The objective of the study was to identify N2O-producing fungi isolated from six qualitatively different sections of an overwintering pasture with substantial cattle impact. 80 out of 164 fungal isolates were considered as N2O-producers in nitrite-containing medium, representing 33 fungal species of 23 different genera. Ability to produce N2O was newly reported in eight genera: Arthrinium, Gibellulopsis, Ilyonectria, Lichtheimia, Paraphaeosphaeria, Purpureocillium, Tolypocladium and Westerdykella. Three levels of fungal N2O-productivity were assigned according to the fraction of nitrite-N transformed into N2O–N: < 1%, 1–10%, over 10%. Fungi capable of high and moderate transformation rates were predominantly isolated from sections under current or past cattle impact, where they contributed with a maximum of 65% of the total N2O emissions. There was no significant effect of cultivation conditions on the fraction of N2O-producing fungi. The results demonstrate that N2O-producing fungi are a common constituent of fungal communities in soils impacted by overwintering cattle.

The use of antibiotics in animal husbandry contributes to the worldwide problem of increasing antibiotic resistance in animal and human pathogens. Intensive animal production is considered an important source of antibiotic resistance genes released to the environment, while the contribution of smaller farms remains to be evaluated. Here we monitor the spread of tetracycline resistance (TC-r) genes at a middle-size conventional dairy farm, where chlortetracycline (CTC, as intrauterine suppository) is prophylactically used after each calving. Our study has shown that animals at the farm acquired the TC-r genes in their early age (1-2 weeks), likely due to colonization with TC-resistant bacteria from their mothers and/or the farm environment. The relative abundance of the TC-r genes tet(W), tet(Q), and tet(M) in fresh excrements of calves was about 1-2 orders of magnitude higher compared to heifers and dairy cows, possibly due to the presence of antibiotic residues in milk fed to calves. The occurrence and abundance of TC-r genes in fresh excrements of heifers and adult cows remained unaffected by intrauterine CTC applications, with tet(O), tet(Q), and tet(W) representing a "core TC-resistome" of the farm, and tet(A), tet(M), tet(Y), and tet(X) occurring occasionally. The genes tet(A), tet(M), tet(Y), and tet(X) were shown to be respectively harbored by Shigella, Lactobacillus and Clostridium, Acinetobacter, and Wautersiella. Soil in the farm proximity, as well as field soil to which manure from the farm was applied, was contaminated with TC-r genes occurring in the farm, and some of the TC-r genes persisted in the field over 3 months following the manure application. Concluding, our study shows that antibiotic resistance genes may be a stable part of the intestinal metagenome of cattle even if antibiotics are not used for growth stimulation, and that smaller dairy farms may also contribute to environmental pollution with antibiotic resistance genes.

The aim of this work was to describe changes in fatty acid profiles of fungi growing under artificial conditions of oxygen depletion. In total, 133 fungal strains belonging to eight orders were isolated from cattle impacted soils and tested. The analysis of the ten most frequent fatty acids revealed significant shift in fatty acids composition as a result of decreasing oxygen level. Taxonomic- as well as aeration-dependent changes in the amounts of fungal biomarker fatty acids (18:1 omega 9 and 18:2 omega 6,9) were found. Therefore, the ratio of these two fatty acids could be considered as an indicator of anaerobic, microaerobic or aerobic conditions in soil. Moreover, fatty acid-based estimation of fungal biomass in soils should be performed as a sum of both biomarker fatty acids and with respect to the soil characteristics as well as to the composition of fungal community. (C) 2015 Elsevier Ltd. All rights reserved.

Antibiotic residues and antibiotic resistance genes originating from animal waste represent environmental pollutants with possible human health consequences. In this study, we addressed the question whether chlortetracycline (CTC) residues in soils can act as selective pressure enhancing the persistence of tetracycline (TC-r) resistance genes in grassland soils receiving cattle feces. We performed a soil microcosm experiment, using 3 grassland soils with different management history, which were incubated with feces from conventionally raised dairy cows. The microcosms included treatments with a low dose (0.2 mg kg(-1)), a high dose (100 mg kg(-1)) and no CTC. The presence and abundance of TC-r genes tet(O), tet(Q) and tet(W) and the intI1 gene coding for class 1 integrase were assessed with real-time PCR after 0, 14, 28, 56 and 86 d of incubation. The genes tet(Q) and intI1 persisted in all feces-containing treatments for at least 28 d, and tet(W) and tet(0) for at least 86 d, though they went close to limits of quantification after 14-28 d in most cases. The soil, but not the dose of CTC, significantly affected the gene persistence. Concluding, certain TC-r genes originating from cattle feces may persist in soil for several months independently from antibiotic selection pressure. (C) 2014 Elsevier Ltd. All rights reserved.

Our current knowledge about the effects of earthworms on microbial communities derives from studies on a few peregrine species, and there is a lack of data for most of the uncommon worms. Therefore, we studied the effects of Allolohophora hrabei (Cernosvitov, 1935), a key earthworm species in fragile fragments of steppe ecosystems in central Europe. We found that the burrowing activity of A. hrabei as well as deposition of casts caused significant differences in the community composition of three domains of soil microorganisms including bacteria, archaea, and fungi. The richness of bacteria and fungi was significantly lower in casts than in the surrounding soil, however, that of archaea showed the opposite trend. Archaea and fungi were less susceptible to the burrowing activity than bacteria; earthworms significantly shifted the composition of bacterial communities between the soil and drilosphere. Deposition of casts was the main factor in affecting soil microorganisms. Cast profiles of all three microbial domains created separate clusters, indicating significantly different communities at all monitoring sites in both years of the study. In general, the activity of A. hrabei can be considered as an important factor that affects the soil microbial communities, and its casts represent a substantial habitat for selected soil microorganisms in steppe grasslands. (C) 2014 Elsevier Ltd. All rights reserved.

The aim of the study was to evaluate the impact of cattle overwintering husbandry on composition of upland grassland soil archaeal, bacterial, and fungal communities by comparative fingerprinting (SSU rRNA denaturing gradient gel electrophoresis (DGGE)) of total (tDNA) and extracellular DNA (eDNA) extracted from three differently impacted soils (severely, moderately, and non-impacted soil) and cattle excrements. Cattle excrements carried a significant amount of viable microorganisms and eDNA, and as a result of its high external returns, the amounts of extractable eDNA in soil increased with cattle impact, being positively correlated with soil microbial biomass and activity. The soil eDNA fraction (2.8 to 5.7 μg g−1 dw) significantly contributed to the soil metagenome, representing 18–31 % of soil tDNA. The largest shift in soil community structure was observed for Archaea, followed by fungi and bacteria, indicating that soil bacteria possess the highest resilience to cattle-induced changes. Cattle excrements showed more diverse bacterial than archaeal and fungal communities. The specific DGGE bands of cattle excrements were also observed in community profiles of cattle-impacted soils, confirming the effect of cattle husbandry on composition of soil microbial communities, probably as a result of introduced rumen-borne microbes. Similar changes were also reflected in the eDNA-derived DGGE profiles, suggesting a higher contribution of bacteria to soil extracellular metagenome than Archaea and fungi. This study provided first evidences about the extracellular mobilome in cattle-impacted soils, carrying also genetic information of Archaea, and its susceptibility to impact by outdoor cattle husbandry.

Fertilizing soils with animal excrements from farms with common antibiotic use represents a risk of disseminating antibiotic resistance genes into the environment. In the case of tetracycline antibiotics, it is not clear, however, whether the presence of antibiotic residues further enhances the gene occurrence in manured soils. We established a microcosm experiment in which 3 farm soils that had no recent history of fertilization with animal excrements were amended on a weekly basis (9 times) with excrements from either an oxytetracycline-treated or an untreated cow. Throughout the study, the concentration of oxytetracycline in excrements from the treated cow was above 500 μg g(-1)dw, whereas no oxytetracycline was detected in excrements from the healthy cow. Both excrements contained tetracycline resistance (TC-r) genes tet(L), tet(M), tet(V), tet(Z), tet(Q) and tet(W). The excrements from the treated cow also contained the tet(B) gene, and a higher abundance of tet(Z), tet(Q) and tet(W). Three weeks after the last excrement addition, the individual TC-r genes differed in their persistence in soil: tet(Q) and tet(B) were not detectable while tet(L), tet(M), tet(Z) and tet(W) were found in all 3 soils. There were, however, no significant differences in the total number, nor in the abundance, of TC-r genes between soil samples amended with each excrement type. The oxytetracycline-rich and the oxytetracycline-free excrement therefore contributed equally to the increase of tetracycline resistome in soil. Our results indicate that other mechanisms than OTC-selection pressure may be involved in the maintenance of TC-r genes in manured soils.

Microscopic soil fungi isolated from arable, grassland and forest soils have been suggested as producers of nitrous oxide (N(2)O). The aim of this work was to screen the capabilities for N(2)O production of microscopic fungi originating in the pasture soils of a cattle overwintering area with three levels of cattle impact intensity. In total, 36 fungal species from 11 genera were isolated during a 2-year study, and production of N(2)O under laboratory conditions was confirmed in 23 species (64%). Species belonging to the genera Fusarium, Penicillium, Monographella, Acremonium, Gibberella, Eurotium, and Pseudallescheria were found to be the most potent N(2)O-producers. Different N(2)O production patterns and wide variations in production rates, ranging from 1 to 150 μg N(2)O-Nd(-1), were observed, resulting in the transformation of 0.2-18.4% of the initial NO(2)(-)-N present in the cultivation medium. The data revealed distinct soil fungal communities in the different sections of the cattle overwintering area, and indicate a significant effect of cattle overwintering on the composition of soil fungal consortia. These observations confirm the importance of soil fungi in total N(2)O fluxes from grazed grassland ecosystems.

The present field study documents substantial changes in the soil microbial community (SMC) and organic matter (SOM) in an upland pasture soil resulting from 10 years of "cattle outdoor over-wintering practice". Soils from a long-term investigated pasture area were compared under three different levels of cattle impact (SI - severe, MI - moderate, NI - no impact). Extended polar lipids analysis (PLA) confirmed a qualitatively new microbial community profile and a several-fold increase of the microbial biomass in the impacted soils (SI) compared to the control NI soil. The new SMC was derived from cattle intestine microorganisms, typical by increased content of archaeal phospholipid ether lipids and by new fatty acids indicative for bacterial and fungal fecal anaerobes. A quality of the SI-SUM, evaluated by the relative content of the pyrolytic fragments profile was more similar to the cattle excrements than to the MI and NI soils, and an organic carbon content of the SI soil was not more than three times higher in comparison to the control NI soil. The quality and quantity of the SOM as well as the SMC in both, the most impacted SI and the control NI soils, were stable in contrast to the moderately impacted MI soil. During the growing season, the MI soil lost 75% of the C-org and 65% of the soil microbial biomass that had accumulated during winter; its aromatic-rich-SOM showed transformation into SOM, enriched by N, P-organic derivates. This transformation was positively correlated to a significant recovery of the actinobacteria and reduction of anaerobic microorganisms during the vegetation season. Results in this study showed that the stability of the soil microbial changes due to the cattle outdoor over-wintering husbandry depended on the stability of the quantitative and qualitative changes of the SOM. (C) 2012 Published by Elsevier B.V.

Ergosterol, a biomarker of living saprotrophic fungi, was obtained from soil by microwave-assisted extraction and determined by GC-MS-MS, HPLC, and GC-FID The GC-MS-MS technique was used to monitor fungal biomass in the soils differently impacted by overwintering cattle The amount of ergosterol in soil increased from 0 93 +/- 0 34 to 29 28 +/- 3 07 mu g g(-1) of soil The detection limits in GC-MS-MS and HPLC methods were ca ten times lower than in GC-FID Whereas GC-MS-MS is the best method for monitoring the complex sterol profile in the environment, the selective MS-MS mode allowed the determination of ergosterol in complex matrices by elimination of coeluting peaks The enrichment of cattle-impacted soils in non-fungal sterols (campesterol, stigmaserol and beta-sitosterol) and stanols (ergostanol, sitostanol) was due to digested plant materials and rumen microflora, suggesting significant biochemical changes in the pasture soils under study

We compared two denaturing gradient gel electrophoresis (DGGE) systems—DCode (Biorad, Hercules, CA, USA) and PhorU (Ingeny, Leiden, NL), performing community level 16S and 18S rRNA gene fragment-PCR-DGGE with total DNA extracted from upland pasture soil used for outdoor cattle husbandry. The methodological evaluation of the DGGE apparatus as parameter influencing DGGE fingerprinting, based on cluster analysis of soil bacterial and fungal community fingerprints, was made in terms of the resulting information about microbial community structures and their response to different degrees of cattle impact. Although the comparative DGGE analysis with different DGGE systems provided similar clustering of microbial community structures in correlation with the degree of cattle impact, our results suggest the DGGE system to be a factor influencing DGGE analysis. To our knowledge this is the first attempt to investigate the hypothetical impact of the DGGE system due to different technical characteristics, recommending the use of one and the same DGGE apparatus throughout an experiment, if the monitoring of microbial community structures requires multiple gel-to-gel analysis.

Overwintering cattle outdoors causes soil surface disturbance, substantial increases of soil N-tot, C-org, and Panda shift in pH to alkaline levels. Since fungi predominate in unfertilized soils with acidic pH and have filamentous hyphae, we hypothesized that changes caused by overwintering cattle outdoors (trampling, excreta returns, and changes in soil chemistry) will lead to suppressed species richness, lower biomass, and alter the structure of fungal communities. The research was conducted on an upland pasture used more than 10 years for cattle overwintering. Both culture-dependent and -independent methods were used for the determination of either fungal species composition (cultivation: DGGE) or biomass (numbers of CFU; concentration of fungal PLEA marker 18:2 omega 6,9). Soils under three different levels of cattle disturbance (S - severe, M - moderate, C - no disturbance/control) were investigated during three subsequent years. In addition, the DGGE analysis of soils was completed by comparison with analysis of fresh cattle excrements (Ex). The composition of fungal communities showed significantly higher richness and a substantial shift in species composition in cattle-disturbed soils (S, M) in comparison to the non-disturbed soil (C). The number of separated DGGE bands was significantly higher in S (30.67 +/- 1.63; mean +/- SD) and M (25.50 +/- 1.64) soils than in the C soil (19.33 +/- 1.75). Sequencing of typical bands revealed common fungal genera - Alternaria, Penicillium, Fusarium, Rhizopus, Isaria, and Metarhizium. Profiles of the S soil were enriched by bands of rumen-born anaerobic fungi (Neocallimastix, Cyllamyces) occurring mainly in profiles of excrements, where relatively low band richness (14.33 +/- 1.15) was observed. The increasing level of cattle disturbance induced an increase in the biomass of complex fungal community over the three-year experimental period from 3.39 +/- 2.11 (mean +/- SD) nmol of fungal PLFA per gram of the C soil to 5.87 +/- 3.16 in the M soil and 9.21 +/- 4.69 in the S soil. Concentrations of soil N-tot and C-org were evaluated as the parameters significantly correlating with biomass as well as composition of the fungal community. (C) 2010 Elsevier Ltd. All rights reserved.

The aim of the pilot study was to describe the impact of outdoor cattle husbandry on the communities of arbuscular mycorrhizal fungi on the overwintering upland pasture (South Bohemia, Czech Republic). We selected three sites with severe, moderate, and light (control) impact intensity. Roots of plant species with a plant cover area 5% were collected at each site. The ratio of nonmycorrhizal plants species decreased with the decreasing impact of cattle. The highest mycorrhizal colonization was found at the control site on a level of the screened plant community (78.57% of root length) as well as on a species level (97.78% of root length of Plantago major L.). At the severely impacted site, 31.67% and 35.56% of root colonization was found on community and species level (Plantago major L.), respectively. Similar results were found also in the length of extraradical mycelium and arbuscules numbers.