16S rDNA-RFLP Research Articles

Comparison of phenotypical and genetic identification of Aeromonas strains isolated from diseased fish

Phenotypicaly identified Aeromonas strains (n=119) recovered mainly from diseased fish were genetically re-identified and the concordance between the results was analysed. Molecular characterization based on the GCAT genus specific gene showed that only 90 (75.6%) strains belonged to the genus Aeromonas. The 16S rDNA-RFLP method identified correctly most of the strains with the exception of a few that belonged to A. bestiarum, A. salmonicida or A. piscicola. Separation of these 3 species was correctly assessed with the rpoD gene sequences, which revealed that 5 strains with the RFLP pattern of A. salmonicida belonged to A. piscicola, as did 1 strain with the pattern of A. bestiarum. Correct phenotypic identification occurred in only 32 (35.5%) of the 90 strains. Only 14 (21.8%) of the 64 phenotypically identified A. hydrophila strains belonged to this species. However, coincident results were obtained in 88% (15/17) of the genetically identified A. salmonicida strains. Phenotypic tests were re-evaluated on the 90 genetically characterized Aeromonas strains and there were contradictions in the species A. sobria for a number of previously published species-specific traits. After genetic identification, the prevailing species were A. sobria, A. salmonicida, A. bestiarum, A. hydrophila, A. piscicola and A. media but we could also identify a new isolate of the recently described species A. tecta. This work emphasizes the need to rely on the 16S rDNA-RFLP method and sequencing of housekeeping genes such as rpoD for the correct identification of Aeromonas strains.

Validation of a partialrpoBgene sequence as a tool for phylogenetic identification of aeromonads isolated from environmental sources

A collection of 50 aeromonads isolated from environmental sources were studied, together with all known Aeromonas nomenspecies, by phenotypic, amplified 16S rDNA restriction analysis (16S rDNA RFLP) and by partial sequence alignment of both 16S rDNA and rpoB genes. Although most of the type strain showed a unique phenotypic pattern, a database constructed on type strain phenotype allowed the identification of only 24% of the isolates. Analysis of 16S rDNA RFLP and the rpoB sequence were almost concordant in identifying environmental isolates at the species level, except for strains belonging to Aeromonas caviae spp., which were not differentiated from Aeromonas aquariorum, nor Aeromonas hydrophila susbsp. dhakensis by 16S rDNA RFLP. In addition, rpoB gene analysis clustered separately a group of isolates found in snails within the A. hydrophila species. In contrast to 16S rDNA RFLP and rpoB, the partial 16S rDNA sequence analysis was weak in resolving species identity. Part of these results, phenotypic and phylogenetic data, showed that Aeromonas molluscorum and Aeromonas sharmana are distant from all other Aeromonas species and that the type species of A. hydrophila subsp. anaerogenes is similar to A. caviae and should be considered synonymous.

Molecular diversity of a native mesorhizobial population of nodulating chickpea (Cicer arietinum L.) in Indian soils

Chickpea plants with nodules were collected from 32 different farmers' fields of eight districts of Haryana state. In total, 137 mesorhizobial isolations were made from these nodules and authenticated. Finally, 50 mesorhizobia were selected based on nodulation test, growth characteristics, and site of sampling. The molecular diversity of the mesorhizobial population was assessed by PCR-amplified ERIC profiles as well as RFLP of 16S rDNA. Considerable molecular diversity in Haryana soils was observed. Chickpea rhizobia were grouped into six different clusters at the 70% similarity level by both methodologies, but clustering of the strains was different. Considering that each cluster represented a mesorhizobial genotype, Haryana soils showed a high richness index (0.46), and RFLP analysis showed that the mesorhizobial genotype (MG) I was present in 38% of nodules, followed by MG III which was detected in 34% of the nodules. The distribution of different MG in Haryana soils showed that all six types of rhizobia were never present in any of the districts; however, a maximum of five types were present in the Bhiwani district. Rhizobial genotype III was invariably present in all the nodule samples tested.

Genetic Diversity of Plant Growth Promoting Rhizobacteria Isolated from Rhizospheric Soil of Wheat Under Saline Condition

In this study, a total of 130 rhizobacteria was isolated from a saline infested zone of wheat rhizosphere, and screened for plant growth promoting (PGP) traits at higher salt (NaCl) concentrations (2, 4, 6, and 8%). The results revealed that 24 rhizobacterial isolates were tolerant at 8% NaCl. Although all the 24 salt tolerable isolates produced indole-3-acetic acid (IAA), while 10 isolates solubilized phosphorus, eight produced siderophore, and six produced gibberellin. However, only three isolates showed the production of 1-aminocyclopropane-1-carboxylate (ACC) deaminase. Diversity was analyzed through 16S rDNA-RFLP, and of these isolates with three tetra cutter restriction enzymes (HaeIII, AluI, and MspI), the representative cluster groups were identified by 16S rDNA sequencing. Bacillus and Bacillus-derived genera were dominant which showed PGP attributes at 8% NaCl concentration. Out of 24 isolates, nitrogen fixing ability (nif H gene) was detected in the two isolates, SU18 (Arthrobacter sp.) and SU48.

GENETIC VARIABILITY OF STOLBUR PHYTOPLASMA IN ANNUAL CROP AND WILD PLANT SPECIES IN SOUTH MORAVIA

SUMMARY The genetic diversity of stolbur phytoplasma isolates was assessed by different molecular typing methods in a wide range of plant hosts in a limited geographical area of the Czech Republic. Plants of Apium graveolens, Capsicum annuum, Solanum lycopersicum, Solanum tuberosum, Amaranthus retroflexus, Calystegia sepium, Cirsium arvense, Convolvulus arvensis, and Datura stramonium displaying symptoms of proliferation, discoloration, and malformation of flower were collected in vegetable crop plots in the vicinity of the village of Lednice (south Moravia) and examined for the presence of phytoplasmas. Using 16S rDNA amplification and RFLP assays, stolbur phytoplasma was detected in some of the symptomatic plants. The genotype of the phytoplasma isolates detected was investigated by restriction pattern or sequence analysis of two non-ribosomal house-keeping genes (tuf and secY) and a recently characterized polymorphic gene (vmp1). According to tuf genotyping, all isolates were of the tufAY-b genotype sensu Langer and Maixner (2004), a genotype known to be associated with bindweed (C. arvensis) reservoirs. A finer isolate differentiation was obtained with vmp1 genotyping as four genetic variants carrying different vmp1 RFLP patterns were found, especially in C. arvensis. In addition, preliminary sampling in other south Moravian localities and secY sequence analyses diclosed also the presence of stolbur isolates different from those previously reported in binweed (C. arvensis) and grapevine.

Aeromonas piscicola sp. nov., isolated from diseased fish

Four Aeromonas strains (S1.2 T, EO-0505, TC1 and TI 1.1) isolated from moribund fish in Spain showed a restriction fragment length polymorphism (RFLP) pattern related to strains of Aeromonas salmonicida and Aeromonas bestiarum but their specific taxonomic position was unclear. Multilocus sequence analysis (MLSA) of housekeeping genes rpoD, gyrB, recA and dnaJ confirmed the allocation of these isolates to an unknown genetic lineage within the genus Aeromonas with A. salmonicida, A. bestiarum and Aeromonas popoffii as the phylogenetically nearest neighbours. Furthermore, a strain biochemically labelled as Aeromonas hydrophila (AH-3), showing a pattern of A. bestiarum based on 16S rDNA-RFLP, also clustered with the unknown genetic lineage. The genes rpoD and gyrB proved to be the best phylogenetic markers for differentiating these isolates from their neighbouring species. Useful phenotypic features for differentiating the novel species from other known Aeromonas species included their ability to hydrolyze elastin, produce acid from l-arabinose and salicin, and their inability to produce acid from lactose and use l-lactate as a sole carbon source. A polyphasic approach using phenotypic characterization, phylogenetic analysis of the 16S rRNA gene and of four housekeeping genes, as well as DNA–DNA hybridization studies and an analysis of the protein profiles by MALDI-TOF-MS, showed that these strains represented a novel species for which the name Aeromonas piscicola sp. nov. is proposed with isolate S1.2 T (=CECT 7443 T, =LMG 24783 T) as the type strain.

Isolation and characterization of culturable bacteria from tropical coastal waters

In this study we isolated and characterized some culturable bacteria from tropical coastal waters of Peninsular Malaysia. We obtained between 0.23 and 1.85 × 103 cfu mL–1 in the Zobell 2216E medium, and cultured 0.04% to 0.12% of total bacterial counts. Different bacterial strains were then selected by 16S rDNA RFLP using four restriction enzymes (DdeI, HhaI, RsaI, and Sau3AI), of which HhaI gave the most RFLP patterns. A total of 54 unique strains were obtained and these were identified by their 16S rDNA gene sequence. These bacterial strains could be divided into five classes: 38 strains of γ-Proteobacteria (61.1%); 3 strains of α-Proteobacteria (5.5%); 2 strains of the Cytophaga-Flavobacterium-Bacteroides group (3.7%); 3 strains of high GC, Gram-positive bacteria (5.5%); and 13 strains of low GC, Gram-positive bacteria (24.1%). These isolates have good potential for further biotechnological studies since about 56% of the isolates exhibited amylase activity, whereas 36% and 18% of the isolates had protease and lipase, respectively. Most (>70%) of the isolates also produced poly-ß-hydroxybutyrate.

Bacteria diversity in paddy field soil by 16S rDNA-RFLP analysis in Ningxia

Bacteria diversity in paddy field soil by 16S rDNA-RFLP analysis in Ningxia

A new 16S rDNA-RFLP method for the discrimination of the accepted species of Arcobacter

The genus Arcobacter that includes some species of clinical interest has been recently enlarged with the addition of several new species. The available molecular techniques for the characterization of Arcobacter spp. are unable to identify all the species included in this genus. We have developed a 16S rDNA-RFLP method able to separate the currently accepted 6 species of Arcobacter, including the 2 hybridization groups of Arcobacter cryaerophilus. The method based on the use of a pair of primers and of the endonuclease MseI has been validated using 12 reference strains (including the type strains) and 75 fresh isolates. All isolates tested produced species-specific RFLP patterns. This easy-to-perform method allows a fast and reliable recognition of the members of this complex genus.

On the identification of clinical Aeromonas by a new restriction fragment length polymorphism of 16S rDNA method

On the identification of clinical Aeromonas by a new restriction fragment length polymorphism of 16S rDNA method

A comparative study on the phylogenetic diversity of culturable actinobacteria isolated from five marine sponge species

A cultivation-based approach was employed to compare the culturable actinobacterial diversity associated with five marine sponge species (Craniella australiensis, Halichondria rugosa, Reniochalina sp., Sponge sp., and Stelletta tenuis). The phylogenetic affiliation of the actinobacterial isolates was assessed by 16S rDNA-RFLP analysis. A total of 181 actinobacterial strains were isolated using five different culture media (denoted as M1-M5). The type of medium exhibited significant effects on the number of actinobacteria recovered, with the highest number of isolates on M3 (63 isolates) and the lowest on M1 (12 isolates). The genera isolated were also different, with the recovery of three genera on M2 and M3, and only a single genus on M1. The number of actinobacteria isolated from the five sponge species was significantly different, with a count of 83, 36, 30, 17, and 15 isolates from S. tenuis, H. rugosa, Sponge sp., Reniochalina sp., and C. australiensis, respectively. M3 was the best isolation medium for recovery of actinobacteria from S. tenuis, H. rugosa, and Sponge sp., while no specific medium preference was observed for the recovery of actinobacteria from Reniochalina sp., and C. australiensis. The RFLP fingerprinting of 16S rDNA genes digested with HhaI revealed six different patterns, in which 16 representative 16S rDNAs were fully sequenced. Phylogenetic analysis indicated that 12 strains belong to the group Streptomyces, three strains belong to Pseudonocardia, and one strain belongs to Nocardia. Two strains C14 (from C. australiensis) and N13 (from Sponge sp.) have only 96.26% and 96.27% similarity to earlier published sequences, and are therefore potential candidates for new species. The highest diversity of three actinobacteria genera was obtained from Sponge sp., though the number of isolates was low. Two genera of actinobacteria, Streptomyces, and Pseudonocardia, were isolated from both S. tenuis and C. australiensis. Only the genus of Streptomyces was isolated from H. rugosa and Reniochalina sp. Sponge species have been demonstrated here to vary as sources of culturable actinobacterial diversity, and the methods for sampling such diversity presented may be useful for improved sampling of such diversity.

Occurrence and distribution of ‘Candidatus Phytoplasma trifolii’ associated with diseases of solanaceous crops in Lebanon

A survey for phytoplasma diseases in tomato and pepper fields in Lebanon was conducted during 2003 and 2004. Tomato plants with stunting, yellowing or purplish leaves, proliferation of laterals buds, hypertrophic calyxes and virescent flowers were found in 25% of the tomato fields surveyed, where they represented 2–8% of the plants. Pepper plants displaying stunting and yellowing of leaves, were found in 27% of the fields and 1–4% of the plants were affected. Phytoplasmas infecting tomato and pepper had identical 16S-rDNA RFLP profiles and sequences. A phytoplasma isolate named PTL was transmitted by dodder from a diseased tomato plant to a periwinkle (Catharanthus roseus) plant in which it induced leaf yellowing, virescence and phyllody. 16S-rDNA phylogenetic analysis classified PTL as a strain of ‘Candidatus Phytoplasma trifolii’.

Diverse bacteria isolated from root nodules of Trifolium, Crotalaria and Mimosa grown in the subtropical regions of China

To analyze the diversity and relationships of rhizobia in the subtropical and tropical zones of China, we characterized 67 bacterial strains isolated from root nodules of five legume species in the genera Trifolium, Crotalaria and Mimosa . PCR-amplified 16S rDNA RFLP, numerical taxonomy, SDS-PAGE of whole cell proteins, sequencing of 16S rDNA and DNA-DNA hybridization grouped the isolates into 17 lineages belonging to Bradyrhizobium, Mesorhizobium, Rhizobium, Sinorhizobium and Burkholderia, as well as a non-symbiotic group of Agrobacterium. The Rhizobium group contained twenty strains isolated from Mimosa pudica, Crotalaria pallida and two species of Trifolium. Fifteen of them were R. leguminosarum. Twenty-one strains isolated from four species of Trifolium, Crotalaria and Mimosa were classified into five groups of Bradyrhizobium, including B. japonicum. Agrobacterium group composed of 20 isolates from Mimosa pudica, C. pallida and Trifolium fragiferum. In addition, several strains of Sinorhizobium and Mesorhizobium associated with Trifolium and Burkholderia associated with Mimosa pudica were also identified. The predominance of Bradyrhizobium in the nodules of Trifolium was a novel finding and it demonstrated that the nodule microsymbionts might be selected by both the geographic factors and the legume hosts.

Genetic diversity of rhizobia associated with Vicia faba in three ecological regions of China

Great genetic diversity was revealed among 75 rhizobal isolates associated with Vicia faba grown in Chinese fields with AFLP, ARDRA, 16S rDNA sequencing, DNA-DNA hybridization, BOX-PCR and RFLP of PCR-amplified nodD and nodC. Most of the isolates were Rhizobium leguminosarum, and six isolates belonged to an unnamed Rhizobium species. In the homogeneity analysis, the isolates were grouped into three clusters corresponding to (1) autumn sowing (subtropical) region where the winter ecotype of V. faba was cultivated, (2) spring sowing (temperate) region where the spring ecotype was grown, and (3) Yunnan province where the intermediate ecotype was sown either in spring or in autumn. Nonrandom associations were found among the nod genotypes, genomic types and ecological regions, indicating an epidemic symbiotic gene transfer pattern among different genomic backgrounds within an ecological region and a relatively limited transfer pattern between different regions. Conclusively, the present results suggested an endemic population structure of V. faba rhizobia in Chinese fields and demonstrated a novel rhizobium associated with faba bean.

Genotypic diversity of rhizobia isolated fromRetama raetam in arid regions of Tunisia

Thirty-five isolates of rhizobia were picked up fromRetama raetam root nodules growing in arid lands of Tunisia. A genotypic approach including PCR-RFLP of 16S rDNA and 16S–23S rDNA was used to study their diversity and their relationships with te n reference strains of rhizobia. Four distinct clusters were defined in numerical analysis of RFLP of 16S rDNA, which related at the 78% similarity level to distinct species ofMesorhizobium, Agrobacterium, Rhizobium andSinorhizobium. More greater variability was detected by analysis of Intergenic Spacers 16S–23S rDNA. The results from both methods used in this study, showed that among all newsolates only three were found to be closely related to species of the genusSinorhizobium.

Conventional Identification of Streptococcus uberis Isolated from Bovine Mastitis in Argentinean Dairy Herds

The objective of this study was to evaluate a conventional scheme for identifying Streptococcus uberis strains isolated from bovine mastitis. Seventy-five gram-positive, catalase-negative cocci were collected from cows with mastitis from 19 dairy herds located in the east-central region of Argentina. Five American Type Culture Collection strains and bovine isolates were identified by the API 20 Strep system and by restriction fragment length polymorphism analysis of 16S rDNA. A conventional scheme based on 11 biochemical tests was selected for identification of Strep. uberis strains: the Christie–Atkins–Munch-Petersen reaction; hydrolysis of Arg, esculin, and sodium hippurate; growth in inulin, mannitol, raffinose, salicin, and sorbitol; and growth at 45°C and in 6.5% NaCl. Reference strains and 25 bovine isolates were classified accurately to the species level by the conventional scheme in a blind assay. Each reference strain and each bovine isolate were identified as belonging to the same species following these 3 methods. The remaining 50 isolates identified as Strep. uberis by the API 20 Strep system and 16S rDNA RFLP were assayed by the conventional scheme. This scheme correctly identified 47 (94%) of 50 isolates as Strep. uberis by comparing their biochemical profile with that of the reference strain. Three (6%) of the 50 isolates were classified as Strep. uberis by the API 20 Strep system and by 16S rDNA RFLP and were identified as Enterococcus faecalis by the conventional scheme. Thirty percent of the Strep. uberis strains showed biochemical profiles identical to the Strep. uberis American Type Culture Collection 27958 strain. Seventy percent of the Strep. uberis strains demonstrated variability compared with the reference strain, resulting in 19 different biochemical profiles. The conventional scheme proposed in this study resulted in a relatively low number of misidentifications and could biochemically identify not only typical, but also atypical Strep. uberis strains. This conventional scheme can be considered an adequate method for identifying Strep. uberis strains isolated from bovine mastitis because of its affordable cost in developing countries, and it may contribute to determining the frequency of isolation of Strep. uberis strains in Argentinean dairy herds.

First report of a phytoplasma associated with abnormal proliferation of cladodes in cactus pear (Opuntia ficus‐indica) in Italy

While Mexico is the main cactus pear-producing country, Italy is the most important producer in the Mediterranean basin. No phytoplasma disease of cactus pear has been reported, despite previous detection of phytoplasmas in related species such as Opuntia tuna (Casper et al ., 1970) and Opuntia linguiformis showing witches’ broom symptoms (Cai et al ., 2002). In 2003, three cactus pear plants showing abnormal growth were observed in the DISTEF collection. The plants showed severe proliferation of cladodes with lack of flowers, fruits and spine production. Viral particles were not observed by transmission electron microscopy in sap from any of the affected plants. Total DNA was extracted from the affected plants and from two symptomless cactus pears as described by Cai et al . (2002). This was used as template for phytoplasma-specific 16S rDNA PCR amplification using one of three universal primer pairs: P1/P7, R16f2/r2 (Lee et al ., 2000) or fU5/rU3 (Lorenz et al. , 1995). DNA preparations from phytoplasma reference strains maintained in periwinkle were used as positive controls. To produce enough amplicon for further characterization by RFLP analysis, nested primer pair R16f2/r2 was used to reamplify P1/P7-primed rDNA products. RFLP analysis was performed with restriction enzymes Alu I, Hha I, Hpa II, Mse I and Taq I. Phytoplasma-specific PCR products were amplified from all three plants showing symptoms with P1/P7 and fU5/rU3 primers by direct PCR, and with R16f2/r2 primers by nested PCR, but symptomless plants were always negative in all three PCR assays. The RFLP patterns obtained from analysis of rDNA amplicons from the former samples were identical to the pattern obtained for reference strain faba bean phyllody phytoplasma, a member of the 16S rDNA RFLP subgroup 16SrII-C. This is the first report of a phytoplasma infecting O. ficus-indica .

A molecular comparison of culturable aerobic heterotrophic bacteria and 16S rDNA clones derived from a deep subsurface sediment

Culture-based techniques have traditionally been the primary tools utilized for studying the microbiology of terrestrial subsurface environments. Recently, nucleic acid-based methods have been employed to further characterize the microbial diversity in subsurface sediments and rocks, but the results have not been related to individual bacteria cultivated from the same environment. Restriction fragment length profiles of 16S rRNA genes derived from bulk community DNA or bacterial isolates were compared to determine the efficacy of PCR-based methods for studying microbial diversity and phylogeny in a deep (188 m) subsurface environment. The phylogenetic relatedness between 16S rRNA genes of enrichment cultures and individual clones was also determined through DNA sequence analysis of 16S rRNA genes. Restriction fragment length profiles from PCR clone libraries accounted for 64% of recovered isolates and 55% of the estimated culturable diversity based upon their 16S rDNA RFLP signatures. DNA sequence comparisons between the 16S rDNA of the most commonly occurring isolates and clones confirmed that similar DNA sequences were contained within the RFLP groups used to categorize the isolates and clones. For 7 of 8 RFLP groups for which DNA sequences were obtained, nearest neighbor assignments corresponded at the genus level but suggested that 16S rDNA sequences from multiple genera were contained within single RFLP profiles. Phylogenetic analysis of 16S rRNA sequences supported the nearest neighbor inferences and indicated that 16S rDNA clones derived from bulk sediment were specifically related to isolates recovered on enrichment plates. This study has shown that a majority of the cultivated aerobic heterotrophic bacteria in a subsurface sediment could be described by 16S rDNA clones obtained from directly extracted DNA, but that PCR-based methods cannot account for all organisms from a given sample. Consequently, a more comprehensive assessment of microbial diversity in subsurface (and probably other) environments can be obtained by using a combination of culture- and molecular-based techniques than by using either method alone.

Restriction fragment length polymorphisms of 16S rDNA and of whole rRNA genes (ribotyping) of Streptococcus iniae strains from the United States and Israel.

Streptococcus iniae (junior synonym S. shiloi) isolated from tilapia and trout in Israel and in the United States were subtyped by restriction length polymorphism (RFLP) based on PCR amplified 16S rDNA and by ribotyping. 16S rDNA RFLP discriminated between S. iniae and other fish pathogens but not between S. iniae strains. HindIII and EcoRI ribotypes of S. iniae discriminated American from Israeli strains rejecting the possibility of an epidemiological link between S. iniae infections in the two countries. Israeli strains isolated from tilapia and trout could not be completely differentiated. The S. iniae ATCC 29178T (T = Type strain) strain, isolated from a freshwater dolphin belonged to a ribotype different from those of all the fish isolates.

Phylogeny and nodulation signal molecule of rhizobial populations able to nodulate common beans—other than the predominant species Rhizobium etli—present in soils from the northwest of Argentina

We examined the bean rhizobia community other than the predominant species Rhizobium etli present in soils of a region that is part of the range occupied by the host in Northwest Argentina, which showed Rep and 16S rDNA RFLP polymorphism. Two populations represented by isolates T29N3L and T44N22P were found to be distinct chromosomal genotypes and closely related to species Rhizobium tropici and Agrobacterium rhizogenes. Their symbiotic genes were analyzed and found to cluster with those from R. tropici as well as with rhizobia isolated from leguminous trees. Three nodulation metabolites produced by T44N22P were detected which are tetra- and pentameric chitocompounds, N-methylated, O-carbamoylated, and N-substituted either by a C 18:0 or C 18:1 acyl chain at their non-reducing end, and all them sulphated at the reducing end. Isolates T29N3L and T44N22P exhibited broad host range but unlike T29N3L, only T44N22P was able to efficiently nodulate Medicago truncatula.