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Preventive measures for microflora and mycotoxin production in foodstuffs.

The conservation of foodstuffs by chemical or physical methods is now practiced on a wide scale. Certain procedures (drying, use of pesticides) are well proved and are used on an industrial scale, although in certain situations they are not without risk to human health. New techniques (controlled atmospheres, irradiation, fumigation) are in use and will undoubtedly see further development for conservation of seeds and cereals in both industrialized and non-industrialized countries. In the latter case, the use of cost-effective fumigants may be of particular value. Preventive measures for the control of molds in foodstuffs are discussed in this report.

Food Contamination↗

Recovery of 1,3-, 2,3-dichloropropenes, 1,2-dibromo-3-chloropropane, and o-, p-dichlorobenzenes from fatty and non-fat foodstuffs by liquid extraction technique.

Food samples including fatty, non-fatty, grain-based, and nongrain-based types were fortified with the following five nematocides and fumigants: 1,3-dichloropropene, 2,3-dichloropropene, 1,2-dibromo-3-chloropropane, o-dichlorobenzene, and p-dichlorobenzene. Then, depending on sample consistency and type, the samples were diluted in, or extracted with organic solvent such as isooctane. A few of the high-fat extracts were passed through Florisil to remove excess fat or endogenous interferences. Analysis of the initial or cleaned up extracts was done by gas chromatography (GC) at 90 degrees C. The dichloropropenes were determined on 20% OV-101 columns with electron-capture and Hall electroconductivity detectors. The dichlorobenzenes and 1,2-dibromo-3-chloropropane, which elute beyond 30 min on the above columns, were determined on 5%-loaded columns using the same detectors. All five analytes were recovered from these techniques. Mean analyte recovery following a direct dilution or extraction was 83%, and following the Florisil cleanup step, was 52%. In 1986, a fumigant survey of about 200 foodstuffs by using this overall technique gave no findings of the five compounds studied here.

Allyl Compounds↗

Mutations in VMK1, a mitogen-activated protein kinase gene, affect microsclerotia formation and pathogenicity in Verticillium dahliae.

Verticillium dahliae is an important soil-borne fungal pathogen that causes vascular wilt diseases in a large variety of important crop plants. Due to its persistence in the soil, control of Verticillium wilt relies heavily on soil fumigation. The global ban on methyl bromide, a highly effective soil fumigant, poses an urgent need to develop alternative control measures against Verticillium wilt; and these might be more forthcoming with a better understanding of the molecular and cellular mechanisms that underpin the pathogenicity of V. dahliae. In this study, we assessed the role in growth, development, and pathogenicity of VMK1, a gene encoding a mitogen-activated protein (MAP) kinase (hence, Verticillium MAP Kinase 1). Disruption of VMK1 via Agrobacterium tumefaciens-mediated transformation, in two V. dahliae isolates, one from lettuce and the other from tomato, resulted in severely reduced virulence in diverse host plants, suggesting that VMK1 is essential for pathogenicity and that the MAP kinase-mediated signaling pathway has a conserved role in fungal pathogenicity. The vmk1 mutants also exhibited reduced conidiation and microsclerotia formation, suggesting that the gene is important for multiple cellular processes.

Agrobacterium tumefaciens↗

Hydroponically cultivated radish fed L-galactono-1,4-lactone exhibit increased tolerance to ozone.

Leaf L-ascorbate content of an ozone (O3)-sensitive radish genotype (Raphanus sativus L. cv. Cherry Belle) was increased 2-fold by feeding hydroponically cultivated plants L-galactono- 1,4-lactone (GalL). Plants were grown in controlled-environment chambers ventilated with charcoal/Purafil-filtered air, and administered one of two O3 fumigation regimes: chronic exposure (75 nmol O3 mol(-1) for 7 h day(-1) for 21 days) and acute exposure (180 nmol O3 mol(-1) for 9 h). Chronic O3 exposure decreased root growth by 11% in plants maintained in pure nutrient solution (-GalL), but resulted in no change in root growth in GalL-fed plants (+GalL). Similarly, GalL-feeding counteracted the negative effects of O3 on CO2 assimilation rate observed in control plants (-GalL). Under acute O3 exposure, GalL-fed plants showed none of the visible symptoms of injury, which were extensive in plants not fed GalL. Leaf CO2 assimilation rate was decreased by acute 03 exposure in both GalL treatments, but the extent of the decline was less marked in GalL-fed plants. No significant changes in stomatal conductance resulted from GalL treatment, so O3 Uptake into leaves was equivalent in + GalL and -GalL plants. Feeding GalL, on the other hand, enhanced the level of ascorbate, and resulted in the maintenance of the redox state of ascorbate under acute O3 fumigation, in both the leaf apoplast and symplast. The effect of GalL treatment on ascorbate pools was consistent with the reduction in O3 damage observed in GalL-fed plants. Attempts to model O3 interception by the ascorbate pool in the leaf apoplast suggested a greater capacity for O3 detoxification in GalL-fed plants, which corresponded with the increase in O3 tolerance observed. However, modelled data for GalL-fed plants suggested that additional constituents of the leaf apoplast may play an important role in the attenuation of environmentally-relevant O3 fluxes.

Air Pollutants↗

Indigenous and introduced arbuscular mycorrhizal fungi contribute to plant growth in two agricultural soils from south-western Australia.

Arbuscular mycorrhizal (AM) fungi occur in all agricultural soils but it is not easy to assess the contribution they make to plant growth under field conditions. Several approaches have been used to investigate this, including the comparison of plant growth in the presence or absence of naturally occurring AM fungi following soil fumigation or application of fungicides. However, treatments such as these may change soil characteristics other than factors directly involving AM fungi and lead to difficulties in identifying the reason for changes in plant growth. In a glasshouse experiment, we assessed the contribution of indigenous AM fungi to growth of subterranean clover in undisturbed cores of soil from two agricultural field sites (a cropped agricultural field at South Carrabin and a low input pasture at Westdale). We used the approach of estimating the benefit of AM fungi by comparing the curvature coefficients (C) of the Mitscherlich equation for subterranean clover grown in untreated field soil, in field soil into which inoculum of Glomus invermaium was added and in soil fumigated with methyl bromide. It was only possible to estimate the benefit of mycorrhizas using this approach for one soil (Westdale) because it was the only soil for which a Mitscherlich response to the application of a range of P levels was obtained. The mycorrhizal benefit (C of mycorrhizal vs. non-mycorrhizal plants or C of inoculated vs. uninoculated plants) of the indigenous fungi corresponded with a requirement for phosphate by plants that were colonised by AM fungi already present in the soil equivalent to half that required by non-mycorrhizal plants. This benefit was independent of the plant-available P in the soil. There was no additional benefit of inoculation on plant growth other than that due to increased P uptake. Indigenous AM fungi were present in both soils and colonised a high proportion of roots in both soils. There was a higher diversity of morphotypes of mycorrhizal fungi in roots of plants grown in the Westdale soil than in the South Carrabin soil that had a history of high phosphate fertilizer use in the field. Inoculation with G. invermaium did not increase the level of colonisation of roots by mycorrhizal fungi in either soil, but it replaced approximately 20% of the root length colonised by the indigenous fungi in Westdale soil at all levels of applied P. The proportion of colonised root length replaced by G. invermaium in South Carrabin soil varied with the level of application of P to the soil; it was higher at intermediate levels of recently added soil P.

Medicago↗

The assessment of air and soil as contributors of some trace metals to vegetable plants. II. Translocation of atmospheric and laboratory-generated cadmium aerosols to and within vegetable plants.

Radish, spinach and lettuce were grown in the field on 109Cd-labelled soil. Efficient transfer to the plant was observed with concentration factors [Cd concn. in plant (dry wt) divided by Cd concn. in soil (dry wt)] of between 0.30 and 2.00. Soil uptake of Cd accounted for only 69-64% of Cd in the plants, the remainder arising from atmospheric deposition. By fumigating radish, lettuce and carrot with Cd aerosol in a wind tunnel and harvesting and analysis subsequent to further growth, translocation of Cd to non-exposed parts of plants was demonstrated. The percentage of deposited Cd which is translocated in the wind-tunnel-fumigated plants is small (0.6-4%), but is larger in plants grown in the field (28-36%) or in a laboratory growth cabinet (12-35%).

Aerosols↗

Mathematical descriptions of accelerated transformation of 1,3-dichloropropene in soil; a microbiological assessment.

The rate of transformation of the soil fumigant (Z)-and (E)-1,3-dichloropropene in moist soil layers was measured at incubation temperatures of 5 degrees C, 10 degrees C and 20 degrees C. 'DD95' was added to four characterized soil layers, in amounts corresponding to realistic field contents after fumigation. Rapid transformation immediately after application was observed in layers with low initial contents (30-300 micrograms/kg dm) and could well be described with a first-order rate model. Incubation at higher doses (5-15 mg/kg dm respectively) showed distinctly different transformation pathways. Degradation curves could well be computed using a microbiological interspective competition (MIC) model for moist soil. The transformation rate is inversely correlated to microorganism population size and growth. Transformation curves described by MIC are characterized by a lag-time, a period of accelerated transformation and a period of decreasing transformation rates. At low temperatures, DT50 values of more than 20 days could be observed. First-order rate computations did not exceed 8 days. The use of different mathematical discriptions for various soil layers and soil temperatures permits simulation of DD95-transformation by microorganisms in the soil profile throughout the growing season.

Allyl Compounds↗

Ozone is not mutagenic in the Tradescantia and tobacco mutagenicity assays.

Ozone fumigation of a double heterozygous chlorophyll mutant Nicotiana tabacum var. xanthi n.c. with concentrations up to 300 nl/l and of a heterozygous Tradescantia clone 4430 with concentrations up to 800 nl/l did not increase the frequency of somatic mutations above the spontaneous levels. However, ozone fumigation at these concentrations led to distinct physiological damage to plant tissues.

Dose-Response Relationship, Drug↗

Effects of sulphur dioxide and nitrogen dioxide, singly and in mixture, on the macroscopic growth of three birch clones.

An investigation of some aspects of the effects of low concentrations of the gases, sulphur dioxide and nitrogen dioxide, singly and in mixture, was made on the growth of three birch clones, two of Betula pendula Roth. (silver birch) and one of Betula pubescens Ehr. (downy birch). Comparative measurements of the growth form and dry mass increment were made over one year in glasshouses supplied with charcoal-filtered ambient air, and SO(2) and NO(2), singly or in mixture, at mean concentrations of 62 ppb (nl litre (-1)) of one or both gases. The main effects were found in those plants that were fumigated with SO(2) singly, and SO(2) and NO(2) together. Both treatments induced premature leaf loss and reduction in mass, especially of roots, the effects increasing over time. The heights and initial leaf areas were maintained, apparently at the expense of other parameters. NO(2), if present singly, had little or no effect, but it tended to enhance the damaging effect of SO(2) when the two were applied together. The different clones showed different degrees of response to the pollutants, but these differences became less marked during the second season of fumigation. The effects found are discussed in relation to the annual growth of trees, particularly birch.

Journal Article↗

Effects of ozone on foliar leaching in Norway spruce (Picea abies L. Karst): confounding factors due to NOx production during ozone generation.

Previous experiments with conifers fumigated with O(3), produced by air-operated electric discharge ozonators, have provided evidence that O(3) increases the leaching of NO(3)(-), NH(4)(+), K(+), Ca(2+), Mg(2+) and some other cations from needles, when the trees are treated with acid mist. This evidence has provided the foundation of the ozone-acid mist hypothesis of spruce decline. We report experiments with Norway spruce saplings fumigated with purified and unpurified O(3). The results show that the accumulation of NO(3)(-) in the needles arises from the rapid deposition of N(2)O(5) and HNO(3) formed from N(2) in the ozonator. An increase in removal of NH(4)(+), Na(+), Ca(2+), Mg(2+), Zn(2+) and Mn(2+) from the needles during soaking in H(2)SO(4), pH3, was also observed, which was related to the increase in NO(3)(-) but was independent of O(3) concentration. It is concluded that results of previous experiments cited in support of the ozone-acid mist hypothesis arose from effects which were at least partly caused by N(2)O(5) produced as a contaminant, and were incorrectly attributed to ozone. Other effects, such as growth stimulations, visible symptons, enhanced frost sensitivity, and infestation by pests or pathogens, which have been attributed to O(3) generated by electric discharge in air, should be interpreted with caution. Future experiments with ozone must eliminate this problem by either using O(2)-driven ozonators, or by purifying the output from air-driven ozonators using cold and/or water traps.

Journal Article↗

Effects of ozone on the yield of spring wheat (Triticum aestivum L., cv. Albis) grown in open-top field chambers.

Spring wheat (Triticum aestivum L., cv. Albis) was grown in the field at a site located in central Switzerland, and exposed to chronic doses of ozone (O(3)) in open-top chambers to study impacts on yield. The experiment was carried out in 1986, 1987 and 1988. The treatments used included charcoal-filtered air (CF), non-filtered air (NF) and non-filtered air to which constant amounts of O(3) (two levels, O(3)-1 and O(3)-2) were added daily from 09.00 until 17.00 local time. Mean solar radiation-weighted O(3) concentrations during the fumigation period were in the range 0.016-0.022 microl litre(-1) (CF), 0.036-0.039 microl litre(-1) (NF), 0.057-0.058 microl litre(-1) (O(3)-1, used in 1987 and 1988 only) and 0.078-0.090 microl litre(-1) (O(3)-2). Fumigation was maintained from the three-leaf stage until harvest. Ambient plots were used as a reference. Plant characteristics examined included straw yield, grain yield, number of grains per head, number of heads per surface area, weight of individual grains and harvest index (ratio of grain weight to total dry weight). Pollutant concentrations and other environmental parameters were monitored continuously inside and outside the chambers. In 1986 and 1987, enclosure mostly increased the values of different parameters, while in 1988, they were decreased. The negative enclosure effect was due to extremely turbulent winds, which caused lodging inside the chambers. In all 3 years, increasing O(3) concentrations negatively affected the parameters studied, except for the number of heads per surface area, which showed no treatment response. Grain yield showed a very sensitive response to O(3). The effect of O(3) on grain yield was due to an effect primarily on grain size and secondarily on grain number. The relative response of grain yield to O(3) was similar in all 3 years, despite year-to-year differences in climatic conditions and enclosure effects. The analysis of the data for combined years revealed an increase of about 10% in grain yield due to air filtration. The corresponding increase in straw yield was only about 3.5%. Exposure-response models were developed for individual years and combined years. It is concluded that, in the study area, ambient O(3) may affect grain yield in spring wheat.

Journal Article↗

Energy and redox status, and carbon allocation in one- to three-year-old spruce needles.

Metabolite pools were determined in lyophilized homogenates of one- to three-year-old needles from 13-year-old spruce trees. These were exposed in open-chambers for five consecutive years to SO(2) (30 microg m(-3)) and O(3) (50 to 180 microg m(-3)), singly and in combination. The samples were taken shortly before bud break (mid-March). The energy (ATP/ADP) and redox status (NAD(P)H/NAD(P)) increased with needle age. This increase was most pronounced in needles exposed to both pollutant gases. To evaluate carbohydrate metabolism, levels of starch, sugars (sucrose, fructose, glucose), sugar phosphates (dihydroxyacetone phosphate, glyceraldehyde 3-phosphate (triose phosphates, TP), fructose 6-phosphate, fructose 2,6-bisphosphate: F2,6BP) and 3-phosphoglyceric acid (PGA) were measured. The concentrations of starch, glucose, fructose and TP on a dry weight basis increased with needle age, while those of sucrose and F2,6BP decreased. Interestingly, the total amount of soluble sugars remained constant in needles from control chambers and large fluctuations mainly occurred after a single application of the pollutants. Needles from fumigated trees generally showed somewhat lower starch and sugar, and fructose 6-phosphate levels. The content of TP increased with needle age while that of sucrose and F2,6BP decreased. This inverse behavior was even more prominent in the presence of both pollutants and is discussed in the context of recent knowledge on the regulation of carbon partitioning. There was a significant increase in the ratio of TP/PGA in samples from the combined exposure treatments. This also indicates an increase in the redox charge and corroborates the data on both the redox status of the pyridine nucleotide system and the significantly increased ATP/ADP ratio. The results are indicative of a switch in metabolism, from anabolic to predominantly catabolic reactions, which is most pronounced in the samples fumigated with both O(3) and SO(2). The data are compared to those obtained from naturally exposed trees in the southern Black Forest (Kälbelescheuer/Haldenhof, near Freiburg, Germany).

Journal Article↗

Mycorrhizal populations and fine root development on Norway spruce exposed to controlled doses of gaseous pollutants and simulated acidic rain treatments.

Effects after long-term fumigation and simulated acidic rain (pH 4.0) treatment in open-top chambers (OTC) during 5 vegetation periods on ectomycorrhizal (EM) frequency, fine root structure and distribution of short roots were recorded for Norway spruce. Simultaneous application of ozone and SO(2) resulted in a significant decrease (P< or =0.01) in the proportion of EM root tips and an increase in the numbers of necrotic short roots. The observed differences were partly due to more than additive effects of ozone and SO(2). On the basis of EM and non-mycorrhizal (NM) short roots in the irrigated controls (OTC-V, pH 5.6, Ah-horizon: EM + NM/cm=0.05) the acid treatments resulted in drastic effects (P< or =0.05) on the branching density and the proportion of EM root tips and NM short roots. Relative numbers of short roots infected by indigenous EM fungi with trees in the fumigation treatments at pH 4.0 were similar: 1.5-1.9 EM tips/cm and differed noticeably from the control: 5.2 EM tips/cm. Again, increasing numbers of necrotic and NM short roots were noted. Parallel measurements by the ingrowth-core technique confirm the argument, that EM formation seems to be a reliable indicator of pollution-induced stress.

Journal Article↗

Effects of ozone, acid mist and soil characteristics on clonal Norway spruce (Picea abies (L.) Karst.)--an introduction to the joint 14 month tree exposure experiment in closed chambers.

This paper introduces a series of publications referring to a single 14-month laboratory study testing the hypothesis that the recent decline of Norway spruce (Picea abies (L.) Karst.) at higher elevations of the Bavarian Forest and comparable forests in medium-range mountains and in the calcareous Alps is caused by an interaction of elevated ozone concentrations, acid mist and site-specific soil (nutritional) characteristics. The effect of climatic extremes, a further important factor, was not included as an experimental variable but was considered by testing of the frost resistance of the experimental plants. Results of these individual studies are presented and discussed in the following 14 papers. Plants from six pre-selected clones of 3-year-old Norway spruce (Picea abies (L.) Karst.) were planted in April 1985 in an acidic soil from the Bavarian Forest, or a calcareous soil from the Bavarian Alps. After a transition period, plants were transferred, in July 1986, into four large environmental chambers and exposed for 14 months to an artificial climate and air pollutant regime based on long-term monitoring in the Inner Bavarian Forest. The climatic exposure protocol followed realistic seasonal and diurnal cycles (summer maximum temperature, 26 degrees C; total mean temperature, 9.8 degrees C; winter minimum, -14 degrees C; mean relative humidity, 70%; maximum irradiance, 500 W m(-2); daylength summer maximum, 17 h; winter minimum, 8 h). Plants were fumigated with ozone, generated from pure oxygen (control: annual mean of 50 microg m(-3); pollution treatment: annual mean of 100 microg m(-3) with 68 episodes of 130-360 microg m(-3) lasting 4-24 h), and background concentrations of SO(2) (22 microg m(-3)) and NO(2) (20 microg m(-3)); windspeed was set at a constant 0.6 m s(-1). Plants were additionally exposed to prolonged episodes of misting at pH 5.6 (control) and pH 3.0 (treatment). Simulation of the target climatic and fumigation conditions was highly reliable and reproducible (temperature +/-0.5 degrees C; rh+/-10%; ozone+/-10 microg m(-3);SO(2) and NO(2)+/-15 microg m(-3)).

Journal Article↗

A small-scale controlled environment chamber for the investigation of the effects of pollutant gases on plants growing at cool or sub-zero temperature.

A new, small-scale controlled environment plant growth chamber is described, which permits accurately controlled temperatures from ambient (c. 20 degrees C) down to -5 degrees C in the day or -15 degrees C at night. The system also allows controlled injection of pollutant gases. The chamber is based on a modified commercial chest freezer with temperature control achieved through by-passing the cooling coils in the inner walls of the freezer. This by-pass is controlled by a BBC microcomputer which gives nominal temperature control to 0.1 degrees C over definable diurnal temperature profiles. Photoperiod is also controlled by the microcomputer. The chamber forms a virtually closed system to which pollutant gases are added by injection from low concentration cylinders, regulated using mass flow controllers. The accuracy with which pollutant concentrations are controlled is increased by filtration of the air-stream after passing over the experimental plants: i.e. a 'single-pass' system is adopted. In operation air temperatures across the chambers are controlled to within 1-1.5 degrees C of the pre-set value over the range 25 to -10 degrees C. Leaf temperatures are 2.5-3.0 degrees C above air temperatures during the day, with a total energy input of c. 180 Wm(-2) from two 400 W metal halide lamps. The lamps provide a photon fluence rate of 270+/-30 micromol quanta PAR m(-2) s(-1) at plant height. During the dark, leaf temperatures deviate less than 1.0 degrees C from air temperature. Soil temperatures are controlled separately from air temperature and are held above zero at all times. To date, fumigation, with NO(2) and SO(2) has been confined to the daytime. At pre-set concentrations of 20 nl litre(-1) variations during fumigation were less than 2.0-2.5 nl litre(-1) and 0.5 nl litre(-1), respectively.

Journal Article↗

Single and interactive effects of low levels of O3, SO2 and NO2 on the growth and yield of spring rape.

During three consecutive seasons (1987-1989), the effects of low-levels of O3, SO2 and NO2 singly and in all possible combinations (NO2 in 1988 and 1989 only) on growth and yield of potted plants of spring rape (Brassica napus L. var. napus, 'callypso') were investigated by means of factorial fumigation experiments in open-top chambers. Plants were exposed from the early vegetative stage of development until seed harvest, to charcoal-filtered air (CF; control) and CF which was supplemented for 8-h per day (8.00-16.00) with O3, for 16-h per day with NO2 (16.00-8.00) and continuously with SO2. Including the controls, the 24-h daily mean concentrations [microg m(-3)] ranged between 6-44 (O3), 9-88 (SO2) and 10-43 (NO2). The corresponding daily mean concentrations during the time of fumigation were 10-121 and 11-60 microg m(-3) for O3 and NO2, respectively. Single effects of O3 on growth and yield parameters were mostly negative and the magnitude of this effect was dependent on the season. O3 reduced plant dry weight by 11.3-18.6% and yield of seeds by 11.4-26.9%. While medium levels of SO2 stimulated the weight of pods up to 33%, higher concentrations (88 microg m(-3)) caused a decline of yield of 12.3%. From the significant interactive effects which were observed, it could be established that SO2 and NO2 alone mostly acted positively, but that their interaction with each other and especially with O3 was antagonistic, as some of the detrimental effects of O3 were mitigated by these pollutants. An important antagonistic effect between SO2 and O3 or NO2 was observed on yield. While 56 microg m(-3) SO2 increased yield by 9.9% compared to the control treatment, it aggravated the yield loss caused by O3 from -16.18% to -21.4%, and it reduced the yield stimulation caused by NO2 from +11.8% to +4.2%. Leaf area was the only parameter which was negatively affected by all pollutants, their joint action being synergistic.

Journal Article↗

Influence of ozone air pollution on plant-herbivore interactions. Part 1: Biochemical changes in ornamental milkweed (Asclepias curassavica L.; asclepiadaceae) induced by ozone.

A series of fumigation experiments was conducted with bloodflower (Asclepias curassavica L.) in continuous-flow stirred reactors (CSTRs) to elucidate the effects of ozone on foliar concentrations of several primary and secondary plant metabolites relevant to herbivores. Plants 8 weeks of age were subjected to different ozone levels ranging from 0 to 134 nl liter(-1) for exposure periods up to 16 days. Leaves were analyzed for concentration of soluble carbohydrates, starch, free amino acids, soluble protein, total phenolics, and total cardenolides. Significant interactions between the linear effects of ozone concentration and exposure time were found for soluble carbohydrates, amino acids, cardenolides and phenolics. No significant treatment effects could be observed on foliar starch and protein concentration. The metabolic responses of plants to fumigation appeared to be altered by overall plant nutrition. It is possible that the metabolic changes observed in the host plant represent important changes in nutritional quality to insects.

Journal Article↗

No general effect of ozone on foliar nutrient concentrations in mature scions of grafted Picea abies trees.

The effect of ozone (< 10, 200, or 400 microg m(-3)), on foliar nutrient concentrations of Picea abies were determined by fumigating potted grafts from mature trees (> 55 and 125 years), representing six clones, in open-top chambers at two locations in Norway. The concentration of nutrients in needles of grafted plants were significantly affected by clone and location. Generally, the concentrations of nutrients were not affected by ozone, but a significant increase in the concentrations of potassium and iron in two of the clones were found. These two clones were the only ones injured (yellow needles) by the fumigation.

Journal Article↗