scholarly journals Development of a multilocus sequence typing method for analysis of Lactobacillus plantarum strains

Microbiology ◽  
2006 ◽  
Vol 152 (1) ◽  
pp. 85-93 ◽  
Author(s):  
Blanca de las Rivas ◽  
Ángela Marcobal ◽  
Rosario Muñoz
Keyword(s):  
Lactobacillus Plantarum ◽  
Multilocus Sequence Typing ◽  
Population Biology ◽  
Decomposition Analysis ◽  
Intergenic Spacer ◽  
Rflp Analysis ◽  
Culture Collection ◽  
Future Application ◽  
Typing Method ◽  
Intergenic Spacer Region

Lactobacillus plantarum is a species of considerable industrial and medical interest. To date, the lack of reliable molecular methods for definite identification at strain level has hindered studies of the population biology of this organism. Here, a multilocus sequence typing (MLST) system for this organism is described, which exploits the genetic variation present in six housekeeping loci to determine the genetic relationship among isolates. The MLST system was established using 16 L. plantarum strains that were also characterized by ribotyping and restriction fragment length polymorphism (RFLP) analysis of the PCR-amplified 16S–23S rDNA intergenic spacer region (ISR). Ribotyping grouped the strains into four groups; however, RFLP analysis of the ISRs showed no differences in the strains analysed. In contrast, MLST had a good discriminatory ability. The sequence analysis of the six genes showed 14 different allelic combinations, with 12 of them represented by only one strain. By using this MLST approach we were able to confirm the identity of two strains deposited in the Spanish Type Culture Collection as different strains. Phylogenetic analysis indicated a panmictic population structure of L. plantarum and split decomposition analysis indicated that recombination plays a role in creating genetic heterogeneity in L. plantarum. As MLST allows precise identification, and easy comparison and exchange of results obtained in different laboratories, the future application of this new molecular method could be useful for the identification of valuable L. plantarum strains.

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

Characterization ofFusarium verticillioides strains by PCR-RFLP analysis of the intergenic spacer region of the rDNA

2005 ◽  
Vol 86 (3) ◽  
pp. 429-435 ◽  
Author(s):  
Belén Patiño ◽  
Salvador Mirete ◽  
Covadonga Vázquez ◽  
Misericordia Jiménez ◽  
M Teresa Rodríguez ◽  
...  
Keyword(s):  
Intergenic Spacer ◽  
Rflp Analysis ◽  
Intergenic Spacer Region ◽  
Pcr Rflp

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 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’.

RFLP analysis of rRNA intergenic spacer regions of 23 isolates of the entomopathogenPaecilomyces farinosus

1997 ◽  
Vol 75 (12) ◽  
pp. 2038-2044 ◽  
Author(s):  
J. S. K. Chew ◽  
D. B. Strongman ◽  
R. M. MacKay
Keyword(s):  
Ribosomal Rna ◽  
Population Biology ◽  
Genetic Relationships ◽  
Intergenic Spacer ◽  
Rflp Analysis ◽  
Restriction Enzyme Digestion ◽  
Paecilomyces Fumosoroseus ◽  
Chain Reaction ◽  
Ecological Requirements ◽  
Polymerase Chain

Genetic relationships between 23 eastern Canadian isolates of the entomopathogen Paecilomyces farinosus (Holm ex S.F. Gray) Brown & Smith were investigated by comparison of DNA fragments produced by restriction enzyme digestion of polymerase chain reaction amplified ribosomal RNA intergenic spacer regions. The variation observed was limited to 40% or less of these regions. All P. farinosus isolates were very dissimilar to isolates of the entomopathogens Beauveria bassiana (Bals.) Vuill. and Paecilomyces fumosoroseus (Wize) Brown & Smith. Seventeen P. farinosus isolates from six different hosts and diverse habitats yielded identical or nearly identical results. Two groups, each with three isolates from two different hosts, were distinct from the main group of isolates. Each of the three P. farinosus groups included some isolates that produced synnemata and some that did not, indicating multiple evolutionary losses of the ability to produce this sporulation structure. We conclude that eastern Canadian P. farinosus, while genetically and phenotypically variable, is not composed largely of strains with strict ecological requirements. Key words: entomopathogenic fungus, population biology, RFLP, ribosomal RNA intergenic spacer.

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.

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