Acinetobacter strains IH9 and OCI1, two rhizospheric phosphate solubilizing isolates able to promote plant growth,constitute a new genomovar of Acinetobacter calcoaceticus

During a screening of phosphate solubilizing bacteria (PSB) in agricultural soils, two strains, IH9 and OCI1, were isolated from the rhizosphere of grasses in Spain, and they showed a high ability to solubilize phosphate in vitro. Inoculation experiments in chickpea and barley were conducted with both strains and the results demonstrated their ability to promote plant growth. The 16S rRNA gene sequences of these strains were nearly identical to each other and to those of Acinetobacter calcoaceticus DSM 30006^T, as well as the strain CIP 70.29 representing genomospecies 3. Their phenotypic characteristics also coincided with those of strains forming the A. calcoaceticus–baumannii complex. They differed from A. calcoaceticus in the utilization of l-tartrate as a carbon source and from genomospecies 3 in the use of d-asparagine as a carbon source. The 16S–23S intergenic spacer (ITS) sequences of the two isolates showed nearly 98% identities to those of A. calcoaceticus, confirming that they belong to this phylogenetic group. However, the isolates appeared as a separate branch from the A. calcoaceticus sequences, indicating their molecular separation from other A. calcoaceticus strains. The analysis of three housekeeping genes, recA, rpoD and gyrB, confirmed that IH9 and OCI1 form a distinct lineage within A. calcoaceticus. These results were congruent with those from DNA–DNA hybridization, indicating that strains IH9 and OCI1 constitute a new genomovar for which we propose the name A. calcoaceticus genomovar rhizosphaerae.     

Characterization of native fluorescent Pseudomonas isolates associated with alfalfa roots in Uruguayan agroecosystems

Establishment of alfalfa crops is continuously threatened by seedling diseases caused by soilborne pathogens. The use of plant beneficial bacteria as inoculants is a feasible and environmentally friendly means to control soil pathogens. Identifying effective plant growth-promoting strains to use on local crops under local environmental conditions requires the screening of large collections of native isolates. A collection of 738 rhizospheric fluorescent Pseudomonas isolates was obtained from alfalfa plants from three agroecological regions representative of Uruguayan agricultural systems. The isolates were evaluated for in vitro pathogen inhibition, biosurfactant production, phosphate solubilization and the presence of genes involved in antibiotic synthesis. Isolates with strong in vitro antagonistic activity toward Pythium debaryanum were more abundant in alfalfa plants established in a previously natural ecosystem while biosurfactant producers were less abundant in that location. A subset of isolates was selected for genotypic characterization by rep-PCR using BOX primers. Twenty-four genotypes were defined, sixteen from a single geographical origin and eight composed of isolates from multiple origins. Genotypic profiles correlated well with phenotypic traits. A subset of isolates was assayed to determine their ability to protect alfalfa against P. debaryanum damping-off and to promote plant growth. Five native Pseudomonas isolates showed significant effects on alfalfa by increasing plant biomass and/or protecting from pathogen infection. Plant growth promoting isolates from each location were genotypically similar. Our work contributes to the knowledge of the phenotypic and genotypic diversity of rhizospheric fluorescent pseudomonads of forage legumes and the frequency of plant growth promoting traits associated with this group of bacteria in different agricultural systems.     

Genotypic and Phenotypic Diversity of phlD-Containing Pseudomonas Strains Isolated from the Rhizosphere of Wheat

Production of 2,4-diacetylphloroglucinol (2,4-DAPG) in the rhizosphere by strains of fluorescent Pseudomonas spp. results in the suppression of root diseases caused by certain fungal plant pathogens. In this study, fluorescent Pseudomonas strains containing phlD, which is directly involved in the biosynthesis of 2,4-DAPG, were isolated from the rhizosphere of wheat grown in soils from wheat-growing regions of the United States and The Netherlands. To assess the genotypic and phenotypic diversity present in this collection, 138 isolates were compared to 4 previously described 2,4-DAPG producers. Thirteen distinct genotypes, one of which represented over 30% of the isolates, were differentiated by whole-cell BOX-PCR. Representatives of this group were isolated from eight different soils taken from four different geographic locations. ERIC-PCR gave similar results overall, differentiating 15 distinct genotypes among all of the isolates. In most cases, a single genotype predominated among isolates obtained from each soil. Thirty isolates, representing all of the distinct genotypes and geographic locations, were further characterized. Restriction analysis of amplified 16S rRNA gene sequences revealed only three distinct phylogenetic groups, one of which accounted for 87% of the isolates. Phenotypic analyses based on carbon source utilization profiles revealed that all of the strains utilized 49 substrates and were unable to grow on 12 others. Individually, strains could utilize about two-thirds of the 95 substrates present in Biolog SF-N plates. Multivariate analyses of utilization profiles revealed phenotypic groupings consistent with those defined by the genotypic analyses.     

Gibbsiella greigii sp. nov., a novel species associated with oak decline in the USA

In 2010, cream-coloured, Gram-negative staining, facultatively anaerobic enterobacteria were isolated from a single black oak tree (Quercus kelloggii) exhibiting decline symptoms in southern California, USA. These 12 isolates were tentatively identified as Gibbsiella quercinecans based on partial gyrB sequencing. Closer examination of the strains using multilocus sequence analysis, based on partial sequences of gyrB, rpoB, infB and atpD genes, and almost complete 16S rRNA gene sequencing suggested that the isolates belong to a novel taxon within the genus Gibbsiella with G. quercinecans as their closest phylogenetic relative. DNA–DNA relatedness studies confirmed that the strains belong to a single taxon in Gibbsiella, which can be differentiated from other members of the genus by several phenotypic traits. Therefore, the name Gibbsiella greigii sp. nov. is proposed for this novel species isolated from symptomatic Q. kelloggii in the USA with FRB 224^T (=LMG 27716^T = NCPPB 4583^T) as the type strain.     

Endophytic occupation of peanut root nodules by opportunistic Gammaproteobacteria

Several bacterial isolates were recovered from surface-sterilized root nodules of Arachis hypogaea L. (peanut) plants growing in soils from Córdoba, Argentina. The 16S rDNA sequences of seven fast-growing strains were obtained and the phylogenetic analysis showed that these isolates belonged to the Phylum Proteobacteria, Class Gammaproteobacteria, and included Pseudomonas spp., Enterobacter spp., and Klebsiella spp. After storage, these strains became unable to induce nodule formation in Arachis hypogaea L. plants, but they enhanced plant yield. When the isolates were co-inoculated with an infective Bradyrhizobium strain, they were even found colonizing pre-formed nodules. Analysis of symbiotic genes showed that the nifH gene was only detected for the Klebsiella-like isolates and the nodC gene could not be amplified by PCR or be detected by Southern blotting in any of the isolates. The results obtained support the idea that these isolates are opportunistic bacteria able to colonize nodules induced by rhizobia.     

Diversity of rhizospheric and endophytic bacteria isolated from dried fruit of Ficus carica

There is currently an increasing demand for the characterization of endophytic bacteria isolated from different parts of plants (rhizosphere, roots, fruit, leaf) in order to improve the organic agriculture practices. The current research was performed to identify both rhizospheric bacteria isolated from the rhizosphere of Ficus carica in three different sites in the north of Tunisia and endophytic bacteria isolated from dried figs. We then characterized them for a diversity of plant growth-promoting (PGP) activities. A collection of 120 isolates from rhizospheric soil and 9 isolates from dried figs was obtained and purified. 16SrDNA gene amplification of rhizospheric bacteria revealed significant diversity and allowed for the assigning of the isolates to 6 phyla: Gammaproteobacteria, Alphaproteobacteria, Betaproteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes. Representative strains of the collection (90 strains) were tested for numerous PGP activities and resistance to abiotic stresses. The most common PGP trait for all bacteria from the three regions was siderophore production (62%), followed by cellulase (38%), then protease activity (37%), then by lipases activity (17%) and lastly by solubilization of phosphates (9%). Twenty -three strains that showed most PGP traits were selected, 8 strains presented 12 or more, and 15 strains displayed between 7 and 11 of 17 PGP activities. The majority of the isolates manifested a possible adaptation to abiotic stress and unfavorable environments. PCR-DGGE analysis of soil rhizosphere of the three sites allowed also for the acquisition of a Cluster analysis of rhizospheric bacterial communities. Our current study identified and characterized for the first time in Tunisia rhizospheric and endophytic bacteria from dried fruit of Ficus carica.     

Isolation and characterization of root-associated bacteria from agricultural crops in the Kavango region of Namibia

Root-associated bacteria can have beneficial effects on their host plants. Microbial products can promote and stimulate plant growth or lead to bioprotection against pathogens. This study aimed to isolate putatively beneficial bacteria from traditional cereals grown by subsistence farmers in the Kavango of Namibia. Bacteria were isolated from surface-sterilized roots of Pennisetum glaucum, Sorghum bicolor, and Zea mays, and subjected to phenotypic and phylogenetic analyses. A total of 44 root-associated bacterial strains were isolated. From 33 distinct isolates, 22 belonged to Firmicutes and Actinobacteria, while 11 were Proteobacteria. Eleven novel phylotypes were among the isolates. Features known to contribute to plant growth-promotion and biocontrol were tested in vitro and revealed promising candidates with multiple beneficial characteristics. This is the first report on the characterization of native isolates associated with important agriculture crops in the Kavango region of Namibia. Such isolates have the potential for application as inoculants adapted to poor soils and local crops. Desiccation-tolerant or sporulating Gram-positive bacteria are of particular interest for this region, which is characterized by a long dry season.     

Intraspecific variation in Burkholderia caledonica: Europe vs. Africa and soil vs. endophytic isolates

The best-known interaction between bacteria and plants is the Rhizobium-legume symbiosis, but other bacteria–plant interactions exist, such as between Burkholderia and Rubiaceae (coffee family). A number of bacterial endophytes in Rubiaceae are closely related to the soil bacterium Burkholderia caledonica. This intriguing observation is explored by investigating isolates from different geographic regions (Western Europe vs. sub-Saharan Africa) and from different niches (free-living bacteria in soil vs. endophytic bacteria in host plants). The multilocus sequence analysis shows five clades, of which clade 1 with two basal isolates deviates from the rest and is therefore not considered further. All other isolates belong to the species B. caledonica, but two genetically different groups are identified. Group A holds only European isolates and group B holds isolates from Africa, with the exception of one European isolate. Although the European and African isolates are considered one species, some degree of genetic differentiation is evident. Endophytic isolates of B. caledonica are found in certain members of African Rubiaceae, but only in group B. Within this group, the endophytes cannot be distinguished from the soil isolates, which indicates a possible exchange of bacteria between soil and host plant.     

Prey Range Characterization, Ribotyping, and Diversity of Soil and Rhizosphere Bdellovibrio spp. Isolated on Phytopathogenic Bacteria

Thirty new Bdellovibrio strains were isolated from an agricultural soil and from the rhizosphere of plants grown in that soil. Using a combined molecular and culture-based approach, we found that the soil bdellovibrios included subpopulations of organisms that differed from rhizosphere bdellovibrios. Thirteen soil and seven common bean rhizosphere Bdellovibrio strains were isolated when Pseudomonas corrugata was used as prey; seven and two soil strains were isolated when Erwinia carotovora subsp. carotovora and Agrobacterium tumefaciens, respectively, were used as prey; and one tomato rhizosphere strain was isolated when A. tumefaciens was used as prey. In soil and in the rhizosphere, depending on the prey cells used, the concentrations of bdellovibrios were between 3 × 10² to 6 × 10³ and 2.8 × 10² to 2.3 × 10⁴ PFU g⁻¹. A prey range analysis of five soil and rhizosphere Bdellovibrio isolates performed with 22 substrate species, most of which were plant-pathogenic and plant growth-enhancing bacteria, revealed unique utilization patterns and differences between closely related prey cells. An approximately 830-bp fragment of the 16S rRNA genes of all of the Bdellovibrio strains used was obtained by PCR amplification by using a Bdellovibrio-specific primer combination. Soil and common bean rhizosphere strains produced two and one restriction patterns for this PCR product, respectively. The 16S rRNA genes of three soil isolates and three root-associated isolates were sequenced. One soil isolate belonged to the Bdellovibrio stolpii-Bdellovibrio starrii clade, while all of the other isolates clustered with Bdellovibrio bacteriovorus and formed two distantly related, heterogeneous groups.     

The effect of plant growth-promoting rhizobacteria on the growth of rice (Oryza sativa L.) cropped in southern Brazilian fields

Background and Aims

Several strains of rhizobacteria may be found in the rhizospheric soil, on the root surface or in association with rice plants. These bacteria are able to colonize plant root systems and promote plant growth and crop yield through a variety of mechanisms. The objectives of this study were to isolate, identify, and characterize putative plant growth-promoting rhizobacteria (PGPR) associated with rice cropped in different areas of southern Brazil.

Methods

Bacterial strains were selectively isolated based on their growth on three selective semi-solid nitrogen-free media. Bacteria were identified at the genus level by PCR-RFLP 16S rRNA gene analysis and partial sequencing methodologies. Bacterial isolates were evaluated for their ability to produce indolic compounds and siderophores and to solubilize phosphate. In vitro biological nitrogen fixation and the ability to produce 1-aminocyclopropane-1-carboxylate deaminase were evaluated for each bacterial isolate used in the inoculation experiments.

Results

In total, 336 bacterial strains were isolated representing 31 different bacterial genera. Strains belonging to the genera Agrobacterium, Burkholderia, Enterobacter, and Pseudomonas were the most prominent isolates. Siderophore and indolic compounds producers were widely found among isolates, but 101 isolates were able to solubilize phosphate. Under gnotobiotic conditions, eight isolates were able to stimulate the growth of rice plants. Five of these eight isolates were also field tested in rice plants subjected to different nitrogen fertilization rates.

Conclusions

The results showed that the condition of half-fertilization plus separate inoculation with the isolates AC32 (Herbaspirillum sp.), AG15 (Burkholderia sp.), CA21 (Pseudacidovorax sp.), and UR51 (Azospirillum sp.) achieved rice growth similar to those achieved by full-fertilization without inoculation, thus highlighting the potential of these strains for formulating new bioinoculants for rice crops.     

Pseudomonas asturiensis sp. nov., isolated from soybean and weeds

Five strains of gram negative bacteria, isolated from soybean (LPPA 221^T, 222 and 223) and weeds (LPPA 816 and 1442), were analyzed by a polyphasic approach. The isolates showed variation in their phenotypic traits and were placed in the Pseudomonas fluorescens lineage, based on 16S rRNA gene sequence phylogeny, as a single but well separated cluster. MLSA analysis based on gyrB and rpoD sequences clustered the strains in a single branch in the Pseudomonas syringae group, and revealed P. viridiflava as closest relative. DNA–DNA hybridizations showed medium levels of DNA–DNA relatedness with the type strain of P. viridiflava (50%) and lower levels (<32%) with other type strains of the P. syringae group, supporting classification within a novel species of the genus Pseudomonas. The strains can be distinguished from species of the P. syringae group by the fatty acid C_17:0 cyclo that is present in a low amount (2.5%) and from P. viridiflava by their inability to assimilate d-tartrate and d-sorbitol, and by the formation of red colonies on TTC medium. For this new species, the name Pseudomonas asturiensis sp. nov. is proposed. The type strain is LPPA 221^T (=LMG 26898^T = CECT 8095^T).     

Isolation and characterization of fenobucarb-degrading bacteria from rice paddy soils

Forty-five fenobucarb-degrading bacteria were isolated from rice paddy soils, and their genetic and phenotypic characteristics were investigated. The isolates were able to utilize fenobucarb as a sole source of carbon and energy. Analysis of the 16S rRNA gene sequence indicated that all the isolates were related to members of the genera Sphingobium and Novosphingobium. Among 45 isolates, 21 different chromosomal DNA fingerprinting patterns were obtained. All these strains exhibited similar growth and degradation patterns on fenobucarb. 2-sec-butylphenol was identified as an intermediate during fenobucarb degradation by HPLC analysis. All of the isolates were able to degrade another carbamate insecticide, carbaryl, and 2-sec-butylphenol, but not other fenobucarb related compounds such as aldicarb and fenoxycarb. Representative strains of the different repetitive extragenic palindromic sequence PCR fingerprint types had one to six plasmids. The plasmid-cured strains lost their degradation abilities, suggesting that fenobucarb degradative genes were on their plasmid DNAs in these strains. When analyzed with PCR amplification using the primers targeting for the previously reported carbamate hydrolase genes, most of the isolates did not exhibit any positive signals for different genes involved in carbamate degradation such as mcd, cahA and cehA genes. This is the first report that microorganisms involved in the degradation of fenobucarb have been isolated and the intermediate of fenobucarb biodegradation was identified.     

Comparison of sampling techniques and different media for the enrichment and isolation of cellulolytic organisms from biogas fermenters

Biogas plants achieve its highest yield on plant biomass only with the most efficient hydrolysis of cellulose. This is driven by highly specialized hydrolytic microorganisms, which we have analyzed by investigating enrichment strategies for the isolation of cellulolytic bacteria out of a lab-scale biogas fermenter. We compared three different cultivation media as well as two different inoculation materials: Enrichment on filter paper in nylon bags (in sacco) or raw digestate. Next generation sequencing of the V3/V4 region of the bacterial 16S rRNA of metagenomic DNA from six different enrichment cultures, each in biological triplicates, revealed an average richness of 48 different OTU’s with an average evenness of 0.3 in each sample. β-Diversity of the bacterial community revealed significant differences between the two sampling techniques or the different media used. The isolation attempt of single cellulolytic organisms resulted in several clonal pure cultures. Regardless which medium or inoculation material, well-known cellulolytic key players such as Clostridium cellulosi, Herbinix hemicellulosilytica and Hungateiclostridium thermocellum were among the isolates. The inoculation material as well as the cultivation conditions are crucial to cultivate the representative cellulolytic organisms. Taking raw digestate as inoculation material and using the same material, filtered and sterilized, for supplementing media allowed to imitate the natural habitat. Pre-enrichment of cellulolytic organisms directly in their natural habitat led to significant advantages concerning high diversity and high abundance of unknown cellulolytic organisms, which is a key factor for the isolation of hitherto unknown species.     

Distribution of phenotypes among Bacillus thuringiensis strains

An extensive collection of Bacillus thuringiensis isolates from around the world were phenotypically profiled using standard biochemical tests. Six phenotypic traits occurred in 20–86% of the isolates and were useful in distinguishing isolates: production of urease (U; 20.5% of isolates), hydrolysis of esculin (E; 32.3% of isolates), acid production from salicin (A; 37.4% of isolates), acid production from sucrose (S; 34.0% of isolates), production of phospholipase C or lecithinase (L; 79.7% of isolates), and hydrolysis of starch (T; 85.8% of isolates). With the exception of acid production from salicin and hydrolysis of esculin, which were associated, the traits assorted independently. Of the 64 possible combinations of these six phenotypic characteristics, 15 combinations accounted for ca. 80% of all isolates, with the most common phenotype being TL (23.6% of isolates). Surprisingly, while the biochemical traits generally assorted independently, certain phenotypic traits associated with the parasporal crystal were correlated with certain combinations of biochemical traits. Crystals that remained attached to spores (which tended to be non-toxic to insects) were highly correlated with the phenotypes that included both L and S. Among the 15 most abundant phenotypes characterizing B. thuringiensis strains, amorphous crystals were associated with TLE, TL, T, and Ø (the absence of positive tested biochemical traits). Amorphous crystal types displayed a distinct bias toward toxicity to dipteran insects. Although all common phenotypes included B. thuringiensis isolates producing bipyramidal crystals toxic to lepidopteran insects, those with the highest abundance of these toxic crystals displayed phenotypes TLU, TLUA, TLUAE, and TLAE.     

Genotypic Diversity and Virulence Characteristics of Clinical and Environmental Vibrio vulnificus Isolates from the Baltic Sea Region

The genetic diversity of Vibrio vulnificus isolates from clinical and environmental sources originating from the Baltic Sea region was evaluated by multilocus sequence typing (MLST), and possible relationships between MLST clusters, potential genotypic and phenotypic traits associated with pathogenicity, and source of isolation were investigated. The studied traits included genotyping of polymorphic loci (16S rRNA, vcg, and pilF), presence/absence of potential virulence genes, including nanA, nab, and genes of pathogenicity regions, metabolic features, hemolytic activity, resistance to human serum, and cytotoxicity to human intestinal cells. MLST generated 35 (27 new) sequence types and divided the 53 isolates (including four reference strains) into two main clusters, with cluster I containing biotype 1 and 2 isolates of mainly environmental origin and cluster II containing biotype 1 isolates of mainly clinical origin. Cluster II isolates were further subdivided into two branches. Branch IIB included isolates from recent cases of wound infections that were acquired at the German Baltic Sea coastline between 2010 and 2011 and isolates from seawater samples of the same regions isolated between 1994 and 2010. Comparing the MLST data with the results of genotyping and phenotyping showed that strains of MLST cluster II possess a number of additional pathogenicity-associated traits compared to cluster I strains. Rapid microbiological methods such as matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry combined with typing of selected virulence-associated traits (e.g., serum resistance, mannitol fermentation, nanA, and pathogenicity region XII) could be used for risk assessment purposes regarding V. vulnificus strains isolated from the Baltic Sea region.     

Comparison of PCR-RFLP and PFGE for determining the clonality of Brucella isolates from human and livestock specimens

Brucellosis is an important zoonotic disease caused by different species of genus Brucella that are pathogenic for humans and a variety of animals. Accurate detection of Brucella spp. infection is important for control of disease. The aim of this study was to comparison of molecular genotyping of Brucella strains by Pulsed-field gel electrophoresis (PFGE) and polymerase chain reaction -Restriction Fragment Length Polymorphism (PCR-RFLP) techniques.Twenty- seven Brucella spp. were isolated from human and animal samples. The isolates identified by conventional microbiological methods and confirmed using PCR for amplification of omp2a gene. Molecular typing of Brucella strains carried out by PCR-RFLP after PstI and PFGE of chromosomal DNA after XbaI enzyme digestion. The omp2a gene PCR Products with different patterns of PCR-RFLP were sequenced.The omp2a gene amplification of all human and animal Brucella isolates were positive for 1100 bp fragment. By PCR-RFLP analysis two genotypes/patterns for human isolates and four genotypes for animal isolates were obtained. In PFGE analysis totally, 7 common clones/clusters and 3 single clones were obtained.The results of this study showed the PFGE method is the more reliable and useful assay for molecular typing of Brucella strains and is more preferred to PCR-RFLP in determination of genetic similarity among human and animal Brucella isolates. The presented data showed PCR-RFLP analysis was not able to differentiate between B. melitensis biovars and vaccine strain.     

Definition of a novel symbiovar (sv. retamae) within Bradyrhizobium retamae sp. nov., nodulating Retama sphaerocarpa and Retama monosperma

In this paper we analyze through a polyphasic approach several Bradyrhizobium strains isolated in Spain and Morocco from root nodules of Retama sphaerocarpa and Retama monosperma. All the strains have identical 16S rRNA genes and their closest relative species is Bradyrhizobium lablabi CCBAU 23086^T, with 99.41% identity with respect to the strain Ro19^T. Despite the closeness of the 16S rRNA genes, the housekeeping genes recA, atpD and glnII were divergent in Ro19^T and B. lablabi CCBAU 23086^T, with identity values of 95.71%, 93.75% and 93.11%, respectively. These differences were congruent with DNA–DNA hybridization analysis that revealed an average of 35% relatedness between the novel species and B. lablabi CCBAU 23086^T. Also, differential phenotypic characteristics of the new species were found with respect to the already described species of Bradyrhizobium. Based on the genotypic and phenotypic data obtained in this study, we propose to classify the group of strains isolated from R. sphaerocarpa and R. monosperma as a novel species named Bradyrhizobium retamae sp. nov. (type strain Ro19^T = LMG 27393^T = CECT 8261^T). The analysis of symbiotic genes revealed that some of these strains constitute a new symbiovar within genus Bradyrhizobium for which we propose the name “retamae”, that mainly contains nodulating strains isolated from Retama species in different continents.     

Multilocus sequence analyses reveal several unnamed Mesorhizobium genospecies nodulating Acacia species and Sesbania sesban trees in Southern regions of Ethiopia

Leguminous trees play an important role in agroforestry in Ethiopia, but studies of their rhizobial symbionts are scarce. In earlier studies, we surveyed natural nodulation of native leguminous trees growing in different agro-ecological zones in Southern Ethiopia, isolated 400 rhizobia, and characterized them based on different phenotypic and genotypic methods. In the present study we characterized 18 strains belonging to the genus Mesorhizobium, isolated from nodules of Acacia abyssinica, A. senegal, A. tortilis and Sesbania sesban. Phylogenetic analysis of nearly full-length 16S rRNA gene grouped the test strains into three distinct clades separated from all currently recognized Mesorhizobium species. Three divergent strains formed separate branches while the other 15 strains formed three distinct groups, genospecies I-III. Grouping of the isolates under study based on the house-keeping genes recA, gyrB, rpoB and gltA were consistent and in agreement with that of 16S rRNA. Similarly phylogenetic relationships based on the symbiosis-related genes nodC, nodA and nifH were generally similar to those shown by the core genes, suggesting that these Acacia and Sesbania symbionts have a long history of separate evolution within Mesorhizobium. Cross inoculation experiments demonstrated a large variation in the ability of the test strains to elicit effective nodules. The Sesbania isolates, occupying a distinct clade in the nodC phylogenetic tree, formed effective nodules only with this host legume. The study strongly suggests that this collection of Mesorhizobium strains comprises several new species, and also indicates the role of the symbiotic genes in determining the host range of these bacteria.     

Biochemical genetic relationship of Thailand and Hawaii isolates of Parastrongylus cantonensis (Nematoda: Angiostrongylidae)

The genetic relationship of the Thailand and Hawaii isolates (strains) of the rat lungworm Parastrongylus (=Angiostrongylus) cantonensis was investigated by gene–enzyme systems using vertical polyacrylamide slab gel electrophoresis. Six gene–enzyme systems were successfully determined, with each being represented by two presumptive loci. Glucose phosphate dehydrogenase, glucose phosphate isomerase, lactate dehydrogenase, malate dehydrogenase and malic enzyme were monomorphic at both loci, and the respective bands exhibited similar mobility in both isolates implying absence of genetic variation.     

Multiresistance of Staphylococcus xylosus and Staphylococcus equorum from Slovak Bryndza cheese

Staphylococcus xylosus, Staphylococcus equorum, and Staphylococcus epidermidis strains were isolated from Bryndza cheese and identified using PCR method. The antimicrobial susceptibility of these strains was assessed using disc diffusion method and broth microdilution method. The highest percentage of resistance was detected for ampicillin and oxacillin, and in contrary, isolates were susceptible or intermediate resistant to ciprofloxacin and chloramphenicol. Fourteen of the S. xylosus isolates (45 %) and eleven of the S. equorum isolates (41 %) exhibited multidrug resistance. None of the S. epidermidis isolate was multiresistant. The phenotypic resistance to oxacillin was verified by PCR amplification of the gene mecA.