The Use of Genus-Specific Amplicon Pyrosequencing to Assess Phytophthora Species Diversity Using eDNA from Soil and Water in Northern Spain

Phytophthora is one of the most important and aggressive plant pathogenic genera in agriculture and forestry. Early detection and identification of its pathways of infection and spread are of high importance to minimize the threat they pose to natural ecosystems. eDNA was extracted from soil and water from forests and plantations in the north of Spain. Phytophthora-specific primers were adapted for use in high-throughput Sequencing (HTS). Primers were tested in a control reaction containing eight Phytophthora species and applied to water and soil eDNA samples from northern Spain. Different score coverage threshold values were tested for optimal Phytophthora species separation in a custom-curated database and in the control reaction. Clustering at 99% was the optimal criteria to separate most of the Phytophthora species. Multiple Molecular Operational Taxonomic Units (MOTUs) corresponding to 36 distinct Phytophthora species were amplified in the environmental samples. Pyrosequencing of amplicons from soil samples revealed low Phytophthora diversity (13 species) in comparison with the 35 species detected in water samples. Thirteen of the MOTUs detected in rivers and streams showed no close match to sequences in international sequence databases, revealing that eDNA pyrosequencing is a useful strategy to assess Phytophthora species diversity in natural ecosystems.     

Allium cepa assay based comparative study of selected vegetables and the chromosomal aberrations due to heavy metal accumulation

Irrigation of industrial effluents may end in the bioaccumulation of various toxic metals and consequent genetic changes in contaminated food crops. To test this hypothesis and extent of genetic modifications, Allium cepa test was performed to food crops viz. tomato (Lycopersicum esculentum) and chili (Capsicum annum) as Allium cepa test is a useful tool to assess genetic variations in plants. Prior to A. cepa test, the plants were exposed to various metal concentrations 125–1000 mg/L in the synthetic wastewater. The extracts of harvested plants were used to grow the root of A. cepa following its standard method. The root tips were fixed, stained and examined under compound microscope (almost 300–400 dividing cells) to check the extent of chromosomal variations during various stages of mitosis. The results revealed various chromosomal abnormalities including laggards, stickiness, vagrant chromosomes, binucleated cells, nuclear lesions, giant cells and c-mitosis at different level of treatment. On the whole, aberrations were increasing with the increasing doses along the positive control. In comparison, chili crop had higher level of aberrations depicting the higher chromosomal changes. Lower mitotic index (MI) with increasing level of doses was also describing the hampered cell division due to increased metal stress. The study is showing that the cell division was ceased with increasing metal stress thus increasing the rate of cell aberrations.     

Chlorinated phenoxyacetic acids and chlorophenols in the modified Allium test

The chlorinated phenoxyacetic acids 2-methyl-4-chlorophenoxyacetic acid (MCPA), 2,4-dichlorophenoxyacetic acid (2,4-D), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) and 2,4,5-T butoxyethyl ester and the chlorophenols 2,4-dichlorophenol and 2,4,5-trichlorophenol were tested for genotoxicity in the modified Allium test, which is based on exposure to the test chemicals of growing onions. The mean length of growing roots were measured and chromosome damage was recorded. Of the substances tested, MCPA was the most toxic and the chlorophenoxyacetic acids were more toxic than the chlorophenols. The lower threshold values for growth retardation were below 0.1 ppm for the acids, approx. at 0.1 ppm for the ester and less than 5 ppm for the phenols. Though a monocotyledon, Allium cepa was sensitive enough to respond to even low concentrations of these dicotyledon-selecting pesticides.     

Molinate biodegradation in soils: natural attenuation versus bioaugmentation

The aims of the present study were to assess the potential of natural attenuation or bioaugmentation to reduce soil molinate contamination in paddy field soils and the impact of these bioremediation strategies on the composition of soil indigenous microbiota. A molinate mineralizing culture (mixed culture DC) was used as inoculum in the bioaugmentation assays. Significantly higher removal of molinate was observed in bioaugmentation than in natural attenuation microcosms (63 and 39 %, respectively) after 42 days of incubation at 22 °C. In the bioaugmentation assays, the impact of Gulosibacter molinativorax ON4^T on molinate depletion was observed since the gene encoding the enzyme responsible for the initial molinate breakdown (harboured by that actinobacterium) was only detected in inoculated microcosms. Nevertheless, the exogenous mixed culture DC did not overgrow as the heterotrophic counts of the bioaugmentation microcosms were not significantly different from those of natural attenuation and controls. Moreover, the actinobacterial clone libraries generated from the bioaugmentation microcosms did not include any 16S rRNA gene sequences with significant similarity to that of G. molinativorax ON4^T. The multivariate analysis of the 16S rRNA DGGE patterns of the soil microcosm suggested that the activity of mixed culture DC did not affect the soil bacterial community structure since the DGGE patterns of the bioaugmentation microcosms clustered with those of natural attenuation and controls. Although both bioremediation approaches removed molinate without indigenous microbiota perturbation, the results suggested that bioaugmentation with mixed culture DC was more effective to treat soils contaminated with molinate.     

Genotoxic potential of the latex from cotton-leaf physicnut (Jatropha gossypiifolia L.)

Jatropha gossypiifolia L. (Euphorbiaceae), popularly known as cotton-leaf physicnut, is a milky shrub notable for its medicinal properties. The present study aimed to evaluate the toxic, cytotoxic and genotoxic effects of the latex of J. gossypiifolia, using Allium cepa L. as test system. Seeds of A. cepa were exposed to five concentrations of the latex (1.25; 2.5; 5; 10 and 20 mL/L) in order to evaluate parameters of toxicity (evaluation of root growth), cytotoxicity (mitotic index frequency) and genotoxicity (frequency of chromosome alterations). The latex showed a significant decrease in root mean growth value as well as mitotic index for the tested concentrations, except for 1.25 mL/L, when compared to results from the negative control. The 1.25, 2.5 and 5 mL/L concentrations induced significant chromo-some adherences, C-metaphases and/or chromosome bridges, as genotoxic effects. The significant frequency of chromosome bridges also indicated mutagenic potential for chromosomes of J. gossypiifolia as discussed in the paper. Considering that the latex is used in popular therapies, and that the test system A. cepa presents good correlation with tests carried out in mammals, it can be pointed out that its use for medicinal purposes may be harmful to human health especially if ingested.     

A feasibility study for the in situ remediation of a former tank farm

A feasibility study of the in situ remediation of a former tank farm (on a petrol station) was made over a period of 150 days at 10 °C. The natural attenuation (which is a sum of the abiotic losses and the natural biodegradation by the indigenous soil microorganisms) and the effect of biostimulation by inorganic nutrient supply were investigated. The contamination was not homogeneously distributed in the seven soil samples investigated. Nutrient addition had no statistically significant effect on hydrocarbon decontamination. A remarkable part of the decontamination had to be attributed to natural attenuation. Soil microbial counts and CO₂ evolution indicated a negative effect of nutrition on the number of microbial hydrocarbon degraders and on soil microbial activity.     

Effect of the Spartina alterniflora Root-Rhizome System on Salt Marsh Soil Denitrifying Bacteria

Nitrous oxide (N₂O) reductase activity was used as an index of the denitrification potential in salt marsh soils. In a short Spartina alterniflora marsh, the seasonal distribution of N₂O reductase activity indicated a causal relationship between S. alterniflora root-rhizome production and the denitrification potential of the soil system. The relationship was not discerned in samples from a tall S. alterniflora marsh. To further examine the in situ plant-denitrifier interaction in the short S. alterniflora marsh, plots with and without living S. alterniflora were established and analyzed for N₂O reductase activity 5 and 18 months later. In the plots without living Spartina there was a significant reduction in the soil denitrification potential after 18 months, indicating that in the SS marsh the denitrifiers are tightly coupled to the seasonal production of below-ground Spartina macroorganic matter. In plots with intact Spartina, the soil denitrification potential was not altered by NH₄NO₃ or glucose enrichment. However, in plots without living Spartina, there were significant changes in soil N₂O reductase activity, thus indicating that the plants can serve as a “buffer” against this form of pulse perturbation.     

Bacillus thuringiensis Isolates from Great Nicobar Islands

Bacillus thuringiensis (Bt) strains were isolated from soil samples of Great Nicobar Islands, one of the “hottest biodiversity hotspots,” where no collection has been characterized previously. The 36 new Bt isolates were obtained from 153 samples analyzed by crystal protein production with light/phase-contrast microscopy, determination of cry gene profile by SDS-PAGE, evaluation of toxicity against Coleopteran, and Lepidopteran insect pests, finally cloning and sequencing. Majority of the isolates showed the presence of 66–35 kDa protein bands on SDS-PAGE while the rest showed >130, 130, 73, and 18 kDa bands. The variations in crystal morphology and mass of crystal protein(s) purified from the isolates of Bt revealed genetic and molecular diversity. Based on the toxicity test, 50 % of isolates were toxic to Ash weevils, 16 % isolates were toxic to cotton bollworm, 38 % isolates were toxic both to ash weevil as well as cotton bollworm, while 11 % of the isolates did not exhibit any toxicity. PCR analysis unveiled prepotency of cry1B- and cry8b-like genes in these isolates. This study appoints the first isolation and characterization of local B. thuringiensis isolates in Great Nicobar Islands. Some of these isolates display toxic potential and, therefore, could be adopted for future applications to control some agriculturally important insect pests in the area of integrated pest management for sustainable agriculture.     

The effect of plant succession on slope stability

The aim of this field investigation was to study the enrichment of biodiversity of the slope at an early phase of succession, initiated by selected pioneers, and to study how this enrichment related to enhancement of the slope stability. Four experimental plots, with differing plant pioneers and number of species (diversity), were designed in order to assess the effects of plant succession on slope stability. Plant growth pattern was assessed by observing the increment in species diversity (number), species frequency and plant biomass. Higher vegetation biomass in a mixed culture situation (LLSS) in the field with Leucaena leucocephala as a pioneer, marked an increase in species diversity after 24 months of observation. In contrast, G (grasses and legume creepers) plot revealed the slowest rate of succession and the lowest above-ground biomass amongst the plots. The mixed-culture plot without L. leucocephala (SS) had also shown a lower biomass, a similar phenomenon observed in a plot grown by L. leucocephala (LL) with low plant diversity. Consequently, these plant growth patterns gave a positive effect on slope stability where the regression study showed that the shear strength was much affected by plant biomass. Meanwhile, throughout the succession process in LLSS plot, root length density reached the highest value amongst the plots, 23 Km m^?3. In relation to this, the saturation level of the slope indicates the unsaturated condition of the soil which resulted in the enhancement of both soil penetrability and soil shear strength of the plot. These attributes reveal a strong positive relationship between the process of natural succession and the stability of slopes.     

Response of a diuron-degrading community to diuron exposure assessed by real-time quantitative PCR monitoring of phenylurea hydrolase A and B encoding genes

A real-time quantitative PCR method was developed to detect and quantify phenlylurea hydrolase genes’ (puhA and puhB) sequences from environmental DNA samples to assess diuron-degrading genetic potential in some soil and sediment microbial communities. In the soil communities, mineralization rates (determined with [ring-¹⁴C]-labeled diuron) were linked to diuron-degrading genetic potentials estimated from puhB number copies, which increased following repeated diuron treatments. In the sediment communities, mineralization potential did not depend solely on the quantity of puhB copies, underlining the need to assess gene expression. In the sediment samples, both puhB copy numbers and mineralization capacities were highly conditioned by whether or not diuron-treated soil was added. This points to transfers of degradative potential from soils to sediments. No puhA gene was detected in soil and sediment DNA extracts. Moreover, some sediments exhibited high diuron mineralization potential even though puhB genes were not detected, suggesting the existence of alternative diuron degradation pathways.     

湘南红壤丘陵区不同植被类型下土壤肥力特征

以自然植被恢复长期定位试验为基础,通过分析自然恢复31a后形成的6个植被类型区(樟树、枫树、梓树、白檵木、唐竹、白茅草)、2个同期种植的人工植被区(湿地松、板栗)以及相邻裸地区0—100 cm土层pH值、有机质及主要养分含量的变化,明确了湘南红壤丘陵区不同植被类型对土壤肥力的影响。结果表明:(1)白茅草和唐竹区的土壤pH值显著高于裸地区,但枫树和白檵木区的土壤酸化明显。(2)土壤有机质、活性有机质、全P、速效P等指标表现为乔木草本灌木,碱解N、全K、速效K表现为灌木乔木草本,全N表现为乔木灌木草本。(3)土壤综合肥力优劣为:枫树区梓树区白檵木区樟树区唐竹区白茅草区湿地松区板栗区裸地区,自然恢复植被比人工植被更有利于土壤肥力的提高。     

Multiple Pathways of Plasmid DNA Transfer in Helicobacter pylori

Many Helicobacter pylori (Hp) strains carry cryptic plasmids of different size and gene content, the function of which is not well understood. A subgroup of these plasmids (e.g. pHel4, pHel12), contain a mobilisation region, but no cognate type IV secretion system (T4SS) for conjugative transfer. Instead, certain H. pylori strains (e.g. strain P12 carrying plasmid pHel12) can harbour up to four T4SSs in their genome (cag-T4SS, comB, tfs3, tfs4). Here, we show that such indigenous plasmids can be efficiently transferred between H. pylori strains, even in the presence of extracellular DNaseI eliminating natural transformation. Knockout of a plasmid-encoded mobA relaxase gene significantly reduced plasmid DNA transfer in the presence of DNaseI, suggesting a DNA conjugation or mobilisation process. To identify the T4SS involved in this conjugative DNA transfer, each individual T4SS was consecutively deleted from the bacterial chromosome. Using a marker-free counterselectable gene deletion procedure (rpsL counterselection method), a P12 mutant strain was finally obtained with no single T4SS (P12ΔT4SS). Mating experiments using these mutants identified the comB T4SS in the recipient strain as the major mediator of plasmid DNA transfer between H. pylori strains, both in a DNaseI-sensitive (natural transformation) as well as a DNaseI-resistant manner (conjugative transfer). However, transfer of a pHel12::cat plasmid from a P12ΔT4SS donor strain into a P12ΔT4SS recipient strain provided evidence for the existence of a third, T4SS-independent mechanism of DNA transfer. This novel type of plasmid DNA transfer, designated as alternate DNaseI-Resistant (ADR) mechanism, is observed at a rather low frequency under in vitro conditions. Taken together, our study describes for the first time the existence of three distinct pathways of plasmid DNA transfer between H. pylori underscoring the importance of horizontal gene transfer for this species.     

Ecological risk assessment of toxic elements,microbial loads,parasites and antinutritional factors in Telfairia occidentalis grown on sewage contaminated soil

The aim of this study was to evaluate the ecological health risk assessment of toxic elements, microbial load, and anti-nutrient constituent build-up in vegetables, Talfairia occidentalis, grown on sewage contaminated soil. Samples of soil and vegetables were taken from farms near the dumpsites (sites A). As a control, samples were taken from an area with no dump sites (sites B). Atomic Absorption Spectrophotometry was used to determine the potentially toxic elements. The basic titrimetric method was used to estimate Phytate and Oxalate in the vegetables in order to assess anti-nutrient constituents. The content of cyanogenic glycosides, tannins, and alkaloids in vegetable samples was determine using a standard method. Total heterotrophic bacteria, E. coli, total coliform, faecal coliform, Staphylococcus aureus, Salmonella, and intestinal parasites were all determined using APHA standard methods. For all samples analyzed, the concentrations of metals – Cr, Zn, Pb, Cu, and Ni in site A were higher than those in site B. The soil and vegetable samples differed from the controls by a significant amount (P <0.05). Vegetable samples from site A were mainly infected with faecal coliform TO (9.7 × 10⁵cfu/g) and other bacteria at levels higher than the recommended levels. In contrast to samples from site B, which were not infected with human intestinal helminth parasites, Ascaris lumbricoides ova and Entamoeba histolytica cryst were present in vegetables in site A samples. The majority of the values obtained for the sewage dumpsite were significantly higher than the values recorded by the World Health Organization (WHO). However, sewage wastes, anthropogenic sources, and atmospheric depositions cause higher concentrations of heavy metals, anti-nutrients, and microbial load around dumpsites, which bioaccumulate in vegetables through uptake from the soil and eventual entry into the food chain. The overall evaluation found that consumption of vegetables grown on sewage soil poses a risk of heavy metals, microbial loads, and anti-nutrients adversely affecting human health.     

Effect of Organic Residues and Liming Materials on Metal Extraction from a Mining-Contaminated Soil

Incubation tests were used to assess the effectiveness of three different organic residues and three different liming materials, alone or in combination, in the remediation of a mine contaminated soil. The organic residues tested were sewage sludge from a municipal wastewater treatment plant (SS), compost from the organic fraction of unsorted municipal solid waste (MSWC), and garden waste compost (GWC), applied at 100 and 200 Mg ha^{? 1}. The liming materials tested were agriculture limestone (6.4 Mg ha^{? 1}), calcium oxide (3.7 Mg ha^{? 1}), and sugar beet sludge (12.2 Mg ha^{? 1}) from the sugar manufacturing process. The soil and mixtures of soil and amendments were adjusted to 70% of the maximum water holding capacity and incubated for 28 days in a controlled-temperature room at 20°C ± 1°C. At the end of the incubation, samples were analyzed for pH, electrical conductivity, organic matter content, CaCl₂-extractable, and' NH4Ac/HAc+ EDTA–extractable metal fractions (Cu, Zn, and Pb). Correlations among the variables and/or similarities among the treatments were identified by principal component analysis and hierarchical cluster analysis. The amendments tested decreased the CaCl₂-extractable Cu and Zn fractions, considered as mobile metal fractions, to below analytical detectable limits, providing organic matter to the soil with levels between 1% and 2% at the end of the experiment, significantly different relatively to the original soil. pH and electrical conductivity reached high values when using 200 Mg ha^{? 1} SS or 200 Mg ha^{? 1} MSWC, with any of the liming materials tested, making these application rates excessive for this particular situation. Furthermore, the treatments using MSWC increased the NH4Ac/HAc+ EDTA–extractable Cu, Pb, and Zn fractions, considered as mobilizable metal fractions, as did the 200 Mg ha^{? 1} SS for Pb and Zn. Considering the overall results, the compost made from garden waste decreased metal solubility in the soil and increased soil pH and organic matter content, without the addition of large amounts of soluble salts, and without increasing the mobilizable metal content. Of the organic materials tested, this was the only one that can be considered adequate for remediation of the contaminated soil under study, at the application rates tested.     

Biochemical degradation pathway of textile dye Remazol red and subsequent toxicological evaluation by cytotoxicity, genotoxicity and oxidative stress studies

Remazol red (RR), a monochloro sulphonated azo dye was degraded up to 97% within 20 min at 40 °C and pH 7 at dye concentration 50 mg l⁻¹ by Pseudomonas aeruginosa BCH. Examination of enzyme status exposed the involvement of various oxidoreductive enzymes viz. laccase, veratryl alcohol oxidase and NADH-DCIP reductase. Analytical studies viz. HPTLC, HPLC, FTIR and GC-MS carried out with dye and dye metabolites formed after dye decolorization confirmed that the decolorization was due to degradation. Based on enzymatic status and GC-MS analysis the possible metabolic pathway followed by bacterial strain for the degradation of RR was proposed. During toxicological scrutiny, cell death was observed in RR treated Allium cepa (A. cepa) root cells. The observed inhibition of catalase (CAT) activity and induction in enzyme activities of sulfur oxide dismutase (SOD) and ascorbate peroxidase (APX) along with raised protein oxidation and lipid peroxidation signified that RR generated the oxidative stress in A. cepa roots. These toxicological studies along with genotoxicity studies using A. cepa roots and phytotoxicity studies using Phaseolus mungo (P. mungo) and Sorghum vulgare (S. vulgare) conclusively designated the toxicity of RR and comparatively less toxic nature of metabolites formed after dye degradation by P. aeruginosa BCH.     

Molecular Method To Assess the Diversity of Burkholderia Species in Environmental Samples

In spite of the importance of many members of the genus Burkholderia in the soil microbial community, no direct method to assess the diversity of this genus has been developed so far. The aim of this work was the development of soil DNA-based PCR-denaturing gradient gel electrophoresis (DGGE), a powerful tool for studying the diversity of microbial communities, for detection and analysis of the Burkholderia diversity in soil samples. Primers specific for the genus Burkholderia were developed based on the 16S rRNA gene sequence and were evaluated in PCRs performed with genomic DNAs from Burkholderia and non-Burkholderia species as the templates. The primer system used exhibited good specificity and sensitivity for the majority of established species of the genus Burkholderia. DGGE analyses of the PCR products obtained showed that there were sufficient differences in migration behavior to distinguish the majority of the 14 Burkholderia species tested. Sequence analysis of amplicons generated with soil DNA exclusively revealed sequences affiliated with sequences of Burkholderia species, demonstrating that the PCR-DGGE method is suitable for studying the diversity of this genus in natural settings. A PCR-DGGE analysis of the Burkholderia communities in two grassland plots revealed differences in diversity mainly between bulk and rhizosphere soil samples; the communities in the latter samples produced more complex patterns.     

The effect of albumin fusion structure on the production and bioactivity of the somatostatin-28 fusion protein in Pichia pastoris

Somatostatin, a natural inhibitor of growth hormone (GH), and its analogs have been used in clinical settings for the treatment of acromegaly, gigantism, thyrotropinoma, and other carcinoid syndromes. However, natural somatostatin is limited for clinical usage because of its short half-life in vivo. Albumin fusion technology was used to construct long-acting fusion proteins and Pichia pastoris was used as an expression system. Three fusion proteins (SS28)₂-HSA, (SS28)₃-HSA, and HSA-(SS28)₂, were constructed with different fusion copies of somatostatin-28 and fusion orientations. The expression level of (SS28)₃-HSA was much lower than (SS28)₂-HSA and HSA-(SS28)₂ due to the additional fusion of the somatostatin-28 molecule. MALDI-TOF mass spectrometry revealed that severe degradation occurred in the fermentation process. Similar to the standard, somatostatin-14, all three fusion proteins were able to inhibit GH secretion in blood, with (SS28)₂-HSA being the most effective one. A pharmacokinetics study showed that (SS28)₂-HSA had a prolonged half-life of 2 h. These results showed that increasing the number of small protein copies fused to HSA may not be a suitable method for improving protein bioactivity.     

Boron and Coumaphos Residues in Hive Materials Following Treatments for the Control of Aethina tumida Murray

In the search of alternatives for controlling Aethina tumida Murray, we recently proposed the BAA trap which uses boric acid and an attractant which mimics the process of fermentation caused by Kodamaea ohmeri in the hive. This yeast is excreted in the feces of A. tumida causing the fermentation of pollen and honey of infested hives and releasing compounds that function as aggregation pheromones to A. tumida. Since the boron is the toxic element in boric acid, the aim of this article is to assess the amount of boron residues in honey and beeswax from hives treated with the BAA trap. For this aim, the amount of bioaccumulated boron in products of untreated hives was first determined and then compared with the amount of boron of products from hives treated with the BAA trap in two distinct climatic and soil conditions. The study was conducted in the cities of Padilla, Tamaulipas, and Valladolid, Yucatan (Mexico) from August 2014 to March 2015. The quantity of boron in honey was significantly less in Yucatan than in Tamaulipas; this agrees with the boron deficiency among Luvisol and Leptosol soils found in Yucatan compared to the Vertisol soil found in Tamaulipas. In fact, the honey from Yucatan has lower boron levels than those reported in the literature. The BAA treatment was applied for four months, results show that the BAA trap does not have any residual effect in either honey or wax; i.e., there is no significant difference in boron content before and after treatment. On the other hand, the organophosphate pesticide coumaphos was found in 100% of wax samples and in 64% of honey samples collected from Yucatan. The concentration of coumaphos in honey ranges from 0.005 to 0.040 mg/kg, which are below Maximum Residue Limit (MRL) allowed in the European Union (0.1 mg/kg) but 7.14% of samples exceeded the MRL allowed in Canada (0.02 mg/kg).     

Revisiting the Dilution Procedure Used To Manipulate Microbial Biodiversity in Terrestrial Systems

It is hard to assess experimentally the importance of microbial diversity in soil for the functioning of terrestrial ecosystems. An approach that is often used to make such assessment is the so-called dilution method. This method is based on the assumption that the biodiversity of the microbial community is reduced after dilution of a soil suspension and that the reduced diversity persists after incubation of more or less diluted inocula in soil. However, little is known about how the communities develop in soil after inoculation. In this study, serial dilutions of a soil suspension were made and reinoculated into the original soil previously sterilized by gamma irradiation. We determined the structure of the microbial communities in the suspensions and in the inoculated soils using 454-pyrosequencing of 16S rRNA genes. Upon dilution, several diversity indices showed that, indeed, the diversity of the bacterial communities in the suspensions decreased dramatically, with Proteobacteria as the dominant phylum of bacteria detected in all dilutions. The structure of the microbial community was changed considerably in soil, with Proteobacteria, Bacteroidetes, and Verrucomicrobia as the dominant groups in most diluted samples, indicating the importance of soil-related mechanisms operating in the assembly of the communities. We found unique operational taxonomic units (OTUs) even in the highest dilution in both the suspensions and the incubated soil samples. We conclude that the dilution approach reduces the diversity of microbial communities in soil samples but that it does not allow accurate predictions of the community assemblage during incubation of (diluted) suspensions in soil.