Removal of toxic metals from sewage sludge by EDTA and hydrodynamic cavitation and use of the sludge as fertilizer.

Sewage sludge (SS) is rich in plant nutrients, including P, N, and organic C, but often contains toxic metals (TMs), which hinders its potential use in agriculture. The efficiency of removal of TMs by washing with ethylenediamine tetraacetate (EDTA), in combination with hydrodynamic cavitation (HC) and the usability of washed sewage sludge as fertilizer were investigated. The environmental risk was assessed. During 8 wash batches an average 35, 68, 47 and 45 % of Pb, Zn, Cd and Cu, respectively, as well as 22 and 5 % Mn and Fe were removed from the SS. The process solutions and EDTA were recycled at a pH gradient of 12.5-2, which was achieved by adding quicklime (CaO) and then acidification by H2SO4, so that no wastewater was produced, only solid waste (ReSoil® method). The quality of the recycled process solutions (they remained unsaturated with salts) and the efficiency of the washing process were maintained across all batches. On average, 46 % of the EDTA was lost during the process and was replenished. The initial leachability of EDTA-mobilized Pb, Zn, Cu, Cr and Fe remaining in the washed SS increased 6-, 17-, 3-, 11- and 11-fold, respectively, but not to hazardous levels except for Zn. After washing, P and K remained in the SS, plant-available P increased 3.3-fold, while total N and C were reduced by 20.28 and 2.44 %, respectively. Washed SS was used as fertilizer in the pot experiment. The yield of Brassica juncea did not improve, the uptake of TMs by the plants and the leaching of TMs from the soil were minimal. Our study highlighted the drawbacks and potential feasibility of the new SS washing method.

Physio-Biochemical Responses of Sweet Cherry Leaf to Natural Cold Conditions.

Trees of the sweet cherry cultivar 'Grace Star' (Prunus avium L.) were exposed to low temperatures without frost for two consecutive nights under natural conditions 36 d after flowering, to study the effects on the physiological properties and metabolic status of leaves. The response was studied by measuring chlorophyll fluorescence and gas exchange parameters and by analyzing chloroplast pigments (i) immediately after exposure, (ii) 24 h and (iii) 48 h later. The first exposure at 2.4 (±0.2) °C and a minimum of 0.8 °C elicited more changes than the second exposure at 4.9 (±0.3) °C and a minimum of 2.4 °C. After the first exposure, the maximum quantum yield of PS II (Fv/Fm), effective quantum efficiency of PS II, net photosynthesis (PN), stomatal conductance (gs), transpiration, and intercellular CO2 concentration were significantly lower, and after the second exposure, the content of chlorophyll b, total chlorophyll, ß-carotene, and lutein were lower. The content of antheraxanthin and zeaxanthin was higher immediately after both exposures, and that of antheraxanthin was also higher 24 h later. Recovery took longer in trees that were exposed twice. Fv/Fm recovered within 48 h, but the de-epoxidation state of the xanthophyll cycle pool, PN, and gs did not reach the level of controls, indicating that the stress effect lasted several days which is probably sufficient to cause fruit drop and reduce yield.

Classification of high-throughput phenotyping data for differentiation among nutrient deficiency in common bean.

The development of automated, image-based, high-throughput plant phenotyping enabled the simultaneous measurement of many plant traits. Big and complex phenotypic datasets require advanced statistical methods which enable the extraction of the most valuable traits when combined with other measurements, interpretation, and understanding of their (eco)physiological background. Nutrient deficiency in plants causes specific symptoms that can be easily detected by multispectral imaging, 3D scanning, and chlorophyll fluorescence measurements. Screening of numerous image-based phenotypic traits of common bean plants grown in nutrient-deficient solutions was conducted to optimize phenotyping and select the most valuable phenotypic traits related to the specific nutrient deficit. Discriminant analysis was used to compare the efficiency of groups of traits obtained by high-throughput phenotyping techniques (chlorophyll fluorescence, multispectral traits, and morphological traits) in discrimination between nutrients [nitrogen (N), phosphorus (P), potassium (K), magnesium (Mg), and iron (Fe)] at early and prolonged deficiency. Furthermore, a recursive partitioning analysis was used to select variables within each group of traits that show the highest accuracy for assigning plants to the respective nutrient deficit treatment. Using the entire set of measured traits, the highest classification success by discriminant function was achieved using multispectral traits. In the subsequent measurements, chlorophyll fluorescence and multispectral traits achieved comparably high classification success. Recursive partitioning analysis was able to intrinsically identify variables within each group of traits and their threshold values that best separate the observations from different nutrient deficiency groups. Again, the highest success in assigning plants into their respective groups was achieved based on selected multispectral traits. Selected chlorophyll fluorescence traits also showed high accuracy for assigning plants into control, Fe, Mg, and P deficit but could not correctly assign K and N deficit plants. This study has shown the usefulness of combining high-throughput phenotyping techniques with advanced data analysis to determine and differentiate nutrient deficiency stress.

Analysis of Morphological Traits, Cannabinoid Profiles, THCAS Gene Sequences, and Photosynthesis in Wide and Narrow Leaflet High-Cannabidiol Breeding Populations of Medical Cannabis.

Cannabis sativa L. is one of the oldest cultivated crops, used in medicine for millennia due to therapeutic characteristics of the phytocannabinoids it contains. Its medicinal properties are highly influenced by the chemotype, that is, the ratio of the two main cannabinoids cannabidiol (CBD) and Δ-9-tetrahydrocannabinol (THC). Based on published data, the chemotype should correlate with plant morphology, genetics, and photosynthetic properties. In this work, we investigated leaf morphology, plant growth characteristics, cannabinoid profiles, THCAS gene sequences, and plant photosynthetic traits in two breeding populations of medical cannabis (MX-CBD-11 and MX-CBD-707). The populations differed significantly in morphological traits. The MX-CBD-11 plants were taller, less branched, and their leaves had narrower leaflets than the bushier, wideleaved MX-CBD-707 plants, and there were significant differences between populations in the dry biomass of different plant parts. Based on these morphological differences, MX-CBD-11 was designated as a narrow leaflet drug type or vernacular "Sativa" type, while MX-CBD-707 was classified as wide leaflet drug type or "Indica" type. Chemical characterisation revealed a discrepancy between the expected chemotypes based on plant morphology; although both populations have high CBD, within each Type II (CBD/THC intermediate) and Type III (CBD dominant) plants were detected. The THCAS gene sequence analysis clustered the plants based on their chemotypes and showed high similarity to the THCAS sequences deposited in NCBI. In silico complementary analysis, using published molecular markers for chemotype determination, showed their low discrimination power in our two populations, demonstrating the genotype dependence of the molecular markers. Basic photosynthetic traits derived from light and CO2 response curves were similar in the populations. However, measurements of gas exchange under chamber conditions revealed higher stomatal conductivity and photosynthesis in MX-CBD-707 plants, which were also characterised by higher day respiration. The results of this study showed that based on visual appearance and some morphological measurements, it is not possible to determine a plant's chemotype. Visually homogenous plants had different cannabinoid profiles and, vice versa, morphologically distinct plants contained similar CBD and THC content. The two chemotypes identified in our experimental plants therefore did not correlate with plant visual appearance, leaf morphometry, and photosynthetic properties of the populations studied. Correlation was only demonstrated with the respect to THCAS sequences, which showed great discrimination power between the chemotypes.

Climatic regulation of leaf and cambial phenology in Quercus pubescens: Their interlinkage and impact on xylem and phloem conduits.

Increased frequency and severity of stressful events affects the growth patterns and functioning of trees which adjust their phenology to given conditions. Here, we analysed environmental effects (temperature, precipitation, VPD and SWC) on the timing of leaf phenology, seasonal stem radial growth patterns, and xylem and phloem anatomy of Quercus pubescens in the sub-Mediterranean in the period 2014-2019, when various adverse weather events occurred, i.e. spring drought in 2015, summer fire in 2016 and summer drought in 2017. Results showed that the timings of leaf and cambium phenology do not occur simultaneously in Q. pubescens, reflecting different environmental and internal constraints. Although year-to-year variability in the timings of leaf and cambial phenology exists, their chronological sequence is fairly fixed. Different effects of weather conditions on different stages of leaf development in spring were observed. Common climatic drivers (i.e., negative effect of hot and dry summers and a positive effect of increasing moisture availability in winter and summer) were found to affect the widths of xylem and phloem increments with more pronounced effect on late formed parts. A legacy effect of the timing of leaf and cambial phenology of the previous growing season on the timing of phenology of the following spring was confirmed. Rarely available phloem data permitted a comprehensive insight into the interlinkage of the timing of cambium and leaf phenology and adjustment strategies of vascular tissues in Mediterranean pubescent oak to various environmental constraints, including frequent extreme events (drought, fire). Our results suggest that predicted changes in autumn/winter and spring climatic conditions for this area could affect the timings of leaf and stem cambial phenology of Q. pubescens in the coming years, which would affect stem xylem and phloem structure and hydraulic properties, and ultimately its performance.

Physiological and Biochemical Responses of Ungrafted and Grafted Bell Pepper Plants (Capsicum annuum L. var. grossum (L.) Sendtn.) Grown under Moderate Salt Stress.

The response of grafted bell pepper plants (Capsicum annuum L. var. grossum (L.) Sendtn.) to salt stress was investigated by analyzing the photosynthetic traits and mineral content of the plants and the metabolic composition of the fruit. The bell pepper variety "Vedrana" was grafted onto the salt-tolerant rootstock "Rocal F1" and grown at two salinities (20 mM and 40 mM NaCl) and control (0 mM NaCl) during the spring-summer period. On a physiological level, similar stomatal restriction of photosynthesis in grafted and ungrafted plants indicated that grafting did not alleviate water balance disturbances under increased salt exposure. Measurements of midday water potential did not show improved water status of grafted plants. The similar metabolic changes in grafted and ungrafted plants were also reflected in similarly reduced fruit yields. Thus, this grafting did not reduce the risk of ionic and osmotic imbalance in pepper plants grown under moderate salt treatment. Changes in the biochemical profiles of the pepper fruit were seen for both added-salt treatments. The fruit phenolic compounds were affected by rootstock mediation, although only for the July harvest, where total phenolics content increased with 40 mM NaCl treatment. Fruit ascorbic acid content increased with the duration of salt stress, without the mediation of the rootstock. The high salt dependence of this quality trait in pepper fruit appears to lead to more limited rootstock mediation effects.

Post-fire effects on development of leaves and secondary vascular tissues in Quercus pubescens.

An increased frequency of fire events on the Slovenian Karst is in line with future climate change scenarios for drought-prone environments worldwide. It is therefore of the utmost importance to better understand tree-fire-climate interactions for predicting the impact of changing environment on tree functioning. To this purpose, we studied the post-fire effects on leaf development, leaf carbon isotope composition (δ13C), radial growth patterns and the xylem and phloem anatomy in undamaged (H-trees) and fire-damaged trees (F-trees) of Quercus pubescens Willd. with good resprouting ability in spring 2017, the growing season after a rangeland fire in August 2016. We found that the fully developed canopy of F-trees reached only half of the leaf area index values measured in H-trees. Throughout the season, F-trees were characterized by higher water potential and stomatal conductivity and achieved higher photosynthetic rates compared to unburnt H-trees. The foliage of F-trees had more negative δ13C values than those of H-trees. This reflects that F-trees less frequently meet stomatal limitations due to reduced transpirational area and more favourable leaf-to-root ratio. In addition, the growth of leaves in F-trees relied more on the recent photosynthates than on reserves due to the fire disturbed starch accumulation in the previous season. Cambial production stopped 3 weeks later in F-trees, resulting in 60 and 22% wider xylem and phloem increments, respectively. A novel approach by including phloem anatomy in the analyses revealed that fire caused changes in conduit dimensions in the early phloem but not in the earlywood. However, premature formation of the tyloses in the earlywood vessels of the youngest two xylem increments in F-trees implies that xylem hydraulic integrity was also affected by heat. Analyses of secondary tissues showed that although xylem and phloem tissues are interlinked changes in their transport systems due to heat damage are not necessarily coordinated.

Impacts of long-term elevated atmospheric CO2 concentrations on communities of arbuscular mycorrhizal fungi.

The ecological impacts of long-term elevated atmospheric CO2 (eCO2 ) levels on soil microbiota remain largely unknown. This is particularly true for the arbuscular mycorrhizal (AM) fungi, which form mutualistic associations with over two-thirds of terrestrial plant species and are entirely dependent on their plant hosts for carbon. Here, we use high-resolution amplicon sequencing (Illumina, HiSeq) to quantify the response of AM fungal communities to the longest running (>15 years) free-air carbon dioxide enrichment (FACE) experiment in the Northern Hemisphere (GiFACE); providing the first evaluation of these responses from old-growth (>100 years) semi-natural grasslands subjected to a 20% increase in atmospheric CO2 . eCO2 significantly increased AM fungal richness but had a less-pronounced impact on the composition of their communities. However, while broader changes in community composition were not observed, more subtle responses of specific AM fungal taxa were with populations both increasing and decreasing in abundance in response to eCO2 . Most population-level responses to eCO2 were not consistent through time, with a significant interaction between sampling time and eCO2 treatment being observed. This suggests that the temporal dynamics of AM fungal populations may be disturbed by anthropogenic stressors. As AM fungi are functionally differentiated, with different taxa providing different benefits to host plants, changes in population densities in response to eCO2 may significantly impact terrestrial plant communities and their productivity. Thus, predictions regarding future terrestrial ecosystems must consider changes both aboveground and belowground, but avoid relying on broad-scale community-level responses of soil microbes observed on single occasions.

Effect of soil water availability on intra-annual xylem and phloem formation and non-structural carbohydrate pools in stem of Quercus pubescens.

Non-structural carbohydrates (NSCs, i.e., starch and soluble sugars) are frequently quantified in the context of tree response to stressful events (e.g., drought), because they serve as a carbon reservoir for growth and respiration, as well as providing a critical osmotic function to maintain turgor and vascular transport under different environmental conditions. We investigated the impact of soil water availability on intra-annual leaf phenology, radial growth dynamics and variation in NSC amounts in the stem of pubescent oak (Quercus pubescens Willd.). from a sub-Mediterranean region. For this purpose, trees growing at two nearby plots differing in bedrock and, consequently, soil characteristics (F-eutric cambisol on eocene flysch bedrock and L-rendzic leptosol on paleogenic limestone bedrock) were sampled. Non-structural carbohydrates were analysed in outer xylem and living phloem (separately for non-collapsed and collapsed parts). Results showed that xylem and phloem increments were 41.6% and 21.2%, respectively, wider in trees from F plot due to a higher rate of cell production. In contrast, the amount of NSCs and of soluble sugars significantly differed among the tissue parts and sampling dates but not between the two plots. Starch amounts were the highest in xylem, which could be explained by the abundance of xylem parenchyma cells. Two clear seasonal peaks of the starch amount were detected in all tissues, the first in September-November, in the period of leaf colouring and falling, and the second in March-April, i.e., at the onset of cambial cell production followed by bud development. The amounts of free sugars were highest in inner phloem + cambium, at the sites of active growth. Soil water availability substantially influenced secondary growth in the stem of Q. pubescens, whereas NSC amounts seemed to be less affected. The results show how the intricate relationships between soil properties, such as water availability, and tree performance should be considered when studying the impact of stressful events on the growth and functioning of trees.

Photosynthetic Traits of Plants and the Biochemical Profile of Tomato Fruits Are Influenced by Grafting, Salinity Stress, and Growing Season.

Changes in the photosynthetic traits of plants and metabolic composition of fruits of two tomato cultivars, grafted onto two rootstocks, grown in three salinity levels were studied in two growing periods during the season. Increased salinity stress conditions lowered water potential, stomatal conductance, and transpiration rate of grafted tomato plants, in both growing periods. Water deficit induced stomatal closure, which resulted in stomatal limitation of photosynthesis. The proline content in tomato leaves increased and was closely correlated with salinity. Some of the quality parameters of tomato fruits were affected by rootstock. The sugar/acid ratio was the highest in fruits of 'Belle'/'Maxifort' grafts. With increasing salt stress conditions from 40 to 60 mM NaCl, the lycopene content increased and ascorbic acid content decreased in fruits of 'Gardel'/'Maxifort' grafts, indicating the ability of this scion/rootstock combination to mitigate the toxicity effect of salinity stress. A higher phenolics concentration in fruits from the first growing period may be an additional indicator of stress, caused by higher temperatures and solar radiation, compared with the later period.

Chronological Sequence of Leaf Phenology, Xylem and Phloem Formation and Sap Flow of Quercus pubescens from Abandoned Karst Grasslands.

Intra-annual variations in leaf development, radial growth, including the phloem part, and sap flow have rarely been studied in deciduous trees from drought-prone environments. In order to understand better the chronological order and temporal course of these processes, we monitored leaf phenology, xylem and phloem formation and sap flow in Quercus pubescens from abandoned karst grasslands in Slovenia during the growing season of 2014. We found that the initial earlywood vessel formation started before bud opening at the beginning of April. Buds started to open in the second half of April and full leaf unfolding occurred by the end of May. LAI values increased correspondingly with leaf development. About 28% of xylem and 22% of phloem annual increment were formed by the time of bud break. Initial earlywood vessels were fully lignified and ready for water transport, indicating that they are essential to provide hydraulic conductivity for axial water flow during leaf development. Sap flow became active and increasing contemporarily with leaf development and LAI values. Similar early spring patterns of xylem sap flow and LAI denoted that water transport in oaks broadly followed canopy leaf area development. In the initial 3 weeks of radial growth, phloem growth preceded that of xylem, indicating its priority over xylem at the beginning of the growing season. This may be related to the fact that after bud break, the developing foliage is a very large sink for carbohydrates but, at the same time, represents a small transpirational area. Whether the interdependence of the chronological sequence of the studied processes is fixed in Q. pubescens needs to be confirmed with more data and several years of analyses, although the 'correct sequence' of processes is essential for synchronized plant performance and response to environmental stress.

Hop (Humulus lupulus L.) response mechanisms in drought stress: Proteomic analysis with physiology.

Drought is one of the major environmental devastating stressors that impair the growth and productivity of crop plants. Despite the relevance of drought stress, changes in physiology and resistance mechanisms are not completely understood for certain crops, including hop (Humulus lupulus L.). In this research the drought response of hop was studied using a conventional physiological approach (gas exchange techniques, fluorescence, relative water content measurements) and proteomic analysis (2D-DIGE). Plants of two cultivars (Aurora and Savinjski golding) were exposed to progressive drought in a pot experiment and analysed at different stress stages (mild, moderate and severe). Measurements of relative water content revealed a hydrostable water balance of hop. Photosynthesis was decreased due to stomatal and non-stomatal limitation to the same extent in both cultivars. Of 28 identified differentially abundant proteins, the majority were down regulated and included in photosynthetic (41%) and sugar metabolism (33%). Fifteen % of identified proteins were classified into the nitrogen metabolism, 4% were related to a ROS related pathway and 7% to other functions.

Bimodal dynamics of primary metabolism-related responses in tolerant potato-Potato virus Y interaction.

BACKGROUND: Potato virus Y (PVY) is a major pathogen that causes substantial economic losses in worldwide potato production. Different potato cultivars differ in resistance to PVY, from severe susceptibility, through tolerance, to complete resistance. The aim of this study was to better define the mechanisms underlying tolerant responses of potato to infection by the particularly aggressive PVY(NTN) strain. We focused on the dynamics of the primary metabolism-related processes during PVY(NTN) infection. RESULTS: A comprehensive analysis of the dynamic changes in primary metabolism was performed, which included whole transcriptome analysis, nontargeted proteomics, and photosynthetic activity measurements in potato cv. Désirée and its transgenic counterpart depleted for accumulation of salicylic acid (NahG-Désirée). Faster multiplication of virus occurred in the NahG-Désirée, with these plants developing strong disease symptoms. We show that while the dynamics of responses at the transcriptional level are extensive and bimodal, this is only partially translated to the protein level, and to the final functional outcome. Photosynthesis-related genes are transiently induced before viral multiplication is detected and it is down-regulated later on. This is reflected as a deficiency of the photosynthetic apparatus at the onset of viral multiplication only. Interestingly, specific and constant up-regulation of some RuBisCO transcripts was detected in Désirée plants, which might be important, as these proteins have been shown to interact with viral proteins. In SA-deficient and more sensitive NahG-Désirée plants, consistent down-regulation of photosynthesis-related genes was detected. A constant reduction in the photochemical efficiency from the onset of viral multiplication was identified; in nontransgenic plants this decrease was only transient. The transient reduction in net photosynthetic rate occurred in both genotypes with the same timing, and coincided with changes in stomatal conductivity. CONCLUSIONS: Down-regulation of photosynthesis-related gene expression and decreased photosynthetic activity is in line with other studies that have reported the effects of biotic stress on photosynthesis. Here, we additionally detected induction of light-reaction components in the early stages of PVY(NTN) infection of tolerant interaction. As some of these components have already been shown to interact with viral proteins, their overproduction might contribute to the absence of symptoms in cv. Désirée.

Effect of EDTA washing of metal polluted garden soils. Part II: Can remediated soil be used as a plant substrate?

In a field experiment on metal contaminated and EDTA-remediated soil we studied plant performance, mycorrhizal associations and prospects of potential re-use of remediated soil as a garden substrate. Two experimental plots of 4 × 1 × 0.3 m were filled, one with remediated and the other with original contaminated soil. Selected cultivars were rotated over the course of 16months. Pb, Zn, Cd and micronutrient plant uptake was measured and their phytoaccessibility was analyzed by the DTPA method. Plant fitness was assessed by chlorophyll fluorescence and gas exchange measurements and evaluation of root colonization were analyzed with mycorrhizal fungi. Remediation reduced Pb and Cd concentrations in roots, green parts and fruits in most of the plants. Phytoaccumulation of Zn was reduced in one half of the cultivars. Some plants suffered from Mn deficiency as total soil Mn was reduced 4-fold and phytoaccessibility of micronutrients Cu, Fe and Mn for 54, 26 and 79%, respectively. Plant biomass was reduced. Photosynthetic parameters of plants grown in original and remediated soil were similar, except for the reduction in Spinacia oleracea. The frequency of mycorrhizal colonization in the roots of Pisum sativum was reduced five-fold and no significant changes were found in Allium cepa roots. Remediation reduced plant uptake of Pb below the concentration stipulated by legislation. Measures to reduce plant accumulation of other toxic metals and to revitalize remediated soil are needed.

Functioning of metal contaminated garden soil after remediation.

The effect of remediation using three EDTA doses (10, 30, 60 mmol kg(-1)) on soil functioning was assessed using column experiment and Brassica rapa. Soil washing removed up to 77, 29 and 72% of metals from soil contaminated with 1378, 578 and 8.5 mg kg(-1) of Pb, Zn and Cd, respectively. Sequential extraction indicated removal from the carbonate soil fraction. Metal oral-accessibility from the stomach phase was reduced by up to 75 and from the small intestine by up to 79% (Pb). Part of metals (up to 0.8% Cd) was lost due to leaching from columns. Remediation reduced toxic metal soil-root transfer by up to 61% but did not prevent metal accumulation in leaves. The fitness of plants grown on EDTA washed soils (gas exchange, fluorescence) was not compromised. Remediation initially reduced the soil DNA content (up to 29%, 30 mmol kg(-1) EDTA) and changed the structure of microbial population.

Local adaptation to soil hypoxia determines the structure of an arbuscular mycorrhizal fungal community in roots from natural CO2 springs.

The processes responsible for producing and maintaining the diversity of natural arbuscular mycorrhizal (AM) fungal communities remain largely unknown. We used natural CO(2) springs (mofettes), which create hypoxic soil environments, to determine whether a long-term, directional, abiotic selection pressure could change AM fungal community structure and drive the selection of particular AM fungal phylotypes. We explored whether those phylotypes that appear exclusively in hypoxic soils are local specialists or widespread generalists able to tolerate a range of soil conditions. AM fungal community composition was characterized by cloning, restriction fragment length polymorphism typing, and the sequencing of small subunit rRNA genes from roots of four plant species growing at high (hypoxic) and low (control) geological CO(2) exposure. We found significant levels of AM fungal community turnover (ß diversity) between soil types and the numerical dominance of two AM fungal phylotypes in hypoxic soils. Our results strongly suggest that direct environmental selection acting on AM fungi is a major factor regulating AM fungal communities and their phylogeographic patterns. Consequently, some AM fungi are more strongly associated with local variations in the soil environment than with their host plant's distribution.

Abundance and diversity of CO2-fixing bacteria in grassland soils close to natural carbon dioxide springs.

Gaseous conditions at natural CO2 springs (mofettes) affect many processes in these unique ecosystems. While the response of plants to extreme and fluctuating CO2 concentrations ([CO2]) is relatively well documented, little is known on microbial life in mofette soil. Therefore, it was the aim of this study to investigate the abundance and diversity of CO2-fixing bacteria in grassland soils in different distances to a natural carbon dioxide spring. Samples of the same soil type were collected from the Stavesinci mofette, a natural CO2 spring which is known for very pure CO2 emissions, at different distances from the CO2 releasing vents, at locations that clearly differed in soil CO2 efflux (from 12.5 to over 200 micromol CO2 m(-2) s(-1) yearly average). Bulk and rhizospheric soil samples were included into analyses. The microbial response was followed by a molecular analysis of cbbL genes, encoding for the large subunit of RubisCO, a carboxylase which is of crucial importance for C assimilation in chemolitoautotrophic microbes. In all samples analyzed, the "red-like" type of cbbL genes could be detected. In contrast, the "green-like" type of cbbL could not be measured by the applied technique. Surprisingly, a reduction of "red-like" cbbL genes copies was observed in bulk soil and rhizosphere samples from the sites with the highest CO2 concentrations. Furthermore, the diversity pattern of "red-like" cbbL genes changed depending on the CO(2) regime. This indicates that only a part of the autotrophic CO2-fixing microbes could adapt to the very high CO2 concentrations and adverse life conditions that are governed by mofette gaseous regime.