Ranking the magnitude of crop and farming system effects on soil microbial biomass and genetic structure of bacterial communities

Biological soil characteristics such as microbial biomass, community structures, activities, and functions may provide important information on environmental and anthropogenic influences on agricultural soils. Diagnostic tools and detailed statistical approaches need to be developed for a reliable evaluation of these parameters, in order to allow classification and quantification of the magnitude of such effects. The DOK long-term agricultural field experiment was initiated in 1978 in Switzerland for the evaluation of organic and conventional farming practices. It includes three representative Swiss farming systems with biodynamic, bio-organic and conventional fertilization and plant protection schemes along with minerally fertilized and unfertilized controls. Effects on microbial soil characteristics induced by the long-term management at two different stages in the crop rotation, i.e. winter wheat after potato or corn, were investigated by analyzing soil bacterial community structures using analysis of PCR-amplified rRNA genes by terminal restriction fragment length polymorphism and ribosomal intergenic spacer analysis. Application of farmyard manure consistently revealed the strongest influence on bacterial community structures and biomass contents. Effects of management and plant protection regimes occurred on an intermediate level, while the two stages in the crop rotation had a marginal influence that was not significant.     

Seasonal and management influences on bacterial community structure in an upland grassland soil

Floristically diverse Nardo-Galion upland grasslands are common in Ireland and the UK and are valuable in agricultural, environmental and ecological terms. Under improvement (inputs of lime, fertiliser and re-seeding), they convert to mesotrophic grassland containing very few plant species. The effects of upland grassland improvement and seasonality on soil microbial communities were investigated at an upland site. Samples were taken at five times in one year in order to observe seasonal trends, and bacterial community structure was monitored using automated ribosomal intergenic spacer analysis (ARISA), a DNA-fingerprinting approach. Differences in soil chemistry and bacterial community structure between unimproved and improved grassland soils were noted. Season was also found to cause mild fluctuations in bacterial community structure, with soil samples from colder months (October and December) more correlated with change in ribotype profiles than samples from warmer months. However, for the majority of seasons clear differences in bacterial community structures from unimproved and improved soils could be seen, indicating seasonal influences did not obscure effects associated with improvement.     

Environmental effects on biofilm bacterial communities: a comparison of natural and anthropogenic factors in New Zealand streams

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Isolation and characterization of 2,4-dichlorophenoxyacetic acid-catabolizing bacteria and their biodegradation efficiency in soil

Bacterial isolates (NJ 10 and NJ 15) capable of degrading the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) were isolated from agricultural soil by enrichment culture technique. The isolates exhibited substantial growth in mineral salt medium supplemented with 0.1–0.5% of 2,4-D as a sole source of carbon and energy. Based on their morphological, cultural and biochemical characteristics, the isolates NJ 10 and NJ 15 have been identified as Pseudomonas species and Pseudomonas aeruginosa, respectively. Biodegradation studies in a soil microcosm enriched with pure cultures of the isolates demonstrated a time-dependent disappearance of 2,4-D from the 100 mg/kg herbicide-amended soil. The HPLC data analysis revealed 96.6 and 99.8% degradation in the soil inoculated with the pure cultures of isolates NJ 10 and NJ 15, respectively with in 20 days of incubation at 30 °C. Both the isolates showed significant solubilization of inorganic phosphate [Ca₃(PO₄)₂] on the specific Pikovskaya's medium.     

Distribution and N2-fixing activity of Frankia strains in relation to soil depth

The qualitative and quantitative distribution of Frankia strains infective on Elaeagnus angustifolia L. from different depths in the same soil (10–20 cm; 30–40 cm; 50–60 cm) were compared. The soil samples collected from a natural stand devoid of Elaeagnaceae were planted with Elaeagnus angustifolia L. seedlings. All plants became nodulated, demonstrating that Frankia strains were present at least down to 60 cm in the soil. The decreased density of Frankia strains was correlated to the decline of soil organic matter content with soil depth. DNA extracted from nodules produced on Elaeagnus angustifolia L. seedlings were polymerase chain reaction (PCR) characterized by amplifying the nif D-K intergenic spacer (IGS), using the polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP) analysis. This showed the presence of seven nif-Hae III profiles within this Frankia community. Diversity of Frankia strains was maintained throughout the soil column, but the relative distribution of strains varied. Some nif-Hae III profiles were only found in the deeper soil, suggesting different selective advantages to withstand the constraints of soil depth. ¹⁵N₂ experiments indicated that all the strains tested had N₂-fixing activity. However, efficiency was not significantly different among nodules of different nif-Hae III profiles. Therefore, N₂-fixing activity does not seem to be the main factor responsible for the different distribution of Frankia strains at different soil depths.     

Monitoring the persistence of Laccaria bicolor as an ectomycorrhizal symbiont of nursery-grown Douglas fir by PCR of the rDNA intergenic spacer

The large-scale inoculation of selected beneficial ectomycorrhizal fungi in forest nurseries has generated renewed interest in the ecology of these symbiotic fungi. However, information on the dissemination and persistence of introduced symbionts is scarce due to the limitation of the current identification methods. To identify ectomycorrhizal fungi on single root tips, we investigated the polymorphism of the PCR-amplified ribosomal DNA intergenic spacer (IGS) from a wide range of ectomycorrhizal fungi. To investigate the reliability of this molecular approach in large-scale surveys, the dissemination and persistence on Douglas fir seedlings of the introduced Laccaria bicolor S238N were assessed in a forest nursery in the Massif Central (France). Several hundred ectomycorrhizas and fruiting bodies were sampled from plots where control and L. bicolor inoculated-Douglas fir seedlings were grown for 1.5 years. PCR typing of mycorrhizas indicated that trees inoculated with L. bicolor S238N remained exclusively colonized by that isolate (or sexually derived isolates) for the entire test period. In contrast, control seedlings were infected by indigenous isolates of Laccaria laccata and Thelephora terrestris. The molecular evidence for the persistence of the introduced mycobiont despite the competition from indigenous isolates of the same species provides further illustration of the potential of exotic species for large-scale microbial application.     

Molecular diversity studies of bacterial communities of oil polluted microbial mats from the Etang de Berre (France)

The biodiversity of microbial mats inhabiting the oil-contaminated lagoon Etang de Berre was determined by molecular approaches. The fingerprint of denaturing gradient gel electrophoresis (DGGE) and automatic ribosomal intergenic spacer analysis (ARISA) of mats exposed to different pollution levels showed specific microbial communities for each site but similar diversity richness. Species composition of the mats were compared by constructing 16S rRNA libraries. Amplified rDNA restriction analysis (ARDRA) of clone libraries confirmed their similar level of diversity richness. Phylogenetic analysis of the 16S rRNA sequences showed that the classes gamma and alpha of Proteobacteria were abundantly present in both sites whereas phylotypes related to the delta-Proteobacteria and to the uncultured WS3 group were mainly found in the site with the highest pollution. Identification of the species involved in oil degradation by combining culture-based approaches and DGGE, showed that enrichment cultures were constituted by members of the Rhodobacterales and species related to Rhodococcus, Sphingomonas, Xanthomonas and Microbacterium, all of them known for their ability to degrade hydrocarbons. Our findings suggest that oil pollution has not affected the biodiversity richness of the mats. However, the populations involved in hydrocarbon degradation represent a minor fraction of the mat communities in the Etang de Berre.     

Morphological and molecular identification of the metacestode parasitizing the liver of rodent hosts in bamboo growing areas of mizoram, northeast India

In Mizoram (Northeast India), rodent outbreaks are known to occur periodically with the onset of bamboo flowering causing a tremendous destruction to food grains and as per the folk belief, often resulting in famine. In an exploratory survey of rodent pests in bamboo growing atreas for their helminth parasite spectrum, metacestodes of tapeworms were frequently encountered infecting the liver lobes and body cavity of the host. The morphological criteria were found to be closely consistent with the metacestode of Taenia species. In molecular characterization of the parasite, the ribosomal DNA (ITS1, ITS2) and mitochondrial COI were amplified and sequenced. Based upon both morphological data and molecular analysis using bioinformatic tools, the metacestode is identified as confirmed to be representing Cysticercus fasciolaris. The adult form of which (Taenia taeniaeformis) commonly occurs in felid and canid mammalian hosts.     

Fate of the herbicides mecoprop, dichlorprop, and 2,4-D in aerobic and anaerobic sewage sludge as determined by laboratory batch studies and enantiomer-specific analysis

Aerobic degradation experiments with the racemic mixtures of mecoprop and dichlorprop revealed that activated sludge collected from the aeration tank of a municipal waste water treatment plant degraded both enantiomers of mecoprop and dichlorprop within 7 days, albeit in an enantioselective manner; the (S) enantiomers were preferentially degraded. Mecoprop, dichlorprop, and 2,4-D were completely metabolized under aerobic conditions, as shown by the 86–98% elimination of dissolved organic carbon. Under anaerobic conditions, the concentration of 2,4-D decreased exponentially with a first-order reaction rate constant of 0.24 per day and without a lag-phase. After an incubation time of 17 days, 2,4-D was completely removed. 2,4-Dichlorophenol was the main metabolite of anaerobic 2,4-D degradation; only traces of 4-chlorophenol were detected. In contrast, the chiral phenoxypropionic acid herbicides mecoprop and dichlorprop persisted under anaerobic conditions during 49 days of incubation.     

KT、2,4-D和NAA对黄独组培苗带芽茎段生长发育的影响

以黄独组培苗带芽茎段为材料,采用植物组织培养和单因子实验法,研究添加KT和2,4-D或KT和NAA组合的MS培养基对黄独带芽茎段再生的影响。结果表明,MS+KT 2.0 mg/L+NAA 0.5 mg/L有利于黄独带芽茎段腋芽萌发和根系生长。     

Relative importance of the effect of 2,4-D,glyphosate, and environmental variables on the soil microbial biomass

Two post-emergence herbicides (glyphosate and 2,4-D) were applied at field application levels to tilled field plots in a mixed cropping area in south-central Alberta. The effects of these chemicals on certain variables associated with microbial biomass and activity were monitored in these plots (as well as corresponding control plots) for 45 days. Glyphosate did not influence any of the microbial variables tested. Addition of 2,4-D significantly influenced all microbial variables investigated but these effects were transient, being detectable only within the first 1–5 days of herbicide addition. The effects of 2,4-D addition on the microbial variables tested, even when significant, were typically small and probably of little ecological consequence especially when spatial and temporal variation in these variables is taken into account.     

Impact of Polyacrylamide Treatment on Sorptive Dynamics and Degradation of 2,4-D and Atrazine in Agricultural Soil

High-molecular-weight, anionic polyacrylamide (PAM) is added to irrigation water to reduce soil erosion during furrow irrigation of crops. The chemical nature of PAM, together with the observation that the polymer can be biotransformed by soil bacteria, led us to question the impact of PAM treatment on the fate of coapplied agrochemicals. The herbicides, atrazine (nonionic) and 2,4-D (anionic), were tested for pesticide sorption, desorption, and degradation in PAM-treated and untreated soils. Sorption of atrazine and 2,4-D in soil was unaffected by PAMtreatment, as was atrazine desorption. However, 2,4-D desorbedmore readily from the PAM-treated soil than from untreated soil. With respect to pesticide degradation, mineralization of the 2,4-D aromatic ring was not impacted by PAM treatment, but decarboxylation of the 2,4-D carboxylic acid side chain was significantly reduced in the PAM-treated soil. Limited mineralization (7 to 10%) of atrazine was observed in both soils. However, in PAM-treated soils atrazine conversion to ¹⁴CO₂ and bound residue components was significantly reduced, and there was an increase in the level of methanol extractable metabolites. These results may indicate that PAM application can alter the environmental fate of some pesticides in soils, especially under the high dose treatment conditions examined in this study.     

Decarboxylase activity involved in methyl ketone production by Staphylococcus carnosus 833, a strain used in sausage fermentation

Outbreaks of Vibrio parahaemolyticus gastroenteritis in the United States (Texas, New York and Pacific Northwest) in 1997-98 emphasized the need to develop molecular methods for identification and differentiation of these organisms. When outbreak isolates were analyzed for the enterobacterial repetitive intergenic consensus sequences, the Texas and New York outbreak isolates had a specific 850-bp DNA fragment that was absent in Pacific Northwest isolates. The 850-bp polymerase chain reaction (PCR) product was found in isolates of serovar O3:K6, which have an unusual potential to spread and cause infections. To develop a specific molecular detection method for serovar O3:K6, the nucleotide sequence of the 850-bp product was determined. The GenBank blast analysis did not show homology with any known Vibrio spp. gene sequences. Two PCR primers were designed to specifically amplify the unique sequences from serovar O3:K6 isolates. Genomic DNA from 10 Texas, eight New York, and seven Pacific Northwest outbreak isolates of V. parahaemolyticus was assayed by PCR. Texas and New York isolates were positive in the PCR assay, giving a 327-bp PCR product as predicted; however, Pacific Northwest isolates were negative, indicating the absence of the target gene. Texas and New York isolates were all serovar O3:K6; the Pacific Northwest isolates were not. The primers were tested with other Vibrio spp. and other closely related species and no amplification of the 327-bp PCR product was found. The PCR method can be used to specifically identify O3:K6 V. parahaemolyticus isolates in less than 6 h.     

2,4-Dichlorophenoxyacetic Acid (2,4-D) Utilization by Delftia acidovorans MC1 at Alkaline pH and in the Presence of Dichlorprop is Improved by Introduction of the tfdK Gene

Growth of Delftia acidovorans MC1 on 2,4-dichlorophenoxyacetic acid (2,4-D) and on racemic 2-(2,4-dichlorophenoxy)propanoic acid ((RS)-2,4-DP) was studied in the perspective of an extension of the strain’s degradation capacity at alkaline pH. At pH 6.8 the strain grew on 2,4-D at a maximum rate (μ_max) of 0.158 h⁻¹. The half-maximum rate-associated substrate concentration (K_s) was 45 μM. At pH 8.5 μ_max was only 0.05 h⁻¹ and the substrate affinity was mucher lower than at pH 6.8. The initial attack of 2,4-D was not the limiting step at pH 8.5 as was seen from high dioxygenase activity in cells grown at this pH. High stationary 2,4-D concentrations and the fact that μ_max with dichlorprop was around 0.2 h⁻¹ at both pHs rather pointed at limited 2,4-D uptake at pH 8.5. Introduction of tfdK from D. acidovorans P4a by conjugation, coding for a 2,4-D-specific transporter resulted in improved growth on 2,4-D at pH 8.5 with μ_max of 0.147 h⁻¹ and K_s of 267 μM. Experiments with labeled substrates showed significantly enhanced 2,4-D uptake by the transconjugant TK62. This is taken as an indication of expression of the tfdK gene and proper function of the transporter. The uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP) reduced the influx of 2,4-D. At a concentration of 195 μM 2,4-D, the effect amounted to 90% and 50%, respectively, with TK62 and MC1. Cloning of tfdK also improved the utilization of 2,4-D in the presence of (RS)−2,4-DP. Simultaneous and almost complete degradation of both compounds occurred in TK62 up to D = 0.23 h⁻¹ at pH 6.8 and up to D = 0.2 h⁻¹ at pH 8.5. In contrast, MC1 left 2,4-D largely unutilized even at low dilution rates when growing on herbicide mixtures at pH 8.5.     

Molecular structure of the Frankia spp. nifD—K intergenic spacer and design of Frankia genus compatible primer

The nifD—K intergenic spacer (IGS) of ArI3 and ACoN24d were found to have a length 265 and 199 nucleotides, respectively. They are markedly less conserved than the two neighbouring genes and have, in some instances, a repeated structure reminiscent of an insertion event The repeated sequence and the IGSs have no detectable homology with sequences in DNA databanks. The IGS has a stem-loop structure with a low folding energy, lower than that between nifH and nifD. No convincing alignment of IGS sequences could be obtained among Frankia strains. Only between ACoN24d and ArI3, which belong to the same genomic species, was the alignment good enough to permit detection of a doubly repeated structure. No promoter could be detected in the IGSs. The putative nifK open reading frame (ORF) in Frankia strain ArI3 has a length of 1587 nucleotides, starting with a GTG codon, preceded by a ribosome binding site of a structure similar to that of nifH (GGAGGN₇). The codon usage was similar to that of previously sequenced Frankia genes with a strong bias toward G- and C-ending codons except in the case of glycine where GGT is frequent. Alignment of the three Frankia nifK sequences (EUN1f; ArI3 and ACoN24d) with those of other nitrogen-fixing bacteria permitted detection of a sequence conserved among the three Frankia strains but absent in the other sequences. A primer targeted to that region in combination with FGPD807-85 amplified the nifD—K IGS sequences of all Frankia strains (except the non-nitrogen-fixing Frankia strains CN3 and AgB1-9) and yet failed to amplify DNA of all other nitrogen–fixing bacteria. Conversely, the failure of primer FGPK700′-92 to amplify Alnus-infective strains could be explained by point mutations in the 3′ part of the primer.     

Effects of<Emphasis Type="Italic"> Vitreoscilla</Emphasis> hemoglobin on the 2,4-dinitrotoluene (2,4-DNT) dioxygenase activity of<Emphasis Type="Italic"> Burkholderia</Emphasis> and on 2,4-DNT degradation in two-phase bioreactors

Expression of vgb, encoding Vitreoscilla hemoglobin (VHb), in Burkholderia strain YV1 was previously shown to improve cell growth and enhance 2,4-dinitrotoluene (2,4-DNT) degradation compared with control strain DNT, especially under hypoxic conditions. In the work reported here, the ratio of 2,4-DNT degraded to oxygen uptake was approximately 5-fold larger for strain YV1 than for strain DNT. The addition of purified VHb to cytosolic fractions of strain DNT increased 2,4-DNT degradation 1.5-fold, compared with 1.1-fold for control bovine Hb, but increased the 2,4-DNT degradation 2.7-fold when added to partially purified 2,4-DNT dioxygenase, compared with 1.3-fold for bovine Hb. This suggests a direct transfer of oxygen from VHb to the oxygenase. In a bioreactor at high 2,4-DNT concentration (using 100 ml oleyl alcohol containing 2 g 2,4-DNT as the second phase) with 1.5 l culture, both strains could remove 0.8 g 2,4-DNT by 120 h; and, under the same conditions in a fed-batch reactor, the degradation increased to 1 g for strain YV1 but not for strain DNT.     

A comparison of bacteria and benthic invertebrates as indicators of ecological health in streams

1. We set out to evaluate the reliability of bacterial communities as an indicator of freshwater ecological health.
2. Samples of epilithic biofilm were taken over a 1-year period from four streams, each impacted by varying degrees of human modification. The bacteria within each sample were characterised using a whole community DNA fingerprinting technique (automated ribosomal intergenic spacer analysis). Spatial and temporal differences in community structure between samples were visualised using multi-dimensional scaling and quantified using permutational multivariate anova . Macrobenthic invertebrates, which are commonly used as indicators of stream ecological health, were also sampled for comparison.
3. Multivariate analysis revealed a clear gradient in macroinvertebrate community structure between sites exposed to increased human impact. Bacterial communities, however, could only distinguish the most impacted site from the remainder.
4. Additional research is required to increase the sensitivity of bacterial community analyses before endorsing their use as an indicator of freshwater ecological health.