scholarly journals Detection, in silico analysis and molecular diversity of phytoplasmas from solanaceous crops in Turkey

2021 ◽  
Vol 58 (No. 1) ◽  
pp. 31-39
Author(s):  
Mustafa Usta ◽  
Abdullah Güller ◽  
Hikmet Murat Sipahioglu
Keyword(s):  
Geographical Area ◽  
Solanum Melongena ◽  
In Silico Analysis ◽  
Rflp Analysis ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Natural Occurrence ◽  
Similarity Coefficients ◽  
Universal Primer ◽  
Virtual Rflp

Phytoplasma-like symptoms of leaf yellowing and calyx malformation were observed in eggplant (Solanum melongena L.), upward leaves and fruit malformation in pepper (Capsicum annuum L.), and aerial tuber formation in potato (S. tuberosum L.) during the survey performed in the late season (August to September) of 2015 and 2016 in Van province (Turkey). A total of 100 samples were tested by nested-PCR using universal primer pairs to assess the sanitary status of the solanaceous crops and to characterise the phytoplasma isolates. Among them, seven samples resulted in a 1.25 kb DNA fragment, and five (two eggplants, two peppers, and one potato) were molecularly characterised (Accession No.: KY579357, KT595210, MF564267, MF564266, and MH683601). BLAST and the virtual restriction fragment length polymorphism (RFLP) analysis of 16S rRNA genes revealed the presence of two distinct phytoplasma infections in solanaceous crops: ‘Candidatus Phytoplasma trifolii’ a member of the clover proliferation group (16SrVI) and subgroup A and ‘Candidatus P. solani’ a member of the stolbur group (16SrXII) and subgroup A. The virtual RFLP analysis and calculated coefficients of RFLP pattern similarities further revealed a remarkable genetic diversity among the ‘Candidatus P. solani’ isolates infecting pepper (similarity coefficient of 0.90) and eggplant (similarity coefficients of 0.98 and 1.00) at the same geographical area. This is the first report of the natural occurrence of ‘Candidadtus P. trifolii’ in potato from the Eastern Anatolia region, Turkey.

scholarly journals Computer-simulated RFLP analysis of 16S rRNA genes: identification of ten new phytoplasma groups

2007 ◽  
Vol 57 (8) ◽  
pp. 1855-1867 ◽  
Author(s):  
Wei Wei ◽  
Robert E. Davis ◽  
Ing-Ming Lee ◽  
Yan Zhao
Keyword(s):  
16S Rrna ◽  
Classification Scheme ◽  
Rflp Analysis ◽  
Plant Diseases ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Similarity Coefficients ◽  
Wide Range

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.

672 IDENTIFICATION OF POLYBACTERIAL 16S RRNA GENES FROM ASCITIC FLUIDS WITH T-RFLP ANALYSIS AND DIRECT SEQUENCING

2012 ◽  
Vol 56 ◽  
pp. S266
Author(s):  
S. Krohn ◽  
J. Hartmann ◽  
A. Brodzinski ◽  
A. Chatzinotas ◽  
S. Bohm ◽  
...  
Keyword(s):  
16S Rrna ◽  
Direct Sequencing ◽  
Rflp Analysis ◽  
16S Rrna Genes ◽  
Rrna Genes

Phenotypic and molecular characterization of chickpea rhizobia isolated from different areas of Tunisia

2007 ◽  
Vol 53 (3) ◽  
pp. 427-434 ◽  
Author(s):  
Boulbaba L’taief ◽  
Bouaziz Sifi ◽  
Maher Gtari ◽  
Mainassara Zaman-Allah ◽  
Mokhtar Lachaâl
Keyword(s):  
Rflp Analysis ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Phenotypic Characteristics ◽  
Cicer Arietinum L ◽  
Mesorhizobium Ciceri ◽  
Length Polymorphisms ◽  
Reference Strains ◽  
Tolerance To Salinity

Several phenotypic markers were used in this study to determine the biodiversity of rhizobial strains nodulating Cicer arietinum L. in various areas of Tunisia. They include symbiotic traits, the use of 21 biochemical substrates, and tolerance to salinity and pH. In addition, restriction fragment length polymorphisms (RFLPs) of PCR-amplified 16S rDNA were compared with those of reference strains. Numeric analysis of the phenotypic characteristics showed that the 48 strains studied fell into three distinct groups. This heterogeneity was highly supported by the RFLP analysis of 16S rRNA genes, and two ribotypes were identified. Chickpea rhizobia isolated from Tunisian soils are both phenotypically and genetically diverse. Results showed that 40 and 8 isolates were assigned, respectively, to Mesorhizobium ciceri and Mesorhizobium mediterraneum .

scholarly journals 454-Pyrosequencing Analysis of Bacterial Communities from Autotrophic Nitrogen Removal Bioreactors Utilizing Universal Primers: Effect of Annealing Temperature

2015 ◽  
Vol 2015 ◽  
pp. 1-12 ◽  
Author(s):  
Alejandro Gonzalez-Martinez ◽  
Alejandro Rodriguez-Sanchez ◽  
Belén Rodelas ◽  
Ben A. Abbas ◽  
Maria Victoria Martinez-Toledo ◽  
...  
Keyword(s):  
Bacterial Diversity ◽  
Nitrogen Removal ◽  
Annealing Temperature ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Universal Primers ◽  
Anammox Bacteria ◽  
Universal Primer ◽  
Optimum Annealing Temperature ◽  
Autotrophic Nitrogen Removal

Identification of anaerobic ammonium oxidizing (anammox) bacteria by molecular tools aimed at the evaluation of bacterial diversity in autotrophic nitrogen removal systems is limited by the difficulty to design universal primers for theBacteriadomain able to amplify the anammox 16S rRNA genes. A metagenomic analysis (pyrosequencing) of total bacterial diversity including anammox population in five autotrophic nitrogen removal technologies, two bench-scale models (MBR and Low Temperature CANON) and three full-scale bioreactors (anammox, CANON, and DEMON), was successfully carried out by optimization of primer selection and PCR conditions (annealing temperature). The universal primer 530F was identified as the best candidate for total bacteria and anammox bacteria diversity coverage. Salt-adjusted optimum annealing temperature of primer 530F was calculated (47°C) and hence a range of annealing temperatures of 44–49°C was tested. Pyrosequencing data showed that annealing temperature of 45°C yielded the best results in terms of species richness and diversity for all bioreactors analyzed.

scholarly journals Analysis of the Endophytic Actinobacterial Population in the Roots of Wheat (Triticum aestivum L.) by Terminal Restriction Fragment Length Polymorphism and Sequencing of 16S rRNA Clones

2004 ◽  
Vol 70 (3) ◽  
pp. 1787-1794 ◽  
Author(s):  
Vanessa M. Conn ◽  
Christopher M. M. Franco
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
16S Rrna ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Rflp Analysis ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Restriction Fragment Length

ABSTRACT The endophytic actinobacterial population in the roots of wheat grown in three different soils obtained from the southeast part of South Australia was investigated by terminal restriction fragment length polymorphism (T-RFLP) analysis of the amplified 16S rRNA genes. A new, validated approach was applied to the T-RFLP analysis in order to estimate, to the genus level, the actinobacterial population that was identified. Actinobacterium-biased primers were used together with three restriction enzymes to obtain terminal restriction fragments (TRFs). The TRFs were matched to bacterial genera by the T-RFLP Analysis Program, and the data were analyzed to validate and semiquantify the genera present within the plant roots. The highest diversity and level of endophytic colonization were found in the roots of wheat grown in a dark loam from Swedes Flat, and the lowest were found in water-repellent sand from Western Flat. This molecular approach detected a greater diversity of actinobacteria than did previous culture-dependent methods, with the predominant genera being Mycobacterium (21.02%) in Swedes Flat, Streptomyces (14.35%) in Red Loam, and Kitasatospora (15.02%) in Western Flat. This study indicates that the soil that supported a higher number of indigenous organisms resulted in wheat roots with higher actinobacterial diversity and levels of colonization within the plant tissue. Sequencing of 16S rRNA clones, obtained using the same actinobacterium-biased PCR primers that were used in the T-RFLP analysis, confirmed the presence of the actinobacterial diversity and identified a number of Mycobacterium and Streptomyces species.

scholarly journals Effect of Temperature on Carbon and Electron Flow and on the Archaeal Community in Methanogenic Rice Field Soil

2000 ◽  
Vol 66 (11) ◽  
pp. 4790-4797 ◽  
Author(s):  
Axel Fey ◽  
Ralf Conrad
Keyword(s):  
Steady State ◽  
Rice Field ◽  
Electron Flow ◽  
Rflp Analysis ◽  
Archaeal Community ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Field Soil ◽  
Rice Field Soil ◽  
Different Temperatures

ABSTRACT Temperature is an important factor controlling CH4production in anoxic rice soils. Soil slurries, prepared from Italian rice field soil, were incubated anaerobically in the dark at six temperatures of between 10 to 37°C or in a temperature gradient block covering the same temperature range at intervals of 1°C. Methane production reached quasi-steady state after 60 to 90 days. Steady-state CH4 production rates increased with temperature, with an apparent activation energy of 61 kJ mol−1. Steady-state partial pressures of the methanogenic precursor H2 also increased with increasing temperature from <0.5 to 3.5 Pa, so that the Gibbs free energy change of H2 plus CO2-dependent methanogenesis was kept at −20 to −25 kJ mol of CH4 −1 over the whole temperature range. Steady-state concentrations of the methanogenic precursor acetate, on the other hand, increased with decreasing temperature from <5 to 50 μM. Simultaneously, the relative contribution of H2 as methanogenic precursor decreased, as determined by the conversion of radioactive bicarbonate to 14CH4, so that the carbon and electron flow to CH4 was increasingly dominated by acetate, indicating that psychrotolerant homoacetogenesis was important. The relative composition of the archaeal community was determined by terminal restriction fragment length polymorphism (T-RFLP) analysis of the 16S rRNA genes (16S rDNA). T-RFLP analysis differentiated the archaeal Methanobacteriaceae,Methanomicrobiaceae, Methanosaetaceae,Methanosarcinaceae, and Rice clusters I, III, IV, V, and VI, which were all present in the rice field soil incubated at different temperatures. The 16S rRNA genes of Rice cluster I andMethanosaetaceae were the most frequent methanogenic groups. The relative abundance of Rice cluster I decreased with temperature. The substrates used by this microbial cluster, and thus its function in the microbial community, are unknown. The relative abundance of acetoclastic methanogens, on the other hand, was consistent with their physiology and the acetate concentrations observed at the different temperatures, i.e., the high-acetate-requiring Methanosarcinaceae decreased and the more modest Methanosaetaceae increased with increasing temperature. Our results demonstrate that temperature not only affected the activity but also changed the structure and the function (carbon and electron flow) of a complex methanogenic system.

scholarly journals Formation of Pseudo-Terminal Restriction Fragments, a PCR-Related Bias Affecting Terminal Restriction Fragment Length Polymorphism Analysis of Microbial Community Structure

2003 ◽  
Vol 69 (5) ◽  
pp. 2555-2562 ◽  
Author(s):  
Markus Egert ◽  
Michael W. Friedrich
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
16S Rrna ◽  
Length Polymorphism ◽  
Fragment Length Polymorphism ◽  
Rflp Analysis ◽  
Restriction Enzymes ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Restriction Fragments ◽  
Restriction Fragment Length

ABSTRACT Terminal restriction fragment length polymorphism (T-RFLP) analysis of PCR-amplified genes is a widely used fingerprinting technique in molecular microbial ecology. In this study, we show that besides expected terminal restriction fragments (T-RFs), additional secondary T-RFs occur in T-RFLP analysis of amplicons from cloned 16S rRNA genes at high frequency. A total of 50% of 109 bacterial and 78% of 68 archaeal clones from the guts of cetoniid beetle larvae, using MspI and AluI as restriction enzymes, respectively, were affected by the presence of these additional T-RFs. These peaks were called “pseudo-T-RFs” since they can be detected as terminal fluorescently labeled fragments in T-RFLP analysis but do not represent the primary terminal restriction site as indicated by sequence data analysis. Pseudo-T-RFs were also identified in T-RFLP profiles of pure culture and environmental DNA extracts. Digestion of amplicons with the single-strand-specific mung bean nuclease prior to T-RFLP analysis completely eliminated pseudo-T-RFs. This clearly indicates that single-stranded amplicons are the reason for the formation of pseudo-T-RFs, most probably because single-stranded restriction sites cannot be cleaved by restriction enzymes. The strong dependence of pseudo-T-RF formation on the number of cycles used in PCR indicates that (partly) single-stranded amplicons can be formed during amplification of 16S rRNA genes. In a model, we explain how transiently formed secondary structures of single-stranded amplicons may render single-stranded amplicons accessible to restriction enzymes. The occurrence of pseudo-T-RFs has consequences for the interpretation of T-RFLP profiles from environmental samples, since pseudo-T-RFs may lead to an overestimation of microbial diversity. Therefore, it is advisable to establish 16S rRNA gene sequence clone libraries in parallel with T-RFLP analysis from the same sample and to check clones for their in vitro digestion T-RF pattern to facilitate the detection of pseudo-T-RFs.

scholarly journals Community Structure of Denitrifiers, Bacteria, and Archaea along Redox Gradients in Pacific Northwest Marine Sediments by Terminal Restriction Fragment Length Polymorphism Analysis of Amplified Nitrite Reductase (nirS) and 16S rRNA Genes

2001 ◽  
Vol 67 (4) ◽  
pp. 1893-1901 ◽  
Author(s):  
Gesche Braker ◽  
Héctor L. Ayala-del-Rı́o ◽  
Allan H. Devol ◽  
Andreas Fesefeldt ◽  
James M. Tiedje
Keyword(s):  
Community Structure ◽  
Restriction Fragment Length Polymorphism ◽  
16S Rrna ◽  
Fragment Length Polymorphism ◽  
Puget Sound ◽  
Rflp Analysis ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Restriction Fragment Length ◽  
Redox Gradients

ABSTRACT Steep vertical gradients of oxidants (O2 and NO3 −) in Puget Sound and Washington continental margin sediments indicate that aerobic respiration and denitrification occur within the top few millimeters to centimeters. To systematically explore the underlying communities of denitrifiers,Bacteria, and Archaea along redox gradients at distant geographic locations, nitrite reductase (nirS) genes and bacterial and archaeal 16S rRNA genes (rDNAs) were PCR amplified and analyzed by terminal restriction fragment length polymorphism (T-RFLP) analysis. The suitablility of T-RFLP analysis for investigating communities of nirS-containing denitrifiers was established by the correspondence of dominant terminal restriction fragments (T-RFs) of nirS to computer-simulated T-RFs ofnirS clones. These clones belonged to clusters II, III, and IV from the same cores and were analyzed in a previous study (G. Braker, J. Zhou, L. Wu, A. H. Devol, and J. M. Tiedje, Appl. Environ. Microbiol. 66:2096–2104, 2000). T-RFLP analysis ofnirS and bacterial rDNA revealed a high level of functional and phylogenetic diversity, whereas the level of diversity ofArchaea was lower. A comparison of T-RFLPs based on the presence or absence of T-RFs and correspondence analysis based on the frequencies and heights of T-RFs allowed us to group sediment samples according to the sampling location and thus clearly distinguish Puget Sound and the Washington margin populations. However, changes in community structure within sediment core sections during the transition from aerobic to anaerobic conditions were minor. Thus, within the top layers of marine sediments, redox gradients seem to result from the differential metabolic activities of populations of similar communities, probably through mixing by marine invertebrates rather than from the development of distinct communities.

scholarly journals Coyotes (Canis latrans) as the Reservoir for a Human Pathogenic Bartonella sp.: Molecular Epidemiology ofBartonella vinsonii subsp. berkhoffii Infection in Coyotes from Central Coastal California

2000 ◽  
Vol 38 (11) ◽  
pp. 4193-4200 ◽  
Author(s):  
Chao-Chin Chang ◽  
Rickie W. Kasten ◽  
Bruno B. Chomel ◽  
Darren C. Simpson ◽  
Carrie M. Hew ◽  
...  
Keyword(s):  
16S Rrna ◽  
Canis Latrans ◽  
Bacterial Genome ◽  
Clinical Signs ◽  
Rflp Analysis ◽  
Domestic Dog ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Intergenic Spacer Region ◽  
Pcr Rflp

Bartonella vinsonii subsp. berkhoffii was originally isolated from a dog suffering infectious endocarditis and was recently identified as a zoonotic agent causing human endocarditis. Following the coyote bite of a child who developed clinical signs compatible with Bartonella infection in Santa Clara County, Calif., this epidemiological study was conducted. Among 109 coyotes (Canis latrans) from central coastal California, 31 animals (28%) were found to be bacteremic with B. vinsonii subsp.berkhoffii and 83 animals (76%) had B. vinsonii subsp. berkhoffii antibodies. These findings suggest these animals could be the wildlife reservoir of B. vinsonii subsp. berkhoffii. PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of the gltA and 16S rRNA genes for these 31 isolates yielded similar profiles that were identical to those of B. vinsonii subsp.berkhoffii. Partial sequencing of the gltA and 16S rRNA genes, respectively, indicated 99.5 and 100% homology between the coyote isolate and B. vinsonii subsp.berkhoffii (ATCC 51672). PCR-RFLP analysis of the 16S-23S intergenic spacer region showed the existence of two different strain profiles, as has been reported in dogs. Six (19%) of 31Bartonella bacteremic coyotes exhibited the strain profile that was identified in the type strain of a canine endocarditis case (B. vinsonii subsp. berkhoffii ATCC 51672). The other 25 bacteremic coyotes were infected with a strain that was similar to the strains isolated from healthy dogs. Based on whole bacterial genome analysis by pulsed-field gel electrophoresis (PFGE) with SmaI restriction endonuclease, there was more diversity in fingerprints for the coyote isolates, which had at least 10 major variants compared to the two variants described for domestic dog isolates from the eastern United States. By PFGE analysis, threeBartonella bacteremic coyotes were infected by a strain identical to the one isolated from three healthy dog carriers. Further studies are necessary to elucidate the mode of transmission of B. vinsonii subsp. berkhoffii, especially to identify potential vectors, and to determine how humans become infected.

scholarly journals Methanogenic Community Dynamics during Anaerobic Utilization of Agricultural Wastes

Acta Naturae ◽  
2012 ◽  
Vol 4 (4) ◽  
pp. 91-97 ◽  
Author(s):  
A. M. Ziganshin ◽  
E. E. Ziganshina ◽  
S. Kleinsteuber ◽  
J. Pröter ◽  
O. N. Ilinskaya
Keyword(s):  
16S Rrna ◽  
Anaerobic Degradation ◽  
Community Dynamics ◽  
Biological Diversity ◽  
Biogas Production ◽  
Rflp Analysis ◽  
Methanogenic Archaea ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene

This work is devoted to the investigation of the methanogenic archaea involved in anaerobic digestion of cattle manure and maize straw on the basis of terminal restriction fragment length polymorphism (TRFLP) analysis of archaeal 16S rRNA genes. The biological diversity and dynamics of methanogenic communities leading to anaerobic degradation of agricultural organic wastes with biogas production were evaluated in laboratory-scale digesters. T-RFLP analysis, along with the establishment of archaeal 16S rRNA gene clone libraries, showed that the methanogenic consortium consisted mainly of members of the genera Methanosarcina and Methanoculleus, with a predominance of Methanosarcina spp. throughout the experiment.

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