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

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 .

2014 ◽  
Vol 2014 ◽  
pp. 1-10 ◽  
Author(s):  
Zineb Benmechernene ◽  
Inmaculada Fernández-No ◽  
Marcos Quintela-Baluja ◽  
Karola Böhme ◽  
Mebrouk Kihal ◽  
...  

Information on the microbiology of camel milk is very limited. In this work, the genetic characterization and proteomic identification of 13 putative producing bacteriocinLeuconostocstrains exhibiting antilisterial activity and isolated from camel milk were performed. DNA sequencing of the 13 selected strains revealed high homology among the 16S rRNA genes for all strains. In addition, 99% homology withLeuconostoc mesenteroideswas observed when these sequences were analysed by the BLAST tool against other sequences from reference strains deposited in the Genbank. Furthermore, the isolates were characterized by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDITOF MS) which allowed for the identification of 2 mass peaks 6242 m/z and 5118 m/z that resulted to be specific to the speciesL. mesenteroides. Remarkably, the phyloproteomic tree provided more intraspecific information ofL. mesenteroidesthan phylogenetic analysis. Accordingly, phyloproteomic analysis groupedL. mesenteroidesstrains into different subbranches, while allL. mesenteroidesisolates were grouped in the same branch according to phylogenetic analysis. This study represents, to our knowledge, the first report on the use of MALDI-TOF MS on the identification of LAB isolated from camel milk.


2012 ◽  
Vol 56 ◽  
pp. S266
Author(s):  
S. Krohn ◽  
J. Hartmann ◽  
A. Brodzinski ◽  
A. Chatzinotas ◽  
S. Bohm ◽  
...  

2004 ◽  
Vol 70 (3) ◽  
pp. 1787-1794 ◽  
Author(s):  
Vanessa M. Conn ◽  
Christopher M. M. Franco

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.


1998 ◽  
Vol 64 (11) ◽  
pp. 4246-4254 ◽  
Author(s):  
Marion Fischer-Le Saux ◽  
Hervé Mauléon ◽  
Philippe Constant ◽  
Brigitte Brunel ◽  
Noël Boemare

ABSTRACT The genetic diversity of symbiotic Xenorhabdus andPhotorhabdus bacteria associated with entomopathogenic nematodes was examined by a restriction fragment length polymorphism analysis of PCR-amplified 16S rRNA genes (rDNAs). A total of 117 strains were studied, most of which were isolated from the Caribbean basin after an exhaustive soil sampling. The collection consisted of 77 isolates recovered from entomopathogenic nematodes in 14 Caribbean islands and of 40 reference strains belonging toXenorhabdus and Photorhabdus spp. collected at various localities worldwide. Thirty distinctive 16S rDNA genotypes were identified, and cluster analysis was used to distinguish the genus Xenorhabdus from the genusPhotorhabdus. The genus Xenorhabdusappears more diverse than the genusPhotorhabdus, and for both genera the bacterial genotype diversity is in congruence with the host-nematode taxonomy. The occurrence of symbiotic bacterial genotypes was related to the ecological distribution of host nematodes.


2020 ◽  
Vol 18 ◽  
pp. 00025
Author(s):  
Dimitriyka Sakalieva

Tomato and pepper are the main vegetable crops cultivated in Bulgaria. Phytoplasma diseases, mainly stolbur, are important plant diseases for these crops in Bulgaria. The goal of the present paper was to verify association of phytoplasmas with the observed disease symptoms in tomato and pepper and to identify the phytoplasmas detected using RFLP analysis of conserved genes and other uncharacterised phytoplasma chromosomal regions. The presence of phytoplasmas was confirmed in all the samples of tomato and pepper showing typical stolbur symptoms. A phytoplasm sample, which caused severe symptoms, showed the same pattern as the reference strain Mol, while all other phytoplasmic reference strains showed different polymorphisms. RFLP profiles were found useful in distinguishing phytoplasmas in stolbur subgroup (16SrXII-A) in natural plant hosts.


2000 ◽  
Vol 66 (11) ◽  
pp. 4790-4797 ◽  
Author(s):  
Axel Fey ◽  
Ralf Conrad

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.


2003 ◽  
Vol 69 (5) ◽  
pp. 2555-2562 ◽  
Author(s):  
Markus Egert ◽  
Michael W. Friedrich

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.


2001 ◽  
Vol 67 (4) ◽  
pp. 1893-1901 ◽  
Author(s):  
Gesche Braker ◽  
Héctor L. Ayala-del-Rı́o ◽  
Allan H. Devol ◽  
Andreas Fesefeldt ◽  
James M. Tiedje

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.


2000 ◽  
Vol 38 (11) ◽  
pp. 4193-4200 ◽  
Author(s):  
Chao-Chin Chang ◽  
Rickie W. Kasten ◽  
Bruno B. Chomel ◽  
Darren C. Simpson ◽  
Carrie M. Hew ◽  
...  

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.


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