY phytoplasma sequences from GenBank (Accession Nos. AF222063 and AY665676, respectively), with the BC isolate also showing 100% identity to a strain of AY, ash witches'-broom phytoplasma (GenBank Accession No. AY566302). AAFC detected phytoplasma DNA in both years in a total of 17 symptomatic plants and 21 asymptomatic plants from different vine varieties in ON, BC, and QC. Positive samples were found to have a 99.0% nt sequence identity to AY subgroup 16SrI-A (GenBank Accession No. AY180956). Sequences were exchanged for confirmation of phytoplasma identity and were deposited in Genbank under Accession Nos. FJ659844 and FJ824597. Phytoplasma strains were identified for all plants in which phytoplasmas were detected. Results show that AY is present in vineyards in the provinces of ON, BC, and QC. To our knowledge, this is the first report of AY being detected in grapevines in Canada.

Molecular data reveals California as the potential source of an invasive leafhopper species, Macrosteles sp. nr. severini, transmitting the aster yellows phytoplasma in Hawaii

AbstractA species of aster leafhopper (Macrosteles sp.) became established in 2001 on Oahu, Hawaii, and through the transmission of the aster yellows phytoplasma, caused devastating losses to the island’s watercress industry. DNA sequence data were analysed from two mitochondrial genes [cytochrome oxidase subunit 1(CO1) and nicotinamide adenine dinucleotide 1 (NADH1)] and one nuclear gene (wingless, Wg) (combined total of 1874 bp) to reconstruct phylogenetic relationships between putative US mainland source populations of aster leafhoppers and those introduced to Hawaii. These data were applied to elucidate the origin(s) and identity of Hawaiian infestations and the amount of genetic diversity within introduced invasive populations. Both phylogenetic search criteria (Bayesian and maximum likelihood models) converged onto similar tree topologies for all three gene regions and suggested that Hawaii infestations represent a single undescribed leafhopper species unrelated to the common aster leafhopper, Macrosteles quadrilineatus. An exact haplotype match was found from a specimen intercepted from watercress shipped to Hawaii from Los Angeles, California, suggesting this region as the potential source for Hawaiian infestations. Two mitochondrial haplotypes were identified in Hawaii suggesting two or perhaps just a single introduction of more than one female.

Association of ‘Candidatus Phytoplasma asteris’ (16SrI group) with yellows of Achyranthes aspera in India

Achyranthes aspera (family Amaranthaceae), commonly known as ‘Latjeera’, is an annual herb that grows wild throughout India. It is an indigenous medicinal plant of Asia and is commonly used by traditional healers for the treatment of malarial fever, dysentery, asthma, hypertension and diabetes (Girach & Khan, 1992). A root extract is also used to treat scorpion and snake bites by Indian tribes. A yellows disease was noticed on a number of A. aspera plants growing as weeds beside a road between Sitapur and Lucknow, U. P., India during February, 2008. The diseased plants showed typical yellows and small leaves symptoms as compared to healthy ones. Samples from plants with symptoms were collected and total DNA was extracted from ~100 mg of leaf tissues employing a phytoplasma enrichment procedure (Ahrens & Seemüller, 1992). Amplicons of ~1·5 kb and ~1·2 kb were produced respectively by direct polymerase chain reaction (PCR) using primers P1/P6 (Deng & Hiruki, 1991) followed by nested PCR using 1:10 diluted first stage products and primers R16F2n/R16R2 (Gundersen & Lee, 1996). Three amplicons of ~1·2 kb were sequenced and consensus sequence data deposited in GenBank (Accession No. EU573926). blast search analysis showed 99% sequence similarities with the members of ‘Candidatus Phytoplasma asteris’ (16SrI group phytoplasma): sugarcane yellows (EU423900); periwinkle little leaf (EU375834, DQ381535); onion yellows (AP006628, D12569); carrot phytoplasma (EU215426, EU215425) and aster yellows phytoplasma (EF489024, AY665676). Phylogenetic analysis of the phytoplasma isolates using MEGA 4·0 tool showed close relationships with isolates of aster yellows ‘Ca. P. asteris’ (16SrI) group indicating the phytoplasma in A. aspera is an isolate of ‘Ca. P. asteris’. Achyranthes aspera has been reported as a host of witches’ broom disease of lime caused by ‘Candidatus Phytoplasma aurantifolia’ in the Sultanate of Oman (Moghal et al., 1998). Previously, ‘Ca. P. asteris’ has been reported in India on Cannabis sativa and Parthenium hysterophorus, but this is a first report of a ‘Ca. P. asteris’ associated with yellows on A. aspera worldwide.

Identification of a phytoplasma from the aster yellows group infecting purple coneflower (Echinacea purpurea) in Slovenia

Purple coneflower (Echinacea purpurea) (Asteraceae family), is a perennial flowering plant native to eastern North America which is widely grown as an ornamental and medicinal plant. Since 2002, phytoplasma-like symptoms have been observed in several gardens in the Savinja valley in Slovenia. Disease incidence (5–30%) has varied between years and increases with plant age. The symptoms seen include plant weakness, leaf yellowing, and floral malformations particularly virescence and phyllody. For molecular identification of the pathogen, the total DNA was isolated from samples of several parts of an infected plant and from a plant without symptoms using a CTAB protocol (Kump & Javornik, 1996). PCR was carried out using three universal 16S rDNA-specific primers P1/P7, Fu5/Ru3 and R16F2n/R16R2. All three primer pairs gave a single PCR products from tissue with symptoms, with the expected lengths of ~1·8, 0·9, and 1·2 kbp, but no products from samples from plants without symptoms. All PCR products were TA cloned (Promega T-easy), sequenced and assembled into a single sequence of a total length of 1840 bp, which was deposited in GenBank (Accession No. EU416172). A blast search revealed 100% sequence identity for the entire contig with poa stunt phytoplasma (DQ640502) and clover phyllody phytoplasma strain CPh (AF222066) sequences, both belonging to the 16SrI-C subgroup of the aster yellows group (AY). Other matches with high sequence similarities (98–99%) also belonged to aster yellows phytoplasma members, e.g. onion yellows phytoplasma (AP006628.2), epilobium phyllody phytoplasma (AY101386), silene virescence phytoplasma (AY744070) and to the Czech strain of purple coneflower phyllody phytoplasma (EF546778). Phytoplasmas belonging to the AY group have been previously identified in purple coneflower in the USA, Canada (Stanosz & Heimann, 1997; Lee et al., 2008) and in the Czech Republic. This is the first report of detection of an isolate of ‘Candidatus Phytoplasma asteris’ (subgroup 16SrI-C) infecting purple coneflower in Slovenia.

ESTABLISHMENT OF A NEW PHYTOPLASMA SUBGROUP, 16SRI-Q, TO ACCOMMODATE A PREVIOUSLY UNDESCRIBED PHYTOPLASMA FOUND IN DISEASED CHERRY IN LITHUANIA

Symptoms of shoot proliferation and abnormally small leaves were observed in diseased sour cherry (Cerasus vulgaris Mill., syn. Prunus cerasus L.) affected by cherry little leaf (ChLL) disease on the Neringa peninsula, Lithuania. Amplification of phytoplasma characteristic 16S rDNA from the diseased cherry indicated infection by a phytoplasma. On the basis of restriction fragment length polymorphism (RFLP) and nucleotide sequence analysis of 16S rDNA amplified by PCR, the ChLL phytoplasma was classified in group 16SrI (aster yellows phytoplasma group), new subgroup 16SrI-Q. Results from phylogenetic analysis of 16S rRNA gene sequences indicated that ChLL phytoplasma was related to ‘Candidatus Phytoplasma asteris’ and may represent a distinct phytoplasma lineage.

Multigene analysis for differentiation of aster yellows phytoplasmas infecting carrots in Serbia

AbstractDuring a survey of large carrot fields in Serbia, plants showing leaf reddening and/or yellowing, adventitious shoot production and reduction in taproot size and quality were observed in a low percentage of plants. To verify phytoplasma association with the described symptoms and to carry out pathogen differentiation, PCR assays followed by restriction fragment length polymorphism (RFLP) analyses and/or sequencing of phytoplasma 16Sr DNA and ribosomal protein genes l22 and s3, tuf, putative aa kinase plus ribosomal recycling factor genes and DNA helicase gene were carried out. Phytoplasmas belonging to 16SrI‐A and 16SrI‐B ribosomal subgroups and to rpI‐A and rpI‐B ribosomal protein subgroups, respectively, were identified by RFLP analyses in 13 of 15 symptomatic plants tested. No amplification was obtained with non‐symptomatic carrot samples. The identification was confirmed by sequence analyses of the phytoplasma genes studied. In two carrot samples, presence of interoperon sequence heterogeneity was detected and phytoplasma strains were identified as belonging to 16SrI group but were not assigned to any 16S rRNA or ribosomal protein subgroup. This research allowed the first molecular identification of phytoplasmas infecting carrot in Serbia using several molecular markers, and it indicates that under field conditions in non‐epidemic outbreaks a certain amount of genetic mutation may occur in conserved genes of these prokaryotes.

AY-WB Phytoplasma Secretes a Protein That Targets Plant Cell Nuclei

The fully sequenced genome of aster yellows phytoplasma strain witches' broom (AY-WB; Candidatus Phytoplasma asteris) was mined for the presence of genes encoding secreted proteins based on the presence of N-terminal signal peptides (SP). We identified 56 secreted AY-WB proteins (SAP). These SAP are candidate effector proteins potentially involved in interaction with plant and insect cell components. One of these SAP, SAP11, contains an N-terminal SP sequence and a eukaryotic bipartite nuclear localization signal (NLS). Transcripts for SAP11 were detected in AY-WB-infected plants. Yellow fluorescence protein (YFP)-tagged SAP11 accumulated in Nicotiana benthamiana cell nuclei, whereas the nuclear targeting of YFP-tagged SAP11 mutants with disrupted NLS was inhibited. The nuclear transport of YFP-SAP11 was also inhibited in N. benthamiana plants in which the expression of importin alpha was knocked down using virus-induced gene silencing (VIGS). Furthermore, SAP11 was detected by immunocytology in nuclei of young sink tissues of China aster plants infected with AY-WB. In summary, this work shows that AY-WB phytoplasma produces a protein that targets the nuclei of plant host cells; this protein is a potential phytoplasma effector that may alter plant cell physiology.