16S rDNA Gene Sequence Research Articles

Morphological, phytochemical and genetic diversity of Ziziphus spina – Christi (L) Des. in South and Southeastern of Iran

Morphological and genetic variations of the Iranian Christ’s-thorn (Ziziphus spina-christi (L.) Desf., Rhamnaceae) give us a knowing perspective to investigate and select elite germplasms for breeding and conservation purposes. In this study, Christ’s-thorn organs and parts of 101 genotypes were sampled from 12 habitats in the South of Iran. In total, 32 qualitative and quantitative characteristics were measured. Analysis of variance was carried out based on completely randomized design and this revealed significant differences for most variables, indicating a large-scale diversity among the genotypes. For instance, fruit weight ranged 0.68–7.7g, fruit length ranged 10.68–27.45mm, seed length 7.22–17.8mm, fruit pulp 2.2–11.4mm and leaf length 16–65.6mm. The genotypes were clustered into three main groups. 5′ Partial 18s rDNA from 9 regions were cloned by specific primers. The partial sequence of 18s rDNA gene in seedling of studied Iranian wild types Christ’s-thorns were shown to be identical with the Ziziphus Spina-Christi 18sr¬DNA gene. Flavonoid quercetin and saponin content were measured for 9 dominant populations. Maximum saponin content was related to the Shahdad sample (large leaves and fruits with least thorn density) with 2.6μgml−1. Maximum flavonoid quercetin content (3.66mgg−1) was related to the Jahrom sample (large leaves and fruits with least thorn density).

Description of a new soft tick species (Acari: Argasidae: Ornithodoros) associated with stream-breeding frogs (Anura: Cycloramphidae: Cycloramphus) in Brazil

In this study, we present a morphological description of immature and adult specimens of Ornithodoros saraivai n. sp., a tick associated with the frog Cycloramphus boraceiensis (Cycloramphidae) at São Sebastião island, located in the São Paulo state seaboard, Brazil. While larvae of O. saraivai are ecologically related to Ornithodoros faccinii, another soft tick associated with cycloramphids, the combination of 7 sternal pairs, 16 dorsal pairs, a pyriform dorsal plate and a partially toothed hypostome constitute unique characters of the O. saraivai larvae. One undetermined nymphal instar and adults of O. saraivai are similar to mature specimens of the Ornithodoros talaje species group; however, the O. saraivai specimens can be recognized by the presence of a robust bean-shaped spiracle with a large spiracular plate and more than two long seta in palpal article I. Identical partial sequences of the mitochondrial 16S rDNA gene confirmed the identity for all collected stages and for two cohorts of laboratory-reared larvae of O. saraivai. A Bayesian and Maximum Parsimony inferred phylogenetic trees support the position of O. saraivai in a clade with O. faccinii, suggesting the existence of an Ornithodoros lineage that evolved in association with amphibians.

Piscirickettsia salmonis infection in cultured lumpfish (Cyclopterus lumpus L.).

A Piscirickettsia salmonis infection was diagnosed in lumpfish (Cyclopterus lumpus L.) juveniles held in a marine research facility on the west coast of Ireland. The main clinical signs and pathology included marked ascites, severe multifocal liver necrosis and severe diffuse inflammation and necrosis of the exocrine pancreas and peri-pancreatic adipose tissue. Numerous Piscirickettsia-like organisms were observed by histopathology in the affected organs, and the bacterial species was characterized by molecular analysis. Sequencing of the partial 16S rDNA gene and internal transcribed spacer region showed the lumpfish sequences to be closely related to previously identified Atlantic salmon (Salmo salar L.) sequences from Ireland. To the authors' knowledge, this is the first detection of P.salmonis in lumpfish worldwide. The infection is considered potentially significant in terms of lumpfish health and biosecurity.

English

Actinomycetes have been demonstrated for plant growth-promotion, antagonistic against plant pathogens and insect pests and biofortification traits in many agriculturally important crops. The present investigation was conducted to characterize probiotic properties of actinomycete(s) isolated from pearl millet flour and batter samples. A media selective and specific were used for isolation, actinomycetes isolation agar (AIA), and the most prominent actinomycete (found abundantly in the AIA plate) was isolated and maintained on AIA slants at 4°C for further investigation. The most prominent actinomycete was characterized for traits including Gram staining, morphology (such as color, margin, size, shape, elevation, form and surface), biochemistry (such as urease, catalase, oxidase, hydrogen sulphide, nitrogen reduction, gelatin liquefaction, starch hydrolysis and carbohydrate utilization), IMViC tests (such as indole, methyl red, Voges Proskauer and citrate utilization), probiotic potentials (such as acid [pH 2, 3], bile [0.5%], NaCl [6 and 9%], phenol tolerance [0.4%]), antibiotic tolerance (such as tetracycline, streptomycin, kanamycin, chloramphenicol, ciprofloxacin, ampicillin, penicillin, erythromycin and vancomycin) and antimicrobial activities against human pathogens (such as Escherichia coli, Staphylococcus aureus and Salmonella typhi). It was possible to isolate only one probiotic actinomycete based on the properties. The sequences of 16s rDNA gene of the actinomycete was matched with Streptomyces species in BLAST analysis. The sequences of the Streptomyces spp. were submitted to NCBI and accession number obtained. This study indicated that the selected Streptomyces spp. could be used to develop new probiotic foods. Key words: Probiotics, pearl millet, Streptomyces species, product development.

Microbial communities in the functional areas of a biofilm reactor with anaerobic–aerobic process for oily wastewater treatment

Microbial communities in the functional areas of biofilm reactors with large height–diameter ratio using the anaerobic-aerobic (A/O) reflux process was investigated to treat heavy oil refinery wastewater without pretreatment. In the process, chemical oxygen demand (COD) and total nitrogen (TN) removal reached 93.2% and 82.8%, and the anaerobic biofilm reactor was responsible for 95% and 99%, respectively. Areas for hydrolysis acidification and acetic acid production, methane production, and COD recovery were obvious in the anaerobic reactor. Among all areas, area for hydrolysis acidification and acetic acid production was the key factor to improve COD removal efficiency. High throughput sequencing of 16S rDNA gene showed that the native community was mainly composed of functional groups for hydrocarbon degradation, syntrophic bacteria union body, methanogenesis, nitrification, denitrification, and sulfate reduction. The deviations between predicted values and actual COD and TN removal were less than 5% in the optimal prediction model.

Molecular evidence and additional morphological characters to distinguish Ornithodoros brodyi and Ornithodoros yumatensis (Ixodida: Argasidae) in their different developmental stages.

Ornithodoros brodyi and Ornithodoros yumatensis are two species distributed in the Americas and associated with bats and caves. Both species have similar morphology, and the diagnostic traits of adults have not been detailed or illustrated accurately. In this study, the independence of both species is validated on the basis of molecular evidence (using partial sequences of 16S rDNA gene), and the morphological differences between them (dentition of the hypostome and traits of individual mammillae) are confirmed through light and scanning electron microscopy. In addition to the above characteristics, we observed other traits that may serve to differentiate both species: dorsal setae are short and thick in O. yumatensis and are thin and moderate in size in O. brodyi. We also observed a conspicuous hood in O. brodyi, which was absent in O. yumatensis. Another characteristic observed is a line of setae, near the end of Tarsi II-IV, which in O. brodyi is formed by less than five setae and in O. yumatensis by more than five. The main morphological difference between larvae of the 2 species is the number of ventral setae [9 (4 circumanal pairs) in O. brodyi and 8 (3 circumanal pairs) in O. yumatensis]. The genetic divergence in 16S rDNA sequences between these two species ranges from 9.7 to 10.6%.

Description of a new species of Pachycheles (Decapoda, Anomura, Porcellanidae) from the southern Caribbean Sea

A new species of porcellanid crab from the southern Caribbean Sea is described. Pachycheles tuerkayi n. sp. has been confused with P. serratus (Benedict, 1901) since the 1950s because the two species are morphologically and ecologically similar and have overlapping distributions in the southern Caribbean. P. tuerkayi n. sp. is restricted to the coasts of Costa Rica, Panamá and Colombia. P. serratus ranges from Puerto Rico and the Virgin Islands to the coasts of Panamá, Colombia and Venezuela. Genetic differences based on DNA sequences of the mitochondrial 16S rDNA gene from the two species surpassed those estimated for geminate porcellanids on each side of the Isthmus of Panamá. Field observations where P. tuerkayi n. sp. and P. serratus overlap indicated that the two species come into contact when sharing the same substrate. The total number of porcellanid species in the western Atlantic rises to 50.

Bacteriomes of the corn leafhopper, Dalbulus maidis (DeLong & Wolcott, 1923) (Insecta, Hemiptera, Cicadellidae: Deltocephalinae) harbor Sulcia symbiont: molecular characterization, ultrastructure, and transovarial transmission

In this study, we surveyed the bacteriome-associated microbiota of the corn leafhopper Dalbulus maidis by means of histological, ultrastructural, and molecular analyses. Amplification and sequencing of 16S rDNA genes revealed that the endosymbiont "Candidatus Sulcia muelleri" (Phylum Bacteroidetes) resides in bacteriomes of D. maidis. Phylogenetic analysis showed that the sequence was closely allied to others found in representatives of the subfamily Deltocephalinae. We failed to amplify other sequences as "Candidatus Nasuia deltocephalinicola," a co-primary symbiont frequently associated to deltocephaline leafhoppers. In addition, a metagenetic analysis carried out in order to investigate the presence of other bacteriome-associated bacteria of D. maidis showed that the sequence of Sulcia accounted for 98.56% of all the sequences. Histological and ultrastructural observations showed that microorganisms harbored in bacteriomes (central syncytium and cytoplasm of uninucleate bacteriocytes) look like others Sulcia described in hemipteran species and they were transovarially transmitted from mother to offspring which is typical of obligate endosymbionts. The only presence of Sulcia in the bacteriomes of D. maidis was discussed.

The diversity and potential function of endophytic bacteria isolated from Kobreasia capillifolia at alpine grasslands on the Tibetan Plateau, China

A total of 50 endophytic bacterial isolates were obtained from Kobreasia capillifolia at alpine grasslands in the Eastern Qilian Mountains on the Tibetan Plateau in China. Based on the sequencing and phylogenetic analysis of 16S rDNA genes, all isolates phylogenetically related closely to Bacillus, Acinetobacter, Stenotrophomonas, Brevundimonas, Arthrobacter, Curtobacterium, Paenibacillus, Plantibacter, Promicromonospora, Serratia, and Microbacterium, among which Bacillus was the predominant genus (47.8% of the total number of endophytic isolates). These isolates possessed different biological functions. In 50 endophytic bacteria, 42 isolates produced indole acetic acid (IAA) on King medium. There were seven isolates showing potency of mineral phosphate solubilization in Pikovaskaia's (PKO) liquid medium. Seven isolates exhibited antagonistic effect against Fusarium avenaceum, Colletotrichum coccodes and Phoma foveata. This was the first report on diversity and plant growth promotion of endophytic bacteria from K. capillifolia on alpine grassland in the Eastern Qilian Mountains, Chain. It is essential for revealing the relationship among plant, microorganism, and the special environment because the potential function of endophytic bacteria made a contribution to the interactions of plants and endophytic bacteria.

English

The present study was conducted to isolate and characterize probiotic properties of bacteria isolated from flour and batter samples of sorghum and pearl millet. A total of five different selective media including plate count agar, yeast glucose chloramphenicol agar, Bifidobacterium agar, actinomycetes isolation agar and de ManRogosa and Sharpe agar were used and the most prominent bacteria (which were found abundantly in the plate) were isolated and maintained on the respective media slants at 4°C for further studies. The bacteria were characterized for various traits including Gram staining, morphology (color, size, shape, elevation, margin, form and surface), biochemistry (urease, catalase, oxidase, hydrogen sulphide, nitrogen reduction, gelatin liquefaction, starch hydrolysis and carbohydrate utilization), IMViC tests (indole, methyl red, Voges Proskauer and citrate utilization), probiotic potentials [acid (pH 2, 3), bile (0.5%), NaCl (6 and 9%)], phenol tolerance [0.4%], antibiotic tolerance (tetracycline, streptomycin, kanamycin, chloramphenicol, ciprofloxacin, ampicillin, penicillin, erythromycin and vancomycin) and antimicrobial activity against human pathogens (Escherichia coli, Staphylococcus aureus and Salmonella typhi). A total of nine probiotic bacterial isolates were short listed based on these traits. The sequences of 16s rDNA gene of the nine isolates were found matched with Bacillus subtilis (two isolates), Bacillus cereus (three isolates), Bacillus pumilus (one isolate), Bacillus amyloliquefaciens (one isolate), Sphingobacterium thalpophilum (one isolate) and Brevibacterium sp. (one isolate) in BLAST analysis. The sequences of the nine bacteria were submitted to NCBI and accession numbers obtained.  This study indicated that the selected bacteria could be exploited to develop new probiotic foods. Key words: Probiotics, prebiotics, sorghum, pearl millet, product development.

Molecular detection of spotted fever group rickettsiae in Amblyomma variegatum ticks from Benin

Spotted fever group (SFG) rickettsiae are obligate intracellular, Gram-negative bacteria transmitted by ticks and causing febrile illness in humans. Despite the presence of suitable tick vectors, the occurrence of SFG rickettsiae has never been investigated in the Republic of Benin (West Africa). In the present study, 910 Amblyomma variegatum ticks collected from 8 different locations in North Eastern Benin were tested for SFG rickettsiae. The samples were first screened for the presence of rickettsial bacteria using 16S rDNA PCR and positive samples were subsequently characterized by ompA PCR. Randomly selected samples among those positive for both assays were subjected to sequencing of 16S rDNA and ompA genes for species identification. The 16S rDNA gene was amplified in 63.4% of the samples (585/910) and the SFG rickettsia-specific ompA gene was detected in 29.4% of the samples (267/910). The prevalence of SFG rickettsiae varied according to the location, and tick gender. Sequence analyses demonstrated the presence of Rickettsia africae and/or closely related species in Benin. These findings extend the geographic distribution of R. africae and spotted fever rickettsioses in Africa. Clinicians in Benin and those treating travellers should be aware of the possibility of SFG rickettsiae infection when they are treating patients with febrile illness.

Preparation of metal-resistant immobilized sulfate reducing bacteria beads for acid mine drainage treatment

Novel immobilized sulfate-reducing bacteria (SRB) beads were prepared for the treatment of synthetic acid mine drainage (AMD) containing high concentrations of Fe, Cu, Cd and Zn using up-flow anaerobic packed-bed bioreactor. The tolerance of immobilized SRB beads to heavy metals was significantly enhanced compared with that of suspended SRB. High removal efficiencies of sulfate (61–88%) and heavy metals (>99.9%) as well as slightly alkaline effluent pH (7.3–7.8) were achieved when the bioreactor was fed with acidic influent (pH 2.7) containing high concentrations of multiple metals (Fe 469 mg/L, Cu 88 mg/L, Cd 92 mg/L and Zn 128 mg/L), which showed that the bioreactor filled with immobilized SRB beads had tolerance to AMD containing high concentrations of heavy metals. Partially decomposed maize straw was a carbon source and stabilizing agent in the initial phase of bioreactor operation but later had to be supplemented by a soluble carbon source such as sodium lactate. The microbial community in the bioreactor was characterized by denaturing gradient gel electrophoresis (DGGE) and sequencing of partial 16S rDNA genes. Synergistic interaction between SRB (Desulfovibrio desulfuricans) and co-existing fermentative bacteria could be the key factor for the utilization of complex organic substrate (maize straw) as carbon and nutrients source for sulfate reduction.

Haplotype variation in the Physa acuta group (Basommatophora): genetic diversity and distribution in Serbia

The genus Physa (= Physella) includes the most abundant and diverse freshwater gastropods native to North America. Due to their invasive nature many species occur throughout the world. The most abundant species, Physa acuta, has been introduced to Europe, Africa, Asia and Australia by human commerce and migrating birds. This species is widely distributed throughout Serbia. The aim of this study was to explore the genetic diversity of P. acuta from Serbia, and to determine the evolutionary relationships among native Physidae populations from North America, Mexico and Cuba and populations from Europe using sequences of the mitochondrial 16S rDNA gene. The ML (Maximum Likelihood) tree revealed two clades within Physidae, and two clades that correspond to the families Planorbidae and Lymnaeidae. In the Physidae clade there are two separate clades: one includes the species Physa spelunca, and the second includes samples of P. acuta. We determined three different haplotypes within specimens from Serbia. One haplotype is genetically closest to species Physa heterostrofa (synonym of P. acuta) from Philadelphia, while the other two are very close to P. acuta specimens from New Mexico. Together with other samples our findings corroborate the notion that we are dealing with one panmictic population of P. acuta and not with several separate species, despite the high genetic diversity between and among the populations. Our results indicate that in the same population in Serbia, there is high genetic distance between samples. Despite the small number of analyzed samples, our findings point to multiple introductions of P. acuta from different locations in America.

Characterization of malolactic bacteria isolated from Aosta Valley wines and evidence of psychrotrophy in some strains.

A survey on indigenous malolactic bacteria populations isolated from wines produced in 13 different wineries of Aosta Valley, the highest vine-growing area in Europe, was carried out in order to characterize the dominant strains in spontaneous malolactic fermentation (MLF) and to reveal the appearance of psychrotrophic ones. Fifty-four isolates were identify by ITS rDNA region analysis and partial sequencing of 16S rDNA gene: 80% were ascribed to Oenococcus oeni while the remaining 20% were attributed to Pediococcus damnosus species. The genetic diversity of 43 O. oeni isolates was investigated through PFGE analysis after ApaI and SfiI restriction: 28 different pulso-types were discriminated at a level of similarity of 90%. In general, the MLF was led by more than one strain simultaneously. No connection between genotype and grape variety or vine-training system or geographical site of isolation was observed. The histidine decarboxylase gene was not found in any isolate. Pediococci proved to be more resistant than oenococci to lysozyme. Three O. oeni strains (2A1, 6A2 and 11A4) were able to develop at 10°C in Petit Rouge wine. Psychrotrophy is a phenotypic trait present in O. oeni species and it may be possible to select strains for the management of MLF in cold climate territories where this biologic transformation is very difficult to control. The natural emergence of strains able to perform the MLF at 10°C in wine is a new finding, interesting because it confirms the ecological ability of O. oeni species to adapt itself to environmental conditions by strain phenotype variations. It can be also a starting point for more sustainable oenological practices, since it would be alternative to the conditioning systems of the tanks or of the wineries where they are costly in terms of investment and energy consumption.

‘Candidatus Rickettsia mendelii’, a novel basal group rickettsia detected in Ixodes ricinus ticks in the Czech Republic

A novel rickettsial sequence in the citrate synthase gltA gene indicating a novel Rickettsia species has been detected in 7 out of 4524 Ixodes ricinus ticks examined within several surveys performed in the Czech Republic from 2005 to 2009. This new Candidatus Rickettsia sp. sequence has been found in 2 nymphs feeding on wild birds (Luscinia megarhynchos and Erithacus rubecula), in a male tick from vegetation, and 4 ticks feeding on a dog (3 males, 1 female tick). Portions of the ompA, ompB, sca4, and htrA genes were not amplifiable in these samples. A maximum likelihood tree of rickettsiae based on comparisons of partial amino acid sequences of citrate synthase and nucleotide sequences of 16S rDNA genes and phylogenetic analysis revealed a basal position of the novel species in the proximity of R. bellii and R. canadensis. The novel species has been named ‘Candidatus Rickettsia mendelii’ after the founder of genetics, Gregor Mendel.

Antifungal Activity of Pseudomonas frederiksbergensis CMAA 1323 Isolated from the Antarctic Hair Grass Deschampsia antarctica

Aims: Epiphytic bacteria, isolated from Deschampsia antarctica, were screened for their potential to inhibit the plant pathogen Botrytis cinerea, the causal agent of gray mold disease of strawberry pseudofruits. This phytopathogenic fungus is more active and the disease is more serious in temperate climate where the temperatures are lower. Methodology: The approach involved the isolation and characterization of bacteria from the phyllosphere of D. antarctica and the evaluation of their antifungal activity. Results: A total of 56 bacterial strains were isolated and only one of them, identified by the sequencing of 16S rDNA gene, as Pseudomonas frederiksbergensis CMAA 1323, inhibited the mycelial growth and the conidial germination of the fungus. Its ethyl acetate extract also inhibited Original Research Article Melo et al.; BMRJ, 14(3): 1-11, 2016; Article no.BMRJ.25314 2 the mycelial growth. Analysis of the bioactive fraction by using liquid chromatography coupled to a mass spectrometry (LC-MS) revealed the presence of cyclo(Pro-Val), a compound belonging to the class of diketopiperazines (DKPs). Conclusion: These findings support the potential use of the psychrophilic bacterium P. frederiksbergensis as promising antifungal agent, and highlight the need for more studies with this bacterium in the biological control of plant pathogenic fungi.

Genetic diversity of cultured populations of giant freshwater prawn (Macrobrachium rosenbergii) in China using mtDNA COI and 16S rDNA markers

Partial sequences of mitochondrial DNA 16S rDNA and COI genes (395 bp and 498 bp respectively) were sequenced from samples of ten cultured populations of Macrobrachium rosenbergii (Giant Freshwater Prawn – GFP) in Zhejiang, Guangdong and Guangxi Provinces in China and two wild populations of GFP from Mekong River and Dongnai River in Viet Nam. Five haplotypes of 16S rDNA were identified in the 360 samples. The wild populations displayed nucleotide diversity (π) of 0.0008 and 0.0003, and genetic diversity (h) of 0.3030 and 0.1310 in the Mekong River and Dongnai River respectively. The cultured populations displayed no significant genetic diversity. COI sequences identified 17 haplotypes based on 21 polymorphic sites. At this marker, the 12 populations showed a range of h from 0.1290 to 0.6940 and π from 0.0003 to 0.0073. The largest genetic distance (Da) among the 12 populations was 0.0065 (between ZJB and BT/DN populations) and the lowest Da was 0.0003 (between GDD and GDA populations). The wild populations had higher genetic diversity than the cultured populations, but three of cultured populations from Zhejiang (ZJA, ZJB and ZJC) had π higher than wild populations, because they originated from Thailand, Bangladesh and the Mekong River in Viet Nam.

A broad-range survey of ticks from livestock in Northern Xinjiang: changes in tick distribution and the isolation of Borrelia burgdorferi sensu stricto.

BackgroundBorreliosis is highly prevalent in Xinjiang Uygur Autonomous Region, China. However, little is known about the presence of Borrelia pathogens in tick species in this region, in addition Borrelia pathogens have not been isolated from domestic animals.MethodsWe collected adult ticks from domestic animals at 19 sampling sites in 14 counties in northern Xinjiang from 2012 to 2014. Ticks were identified to species by morphology and were molecularly analysed by sequences of mitochondrial 16S rDNA gene; 4–8 ticks of each species at every sampling site were sequenced. 112 live adult ticks were selected for each species in every county, and were used to culture Borrelia pathogens; the genotypes were then determined by sequences of the 5S-23S rRNA intergenic spacer and the outer surface protein A (ospA) gene.ResultsA total of 5257 adult ticks, belonging to four genera and seven species, were collected. Compared with three decades ago, the abundance of the five common tick species during the peak ixodid tick season has changed. Certain tick species, such as Rhipicephalus turanicus (Rh. turanicus), was found at Jimusaer, Yining, Fukang, and Chabuchaer Counties for the first time. Additionally, the sequence analyses showed that the Hyalomma asiaticum (Hy. asiaticum), Haemaphysalis punctata (Ha. punctata), and Dermacentor marginatus (D. marginatus) that were collected from different sampling sites (≥3 sites) shared identical 16S rDNA sequences respectively. For the tick species that were collected from the same county, such as Hy. asiaticum from Shihezi County and Rh. turanicus from Yining County, their 16S rDNA sequences showed genetic diversity. In addition, sixteen Borrelia isolates were found in Hy. asiaticum, Ha. punctata, D. marginatus and Rh. turanicus, which infested cattle, sheep, horse and camel in Yining, Chabuchaer, Shihezi and Shawan Counties. All of the isolates were genetically identified as B. Burgdorferi sensu stricto.ConclusionsWarmer and wetter climate may have contributed to the altered distribution and abundance of the five most common ticks in northern Xinjiang. The genetic analyses showed that certain tick species, such as Hy. asiaticum or Rh. turanicus, exhibit genetic commonness or diversity. Additionally, this study is the first to isolate B. burgdorferi sensu stricto in Hy. asiaticum asiaticum, H. punctata, D. nuttalli and D. marginatus ticks from domestic animals. These ticks may transmit borreliosis among livestock.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-015-1021-0) contains supplementary material, which is available to authorized users.

Quantification of the potential biocontrol and direct plant growth promotion abilities based on multiple biological traits distinguish different groups of Pseudomonas spp. isolates

Members of the Pseudomonas genus colonize the rhizosphere of different plant species and display plant-probiotic traits. Therefore, they are interesting candidates for biological agricultural inputs to stimulate plant development and/or promote crop health. We have generated a collection of 19 isolates of pseudomonads obtained from either bulk soil or the rhizosphere of healthy individuals of major extensive crops from different plots under no-till management located in Argentina. Isolates were selected for their ability to antagonize several fungal pathogens recovered from infected soybean and maize plants. Partial sequencing of 16S rDNA, oprF and rpoB genes positioned isolates within all major pseudomonads groups. Most isolates colonized the rhizosphere of soybean and maize seedlings without affecting germination or development. Members of the Pseudomonas chlororaphis subgroup, and one isolate of the Pseudomonas putida subgroup, displayed the widest antifungal spectrum and strongest antagonistic potential. A principal component analysis based on the outcome of quantitative and qualitative tests related to biocontrol of fungal pathogens, direct plant growth promotion, and other root colonization-related traits, showed that isolates with the highest antagonistic potential and mostly of soil origin were grouped together, whereas rhizospheric isolates and those with strong flagellar-dependent motility, exoprotease production and biofilm development, were clustered in a separate group. Interestingly, quantitative comparison of newly developed biocontrol and direct growth promotion indices revealed that these two plant probiotic traits are rather associated to different set of isolates, and rarely present in the same pseudomonad strains. These indices could be employed to screen and categorize isolates by their potential to act as a biocontrol or a biofertilizer agent.

Isolation of previously uncultivable bacteria from a nickel contaminated soil using a diffusion-chamber-based approach

A cultivable portion of the bacterial community exhibits important metabolic activities in metal-contaminated soils. We aimed to isolate, identify and characterise hardly cultivable and previously uncultured bacteria (with similarity<97% to closest relatives) from Ni-contaminated soil in Slovakia by a diffusion-chamber-based approach. Screened bacteria were either directly grown on minimal solid media or passaged through four generations of diffusion chamber. The phylogeny was reconstructed using partial sequences of 16S rDNA genes from separated isolates inoculum and PCR-DGGE analysis. A total of 260 isolates obtained from both, diffusion-chamber-based approach and traditional cultivation techniques, represented 108 species belonging to 6 bacterial phyla: Actinobacteria, Bacteroidetes, Firmicutes, α-, β- and γ-Proteobacteria, and one yet unclassified species. Up to 51 discrete bands from PCR-DGGE analysis, representing different taxa were assigned to same phyla. 29.6% of the isolates and 56.9% of the bands were found as previously uncultivable bacteria, and 8.5% of the isolates and 19.6% of the bands were found with similarity<97% to their closest relatives. The phylogeny was also reconstructed with partial sequences of selected heavy-metal resistance genes. In 14 of previously uncultured β- or γ-Proteobacteria nccA-like gene products were identified. Majority of them were assigned to a system of transmembrane metal pumps. All these isolates were resistant to Ni, Co, Zn, Cu and Cd ions. The comparison of diffusion-chamber approach with traditional cultivation revealed that diffusion chamber cultivation produced more isolates of a real phylogenetic novelty and led to in vitro cultivation of poorly cultivable species.