The presence of Bacillus thuringiensis (Bt) protein in earthworms Eisenia fetida has no deleterious effects on their growth and reproduction

Earthworms Eisenia fetida, bred in substances with stover of two genetically-engineered Bacillus thuringiensis (Bt) corns (5422Bt1 (Event Bt11) and 5422CBCL (MON810)) expressing Cry1Ab and their near-isogenic non-Bt corn (5422), were used to investigate the non-target effects of Bt corn on soil-dwelling organisms. Cry1Ab concentrations in substances, casts and guts of E. fetida were also investigated by Enzyme-linked immunosorbent assay (ELISA). More than 90% individuals of E. fetida survived over a period of 30 d, irrespective of whether they received Bt corn or non-Bt corn. Compared to 5422 treatments, significantly higher relative growth rate and more number of new offspring and cocoons of E. fetida were found in 5422Bt1 and 5422CBCL treatments. These results were unlikely to be directly caused by Cry1Ab released from Bt corns but rather by differences in other factors of plants such as plant components (soluble sugar, total organic carbon, total protein and available phosphorus of Bt corns were more than 5422). ELISA results indicated immunoreactive Cry1Ab was detectable in substances, and the casts, guts of E. fetida from Bt corns treatments, of which the highest levels were detected in substances under the corresponding experimental conditions. With the increase of treated time, a strong decline was observed in Cry1Ab from substances and casts of E. fetida, whereas Cry1Ab in guts of E. fetida from 5422Bt1 treatments gradually increased and that from 5422CBCL treatments increased between 14 and 30 d. Therefore, the presence of Cry1Ab in E. fetida had no deleterious effects on their growth and reproduction.     

Fine root dynamics of mature European beech (Fagus sylvatica L.) as influenced by elevated ozone concentrations

Fine root dynamics (diameter < 1 mm) in mature Fagus sylvatica, with the canopies exposed to ambient or twice-ambient ozone concentrations, were investigated throughout 2004. The focus was on the seasonal timing and extent of fine root dynamics (growth, mortality) in relation to the soil environment (water content, temperature). Under ambient ozone concentrations, a significant relationship was found between fine root turnover and soil environmental changes indicating accelerated fine root turnover under favourable soil conditions. In contrast, under elevated ozone, this relationship vanished as the result of an altered temporal pattern of fine root growth. Fine root survival and turnover rate did not differ significantly between the different ozone regimes, although a delay in current-year fine root shedding was found under the elevated ozone concentrations. The data indicate that increasing tropospheric ozone levels can alter the timing of fine root turnover in mature F. sylvatica but do not affect the turnover rate.     

Ozone exposure of field-grown winter wheat affects soil mesofauna in the rhizosphere

A 2-year open-top chamber experiment with field-grown winter wheat (Triticum aestivum L. cv. Astron) was conducted to examine the effects of ozone on plant growth and selected groups of soil mesofauna in the rhizosphere. From May through June in each year, plants were exposed to two levels of O₃: non-filtered (NF) ambient air or NF+ 40 ppb O₃ (NF+). During O₃ exposure, soil sampling was performed at two dates according to different plant growth stages. O₃ exposure reduced above- and below-ground plant biomass in the first year, but had little effect in the second year. The individual density of enchytraeids, collembolans and soil mites decreased significantly in the rhizosphere of plants exposed to NF+ in both years. Differences were highest around anthesis, i.e. when plants are physiologically most active. The results suggest that elevated O₃ concentrations may influence the dynamic of decomposition processes and the turnover of nutrients.     

Effects of chronic elevated ozone exposure on gas exchange responses of adult beech trees (Fagus sylvatica) as related to the within-canopy light gradient

The effects of elevated O₃ on photosynthetic properties in adult beech trees (Fagus sylvatica) were investigated in relation to leaf mass per area as a measure of the gradually changing, within-canopy light availability. Leaves under elevated O₃ showed decreased stomatal conductance at unchanged carboxylation capacity of Rubisco, which was consistent with enhanced δ¹³C of leaf organic matter, regardless of the light environment during growth. In parallel, increased energy demand for O₃ detoxification and repair was suggested under elevated O₃ owing to enhanced dark respiration. Only in shade-grown leaves, light-limited photosynthesis was reduced under elevated O₃, this effect being accompanied by lowered F_v/F_m. These results suggest that chronic O₃ exposure primarily caused stomatal closure to adult beech trees in the field regardless of the within-canopy light gradient. However, light limitation apparently raised the O₃ sensitivity of photosynthesis and accelerated senescence in shade leaves.     

Tibouchina pulchra (Cham.) Cogn., a native Atlantic Forest species, as a bio-indicator of ozone: visible injury

Tibouchina pulchra saplings were exposed to carbon filtered air (CF), ambient non-filtered air (NF) and ambient non-filtered air+40 ppb ozone (NF+O3) 8 h per day during two months. The AOT40 values at the end of the experiment were 48, 910 and 12,895 ppb h(-1), respectively, for the three treatments. After 25 days of exposure (AOT40=3871 ppb h(-1)), interveinal red stippling appeared in plants in the NF+O3 chamber. In the NF chamber, symptoms were observed only after 60 days of exposure (AOT40=910 ppb h(-1)). After 60 days, injured leaves per plant corresponded to 19% in NF+O3 and 1% in the NF treatment; and the average leaf area injured was 7% within the NF+O3 and 0.2% within the NF treatment. The extent of leaf area injured (leaf injury index) was mostly explained by the accumulated exposure of ozone (r2=0.89; p<0.05).     

Combined effects of elevated CO2 and natural climatic variation on leaf spot diseases of redbud and sweetgum trees

Atmospheric CO₂ concentrations are predicted to double within the next century and alter climate regimes, yet the extent that these changes will affect plant diseases remains unclear. In this study conducted over five years, we assessed how elevated CO₂ and interannual climatic variability affect Cercospora leaf spot diseases of two deciduous trees. Climatic data varied considerably between the five years and altered disease expression. Disease incidence and severity for both species were greater in years with above average rainfall. In years with above average temperatures, disease incidence for Liquidambar styraciflua was decreased significantly. When significant changes did occur, disease incidence and severity always increased under elevated CO₂. Chlorophyll fluorescence imaging of leaves revealed that any visible increase in disease severity induced by elevated CO₂ was mitigated by higher photosynthetic efficiency in the remaining undamaged leaf tissue and in a halo surrounding lesions.     

Will photosynthetic capacity of aspen trees acclimate after long-term exposure to elevated CO2 and O3?

Photosynthetic acclimation under elevated carbon dioxide (CO₂) and/or ozone (O₃) has been the topic of discussion in many papers recently. We examined whether or not aspen plants grown under elevated CO₂ and/or O₃ will acclimate after 11 years of exposure at the Aspen Face site in Rhinelander, WI, USA. We studied diurnal patterns of instantaneous photosynthetic measurements as well as A/C_i measurements monthly during the 2004-2008 growing seasons. Our results suggest that the responses of two aspen clones differing in O₃ sensitivity showed no evidence of photosynthetic and stomatal acclimation under either elevated CO₂, O₃ or CO₂ + O₃. Both clones 42E and 271 did not show photosynthetic nor stomatal acclimation under elevated CO₂ and O₃ after a decade of exposure. We found that the degree of increase or decrease in the photosynthesis and stomatal conductance varied significantly from day to day and from one season to another.     

Nitrogen mediates above-ground effects of ozone but not below-ground effects in a rhizomatous sedge

Ozone and atmospheric nitrogen are co-occurring pollutants with adverse effects on natural grassland vegetation. Plants of the rhizomatous sedge Carex arenaria were exposed to four ozone regimes representing increasing background concentrations (background-peak): 10-30, 35-55, 60-80 and 85-105 ppb ozone at two nitrogen levels: 12 and 100 kg N ha⁻¹ yr⁻¹. Ozone increased the number and proportion of senesced leaves, but not overall leaf number. There was a clear nitrogen × ozone interaction with high nitrogen reducing proportional senescence in each treatment and increasing the ozone dose (AOT40) at which enhanced senescence occurred. Ozone reduced total biomass due to significant effects on root biomass. There were no interactive effects on shoot:root ratio. Rhizome tissue N content was increased by both nitrogen and ozone. Results suggest that nitrogen mediates above-ground impacts of ozone but not impacts on below-ground resource translocation. This may lead to complex interactive effects between the two pollutants on natural vegetation.     

The impact of ambient ozone on mountain spruce forests in the Czech Republic as indicated by malondialdehyde

Malondialdehyde (MDA), a product of lipid peroxidation and biomarker of oxidative stress, is measured over the long term in spruce Picea abies needles under real conditions in three Czech mountain border areas. The trends presented collate the MDA content in spruce needles with ambient ozone, temperature and precipitation as casual, and defoliation as a subsequent factor for the period 1994-2006. We have found the overall decreasing trends in MDA and defoliation. The highest MDA and defoliation are recorded in the Jizerske, the lowest in the Krusne hory Mts. Out of the examined variables the MDA is predicted best by mean temperature in vegetation season, median of O₃ concentrations and AOT40; these three variables account for 34% of MDA1 and 36% of MDA2 variability. Our hypothesis that higher ambient O₃ exposure results in higher MDA contents in P. abies needles under real conditions has not been approved.     

Ozone fumigation (twice ambient) reduces leaf infestation following natural and artificial inoculation by the endophytic fungus Apiognomonia errabunda of adult European beech trees

In 2006, a controlled infection study was performed in the ‘Kranzberger Forst’ to address the following questions: (1) Will massive artificial inoculation with Apiognomonia errabunda override the previously observed inhibitory effect of chronic ozone? (2) Can biochemical or molecular markers be detected to account for the action of ozone? To this end six adult beech trees were chosen, three ozone fumigated (2× ozone) and three control trees (ambient = 1× ozone). Spore-sprayed branches of sun and shade crown positions of each of the trees, and uninoculated control branches, were enclosed in 100-L plastic bags for one night to facilitate infection initiation. Samples were taken within a five-week period after inoculation. A. errabunda infestation levels quantified by real-time PCR increased in leaves that were not fumigated with additional ozone. Cell wall components and ACC (ethylene precursor 1-amino cyclopropane-1-carboxylic acid) increased upon ozone fumigation and may in part lead to the repression of fungal infection.     

Species-specific effects of elevated ozone on wetland plants and decomposition processes

Seven species from two contrasting wetlands, an upland bog and a lowland rich fen in North Wales, UK, were exposed to elevated ozone (150 ppb for 5 days and 20 ppb for 2 days per week) or low ozone (20 ppb) for four weeks in solardomes. The rich fen species were: Molinia caerulea, Juncus subnodulosus, Potentilla erecta and Hydrocotyle vulgaris and the bog species were: Carex echinata, Potentilla erecta and Festuca rubra. Senescence significantly increased under elevated ozone in all seven species but only Molinia caerulea showed a reduction in biomass under elevated ozone. Decomposition rates of plants exposed to elevated ozone, as measured by carbon dioxide efflux from dried plant material inoculated with peat slurry, increased for Potentilla erecta with higher hydrolytic enzyme activities. In contrast, a decrease in enzyme activities and a non-significant decrease in carbon dioxide efflux occurred in the grasses, sedge and rush species.     

A comparison of two stomatal conductance models for ozone flux modelling using data from two Brassica species

In this study we tested and compared a multiplicative stomatal model and a coupled semi-empirical stomatal-photosynthesis model in their ability to predict stomatal conductance to ozone (g_st) using leaf-level data from oilseed rape (Brassica napus L.) and broccoli (Brassica oleracea L. var. italica Plenck). For oilseed rape, the multiplicative model and the coupled model were able to explain 72% and 73% of the observed g_st variance, respectively. For broccoli, the models were able to explain 53% and 51% of the observed g_st variance, respectively. These results support the coupled semi-empirical stomatal-photosynthesis model as a valid alternative to the multiplicative stomatal model for O₃ flux modelling, in terms of predictive performance.     

Factors Affecting Ozone Sensitivity and Susceptibility Of Cotton Plants

Cotton plants were investigated to ascertain the growth period of maximum sensitivity to ozone. Plants were grown from seed in the greenhouse to obtain a two-day interval age series to about three months old. Before fumigation with 0.2 to 1.0 ppm ozone for 1 hr, plants were preconditioned under standard growth chamber conditions with day temperatures of 32°, night temperatures of 21°, relative humidity of 50%, and a light intensity of 6 X 10⁴ ergs/cm²/sec. Also, stomatal opening was monitored with a resistance hygrometer. Ozone damage to leaves was assessed by rating per cent visible damage with an arbitrary scale. Approximately 0.6-0.7 ppm ozone exposure for 1 hr was necessary for visible damage. Leaves were susceptible to ozone when about three-quarters fully expanded. Leaves appearing on older plants became progressively less sensitive. Diurnally, susceptible leaves were only sensitive after several hours in the light and then lost sensitivity toward the end of a normal day. The two most critical questions concerning the nature of ozone sensitivity and susceptibility are: (7) what is the nature of susceptibility, and (2) what is the nature of sensitivity of susceptible leaves?     

Seasonal differences and within-canopy variations of antioxidants in mature spruce (Picea abies) trees under elevated ozone in a free-air exposure system

The effect of free-air ozone fumigation and crown position on antioxidants were determined in old-growth spruce (Picea abies) trees in the seasonal course of two consecutive years (2003 and 2004). Levels of total ascorbate and its redox state in the apoplastic washing fluid (AWF) were increased under double ambient ozone concentrations (2xO3), whilst ascorbate concentrations in needle extracts were unchanged. Concentrations of apoplastic and symplastic ascorbate were significantly higher in 2003 compared to 2004 indicating a combined effect of the drought conditions in 2003 with enhanced ozone exposure. Elevated ozone had only weak effects on total glutathione levels in needle extracts, phloem exudates and xylem saps. Total and oxidised glutathione concentrations were higher in 2004 compared to 2003 and seemed to be more affected by enhanced ozone influx in the more humid year 2004 compared to the combined effect of elevated ozone and drought in 2003 as observed for ascorbate.     

Effects of ozone on chlorophyll and quantum yield of tobacco (Nicotiana tabacum L.) varieties

Plants of Bel-W3 and of seven commercial tobacco varieties (Nicotiana tabacum L.) were exposed to two relatively low ozone concentrations (90 or 135 ppb) for 20 consecutive days, for 8 h per day. Ozone caused necrotic and chlorotic spots, acceleration of leaf senescence, depression of photosynthetic mechanism, chlorophyll diminution and greater destruction of chl a than of chl b. The higher sensitivity of chl a was also confirmed by exposure of segments of leaves in test tubes to high ozone concentration (>1000 ppb) as well as by bubbling of ozone in extracts of chlorophyll in vitro. The quantum yield (QY) of photosynthesis was positively correlated with the chlorophyll content and negatively correlated with the visible injury and the chl b/a ratio.     

Growth and photosynthetic responses to copper in wild grapevine

The present study evaluates the tolerance and accumulation potential of Vitis vinifera ssp. sylvestris under moderate and high external Cu levels. A greenhouse experiment was conducted in order to investigate the effects of a range of external Cu concentrations (0–23 mmol L⁻¹) on growth and photosynthetic performance by measuring gas exchange, chlorophyll fluorescence parameters and photosynthetic pigments. We also measured the total copper, nitrogen, phosphorus, sulphur, calcium, magnesium, iron, potassium and sodium concentrations in the plant tissues. All the experimental plants survived even with external Cu concentrations as high as 23 mmol L⁻¹ (1500 mg Cu L⁻¹), although the excess of metal resulted in a biomass reduction of 35%. The effects of Cu on growth were linked to a reduction in net photosynthesis, which may be related to the effect of the high concentration of the metal on photosynthetic electron transport. V. vinifera ssp. sylvestris survived with leaf Cu concentrations as high as 80 mg kg⁻¹ DW and growth parameters were unaffected by leaf tissue concentrations of 35 mg Cu kg⁻¹ DW. The results of our study indicate that plants of V. vinifera ssp. sylvestris from the studied population are more tolerant to Cu than the commercial varieties of grapevine that have been studied in the literature, and could constitute a basis for the genetic improvement of Cu tolerance in grapevine.     

Enhanced ozone strongly reduces carbon sink strength of adult beech (Fagus sylvatica) - Resume from the free-air fumigation study at Kranzberg Forest

Ground-level ozone (O₃) has gained awareness as an agent of climate change. In this respect, key results are comprehended from a unique 8-year free-air O₃-fumigation experiment, conducted on adult beech (Fagus sylvatica) at Kranzberg Forest (Germany). A novel canopy O₃ exposure methodology was employed that allowed whole-tree assessment in situ under twice-ambient O₃ levels. Elevated O₃ significantly weakened the C sink strength of the tree-soil system as evidenced by lowered photosynthesis and 44% reduction in whole-stem growth, but increased soil respiration. Associated effects in leaves and roots at the gene, cell and organ level varied from year to year, with drought being a crucial determinant of O₃ responsiveness. Regarding adult individuals of a late-successional tree species, empirical proof is provided first time in relation to recent modelling predictions that enhanced ground-level O₃ can substantially mitigate the C sequestration of forests in view of climate change.     

Leaf phenolic compounds in red clover (Trifolium pratense L.) induced by exposure to moderately elevated ozone

Red clover (Trifolium pratense L.), an important feed crop in many parts of the world, is exposed to elevated ozone over large areas. Plants can limit ozone-induced damages by various defence mechanisms. In this work, changes in the concentrations of antioxidant phenolic compounds induced by slightly elevated levels of ozone were determined in red clover leaves by high-performance liquid chromatography and mass spectrometry. 31 different phenolics were identified and the most abundant isoflavones and flavonoids were biochanin A glycoside malonate (G-M), formononetin-G-M and quercetin-G-M. Elevated ozone (mean 32.4 ppb) increased the total phenolic content of leaves and also had minor effects on the concentrations of individual compounds. Elevated ozone increased the net photosynthesis rate of red clover leaves before visible injuries by 21-23%. This study thus suggests that the concentrations of phenolics in red clover leaves change in response to slightly elevated ozone levels.