Single-strand Conformation Polymorphism Profiles Research Articles

Description of the structural diversity of rumen microbial communities in vitro using single-strand conformation polymorphism profiles

Changes of the rumen microbial community structure, as it can be established with a rumen simulation technique (RUSITEC) were studied using PCR and single-strand conformation polymorphism (SSCP) of small subunit rDNA genes (SSU rDNA). Four total mixed rations were incubated and two ammonia levels in the artificial saliva were applied. Three replicated vessels were used for each treatment. Mixed microbial fractions were isolated by stepwise centrifugation from the liquid fraction (reference microbes, RM) and from the solids of the feed residues (solid-associated microbes, SAM). PCR-primers targeting archaea, fibrobacter, clostridia, and bacteria, respectively, were applied to represent the individual taxonomic groups by SSCP profiles. These SSCP profiles were converted into a binary matrix and distances among treatments were visualised by non-metric multidimensional scaling. Between replicates belonging to one treatment only small differences were found, indicating a high reproducibility of the RUSITEC and the chosen SSCP method. The ammonia concentration seems to be affecting the SSCP profiles. Great differences occurred between RM and SAM, especially for profiles targeting bacteria and clostridia. Differences in the profiles of RM were also found between mixed rations that contained the same feedstuffs in different ratios and between rations with similar nutrient content but based on different feedstuffs. In conclusion, the PCR-SSCP-based technique in conjunction with non-metric multidimensional scaling was sufficiently sensitive to detect and compare changes in composition of rumen microbial community structure in vitro as affected by diet and other environmental factors.

Electrophoretic analysis of sequence variability in three mitochondrial DNA regions for ascaridoid parasites of human and animal health significance

Sequence variability in three mitochondrial DNA (mtDNA) regions, namely cytochrome c oxidase subunit 1 (cox1), NADH dehydrogenase subunits 1 and 4 (nad1 and nad4), among and within Toxocara canis, T. cati, T. malaysiensis, T. vitulorum and Toxascaris leonina from different geographical origins was examined by a mutation-scanning approach. A portion of the cox1 gene (pcox1), a portion of the nad1 and nad4 genes (pnad1 and pnad4) were amplified separately from individual ascaridoid nematodes by polymerase chain reaction and the amplicons analyzed by single-strand conformation polymorphism (SSCP). Representative samples displaying sequence variation in SSCP profiles were subjected to sequencing in order to define genetic markers for their specific identification and differentiation. While the intra-specific sequence variations within each of the five ascaridoid species were 0.2-3.7% for pcox1, 0-2.8% for pnad1 and 0-2.3% for pnad4, the inter-specific sequence differences were significantly higher, being 7.9-12.9% for pcox1, 10.7-21.1% for pnad1 and 12.9-21.7% for pnad4, respectively. Phylogenetic analyses based on the combined sequences of pcox1, pnad1 and pnad4 revealed that the recently described species T. malaysiensis was more closely related to T. cati than to T. canis. These findings provided mtDNA evidence for the validity of T. malaysiensis and also demonstrated clearly the usefulness and attributes of the mutation-scanning sequencing approach for studying the population genetic structures of these and other nematodes of socio-economic importance.

Contributions of fermentative acidogenic bacteria and sulfate-reducing bacteria to lactate degradation and sulfate reduction

The roles of fermentative acidogenic bacteria and sulfate-reducing bacteria (SRB) in lactate degradation and sulfate reduction in a sulfidogenic bioreactor were investigated by traditional chemical monitoring and culture-independent methods. A continuously stirred tank reactor fed with synthetic wastewater containing lactate and SO 4 2 - at 35 °C, 10 h of hydraulic retention time was used. The results showed that sulfate removal efficiency reached 99%, and sulfide and acetate were the main end products after 20 d of operation. 16S rRNA gene based clone libraries and single-strand conformation polymorphism profiles demonstrated that the proportion of SRB increased from 16% to 95%, and that Desulfobulbus spp., Desulfovibrio spp., Pseudomonas spp. and Clostridium spp. formed a stable, dominant community structure. The decreasing COD/ SO 4 2 - ratio had little effect on the community pattern except that Pseudomonas spp. and Desulfobulbus spp. increased slightly. The addition of molybdate to the influent significantly changed the microbial community, sulfate removal efficiency and the pattern of end products. Clostridium spp., Bacteroides spp. and Ruminococcus spp. became the dominant community members. The main end products switched from acetate to ethanol and then to propionate with the oxidation–reduction potentials increasing from −420 to −290 mV. A lactate degradation pathway was deduced: lactate served as the electronic donor for Desulfovibrio spp., or was fermented by Clostridium spp. and Bacteroides spp. to produce propionate or ethanol, which were subsequently utilized by Desulfobulbus spp. and Desulfovibrio spp. The acidotrophic SRB oxidized part of the acetate finally.

Microbial community structure in different wastewater treatment processes characterized by single-strand conformation polymorphism (SSCP) technique

In order to investigate microbial community structures in different wastewater treatment processes and understand the relationship between the structures and the status of processes, the microbial community diversity, variety and distribution in five wastewater treatment processes were studied by a culture-independent genetic fingerprinting technique single-strand conformation polymorphism (SSCP). The five processes included denitrifying and phosphate-removal system (diminished N), Chinese traditional medicine wastewater treatment system (P), beer wastewater treatment system (W), fermentative biohydrogen-producing system (H), and sulfate-reduction system (S). The results indicated that the microbial community profiles in the wastewater bioreactors with the uniform status were very similar. The diversity of microbial populations was correlated with the complexity of organic contaminants in wastewater. Chinese traditional medicine wastewater contained more complex organic components; hence, the population diversity was higher than that of simple nutrient bioreactors fed with molasses wastewater. Compared with the strain bands in a simulated community, the relative proportion of some functional microbial populations in bioreactors was not dominant. Fermentative biohydrogen producer Ethanoligenens harbinense in the better condition bioreactor had only a 5% band density, and the Desulfovibrio sp. in the sulfate-reducing bioreactor had less than 1.5% band density. The SSCP profiles could identify the difference in microbial community structures in wastewater treatment processes, monitor some of the functional microbes in these processes, and consequently provide useful guidance for improving their efficiency.

The use of SSCP analysis in the assessment of phytoplasma gene variability

Single-strand conformation polymorphism (SSCP) analysis is a broadly used technique for detecting mutations. The aim of this work was to assess the applicability of SSCP as a new tool for the detection of the molecular variability of uncultivable mollicutes — phytoplasmas. Three phytoplasma regions were investigated: 16S rDNA, tuf gene, and dnaB gene. Fragments amplified by PCR were subjected to SSCP under conditions optimized for each fragment length. In all of the analyzed regions, SSCP revealed the presence of polymorphism undetected by routine RFLP analyses. Reliability of the method was confirmed by the multiple alignments and phylogenetic analyses of representative sequences showing different SSCP profiles.

Dynamic characteristics of bacterial community in a sulphate-reducing bioreactor fed with acetate and intermittent ethanol

Acetotrophic Sulphate-Reducing Bacteria (ASRB) were enriched in a sulphidogenic Continuous Stirred Tank Reactor (CSTR) fed with acetate/ethanol and diagnosed by Single-Strand Conformation Polymorphism (SSCP) and 16S rRNA gene clone library. The CSTR was run for 65 days with synthetic wastewater containing sulphate and acetate with intermittent ethanol at hydraulic retention time of 10 h. The Sulphate-Removal Rate (SRR) reached to 3.8 g/(L?day) after 35 days of start-up. The SSCP profiles of bacterial community changed rapidly at the beginning of start-up before Desulphococcus sp., Desulphomicrobium sp., Aminomonas and Anaerolinea formed a stable community. Bacterial diversity decreased when the ethanol in the influent was replaced by acetate of equal Chemical Oxygen Demand (COD). Desulphomicrobium sp. was enriched and found to have a significant role in acetate utilisation. When ethanol was re-added instead of acetate of equal COD strength, microbial diversity increased and the Desulphomicrobium band of SSCP profiles became weak. At the later start-up stage, the 16S rRNA gene clone library indicated the presence of bacteria belonging to six different known phyla and sequences with similarities to those of sulphate-reducing bacteria accounted for 22%. The SSCP band sequences revealed that the characteristics of bacterial community populations resembled those of clone library sequences. The present study showed the addition of ethanol to influent would enhance bacterial diversity and SRR, ASRB could be enriched and Desulphomicrobium sp. was the primary source of acetate oxidation.

Diversity responses of rumen microbial communities to Fusarium‐contaminated feed, evaluated with rumen simulating technology

The mycotoxin-producing fungus Fusarium culmorum causes major feed spoilages in agricultural livestock, but effects of F. culmorum-contaminated feed on the structural diversity of the rumen-inhabiting microbial community are not understood. Avoiding animal experiments, this study was conducted with the rumen simulating technique (Rusitec). Small subunit (SSU) rRNA gene copy numbers of bacteria and archaea, determined by quantitative polymerase chain reaction (PCR), indicated no differences between contaminated and non-contaminated digested feed, but fungal copy numbers, not attributable to F. culmorum itself, were elevated approximately fourfold in the contaminated feed, with 2.3 x 10(9) g(-1) dry weight. Single-strand conformation polymorphism profiles of PCR-amplified partial SSU rRNA genes revealed a single but clear difference between contaminated and non-contaminated feed in profiles encompassing the phylogenetic clusters of Fibrobacteriales and Clostridiales. Minor quantitative differences were also seen in profiles of archaea and fungi. Positive correlations were found between fungal rRNA gene copy numbers and the degradability of different nutrients, but there was no correlation with degradation rates of the major mycotoxin contaminant deoxynivalenol. Thus, the diversity responses of the microbial community to F. culmorum-contaminated feed were caused by a fungus-induced, altered feed quality rather than by direct mycotoxicity.

Thermophilic methanogenic Archaea in compost material: Occurrence, persistence and possible mechanisms for their distribution to other environments

Since compost is widely used as soil amendment and the fact that during the processing of compost material high amounts of microorganisms are released into the air, we investigated whether compost may act as a carrier for thermophilic methanogens to temperate soils. All eight investigated compost materials showed a clear methane production potential between 0.01 and 0.98 μmol CH 4 g dw −1 h −1 at 50 °C. Single strand conformation polymorphism (SSCP) and cloning analysis indicated the presence of Methanosarcina thermophila, Methanoculleus thermophilus, and Methanobacterium formicicum. Bioaerosols collected during the turning of a compost pile showed both a highly similar SSCP profile compared to the corresponding compost material and clear methane production during anoxic incubation in selective medium at 50 °C. Both observations indicated a considerable release of thermophilic methanogens into the air. To analyse the persistence of compost-borne thermophilic methanogens in temperate oxic soils, we therefore studied their potential activity in compost and compost/soil mixtures, which was brought to a meadow soil, as well as in an agricultural soil fertilised with compost. After 24 h anoxic incubation at 50 °C, all samples containing compost showed a clear methanogenic activity, even 1 year after application. In combination with the in vitro observed resilience of the compost-borne methanogens against desiccation and UV radiation we assume that compost material acts as an effective carrier for the distribution of thermophilic methanogens by fertilisation and wind.

Identification of anisakid nematodes with zoonotic potential from Europe and China by single-strand conformation polymorphism analysis of nuclear ribosomal DNA

Using genetic markers defined previously in the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA (rDNA), isotopic, and non-isotopic polymerase-chain-reaction-coupled single-strand conformation polymorphism (SSCP) were utilized to identify each of three anisakid species [Anisakis simplex (s.l.), Contracaecum osculatum (s.l.), and Hysterothylacium aduncum] from different host species and geographical locations in Poland and Sweden. While subtle microheterogeneity was observed within each of Anisakis simplex (s.l.) and H. aduncum, distinct SSCP profiles were displayed for each of the three species, allowing identification and differentiation of the three taxa. Subsequent sequencing of the ITS-1 and ITS-2 rDNA revealed that A. simplex (s.l.) represented Anisakis simplex s.s. and Contracaecum osculatum (s.l.) represented C. osculatum C. Application of the non-isotopic SSCP assay of ITS-2 to larval anisakid samples from different hosts and geographical locations in China revealed three distinct SSCP profiles, one of which was consistent with that of A. simplex (s.l.), and the other two had different SSCP profiles from that of C. osculatum C and H. aduncum. Sequencing of the ITS-1 and ITS-2 rDNA for representative Chinese anisakid samples examined revealed three anisakid species in China, i.e., Anisakis typica, Anisakis pegreffii, and Hysterothylacium sp. These molecular tools will be useful for identification and investigation of the ecology of anisakid nematodes in China and elsewhere.

Classification of Mycoplasma synoviae strains using single-strand conformation polymorphism and high-resolution melting-curve analysis of the vlhA gene single-copy region

Mycoplasma synoviae is an economically important pathogen of poultry worldwide, causing respiratory infection and synovitis in chickens and turkeys. Identification of M. synoviae isolates is of critical importance, particularly in countries in which poultry flocks are vaccinated with the live attenuated M. synoviae strain MS-H. Using oligonucleotide primers complementary to the single-copy conserved 5' end of the variable lipoprotein and haemagglutinin gene (vlhA), amplicons of approximately 400 bp were generated from 35 different M. synoviae strains/isolates from chickens and subjected to mutation scanning analysis. Analysis of the amplicons by single-strand conformation polymorphism (SSCP) revealed 10 distinct profiles (A-J). Sequencing of the amplicons representing these profiles revealed that each profile related to a unique sequence, some differing from each other by only one base-pair substitution. Comparative high-resolution melting (HRM) curve analysis of the amplicons using SYTO 9 green fluorescent dye also displayed profiles which were concordant with the same 10 SSCP profiles (A-J) and their sequences. For both mutation detection methods, the Australian M. synoviae strains represented one of the A, B, C or D profiles, while the USA strains represented one of the E, F, G, H, I or J profiles. The results presented in this study show that the PCR-based SSCP or HRM curve analyses of vlhA provide high-resolution mutation detection tools for the detection and identification of M. synoviae strains. In particular, the HRM curve analysis is a rapid and effective technique which can be performed in a single test tube in less than 2 h.

Characterization of the predominant bacterial population of different mangrove rhizosphere soils using 16S rRNA gene-based single-strand conformation polymorphism (SSCP)

Variations in chemical parameters and bacterial populations in mangrove rhizosphere samples were noted for different sites. The C, N, P and K contents as well as pH, EC and salinity showed variation between sites. Significant differences in soil properties were also found in sampling sites. Two types of soil were noted among sites. Guesthouse had significantly higher organic matter and nutrient content (N) than other three sites suggesting that human discharges, litter deposition and surface runoff were major nutrient inputs. This contaminated site was located at the landward edges. Positive correlations between organic matter, N, P and K contents were found suggesting that these nutrients were from similar input sources. Effects of sampling sites on microbial diversity were also analyzed via SSCP. Porteresia coarctata and Rhizophora mucronata did not show any variation in the banding patterns among replicates sampled in short distance within site. But Sonneratia apetala showed variation among replicates sampled in distance within site. A significant variation was noted in the SSCP profile among replicates between sites. The majority of dominant SSCP band sequences were related to bacterial genera of root and root-free soil environments, namely Bacillus, Planococcus, Planomicrobium, low G+C Gram-positive bacterium, glacial ice bacterium and unidentified bacteria. In the analysis of 16S rRNA sequences, members belonging to the phylum Firmicutes dominated the sequence collection. The phylogenetic analysis of 16S rRNA gene sequences showed close relationships to a wide range of clones or bacterial species of phylum Firmicutes and unidentified bacteria.

Stability of microbial communities in goat milk during a lactation year: Molecular approaches

The microbial communities in milks from one herd were evaluated during 1-year of lactation, using molecular methods to evaluate their stability and the effect of breeding conditions on their composition. The diversity of microbial communities was measured using two approaches: molecular identification by 16S and 18S rDNA sequencing of isolates from counting media (two milks), and direct identification using 16S rDNA from clone libraries (six milks). The stability of these communities was evaluated by counting on selective media and by Single Strand Conformation Polymorphism (SSCP) analysis of variable region V3 of the 16S rRNA gene and variable region V4 of the 18S rRNA gene. One hundred and eighteen milk samples taken throughout the year were analyzed. Wide diversity among bacteria and yeasts in the milk was revealed. In addition to species commonly encountered in milk, such as Lactococcus lactis, Lactococcus garvieae, Enterococcus faecalis, Lactobacillus casei, Leuconostoc mesenteroides, Staphylococcus epidermidis, Staphylococcus simulans, Staphylococcus caprae, Staphylococcus equorum, Micrococcus sp., Kocuria sp., Pantoea agglomerans and Pseudomonas putida, sequences were affiliated to other species only described in cheeses, such as Corynebacterium variabile, Arthrobacter sp., Brachybacterium paraconglomeratum, Clostridium sp. and Rothia sp. Several halophilic species atypical in milk were found, belonging to Jeotgalicoccus psychrophilus, Salinicoccus sp., Dietza maris, Exiguobacterium, Ornithinicoccus sp. and Hahella chejuensis. The yeast community was composed of Debaryomyces hansenii, Kluyveromyces lactis, Trichosporon beigelii, Rhodotorula glutinis, Rhodotorula minuta, Candida pararugosa, Candida intermedia, Candida inconspicua, Cryptococcus curvatus and Cryptococcus magnus. The analyses of microbial counts and microbial SSCP profiles both distinguished four groups of milks corresponding to four periods defined by season and feeding regime. The microbial community was stable within each period. Milks from winter were characterized by Lactococcus and Pseudomonas, those from summer by P. agglomerans and Klebsiella and those from autumn by Chryseobacterium indologenes, Acinetobacter baumanii, Staphylococcus, Corynebacteria and yeasts. However, the composition of the community can vary according to factors other than feeding. This study opens new investigation fields in the field of raw milk microbial ecology.

Preferential accumulation of severe variants of Citrus tristeza virus in plants co-inoculated with mild and severe variants

The viral population in sweet orange plants, either healthy or pre-inoculated with the asymptomatic isolate of Citrus tristeza virus (CTV) T32, and then graft- or aphid-inoculated with the stem-pitting isolate T318, was characterized with respect to symptom expression, reaction with monoclonal antibody MCA13, single-strand conformation polymorphism (SSCP) of genes p18 and p20, bi-directional RT-PCR, and dot-blot hybridisation. All plants inoculated with T318, with or without pre-inoculation, showed stem pitting, reacted with MCA13, had the SSCP profile characteristic of this isolate, and in bi-directional RT-PCR yielded a 450-bp DNA product associated with severe isolates, indicating that T32 afforded no protection against T318. The latter isolate had two main sequence variants, the minor one of which was indistinguishable from the main T32 sequence, and both were detected in most plants that were graft-inoculated with T318. However, the T32 variant was not detected in plants that were aphid-inoculated only with T318 and also showed stem pitting. This suggested an association of symptoms with the major T318 sequence and preferential transmission of this variant by aphids. The T318-specific variant accumulated more than the T32 variant in plants in which both were replicating, suggesting a higher fitness of the former. Our results clearly emphasize the potential threat of severe CTV variants in areas where mild isolates are presently predominant.

Differences in the rhizosphere bacterial community of a transplastomic tobacco plant compared to its non-engineered counterpart

Cultivation-independent analyses were carried out to compare the bacterial community structure found in the rhizospheres of a transplastomic tobacco plant carrying the antibiotic resistance marker-gene aadA and its non-engineered parental line. PCR- and reverse transcriptase PCR-amplifications of 16S rRNA and their corresponding genes were carried out with primers targeting the domain Bacteria. The diversity of PCR-products amplified from total nucleic acids extracted from rhizospheres of 10-week-old plants, which had been grown in potting soil in the greenhouse, was visualized by genetic profiling using the single-strand conformation polymorphism (SSCP) technique. The SSCP profiles generated from DNA extracted with two different protocols, one including total RNA and the other only DNA, did not show any differences. The SSCP profiles amplified from RNA and DNA were also highly similar to each other, indicating that the dominant bacteria detected were metabolically active. High similarities were seen between the SSCP profiles from the transplastomic and the non-engineered plants, except for a single band that consistently occurred with samples from the non-engineered plants (six replicates), but not, or only weakly, with their engineered counterparts. DNA sequencing and database analysis revealed that the partial rRNA gene matched to a Flavobacterium sp. Other bands of the SSCP-profiles, related to Burkholderia and Bordetella were variable between individual plants but not affected by the transplastomic modification. Thus, the transplastomic modification caused a relative decline of a specific Flavobacterium population but not of other bacteria. Further studies including additional tobacco cultivars, soils and conditions of cultivation would be desirable, to elucidate the ecological importance of this difference.

Bighorn Sheep, a New Host Record for Parelaphostrongylus odocoilei (Nematoda: Protostrongylidae)

Larval nematodes with a dorsal spine on the tail were recovered from fecal samples of California bighorn sheep (Ovis canadensis californiana) in northeastern Washington State, USA. The identity of these dorsal-spined larvae (DSL) was established by single-strand conformation polymorphism (SSCP) analyses of a partial fragment of the first internal transcribed spacer of the ribosomal DNA. The SSCP profiles of individual DSL from bighorn sheep were compared with those of DSL of five protostrongylid species (Parelaphostrongylus andersoni, P odocoilei, P. tenuis, Elaphostrongylus rangiferi, and Muellerius capillaris) but were identical to only those of P. odocoilei. This study represents the first confirmed identification of P. odocoilei in bighorn sheep.

Rapid Screening of Fluoroquinolone Resistance Determinants in Streptococcus pneumoniae by PCR-Restriction Fragment Length Polymorphism and Single-Strand Conformational Polymorphism

A rapid method, using PCR-restriction fragment length and single-strand conformation polymorphism (SSCP), was applied to screen for mutations of the fluoroquinolone resistance determinants in Streptococcus pneumoniae. One hundred nonduplicate Streptococcus pneumoniae isolates with ciprofloxacin MICs of > or = 4.0 microg/ml from the Prince of Wales Hospital, Hong Kong, years 2000 to 2003, were examined. For each isolate, PCR amplicons of quinolone resistance-determining regions (QRDRs) of gyrA, gyrB, parC, and parE genes were digested with AluI, HinfI, Sau3AI, and MspI, respectively, and analyzed by SSCP. Each SSCP pattern was given a number, and each isolate obtained a four-digit code, e.g., 1111, that represented the SSCP profile. The SSCP patterns were correlated to mutations characterized from sequence analyses of PCR amplicons. The most common SSCP profile obtained was no. 5232 (40%), which included strains with two amino acid substitutions in the ParC (Lys-137-Asn) and ParE (Ile-460-Val) genes, followed by the SSCP profile 5223 (17%), which included strains with amino acid substitutions in the ParE (Ile-460-Val) gene only. Ten isolates (10%) with amino acid substitutions at GyrA and ParE (+/-ParC) genes were resistant to levofloxacin with a MIC of > or = 16 microg/ml. Other SSCP profiles were unique in distinguishing the common amino acid substitutions in GyrA (Ser-81-Phe) and ParC (Lys-137-Asn, Ser-79-Phe plus Lys-137-Asn, Asp-83-Asn plus Lys-137-Asn, Ser-79-Phe, and Glu-96-Asp). SSCP analysis of restricted fragments generated patterns that were highly discriminative for mutations present in the QRDRs of gyrA, gyrB, parC, and parE. This method provides a database of high resolution profiles on these mutations and allows rapid screening for new mutations of the fluoroquinolone resistance genes.

Genetic profiling of bacterial communities from the rhizospheres of ozone damaged Malva sylvestris ( Malvaceae)

The phytotoxicity of tropospheric ozone at elevated concentrations can cause severe effects on plant vegetation. This study analyzed whether ozone-damaged plants transmit signals into the soil and thereby affect the structural diversity of bacteria inhabiting the rhizosphere. To generate highly stressful conditions, Malva sylvestris was raised from seeds under ozone exposure. Additionally, the plant age at the onset of ozone-stress was varied. Rhizosphere bacterial communities were analyzed by single-strand conformation polymorphism (SSCP) of PCR amplified partial 16S ribosomal RNA genes. None of these vigorous stress conditions caused a change in the genetic profiles targeting Bacteria with primers hybridizing to highly conserved regions in the 16S rRNA genes, despite visibly ozone-triggered injuries of the leaves and a reduced root biomass. Differences in the SSCP profiles however were observed between plants of different ages. In a more realistic scenario, plants raised in the greenhouse under normal atmospheric conditions were exposed to ozone episodes as they can occur during summer in industrialized countries of the northern hemisphere. To increase the sensitivity of detection, genetic profiles representing Alphaproteobacteria, Actinobacteria and Pseudomonas were analyzed separately in addition to those of Bacteria. None of the profiles showed any ozone-related alterations. In summary, the results of this study indicated that, even when the plants were visibly injured by ozone, the stress was not transmitted to the soil in a way that affected the structural diversity of the dominant rhizosphere bacterial community.

Sscp analysis of variations in haplotypes of citrus tristeza virus isolated from yuzu (Citrus junos) in geographically separate regions of Korea

Yuzu (Cittus junos) trees were examined from six geographically separate provinces in the Republic of Korea, including four islands (Geoje, Namhae, Wan, and Jeju), 1 peninsula (Goheung), and 1 inland area (Boseong). The population of sequence variants of citrus tristeza virus (CTV) was isolated and analyzed by single-strand conformation polymorphism (SSCP) analysis of cDNA from thep20 gene. SSCP profiles of 65 PCR products showed different band patterns but with similar intensities. Sixteen haplotypes were subgrouped according to their SSCP profiles and severity of symptoms. Their genomes were sequenced and compared. DNA analysis of thep20 genes revealed nucleotide identities ranging from 88-99.8%. Based on SSCP analysis, the pathologically mild isolates of CTV yielded two to three DNA bands, whereas the most virulent isolates contained more than two bands. Comparisons of these physically separate haplotypes suggest that CTV isolates with multiple SSCP profiles could have arisen as a result of a mixed infection and genetic recombination of two divergent isolates. Plants with severe disease symptoms, such as stem pitting, closely corresponded to a CTV strain showing typical SSCP profiles in Florida (USA) and Japan.

Bacterial diversity in a finished compost and vermicompost: differences revealed by cultivation-independent analyses of PCR-amplified 16S rRNA genes

Bacterial communities are important catalysts in the production of composts. Here, it was analysed whether the diversity of bacteria in finished composts is stable and specific for the production process. Single-strand conformation polymorphism (SSCP) based on polymerase chain reaction amplified partial 16S rRNA genes was used to profile and analyse bacterial communities found in total DNA extracted from finished composts. Different batches of compost samples stored over a period of 12 years and a 1-year-old vermicompost were compared to each other. According to digital image analysis, clear differences could be detected between the profiles from compost and vermicompost. Differences between three different periods of compost storage and between replicate vermicompost windrows were only minor. A total of 41 different 16S rRNA genes were identified from the SSCP profiles by DNA sequencing, with the vast majority related to yet-uncultivated bacteria. Sequences retrieved from compost mainly belonged to the phyla Actinobacteria and Firmicutes. In contrast, vermicompost was dominated by bacteria related to uncultured Chloroflexi, Acidobacteria, Bacteroidetes and Gemmatimonadetes. The differences were underscored with specific gene probes and Southern blot hybridizations. The results confirmed that different substrates and composting processes selected for specific bacterial communities in the finished products. The specificity and consistency of the bacterial communities inhabiting the compost materials suggest that cultivation-independent bacterial community analysis is a potentially useful indicator to characterize the quality of finished composts in regard to production processes and effects of storage conditions.

Effect of elevated tropospheric ozone on the structure of bacterial communities inhabiting the rhizosphere of herbaceous plants native to Germany.

Current elevated concentrations of ozone in the atmosphere, as they are observed during summer seasons, can cause severe effects on plant vegetation. This study was initiated to analyze whether ozone-stressed plants also transfer signals below ground and thereby alter the bacterial community composition in their rhizospheres. Herbaceous plants, native to Germany, with tolerance (Anthoxanthum odoratum, Achillea millefolium, Poa pratensis, Rumex acetosa, and Veronica chamaedrys) and sensitivity (Matricaria chamomilla, Sonchus asper, and Tanacetum vulgare) to ozone, raised in the greenhouse, were exposed in open-top chambers to two different ozone regimes, i.e., "summer stress" and a normal ozone background. DNA of bacterial cells from the rhizospheres was directly extracted, and partial sequences of the 16S rRNA genes were PCR amplified with primers targeting the following phylogenetic groups: Bacteria, alpha-Proteobacteria, Actinobacteria, and Pseudomonas, respectively. The diversity of the amplified products was analyzed by genetic profiling based on single-strand conformation polymorphism (SSCP). Neither the tolerant nor the sensitive plants, the latter with visible above-ground damage, showed ozone-induced differences in any of the SSCP profiles, with the single exception of Actinobacteria-targeted profiles from S. asper. To increase the stress, S. asper was germinated and raised in the continuous presence of an elevated level of ozone. SSCP profiles with Bacteria-specific primers combined with gene probe hybridizations indicated an ozone-related increase in a Xanthomonas-related 16S rRNA gene and a decrease in the respective gene from the plant plastids. The fact that only this latter unrealistic scenario caused a detectable effect demonstrated that ozone stress has a surprisingly small effect on the structural diversity of the bacterial community in rhizospheres.