scholarly journals First Report of Oat as Host of a Phytoplasma Belonging to Group 16SrI, Subgroup A

Plant Disease ◽  
2002 ◽  
Vol 86 (4) ◽  
pp. 443-443 ◽  
Author(s):  
R. Jomantiene ◽  
R. E. Davis ◽  
A. Alminaite ◽  
D. Valiunas ◽  
R. Jasinskaite
Keyword(s):  
16S Rdna ◽  
Plant Species ◽  
Nested Pcr ◽  
Sequence Similarity ◽  
Intergenic Spacer ◽  
Rflp Analysis ◽  
23S Rrna ◽  
Universal Primer ◽  
Intergenic Spacer Region ◽  
Group I

Diseased plants of oat (Avena sativa L.) exhibiting abnormal proliferation of spikelets were observed in the field in Raseniai, Lithuania. The possible association of a phytoplasma with the disease, termed oat proliferation (OatP), was determined using polymerase chain reaction (PCR) for amplification of phytoplasmal ribosomal (r) RNA gene (rDNA) sequences from template DNA extracted from the diseased oats. DNA extractions and nested PCRs were conducted as previously described (2). In the nested PCRs, the first reaction was primed by phytoplasma-universal primer pair P1/P7, and the second (nested) PCR was primed by primer pair R16F2n/R16R2 (F2n/R2). Phytoplasmal rDNA was amplified in the nested PCR, indicating that the plants contained a phytoplasma, designated oat proliferation (OatP) phytoplasma. The OatP phytoplasma was identified and classified according to the system of Lee et al. (2) through restriction fragment length polymorphism (RFLP) analysis of 16S rDNA amplified in the PCR primed by F2n/R2. On the basis of collective RFLP patterns of the 16S rDNA, the OatP phytoplasma was classified as a member of group 16SrI (group I, aster yellows phytoplasma group). The RFLP patterns of the 16S rDNA were indistinguishable from those of 16S rDNA from tomato big bud (BB) phytoplasma and other phytoplasmas classified in group I, subgroup A (subgroup I-A, tomato big bud phytoplasma subgroup). The 1.8-kbp rDNA product of PCR primed by primer pair P1/P7 was cloned, and its nucleotide sequence was determined. The sequence was deposited in GenBank under Accession No. AF453416. Results from putative restriction site analysis of the cloned and sequenced rDNA were in excellent agreement with the results from enzymatic RFLP analysis of uncloned rDNA from OatP-diseased oat plants. Sequence similarity between the 1.8-kbp rDNA of OatP phytoplasma and that of BB phytoplasma (GenBank No. AF222064) was 99.2%; 9 of the 14 base changes were in the 16S-23S rRNA intergenic spacer region. The base differences in rDNA may signal that the OatP and BB phytoplasmas are mutually distinct in their biologies. Phytoplasmas classified in subgroup I-A have previously been reported in a broad range of plant species in North America and Europe, although there are no previous definitive reports of oat as a host of a subgroup I-A phytoplasma (3,4). In 1977, Fedotina (1) reported electron microscopy of a mycoplasma-like organism (phytoplasma) in pseudorosette-diseased oat plants in Siberia, but the identity of that phytoplasma remains unknown. Subgroup I-A phytoplasma strains are geographically widespread and have been found in numerous plant species (3,4). The discovery reported here, of a subgroup I-A phytoplasma in diseased oats in Lithuania, provokes questions concerning possible impacts of this phytoplasma on oat cultivation in central Europe and other regions. References: (1) V. L. Fedotina. Arch. Phytopathol. Pflanzenschutz 13:177, 1977. (2) I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (3) C. Marcone et al. Int. J. Syst. Evol. Microbiol. 50:1703, 2000. (4) D. Valiunas et al. Plant Dis. 85:804, 2001.

scholarly journals First Report of a Group 16SrI, Subgroup B, Phytoplasma in Diseased Epilobium hirsutum in the Region of Tallin, Estonia

Plant Disease ◽  
2002 ◽  
Vol 86 (10) ◽  
pp. 1177-1177 ◽  
Author(s):  
A. Alminaite ◽  
R. E. Davis ◽  
D. Valiunas ◽  
R. Jomantiene
Keyword(s):  
16S Rdna ◽  
Sequence Similarity ◽  
Intergenic Spacer ◽  
Rflp Analysis ◽  
Nested Polymerase Chain Reaction ◽  
Universal Primer ◽  
Aster Yellows ◽  
First Report ◽  
Intergenic Spacer Region ◽  
Aster Yellows Phytoplasma

Symptoms of phyllody of flowers and general plant yellowing indicating possible phytoplasma infection were observed in diseased plants of hairy willow-weed (Epilobium hirsutum L., family Onagraceae) growing in a meadow at Harku Village near Tallin, Estonia. DNA was extracted from diseased E. hirsutum using a Genomic DNA Purification Kit (Fermentas AB, Vilnius, Lithuania) and used as a template in nested polymerase chain reaction (PCR). Ribosomal (r) DNA was initially amplified in PCR primed by phytoplasma universal primer pair P1/P7 (4) and reamplified in PCR primed by nested primer pair 16SF2n/16SR2 (F2n/R2) (1) as previously described (2). Products of 1.8 kbp and 1.2 kbp were obtained in PCR primed P1/P7 and F2n/R2, respectively, from all four symptomatic plants examined. These data indicated that the diseased E. hirsutum plants were infected by a phytoplasma, termed epilobium phyllody (EpPh) phytoplasma. The 16S rDNA amplified in PCR primed by nested primer pair F2n/R2 was subjected to restriction fragment length polymorphism (RFLP) analysis using restriction endonucleases AluI, MseI, HpaI, HpaII, HhaI, RsaI, HinfI, and HaeIII (Fermentas AB). On the basis of the collective RFLP profiles, EpPh phytoplasma was classified in group 16SrI (aster yellows phytoplasma group), subgroup B (aster yellows phytoplasma subgroup), according to the phytoplasma classification scheme of Lee et al. (3). The 1.8-kbp rDNA product of P1/P7-primed PCR, which included 16S rDNA, 16S-23S intergenic spacer region, and the 5′ -end of 23S rDNA, was cloned in Escherichia coli using the TOPO TA Cloning Kit (Invitrogen, Carlsbad, Ca) according to manufacturer's instructions and sequenced. The sequence was deposited in the GenBank database as Accession No. AY101386. This nucleotide sequence shared 99.8% sequence similarity with a comparable rDNA sequence (GenBank Accession No. AF322644) of aster yellows phytoplasma AY1, a known subgroup 16SrI-B strain. The EpPh phytoplasma sequence was highly similar (99.9%) to operons rrnA (GenBank Accession No. AY102274) and rrnB (GenBank Accession No. AY102273) from Valeriana yellows (ValY) phytoplasma infecting Valeriana officinalis plants in Lithuania. ValY phytoplasma was found to exhibit rRNA interoperon sequence heterogeneity (D. Valiunas, unpublished data). To our knowledge, this is the first report to reveal E. hirsutum as a host of phytoplasma and to demonstrate the occurrence of a plant pathogenic mollicute in the northern Baltic region. References: (1) D. E. Gundersen and I.-M. Lee. Phytopathol. Mediterr. 35:144, 1996. (2) R. Jomantiene et al. HortScience 33:1069, 1998. (3) I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (4) B. Schneider et al. Phlogenetic classification of plant pathogenic mycoplasma-like organisms or phytoplasmas. Page 369 in: Molecular and Diagnostic Procedures in Mycoplasmology, Vol 1, R. Razin, and J. G. Tully eds. Academic Press, San Diego, 1995.

scholarly journals Maize (Zea mays L.): A New Host for Ligustrum witches’ Broom Phytoplasma

Pathogens ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 723
Author(s):  
Behçet Kemal Çağlar ◽  
Serkan Pehlivan ◽  
Ekrem Atakan ◽  
Toufic Elbeaino
Keyword(s):  
Phylogenetic Analyses ◽  
Intergenic Spacer ◽  
23S Rrna ◽  
Rrna Gene ◽  
Universal Primer ◽  
New Host ◽  
Intergenic Spacer Region ◽  
Distant Relationship ◽  
Molecular Information ◽  
Corn Plants

In the 2019–2020 growing season, two corn fields located in İmamoğlu town (Adana Province, Turkey) were surveyed following the appearance of phytoplasma-like symptoms on maize plants. A total of 40 samples were collected and tested in first-round and nested PCR using universal primer pairs P1/P7 and R16F2n/R16R2, respectively. All maize-diseased plants reacted positively, whilst no PCR amplifications were obtained from asymptomatic plants. Blast sequence analysis of R16F2n/R16R2-primed amplicons from different maize isolates showed 99.2% to 100% of identity with the 16S rRNA gene of Ligustrum witches’ broom phytoplasma (LiWBP). To gain additional molecular information on the 16S ribosomal RNA and 23S rRNA intergenic spacer region of LiWBP, not identified previously, the P1/P7-primed amplicons were also sequenced and analyzed. The results show that maize isolates from Turkey share 99.6% to 100% of identity among them, whereas the highest identity found (91%) was with members of groups 16SrII and 16SrXXV (peanut and tea witches’ broom groups, respectively). This distant relationship between LiWBP and members of 16SrII and XXV was also confirmed by RFLP and phylogenetic analyses. This is the first finding of LiWBP on maize in nature, where it was found responsible for phyllody disease of corn plants in Turkey. The additional molecular information acquired in this study on the 16S–23S rRNA intergenic spacer region of LiWBP further corroborates its distant relationship to any other phytoplasma groups.

scholarly journals Corn with Symptoms of Reddening: New Host of Stolbur Phytoplasma

Plant Disease ◽  
2006 ◽  
Vol 90 (10) ◽  
pp. 1313-1319 ◽  
Author(s):  
B. Duduk ◽  
A. Bertaccini
Keyword(s):  
16S Rdna ◽  
Intergenic Spacer ◽  
Rflp Analysis ◽  
Gene Sequences ◽  
New Host ◽  
Intergenic Spacer Region ◽  
New Finding ◽  
Polymerase Chain ◽  
Stolbur Phytoplasma ◽  
Uncertain Etiology

Recurrent epiphytotic outbreaks of a disease of uncertain etiology known as reddening of corn (Zea mays) have occurred in some areas of Serbia during the last 50 years. Affected plants show early and abnormal ripening, dry precociously, and have poor, shriveled grains. Using molecular tools, phytoplasmas were detected in diseased plants and their identity was subsequently deduced as a subgroup 16SrXII-A strain by a variety of supporting assays involving restriction fragment length polymorphism (RFLP) analysis of polymerase chain reaction (PCR)-amplified 16S rDNA and tuf gene sequences, selective amplification of phytoplasma DNA using primer pair G35p/m, similarity of 16-23S intergenic spacer region (SR) sequences, and similarity and phylogenetic analysis of 16S rDNA gene sequences. Presence of stolbur phytoplasmas in corn with reddening symptoms is a new finding not only for Serbia: it is the first report of stolbur phytoplasma in this species worldwide.

scholarly journals Isolation of Bartonella species from rodents in Taiwan including a strain closely related to ‘Bartonella rochalimae’ from Rattus norvegicus

2008 ◽  
Vol 57 (12) ◽  
pp. 1496-1501 ◽  
Author(s):  
Jen-Wei Lin ◽  
Chun-Yu Chen ◽  
Wan-Ching Chen ◽  
Bruno B. Chomel ◽  
Chao-Chin Chang
Keyword(s):  
Rattus Norvegicus ◽  
Phylogenetic Analyses ◽  
Intergenic Spacer ◽  
Rflp Analysis ◽  
23S Rrna ◽  
Human Pathogens ◽  
Bartonella Species ◽  
Intergenic Spacer Region ◽  
Pcr Rflp ◽  
Rodent Populations

An increasing number of Bartonella species originally isolated from small mammals have been identified as emerging human pathogens. During an investigation of Bartonella infection in rodent populations carried out in Taiwan in 2006, a total of 58 rodents were tested. It was determined that 10.3 % (6/58) of the animals were Bartonella bacteraemic. After PCR/RFLP analysis, four isolates were identified as Bartonella elizabethae and one isolate as Bartonella tribocorum. However, there was one specific isolate with an unrecognized PCR/RFLP pattern. After further sequence and phylogenetic analyses of the gltA, ftsZ and rpoB genes, and the 16S–23S rRNA intergenic spacer region, the results indicated that this specific isolate from Rattus norvegicus was closely related to human pathogenic ‘Bartonella rochalimae’. Further studies need to be conducted to evaluate whether this rodent species could be a reservoir for ‘B. rochalimae’.

scholarly journals First Report of Barley as Host of a Phytoplasma Belonging to Group 16SrI, Subgroup B, and Ribosomal Protein Subgroup rpI-B in Lithuania

Plant Disease ◽  
2005 ◽  
Vol 89 (3) ◽  
pp. 339-339 ◽  
Author(s):  
L. Urbanaviciene ◽  
R. Jomantiene ◽  
R. E. Davis
Keyword(s):  
Ribosomal Protein ◽  
16S Rdna ◽  
Rflp Analysis ◽  
Gene Sequences ◽  
Universal Primer ◽  
Group I ◽  
Pcr Product ◽  
Polymerase Chain ◽  
Template Dna ◽  
Aster Yellows Phytoplasma

Numerous diseased plants of barley (Hordeum vulgaris L.) exhibiting twisted, abnormally thin and yellowed awns, reduced spikelets, and general stunting and yellowing were observed in fields in the Vilnius and Kaisiadorys regions of Lithuania. The possible association of a phyto-plasma with the disease, termed barley deformation (BaDef), was assessed using polymerase chain reaction (PCR). Three phytoplasma universal primer pairs (P1/P7, R16F2n/R16R2, and rpF1/rpR1) (1,2,4) were employed to amplify ribosomal (r) RNA gene (rDNA) and ribosomal protein (rp) gene sequences. Template DNA extractions and PCR (direct and nested) were conducted as previously described (4). Although DNA was amplified in PCRs containing template extracted from diseased plants, no amplification was observed in PCRs containing DNA from symptomless plants sampled from the same fields. The BaDef phytoplasma was identified and classified according to Lee et al. (4) through restriction fragment length polymorphism (RFLP) analysis of 1.2-kbp 16S rDNA amplified in the PCR primed by primer pair R16F2n/R16R2 and analysis of the 1.2-kbp rp gene sequences amplified in PCR primed by primer pair rpF1/rpR1. On the basis of collective RFLP patterns of amplified 16S rDNA and rp gene sequences, the BaDef phytoplasma was classified as a member of group 16SrI (group I, aster yellows phytoplasma group), subgroup B (16SrI-B), and rp subgroup rpI-B. Ribosomal protein subgroup B was distinguished from other rp subgroups on the basis of the presence of a recognition site for HpaII. The 1.8-kbp rDNA product of PCR primed by P1/P7 and the 1.2-kbp rpF1/rpR1 PCR product were cloned and sequenced, and the sequences were deposited in GenBank under Accession No. AY734453 for the BaDef 16S rDNA and Accession No. AY735448 for the BaDef rp gene sequence. Previously, only oat proliferation (OatP) phytoplasma, a member of subgroup 16SrI-A, had been characterized in a cereal crop (Avena sativa L.) in Europe (3); BaDef is another phytoplasmal disease threatening cereal crops in the region. References: (1) S. Deng and D. Hiruki. J. Microbiol. Methods 14:53, 1991. (2) D. E. Gundersen and I. M. Lee. Phytopathol. Mediterr. 35:144, 1996. (3) R. Jomantiene et al. Plant Dis. 86:443, 2002. (4) I. M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998.

scholarly journals Detection of XIIA phytoplasma group on cultivar Zupljanka in Zupa vineyard region by RFLP analysis of 16s rDNA sequences

Genetika ◽  
2010 ◽  
Vol 42 (1) ◽  
pp. 145-153
Author(s):  
Dragana Josic ◽  
Slobodan Kuzmanovic ◽  
Sasa Stojanovic ◽  
Goran Aleksic ◽  
Snezana Pavlovic ◽  
...  
Keyword(s):  
16S Rdna ◽  
Direct Sequencing ◽  
Intergenic Spacer ◽  
Rflp Analysis ◽  
Restriction Enzymes ◽  
Intergenic Spacer Region ◽  
Region Detection ◽  
Rdna Sequences ◽  
16S Rdna Sequences ◽  
Stolbur Phytoplasma

'Bois noir' (BN) is an important grapevine disease associated with phytoplasmas belonging to ribosomal subgroup 16SrXII-A. Phytoplasmas cause diseases in several hundred plant species. The number of infected cultivars is growing each year and it is important to follow the spreading of the phytoplasma in the different regions and identify which strains are present in specific regions on specific cultivars. Phytoplasmas are identified and classified based on direct sequencing of phytoplasma 16S rDNA or the 16S to 23S intergenic spacer region, but this approach is not always practical when a large number of unknown phytoplasmas is to be analyzed. Classification by RFLP analysis has provided a simple and rapid method that can be used to differentiate and identify a large number of unclarified phytoplasmas. Our objective was to investigate presence of phytoplasmas of 16SrXII-A group (Stolbur) in Zupa vineyard region. Detection was based on RFLP analysis of 16s rDNA sequences using four restriction enzymes: Tru1I, AluI, KpnI and TaqI. We identified phytoplasmas of XIIA group on two of three investigated cultivars (Zupljanka and Frankovka, but not on Plovdina) in the Zupa vineyard regions (Gornje Rataje and Tules locality). This is the first report of Stolbur phytoplasma on cv. Zupljanka in Zupa region.

Sequence Length Variation of Internal Genic Space of 16S rDNA-23S rDNA in Bacterium with High Yield of Hydrogen Production

2011 ◽  
Vol 183-185 ◽  
pp. 1413-1416
Author(s):  
Yong Feng Li ◽  
Yi Xuan Wang ◽  
Lu Wang ◽  
Zhan Qing Wang
Keyword(s):  
16S Rdna ◽  
Pcr Amplification ◽  
Intergenic Spacer ◽  
Rrna Genes ◽  
High Yield ◽  
23S Rrna ◽  
Sequence Length ◽  
Rrna Gene ◽  
Length Variation ◽  
Intergenic Spacer Region

To develop the identification of species for fermentative biohydrogen-producing bacterium, scholars have found a method which is based on PCR amplification of the 16S rRNA gene (rDNA)-23S rDNA intergenic regions. In the study, a large fragment of the rDNA operon, including the 16S rDNA, the intergenic spacer region (ISR) and approximately 2000 bases of the 23S rDNA, were polymerasechain reaction (PCR) amplified. The PCR amplification of the genomic DNA of Leptonema ilk strain 3055 using primers directed against conserved regions of the rRNA operon provided evidence that the 16S and 23S rRNA genes were linked via an intergenic spacer region. The sequencing of the intergenic spacer region indicated that it was 435 nucleotides in length and sequence similarity searches revealed that it bore no homology to any known sequences including tRNA available in databases.

scholarly journals First Report of Aster Yellows-Related Subgroup I-A Phytoplasma Strains in Carrot, Phlox, Sea-Lavender, Aconitum, and Hyacinth in Lithuania

Plant Disease ◽  
2001 ◽  
Vol 85 (7) ◽  
pp. 804-804 ◽  
Author(s):  
D. Valiunas ◽  
A. Alminaite ◽  
J. Staniulis ◽  
R. Jomantiene ◽  
R. E. Davis
Keyword(s):  
16S Rdna ◽  
Nested Pcr ◽  
Sequence Similarity ◽  
Rflp Analysis ◽  
Plant Host ◽  
Aster Yellows ◽  
Mature Leaves ◽  
Young Leaves ◽  
Sea Lavender ◽  
The Baltic

Phytoplasma strains that belong to group 16SrI (aster yellows phytoplasma group), subgroup A (I-A, North American tomato big bud phytoplasma subgroup) were discovered in diverse plant species in Lithuania. Plants in which the strains were found exhibited symptoms characteristic of infections by phytoplasma. Carrot (Daucus sativus) with carrot proliferation disease exhibited symptoms of proliferation of the crown, chlorosis of young leaves, and reddening of mature leaves. Diseased phlox (Phlox paniculata) exhibited symptoms of virescence and leaf chlorosis. Diseased sea-lavender (Limonium sinuatum) exhibited abnormal proliferation of shoots, chlorosis of young leaves, reddening of mature leaves, and degeneration of flowers. Diseased hyacinth (Hyacinthus orientalis) exhibited chlorosis of leaves and undeveloped flowers. Diseased Aconitum sp. exhibited proliferation of shoots. Phytoplasma-characteristic ribosomal (r) DNA was detected in the plants by use of the polymerase chain reaction (PCR). The rDNA was amplified in PCR primed by primer pair P1/P7 and reamplified in nested PCR primed by primer pair R16F2n/R16R2 (F2n/R2), as previously described (1). The phytoplasmas were classified through restriction fragment length polymorphism (RFLP) analysis of 16S rDNA, amplified in the nested PCR primed by F2n/R2, using single endonuclease enzyme digestion with AluI, MseI, KpnI, HhaI, HaeIII, HpaI, HpaII, RsaI, HinfI, TaqI, and Sau3AI. Collective RFLP patterns indicated that all detected phytoplasma strains were affiliated with subgroup I-A. The 16S rDNA amplified from the phytoplasma (CarrP phytoplasma) in diseased carrot was cloned in Escherichia coli, sequenced, and the sequence deposited in the GenBank data library (GenBank accession no. AF291682). The 16S rDNAs of CarrP and tomato big bud (GenBank acc. no. AF222064) phytoplasmas shared 99.8% nucleotide sequence similarity. Phytoplasmas belonging to group 16SrIII (3), group 16SrV (D. Valiunas, unpublished data), and subgroup I-C in group 16SrI (2,3) occur in Lithuania. This report records the first finding of a subgroup I-A phytoplasma in the Baltic region and expands the known plant host range of this phytoplasma subgroup. References: (1) R. Jomantiene et al. Int. J. Syst. Bacteriol. 48:269, 1998. (2) Jomantiene et al. Phytopathology 90:S39, 2000. (3) Staniulis et al. Plant Dis. 84:1061, 2000.

Characterization of Fusarium spp. isolates by PCR-RFLP analysis of the intergenic spacer region of the rRNA gene (rDNA)

2006 ◽  
Vol 106 (3) ◽  
pp. 297-306 ◽  
Author(s):  
A. Llorens ◽  
M.J. Hinojo ◽  
R. Mateo ◽  
M.T. González-Jaén ◽  
F.M. Valle-Algarra ◽  
...  
Keyword(s):  
Intergenic Spacer ◽  
Rflp Analysis ◽  
Rrna Gene ◽  
Fusarium Spp ◽  
Intergenic Spacer Region ◽  
Pcr Rflp

Genomic fingerprinting ofFrankiamicrosymbionts fromCeanothuscopopulations using repetitive sequences and polymerase chain reactions

1999 ◽  
Vol 77 (9) ◽  
pp. 1220-1230 ◽  
Author(s):  
Soon-Chun Jeong ◽  
David D Myrold
Keyword(s):  
Dna Sequencing ◽  
Dna Analysis ◽  
Repetitive Sequences ◽  
Intergenic Spacer ◽  
Rrna Genes ◽  
23S Rrna ◽  
Genomic Fingerprinting ◽  
Chain Reactions ◽  
Intergenic Spacer Region ◽  
Polymerase Chain

Specificity between Ceanothus species and their microsymbionts, Frankia, were investigated with nodules collected from three geographically separated copopulations of Ceanothus species. Nodules were analyzed using DNA sequencing and repetitive sequence polymerase chain reaction (rep-PCR) techniques. DNA sequencing of the intergenic spacer region between 16S and 23S rRNA genes suggested that Ceanothus-microsymbiotic Frankia are closely related at the intraspecific level. Diversity of the microsymbionts was further analyzed by genomic fingerprinting using repetitive sequences and PCR. A newly designed direct repeat (DR) sequence and a BOX sequence were used as PCR primers after justification that these primers can generate Frankia-specific fingerprints from nodule DNA. Analysis of the nodules using BOX- and DR-PCR showed that Ceanothus-microsymbiotic Frankia exhibited less diversity within each copopulation than among copopulations. These data suggested that geographic separation plays a more important role for divergence of Ceanothus-microsymbiotic Frankia than host plant.Key words: Frankia, Ceanothus, rep-PCR, diversity.

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