Soil microbial community structures are shaped by agricultural systems revealing little temporal variation.

Many studies in soil microbial ecology are undertaken with a single sampling event, with the influence of temporal progression rarely being considered. Under field conditions, soil samples were taken from different agricultural systems; a sown grassland to maize rotation (MC), an intensively managed permanent grassland (INT), as well as extensively managed permanent grasslands with high (EXT_HP), low to sufficient (EXT_LP) and deficient available P (EXT_DP), six times throughout the 2017 growing season. Thus, this study aimed to determine if any differences in soil microbiome structures between both sharply contrasting (MC - INT - EXT), slightly differing (EXT_HP - EXT_DP) and quite similar (EXT_HP - EXT_LP and EXT_LP - EXT_DP) agricultural systems persist through changing growth conditions within the growing season. For both fungal and bacterial community structure, the influence of agricultural system (CV = 0.256, P < 0.001 and CV = 0.145, P < 0.01, respectively) was much greater than that of temporal progression (√CV = 0.065 and 0.042, respectively, both P < 0.001). Importantly, nearly all agricultural systems persistently harbored significantly distinct fungal community structures across each of the six sampling events (all at least P < 0.05). There were not as many pairwise differences in bacterial community structure between the agricultural systems, but some did persist (MC and EXT_HP âˆ¼ EXT_DP, all P < 0.001). Additionally, persistent indicator fungal OTUs (IndVal >0.7, P ≤ 0.05) associated to each agricultural system (except EXT_LP) were found in each of the six sampling events. These results highlight the temporal stability of pairwise differences in soil microbiome structures between established agricultural systems through changing plant growth conditions, even between those with a comparable management regime. This is a highly relevant finding in informing the sampling strategy of studies in soil microbial ecology as well as for designing efficient soil biodiversity monitoring systems.

Small-scale agricultural grassland management can affect soil fungal community structure as much as continental scale geographic patterns.

A European transect was established, ranging from Sweden to the Azores, to determine the relative influence of geographic factors and agricultural small-scale management on the grassland soil microbiome. Within each of five countries (factor 'Country'), which maximized a range of geographic factors, two differing growth condition regions (factor 'GCR') were selected: a favorable region with conditions allowing for high plant biomass production and a contrasting less favorable region with a markedly lower potential. Within each region, grasslands of contrasting management intensities (factor 'MI') were defined: intensive and extensive, from which soil samples were collected. Across the transect, 'MI' was a strong differentiator of fungal community structure, having a comparable effect to continental scale geographic factors ('Country'). 'MI' was also a highly significant driver of bacterial community structure, but 'Country' was clearly the stronger driver. For both, 'GCR' was the weakest driver. Also at the regional level, strong effects of MI occurred on various measures of the soil microbiome (i.e. OTU richness, management-associated indicator OTUs), though the effects were largely regional-specific. Our results illustrate the decisive influence of grassland MI on soil microbial community structure, over both regional and continental scales, and, thus, highlight the importance of preserving rare extensive grasslands.

Overall bioburden by total colony count does not predict the presence of pathogens with high clinical relevance in hospital and community environments.

BACKGROUND: Healthcare-associated infections (HAIs) affect millions of patients, increasing morbidity and mortality. Pathogens of HAIs originate from both the patient's own flora and the environment, including multi-drug-resistant organisms. AIMS: To determine the bioburden on different types of high-touch surfaces, and to identify cultures to species level and stratify strains into those of low and high clinical relevance. DESIGN: Association between bioburden and presence of pathogens of high clinical relevance (PHCR) in a tertiary care centre and urban environment. METHODS: The overall bioburden measured by total colony count (TCC) was assessed using tryptic soy agar contact plates and two selective agars to improve detection of PHCR. Isolates were routinely identified to species level using matrix-assisted laser desorption/ionization - time of flight mass spectrometry (MALDI-TOF). The definition of PHCR was based on listings outlined by the Centers for Disease Control and Prevention. FINDINGS: In total, 1431 contact plates were processed from 477 surfaces: 153 from hospitals and 324 from publicly accessible institutions or devices. At least one PHCR was identified from cultures from 73 samples. TCC was found to be poorly correlated with the presence of PHCR. CONCLUSION: TCC poorly predicted the presence of PHCR, rendering the results from environmental sampling difficult to interpret. MALDI-TOF enables the identification of large numbers of isolates from the environment at low cost. Further studies on environmental contamination should use MALDI-TOF to identify all pathogens grown.

Association of time under immunosuppression and different immunosuppressive medication on periodontal parameters and selected bacteria of patients after solid organ transplantation.

BACKGROUND: Aim of this study was to investigate the association of the time under immunosuppression and different immunosuppressive medication on periodontal parameters and selected periodontal pathogenic bacteria of immunosuppressed patients after solid organ transplantation (SOT). MATERIAL AND METHODS: 169 Patients after SOT (lung, liver or kidney) were included and divided into subgroups according their time under (0-1, 1-3, 3-6, 6-10 and >10 years) and form of immunosuppression (Tacrolimus, Cyclosporine, Mycophenolate, Glucocorticoids, Sirolimus and monotherapy vs. combination). Periodontal probing depth (PPD) and clinical attachment loss (CAL) were assessed. Periodontal disease severity was classified as healthy/mild, moderate or severe periodontitis. Subgingival biofilm samples were investigated for eleven selected potentially periodontal pathogenic bacteria using polymerasechainreaction. RESULTS: The mean PPD and CAL as well as prevalence of Treponema denticola and Capnocytophaga species was shown to be different but heterogeneous depending on time under immunosuppression (p<0.05). Furthermore, only the medication with Cyclosporine was found to show worse periodontal condition compared to patients without Cyclosporine (p<0.05). Prevalence of Porphyromonas gingivalis, Tannerella forsythia and Fusobacterium nucleatum was reduced and prevalence of Parvimonas micra and Capnocytophaga species was increased in patients under immunosuppression with Glucocorticoids, Mycophenolate as well as combination therapy. CONCLUSION: Time under and form of immunosuppression might have an impact on the clinical periodontal and microbiological parameters of patients after SOT. Patients under Cyclosporine medication should receive increased attention. Differences in subgingival biofilm, but not in clinical parameters were found for Glucocorticoids, Mycophenolate and combination therapy, making the clinical relevance of this finding unclear.

Marker-trait association analysis for agronomic and compositional traits in sainfoin (Onobrychis viciifolia).

Sainfoin (Onobrychis viciifolia) is a perennial forage legume with great potential for use in sustainable agriculture due to its low input requirements, good drought tolerance, and production of forage rich in polyphenolic compounds, which are beneficial for animal health. However, its distribution and cultivation are limited due to its moderate agronomic performance and a general lack of well adapted, highly yielding cultivars. Faster progress in breeding is imperative, but is often hampered by the complex inheritance of traits and limited knowledge on the genetic composition of this tetraploid, outbreeding species. Molecular genetic tools might aid phenotypic selection; however, to date no information on marker-trait associations is available for sainfoin. Hence, the goal of the present study was to detect marker-trait associations in a biparental F1 population. Single plants were screened for recently developed genetic markers and phenotyped for important agronomic traits and concentrations of different polyphenolic compounds. Significant trait-associated markers (TAM) were detected for plant height (11), plant vigor (1), and seed yield (7). These three traits were positively correlated with each other and shared some TAMs. Correlations among markers suggested that two independent loci control these three vigor-related traits. One additional, independent TAM was detected for the share of prodelphinidins in total condensed tannins. Our results provide insight into the genetic control of important traits of sainfoin, and the TAMs reported here could assist selection in combination with phenotypic assessment.

A PCR-based tool for cultivation-independent detection and quantification of Metarhizium clade 1.

The entomopathogenic fungus Metarhizium anisopliae and sister species are some of the most widely used biological control agents for insects. Availability of specific monitoring and quantification tools are essential for the investigation of environmental factors influencing their environmental distribution. Naturally occurring as well as released Metarhizium strains in the environment traditionally are monitored with cultivation-dependent techniques. However, specific detection and quantification may be limited due to the lack of a defined and reliable detection range of such methods. Cultivation-independent PCR-based detection and quantification tools offer high throughput analyses of target taxa in various environments. In this study a cultivation-independent PCR-based method was developed, which allows for specific detection and quantification of the defined Metarhizium clade 1, which is formed by the species Metarhizium majus, Metarhizium guizhouense, Metarhizium pingshaense, Metarhizium anisopliae, Metarhizium robertsii and Metarhiziumbrunneum, formerly included in the M. anisopliae cryptic species complex. This method is based on the use of clade-specific primers, i.e. Ma 1763 and Ma 2097, that are positioned within the internal transcribed spacer regions 1 and 2 of the nuclear ribosomal RNA gene cluster, respectively. BLAST similarity searches and empirical specificity tests performed on target and non-target species, as well as on bulk soil DNA samples, demonstrated specificity of this diagnostic tool for the targeted Metarhizium clade 1. Testing of the primer pair in qPCR assays validated the diagnostic method for specific quantification of Metarhizium clade 1 in complex bulk soil DNA samples that significantly correlated with cultivation-dependent quantification. The new tool will allow for highly specific and rapid detection and quantification of the targeted Metarhizium clade 1 in the environment. Habitat with high Metarhizium clade 1 densities can then be analyzed for habitat preferences in greater detail using cultivation-dependent techniques and genetic typing of isolates.

Development of a single-nucleotide polymorphism (SNP) assay for genotyping of Pandora neoaphidis.

Pandora neoaphidis (Entomophthoromycotina, Entomophthorales) is one of the most important fungal pathogens of aphids with great potential as a biological control agent. Development of tools that allow high-resolution monitoring of P. neoaphidis in the environment is a prerequisite for the successful implementation of biological control strategies. In this study, a single-nucleotide polymorphism (SNP) assay was developed. The assay targets 13 SNPs identified in 6 genomic regions including the largest subunit of nuclear RNA polymerase II (RPB1) gene, the second-largest subunit of nuclear RNA polymerase II (RPB2) gene, the ß-tubulin (BTUB) gene, the elongation factor 1α-like (EFL) gene, the large subunit (LSU) rRNA gene, and the small subunit (SSU) rRNA gene together with the internal transcribed spacer (ITS). The assay allowed the discrimination of 15 different SNP profiles among 19 P. neoaphidis isolates and 4 P. neoaphidis-infected cadavers. Results showed that the assay is applicable to DNA extracted from infected aphids allowing genotyping of the fungus without cultivation. The SNP assay provides an efficient tool for investigation of population structures and dynamics of P. neoaphidis, as well as its persistence and epidemiology in agro-ecosystems. Furthermore, it constitutes a powerful approach for monitoring potential biological control strains of P. neoaphidis in the environment.

A PCR-based tool for the cultivation-independent monitoring of Pandora neoaphidis.

Pandora neoaphidis is one of the most important fungal pathogens of aphids and has a great potential for use in biocontrol. Little is known on how this fungus persists in an area and in particular on its overwintering strategies. It is hypothesized that natural areas play an important role for survival and that soil may serve as a source of inoculum for new aphid populations in spring. To test these hypotheses, a cultivation-independent PCR-based diagnostic tool was developed, that allows the detection of P. neoaphidis in the environment. Two P. neoaphidis specific PCR primer pairs were designed, targeting sequences in the ribosomal RNA gene cluster. Specificity of both primer pairs was demonstrated with P. neoaphidis and non-target close entomophthoralean relatives. Moreover, single amplicons of expected sizes were obtained with both primer pairs from various environmental sample types, including aphid cadavers, plant material, and soil. The PCR-based diagnostic tool was applied to investigate the persistence of P. neoaphidis in soil samples obtained in 2004/2005 from a nettle field harboring infected aphids in fall 2004. P. neoaphidis was detected in every sample collected in November 2004 and March 2005, suggesting an overwintering stage of P. neoaphidis in top soil layers. The developed cultivation-independent PCR-based tool will be valuable for further investigation of the ecology of P. neoaphidis and for the development and future implementation of management strategies against aphids involving conservation biocontrol.

Clinician response to Candida organisms in the urine of patients attending hospital.

The epidemiology of 54 episodes of candiduria with respect to clinical risk factors, species of Candida and physician response to the isolation of Candida in urine were studied in an observational survey over 3 months. Candida spp. were isolated from 4.7% of positive urine cultures. Common predisposing conditions included antibiotic use (74.1%), urinary drainage devices (57.4%), surgery (51.9%), intensive care unit (ICU) or high-dependency care unit (HDU) admission (42.6%) and urinary tract (UT) disease (18.5%). Upper UT infection was uncommon (n = 3). Of 65 Candida isolates, C. albicans predominated (85.2%), followed by C. glabrata (27.8%) and other Candida spp. (6.2%). All isolates were susceptible to fluconazole, itraconazole, voriconazole, amphotericin and caspofungin. Indwelling urinary catheters were removed in 76.2% of episodes. Antifungal therapy was initiated in 33.3% of cases independently of patient symptoms, underlying disease or Candida colony count. Patients in ICU/HDUs were significantly more likely to receive antifungal agents than those outside these units (p < 0.001). Fluconazole was the most common drug prescribed (77.8%). Clearance of candiduria occurred independently of antifungal therapy (p = 0.60). Physicians often did not follow up a positive urine result for Candida. Efforts to increase clinician awareness of current recommendations for managing candiduria and further study to elucidate specific risk factors in defined patient populations are warranted.

Changes in lead availability affect bacterial community structure but not basal respiration in a microcosm study with forest soils.

This study investigates the effects of Pb during time on the bacterial communities of forest soils using water-extractable Pb concentrations in the soil solution as predictors of Pb bioavailability. In a microcosm experiment we applied increasing concentrations of Pb(NO(3))(2) solutions (0.5, 2, 8, 32 mM) to 5 forest soils of pH<5 and to a calcareous soil of pH>6.5. Sampling of the microcosms was performed after 3, 30 and 90 days of incubation. Community analysis included basal respiration rates and changes in the structure of the bacterial communities through T-RFLP fingerprinting. We also investigated functional stability in terms of resistance, expressed as the effects on basal respiration after 3 days of incubation, and of resilience, expressed as the recovery of bacterial community structure and of respiration rates after 90 days of incubation. Water-extractable Pb increased with time in most of the soils, in parallel with an increase of water-extractable dissolved organic carbon (DOC). The increased concentrations slightly affected bacterial community structure, although OTU (operational taxonomic unit) richness was not significantly reduced with Pb concentrations in any of the soils. The highest Pb treatment (32 mM) caused significant effects on basal respiration in some of the acidic soils, but no clear trend was observed in relation to increased Pb bioavailability with time. Resistance to Pb additions was evident in five of the six soils, but only two showed resilience after 90 days. This is the first study showing the effects of time on Pb bioavailability in soils and on the resulting reactions of the soil microbial communities.

Thyrotoxicosis-induced prinzmetal variant angina.

We describe a postmenopausal women with new onset of variant angina caused by thyrotoxicosis due to Graves' disease. During exercise bicycle ergometry at 50 Watts, the patient developed typical angina with ST segment elevation in the precordial leads. A coronary angiogram revealed normal coronary arteries. Graves' disease with overt hyperthyroidism was diagnosed. After achieving an euthyroid state with administration of propylthiouracil, the symptoms resolved completely and the patient had a normal exercise capacity without electrocardiographic changes. Thus, we conclude that in patients with thyrotoxicosis, variant angina and normal coronary arteries, restoration of normal thyroid function may be curative.

Optimization of bulked AFLP analysis and its application for exploring diversity of natural and cultivated populations of red clover.

Landraces and wild populations of red clover (Trifolium pratense L.) may represent a significant yet poorly characterized genetic resource of temperate grasslands. A bulking strategy with amplified fragment length polymorphism (AFLP) markers was optimized to characterize 120 red clover populations in 6 different groups: Swiss wild clover populations, Mattenklee landraces, Mattenklee cultivars, field clover cultivars, Dutch wild clover populations, and Dutch landraces. Analysis of 2 bulked samples/population consisting of 20 plants each with12 AFLP primer combinations was found optimal for determining genetic diversity and relationships within and among red clover populations and groups. Swiss wild clover populations were clearly separated from all other red clover groups and variability within and among populations was shown to be particularly high in wild clover populations and Mattenklee landraces, emphasising their value as genetic resources for improvement of red clover cultivars, as well as for conservation and restoration of biodiversity. This study shows that the ancestry of red clover landraces is primarily found in introduced cultivars rather than in natural wild clover populations. In addition, the methodological considerations presented here may help improve diversity analyses using bulked samples.

Swiss Mattenklee landraces, a distinct and diverse genetic resource of red clover (Trifolium pratense L.).

Genetic variability within and among 19 landraces and cultivars of red clover ( Trifolium pratense L.) was investigated by means of amplified fragment length polymorphism (AFLP) analysis in order to assess the potential value of Swiss Mattenklee landraces as genetic resources for plant breeding and the preservation of biodiversity. Populations were classified into three groups according to their origin and agronomic features: Mattenklee landraces (8), Mattenklee cultivars (8) and field clover cultivars (3). Analysis of molecular variance based on 276 polymorphic AFLP markers revealed 80% of total variability to be due to variability within populations while 12% were attributed to variability among groups. Stepwise discriminant analysis identified a subset of 126 AFLP markers which best separated individual plants into the three respective groups. Genetic distances between populations were considerably larger among groups than among populations within the same group, providing further evidence for the genetic distinction between Mattenklee landraces, Mattenklee cultivars and field clover cultivars. AFLP markers identified two landrace clusters, containing three and four populations respectively, which, together with one additional landrace, may sufficiently represent the genetic variability of all eight landraces investigated. The results of this study strongly suggest that Swiss Mattenklee landraces form a genetically distinct group of red clover. The data obtained provide criteria on how to efficiently manage, preserve and exploit Mattenklee germplasm.

A strategy for optimizing quality and quantity of DNA extracted from soil.

The efficiency of a bead beating method was studied in detail with regard to a variety of factors including beating time and speed, volume and temperature of the buffer, as well as amount and type of beads employed. The results presented here reveal that all of these parameters have a significant effect on yield and quality of DNA extracted from soils. Precise adjustment of extraction conditions allows for significantly higher yields of high quality DNA from soils than previously reported. We further evaluated the effect of the extraction conditions on the apparent soil microbial community structures, as observed by polymerase chain reaction (PCR) and RFLP. Differences in the fingerprints of DNA extracted under different conditions suggest that results could be biased when using gentle extraction procedures. Based on multiple subsequent extractions using very harsh extraction conditions, we propose a protocol for the quantification of the total DNA content in soils. Extractions from six soils of different texture and chemical characteristics with selected bead beating protocols revealed that the quality (fragment size and purity) of the extracted DNA was generally very good, but also depended on the soil characteristics. While a single, general protocol for optimal DNA recovery from all soils cannot be given, this study provides detailed guidelines on how to optimize the general method to obtain optimal DNA from individual soils.

Dissection autoradiography: a screening technique using storage phosphor autoradiography to detect the biodistribution of radiolabelled compounds.

INTRODUCTION: This study reports an alternative, rapid, whole body autoradiography technique which utilises storage-phosphor imaging technology. Conventionally, tissue or whole body sections have been used to examine the distribution of radiolabelled test compounds. However, the information acquired relates only to the sections examined, and the amount of radioactivity within the whole organ cannot be quantified. We have developed a rapid semi-quantitative technique that produces a concise visual representation of the distribution of the isotope throughout the entire animal: dissection autoradiography (DAR). METHODS: By dissecting a mouse which has been administered 14C-labelled methotrexate (MTX) and drying the tissues on a gel dryer, whole organs and aliquots of body fluids can be exposed to a phosphor imaging plate. The data obtained was analysed with the software associated with the phosphor imaging system and, by using 14C standards, the amount of 14C per total organ or tissue was quantified relative to other samples. Another widely used method to detect radiolabelled material in vivo is tissue solubilisation (TS) followed by liquid scintillation counting (LSC). This conventional method was compared with DAR. RESULTS: The new technique described in this communication was found to have a high level of reproducibility (R(2)= 88-95%). Whilst DAR was less sensitive than TS and LSC, trends over time in the biodistribution of 14C-MTX throughout most tissues were consistent between techniques. DISCUSSION: Whilst TS and LSC was a more sensitive technique, it was labour intensive and expensive in terms of consumables and time when compared with DAR. Dissection autoradiography has the potential to be used to screen quickly large numbers of samples in the biodistribution studies of various conjugates, isomers, derivatives or formulations of a parent compound, following a variety of routes of administration.

Temporal and Spatial Distribution of the nifH Gene of N(2) Fixing Bacteria in Forests and Clearcuts in Western Oregon.

Decomposition of plant litter is a primary mechanism of nutrient recycling and redistribution in most terrestrial ecosystems. Previously we demonstrated by a nested PCR protocol that 20 distinctive nifH (the gene encoding nitrogenase reductase) HaeIII restriction fragment length polymorphism (RFLP) patterns were derived from bulk DNA associated with samples of plant litter and soil collected at one Douglas Fir (DF) forest [33]. Five of the nifH DNA patterns (II-VI) were dominant types in DF litter with characteristic fragments of 237-303 bp length, whereas samples from soil contained primarily seven other patterns 131-188 bp length (IX-XV). Here we report that the 237-303 bp fragments characteristic for forest litter could generally not be detected in plant litter or soil samples collected in clearcuts that adjoin the forest sites. The same fragments (237-303 bp) were also found in the litter at this DF forest site over 16 months and were consistently found in litter at 12 other DF forest or recent (<2 yrs) clearcut sites. However, trace to none of these fragments were detected in 6 clearcut (5-10 yrs) or different forest types (oak, alder) collected over a 200 km east-west direction in western Oregon, USA. Data suggest that the logging practice in DF forests that creates a clearcut removes a unique gene pool of nitrogen-fixing microorganisms. These organisms could potentially contribute more to nitrogen fixation in forest litter than litter from natural or invasive plants that grow in clearcuts [26].

Substrate preference profiles of proteases released by allergenic pollens.

BACKGROUND: Pollens are important triggers for allergic asthma and seasonal rhinitis. We have recently reported that proteases released by major allergenic pollens can injure airway epithelial cells in vitro. Disruption of epithelial integrity by proteases released following deposition of pollens on mucosal surfaces could promote sensitization and induce inflammation. OBJECTIVE: To compare protease activities released by allergenic pollens of various genera. METHODS: We used a rapid microassay which quantifies cleavage of dipeptide ester substrates to characterize the substrate preference profiles of serine proteases in diffusates of the pollens of perennial ryegrass (Lolium perenne), Kentucky blue grass (Poa pratensis), Bermuda grass (Cynodon dactylon), Western ragweed (Ambrosia spp.), white birch (Betula spp.) and Sydney golden wattle (Acacia longifolia). RESULTS: Comparison of the profiles revealed notable differences as well as similarities between serine protease activities released by these pollens. Diffusates of Kentucky blue grass pollen exhibited very high substrate preference for arginine and lysine. For other pollens, cleavage of the cysteine substrate was usually the most rapid and was associated with marked preference for leucine and methionine. There was considerable variation between these pollens in the rates of cleavage of the histidine substrate. In addition, we observed high rates of cleavage of arginine and lysine substrates by Acacia pollen diffusate. CONCLUSION: At least two dominant patterns of substrate preference are identifiable in the mixtures of proteases released by hydrated pollens. Purification of the proteases responsible for these patterns of activity will facilitate investigation of their role in airway epithelial injury and allergic disease.

Immobilization of highly concentrated cell suspensions using the laminar jet breakup technique

The controlled breakup of a disturbed jet is a well-known technique for monodisperse particle production and has been well-investigated for Newtonian and viscoelastic fluids. For the immobilization of cells in beads of a hydrogel it has been observed that there is a maximum cell concentration beyond which a monodisperse bead distribution cannot be reached. Higher concentrations lead to irregular jet breakup and thus to droplet coalescence forming beads with double or triple volume. This maximum cell concentration has been investigated for different operating conditions using latex beads of different diameters as a model substance for cells and baker's yeast as a comparison. The maximum cell concentration where a monodisperse size distribution could still be achieved was increased using an electrostatic droplet dispersion system.

Fingerprinting of mixed bacterial strains and BIOLOG gram-negative (GN) substrate communities by enterobacterial repetitive intergenic consensus sequence-PCR (ERIC-PCR).

PCR-based genomic fingerprinting by use of enterobacterial repetitive intergenic consensus primers (ERIC-PCR) was evaluated for its use in fingerprinting DNA of mixed Gram-negative bacterial strains and BIOLOG Gram-negative (GN) microplate substrate communities. ERIC-PCR fingerprints of six different pure bacterial strains and a combined mixture of the strains were compared with fingerprints obtained by two more established methods: amplified ribosomal DNA restriction analysis (ARDRA) and random amplified polymorphic DNA analysis (RAPD-PCR). The ERIC-PCR fingerprint of the mixed strains was highly reproducible and was more species-specific and representative of the individual strain fingerprints than the ARDRA and RAPD-PCR fingerprints, respectively. ERIC-PCR fingerprinting of model and rhizosphere BIOLOG GN substrate communities also provided clearly distinguishable fingerprints. Results of this study suggest that ERIC-PCR represents a rapid and highly discriminating method for fingerprinting DNA of mixed Gram-negative bacterial strains and BIOLOG GN substrate communities.

Comparison of Parental and Transgenic Alfalfa Rhizosphere Bacterial Communities Using Biolog GN Metabolic Fingerprinting and Enterobacterial Repetitive Intergenic Consensus Sequence-PCR (ERIC-PCR).

> Abstract Rhizosphere bacterial communities of parental and two transgenic alfalfa (Medicago sativa L.) of isogenic background were compared based on metabolic fingerprinting using Biolog GN microplates and DNA fingerprinting of bacterial communities present in Biolog GN substrate wells by enterobacterial repetitive intergenic consensus sequence-PCR (ERIC-PCR). The two transgenic alfalfa expressed either bacterial (Bacillus licheniformis) genes for alpha-amylase or fungal (Phanerochaete chrysosporium) genes for Mn-dependent lignin peroxidase (Austin S, Bingham ET, Matthews DE, Shahan MN, Will J, Burgess RR, Euphytica 85:381-393). Cluster analysis and principal components analysis (PCA) of the Biolog GN metabolic fingerprints indicated consistent differences in substrate utilization between the parental and lignin peroxidase transgenic alfalfa rhizosphere bacterial communities. Cluster analysis of ERIC-PCR fingerprints of the bacterial communities in Biolog GN substrate wells revealed consistent differences in the types of bacteria (substrate-specific populations) enriched from the rhizospheres of each alfalfa genotype. Comparison of ERIC-PCR fingerprints of bacterial strains obtained from substrate wells to substrate community ERIC-PCR fingerprints suggested that a limited number of populations were responsible for substrate oxidation in these wells. Results of this study suggest that transgenic plant genotype may affect rhizosphere microorganisms and that the methodology used in this study may prove a useful approach for the comparison of bacterial communities.