Amplified Ribosomal DNA Restriction Analysis Groups Research Articles

Plant growth-promoting characteristics of halotolerant endophytic bacteria isolated from Sporobolus specatus (Vahr) Kunth and Cyperus laevigatus L. of Ethiopian rift valley lakes.

Understanding plant microbes' intimate relationship and search for beneficial microbes is a sustainable alternative to improve plant growth and yield under a wide range of biotic and abiotic stress conditions. More than 20% of the total global agricultural land is affected by salinity. High salinity challenges crop plants by affecting several metabolic pathways and decreasing plant growth and yield. Unlike chemical fertilizers and pesticides, endophytic microbes offer an eco-friendly approach to increasing crop yield via various metabolites during salinity stress. The objective of this study was to isolate and characterize endophytic halotolerant bacterial isolates from haloalkaliphytes, investigate their plant growth-promoting (PGP) properties and tolerance for various stress conditions. Sporobolus specatus (Vahr) Kunth and Cyperus laevigatus L. grass samples were collected from the shores of two Ethiopian soda lakes (Lakes Abijata, and Chitu, respectively). A total of 167 halotolerant endophytic bacterial isolates, that clustered into 21 ARDRA (Amplified ribosomal DNA restriction analysis) groups, affiliated to members of 11 bacterial genera, namely Halomonas, Agrobacterium, Exiguobacterium, Jonesia, Stenotrophomonas, Pseudomonas, Alishewanella, Kosakonia, Bacillus, Paracoccus and Pannonibacter, were identified based on 16S rRNA sequencing. Most of the strains were able to produce IAA (indole-3-acetic acid) and hydrogen cyanide, grow on a nitrogen-free medium and solubilize phosphate. In vitro tolerance tests reveal that isolates were tolerant to: 5.0-15% NaCl, up to 40% PEG 6000, temperatures up to 50°C, and pH 5-11. These characteristics of the isolates indicate their potential PGP application under various plant stress conditions.

Genetic diversity of Rhizobium spp. isolated from soil samples of Bangladesh

Rhizobia are Gram-negative soil-inhabiting bacteria commonly found in nodules of leguminous plants. These bacteria exclusively fix atmospheric nitrogen into ammonia, which is convertible to urea to serve natural fertilizer to the plants. However, rhizobial microbiota of Bangladesh have been studied poorly. Here, twenty isolates were identified as Rhizobium through observation of cultural characteristics, morphological characteristics and different biochemical tests. Isolates differed from one another in terms of their stress response characteristics like salt-stress tolerance,temperature tolerance, antibiotic susceptibility, and pH. Most of the isolates were resistant to ampicillin and erythromycin and streptomycin; and were sensitive to kanamycin and ciprofloxacin. Isolates were clustered into three genotypic groups according to the banding patterns of Amplified Ribosomal DNA Restriction Analysis (ARDRA). All the strains in ARDRA group 1 were Rhizobium azibense, and the others were Rhizobium bangladeshense and Rhizobium qilianshanense. Future studies would validate their capacity of nitrogen fixation and the scope of improvement of these strains to increase their efficiency of nitrogen fixation.
 Bangladesh J Microbiol, Volume 36 Number 1 June 2019, pp 07-10

Molecular diversity of phosphate solubilizing bacteria isolated from the rhizosphere of chickpea, mustard and wheat

Molecular diversity of native phosphate solubilizing bacteria from rhizosphere of important crops like chickpea ( Cicer arietinum ), mustard ( Brassica campestris ) and wheat ( Triticium aestivum ) grown in Haryana was studied A total of 193 isolates were selected from the rhizosphere of chickpea (76), mustard (68) and wheat (49) based on zone of P-solubilization. These were screened for P-solubilization on solid as well as liquid Pikovskaya's medium. All the isolates showed large variations in P-solubilization.On the basis of the 20 biochemical tests, a dendrogram was constructed using UPGMA with which all the PSB isolates were clustered in nine major groups. The isolates from chickpea, mustard and wheat rhizosphere were randomly distributed. Twenty three PSB isolates representing nine biochemical groups were selected and genetic diversity was determined by amplified ribosomal DNA restriction analysis (ARDRA) using Hinf 1 and Msp 1. The banding pattern was analyzed using NT-SYS programme and a dendrogram was constructed. The isolates were divided into 10 ARDRA groups at 85% similarity level. Majority of the isolates fell in one group while the remaining isolates constituted the separate groups. PSB from different crops rhizosphere were randomly distributed. Biochemical groups did not correlate to the ARDRA groups.

The bacterial community associated with Tetraselmis suecica outdoor mass cultures

Interactions between algae and associated bacterial communities in the phycosphere depend on many factors such as culture age, nutrient availability, and antibiotic production and greatly influence algal survival and growth. The microbial community associated with the marine microalga Tetraselmis suecica F&M-M33 in a laboratory culture and an outdoor mass culture, set up from the laboratory one, run during a whole year was investigated in different seasons through isolation in pure culture of bacteria, amplified ribosomal DNA restriction analysis (ARDRA) of all the isolates, and terminal restriction fragment length polymorphism (T-RFLP) analysis. The total number of bacterial isolates was 152, which clustered in thirty-six 16S rDNA groups by ARDRA. Sequencing of a representative of each ARDRA group permitted identification of bacterial genera never reported before in association with microalgae in outdoor mass cultures, although most of them were previously found to be associated with the marine environment (e.g., seawater, sea sediments). T-RFLP analysis revealed that spring and autumn bacterial community profiles were closely related, while the bacterial laboratory community was considerably different (less than 50 % similarity) from that associated with the outdoor culture in different seasons. T-RFLP results suggest the presence of a core of bacteria that are closely associated with the alga and of a part of the community which varies seasonally.

Phylogenetic appraisal of antagonistic, slow growing actinomycetes isolated from hypersaline inland solar salterns at Sambhar salt Lake, India

Inland solar salterns established in the vicinity of Sambhar Lake are extreme saline environments with high salinity and alkalinity. In view of the fact that microbes inhabiting such extreme saline environments flourish the contemporary bioprospecting, it was aimed to selectively isolate slow growing and rare actinomycetes from the unexplored solar salterns. A total of 14 slow growing actinomycetes were selectively isolated from three composite soil samples of inland solar salterns. Among the isolates, four groups were formed according to similarity of the banding patterns obtained by amplified ribosomal DNA restriction analysis (ARDRA). A subset of representative isolates for each ARDRA group was identified using 16S rDNA sequence based phylogenetic analysis and subsequently the entire isolates were assigned under three different genera; Streptomyces, Pseudonocardia, and Actinoalloteichus. The genus Streptomyces was found to be the dominant among the isolates. Furthermore, rare actinomycete genus Actinoalloteichus was isolated for the first time from solar saltern. Determination of salt-tolerance revealed that certain level of salt-tolerance and moderate halophilism occurs among the actinomycetes isolated from the inland salterns. In addition, all the acinomycetes were screened in two levels to unravel their ability to produce antimicrobial compounds. Significant antimicrobial activity was found among the actinomycetes against a range of bacteria and fungi to worth further characterization of these persuasive actinomycetes and their antimicrobial secondary metabolites. In a nutshell, this study offered a first interesting insight on occurrence of antagonistic rare actinomycetes and streptomycetes in inland solar salterns associated with Sambhar salt Lake.

Phylotype diversity in a benthic cyanobacterial mat community on King George Island, maritime Antarctica.

Cyanobacterial 16S ribosomal RNA gene diversity was examined in a benthic mat on Fildes Peninsula of King George Island (62º09'54.4''S, 58º57'20.9''W), maritime Antarctica. Environmental DNA was isolated from the mat, a clone library of PCR-amplified 16S rRNA gene fragments was prepared, and amplified ribosomal DNA restriction analysis (ARDRA) was done to assign clones to seven groups. Low cyanobacterial diversity in the mat was suggested in that 83% of the clones were represented by one ARDRA group. DNA sequences from this group had high similarity with 16S rRNA genes of Tychonema bourrellyi and T. bornetii isolates, whose geographic origins were southern Norway and Northern Ireland. Cyanobacterial morphotypes corresponding to Tychonema have not been reported in Antarctica, however, this morphotype was previously found at Ward Hunt Lake (83ºN), and in western Europe (52ºN). DNA sequences of three of the ARDRA groups had highest similarity with 16S rDNA sequences of the Tychonema group accounting for 9.4% of the clones. Sequences of the remaining three groups (7.6%) had highest similarity with 16S rRNA genes of uncultured cyanobacteria clones from benthic mats of Lake Fryxell and fresh meltwater on the McMurdo Ice Shelf.

Molecular identification and pectate lyase production by Bacillus strains involved in cocoa fermentation

We have previously reported the implication of Bacillus in the production of pectinolytic enzymes during cocoa fermentation. The objective of this work was to identify the Bacillus strains isolated from cocoa fermentation and study their ability to produce pectate lyase (PL) in various growth conditions. Ninety-eight strains were analyzed by Amplified Ribosomal DNA Restriction Analysis (ARDRA). Four different banding patterns were obtained leading to the clustering of the bacterial isolates into 4 distinct ARDRA groups. A subset of representative isolates for each group was identified by 16S rRNA gene partial sequencing. Six species were identified: Bacillus subtilis, Bacillus pumilus, Bacillus sphaericus, Bacillus cereus, Bacillus thuringiensis, together with Bacillus fusiformis which was isolated for the first time from cocoa fermentation. The best PL producers, yielding at least 9 U/mg of bacterial dry weight, belonged to B. fusiformis, B. subtilis, and B. pumilus species while those belonging to B. sphaericus, B. cereus and B. thuringiensis generally showed a low level of activity. Two kinds of PL were produced, as revealed by isoelectrofocusing: one with a pI of 9.8 produced by B. subtilis and B. fusiformis, the other with a pI of 10.5 was produced by B. pumilus. Strains yielded about 2 fold more PL in a pectic compound medium than in glucose medium and maximum enzyme production occurred in the late stationary bacterial growth phase. Together all these results indicate that PL production in the bacilli studied is modulated by the growth phase and by the carbon source present in the medium.

Genetic diversity of siderophore-producing bacteria of tobacco rhizosphere.

The genetic diversity of siderophore-producing bacteria of tobacco rhizosphere was studied by amplified ribosomal DNA restriction analysis (ARDRA), 16S rRNA sequence homology and phylogenetics analysis methods. Studies demonstrated that 85% of the total 354 isolates produced siderophores in iron limited liquid medium. A total of 28 ARDRA patterns were identified among the 299 siderophore-producing bacterial isolates. The 28 ARDRA patterns represented bacteria of 14 different genera belonging to six bacterial divisions, namely β-, γ-, α-Proteobacteria, Sphingobacteria, Bacilli, and Actinobacteria. Especially, γ-Proteobacteria consisting of Pseudomonas, Enterobacter, Serratia, Pantoea, Erwinia and Stenotrophomonas genus encountered 18 different ARDRA groups. Results also showed a greater siderophore-producing bacterial diversity than previous researches. For example, Sphingobacterium (isolates G-2-21-1 and G-2-27-2), Pseudomonas poae (isolate G-2-1-1), Enterobacter endosymbiont (isolates G-2-10-2 and N-5-10), Delftia acidovorans (isolate G-1-15), and Achromobacter xylosoxidans (isolates N-46-11HH and N-5-20) were reported to be able to produce siderophores under low-iron conditions for the first time. Gram-negative isolates were more frequently encountered, with more than 95% total frequency. For Gram-positive bacteria, the Bacillus and Rhodococcus were the only two genera, with 1.7% total frequency. Furthermore, the Pseudomonas and Enterobacter were dominant in this environment, with 44.5% and 24.7% total frequency, respectively. It was also found that 75 percent of the isolates that had the high percentages of siderophore units (% between 40 and 60) belonged to Pseudomonas. Pseudomonas sp. G-229-21 screened out in this study may have potential to apply to low-iron soil to prevent plant soil-borne fungal pathogen diseases.

Bioremediation potential of a tropical soil contaminated with a mixture of crude oil and production water.

A typical tropical soil from the northeast of Brazil, where an important terrestrial oil field is located, was accidentally contaminated with a mixture of oil and saline production water. To study the bioremediation potential in this area, molecular methods based on PCR-DGGE were used to determine the diversity of the bacterial communities in bulk and in contaminated soils. Bacterial fingerprints revealed that the bacterial communities were affected by the presence of the mixture of oil and production water, and different profiles were observed when the contaminated soils were compared with the control. Halotolerant strains capable of degrading crude oil were also isolated from enrichment cultures obtained from the contaminated soil samples. Twenty-two strains showing these features were characterized genetically by amplified ribosomal DNA restriction analysis (ARDRA) and phenotypically by their colonial morphology and tolerance to high NaCl concentrations. Fifteen ARDRA groups were formed. Selected strains were analyzed by 16S rDNA sequencing, and Actinobacteria was identified as the main group found. Strains were also tested for their growth capability in the presence of different oil derivatives (hexane, dodecane, hexadecane, diesel, gasoline, toluene, naphthalene, o-xylene, and p-xylene) and different degradation profiles were observed. PCR products were obtained from 12 of the 15 ARDRA representatives when they were screened for the presence of the alkane hydroxylase gene (alkB). Members of the genera Rhodococcus and Gordonia were identified as predominant in the soil studied. These genera are usually implicated in oil degradation processes and, as such, the potential for bioremediation in this area can be considered as feasible.

Method for reliable isolation of Lactobacillus sakei strains originating from Tunisian seafood and meat products

In Tunisia, several food products derived from meat or seafood are naturally processed, without any addition of bacterial starters. Such fermented, dried-cured, salted, or marinated products, as well as the raw meat or fish may thus provide a source to isolate the natural microflora colonizing such environments. We isolated lactic acid bacteria from a representative range of flesh-foods sold or manufactured in different parts of Tunisia, and selectively searched for Lactobacillus sakei, a lactic acid bacterium potentially useful as starter or protective culture. Eighty six (86) strains were isolated from various seafood (anchovy, sardine, sole, mullet, and octopus), or meat (pork, veal, beef, sheep, chicken, and turkey) products that were either fresh, or transformed by different traditional processes. Several methods were used in order to develop a rapid and reliable protocol for the direct identification of L. sakei. Amplified ribosomal DNA restriction analysis (ARDRA) classified the various isolates into 9 distinct groups. Search for the presence of the L. sakei specific katA gene indicated that all positive isolates were grouped in the same ARDRA group. Sequencing of 16S rDNA confirmed those isolates as L. sakei. Those 22 different L. sakei strains represent 25.6% of the total isolates, while other isolates found in the different ARDRA groups were tentatively ascribed to Lactobacillus plantarum, Lactococcus lactis/garviae, Enterococcus avium, Streptococcus parauberis, Hafnia alvei, Pediococcus pentosaceus, and Lactobacillus curvatus through 16S rDNA sequencing. A fast and reliable method to isolate and discriminate L. sakei from complex food environments is proposed.

Identification of the bacterial community of maple sap by using amplified ribosomal DNA (rDNA) restriction analysis and rDNA sequencing.

The bacterial community of maple sap was characterized by analysis of samples obtained at the taphole of maple trees for the 2001 and 2002 seasons. Among the 190 bacterial isolates, 32 groups were formed according to the similarity of the banding patterns obtained by amplified ribosomal DNA restriction analysis (ARDRA). A subset of representative isolates for each ARDRA group was identified by 16S rRNA gene fragment sequencing. Results showed a wide variety of organisms, with 22 different genera encountered. Pseudomonas and Ralstonia, of the gamma- and beta-Proteobacteria, respectively, were the most frequently encountered genera. Gram-positive bacteria were also observed, and Staphylococcus, Plantibacter, and Bacillus were the most highly represented genera. The sampling period corresponding to 50% of the cumulative sap flow percentage presented the greatest bacterial diversity according to its Shannon diversity index value (1.1). gamma-Proteobacteria were found to be dominant almost from the beginning of the season to the end. These results are providing interesting insights on maple sap microflora that will be useful for further investigation related to microbial contamination and quality of maple products and also for guiding new strategies on taphole contamination control.

Genetic diversity of carbofuran-degrading soil bacteria.

The genetic diversity of 128 carbofuran-degrading bacteria was determined by ARDRA (amplified ribosomal DNA restriction analysis) of 16S rDNA and restriction fragment length polymorphism analysis of the 16S-23S rDNA spacer region (IGS) using five endonucleases. The isolates were distributed in 26 distinct ARDRA groups and 45 IGS types revealing a high level of microbial diversity confirmed by ARDRA clustering and sequencing of 16S rDNA. The occurrence of a methylcarbamate-degrading gene (mcd) was monitored by polymerase chain reaction amplification using specific primers. The mcd gene was detected only in 58 bacteria and there was no clear relationship between the presence of this gene and the phylogenetic position of the strain.

Genetic diversity of carbofuran-degrading soil bacteria

The genetic diversity of 128 carbofuran-degrading bacteria was determined by ARDRA (amplified ribosomal DNA restriction analysis) of 16S rDNA and restriction fragment length polymorphism analysis of the 16S–23S rDNA spacer region (IGS) using five endonucleases. The isolates were distributed in 26 distinct ARDRA groups and 45 IGS types revealing a high level of microbial diversity confirmed by ARDRA clustering and sequencing of 16S rDNA. The occurrence of a methylcarbamate-degrading gene ( mcd) was monitored by polymerase chain reaction amplification using specific primers. The mcd gene was detected only in 58 bacteria and there was no clear relationship between the presence of this gene and the phylogenetic position of the strain.

Effect of field inoculation with Sinorhizobium meliloti L33 on the composition of bacterial communities in rhizospheres of a target plant (Medicago sativa) and a non-target plant (Chenopodium album)-linking of 16S rRNA gene-based single-strand conformation polymorphism community profiles to the diversity of cultivated bacteria.

Fourteen weeks after field release of luciferase gene-tagged Sinorhizobium meliloti L33 in field plots seeded with Medicago sativa, we found that the inoculant also occurred in bulk soil from noninoculated control plots. In rhizospheres of M. sativa plants, S. meliloti L33 could be detected in noninoculated plots 12 weeks after inoculation, indicating that growth in the rhizosphere preceded spread into bulk soil. To determine whether inoculation affected bacterial diversity, 1,119 bacteria were isolated from the rhizospheres of M. sativa and Chenopodium album, which was the dominant weed in the field plots. Amplified ribosomal DNA restriction analysis (ARDRA) revealed plant-specific fragment size frequencies. Dominant ARDRA groups were identified by 16S rRNA gene nucleotide sequencing. Database comparisons indicated that the rhizospheres contained members of the Proteobacteria (alpha, beta, and gamma subgroups), members of the Cytophaga-Flavobacterium group, and gram-positive bacteria with high G+C DNA contents. The levels of many groups were affected by the plant species and, in the case of M. sativa, by inoculation. The most abundant isolates were related to Variovorax sp., Arthrobacter ramosus, and Acinetobacter calcoaceticus. In the rhizosphere of M. sativa, inoculation reduced the numbers of cells of A. calcoaceticus and members of the genus Pseudomonas and increased the number of rhizobia. Cultivation-independent PCR-single-strand conformation polymorphism (SSCP) profiles of a 16S rRNA gene region confirmed the existence of plant-specific rhizosphere communities and the effect of the inoculant. All dominant ARDRA groups except Variovorax species could be detected. On the other hand, the SSCP profiles revealed products which could not be assigned to the dominant cultured isolates, indicating that the bacterial diversity was greater than the diversity suggested by cultivation.