Sequence heterogeneity in the two 16S rRNA genes of Phormium yellow leaf phytoplasma.
ABSTRACT: Phormium yellow leaf (PYL) phytoplasma causes a lethal disease of the monocotyledon, New Zealand flax (Phormium tenax). The 16S rRNA genes of PYL phytoplasma were amplified from infected flax by PCR and cloned, and the nucleotide sequences were determined. DNA sequencing and Southern hybridization analysis of genomic DNA indicated the presence of two copies of the 16S rRNA gene. The two 16S rRNA genes exhibited sequence heterogeneity in 4 nucleotide positions and could be distinguished by the restriction enzymes BpmI and BsrI. This is the first record in which sequence heterogeneity in the 16S rRNA genes of a phytoplasma has been determined by sequence analysis. A phylogenetic tree based on 16S rRNA gene sequences showed that PYL phytoplasma is most closely related to the stolbur and German grapevine yellows phytoplasmas, which form the stolbur subgroup of the aster yellows group. This phylogenetic position of PYL phytoplasma was supported by 16S/23S spacer region sequence data.
Project description:In January 2012, spreading type petunia cv. Wave Pink plants showing an abnormal growth habit of sprouting unusual multiple plantlets from the lateral buds were collected from a greenhouse in Gwacheon, Gyeonggi Province, Korea. The presence of phytoplasma was investigated using PCR with the primer pairs P1/P6, and R16F1/R1 for nested-PCR. In the nested PCR, 1,096 bp PCR products were obtained, and through sequencing 12 Pet-Stol isolates were identified. Comparison of the nucleotide sequences of 16S rRNA gene of the 12 Pet-Stol isolates with other phytoplasmas belonging to aster yellows or Stolbur showed that Pet-Stol isolates were members of Stolbur. The presence of phytoplasma in petunia was also confirmed by microscopic observation of the pathogens. In this study, Stolbur phytoplasma was identified from spreading type petunia cultivars by sequence analysis of 16S rRNA gene of phytoplasma and microscopic observation of phytoplasma bodies. This is the first report of Stolbur phytoplasma in commercial Petunia hybrida cultivars.
Project description:In order to develop a diagnostic tool to identify phytoplasmas and classify them according to their phylogenetic group, we took advantage of the sequence diversity of the 16S-23S intergenic spacer regions (SRs) of phytoplasmas. Ten PCR primers were developed from the SR sequences and were shown to amplify in a group-specific fashion. For some groups of phytoplasmas, such as elm yellows, ash yellows, and pear decline, the SR primer was paired with a specific primer from within the 16S rRNA gene. Each of these primer pairs was specific for a specific phytoplasma group, and they did not produce PCR products of the correct size from any other phytoplasma group. One primer was designed to anneal within the conserved tRNA(Ile) and, when paired with a universal primer, amplified all phytoplasmas tested. None of the primers produced PCR amplification products of the correct size from healthy plant DNA. These primers can serve as effective tools for identifying particular phytoplasmas in field samples.
Project description:During a survey performed in sapota orchards of India, from 2015 to 2018, symptoms of phyllody, little leaf, flat stem and witches' broom were observed in three states: Karnataka, Kerala and Tripura. The association of phytoplasmas was confirmed in all the symptomatic sapota samples by using nested PCR specific primers (P1/P7, R16F2n/R16R2 and 3Far/3Rev) with amplification of fragments of ~ 1.25 kb and ~ 1.3 kb. Association of three phytoplasma groups, aster yellows with flat stem from Tripura (Lembucherra), clover proliferation with phyllody symptoms at Karnataka (Bengaluru) and bermuda grass white leaf with flat stem and little leaf from Kerala (Thiruvananthapuram) and Tripura (Cocotilla) were confirmed by <i>16S rRNA</i> gene sequence comparison analysis. Virtual RFLP analysis of <i>16S rRNA</i> gene sequences using pDRAW32 further classified the sapota phytoplasma isolates into 16SrI-B, 16SrVI-D and 16SrXIV-A subgroups. This is the first report on identification of three phytoplasma groups in sapota in world.
Project description:Studies of phytoplasma-insect vector interactions and epidemiological surveys of plant yellows associated with the stolbur phytoplasma (StolP) require the identification of relevant candidate genes and typing markers. A recent StolP genome survey identified a partial coding sequence, SR01H10, having no homologue in the "Candidatus Phytoplasma asteris" genome but sharing low similarity with a variable surface protein of animal mycoplasmas. The complete coding sequence and its genetic environment have been fully characterized by chromosome walking. The vmp1 gene encodes a protein of 557 amino acids predicted to possess a putative signal peptide and a potential C-terminal transmembrane domain. The mature 57.8-kDa VMP1 protein is likely to be anchored in the phytoplasma membrane with a large N-terminal hydrophilic part exposed to the phytoplasma cell surface. Southern blotting experiments detected multiple sequences homologous to vmp1 in the genomes of nine StolP isolates. vmp1 is variable in size, and eight different vmp1 RsaI restriction fragment length polymorphism types could be distinguished among 12 StolP isolates. Comparison of vmp1 sequences revealed that insertions in largest forms of the gene encode an additional copy of a repeated domain of 81 amino acids, while variations in 11-bp repeats led to gene disruption in two StolP isolates. vmp1 appeared to be much more variable than three housekeeping genes involved in protein translation, maturation, and secretion and may therefore be involved in phytoplasma-host interactions.
Project description:The present study examined the effects of <i>Candidatus</i> Phytoplasma solani infection on antioxidative metabolism in leaves and roots of carrot (<i>Daucus carota</i> L.). Disease symptoms appeared at the end of June in the form of the chlorosis on some of the leaves, which became intensely red one week later, while the previously healthy leaves from the same branch becme chlorotic. A few days later, all leaves from the infected leaf branch were intensely red. Infected plants also had slower growth compared to the healthy ones with fewer leaf branches developed. The roots of infected plants were less developed, seared, or gummy with or without brown-colored root hair. The presence of the pathogen was detected by sequencing the 16S rRNA. National Center for Biotechnology Information (NCBI) BLAST analyses of the obtained sequence revealed 100% identity of tested strain with deposited Ca. Phytoplasma solani strains from various countries and hosts, all belonging to the "stolbur" group (16SrXII-A). Identity of 99.74% was found when the tested Serbian strain (MF503627) was compared with the reference stolbur strain STOL11 (AF248959). The oxidative damage of membranes in carrot cells was accompanied by a decrease in the content of photosynthetic pigments. Furthermore, for the determination of specific scavenging properties of the extracts, in vitro antioxidant assay was performed. In phytoplasma-infected carrot leaves, there was a greater reduction in the level of glutathione content (GSH); however; flavonoids and anthocyanidins seem to be responsible for the accompanied increased antioxidative capacity against hydroxyl radical and hydrogen peroxide.
Project description:Bois noir (BN) associated with '<i>Candidatus</i> Phytoplasma solani' (Stolbur) is regularly found in Austrian vine growing regions. Investigations between 2003 and 2008 indicated sporadic presence of the confirmed disease vector <i>Hyalesthes obsoletus</i> and frequent infections of bindweed and grapevine. Infections of nettles were rare. In contrast present investigations revealed a mass occurrence of <i>H. obsoletus</i> almost exclusively on stinging nettle. The high population densities of <i>H. obsoletus</i> on <i>Urtica dioica</i> were accompanied by frequent occurrence of '<i>Ca.</i> P. solani' in nettles and planthoppers. Sequence analysis of the molecular markers <i>secY, stamp, tuf</i> and <i>vmp1</i> of stolbur revealed a single genotype named CPsM4_At1 in stinging nettles and more than 64 and 90 % abundance in grapevine and <i>H. obsoletus</i>, respectively. Interestingly, this genotype showed tuf b type restriction pattern previously attributed to bindweed associated '<i>Ca.</i> P. solani' strains, but a different sequence assigned as tuf b2 compared to reference tuf b strains. All other marker genes of CPsM4_At1 clustered with tuf a and nettle derived genotypes verifying distinct nettle phytoplasma genotypes. Transmission experiments with <i>H. obsoletus</i> and <i>Anaceratagallia ribauti</i> resulted in successful transmission of five different strains including the major genotype to <i>Catharanthus roseus</i> and in transmission of the major genotype to <i>U. dioica</i>. Altogether, five nettle and nine bindweed associated genotypes were described. Bindweed types were verified in 34 % of grapevine samples, in few positive <i>Reptalus panzeri</i>, rarely in bindweeds and occasionally in <i>Catharanthus roseus</i> infected by <i>H. obsoletus</i> or <i>A. ribauti</i>. '<i>Candidatus</i> Phytoplasma convolvuli' (bindweed yellows) was ascertained in nettle and bindweed samples.
Project description:Geographically diverse samples from strawberry exhibiting symptoms of Strawberry Green Petal (SbGP), periwinkle plants with virescence, and blackberry, blueberry, and raspberry plants displaying yellowing and inedible fruits, were assayed for the presence of phytoplasma DNA. PCR targeting the 16S rRNA-encoding gene and chaperonin-60 (cpn60) showed that the plants were infected with phytoplasma subgroup16SrXIII-(A/I)I (SbGP/MPV). To examine the geographic distribution of this pathogen in Mexico, we designed an array of cpn60-targeted molecular diagnostic assays for SbGP/MPV phytoplasma. A fluorescent microsphere hybridization assay was designed that was capable of detecting SbGP/MPV phytoplasma in infected plant tissues, successfully differentiating it from other known phytoplasma cpn60 UT sequences, while identifying a double infection with SbGP/MPV and aster yellows (16SrI) phytoplasma. Two quantitative assays, quantitative real-time PCR (qRT-PCR) and droplet digital PCR (ddPCR), gave similar results in infected samples. Finally, a loop-mediated isothermal amplification (LAMP) assay provided rapid detection of SbGP/MPV phytoplasma DNA. Application of these assays revealed that SbGP/MPV phytoplasma is widely distributed in Central Mexico, with positive samples identified from eleven localities within three states separated by hundreds of kilometres. These results also provide tools for determining the presence and geographic distribution of this pathogen in plant and insect samples in other localities.
Project description:Samples from toria plants (Brassica rapa L. subsp. dichotoma (Roxb.)) exhibiting phyllody, virescence, witches broom, extensive malformation of floral parts, formation of bladder like siliquae and flower sterility were collected from four different locations in India. Sequencing and phylogenetic analysis of the 16S rRNA, a part of 23S rRNA, partial sec A genes, rp gene and 16S-23S intergenic spacer region indicated that the phytoplasmas associated with toria phyllody (TP) symptoms were identical and belonged to 16SrIX phytoplasma Pigeon pea witches'-broom (PPWB) group. The iPhyClassifier generated virtual RFLP pattern of 1.25 kb 16S rDNA sequences indicated that TP phytoplasma belongs to 16SrIX-C phytoplasma subgroup. Complete 23S rRNA gene of TP phytoplasma had 2,787 nucleotides and is the first sequence of 16SrIX phytoplasma group. Restriction digestion of 16S rDNA and 23S rDNA PCR products has also shown that TP phytoplasmas from all the four locations in India were identical. Toria is a previously unreported host for a phytoplasma in16SrIX-C subgroup.
Project description:Here, we investigate the endosymbiotic microbiota of the Macrosteles leafhoppers M. striifrons and M. sexnotatus, known as vectors of phytopathogenic phytoplasmas. PCR, cloning, sequencing, and phylogenetic analyses of bacterial 16S rRNA genes identified two obligate endosymbionts, "Candidatus Sulcia muelleri" and "Candidatus Nasuia deltocephalinicola," and five facultative endosymbionts, Wolbachia, Rickettsia, Burkholderia, Diplorickettsia, and a novel bacterium belonging to the Rickettsiaceae, from the leafhoppers. "Ca. Sulcia muelleri" and "Ca. Nasuia deltocephalinicola" exhibited 100% infection frequencies in the host species and populations and were separately harbored within different bacteriocytes that constituted a pair of coherent bacteriomes in the abdomen of the host insects, as in other deltocephaline leafhoppers. Wolbachia, Rickettsia, Burkholderia, Diplorickettsia, and the novel Rickettsiaceae bacterium exhibited infection frequencies at 7%, 31%, 12%, 0%, and 24% in M. striifrons and at 20%, 0%, 0%, 20%, and 0% in M. sexnotatus, respectively. Although undetected in the above analyses, phytoplasma infections were detected in 16% of M. striifrons and 60% of M. sexnotatus insects by nested PCR of 16S rRNA genes. Two genetically distinct phytoplasmas, namely, "Candidatus Phytoplasma asteris," associated with aster yellows and related plant diseases, and "Candidatus Phytoplasma oryzae," associated with rice yellow dwarf disease, were identified from the leafhoppers. These results highlight strikingly complex endosymbiotic microbiota of the Macrosteles leafhoppers and suggest ecological interactions between the obligate endosymbionts, the facultative endosymbionts, and the phytopathogenic phytoplasmas within the same host insects, which may affect vector competence of the leafhoppers.
Project description:Aster Yellows phytoplasma (AYp; 'Candidatus Phytoplasma asteris') is an obligate bacterial pathogen that is the causative agent of multiple diseases in herbaceous plants. While this phytoplasma has been examined in depth for its disease characteristics, knowledge about the spatial and temporal dynamics of pathogen spread is lacking. The phytoplasma is found in plant's phloem and is vectored by leafhoppers (Cicadellidae: Hemiptera), including the aster leafhopper, Macrosteles quadrilineatus Forbes. The aster leafhopper is a migratory insect pest that overwinters in the southern United States, and historical data suggest these insects migrate from southern overwintering locations to northern latitudes annually, transmitting and driving phytoplasma infection rates as they migrate. A more in-depth understanding of the spatial, temporal and genetic determinants of Aster Yellows disease progress will lead to better integrated pest management strategies for Aster Yellows disease control. Carrot, Daucus carota L., plots were established at two planting densities in central Wisconsin and monitored during the 2018 growing season for Aster Yellows disease progression. Symptomatic carrots were sampled and assayed for the presence of the Aster Yellows phytoplasma. Aster Yellows disease progression was determined to be significantly associated with calendar date, crop density, location within the field, and phytoplasma subgroup.