Correspondence G. Firrao firrao{at}uniud.it
The trivial name phytoplasma has been adopted to collectively name wall-less, non-helical prokaryotes that colonize plant phloem and insects, which were ...formerly known as mycoplasma-like organisms. Although phytoplasmas have not yet been cultivated in vitro , phylogenetic analyses based on various conserved genes have shown that they represent a distinct, monophyletic clade within the class Mollicutes . It is proposed here to accommodate phytoplasmas within the novel genus Candidatus ( Ca. ) Phytoplasma. Given the diversity within Ca. Phytoplasma, several subtaxa are needed to accommodate organisms that share <97·5 % similarity among their 16S rRNA gene sequences. This report describes the properties of Ca. Phytoplasma, a taxon that includes the species Ca. Phytoplasma aurantifolia (the prokaryote associated with witches'-broom disease of small-fruited acid lime), Ca. Phytoplasma australiense (associated with Australian grapevine yellows), Ca. Phytoplasma fraxini (associated with ash yellows), Ca. Phytoplasma japonicum (associated with Japanese hydrangea phyllody), Ca. Phytoplasma brasiliense (associated with hibiscus witches'-broom in Brazil), Ca. Phytoplasma castaneae (associated with chestnut witches'-broom in Korea), Ca. Phytoplasma asteris' (associated with aster yellows), Ca. Phytoplasma mali (associated with apple proliferation), Ca. Phytoplasma phoenicium (associated with almond lethal disease), Ca. Phytoplasma trifolii (associated with clover proliferation), Ca. Phytoplasma cynodontis' (associated with Bermuda grass white leaf), Ca. Phytoplasma ziziphi (associated with jujube witches'-broom), Ca. Phytoplasma oryzae (associated with rice yellow dwarf) and six species-level taxa for which the Candidatus species designation has not yet been formally proposed (for the phytoplasmas associated with X-disease of peach, grapevine flavescence dorée, Central American coconut lethal yellows, Tanzanian lethal decline of coconut, Nigerian lethal decline of coconut and loofah witches'-broom, respectively). Additional species are needed to accommodate organisms that, despite their 16S rRNA gene sequence being >97·5 % similar to those of other Ca. Phytoplasma species, are characterized by distinctive biological, phytopathological and genetic properties. These include Ca. Phytoplasma pyri (associated with pear decline), Ca. Phytoplasma prunorum (associated with European stone fruit yellows), Ca. Phytoplasma spartii (associated with spartium witches'-broom), Ca. Phytoplasma rhamni (associated with buckthorn witches'-broom), Ca. Phytoplasma allocasuarinae (associated with allocasuarina yellows), Ca. Phytoplasma ulmi (associated with elm yellows) and an additional taxon for the stolbur phytoplasma. Conversely, some organisms, despite their 16S rRNA gene sequence being <97·5 % similar to that of any other Ca. Phytoplasma species, are not presently described as Candidatus species, due to their poor overall characterization.
Abbreviations: Ca. , Candidatus ; IRPCM, International Research Programme for Comparative Mycoplasmology
Published online ahead of print on 13 February 2004 as DOI 10.1099/ijs.0.02854-0.
This paper is dedicated to the memory of Monique Garnier-Semancik, fine scientist and friend.
Phytoplasma research begins to bloom (75). Indeed, this review shows that substantial progress has been made with the identification of phytoplasma effectors that alter flower development, induce ...witches' broom, affect leaf shape, and modify plant-insect interactions. Phytoplasmas have a unique life cycle among pathogens, as they invade organisms of two distinct kingdoms, namely plants (Plantae) and insects (Animalia), and replicate intracellularly in both. Phytoplasmas release effectors into host cells of plants and insects to target host molecules, and in plants these effectors unload from the phloem to access distal tissues and alter basic developmental processes. The effectors provide phytoplasmas with a fitness advantage by modulating their plant and insect hosts. We expect that further research on the functional characterization of phytoplasma effectors will generate new knowledge that is relevant to fundamental aspects of plant sciences and entomology, and for agriculture by improving yields of crops affected by phytoplasma diseases.
Phytoplasmas are a group of bacteria that are associated with hundreds of plant diseases. Due to their economical importance and the difficulties involved in the experimental study of these obligate ...pathogens, genome sequencing and comparative analysis have been utilized as powerful tools to understand phytoplasma biology. To date four complete phytoplasma genome sequences have been published. However, these four strains represent limited phylogenetic diversity. In this study, we report the shotgun sequencing and evolutionary analysis of a peanut witches'-broom (PnWB) phytoplasma genome. The availability of this genome provides the first representative of the 16SrII group and substantially improves the taxon sampling to investigate genome evolution. The draft genome assembly contains 13 chromosomal contigs with a total size of 562,473 bp, covering ∼90% of the chromosome. Additionally, a complete plasmid sequence is included. Comparisons among the five available phytoplasma genomes reveal the differentiations in gene content and metabolic capacity. Notably, phylogenetic inferences of the potential mobile units (PMUs) in these genomes indicate that horizontal transfer may have occurred between divergent phytoplasma lineages. Because many effectors are associated with PMUs, the horizontal transfer of these transposon-like elements can contribute to the adaptation and diversification of these pathogens. In summary, the findings from this study highlight the importance of improving taxon sampling when investigating genome evolution. Moreover, the currently available sequences are inadequate to fully characterize the pan-genome of phytoplasmas. Future genome sequencing efforts to expand phylogenetic diversity are essential in improving our understanding of phytoplasma evolution.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Phytoplasmas are specialised bacteria that are obligate parasites of plant phloem tissue and insects. These bacteria have resisted all attempts of cell-free cultivation. Genome research is of ...particular importance to analyse the genetic endowment of such bacteria. Here we review the gene content of the four completely sequenced ‘Candidatus Phytoplasma’ genomes that include those of ‘Ca. P. asteris’ strains OY-M and AY-WB, ‘Ca. P. australiense,’ and ‘Ca. P. mali’. These genomes are characterized by chromosome condensation resulting in sizes below 900 kb and a G + C content of less than 28%. Evolutionary adaption of the phytoplasmas to nutrient-rich environments resulted in losses of genetic modules and increased host dependency highlighted by the transport systems and limited metabolic repertoire. On the other hand, duplication and integration events enlarged the chromosomes and contribute to genome instability. Present differences in the content of membrane and secreted proteins reflect the host adaptation in the phytoplasma strains. General differences are obvious between different phylogenetic subgroups. ‘Ca. P. mali’ is separated from the other strains by its deviating chromosome organization, the genetic repertoire for recombination and excision repair of nucleotides or the loss of the complete energy-yielding part of the glycolysis. Apart from these differences, comparative analysis exemplified that all four phytoplasmas are likely to encode an alternative pathway to generate pyruvate and ATP.
The focus of this research was the development and evaluation of different complex liquid and solid media for the isolation and growth of phytoplasma strains infecting grapevine plants. Previously ...reported media supporting phytoplasma isolation are commercial and not easy to modify in order to improve performance and selectivity towards obtaining pure cultures of ‘Candidatus Phytoplasma’ species. Three media (Piv®, CB and MB) were therefore evaluated for phytoplasma isolation and colony formation under microaerophilic growing conditions, using grapevine canes from plants showing yellows symptoms, and infected by “flavescence dorée”, “bois noir” and aster yellows phytoplasmas as sources. The newly developed methodology was applied for two years at three sample collection times. Broad applicability and a good repeatability in supporting phytoplasma colony formation were obtained in Pivs® and CBs media. While the MB medium did not support phytoplasma isolation and growth, the CB media support a phytoplasma growth comparable to the one obtained in the previously reported media. This medium has the advantage of a formulation that allow its modification to implement specificity towards selective phytoplasma growth. Moreover preliminary trials on serial dilutions and tetracycline addition confirmed some phytoplasma growth behaviours.
•Phytoplasmas were isolated from infected field-collected grapevine samples.•Complex media for phytoplasma isolation and growth were tested.•Unreported microaerophilic conditions are settled for phytoplasma plate colony growth.•“Flavescence dorée”, “bois noir” and aster yellows phytoplasma colonies are obtained from field infected materials.•Phytoplasma presence in colonies was detected by nested-PCR and sequencing on two genes.
Molecular Plant Pathology Laboratory, USDA-Agricultural Research Service, Beltsville, MD 20705, USA
Correspondence Yan Zhao yan.zhao{at}ars.usda.gov
Phytoplasmas, the causal agents of numerous plant ...diseases, are insect-vector-transmitted, cell-wall-less bacteria descended from ancestral low-G+C-content Gram-positive bacteria in the Bacillus – Clostridium group. Despite their monophyletic origin, widely divergent phytoplasma lineages have evolved in adaptation to specific ecological niches. Classification and taxonomic assignment of phytoplasmas have been based primarily on molecular analysis of 16S rRNA gene sequences because of the inaccessibility of measurable phenotypic characters suitable for conventional microbial characterization. In the present study, an interactive online tool, i PhyClassifier, was developed to expand the efficacy and capacity of the current 16S rRNA gene sequence-based phytoplasma classification system. i PhyClassifier performs sequence similarity analysis, simulates laboratory restriction enzyme digestions and subsequent gel electrophoresis and generates virtual restriction fragment length polymorphism (RFLP) profiles. Based on calculated RFLP pattern similarity coefficients and overall sequence similarity scores, i PhyClassifier makes instant suggestions on tentative phytoplasma 16Sr group/subgroup classification status and Candidatus Phytoplasma species assignment. Using i PhyClassifier, we revised and updated the classification of strains affiliated with the peach X-disease phytoplasma group. The online tool can be accessed at http://www.ba.ars.usda.gov/data/mppl/iPhyClassifier.html .
Abbreviations: RFLP, restriction fragment length polymorphism
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of Montana potato purple top phytoplasma PPT-MT117-1, pecan bunch phytoplasma PB1, goldenrod yellows phytoplasma GR1 and Alaska potato purple top phytoplasma PPT-AK6 determined in this study are FJ226074 , FJ376626 , FJ376627 and FJ376629 .
Similarity coefficients derived from analysis of virtual RFLP patterns of 16S rRNA F2nR2 sequences from phytoplasma strains in the peach X-disease group (16SrIII) are available as supplementary material with the online version of this paper.
Potato purple top wilt (PPT) is a devastating disease that occurs in various regions of North America and Mexico. At least three distinct phytoplasma strains belonging to three different phytoplasma ...groups (16SrI, 16SrII and 16SrVI) have been associated with this disease. A new disease with symptoms similar to PPT was recently observed in Texas and Nebraska, USA. Two distinct phytoplasma strain clusters were identified. One belongs to the 16SrI phytoplasma group, subgroup A, and the other is a novel phytoplasma that is most closely related to, and shares 96.6 % 16S rRNA gene sequence similarity with, a member of group 16SrXII. Phylogenetic analysis of 16S rRNA gene sequences of the novel PPT-associated phytoplasma strains, previously described 'Candidatus Phytoplasma' organisms and other distinct unnamed phytoplasmas indicated that the novel phytoplasma, termed American potato purple top wilt (APPTW) phytoplasma, represents a distinct lineage and shares a common ancestor with stolbur phytoplasma, 'Candidatus Phytoplasma australiense', 'Candidatus Phytoplasma japonicum', 'Candidatus Phytoplasma fragariae', bindweed yellows phytoplasma (IBS), 'Candidatus Phytoplasma caricae' and 'Candidatus Phytoplasma graminis'. On the basis of unique 16S rRNA gene sequences and biological properties, it is proposed that the APPTW phytoplasma represents 'Candidatus Phytoplasma americanum', with APPTW12-NE as the reference strain.
One of the most important themes in agricultural science is the identification of virulence factors involved in plant disease. Here, we show that a single virulence factor, tengu-su inducer (TENGU), ...induces witches' broom and dwarfism and is a small secreted protein of the plant-pathogenic bacterium, phytoplasma. When tengu was expressed in Nicotiana benthamiana plants, these plants showed symptoms of witches' broom and dwarfism, which are typical of phytoplasma infection. Transgenic Arabidopsis thaliana lines expressing tengu exhibited similar symptoms, confirming the effects of tengu expression on plants. Although the localization of phytoplasma was restricted to the phloem, TENGU protein was detected in apical buds by immunohistochemical analysis, suggesting that TENGU was transported from the phloem to other cells. Microarray analyses showed that auxin-responsive genes were significantly down-regulated in the tengu-transgenic plants compared with GUS-transgenic control plants. These results suggest that TENGU inhibits auxin-related pathways, thereby affecting plant development.
Molecular mechanisms underlying phytoplasma interactions with host plants are largely unknown. In this study attempts were made to identify effectors of three phytoplasma strains related to ‘Ca. P. ...aurantifolia’, crotalaria phyllody (CrP), faba bean phyllody (FBP), and witches’ broom disease of lime (WBDL), using information from draft genome of peanut witches’ broom phytoplasma. Seven putative effectors were identified in WBDL genome (SAP11, SAP21, Eff64, Eff115, Eff197, Eff211 and EffSAP67), five (SAP11, SAP21, Eff64, Eff99 and Eff197) in CrP and two (SAP11, Eff64) in FBP. No homologs to Eff64, Eff197 and Eff211 in phytoplasmas of other phylogenetic groups were found. SAP11 and Eff64 homologs of ‘Ca. P. aurantifolia’ strains shared at least 95.9% identity and were detected in the three phytoplasmas, supporting their role within the group. Five of the putative effectors (SAP11, SAP21, Eff64, Eff115, and Eff99) were transcribed from total RNA extracts of periwinkle plants infected with these phytoplasmas. Transcription profiles of selected putative effectors of CrP, FBP and WBDL indicated that SAP11 transcripts were the most abundant in the three phytoplasmas. SAP21 transcript levels were comparable to those of SAP11 for CrP and not measurable for the other phytoplasmas. Eff64 had the lowest transcription level irrespective of sampling date and phytoplasma isolate. Eff115 transcript levels were the highest in WBDL infected plants. This work reports the first sequence information for 14 putative effectors in three strains related to ‘Ca. P. aurantifolia’, and offers novel insight into the transcription profile of five of them during infection of periwinkle.
Phytoplasma detection and identification is primarily based on PCR followed by restriction fragment length polymorphism analysis. This method detects and differentiates phytoplasmas including those ...not yet identified. The protocol describes the application of this method for identification of phytoplasmas at 16S rRNA (16Sr) group and 16Sr subgroup levels on amplicons and also in silico on the same sequences.
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