[Show abstract][Hide abstract] ABSTRACT: Spiroplasma turonicum was isolated from a Haematopota sp. fly in France. We report the nucleotide sequence of the circular chromosome of strain Tab4c(T). The genome information will facilitate evolutionary studies of spiroplasmas, including symbionts of insects and ticks and pathogens of plants, insects, crustaceans, and humans.
[Show abstract][Hide abstract] ABSTRACT: Spiroplasma kunkelii
causes corn stunt disease of
L. in the Americas. Here, we report the nucleotide sequence of the 1,463,926-bp circular chromosome and four plasmids of strain CR2-3x. This information will facilitate studies of
pathogenicity and evolutionary adaptations to transkingdom parasitism in plants and insect vectors.
[Show abstract][Hide abstract] ABSTRACT: Few studies have determined the presence of phytoplasmas in important crops in Puerto Rico. Disease
symptoms resembling those caused by phytoplasmas were observed in pigeon pea (Cajanus cajan), periwinkle (Catharanthus roseus), tabebuia (Tabebuia heterophylla), Spanish lime (Melicoccus bijugatus), ixora (Ixora coccinea), mango (Mangifera indica), cactus (Opuntia spp.), citrus trees (Citrus spp.), and coffee (Coffea arabica). Sixty-two samples from these species were tested using conventional PCR to amplify the 16S rRNA and ribosomal protein genes (rpIVrpsC). Fifty-one percent of the tested samples (corresponding
to periwinkle, pigeon pea, citrus, coffee and tabebuia) were positive for phytoplasmas, with amplicons of 0.8 (16S rRNA gene) and 1.2 kb (rpIV-rpsC genes), depending upon primers used in PCRs. For both genetic loci, DNA sequences showed 99 % identity with pigeon pea witches’ broom phytoplasma (PPWB). Due to the lack of studies of potential insect vectors, common Auchenorrhyncha species were sweep-collected
from pigeon pea and citrus and tested for phytoplasma. Of nine insect genera collected, Empoasca kraemeri
(Cicadellidae), Melormenis antillarum (Flatidae), and Colpoptera maculifrons (Issidae) were positive for PPWB based on results from conventional PCR and DNA sequence analysis. The findings indicate that these insects fed upon the aforementioned plant species, ingesting contents of phloem, and may act as potential phytoplasma vectors in the field. These are first reports of PPWB phytoplasma infections in citrus species (C. sinensis and C. limon), coffee, periwinkle and tabebuia, and in insects (E. kraemeri, M. antillarum and C. maculifrons) for Puerto Rico.
[Show abstract][Hide abstract] ABSTRACT: Xylella fastidiosa causes bacterial leaf scorch in many landscape trees including elm, oak, sycamore and mulberry, but methods for specific identification of a particular tree host species-limited strain or differentiation of tree-specific strains are lacking. It is also unknown whether a particular landscape tree-infecting X. fastidiosa strain is capable of infecting multiple landscape tree species in an urban environment. We developed two PCR primers specific for mulberry-infecting strains of X. fastidiosa based on the nucleotide sequence of a unique open reading frame identified only in mulberry-infecting strains among all the North and South American strains of X. fastidiosa sequenced to date. PCR using the primers allowed for detection and identification of mulberry-infecting X. fastidiosa strains in cultures and in samples collected from naturally infected mulberry trees. In addition, no mixed infections with or non-specific detections of the mulberry-infecting strains of X. fastidiosa were found in naturally X. fastidiosa-infected oak, elm and sycamore trees growing in the same region where naturally infected mulberry trees were grown. This genotype-specific PCR assay will be valuable for disease diagnosis, studies of strain-specific infections in insects and plant hosts, and management of diseases caused by X. fastidiosa. Unexpectedly but interestingly, the unique open reading frame conserved in the mulberry-infecting strains in the U. S. was also identified in the recently sequenced olive-associated strain CoDiRO isolated in Italy. When the primer set was tested against naturally infected olive plant samples collected in Italy, it allowed for detection of olive-associated strains of X. fastidiosa in Italy. This PCR assay, therefore, will also be useful for detection and identification of the Italian group of X. fastidiosa strains to aid understanding of the occurrence, evolution and biology of this new group of X. fastidiosa strains.
PLoS ONE 06/2015; 10(6):e0129330. DOI:10.1371/journal.pone.0129330 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In China, potato is widely cultivated economic crop. Recently, potato diseases with characteristic symptoms of phytoplasma infections were found in potato fields. In 2006 and 2007, samples exhibiting symptoms including rosette and upright growth, upward rolling, yellowing and purpling of leaves, shortened and thickened internodes and formation of aerial tubers were collected from plants in Yunnan and Inner Mongolia and analyzed for the presence of phytoplasmas. DNA was extracted from tissues of 63 symptomatic and 10 asymptomatic plants. Phytoplasma 16S rRNA was amplified by PCR with primer pair P1/P7, followed by nested PCR with P1A/P7A, P1A/16S-SR or R16F2n/R16R2n. Twenty nine symptomatic plants (46 %), but no asymptomatic plants, tested positive for phytoplasmas. Nested PCR products were cloned and sequenced. Sequence analysis indicated that the phytoplasmas from diseased potatoes shared 98.1–99.8 % similarity with ‘Candidatus Phytoplasma fragariae’ (16SrXII-E) and other strains in 16SrXII subgroups. RFLP and phylogenetic analyses also indicated that phytoplasmas of group 16SrXII were associated with phytoplasma infected potatoes in China; these strains are most closely related to subgroup 16SrXII-E. Our results showed that five strains belonged to 16SrXII-E; 11 strains were designated as a new 16SrXII subgroup, 16SrXII-I; and subgroup affiliations of all others were not determined. The genetic diversity of the strains was corroborated by sequence analysis of ribosomal protein genes, the elongation factor Tu gene (tuf) and the pre-protein translocase membrane subunit gene (secY). The results illustrated the complexity and diversity of phytoplasmas associated with potatoes in China.
European Journal of Plant Pathology 06/2015; 142(2). DOI:10.1007/s10658-015-0616-9 · 1.49 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Phytoplasmas are unculturable, cell wall-less bacteria that parasitize plants and insects. This transkingdom life cycle requires rapid responses to vastly different environments including transitions from plant phloem sieve elements to various insect tissues and alternations among diverse plant hosts. Features enabling such flexibility in other microbes include simple sequence repeats (SSRs) -- mutation-prone, phase variable short DNA tracts that function as "evolutionary rheostats" and enhance rapid adaptations. To gain insights into the occurrence, distribution, and potentially functional roles of SSRs in phytoplasmas, we performed computational analysis on genomes of five completely sequenced phytoplasma strains, including 'Candidatus Phytoplasma asteris'-related strains OYM and AYWB, 'Candidatus Phytoplasma australiense'-related strains CBWB and SLY, and 'Candidatus Phytoplasma mali'-related strain AP-AT. The overall density of SSRs in phytoplasma genomes was higher than in representative strains of other prokaryotes. While mono- and tri-nucleotide SSRs were significantly overrepresented in the phytoplasma genomes, di-nucleotide SSRs, and other higher order SSRs were underrepresented. The occurrence and distribution of long SSRs in the prophage islands and phytoplasma-unique genetic loci indicated that SSRs played a role in compounding the complexity of sequence mosaics in individual genomes and in increasing allelic diversity among genomes. Findings from computational analyses were further complemented by an examination of SSRs in varied additional phytoplasma strains, with a focus on potential contingency genes. Some SSRs were located in regions that could profoundly alter the regulation of transcription and translation of affected genes, and/or composition of protein products.
International Journal of Systematic and Evolutionary Microbiology 04/2015; 65(8). DOI:10.1099/ijs.0.000273 · 2.51 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Phytoplasmas are a diverse but phylogenetically coherent group of cell wall-less bacteria affiliated with the class Mollicutes. Due to difficulties in establishing axenic culture, phytoplasmas were assigned to a provisional genus, 'Candidatus Phytoplasma', and the genus was embraced within the order Acholeplasmatales. However, phytoplasmas differ significantly from acholeplasmas in their habitat specificities, life modes, metabolic capabilities, genomic architectures, and phylogenetic positions. This communication describes unique ecological, nutritional, biochemical, genomic, and phylogenetic properties that distinguish phytoplasmas from acholeplasmas and all other taxa in the class Mollicutes. Since such distinguishing properties of the phytoplasmas are not referable to the descriptions of the order Acholeplasmatales and of all other existing orders, namely Mycoplasmatales, Entomoplasmatales, and Anaeroplasmatales, this communication raises questions concerning whether 'Ca. Phytoplasma' should be retained in the order Acholeplasmatales or whether a new provisional order and a new provisional family should be erected to accommodate the genus 'Candidatus Phytoplasma'.
International Journal of Systematic and Evolutionary Microbiology 01/2015; 65(Pt 3). DOI:10.1099/ijs.0.000050 · 2.51 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Few studies have determined the presence of phytoplasma from important crops in Puerto Rico. Disease symptoms resembling those caused by phytoplasma were observed in different plant species such as pigeon pea (Cajanus cajan), periwinkle (Catharanthus roseus), tabebuia (Tabebuia heterophylla), Spanish lime (Melicoccus bijugatus), ixora (Ixora coccinea), mango (Mangifera indica), cactus (Opuntia spp.), citrus trees (Citrus spp.), and coffee (Coffea arabica). Sixty-two plant samples from these species were tested using end point PCR with universal and specific primers (i.e., nested PCR) that prime amplification of the 16S rDNA and ribosomal protein genes (rpIV-rpsC). Fifty-one percent of the samples tested corresponding to periwinkle, pigeon pea, citrus, coffee and tabebuia were positive for phytoplasmas with amplicons of 0.8 and 1.2kb, respectively, depending upon the primers used in PCRs. In both cases the DNA sequences showed 99% of identity with pigeon pea witches’-broom phytoplasma (PPWB) and by restriction patterns (RLFP) obtained from these samples belonged to group 16SrIX. Due to the lack of studies of potential insect vectors, common auchenorrhyncha species were sweep-collected from pigeon pea and citrus and tested for phytoplasma. Of nine insect genera collected, Empoasca kraemeri, Melornemis antillarum and Colpoptera maculifrons were positive for PPWB phytoplasma based on results from conventional PCR and DNA sequence analysis. These findings indicate that these insects fed upon the aforementioned plant species, and may act as potential phytoplasma vectors in the field. Finally, specific primers were designed for qPCR assay to amplify a 102-bp region of the 16S rDNA gene from samples with low level infections of phytoplasma. By the SYBR® Green method, the melting temperature (Tm) recorded in positive samples was 82.3oC. These primers amplified and identified DNA of phytoplasma belonging to the groups and subgroups 16SrV-A, 16SrIII-H, 16SrII-D, 16SrV-C, 16SrII-C, 16SrVI-A, 16SrXII-A and 16SrIX-C.
APS Caribbean Division Meeting, 2014 at the Sugar Bay Resort and Spa in the U.S. Virgin Islands.; 07/2014
[Show abstract][Hide abstract] ABSTRACT: Xylella fastidiosa causes bacterial leaf scorch in landscape trees including sycamore. We determined the draft genome of X. fastidiosa strain Sy-Va, isolated in Virginia from a sycamore tree displaying leaf scorch symptoms. The Sy-VA genome contains 2,477,829 bp,
and has a G+C content of 51.64 mol%.
[Show abstract][Hide abstract] ABSTRACT: In this study, the taxonomic position and group classification of the phytoplasma associated with a lethal yellowing-type disease (LYD) of coconut (Cocos nucifera L.) in Mozambique were addressed. Pairwise sequence similarity values based on alignment of near full-length 16SrRNA genes (1530 bp) revealed that the Mozambique coconut phytoplasma (LYDM) shared 100% identity with a comparable sequence derived from a phytoplasma strain (LDN), responsible for Awka wilt disease of coconut in Nigeria, and shared 99-99.6% identity with 16S rRNA sequences from strains associated with Cape St. Paul wilt (CSPW) disease of coconut in Ghana and Côte d'Ivoire. Similarity scores further determined the 16S rRNA gene of LYDM phytoplasma to share <97.5% sequence identity with all prior descriptions of 'Ca. Phytoplasma' species. Presence of unique regions in the 16S rRNA distinguished LYDM phytoplasma from all currently described 'Candidatus Phytoplasma' species, justifying its recognition as reference strain of a novel taxon, 'Candidatus Phytoplasma palmicola'. Virtual restriction fragment length polymorphism (RFLP) profiles of the F2n/R2 portion (1251 bp) of the 16S rRNA gene and pattern similarity coefficient values delineated coconut LYDM phytoplasma strains from Mozambique as new members of established group 16SrXXII, subgroup A (16SrXXII-A). Similarity coefficients of 0.97 were obtained for comparisons between subgroup 16SrXXII-A strains and CSPW phytoplasmas from Ghana and Côte d'Ivoire. On this basis, CSPW phytoplasma strains were designated as members of a new subgroup, 16SrXXII-B.
International Journal of Systematic and Evolutionary Microbiology 02/2014; 64(Pt 6). DOI:10.1099/ijs.0.060053-0 · 2.51 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The basidiomycete Moniliophthora roreri is the causal agent of Frosty pod rot (FPR) disease of cacao (Theobroma cacao), the source of chocolate, and FPR is one of the most destructive diseases of this important perennial crop in the Americas. This hemibiotroph infects only cacao pods and has an extended biotrophic phase lasting up to sixty days, culminating in plant necrosis and sporulation of the fungus without the formation of a basidiocarp.
We sequenced and assembled 52.3 Mb into 3,298 contigs that represent the M. roreri genome. Of the 17,920 predicted open reading frames (OFRs), 13,760 were validated by RNA-Seq. Using read count data from RNA sequencing of cacao pods at 30 and 60 days post infection, differential gene expression was estimated for the biotrophic and necrotrophic phases of this plant-pathogen interaction. The sequencing data were used to develop a genome based secretome for the infected pods. Of the 1,535 genes encoding putative secreted proteins, 1,355 were expressed in the biotrophic and necrotrophic phases. Analysis of the data revealed secretome gene expression that correlated with infection and intercellular growth in the biotrophic phase and invasive growth and plant cellular death in the necrotrophic phase.
Genome sequencing and RNA-Seq was used to determine and validate the Moniliophthora roreri genome and secretome. High sequence identity between Moniliophthora roreri genes and Moniliophthora perniciosa genes supports the taxonomic relationship with Moniliophthora perniciosa and the relatedness of this fungus to other basidiomycetes. Analysis of RNA-Seq data from infected plant tissues revealed differentially expressed genes in the biotrophic and necrotrophic phases. The secreted protein genes that were upregulated in the biotrophic phase are primarily associated with breakdown of the intercellular matrix and modification of the fungal mycelia, possibly to mask the fungus from plant defenses. Based on the transcriptome data, the upregulated secreted proteins in the necrotrophic phase are hypothesized to be actively attacking the plant cell walls and plant cellular components resulting in necrosis. These genes are being used to develop a new understanding of how this disease interaction progresses and to identify potential targets to reduce the impact of this devastating disease.
[Show abstract][Hide abstract] ABSTRACT: Wall-less bacteria known as phytoplasmas are obligate transkingdom parasites and pathogens of plants and insect vectors. These unusual bacteria possess some of the smallest genomes known among pathogenic bacteria, and have never been successfully isolated in artificial culture. Disease symptoms induced by phytoplasmas in infected plants include abnormal growth and often severe yellowing of leaves, but mechanisms involved in phytoplasma parasitism and pathogenicity are little understood. A phage based genomic island (sequence variable mosaic, SVM) in the genome of Malaysian periwinkle yellows (MPY) phytoplasma harbors a gene encoding membrane-targeted proteins, including a putative phospholipase (PL), potentially important in pathogen-host interactions. Since some phytoplasmal disease symptoms could possibly be accounted for, at least in part, by damage and/or degradation of host cell membranes, we hypothesize that the MPY phytoplasma putative PL is an active enzyme. To test this hypothesis, functional analysis of the MPY putative pl gene-encoded protein was carried out in vitro after its expression in bacterial and yeast hosts. The results demonstrated that the heterologously expressed phytoplasmal putative PL is an active lipolytic enzyme and could possibly act as a pathogenicity factor in the plant, and/or insect, host.
Microbiological Research 09/2013; 169(5-6). DOI:10.1016/j.micres.2013.08.007 · 2.56 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The iPhyClassifier is an internet-based research tool for quick identification and classification of diverse phytoplasmas. The iPhyClassifier simulates laboratory restriction enzyme digestions and subsequent gel electrophoresis and generates virtual restriction fragment length polymorphism (RFLP) patterns. Based on RFLP pattern similarity coefficient scores, the iPhyClassifier gives instant suggestions on group and subgroup classification status of the phytoplasma strains under study. The iPhyClassifier also aligns the query sequences with that of reference strains of all previously described 'Candidatus Phytoplasma' species, -calculates sequence similarity scores, and assigns the phytoplasmas under study into respective 'Ca. Phytoplasma' species as related strains according to the guidelines set forth by the Phytoplasma Taxonomy Group of the International Research Program on Comparative Mycoplasmology. Additional functions of the iPhyClassifier include delineation of potentially new phytoplasma groups and subgroups as well as new 'Ca. Phytoplasma' species. This chapter describes the program components, the operational procedure, and the underlying principles of the iPhyClassifier operation. The chapter also provides hints on how to interpret the results.
[Show abstract][Hide abstract] ABSTRACT: Detection of pathogen DNA by polymerase chain reaction (PCR) assays is the most widely used method for diagnosing phytoplasma diseases. Reliable and efficient detection of phytoplasmas, especially in woody perennial plants, is challenging due to the unusually low abundance and sporadic distribution of phytoplasmas within infected host tissues. Detection success depends largely upon the host species and sampling procedures and, to a lesser extent, on the protocol used for DNA extraction. Here we describe a simple, straightforward, nondestructive stem sampling protocol to confirm phytoplasma infection of palms and other arborescent monocots of large stature. The protocol requires minimal processing of excised tissues and yields phytoplasma DNA preparations in suitable quantity for reliable detection by nested PCR assays.
[Show abstract][Hide abstract] ABSTRACT: A TaqMan-based real-time PCR assay was developed for specific detection of strains of X. fastidiosa causing oleander leaf scorch. The assay uses primers WG-OLS-F1 and WG-OLS-R1 and the fluorescent probe WG-OLS-P1, designed based on unique sequences found only in the genome of oleander strain Ann1. The assay is specific, allowing detection of only oleander-infecting strains, not other strains of X. fastidiosa nor other plant-associated bacteria tested. The assay is also sensitive, with a detection limit of 10.4fg DNA of X. fastidiosa per reaction in vitro and in planta. The assay can also be applied to detect low numbers of X. fastidiosa in insect samples, or further developed into a multiplex real-time PCR assay to simultaneously detect and distinguish diverse strains of X. fastidiosa that may occupy the same hosts or insect vectors. Specific and sensitive detection and quantification of oleander strains of X. fastidiosa should be useful for disease diagnosis, epidemiological studies, management of oleander leaf scorch disease, and resistance screening for oleander shrubs.
[Show abstract][Hide abstract] ABSTRACT: X-disease is one of the most serious diseases known in peach (Prunus persica). Based on RFLP analysis of 16S rRNA gene sequences, peach X-disease phytoplasma strains from eastern and western United States and eastern Canada were classified in 16S rDNA RFLP group 16SrIII, subgroup A. Phylogenetic analyses of 16S rRNA gene sequences revealed that the X-disease phytoplasma strains formed a distinct subclade within the phytoplasma clade and supported the hypothesis that they represent a lineage distinct from those of previously described 'Candidatus Phytoplasma' species. Nucleotide sequence alignments revealed that all studied X-disease phytoplasma strains shared less than 97.5 % similarity of 16S rDNA with previously described 'Candidatus Phytoplasma' species. Based on unique properties of DNA, we propose recognition of X-disease phytoplasma strain PX11CT1R as representative of a novel taxon, 'Candidatus Phytoplasma pruni'. Results from nucleotide and phylogenetic analyses of secY and ribosomal protein (rp) gene sequences provided additional molecular markers of the 'Candidatus Phytoplasma' lineage. We propose that the term 'Candidatus Phytoplasma pruni' be applied to phytoplasma strains whose 16S rRNA gene sequences contain the oligonucleotide sequences of unique regions that are designated in the formally published description of the species. Such strains include X-disease phytoplasma and - within the tolerance of a single base difference in one unique sequence - peach rosette, peach red suture, and little peach phytoplasmas. We further propose that secY, rp, and other genetic loci from the reference strain of a species, and where possible oligonucleotide sequences of unique regions of those genes that distinguish species within a given 16Sr group, be incorporated in amended descriptions and as part of future descriptions of 'Candidatus Phytoplasma' species.
International Journal of Systematic and Evolutionary Microbiology 07/2012; 63(Pt 2). DOI:10.1099/ijs.0.041202-0 · 2.51 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This study addressed the taxonomic position and group classification of a phytoplasma responsible for virescence and phyllody symptoms in naturally diseased Madagascar periwinkle plants in western Malaysia. Unique regions in the 16S rRNA gene from the Malaysian periwinkle virescence (MaPV) phytoplasma distinguished the phytoplasma from all previously described 'Candidatus Phytoplasma' species. Pairwise sequence similarity scores, calculated through alignment of full-length 16S rRNA gene sequences, revealed that the MaPV phytoplasma 16S rDNA shared 96.5% or less sequence similarity with that of previously described 'Ca. Phytoplasma' species, justifying the recognition of the MaPV phytoplasma as a reference strain of a novel taxon, 'Candidatus Phytoplasma malaysianum'. The 16S rDNA F2nR2 fragment from the MaPV phytoplasma exhibited a distinct restriction fragment length polymorphism (RFLP) profile and the pattern similarity coefficient values were lower than 0.85 with representative phytoplasmas classified in any of the 31 previously delineated 16Sr groups; therefore, the MaPV phytoplasma was designated member of a new 16Sr group, 16SrXXXII. Phytoplasmas affiliated with this novel taxon and new group included diverse strains infecting periwinkle, coconut palm, and oil palm in Malaysia. Three phytoplasmas were characterized as representatives of three distinct subgroups, 16SrXXXII-A, 16SrXXXII-B, and 16SrXXXII-C, respectively.
International Journal of Systematic and Evolutionary Microbiology 04/2012; 63(Pt 2). DOI:10.1099/ijs.0.041467-0 · 2.51 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The number of newly discovered phytoplasma strains has increased dramatically in the last decade. For epidemiological studies and for international quarantine, appropriate molecular tools are essential to identify and distinguish phytoplasmas at taxonomic ranks at and below ribosomal group level. Currently, use of RFLP analysis of the secY gene is one of the finest tools available to achieve the resolving power needed for fine differentiation of closely related phytoplasmas. In the present study, we developed a sytem for computer-simulated SNP-based analysis of secY sequences that incorporates SNPs located both within and outside of restriction sites, increasing the number of characters beyond those used in RFLP analyses. With the inclusion of the additional informative characters, the SNP-based system further enhances strain separation and characterization. This new system will pro-vide additional molecular markers and should aid identification and characterization of strains that are ecologically distinct and/or originate from different geographical regions.