Urine patches in pastures rank among the highest sources of the greenhouse gas nitrous oxide (N 2 O) from animal production systems. Previous laboratory studies indicate that N 2 O emissions for urine-N in pastures may increase with a factor five or eight in combination with soil compaction and dung, respectively. These combinations of urine, compaction and dung occur regularly in pastures, especially in socalled camping areas. The aims of this study were (i) to experimentally quantify the effect of compaction and dung on emission factors of N 2 O from urine patches under field conditions; (ii) to detect any seasonal changes in emission from urine patches; and (iii) to quantify possible effects of urine concentration and -volume. A series of experiments on the effects of compaction, dung, urine-N concentration and urine volume was set up at a pasture on a sandy soil (typic Endoaquoll) in Wageningen, the Netherlands. Artificial urine was applied 8 times in the period August 2000-November 2001, and N 2 O emissions were monitored for a minimum of 1 month after each application. The average emission factor for urine-only treatments was 1.55%. Over the whole period, only soil compaction had a clear significant effect, raising the average N 2 O emissions from urine patches from 1.30% to 2.92% of the applied N. Dung had no consistent effect; although it increased the average emissions from 1.60% to 2.82%, this was clearly significant (P < 0.01) for only one application date and marginally significant (P ¼ 0.054) for the whole experiment. Both compaction and dung increased water-filled pore space (WFPS) of the topsoil for a more prolonged time than high urine volumes. No effect of amount of urine-N or urine volume on N 2 O emissions relative to added N was detected for the whole experiment. There were clear differences between application dates, with highest emissions for urine-only treatments of 4.25% in October, 2000, and lowest of )0.11% in June, 2001. Emissions peaked at 60-70% WFPS, and decreased rapidly with both higher and lower WFPS. We conclude that compaction leads to a considerable increase in the N 2 O emissions under field conditions, mainly through higher WFPS. Dung addition may have the same effect, although this was not consistent throughout our experiment. Seasonal variations seemed mainly driven by differences in WFPS. Based on this study, mitigation strategies should focus on minimizing the grazing period with wet conditions leading to WFPS > 50%, avoiding camping areas in pastures, and on avoiding grazing under moist soil conditions. Greenhouse gas budgets for grazing conditions should include the effects of soil compaction and dung to represent actual emissions.

SignificanceFungi are a broad class of organisms that play crucial roles in a wide variety of natural and industrial processes. Some are also harmful, destroying crops or infecting immunocompromised patients. Many fungi, at some point during their life cycle, contain two different nuclei, each with different genetic content. We examine the regulation of genes from these nuclei in a mushroom-forming fungus. We find that these nuclei contribute differently to the regulation of the fungal cells, and may therefore have a different impact on their environment. Furthermore, these differences change throughout the development of different tissues. This work contributes to our understanding of fungal physiology by examining this process.

Agaricus bisporus mushrooms are commercially produced on a microbe rich compost. Here, fungal and bacterial biomass was quantified in compost with and without colonization by A. bisporus. Chitin content, indicative of total fungal biomass, increased during a 26-day period from 576 to 779 nmol N-acetylglucosamine g−1 compost in the absence of A. bisporus (negative control). A similar increase was found in the presence of this mushroom forming fungus. The fungal phospholipid-derived fatty acid (PLFA) marker C18:2ω6, indicative of the living fraction of the fungal biomass, decreased from 575 to 280 nmol g−1 compost in the negative control. In contrast, it increased to 1200 nmol g−1 compost in the presence of A. bisporus. Laccase activity was absent throughout culturing in the negative control, while it correlated with the fungal PLFA marker in the presence of A. bisporus. PLFA was also used to quantify living bacterial biomass. In the negative control, the bacterial markers remained constant at 3000–3200 nmol PLFA g−1 compost. In contrast, they decreased to 850 nmol g−1 compost during vegetative growth of A. bisporus, implying that bacterial biomass decreased from 17.7 to 4.7 mg g−1 compost. The relative amount of the Gram positive associated PLFA markers a15:0 and a17:0 and the Gram negative PLFA associated markers cy17:0 and cy19:0 increased and decreased, respectively, suggesting that Gram negative bacteria are more suppressed by A. bisporus. Together, these data indicate that fungal biomass can make up 6.8% of the compost after A. bisporus colonization, 57% of which being dead. Moreover, results show that A. bisporus impacts biomass and composition of bacteria in compost.Electronic supplementary materialThe online version of this article (doi:10.1186/s13568-016-0304-y) contains supplementary material, which is available to authorized users.

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Malassezia spp. are part of the normal human and animal mycobiota but are also associated with a variety of dermatological diseases. The absence of a transformation system hampered studies to reveal mechanisms underlying the switch from the non-pathogenic to pathogenic life style. Here we describe, a highly efficient Agrobacterium-mediated genetic transformation system for Malassezia furfur and M. pachydermatis. A binary T-DNA vector with the hygromycin B phosphotransferase (hpt) selection marker and the green fluorescent protein gene (gfp) was introduced in M. furfur and M. pachydermatis by combining the transformation protocols of Agaricus bisporus and Cryptococcus neoformans. Optimal temperature and co-cultivation time for transformation were 5 and 7days at 19°C and 24°C, respectively. Transformation efficiency was 0.75-1.5% for M. furfur and 0.6-7.5% for M. pachydermatis. Integration of the hpt resistance cassette and gfp was verified using PCR and fluorescence microscopy, respectively. The T-DNA was mitotically stable in approximately 80% of the transformants after 10 times sub-culturing in the absence of hygromycin. Improving transformation protocols contribute to study the biology and pathophysiology of Malassezia.