Robert E Davis

Institute of Biotechnology Vilnius University, Vil'nyus, Vilniaus Apskritis, Lithuania

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Publications (42)74.85 Total impact

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    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; · 2.11 Impact Factor
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    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.
    BMC Genomics 02/2014; 15(1):164. · 4.40 Impact Factor
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    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%.
    Genome announcements. 01/2014; 2(4).
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    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; · 1.99 Impact Factor
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    ABSTRACT: Phytoplasmas classified in group 16SrXII infect a wide range of plants and are transmitted by polyphagous planthoppers of the family Cixiidae. Based on 16S rRNA gene sequence identity and biological properties, group 16SrXII encompasses several species, including 'Candidatus Phytoplasma australiense', 'Ca. Phytoplasma japonicum', and 'Ca. Phytoplasma fragariae'. Other group 16SrXII phytoplasma strains are associated with stolbur disease in wild and cultivated herbaceous and woody plants and with bois noir disease in grapevines (Vitis vinifera L.). Such latter strains have been informally proposed to represent a separate species, 'Candidatus Phytoplasma solani', but a formal description of this taxon has not previously been published. In the present work, stolbur disease strain STOL11 was distinguished from reference strains of previously described 'Ca. Phytoplasma' species based on 16S rRNA gene sequence (16S rDNA) similarity and a unique signature sequence in 16S rDNA. Other stolbur and bois noir associated ('Ca. Phytoplasma solani') strains shared >99% 16S rDNA sequence similarity with strain STOL11 and contained the signature sequence. 'Ca. Phytoplasma solani' is the only phytoplasma known to be transmitted by Hyalesthes obsoletus. Insect vectorship and molecular characteristics are consistent with the concept that diverse 'Ca. Phytoplasma solani' strains share common properties and represent an ecologically distinct gene pool. Phylogenetic analyses of 16S rRNA, tuf, secY, and rplV-rpsC gene sequences supported this view and yielded congruent trees on which 'Ca. Phytoplasma solani' strains formed, within the group 16SrXII clade, a monophyletic subclade that was most closely related to, but distinct from, that of 'Ca. Phytoplasma australiense'-related strains. Based on distinct molecular and biological properties, stolbur and bois noir associated strains are proposed to represent a novel species level taxon, 'Ca. Phytoplasma solani'; STOL11 is designated the reference strain.
    INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY 01/2013; 63:2879-2894. · 2.11 Impact Factor
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    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.
    Methods in molecular biology (Clifton, N.J.) 01/2013; 938:147-58. · 1.29 Impact Factor
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    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.
    Methods in molecular biology (Clifton, N.J.) 01/2013; 938:329-38. · 1.29 Impact Factor
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    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.
    Journal of microbiological methods 11/2012; · 2.43 Impact Factor
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    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; · 2.11 Impact Factor
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    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; · 2.11 Impact Factor
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    ABSTRACT: Symptoms of abnormal proliferation of shoots resulting in formation of witches'-broom growths were observed on diseased plants of passion fruit (Passiflora edulis f. flavicarpa Deg.) in Brazil. RFLP analysis of 16S rRNA gene sequences amplified in PCRs containing template DNAs extracted from diseased plants collected in Bonito (Pernambuco) and Viçosa (Minas Gerais) Brazil, indicated that such symptoms were associated with infections by two mutually distinct phytoplasmas. One phytoplasma, PassWB-Br4 from Bonito, represents a new subgroup, 16SrIII-V, in the X-disease phytoplasma group ('Candidatus Phytoplasma pruni'-related strains). The second phytoplasma, PassWB-Br3 from Viçosa, represents a previously undescribed subgroup in group 16SrVI. Phylogenetic analyses of 16S rRNA gene sequences were consistent with the hypothesis that strain PassWB-Br3 is distinct from previously described 'Ca. Phytoplasma' species. Nucleotide sequence alignments revealed that strain PassWB-Br3 shared less than 97.5 % 16S rRNA gene sequence similarity with previously described 'Ca. Phytoplasma' species. The unique properties of its DNA, in addition to natural host and geographical occurrence, support the recognition of strain PassWB-Br3 as a representative of a novel taxon, 'Candidatus Phytoplasma sudamericanum'.
    INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY 06/2011; 62(Pt 4):984-9. · 2.11 Impact Factor
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    ABSTRACT: An azalea little leaf (AzLL) disease characterised by abnormally small leaves, yellowing and witches'-broom growth symptoms was observed in suburban Kunming, southwest China. Transmission electron microscopic observations of single-membrane-bound, ovoid to spherical bodies in phloem sieve elements of diseased plants and detection of phytoplasma-characteristic 16S rRNA gene sequence in DNA samples from diseased plants provided evidence linking the disease to infection by a phytoplasma. Results from restriction fragment length polymorphism, phylogenetic and comparative structural analyses of multiple genetic loci containing 16S rRNA, rpsS, rplV, rpsC and secY genes indicated that the AzLL phytoplasma represented a distinct, new 16Sr subgroup lineage, designated as 16SrI-T, in the aster yellows phytoplasma group. The genotyping also revealed that the AzLL phytoplasma represented new rp and secY gene lineages [rp(I)-P and secY(I)-O, respectively]. Phylogenetic analyses of secY and rp gene sequences allowed clearer distinctions between AzLL and closely related strains than did analysis of 16S rDNA.
    Annals of Applied Biology 03/2011; 158(3):318 - 330. · 2.15 Impact Factor
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    ABSTRACT: Pear (Pyrus communis L.) is a nutrient-dense fruit with strong consumer demand and high commercial value. However, most cultivated pear varieties are often susceptible to diseases caused by fungi, bacteria, and viruses. The purpose of the present study was to establish an efficient genetic transformation and regeneration protocol, paving the way for genetic engineering of pear cultivars with enhanced disease resistance. Major factors that influence transformation and regeneration were examined and optimal conditions were established for efficient transformation from leaf explants of ‘Old Home’, a valuable pear interstem and rootstock. High transformation efficiency was achieved largely due to an improved infection/transformation induction strategy. Co-cultivation of Agrobacterium cells and leaf segments on a liquid induction medium yielded a fivefold increase in transformation frequency. Southern hybridization analysis revealed presence of reporter gene uidA in the genomic DNA samples from independent transgenic plants, confirming the integration of the transgene in recipient pear genomes. The stability of T-DNA integration was evaluated by the consistent presence of the Km selectable marker and the expression pattern of the introduced reporter gene uidA was analyzed by GUS histochemical assay. KeywordsPear rootstock– Agrobacterium tumefaciens –GUS–Phloem-specific expression
    Acta Physiologiae Plantarum 01/2011; 33(2):383-390. · 1.31 Impact Factor
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    ABSTRACT: 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, iPhyClassifier, was developed to expand the efficacy and capacity of the current 16S rRNA gene sequence-based phytoplasma classification system. iPhyClassifier 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, iPhyClassifier makes instant suggestions on tentative phytoplasma 16Sr group/subgroup classification status and 'Candidatus Phytoplasma' species assignment. Using iPhyClassifier, 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.
    International journal of systematic and evolutionary microbiology 08/2009; 59(Pt 10):2582-93. · 2.11 Impact Factor
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    ABSTRACT: Salt cedar trees with pronounced witches'-broom symptoms were observed in their natural habitat in China. 16S rRNA gene sequences unique to phytoplasmas were detected in every DNA sample extracted from stem and leaf tissues of the symptomatic trees, revealing a direct association between phytoplasma infection and the salt cedar witches'-broom (SCWB) disease. Phylogenetic analysis of the SCWB phytoplasma 16S rRNA gene sequence indicated that the SCWB phytoplasma belonged to a subclade consisting of several mutually distinct 'Candidatus Phytoplasma' taxa including 'Ca. Phytoplasma prunorum', 'Ca. Phytoplasma mali', 'Ca. Phytoplasma pyri' and 'Ca. Phytoplasma spartii'. Pairwise sequence similarity scores calculated from an alignment of near full-length 16S rRNA genes revealed that SCWB phytoplasma shared 96.6 % or less sequence similarity with each previously described or proposed 'Ca. Phytoplasma' taxon, justifying the recognition of SCWB phytoplasma as a novel taxon, 'Candidatus Phytoplasma tamaricis'. The distinct virtual RFLP pattern derived from the SCWB phytoplasma 16S rRNA gene sequence, together with its lower-than-threshold similarity coefficient values with RFLP patterns of any of the 29 previously established groups, supported the recognition of a new 16Sr group, designated 16SrXXX, salt cedar witches'-broom phytoplasma group.
    International journal of systematic and evolutionary microbiology 08/2009; 59(Pt 10):2496-504. · 2.11 Impact Factor
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    ABSTRACT: Phytoplasmas are cell wall-less bacteria that cause numerous diseases in several hundred plant species. During adaptation to transkingdom parasitism in diverse plant and insect hosts, phytoplasma evolution has given rise to widely divergent lineages. Since phytoplasmas cannot be cultured in a cell-free medium, measurable phenotypic characters suitable for conventional microbial classification are mostly inaccessible. Currently, phytoplasma differentiation and classification are mainly dependent on restriction fragment length polymorphism (RFLP) analysis of 16S rRNA gene sequences. Extending our recent efforts in the exploitation of computer-simulated 16S rRNA gene RFLP analysis and virtual gel plotting for rapid classification of phytoplasmas, we have developed a Perl program for automated RFLP pattern comparison and similarity coefficient calculation. This program streamlines virtual RFLP pattern analysis and has led to the establishment of a criterion for phytoplasma 16Sr subgroup classification and to the delineation of new and distinct subgroup lineages in the clover proliferation phytoplasma group (16SrVI).
    International journal of systematic and evolutionary microbiology 11/2008; 58(Pt 10):2368-77. · 2.11 Impact Factor
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    Wei Wei, Robert E Davis, Rasa Jomantiene, Yan Zhao
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    ABSTRACT: Mobile genetic elements have impacted biological evolution across all studied organisms, but evidence for a role in evolutionary emergence of an entire phylogenetic clade has not been forthcoming. We suggest that mobile element predation played a formative role in emergence of the phytoplasma clade. Phytoplasmas are cell wall-less bacteria that cause numerous diseases in plants. Phylogenetic analyses indicate that these transkingdom parasites descended from Gram-positive walled bacteria, but events giving rise to the first phytoplasma have remained unknown. Previously we discovered a unique feature of phytoplasmal genome architecture, genes clustered in sequence-variable mosaics (SVMs), and suggested that such structures formed through recurrent, targeted attacks by mobile elements. In the present study, we discovered that cryptic prophage remnants, originating from phages in the order Caudovirales, formed SVMs and comprised exceptionally large percentages of the chromosomes of 'Candidatus Phytoplasma asteris'-related strains OYM and AYWB, occupying nearly all major nonsyntenic sections, and accounting for most of the size difference between the two genomes. The clustered phage remnants formed genomic islands exhibiting distinct DNA physical signatures, such as dinucleotide relative abundance and codon position GC values. Phytoplasma strain-specific genes identified as phage morons were located in hypervariable regions within individual SVMs, indicating that prophage remnants played important roles in generating phytoplasma genetic diversity. Because no SVM-like structures could be identified in genomes of ancestral relatives including Acholeplasma spp., we hypothesize that ancient phage attacks leading to SVM formation occurred after divergence of phytoplasmas from acholeplasmas, triggering evolution of the phytoplasma clade.
    Proceedings of the National Academy of Sciences 09/2008; 105(33):11827-32. · 9.81 Impact Factor
  • Hong Cai, Wei Wei, Robert E Davis, Hairu Chen, Yan Zhao
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    ABSTRACT: Phytoplasmas were detected in cactus (Opuntia species) plants exhibiting witches'-broom disease symptoms in Yunnan Province, south-western China. Comparative and phylogenetic analyses of 16S rRNA gene sequences indicated that an overwhelming majority of the cactus-infecting phytoplasmas under study belonged to the peanut witches'-broom phytoplasma group (16SrII). Genotyping through use of computer-simulated restriction fragment length polymorphism (RFLP) analysis of 16S rRNA genes revealed a remarkable genetic diversity among these cactus-infecting phytoplasma strains. Based on calculated coefficients of RFLP pattern similarities, seven new 16SrII subgroups were recognized, bringing the total of described group 16SrII subgroups to 12 worldwide. Geographical areas differed from one another in the extent of genetic diversity among cactus-infecting phytoplasma strains. The findings have implications for relationships between ecosystem distribution and the emergence of group 16SrII subgroup diversity.
    International journal of systematic and evolutionary microbiology 06/2008; 58(Pt 6):1448-57. · 2.11 Impact Factor
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    ABSTRACT: SummaryA polymerase chain reaction (PCR) protocol, previously designed for amplification of a DNA fragment from aster yellows mycoplasmalike organism (MLO), was employed to investigate the detection of MLO DNA in field-collected and in vitro micropropagated plants. PCR with template DNA extracted from symptomatic, naturally-infected samples of Brassica, Chrysanthemum and Hydrangea, each yielded a DNA band corresponding to 1.0 Kbp. However, no DNA product was observed when either infected Ranunculus (with phyllody disease) or Gladiolus with (symptoms of ‘germs fins’) was used as source of template nucleic acid for PCR; further experiments indicated absence of target DNA in the case of Ranunculus and the presence of substances in Gladiolus which inhibited the PCR. The MLO-specific DNA was detected by PCR using less than 95 pg of total nucleic acid (equivalent to total nucleic acid from 1.9, ug tissue) in the case of field-collected Hydrangea and less than 11.4 pg of nucleic acid (equivalent to total nucleic acid from 19 ng of tissue) in the case of field-collected Brassica. The findings illustrate highly sensitive detection of MLOs in both field-grown and in vitro micropropagated infected plants.
    Annals of Applied Biology 02/2008; 121(3):593 - 599. · 2.15 Impact Factor
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    Wei Wei, Robert E Davis, Ing-Ming Lee, Yan Zhao
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    ABSTRACT: Phytoplasmas are cell wall-less bacteria that cause numerous plant diseases. As no phytoplasma has been cultured in cell-free medium, phytoplasmas cannot be differentiated and classified by the traditional methods which are applied to culturable prokaryotes. Over the past decade, the establishment of a phytoplasma classification scheme based on 16S rRNA restriction fragment length polymorphism (RFLP) patterns has enabled the accurate and reliable identification and classification of a wide range of phytoplasmas. In the present study, we expanded this classification scheme through the use of computer-simulated RFLP analysis, achieving rapid differentiation and classification of phytoplasmas. Over 800 publicly available phytoplasma 16S rRNA gene sequences were aligned using the CLUSTAL_X program and the aligned 1.25 kb fragments were exported to pDRAW32 software for in silico restriction digestion and virtual gel plotting. Based on distinctive virtual RFLP patterns and calculated similarity coefficients, phytoplasma strains were classified into 28 groups. The results included the classification of hundreds of previously unclassified phytoplasmas and the delineation of 10 new phytoplasma groups representing three recently described and seven novel putative 'Candidatus Phytoplasma' taxa.
    International journal of systematic and evolutionary microbiology 09/2007; 57(Pt 8):1855-67. · 2.11 Impact Factor

Publication Stats

366 Citations
74.85 Total Impact Points

Institutions

  • 2013
    • Institute of Biotechnology Vilnius University
      Vil'nyus, Vilniaus Apskritis, Lithuania
    • University of Milan
      Milano, Lombardy, Italy
  • 2003–2013
    • United States Department of Agriculture
      • Agricultural Research Service (ARS)
      Washington, D. C., DC, United States
  • 2012
    • Putra University, Malaysia
      Putrajaya, Putrajaya, Malaysia
  • 2011
    • University of Maryland, College Park
      • Institute for Bioscience and Biotechnology Research
      College Park, MD, United States
  • 2008
    • Yunnan Agricultural University
      Panlong, Shaanxi, China
  • 2006–2007
    • Institute of Botany
      Vil'nyus, Vilniaus Apskritis, Lithuania