Biogeography and Degree of Endemicity of Fluorescent Pseudomonas Strains in Soil

Fluorescent Pseudomonas strains were isolated from 38 undisturbed pristine soil samples from 10 sites on four continents. A total of 248 isolates were confirmed as Pseudomonas sensu stricto by fluorescent pigment production and group-specific 16S ribosomal DNA (rDNA) primers. These isolates were analyzed by three molecular typing methods with different levels of resolution: 16S rDNA restriction analysis (ARDRA), 16S-23S rDNA intergenic spacer-restriction fragment length polymorphism (ITS-RFLP) analysis, and repetitive extragenic palindromic PCR genomic fingerprinting with a BOX primer set (BOX-PCR). All isolates showed very similar ARDRA patterns, as expected. Some ITS-RFLP types were also found at every geographic scale, although some ITS-RFLP types were unique to the site of origin, indicating weak endemicity at this level of resolution. Using a similarity value of 0.8 or more after cluster analysis of BOX-PCR fingerprinting patterns to define the same genotypes, we identified 85 unique fluorescent Pseudomonas genotypes in our collection. There were no overlapping genotypes between sites as well as continental regions, indicating strict site endemism. The genetic distance between isolates as determined by degree of dissimilarity in BOX-PCR patterns was meaningfully correlated to the geographic distance between the isolates' sites of origin. Also, a significant positive spatial autocorrelation of the distribution of the genotypes was observed among distances of <197 km, and significant negative autocorrelation was observed between regions. Hence, strong endemicity of fluorescent Pseudomonas genotypes was observed, suggesting that these heterotrophic soil bacteria are not globally mixed.     

Biogeography and degree of endemicity of fluorescent Pseudomonas strains in soil

Fluorescent Pseudomonas strains were isolated from 38 undisturbed pristine soil samples from 10 sites on four continents. A total of 248 isolates were confirmed as Pseudomonas sensu stricto by fluorescent pigment production and group-specific 16S ribosomal DNA (rDNA) primers. These isolates were analyzed by three molecular typing methods with different levels of resolution: 16S rDNA restriction analysis (ARDRA), 16S-23S rDNA intergenic spacer-restriction fragment length polymorphism (ITS-RFLP) analysis, and repetitive extragenic palindromic PCR genomic fingerprinting with a BOX primer set (BOX-PCR). All isolates showed very similar ARDRA patterns, as expected. Some ITS-RFLP types were also found at every geographic scale, although some ITS-RFLP types were unique to the site of origin, indicating weak endemicity at this level of resolution. Using a similarity value of 0.8 or more after cluster analysis of BOX-PCR fingerprinting patterns to define the same genotypes, we identified 85 unique fluorescent Pseudomonas genotypes in our collection. There were no overlapping genotypes between sites as well as continental regions, indicating strict site endemism. The genetic distance between isolates as determined by degree of dissimilarity in BOX-PCR patterns was meaningfully correlated to the geographic distance between the isolates' sites of origin. Also, a significant positive spatial autocorrelation of the distribution of the genotypes was observed among distances of <197 km, and significant negative autocorrelation was observed between regions. Hence, strong endemicity of fluorescent Pseudomonas genotypes was observed, suggesting that these heterotrophic soil bacteria are not globally mixed.     

Pristine soils mineralize 3-chlorobenzoate and 2,4-dichlorophenoxyacetate via different microbial populations.

Biodegradation of two chlorinated aromatic compounds was found to be a common capability of the microorganisms found in the soils of undisturbed, pristine ecosystems. We used 2,4-dichlorophenoxyacetate (2,4-D) and 3-chlorobenzoate (3CBA) as enrichment substrates to compare populations of degrading bacteria from six different regions making up two ecosystems. We collected soil samples from four Mediterranean (California, central Chile, the Cape region of South Africa, and southwestern Australia) and two boreal (northern Saskatchewan and northwestern Russia) ecosystems that had no direct exposure to pesticides or to human disturbance. Between 96 and 120 samples from each of the six regions were incubated with 50 ppm of [U-14C]2,4-D or [U-14C]3CBA. Soils from all regions samples mineralized both 2,4-D and 3CBA, but 3CBA was mineralized without a lag period, while 2,4-D was generally not mineralized until the second week. 3CBA degradative capabilities were more evenly distributed spatially than those for 2,4-D. The degradative capabilities of the soils were readily transferred to fresh liquid medium. 3CBA degraders were easily isolated from most soils. We recovered 610 strains that could release carbon dioxide from ring-labeled 3CBA. Of these, 144 strains released chloride and degraded over 80% of 1 mM 3CBA in 3 weeks or less. In contrast, only five 2,4-D degraders could be isolated, although a variety of methods were used in an attempt to culture the degraders. The differences in the distribution and culturability of the bacteria responsible for 3CBA and 2,4-D degradation in these ecosystems suggest that the two substrates are degraded by different populations. We also describe a 14C-based microtiter plate method that allows efficient screening of a large number of samples for biodegradation activity.     

Effect of restriction endonucleases on assessment of biodiversity of cultivable polar marine planktonic bacteria by amplified ribosomal DNA restriction analysis

To choose a suitable restriction endonuclease for quick assessment of bacterial diversity in polar environments by ARDRA, we investigated the effect of restriction enzymes on ARDRA patterns of cultivable marine planktonic bacteria isolated from polar region. Thirty-three isolates were analyzed by ARDRA using five enzymes (HinfI, HaeIII, AluI, and the mix AfaI/MspI), respectively, resulting in different groups, each group corresponding to a particular genotype. A comparison of the ARDRA patterns was carried out, and phylogenetic position of all thirty-three bacteria was obtained by 16S rDNA sequencing. Consistent with phylogenetic analysis, ARDRA pattern comparison revealed that AluI, being sensitive and reliable enough to generate species-specific patterns, was a suitable restriction enzyme used for evaluating bacterial diversity, suggesting a combination of ARDRA with AluI and 16S rDNA sequencing can provide a simple, fast and reliable means for bacterial identification and diversity assessment in polar environments.     

Frequency and Biodiversity of 2,4-Diacetylphloroglucinol-Producing Bacteria Isolated from the Maize Rhizosphere at Different Stages of Plant Growth

A Pseudomonas 2,4-diacetylphloroglucinol (DAPG)-producing population that occurred naturally on the roots, in rhizosphere soil of Zea mays and in the nonrhizosphere soil was investigated in order to assess the microbial diversity at five stages of plant growth. A total of 1,716 isolates were obtained, and 188 of these isolates were able to produce DAPG. DAPG producers were isolated at each stage of plant growth, indicating that the maize rhizosphere is colonized by natural DAPG producers throughout development. The frequency of DAPG producers was very low in the first stage of plant growth and increased over time. An analysis of the level of biodiversity of the DAPG producers at the species level was performed by comparing the AluI restriction patterns of the 16S ribosomal DNAs (rDNAs) amplified by PCR from 167 isolates. This comparison allowed us to cluster the isolates into four amplified rDNA restriction analysis (ARDRA) groups, and the main group (ARDRA group 1) contained 89.8% of the isolates. The diversity of the 150 isolates belonging to ARDRA group 1 was analyzed by the random amplified polymorphic DNA (RAPD) technique. An analysis of RAPD patterns by a molecular variance method revealed that there was a high level of genetic diversity in this population and that the genetic diversity was related to plant age. Finally, we found that some of the DAPG producers, which originated from all stages of plant growth, had the same genotype. These DAPG producers could be exploited in future screening programs for biocontrol agents.     

Evaluation of different PCR-based DNA fingerprinting techniques for assessing the genetic variability of isolates of the fungus Epicoccum nigrum

F. ARENAL, G. PLATAS, J. MARTÍN, O. SALAZAR and FERNANDO PELÁEZ.1999.Thirty-six strains of the fungus Epicoccum nigrum , isolated from different substrata and ecosystems of Europe, America and Africa, were analysed using 14 molecular markers included in 5 different genetic fingerprinting techniques: AP-PCR, tDNA-PCR, microsatellite-primed PCR, ARDRA and AFLP. All of the techniques used were able to differentiate the isolates, showing a high genetic diversity within the species. However, the different techniques detected different levels of similarity among the strains; ARDRA shows the most homogeneous results whilst AP-PCR shows the most heterogeneous. The similarity indices achieved for each strain were compared for the different techniques. The distribution obtained by microsatellite-primed PCR was similar to those shown by AP-PCR techniques. tDNA-PCR and AFLP rendered similar distributions, and ARDRA showed remarkably different results from the other techniques. The results also reveal the lack of an overall correlation between geographical or ecological origin of the isolates and their genotypes.     

Characterization of Hepatitis C Virus Genotypes by Direct Sequencing of HCV 5′UTR Region of Isolates from Saudi Arabia

The current study was designed to determine the Hepatitis C Virus (HCV) genotypes in a representative sample of HCV chronically infected patients in Saudi Arabia. All HCV isolates were genotyped by sequencing of the 5′UTR region and newly identified HCV isolates were identified. Specific universal primers targeting 5′UTR region were used for both amplification and sequencing of all isolates that resulted in 244 bp fragment which represent about 80% of 5′UTR region. Most of HCV isolates in this study were genotype 4 (76.4%) where only few isolates were recognized as genotype 1 (19.6%). All results were compared to HCV reference sequences from LOS ALAMOS HCV database, considering only the complete full genomes for the main phylogenetic analysis. Sequences that showed maximum identity (98% –100%) were selected. Most isolates were identical with HCV genotype 4 references. Some isolates were similar to different subtypes of HCV genotypes 4, 1 and 6. Phylogenetic analysis showed resemblance of most isolates to similar ones from the Far East, North America and Egypt. Using sequence Weblogo, Alignment analysis of isolated HCV genotypes 4 and 1 showed 92% and 95.5% nucleotide conservation, respectively. There was no predominant nucleotide in the varied sites, in both genotypes. All isolated sequences were submitted to GenBank database.     

Restriction fragment length polymorphism analysis of 16S rDNA and low molecular weight RNA profiling of rhizobial isolates from shrubby legumes endemic to the Canary islands

Thirty-six strains of slow-growing rhizobia isolated from nodules of four woody legumes endemic to the Canary islands were characterised by 16S rDNA PCR-RFLP analyses (ARDRA) and LMW RNA profiling, and compared with reference strains representing Bradyrhizobium japonicum, B. elkanii, B. liaoningense, and two unclassified Bradyrhizobium sp. (Lupinus) strains. Both techniques showed similar results, indicating the existence of three genotypes among the Canarian isolates. Analysis of the combined RFLP patterns obtained with four endonucleases, showed the existence of predominant genotype comprising 75% of the Canarian isolates (BTA-1 group) and the Bradyrhizobium sp. (Lupinus) strains. A second genotype was shared by nine Canarian isolates (BGA-1 group) and the B. japonicum and B. liaoningense reference strains. The BES-5 strain formed an independent group, as also did the B. elkanii reference strains. LMW RNA profile analysis consistently resolved the same three genotypes detected by 16S ARDRA among the Canarian isolates, and suggested that all these isolates are genotypically more related to B. japonicum than to B. elkanii or B. liaoningense. Cluster analysis of the combined 16S ARDRA and LMW RNA profiles resolved the BTA-1 group with the Bradyrhizobium sp. (Lupinus) strains, and the BES-5 isolate, as a well separated sub-branch of the B. japonicum cluster. Thus, the two types of analyses indicated that the isolates related to BTA-1 conform a group of bradyrhizobial strains that can be clearly distinguishable from representatives of the tree currently described Bradyrhizobium species. No correlation between genotypes, host legumes, and geographic location was found.     

Frequency and biodiversity of 2,4-diacetylphloroglucinol-producing bacteria isolated from the maize rhizosphere at different stages of plant growth

A Pseudomonas 2,4-diacetylphloroglucinol (DAPG)-producing population that occurred naturally on the roots, in rhizosphere soil of Zea mays and in the nonrhizosphere soil was investigated in order to assess the microbial diversity at five stages of plant growth. A total of 1,716 isolates were obtained, and 188 of these isolates were able to produce DAPG. DAPG producers were isolated at each stage of plant growth, indicating that the maize rhizosphere is colonized by natural DAPG producers throughout development. The frequency of DAPG producers was very low in the first stage of plant growth and increased over time. An analysis of the level of biodiversity of the DAPG producers at the species level was performed by comparing the AluI restriction patterns of the 16S ribosomal DNAs (rDNAs) amplified by PCR from 167 isolates. This comparison allowed us to cluster the isolates into four amplified rDNA restriction analysis (ARDRA) groups, and the main group (ARDRA group 1) contained 89.8% of the isolates. The diversity of the 150 isolates belonging to ARDRA group 1 was analyzed by the random amplified polymorphic DNA (RAPD) technique. An analysis of RAPD patterns by a molecular variance method revealed that there was a high level of genetic diversity in this population and that the genetic diversity was related to plant age. Finally, we found that some of the DAPG producers, which originated from all stages of plant growth, had the same genotype. These DAPG producers could be exploited in future screening programs for biocontrol agents.     

Genotypic Distribution of Hepatitis C Virus in Thailand and Southeast Asia

The majority of hepatitis C virus (HCV) infection results in chronic infection, which can lead to liver cirrhosis and hepatocellular carcinoma. Global burden of hepatitis C virus (HCV) is estimated at 150 million individuals, or 3% of the world’s population. The distribution of the seven major genotypes of HCV varies with geographical regions. Since Asia has a high incidence of HCV, we assessed the distribution of HCV genotypes in Thailand and Southeast Asia. From 588 HCV-positive samples obtained throughout Thailand, we characterized the HCV 5’ untranslated region, Core, and NS5B regions by nested PCR. Nucleotide sequences obtained from both the Core and NS5B of these isolates were subjected to phylogenetic analysis, and genotypes were assigned using published reference genotypes. Results were compared to the epidemiological data of HCV genotypes identified within Southeast Asian. Among the HCV subtypes characterized in the Thai samples, subtype 3a was the most predominant (36.4%), followed by 1a (19.9%), 1b (12.6%), 3b (9.7%) and 2a (0.5%). While genotype 1 was prevalent throughout Thailand (27–36%), genotype 3 was more common in the south. Genotype 6 (20.9%) constituted subtype 6f (7.8%), 6n (7.7%), 6i (3.4%), 6j and 6m (0.7% each), 6c (0.3%), 6v and 6xa (0.2% each) and its prevalence was significantly lower in southern Thailand compared to the north and northeast (p = 0.027 and p = 0.030, respectively). Within Southeast Asia, high prevalence of genotype 6 occurred in northern countries such as Myanmar, Laos, and Vietnam, while genotype 3 was prevalent in Thailand and Malaysia. Island nations of Singapore, Indonesia and Philippines demonstrated prevalence of genotype 1. This study further provides regional HCV genotype information that may be useful in fostering sound public health policy and tracking future patterns of HCV spread.     

Succession and convergence of biofilm communities in fixed-film reactors treating aromatic hydrocarbons in groundwater.

Community composition, succession, and performance were compared in three fluidized bed reactors (FBR) operated to test preemptive colonization and the influence of toluene compared with a mixture of benzene, toluene, and p-xylene (BTX) as feeds. One reactor was inoculated with toluene-degrading strains Pseudomonas putida PaW1, Burkholderia cepacia G4, and B. pickettii PKO1. PaW1 outcompeted the other two strains. When groundwater strains were allowed to challenge the steady-state biofilm developed by inoculated strains, they readily displaced the inoculated strains and further reduced the toluene effluent concentration from 0.140 to 0.063 mg/liter for 98% removal. Amplified ribosomal DNA restriction analysis (ARDRA) of reactor community DNA showed a succession of populations to a pattern that was stable for at least 4 months of operation. Parallel reactors fed toluene and BTX but inoculated directly from groundwater had the same treatment performance and the same ARDRA profiles as each other and as the seeded reactor once the groundwater community took over. Convergence and stability of populations were confirmed by genotype analysis of 120 isolates taken from all reactors and at several times. Ninety percent of the isolates were of 4 of the 12 genotypes found, and their ARDRA patterns accounted for most of the community ARDRA patterns. Estimates of the maximum specific growth rates (mu max), half-saturation constants (K(m)), and maximum substrate utilization rates (Vmax) of the 12 genotypes isolated revealed a rather high diversity of toluene use kinetics even though the toluene in the feed was constant. The climax populations, however, generally showed kinetic parameters indicative of greater competitiveness than the inocula. rRNA sequence analysis of three codominant strains showed them to be members of the alpha, beta, and gamma subdivisions of the Proteobacteria. Two were similar to Comamonas and Pseudomonas putida, but the member of the alpha group was somewhat distant from any organism in the rRNA database. The convergence of communities to the same composition from three different starting conditions and their constancy over several months suggests that a rather stable community was selected.     

内蒙古锡林浩特地区嗜盐古菌多样性的研究

从内蒙古锡林浩特地区3个不同的盐湖中共分离到165株古菌,通过ARDRA分析后得到不同的类群,从各个类群中随机选取1~2个代表菌株进行16S rDNA序列测定和系统发育的分析。结果表明分离的菌株分布在Halorubrum,Natronococcus,Natronorubrum,Haloterrigena,Halorhabdus,Halobiforma,Haloarcula,Haloferax8个属和另外两个分支中,表现了锡林浩特地区嗜盐古菌的多样性。部分菌株的16S rDNA序列同源性低于97%,可能是潜在的新属或新种,代表了该地区嗜盐古菌的独特类型。     

Mathematical estimations of hyper-ammonia producing ruminal bacteria and evidence for bacterial antagonism that decreases ruminal ammonia production(1)

Bacteria capable of denitrification are spread among phylogenetically diverse groups. In the present investigation, molecular methods (amplified ribosomal DNA restriction analysis (ARDRA) and partial 16S rDNA gene sequencing) were used to determine the genetic diversity of culturable denitrifying soil bacteria. The purpose of this work was to study the microbial density and diversity of denitrifying communities isolated from two luvisols and a rendosol. The denitrifying bacterial density was significantly higher in the two luvisols (3x10(6) and 4x10(6) bacteria g(-1) dry soil) than in the rendosol (4x10(5) bacteria g(-1) dry soil). Denitrifying isolates from soils were grouped according to the similarity of their restriction patterns into 26 ARDRA types. Interestingly ARDRA analysis suggests that some denitrifying isolates are specific to a soil type while others seem to be geographically widespread. The number of individual isolates found in each ARDRA type appeared to be highly variable between the two sampling dates but some denitrifying types were capable of persisting in soil. The tree obtained from the partial sequences revealed five major branches exhibiting highest identity to the following genera: (i) Burkholderia-Ralstonia, (ii) Pseudomonas, (iii) Xanthomonas-Frateuria, (iv) Bacillus and (v) Streptomyces. Our 16S rDNA-based analysis clearly reveals broad diversity exceeding that previously described in the literature.     

Biodiversity of Geodermatophilaceae isolated from altered stones and monuments in the Mediterranean basin

An investigation was made into the occurrence and biodiversity of Geodermatophilaceae on 78 samples of altered stone surfaces from 24 monuments and natural stones in the Mediterranean basin; it was found that the total microbial counts ranged between 0 and 10⁷ cfu g⁻¹ dry weight. Members of the Geodermatophilaceae family were isolated from 22 of the 78 samples examined, with the incidence of Geodermatophilaceae colonies in the cultivable population ranging from 1% to 100%. The highest percentage was found in six samples of markedly deteriorated stone. Sixty-five strains randomly isolated from the plates were clustered in six different groups by amplified 16S rDNA restriction analysis (ARDRA) using five different restriction enzymes. Twenty-five strains, representing all the ARDRA haplotypes, were characterized further by partial sequencing (350–550 bp) of the 16S rDNA and by analysing 76 morphological, metabolic and physiological properties. The strains were associated with three well-separated clusters of the genera Geodermatophilus , Blastococcus and Modestobacter . On the basis of 16S rDNA sequence and ARDRA analysis, only two strains were found to be related to the two reference strains of Geodermatophilus . All the others could be grouped with Blastococcus aggregatus (19 strains) or the Antarctic species Modestobacter multiseptatus (44 strains), suggesting that it is these two groups, rather than Geodermatophilus , that tend to colonize the stone surfaces, and that Modestobacter -like strains are also found in temperate/Mediterranean climates. From the BOX-polymerase chain reaction (PCR) data, it can be seen that the Modestobacter -like strains, belonging to the most represented ARDRA haplotype (haplotype B, 34 strains), are very polymorphic and that, over a stone surface, there is a wide genetic diversity at the microsite level.     

Allelic variation in the cg2 gene does not correlate with chloroquine resistance among Indian Plasmodium falciparum isolates

The cg2 gene of Plasmodium falciparum has been proposed to be associated with chloroquine resistance. Here we describe PCR amplification and sequencing of all the four repeat regions (kappa (κ), gamma (γ), psi (φ) and omega (ω)) of this gene, from Indian isolates. There were variant forms for each of these repeat regions (two for κ and γ, and three for φ and ω) among the 123 Indian isolates of P. falciparum. Among these isolates certain forms of φ and ω repeats were uniquely present while some of the reported forms of the κ and ω repeats were absent. The pattern of combination of all four repeat regions of cg2 gene (genotype) was analysed from 52 isolates. A total of 11 different genotypes were observed among these cases, of which 10 were unique to Indian isolates. Certain genotypes were more common than others. The nucleotide sequencing of all the four repeat regions revealed that Indian isolates have some unique repeating units within the γ and ω domains. Altogether, the PCR and sequencing results showed that there was an unrelatedness between cg2 repeats and chloroquine resistance.     

Genetic Diversity and Biological Control Activity of Novel Species of Closely Related Pseudomonads Isolated from Wheat Field Soils in South Australia

Rhizobacteria closely related to two recently described species of pseudomonads, Pseudomonas brassicacearum and Pseudomonas thivervalensis, were isolated from two geographically distinct wheat field soils in South Australia. Isolation was undertaken by either selective plating or immunotrapping utilizing a polyclonal antibody raised against P. brassicacearum. A subset of 42 isolates were characterized by amplified 16S ribosomal DNA restriction analysis (ARDRA), BIOLOG analysis, and gas chromatography-fatty acid methyl ester (GC-FAME) analysis and separated into closely related phenetic groups. More than 75% of isolates tested by ARDRA were found to have >95% similarity to either Pseudomonas corrugata or P. brassicacearum-P. thivervalensis type strains, and all isolates had >90% similarity to either type strain. BIOLOG and GC-FAME clustering showed a >70% match to ARDRA profiles. Strains representing different ARDRA groups were tested in two soil types for biological control activity against the soilborne plant pathogen Gaeumannomyces graminis var. tritici, the causative agent of take-all of wheat and barley. Three isolates out of 11 significantly reduced take-all-induced root lesions on wheat plants grown in a red-brown earth soil. Only one strain, K208, was consistent in reducing disease symptoms in both the acidic red-brown earth and a calcareous sandy loam. Results from this study indicate that P. brassicacearum and P. thivervalensis are present in Australian soils and that a level of genetic diversity exists within these two novel species but that this diversity does not appear to be related to geographic distribution. The result of the glasshouse pot trial suggests that some isolates of these species may have potential as biological control agents for plant disease.     

Molecular phylogeny of the psittacid herpesviruses causing Pacheco's disease: correlation of genotype with phenotypic expression

Fragments of 419 bp of the UL16 open reading frame from 73 psittacid herpesviruses (PsHVs) from the United States and Europe were sequenced. All viruses caused Pacheco's disease, and serotypes of the European isolates were known. A phylogenetic tree derived from these sequences demonstrated that the PsHVs that cause Pacheco's disease comprised four major genotypes, with each genotype including between two and four variants. With the exception of two viruses, the serotypes of the virus isolates could be predicted by the genotypes. Genotypes 1 and 4 corresponded to serotype 1 isolates, genotype 2 corresponded to serotype 2 isolates, and genotype 3 corresponded to serotype 3 isolates. The single serotype 4 virus mapped to genotype 4. DNA from a virus with a unique serotype could not be amplified with primers that amplified DNA from all other PsHVs, and its classification remains unknown. Viruses representing all four genotypes were found in both the United States and Europe, and it was therefore predicted that serotypes 1, 2, and 3 were present in the United States. Serotype 4 was represented by a single European isolate that could not be genetically distinguished from serotype 1 viruses; therefore, the presence of serotype 4 in the United States could not be predicted. Viruses of genotype 4 were found to be the most commonly associated with Pacheco's disease in macaws and conures and were least likely to be isolated in chicken embryo fibroblasts in the United States. All four genotypes caused deaths in Amazon parrots, but genotype 4 was associated with Pacheco's disease only in Amazons in Europe. Genotypes 2, 3, and 4, but not 1, were found in African grey parrots. Although parrots from the Pacific distribution represent a relatively small percentage of the total number of birds with Pacheco's disease, all four genotypes were found to cause disease in these species.     

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 γ- and β-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). γ-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.     

Restriction fragment length polymorphisms and sequence analysis: an approach for genotyping infectious pancreatic necrosis virus reference strains and other aquabirnaviruses isolated from northwestern Spain

Reference strains of infectious pancreatic necrosis virus resembling the 10 recognized serotypes and local isolates of aquabirnaviruses isolated in northwestern Spain from reservoirs (mollusks) and from asymptomatic and carrier cultured fish were genotyped by restriction fragment length polymorphism (RFLP) and nucleic acid sequence analyses. The RFLP analysis yielded seven genogroups, each of which was clearly correlated with a serotype. Sequence analysis of the three open reading frames provided quite similar results in terms of genogrouping. Based on the results of this study and in order to unify the two types of assays, we propose placing aquabirnaviruses into six genogroups, four of which can be subdivided into two genotypes based on a two-step restriction analysis. The genotyping corresponds with serotyping as follows: genogroup I includes two genotypes corresponding to serotypes A9 (genotype I.1) and A1 (genotype I.2); genogroup II corresponds to serotype A3; genogroup III includes genotypes III.1 (serotype A2) and III.2 (serotype B1); genogroups IV and V include two genotypes, each corresponding to serotypes A5, A6, A7, and A8 (genotypes IV.1, IV.2, V.1, and V.2, respectively);and genogroup VI corresponds to serotype A4. As expected, most local isolates belonged to genotype III.1 and genogroup II. However, a few local isolates corresponded to the American types of genogroup I. Finally, based on the results of this study and due to its simplicity, the two-step restriction analysis assay is proposed as a method for typing new isolates of aquabirnaviruses, and the results correspond to the results of conventional serotyping.