Targeting allergenic fungi in agricultural environments aids the identification of major sources and potential risks for human health.

Fungi are, after pollen, the second most important producers of outdoor airborne allergens. To identify sources of airborne fungal allergens, a workflow for qPCR quantification from environmental samples was developed, thoroughly tested, and finally applied. We concentrated on determining the levels of allergenic fungi belonging to Alternaria, Cladosporium, Fusarium, and Trichoderma in plant and soil samples from agricultural fields in which cereals were grown. Our aims were to identify the major sources of allergenic fungi and factors potentially influencing their occurrence. Plant materials were the main source of the tested fungi at and after harvest. Amounts of A. alternata and C. cladosporioides varied significantly in fields under different management conditions, but absolute levels were very high in all cases. This finding suggests that high numbers of allergenic fungi may be an inevitable side effect of farming in several crops. Applied in large-scale studies, the concept described here may help to explain the high number of sensitization to airborne fungal allergens.

Drought and air warming affects abundance and exoenzyme profiles of Cenococcum geophilum associated with Quercus robur, Q. petraea and Q. pubescens.

The present study aimed to elucidate the influence of drought and elevated temperature on relative abundance and functioning of the ectomycorrhizal fungus Cenococcum geophilum on three oak species differing in adaptation to a warm and dry climate. The experiment QUERCO comprised three Quercus species (Q. robur, Q. petraea, Q. pubescens) grown for 3 years under four treatments: elevated air temperature, drought, a combination of the two, and control. Fine root samples were analysed for relative abundance and potential extracellular enzyme activities of ectomycorrhizae of C. geophilum, a fungal species known to be drought resistant. The relative abundance of C. geophilum on the roots of the oak species was significantly increased by temperature, decreased by drought, but unchanged in the combined treatment compared to the control. Although the extent of treatment effects differed among oak species, no significant influence of tree species on relative abundance of C. geophilum was detected. Exoenzyme activities of C. geophilum on Q. robur and Q. petraea (but not Q. pubescens) significantly increased in the combined treatment, but for all oak species were reduced under drought and air warming alone compared to the control. There was a significant negative correlation between abundance of C. geophilum and its leucine aminopeptidase activity. As this enzyme is not frequent among ectomycorrhizal fungi, this emphasises the functional importance of C. geophilum in the ectomycorrhizal community. Our results indicate that increased temperature and drought will influence the relative abundance and enzyme activity of C. geophilum. However, both the Quercus species and C. geophilum tolerated warming and strong drought.

Short term effects of ozone on the plant-rhizosphere-bulk soil system of young beech trees.

Plant growth largely depends on microbial community structure and function in the rhizosphere. In turn, microbial communities in the rhizosphere rely on carbohydrates provided by the host plant. This paper presents the first study on ozone effects in the plant-rhizosphere-bulk soil system of 4-year-old beech trees using outdoor lysimeters as a research platform. The lysimeters were filled with homogenized soil from the corresponding horizons of a forest site, thus minimizing field heterogeneity. Four lysimeters were treated with ambient ozone (1 x O3) and four with double ambient ozone concentrations (2 x O3; restricted to 150 ppb). In contrast to senescence, which was almost unaffected by ozone treatment, both the photochemical quantum yield of photosystem II (PSII) and leaf gas exchange were reduced (11 - 45 %) under the elevated O3 regime. However, due to large variation between the plants, no statistically significant O3 effect was found. Even though the amount of primary metabolites, such as sugar and starch, was not influenced by elevated O3 concentrations, the reduced photosynthetic performance was reflected in leaf biochemistry in the form of a reduction in soluble phenolic metabolites. The rhizosphere microbial community also responded to the O3 treatment. Both community structure and function were affected, with a tendency towards a lower diversity and a significant reduction in the potential nutrient turnover. In contrast, litter degradation was unaffected by the fumigation, indicating that in situ microbial functionality of the bulk soil did not change.

Mycorrhizosphere responsiveness to atmospheric ozone and inoculation with Phytophthora citricola in a phytotron experiment with spruce/beech mixed cultures.

The aim was to analyze functional changes in the mycorrhizosphere (MR) of juvenile spruce and beech grown in a mixture under ambient and twice ambient ozone and inoculated with the root pathogen Phytophthora citricola. The phytotron experiment was performed over two vegetation periods, adding the pathogen at the end of the first growing season. Root biomass data suggest that the combined treatment affected spruce more than beech and that the negative influence of ozone on stress tolerance against the root pathogen P. citricola was greater for spruce than for beech. In contrast, beech was more affected when the pathogen was the sole stressor. The functional soil parameter chosen for studies of MR soil samples was activity of extracellular enzymes. After the first year of ozone exposure, MR soil samples of both species showed increased activity of almost all measured enzymes (acid phosphatase, chitinase, beta-glucosidase, cellobiohydrolase) in the O3 treatment. Species-specific differences were observed, with a stronger effect of P. citricola on beech MR and a stronger ozone effect on spruce MR. In the second year, the effects of the combined treatment (ozone and P. citricola) were a significant increase in the activity of most enzymes (except cellobiohydrolase) for both tree species. The results indicated that responsiveness of MR soils towards ozone and P. citricola was related to the severity of infection of the plants and the reduction of belowground biomass, suggesting a strong, direct influence of plant stress on MR soil enzyme activity. Additional research is needed using different species and combined stresses to determine the broader ecological relevance of shifts in rhizosphere enzymes.

A rapid and highly sensitive method for measuring enzyme activities in single mycorrhizal tips using 4-methylumbelliferone-labelled fluorogenic substrates in a microplate system.

A microplate fluorimetric assay was developed for measuring potential activities of extracellular enzymes of individual ectomycorrhizal (EM) roots using methylumbelliferone (MU)-labelled fluorescent substrate analogues and microsieves to minimise damage due to manipulation of excised mycorrhizal roots. Control experiments revealed that enzyme activities remained stable over the whole time of the experiment suggesting a strong affinity of the studied enzymes to the fungal cell walls. The same mycorrhizal tips thus could be used repeatedly for enzyme detection and subsequently analysed for the projection area by automated image analysis. The developed system was evaluated on four different EM species measuring pH optimum and substrate saturation of phosphatase, chitinase and beta-glucosidase. The four EM species studied were Lactarius subdulcis, Russula ochroleuca, Cortinarius obtusus and Xerocomus cf. chrysenteron. Depending upon the enzyme, each species exhibited different levels of enzymatic activities as well as enzyme kinetics and showed also differences in pH optima.

Xerocomus badius - Picea abies, an ectomycorrhiza of high activity and element storage capacity in acidic soil.

Mycorrhizas were collected from three Norway spruce (Picea abies) stands in southwest Germany, sorted on the morphotype level and analysed by fluorescein diacetate vital fluorescence staining and the accumulation of elements using inductively coupled plasma-atomic emission spectrometry (ICP-AES) and electron energy-loss spectroscopy (EELS). Xerocomus badius - Picea abies mycorrhizas showed a higher frequency of active hyphal sheaths and a higher potential to store nitrogen, phosphorus, potassium, magnesium, iron and zinc than other mycorrhizal types. Phosphorus and nitrogen were localized by EELS in vacuolar bodies which occurred consistently in the sheath of X. badius mycorrhizas. The results indicate that X. badius is well adapted to acidic stands and that its mycorrhizas are very efficient in uptake and storage of macronutrients.

Characterization and identification of black alder ectomycorrhizas by PCR/RFLP analyses of the rDNA internal transcribed spacer (ITS).

The identity of black alder (Alnus glutinosa (L.) Gaertn.) ectomycorrhizas was investigated using PCR/RFLP analysis of the ITS region from 16 morphotypes sampled at a 60-yr-old black alder stand. A comparison was made with restriction patterns from sporocarps of 28 mycobionts, of which 16 originated from the same stand, the remaining 12 came from two geographically distant alder stands. Eight of the mycorrhizal types could thus be identified, whereas eight mycorrhizal types remained unidentified. The identified mycorrhizas belonged to the genera Russula, Lactarius, Naucoria and Cortinarius. Four of the identified ectomycorrhizal types had identical PCR/RFLP profiles to corresponding fruit bodies from all investigated stands with no detectable intraspecific variation, despite the geographical distance of c.300 km between the sampling locations. By contrast, intraspecific variation between sporocarps from the different locations was detected in Paxillus rubicundulus, mycorrhizas of which were not found. The diversity of fruiting alder mycobionts at the main experimental plot only partly matched the diversity observed from mycorrhizas when comparing their PCR/RFLP profiles. The results are discussed regarding sampling techniques, PCR/RFLP analyses and ecological aspects.