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Plant response to variable ozone regimes of constant dosage Bicak, Charles Ray 1978

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PLANT RESPONSE TO VARIABLE OZONE REGIMES OF CONSTANT DOSAGE by CHARLES JAY BICAK B.Sc. (Ed.) Kearney State College, 1974 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN THE FACULTY OF GRADUATE STUDIES in the Department of Plant Science We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH May, 1978 (c!) Charles Jay Bicak, COLUMBIA 1978 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of Brit ish Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Plant Science The University of Brit ish Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date May 25, 1978 i ABSTRACT Most air pollutant investigations in controlled environments have utilized the conventional "steady state" exposure, yet air pollutant con-centrations are rarely static in ambient conditions. Peak concentrations in air pollutant exposures vary in magnitude and occurrence despite equivalent doses and are likely to result in different degrees of injury to plants. The results of experiments involving 5 treatment regimes with a single peak concentration,.that varied in magnitude and occur-rence while treatment doses remained equivalent, confirm this hypothesis and demonstrate that the levels of injury to bush beans (Phaseolus  vulgaris)and radish (Raphanus sativus) may vary from 15 to 85% of the leaf area. An absolute injury assessment technique was developed for determining percent necrosis in the 5 treatments, with the same two crop species responding in a s.imijar manner to the various constant dosage regimes. A simple workable model is presented that incorporates stomatal diffusive resistance along with the various exposure components, including cumulative dose, maximum concentration, and the interval of time during which the peak concentration was administered. Preliminary investigations failed to confirm an interaction between ozone and greenhouse whitefly in bush beans. i i TABLE OF CONTENTS Page ABSTRACT i TABLE OF CONTENTS i i LIST OF TABLES i i i LIST OF FIGURES iv ACKNOWLEDGEMENTS vi 1 INTRODUCTION 1 2 LITERATURE REVIEW 5 3 RE-EXAMINATION OF OZONE-WHITEFLY INTERACTION 24 I Introduction 24 II Materials and Methods 25 III Results and Discussion 28 4 INJURY ASSESSMENT IN PLANT LEAVES EXPOSED TO OZONE 31 I Introduction 31 II Materials and Methods 33 III Results and Discussion 34 5 VARIABLE OZONE DOSE APPLICATION 43 I Introduction 43 II Materials and Methods 50 Continuous Stomatal Measurement 50 Growth of Radish 53 III Results and Discussion 54 Plant Injury and Exposure Components 54 Dose Response 76 Growth of Radish 85 Injury Prediction 89 CONCLUSION 102 LITERATURE CITED 104 APPENDIX 115 i i i LIST OF TABLES Table Page 1 Experiment 1 - Ozone and Whitefly Exposure Treatments 26 2 Experiment 2 - Ozone and Whitefly Exposure Treatments 27 3a Experiment 1 - Plant Injury 28 3b Experiment 2 - Plant Injury Assessed by Reflectance Spectrophotometry 29 4 Percent Absolute Injury (Beans) 35 5 Percent Visual Injury (Beans) 36 6 Percent Absolute Injury (Radish) 37 7 Percent Visual Injury (Radish) 38 8 Percent Injury and Over-Estimation 39 9 Ozone Treatments Administered to Bean and Radish Plants 51 10 Ozone Injury with Various Constant Dosage Regimes 54 11 Percent Injury and Related Mean Stomatal Resistance 59 12 Stomatal Resistance at C v and Subsequent Injury Levels 64 max 13 Cumulative Ozone Dose 70 14 Single Stomatal Resistance Measurements in Beans 24 hrs After the Termination of Ozone Exposure 85 i v LIST OF FIGURES Figure Page 1 Scatter Diagram of Injury Levels in Raphanus sativ u s. Experiment 1. 41 2 Scatter Diagram of Injury Levels in Raphanus sativus. Experiment 2. 42 3 Comparison of Ozone Measurement in the Lower Mainland Air Basin, Vancouver, B.C. 44 4 Stomatal Resistance Through a 12 hour Photoperiod with Beans 56 5 Stomatal Resistance Through a 12 hour Photoperiod with Radish 57 6 a-d Stomatal Resistance and Ozone Treatment Patterns in Beans Over Time 60-65 7 a-d Stomatal Resistance and Ozone Treatment Patterns in Radish Over Time 66-69 8 Changes in r s in Beans with Increases in Cumulative Dose in Time (Steady State Exposure) 72 9 Changes in r s in Beans with Increases in Cumulative Dose in Time (Middle Peak or Bell Curve Exposure) 73 10 Changes in r s in Beans with Increases in Cumulative Dose in Time (Late or Step Up to Peak Exposure) 74 11 Changes in r s in Beans with Increases in Cumulative Dose in Time (Early or Step Down Peak Exposure) 75 12 Changes in r s in Radish with Increases in Cumulative Dose in Time (Steady State Exposure) 77 13 Changes in r s in Radish with Increases in Cumulative Dose in Time (Middle Peak or Bell Curve Exposure) 78 14 Changes in r s in Radish with Increases in Cumulative Dose in Time (Late or Step Up to Peak Exposure) 79 15 Changes in rs.in Radish with Increases in Cumulative Dose in Time (Early or Step Down from Peak Exposure) 80 16 Ozone Flux Defined as Stomatal Conductance (1/rs) Multiplied by Ozone Concentration (C) in Beans 82 17 Ozone Flux Defined as Stomatal Conductance (1/rs) Multiplied by Ozone Concentration (C) in Radish 83 V Figure Page 18 Patterns of Dry Weight Change in Radish with Increasing Injury. Experiment 1. 86 19 Patterns of Dry Weight Change in Radish with Increasing Injury. Experiment 2. 87 20a Ozone Flux at the Time of Peak Concentration in Beans 90 20b Ozone Flux at the Time of Peak Concentration in Beans 91 21a Ozone Flux at the Time of Peak Concentration in Beans 92 21b Ozone Flux at the Time of Peak Concentration in Beans 93 : 22 Injury Prediction in Beans 95 23 Injury Prediction in Radish 96 24 Injury Prediction in Beans 98 25 Injury Prediction in Beans 99 26' Injury Prediction in Radish 101 vi ACKNOWLEDGEMENTS I am grateful to Dr. Victor C. Runeckles for his invaluable guidance and constructive criticism throughout this work. I am also thankful for the suggestions and assistance of my committee members, Dr. P.A. Jolliffe and Dr. R.H. Elliott. Thanks are extended to I. Derics, A. Herath and A. Hoda for their technical help. I am also grateful to my wife, Marylin, for her consistent support and encouragement. Finally, I acknowledge the financial assistance of the National Research Council through grants to Dr. V.C. Runeckles and five teaching assistantships from the Department of Plant Science. 1 CHAPTER 1 INTRODUCTION Man-made air pollution has been a constituent of the atmosphere since the advent of the industrial age in 19th century Europe and the United States. Prior to 1940, particulate matter and sulfur dioxide were the primary recognized air pollutants. Air pollution problems have escalated in the past thirty years. Coping with the environmental impact and effects of toxic gases upon vegetation and health has become one of the more pressing concerns of the second half of the 20th century. Photochemical smog is one of the more recent contributions to air pollution, of which ozone is a major component. The motor vehicle and industrial use of petroleum products are the principal indirect sources of ozone. Several reviews have dealt with ozone and its effects on plant systems (Jacobson and Hill, 1970; Treshow, 1970; Heggestad and Heck, 1971). Most of the effects attributable to ozone are detrimental and relate to exposures to concentrations of the toxic gases which are demonstrably harmful. Ozone has been implicated in plant biochemical and physiological alterations that result in leaf tissue necrosis, chlorosis, and reduced growth. The response of plants to ozone is dependent upon concentration of the air pollutant and duration of exposure. In addition, many environmental variables such as light, temperature, relative humidity, edaphic factors, nutrition, infection with pathogens, and insect infestation modify plant response to ozone. Air pollutants may interact with biological agents such as pathogens 2 and insects as well as with components of the physical environment of plants. Among the plant responses to pathogenesis and insect attack are enhanced senescence and abscission of plant organs (Galil, 1968; Heagle, 1973). While considerable work has been undertaken on air pol-lutant-pathogen-host plant interaction, as reviewed by Heagle (1973), relatively l i t t l e attention has been paid to air pollutant-insect-host plant systems. Runeckles and Rosen (1976) reported elevated injury levels in bush beans exposed to both ozone and greenhouse whitefly. Since the injury observed was greater than the sum of the ozone and insect effects induced in separate treatments, they reported an apparent synergism between ozone and the insects. Since enhanced ethylene production has been reported following insect infestation (Williamson, 1950; Galil, 1968) and as a consequence of exposure of plants to ozone (Craker, 1971) and since ethylene is known to induce senescence in plant tissues, the observed synergism could have been the result of enhanced ethylene production. Plant response to ozone has been determined in terms of biochemical and physiological changes, or in terms of visual injury. Most ozone injury assessment techniques involve subjective rating systems in which visual estimates of the amount of injury (usually necrosis) present on affected organs are assigned scores on scales which range numerically from zero to complete necrosis. Such systems have been employed since the development of threshold models (O'Gara, 1922) and have been modified over time (Thomas and Hi l l , 1935; Zahn, 1963). A reliable, absolute injury assessment technique is essential as a check upon visual observations. In addition to the inherent subjectivity of the visual system there are differences in relative sensitivity from the lower to the upper end of the injury spectrum. 3 The disparity in injury assessment with identical treatments from differ-ent laboratories emphasizes the necessity for an objective technique. Further problems arise in the interpretation of laboratory exposures in terms of their relevance to field conditions because inadequate con-sideration is given to the details of dosage (the integral of^concentration over time). Most controlled environment studies of the effects of air pollutants on plants are characterized by conventional steady state con-ditions in which pollutant concentrations are maintained at a fixed level throughout the treatment period. In contrast, air pollutant concentrations under field conditions are continuously fluctuating. Such fluctuations are induced by variations in air pollutant sources and sinks and by changes in environmental conditions, especially atmospheric turbulence. Hence, there is a dearth of information pertinent to ozone fumigations that more nearly approximate diurnal and day to day fluctuations. Although the potential influence of other environmental stresses has been considered in conjunction with ozone, few workers have pursued the kinetic dose response concept. In addition, the bulk of the studies have focussed on threshold res- . ponses, curve-fitting, and the development of threshold response equations, with l i t t l e emphasis on injury prediction or even the involvement of degree of injury as a variable. The notable exceptions are Larsen and Heck (1976) and Oshima (1974). In most studies, observed plant response has been related to input concentrations of pollutants and to concentrations measured at some arbi-trarily chosen location within the exposure chamber. The materials of construction used in such chambers, the plant canopies themselves, soil, 4 and pots all possess air pollutant sorptive properties (Hill, 1971; Thorne and Hanson, 1972; Resh and Runeckles, 1973). Differences between input concentrations and leaf surface concentrations have been verified by sorptive studies and yet in few cases has any attention been paid to the obvious - that response must relate to "effective" dose or actual dose within the plant, and that this may be considerably different from that suggested by the input or ambient concentration. . An injury prediction model must therefore incorporate an approximation of gaseous air pollution flow at the leaf surface. In the light of the foregoing, the objectives of the present study were therefore: - To re-examine the ozone-whitefly interaction described by Runeckles and Rosen (1976); - To develop an objective, quantitative method of measuring necrotic injury and to compare it with visual rating techniques; - To determine the effects of various constant dosage treatments involving peak concentrations occurring at different times within the fumigation period. This investigation led in turn to an attempt at developing a simple pre-diction model. The results of these investigations are presented sequen-tially in the four sections which follow a general review of the pertinent literature. Because the different objectives called for the use of some-what different procedures, the specific methods and materials employed are for the most part described separately in each section. 5 CHAPTER 2 LITERATURE REVIEW Ozone has been recognized as an important constituent of air pollution in the urban atmosphere. A large proportion of air pollution injury in North America is attributable to ozone as a constituent of the type of air pollution known as photochemical oxidant or photochemical smog. Oxides of nitrogen together with ultraviolet irradiation are prerequisites for such ozone formation. Nitric oxide (NO) is an important by-product of combustion processes, and reacts with oxygen in the air to form nitrogen dioxide (NO2). In the presence of ultraviolet light, NO2 reacts with oxygen to yield ozone, reforming nitric oxide in the process. In the pre-sence of hydrocarbons, the process becomes cyclical, and ozone is produced in increasing quantities. This dynamic equilibrium can be expressed as: (Altshuller, 1965). Haagen-Smit et al_. (1952) were the first to report the influence of some component of the atmosphere in the Los Angeles area of California upon plant tissue. They observed both necrotic lesions and a chlorotic condition in plants exposed to that mixture of pollutants which had been termed "smog". Richards ejt al_. (1958) identified ozone as an important photochemical phytotoxicant present in smog that was responsible for injury to grape leaf tissue in southern California. In 1959 Heggestad and Middleton concluded that ozone was the cause of widespread injury to tobacco 2* NO + 03 6 in the eastern United States. Similar conclusions were reached by Macdowall and his coworkers with regard to tobacco injury in southern Ontario (Macdowall et al_., 1964). Since that time, ozone has been iden-tified as a phytotoxic agent in the air in many parts of the world including the United Kingdom, Holland, Japan, and Mexico. Effects on vegetation range from local necrotic lesions and chlorosis, especially of the leaves, to enhanced senescence and reduction in growth. However, Bennett et a]_. (1974) have suggested that at very low levels, stimulation of growth may also occur. Air pollutant injury is usually classified as either acute or chronic. Acute injury results from short term, high concentration exposures, while chronic injury occurs as a result of long term, low concentration exposures. An important distinction between injury and damage has been made by Guderian et al_. (1960). "Damage" refers to the visible effects of an air pollutant upon a plant, especially a crop species, which affects the current market value. "Injury" is a broad term encompassing all plant responses over an extended period of time regardless of whether the affected plants show obvious visible symptoms or not. Wolozon and Landau (1966), however, note the complexity in differentiating between damage and injury. Using the examples of three crops that respond dif-ferently to the air pollutant sulfur dioxide (spinach, fodder turnips, and ornamentals), they point out that although injury to fodder turnip might not result in a significant loss in marketability, nutritional value would decline even though there would be no "damage". On the other hand, damage would result from S0? exposures in both spinach and ornamentals, 7 although the loss in market value would occur for different reasons. Injury in spinach would cause a decline in nutritional value that would not be immediately apparent in market value but would also result in blemished leaves which would reduce marketable value. Alternatively, injury in ornamentals might well cause discoloration or stunting, for example, over an extended period of time, and hence make the plants un-marketable. A number of investigators have examined the effects of ozone on various crop and native species of plants. Crop species which are sen-sitive to ozone have been catalogued by several reviewers (Hill et al., 1970; Treshow, 1970). Among the most sensitive are alfalfa (Medicago  sativa L. var. Ranger), bean (Phaseolus vulgaris L.), wheat (Triticum  aestivium L. var. Lemki), oat (Avena sativa L. var. Overland), onion (Al1ium cepa L.), radish (Raphanus sativus L.), grape (Vitis vinifera L.), pine (Pinus strobus L. eastern white), and weeping willow (Salix babylonica L.). The varietal distinction is often necessary because genetic factors are important in causing variations in sensitivity within a species (Heck, 1968). A broad spectrum of plants are subject to injury induced by ozone. Ozone levels of from 0.04 to 0.10 ppm (parts per million, vol./vol.) are usually necessary to produce visible injury in sensitive plants following exposures lasting a few hours. Ozone injury is characterized by one or more of four lesion types. These symptoms are a manifestation of primary site attack by ozone in the leaf (Hill et a l . , 1970). In the first type of response, pigmented lesions are formed. 8 Ledbetter ejt al_. (1959) noted that these lesions are sharply delineated, small and dot-like, and may occur in plants ranging from dedicuous trees and shrubs to some herbaceous species. The lesions may be dark brown, red, or black. The "injury" is localized within the palisade paren-chyma in the upper portion of the leaf mesophyll tissue and results from an enhancement of anthocyanin production. The veins often remain unaf-fected with their orientation frequently dictating the angular shape of the interveinal lesions. A second injury symptom is upper surface or bifacial bleaching invol-ving the palisade parenchyma cells within the leaf. Palisade cells and occasionally upper epidermal cells collapse. Connections are retained between collapsed cells and the uninjured cells above and below them. Gray, off-white or beige colors often result. Veins are rarely affected. As opposed to pigmented lesions, the necrotic spots are usually irregular in shape. Bifacial necrosis is a third form of injury. Initial symptoms include the occurrence of water-soaked areas below the leaf surface. Eventually all the leaf tissue within the lesion is killed and the affected part of the leaf dries and becomes paper thin. Coloration of the dried tissue is variable, ranging from white to dark brown. Usually, however, the injured area is darker in color than unaffected leaf tissue. As a consequence of extensive injury of this type, leaves often acquire a permanent bronzed appearance over time. The fourth form of injury is chlorosis. When lesions occur, they are yellow to light-green in color and range from a few cells to a milli-meter or more in diameter. Occasionally the lesions merge to lend a 9 widespread and mottled appearance to the leaf tissue. Many of the cells remain alive but, as Hill e_t al_. (1961) have noted, there is a disruption of chloroplast membranes and a reduction in chlorophyll content. Visible injury symptoms in plant tissue are the ultimate indication of physiological alterations. Low levels of air pollutants may be func-tional in altering plant metabolism without markedly affecting plant morphology. Todd et al_. (1958) described this "hidden injury" as a reduction in nutrient content and plant growth processes with no change in physical appearance following air pollutant exposure. Such "hidden injury" or "subtle effects" have been observed in other stress situations, such as nutrient deficiency (Bouria and Dowling, 1966). Because of the complex responses elicited in plant systems, the study of air pollutants has evolved from descriptive investigations to mechanism-oriented investigations. Researchers began to investigate ozone entry into the plant and its subsequent modification of tissue biochemicals. As with all gases, gaseous air pollutants enter the plant mainly via the stomates. The complexity of stomatal function has been emphasized in a recent review by Raschke (1975). Many workers have investigated the osmotic changes involved in stomatal movement. Theories of operation have ranged from starch-sugar interconversions in guard cells (Scarth, 1932) to K+ involvement in opening and closure (Imamura, 1943; Fijuno, 1959; Fischer, 1968). Air pollutants themselves influence stomatal aper-ature. Furthermore, a host of environmental factors modifies the influence of ozone and other pollutants upon stomates. Meidner and Mansfield (1968) stress the importance of light, carbon dioxide, temperature, water supply, relative humidity, and other environmental factors in conjunction with 10 an! air pollutant in modifying stomatal response. It has been noted that pretreatment of onion, Allium cepa L. with low levels of ozone stimulates stomatal closure and affords a mechanism for resistance (Engle and Gabelman, 1966). Once i t is within the sub-stomatal cavity, ozone reacts with con-stituents of the cells it contacts. Heath (1975) has discussed the prominent biochemical components of the cell with which ozone may interact. There is probably l i t t l e reactivity of ozone with the cell wall, although Ordin ejt a]_. (1969) have noted a depression of the UDP-glucose polysaccharide synthetase system following exposure to ozone. Sufficient evidence, however, is lacking to implicate ozone in reaction with the cell wall directly. On the other hand, a significant amount of literature exists per-tinent to the interaction of ozone with the plasmalemma. An in vitro modification of plasmalemma constituents was noted following exposure to ozone by Mudd ejt al_. (1973). They observed changes in the amino acids of the plasmalemma proteins, including cysteine, methionine, tryptophan, tyrosine, histidine, and phenylalanine. Changes have also been observed in proteins, unsaturated fatty acids, and sulfhydryl-containing constituents of the plasmalemma. Evans and Ting (1973), Perchorowicz and Ting (1974), and Chimiklis and Heath (1975) noted distinct alterations in permeability of the plasmalemma following ozone exposure. The latter researchers observed an efflux of K+ ions from the unicellular algae, Chlorella  sorokiniana. Such a response to ozone occurs in a matter of seconds. Chimiklis and Heath suggest, therefore, that the ozone exposure induces development of primary symptoms, characterized by cell membrane alterations, 11 as opposed to secondary cellular injury. The membrane alteration may be reversible; the cells were found to resume exponential growth following exposure to 26 u moles of ozone per liter for 10 minutes. K+ efflux also ceased shortly after the removal of ozone. It has been noted by many workers, Dugger and Ting (1970), for example, that developmental age is a crucial factor in some species in determining extent of injury following ozone exposure, with maximum sus-ceptibility being reached between the times of maximum rate of leaf growth and the attainment of maximum leaf size. Chimiklis and Heath (1975) contend that high turgor in plants just prior to full leaf expansion physically stretches the plasmalemma. This in turn may expose more reac-tive sites to ozone. Hence, a greater injury results in plants in this developmental stage. Furthermore, Heath ejt al^. (1974) have reported elevated K+ levels in Phaseolus just prior to complete expansion of leaves. The stomatal closure observed in many laboratories during exposures to ozone (Koritz and Went, 1953; Seidman et al,., 1965; Heck et al_., 1965; Hill and Littlefield, 1969) may be a consequence of the loss of K+, hence turgor, from guard cells. In addition, Mansfield and Majernik (1970) have noted that the concentration of CO2 increases as the concentration of other pollutants increases. They hypothesize that this elevation may afford the plant some protection from the pollutant gas. Changes in cytoplasmic biochemicals have also been reported in plant tissues exposed to ozone, for example, increases in y-aminobutyrate and decreases in glutamate (Tomlinson and Rich, 1967), increases in glycine, aspartate, glutamate, asparagine, B-alanine, threonine, serine, valine, 12 leucine, isoleucine, lysine, histidine, y-aminobutyric acid (Ting and Mukerji, 1971), and alterations in chloroplast morphology and metabolism (Coulson and Heath, 1974). If the cell membrane is the first structure within the leaf exposed to ozone, changes in the permeability of the plasmalemma and in related cytoplasmic constituents are indicative of primary ozone reaction in the plant. Alterations within the cell such as the oxidation of sulf-hydryls (Mudd, 1973), changes in the levels of amino acids and proteins over time (Tingey et al_., 1973), and changes in the levels of reducing sugars over time (Tingey et al., 1973) may be indicative of secondary alterations in plant systems. There is insufficient evidence at present, however, to suggest a distinct categorization of primary and secondary effects of ozone within plants. Air pollutant effects in plants are often coupled with other injury causing agents such as pathogens and insects. Heagle (1973) has reviewed the research pertinent to the interaction of air pollutants and plant parasites (pathogens and insects). Some work has been done related to the biological interaction of air pollutants with fungi, bacteria, and viruses (reviewed by Heagle, 1973), but li t t l e is known about the inter-action of air pollutants with insects. Of the reports in the literature, most focus upon the effect of air pollutants and insects upon conifers. Cobb et_ al_. (1968) reported that Ponderosa pines injured by ozone were attacked by large populations of bark beetles, but the nutritional status of the plants was insufficient to maintain those populations. An increased susceptibility to bark beetle infestation has also bee reported in coniferous trees weakened by exposure to sulfur dioxide (Scheffer and 13 Hedgcock, 1955). Rosen and Runeckles (1976) have observed a marked increase in injury levels in bush bean (Phaseolus vulgaris L.) exposed to both ozone and whitefly infestation. This synergism may be due to increased entry of the pollutant gas through damaged leaf tissue. Alternatively, the interaction of ozone and whitefly may evoke a chemical response in the plant, notably a release of ethylene. The stimulation of ethylene production has been reported subsequent to ozone fumigation (Craker, 1971) and insect infestation (Abeles, 1973). Stress ethylene production has been identified as a general wound response in many plants. There may be a response due to.insect infestation and ozone that is analogous to pathogen (especially rust fungi) parasitism and ozone, which is known to stimulate ethylene production. Among the biological actions of ethylene is the acceleration of senescence and plant tissue death. In all studies of plant response to air pollutants, the availability of methods of assessing injury is an essential prerequisite. The methods for the assessment of plant response to air pollutants may be divided into three groups: those which depend upon visual foliar injury; those which involve measurements of plant growth, yield, and development; and those which focus on physiological and biochemical responses (Tingey e_t al_., 1978). Four visual assessment techniques are commonly used. The first is a ranking system in which plants are rated and assigned to an injury group usually designated numerically, 0-5 for example, and is based upon simil-arity in extent of injury in plants within a group. Such a rating system is actually a division of plants by a predetermined percentage range. 14 The second technique is simply the use of percentage increments to attempt to be more precise with percent injury often based upon 5% increments. A third method involves measurement of the incidence of injury or the averaging of the number of injured leaves on replicate plants that have been affected following air pollutant exposure (Menser, 1963). A final technique is remote sensing which involves the interpre-tation of aerial photographs of vegetation exposed to air pollutants. All four procedures are characterized by inherent subjectivity. It is possible to develop a high degree of uniformity among workers within a laboratory, but unfortunately the interchange of illustrative materials is inadequate in an attempt to compare results of several laboratories. Perhaps the remote sensing technique provides the most objective method for visually assessing air pollutant injury, since it has the potential for being developed into a reproducible and objective method. It must be borne in mind, however, that aerial photographs vary with the time of year, other stress factors may initiate injury symptoms similar to air pollutants, specific air pollutants cannot be identified, and ground checks are necessary for verification of an air pollutant effect (Van Genderen, 1974). Todd and Arnold (1961) described a comparison between a visual injury rating system and analysis of chlorophyll content and fresh weight changes in pinto beans. It was concluded that a visual rating system tended to overestimate injury levels. Macdowall (1971) has confirmed this tendency. Analysis of changes in chlorophyll content and fresh weight were considered more accurate than visual assessment in the estimation of leaf tissue injury. 15 Wolozin and Landau (1966) refer to the distinction between damage and injury assessment. Attention to immediately visible, acute effects is often essential in determining damage to crop plants, but the more subtle (hidden) long term effects are not easily discernible by visual assessment techniques. This difficulty led Runeckles and Resh (1975) to develop their technique for the assessment of chronic ozone injury based upon reflectance spectrophotometry, since they found that measurement of extracted chlorophyll lacked sufficient sensitivity because rela-tively few of the chlorophyll containing cells in the leaf were affected. In spite of the limitations imposed by subjective assessment of injury, however, such procedures continue to be the most commonly used methods for obtaining quantitative data. Not all experimentation has been concerned with the acquisition of data assessing the extent of injury. A notable exception is that which has focussed on dose-response relationships. In many cases, such relation-ships have been examined in terms of threshold response, where the "threshold" is arbitrarily defined as that combination of concentration and time of exposure which is just sufficient to cause the development of minimal visual symptoms. However, slight variations in exposure time, concentration, and other environmental factors may result in relatively large changes in extent of injury. Heck (1968) has made some generalizations about factors influencing oxidant injury: 1) plants grown under a short photoperiod are more sensitive to ozone than those grown under a long photoperiod; 2) a long period of darkness just before exposure increases sensitivity; 3) low temperatures prior to exposure reduce sensitivity; 4) drought 16 conditions render plants less sensitive, and 5 ) low soil nutrition tends to confer a greater sensitivity upon plants. Time of day, devel-opmental age of the plants, and the presence of other biotic and abiotic stresses also modify injury response, as noted earlier. Pretreatment with sub-acute doses of ozone may also have a profound effect on the nature and magnitude of the response of plants to subsequent acute doses, as reported by Runeckles and Rosen ( 1 9 7 4 , 1977 ) and Heagle and Heck ( 1 9 7 4 ) . The influence of dosage (time x concentration) on the extent of ozone injury is thus contingent upon the state of the plant at the time of exposure, and this in turn is dependent upon a wide range of external and temporal factors. The relation of dose and plant injury is of critical importance in the interpretation of plant response. The term "dose" or "dosage" is defined as the concentration of a pollutant multiplied by the exposure time (Munn, 1 9 7 0 ) . The concept of contaminant dosage was first considered in the gase-ous air pollutant threshold model developed by O'Gara ( 1 9 2 2 ) : (C - CR) t = K, where CR = the irritation threshold concentrations, C = the given concen-tration, greater than CR, t = the time of exposure, and K = a constant. Modifications upon the O'Gara model by Thomas and Hill ( 1 9 3 5 ) : y = a - bx, where y = the percentage of the full yield, x = the percentage of the leaf area destroyed, b = yield as a fraction of control yield when the leaf tissue is totally destroyed, and a = a constant near to 1 0 0 ; and Zahn ( 1 9 6 3 ) : t R = K-|P-| ^ | Q ^ 1 ^ ) » w n e r e P-| = a resistance factor that attempts R to account for varying external growth factors in the plant system; also incorporated the dosage concept. Guderian et aK ( 1960 ) reported that the threshold of injury was best described by an 17 exponential relationship which incorporates a weighting of concentration rather than time: tR = K.e"a ^ " ^ , where K = the vegetation life-time, a = the "biological complex factor", and tR = the fumigation time in hours needed to reach the threshold. Two primary factors were con-sidered in their work: exposure-concentration, frequency, and duration; and individual plant resistance to an air pollutant. In addition to these studies aimed at defining threshold response curves, a few workers have attempted to develop models which include degree of injury as a variable. Work by Middleton (1956-57) and Mukammal (1964) suggested a linear dose-injury response to ozone in plant systems. Mukammal, using field data rather than information from exposure chamber studies, derived a relationship similar to that of Middleton by multiplying oxidant dose by an evapotranspiration factor. Investigation by Heck ejt al_. (1966), however, over a wider range of concentrations and times of exposure, yielded a sigmoidal dose-injury relationship for tobacco and pinto bean. Heck and his colleagues have concluded that an injury response cannot be defined simply in terms of dose. Consideration must be given to the dose components - time and concentration. In this context, four important observations were made by Heck et al_.: 1) the injury threshold concen-tration of ozone is time-dependent, for exposures up to approximately 4 hours; 2) concentrations of up to perhaps 0.06 ppm may be accompanied by a time lag following exposure before injury symptom development occurs; 3) i f the ozone concentration is at least 0.2 ppm, injury response is rapid in short time intervals, and 4) injury response changeswith small concentration changes is rapid after an exposure of more than an hour. These observations substantiate the sigmoidal versus linear 18 dose-injury relationships found in tobacco and pinto beans. In 1976, Larsen and Heck proposed a model for expressing plant injury based upon pollutant concentration and exposure duration: t. D C = Mg hrSg t , where C = the pollutant concentration, Mg hr = the geometric mean concentration for a given exposure, Sg.= the standard geometric deviation, Z = the number of standard deviations that the percentage of leaf injury is from the median, t = the exposure duration in duration. It was hours, p =.the.slope' of the logarithmic plot of ozone coneentration and exposure/ suggested that a constant percentage of leaf surface would be injured by a pollutant concentration that is inversely related to the duration of exposure raised to an exponent. They suggested that percent leaf injury was lognormally related to the duration of exposure. This may be taken to mean that extremely low doses elicit no response and high doses are characterized by a maximal response. Wolozen and Landau (1966) proposed an injury determination model in which an injury index (for example, percentage of leaf destruction) is termed a dependent variable, y, and a number of independent variables, x, contribute to the extent of plant injury. Other injury and damage assessment models have been proposed as well (Oshima, 1974; Liu and Yu, 1977). Virtually all the assessment models reported in the literature have been based upon acute gaseous air pollutant levels because visible, discrete injury symptoms can be produced. The majority of these models are based upon experimentation involving the use of exposure chambers differing widely in their complexity and ability to simulate field conditions. Furthermore, rarely have the investigators given any indication, of recognizing that the ambient 19 concentrations which they maintained within their chambers may bear lit t l e relation to the actual concentration impinging upon the reactive sites within the leaf tissues. Runeckles (1974) has referred to the latter as "effective" dose, since it is obvious that the degree of response, whether it be visible or not, will be dictated ultimately by the amount.of the gaseous pollutant that enters the leaf, rather than by the concentration of the pollutant measured at some arbitrary distance outside the leaf. Studies of the uptake of pollutant gases have been dominated by two considerations. Thus, the role of plants as sinks for pollutants has been the focus of much of the work of Hill and his associates at Utah State University (Hill, 1971). Determination of the rates of uptake of pollutants by leaves and other surfaces has been the objective of others, usually with a view to determining the rate of accumulation of pollutants such as sulfur dioxide or fluorides. Hill ejt al_. (1971) devised a chamber system to measure the absorp-tion of gaseous pollutants under controlled environmental conditions. Having established a predetermined concentration of pollutant, gas exchange between the chamber atmosphere and a plant canopy, was monitored by measuring the amount of pollutant required to maintain the predeter-mined level. Pollutant was added to the chamber at the same rate that it was removed. Hence, the rate of removal was measured by the rate of addition. Hill ejt a]_. determined an ozone removal rate from an alfalfa -1 -2 -1 (Medicago sativa) canopy of 10 u l min m pphm . Under different chamber conditions, rectangular plexiglass chambers 120 cm long by 34 cm wide, Runeckles (1974) calculated an ozone uptake rate by kidney bean 20 (Phaseolus vulgaris) that was comparable to that of Hill et al.; -1 -2 -1 10.4 pi min m pphm . Consideration was given in the latter work to inlet and outlet concentrations of a pollutant in a chamber in the presence and absence of plants. Actual uptake of the pollutant could thus be distinguished from absorption or adsorption to chamber parts, pots, or soil. Effective dosage, or leaf boundary layer gas concentration, is a more precise indication of critical concentration than the ambient dosage or gross fumigation concentration. Thorne and Hanson (1972) compared the abilities of a number of plant species to absorb ozone. Their techniques were similar to those of Hill (1971) in which chamber inlet and outlet gas concentrations were measured. They calculated velocity of pollutant deposition -2 y _ Ozone sorbed/min/cm of leaf surface 9 Ozone concentration in yl/cm3 which includes both absorption and adsorption within the plant canopy. It is noteworthy that Thorne and Hanson (1972) observed a strong positive correlation between ozone deposition velocity and transpiration rate in leaf tissue of several species. Transpiration data alone, however, are inadequate in predicting ozone uptake by leaf tissue although they do permit an approximation of potential ozone sorption by leaf tissue. Environmental factors such as light, soil water potential, temperature, etc., have their own effects on transpiration and stomatal opening. Work by Rich et al_. (1970) compared well with the positive correlation between ozone deposition and apparent transpiration observed by Thorne and Hanson. They noted that the degree of ozone injury in tobacco in the field could 21 be correlated with ambient ozone concentrations only when atmospheric water content and plant water requirements were given consideration. This work recalls that of Mukammal (1964) in which a theoretical evapo-transpiration factor was used to modify an air pollutant dose. These studies tend to confirm a crucial role of stomatal function in uptake of ozone. Variations in temperature, light intensity, soil moisture, etc., independently modify stomatal resistance and hence aperture. The effects of vertical air pollutant gradients and air velocity upon pollutant injury to plants have been investigated. Heagle et al. (1971) concluded that air velocities had lit t l e or no effect on ozone injury to oat and cucumber. Their work, however, was conducted under the limitations of growth chambers that do not simulate ambient wind speeds, turbulence, mixing, etc., and may be discounted, particularly in the light of the observations of Ashenden and Mansfield (1977). These workers provided unequivocal evidence of the greater impact of a given concen-tration of on the growth of ryegrass (Lolium perenne) when the wind speed within the exposure chamber in which the experiments were conducted was increased from 0.17 to 0.42 oms'V (0.37 to 0.93 mph). They attribute the observations to the reduction of the boundary layer thickness at the higher wind speed, with the consequent increases in uptake and "effective" dose. Gradients in air pollutant concentration (van Dop, 1977; Runeckles et aT., 1978) and variations in wind speed (Slinn, 1977) are important determinants in the rate and amount of gaseous deposition at a leaf surface. Bennett and Hill (1973) note that it appears as though most ozone is taken up at the plant canopy surface. Pollutant uptake occurs primarily 22 through stomata. Stomatal response to environmental stresses including gaseous pollutants was reviewed earlier. It is crucial that any model for gaseous pollutant sorption include consideration for stomatal status. Distinct differences exist between wind velocity and pollutant concentration above, within, and below a plant canopy. Such variations coupled with variations in canopy densities and geometric properties are viable contributions to changes in stomata and the poten-tial pollutant sink properties of plants. Pollutant uptake models have been proposed in recent years. Bennett ejt al_. (1973) devised a model based upon an electrical analogue simu-lation. This concept has long been used in characterizing CO^  and H^O exchange in leaf systems. A rearrangement of Fick's law of diffusion yields the following equation: where q is the quantity of gas diffusing per unit time, t, across a given cross-sectional area, A, in a system, driven by a concentration gradient, C, along a pathway, X. Q then represents the flow or flux of the pol-lutant gas. D is the diffusion coefficient of the specific gas under consideration. As noted by Bennett et , this equation is mathemati-cally equivalent to Ohm's law: Pollutant solubility, surface characteristics, and external pollutant concentrations are given consideration in this model. Use of the equation derived by Bennett ejt al_., is limited in that it is necessary to know pollutant fluxes, In addition, Runeckles (1974) Q (Aq/AAt) = AC AX/D Q (flux) = 0(potential difference) R(resistance to transfer) 23 has observed that the model was developed with emphasis upon the "sink" properties of plants as opposed to effective dosage. It is the effective dosage, or leaf boundary layer concentration, and variations in that concentration that are important in predictive modelling and approxi-mating flux. Outside the laboratory, and under natural field conditions, concen-trations of air pollutants are almost invariably continuously changing from second to second, minute to minute, and hour to hour. Long term monitoring studies, for example Sagert and Tennis (1975), have confirmed variations in the concentrations of ozone and other air pollutants diurnally and from day to day. However, li t t l e attention has been paid to the effect of such variations in a given dose application. Virtually all controlled gaseous pollutant studies are conducted under "steady state" conditions. That is, the air pollutant concentration remains the same throughout a treatment time interval. McLaughlin et_ al_. (1976) have commented upon the unrealistic nature of consistent pollutant fumigation studies since variations in pollutant concentrations occur daily. In their work, a programmable system was used to approximate the fluctu-ations observed in ambient SO2 concentrations. It has been observed that there often occurs a higher potential toxicity with short term, high concentration air pollutant exposures when contrasted with longer exposures at lower levels. This may result despite equivalent dose applications (Webster, 1967). Extrapolation from controlled "steady state" conditions to variable ambient conditions in many instances becomes meaningless. 24 CHAPTER 3 RE-EXAMINATION OF OZONE-WHITEFLY INTERACTION  I INTRODUCTION Research pertinent to the interrelationships between air pollutants, phytophagous insects, and plants has been very limited. Rosen and Runeckles (1976) reported an ozone-greenhouse whitefly interaction on bush beans under laboratory conditions. An apparent synergism was observed between ozone at concentrations below the acute injury threshold and whitefly infestation in which severe injury, manifested as necrosis and chlorosis, resulted from the combination of the treatments. While the ozone-induced enhancement of the severity of injury caused by insects was readily observed visually, measurement of the spectral reflectance of affected leaves by the method described by Runeckles and Resh (1974) provided quantitative evidence for a two-fold increase in injury to leaves subjected to both ozone and whitefly as opposed to either ozone or whitefly alone. Two explanations were suggested to account for this phenomenon: first, the apparent synergism may be due to increased entry of the pollutant gas through leaf tissue mechanically damaged by the insect, and second, and perhaps more likely, the possibility that the presence of both insect and ozone injury induced an accelerated rate of ethylene production within the leaves which caused an increased chlorosis as a result of accelerated senescence. Stress ethylene production in injured or dying plant tissue was first reported by Williamson and Dimock (1953). Increases in ethylene production 25 have been reported following insect infestation (Craker, 1971; Abeles, 1973; Williamson, 1950), chemical trauma (Abeles, 1967), mechanical wounding (Galil, 1968; Hall, 1951), temperature extremes (Vines et al., 1968; Young and Meredith, 1971), disease (Ross and Williamson, 1951; Balazs, 1969), drought (McMichael, 1972), and gamma irradiation (Maxie, 1966). Preliminary investigations by Craker (1971) were followed by more extensive studies by Tingey and Standley (1975) which revealed an elevated ethylene production level in a wide range of plants exposed to ozone. The increased rate of ethylene production persisted for as long as 24 hours in some cases. Craker (1971) observed that differences in ethylene production between ozone-treated and control plants could be detected one hour following ozone treatment. However, neither Craker (1971) nor Tingey and Standley (1975) used ozone dosages as low as those used by Rosen and Runeckles (1976). II MATERIALS AND METHODS Phaseolus vulgaris L. (Pure Gold Wax var.) seed was sown in 12 cm pots containing soil, and kept in the greenhouse until the plants were 14 days old. Treatments were conducted in controlled environment chambers (Model E7, Controlled Environments Ltd., Winnipeg) which provided 0.65 m of growing area. Air entering the chamber passed through a charcoal filter and with both entry ports in the fully open position, approximately 1.95 cubic meters per minute were flushed through the system giving 26 approximately two air changes per minute. Wind speed through the plant canopy was 0.8 m sec"^, vertically upwards. The photoperiod was 12 hours (7.00 a.m. to 7.00 p.m.) during which the light intensity at the leaf was 3.4 x 10^  u W cm~^  from 400 to 700 nm. Temperatures were 25°C (day) and 17°C (night). Ozone was generated by passing a stream of compressed oxygen through a high voltage static discharge tube with the ozone generation maintained at a constant level by means of a voltage regulator in the line. Ozone concentrations were monitored with a microcoulomb ozone sensor (Mast Model 724-2 Ozone Meter, Mast Development Company, Davenport, Iowa). Ozone fumigations were for 8 hours in each of either 2 (Experiment 1) or 3 (Experiment 2) successive days. In the first of two experiments, the treatments were as shown in Table 1, using ten 14-day-old bean plants per treatment. Table 1. Treatments. Experiment 1. Treatment Exposure Conditions 1 Filtered air 2 Filtered air Ozone, 0.05 ppm (2 x 8 hours) 3 Filtered air Greenhouse Whitefly 4 Filtered air Ozone, 0.05 ppm (2x8 hours) Greenhouse Whitefly Trialeurodes vaporariorum (Greenhouse Whitefly) was readily available in the U.B.C. greenhouses as it infests the abaxial surface of leaves in 27 a wide variety of plants. Fifty insects were placed on each plant and, if left undisturbed, the population remained relatively constant throughout the treatment period. Symptoms were evaluated 4 days after exposure by examining the two primary leaves of each plant. A visual rating system was employed that ranged from 0 to 5. If there were no symptoms, a zero was recorded. The other numerical designations were indicative of an injury type: 1-chlorosis; 2-light necrotic stipple; 3-severe necrotic stipple; 4-necrotic fleck, and 5-widespread necrosis. In the second experiment, the treatments were as shown in Table 2. Table 2. Treatments: Experiment 2. Treatment Exposure Conditions 1 Filtered air 2 Filtered air Ozone, 0.02 ppm (3x8 hours) 3 Filtered air Greenhouse Whitefly 4 Filtered air Ozone, 0.02 ppm (3x8 hours) Greenhouse Whitefly Reflectance measurements were made using a Perkin-Elmer Coleman 124 spectrophotometer equipped with a reflectance integrating sphere (according to Runeckles and Resh, 1974). The machine was ajusted to 100 percent reflectance with a gypsum blank at 550 nm because this is the approximate wavelength of maximum leaf tissue reflectance (Gates et al_., 1965). Symptoms were again evaluated 4 days after exposure, Reflectance measure-ments were made on disks of tissue, one from each primary leaf of each 28 plant (20 measurements per treatment). Concentration and dosage were greater in Experiment 1 (0.05 ppm over 2 x 8 hours) than in Experiment 2 (0.02 ppm over 3 x 8 hours). The shorter term, higher concentration exposure was implemented to attempt to confirm an ozone-insect interaction and the slightly longer term, low concentration exposure was designed to more closely approximate ambient levels in the Point Grey vicinity of Vancouver, B.C. Although insect populations were not examined intensively, l i t t l e change, if any, was noted visually among treatments in the two experiments. I l l RESULTS AND DISCUSSION Injury assessment in Experiment 1 indicated an elevated injury level in those plants exposed to both ozone and whitefly, as shown in Table 3a. There were no visible differences between plants treated in filtered air in the absence of whitefly and those grown in ambient air in the greenhouse in the absence of whitefly. Table 3a. Experiment 1. Plant Injury. Treatment Injury Number O3 Insect 1 - - 0 2 + - 2 3 - + 3 4 + + 4 29 Exposure to.ozone (Treatment 2) resulted in 1ight -stipple while whitefly infestation in filtered air resulted in severe stipple. The elevated injury (necrosis) in the presence of both stresses may be additive but' the information is inadequate to suggest a synergism. Aware of the inherent subjectivity in the visual rating system, reflectance spectrophotometry was utilized in the quantification of injury in the second experiment.. An analysis of variance revealed a significant difference among reflectance means at the 0.05 level (Table 3b). Table3b. Experiment 2. Injury Assessed by Reflectance Spectrophotometry. Treatment % Reflectance (550 nm) Number 0^  Insect 1 19 2 + - 26 3 - + 19 4 + + 25 S.E. = 0.27 Since increased reflectance from the abaxial surface is indicative of loss of chlorophyll from palisade tissue and hence, increased injury, it is apparent that under these low dosage conditions ozone alone appears to be more injurious than ozone with whiteflies. The Student-Newman-Keuls test, however, showed the difference was not significant at the 0.05 level. These preliminary investigations tended to raise questions not par-ticularly pertinent to an air pollutant-insect interaction, but rather about injury assessment techniques and variations in exposure conditions. Neither experiment confirmed the synergism reported by Rosen and Runeckles 30 (1976), although in Experiment 1 an additive effect was clear. However, they exposed their plants to six 6-hour treatments with 0.02 ppm 0^  (total dosage, 0.72 ppmh), in contrast to the two 8-hour exposures to 0.05 ppm Og of Experiment 1 (total dosage, 0.8 ppmh) and the three 8-hour exposures to 0.02 ppm 0^  of Experiment 2 (total dosage, 0.48 ppmh). Differences in the two injury assessment methods and in the apparent plant response to variable ozone concentrations and total doses emphasizes the importance of: 1) an absolute injury assessment technique, and 2) clarification of the role of the dosage components (concentration and exposure duration) in determining plant injury. Hence, no further work on the ozone-whitefly interaction was conducted, and attention was turned to two aspects: investigations of the dose-response of bean plants to ozone, and studies aimed at determining a precise relationship between visual assessment of injury and measurements of the actual areas of necrosis. Further studies on the ozone-whitefly interaction, however, were planned. They are described in the Appendix, 31 CHAPTER 4 INJURY ASSESSMENT IN PLANT LEAVES EXPOSED TO OZONE  I INTRODUCTION Results in Chapter 3 led to the necessity for an absolute injury assess-ment technique that could serve as a check for methods of visual estimation of foliar injury. Of the injury assessment methods currently in use, visual estimation of injury is most commonly employed. Measurement of various physiological and biochemical processes in plants has often indicated a poor correlation between visual injury rating systems and actual plant injury (Tingey, 1974). Todd and Arnold (1961) have suggested that measurement of changes in chloro-phyll content and fresh weight more accurately describe the inhibition of normal leaf metabolism following ozone exposure. Runeckles and Resh (1975) have shown that reflectance spectrophotometry of the adaxial leaf surface provides a technique that is more sensitive than chlorophyll extraction in the resolution of visible differences in chronic ozone injury. Such injury is confined primarily to the palisade mesophyll tissue just beneath the upper leaf surface (Ledbetter e_t al_., 1959). Analysis of the effects of ozone on plant tissue at the cellular level has also been suggested as a measure of injury. Chimiklis and Heath (1975) have noted an efflux of K+ from the unicellular algae, Chi ore!la sorokiniana, upon interaction of ozone with the plant cell membrane. They postulate that K+ leakage could serve as a rapid indicator of plant cell sensitivity to an ozone stress. A host of other metabolic processes such as ozone and 32 lysozymic reaction (Leh and Mudd, 1974), pigment production (Howell, 1974) and water and salt transport (Ting ejt al_., 1974) has been investigated. Depression in soluble sugar levels, amino acid levels, and phenolic meta-bolites have been reported immediately following exposure to ozone (Tingey, 1974). Responses of plants within 24 hours indicated increased levels of these metabolites in contrast to controls. Investigation of the effects of ozone upon a single plant metabolite provides an incomplete picture of the role of ozone in the cell. It is uncertain whether the chemical changes are associated with early reactions to ozone or are delayed conse-quences (secondary effects) of cell injury. Field examination of a complete spectrum of plant metabolites is impractical in injury assessment. With all these conflicting choices of assessment procedures, none offers the simplicity of visual assessment, either in terms of extent of necrosis, or of the occurrence and number of necrotically injured leaves on a plant, particularly for field use. Nevertheless, in the case of visual assessment of necrotic area, i t is important to obtain a measure of any subjective biases which may exist in those conducting the evaluations. Hence, i t is important that a method of measuring necrotic area accurately be available for comparative purposes, and even as an aid in the process of training those who use visual assessment techniques. This chapter is con-cerned with the development of such an absolute measurement technique and its use in determining the reliability of subjective data. 33 II MATERIALS AND METHODS Phaseolus vulgaris (Pure Gold Wax) and Raphanus sativus (Cherry Belle) were grown as described previously (Chapter 3). The experimental conditions, using growth chambers, were as described in Chapter 3 except that ozone levels were monitored with a Chemilumenescerce Ozone Analyzer (Monitor Labs Model 8410) connected to a Mast Development Company (Model 80A) strip-chart recorder. Under these experimental conditions i t was found that 14-day old bean plants fumigated in an atmosphere of 0.4 ppm ozone for 7 hours yielded discrete necrotic lesions two days after exposure. An exposure to 0.2 ppm for 7 hours yielded similar lesions on radish two days after exposure. A complete, closed canopy (Shimwell, 1972) was obtained within the chambers with either 42 bean plants or 84 radish plants. Light intensity 4 2 at the leaf canopy was 3.4 x 10 yW/cm between 400-750 nm (measured with an Isco Model SR spectroradiometer). Two experiments each were conducted with beans and radishes with various types of exposure to a total dose of 2.8 ppm-h (bean) or 1.4 ppm-h (radish); the treatments lasted 7 hours, and are described in detail in Chapter 5. The different types of application were not devised specifically to test the accuracy of the visual rating system, but rather for elucidation of specific dose-response questions and involved exposure to peak concentrations at different times throughout the 7 hour treatment. Visual estimates of foliar injury were based upon percentage leaf area necrosis (% LAN) in 5% increments (Runeckles and Rosen, 1974). In order to relate these visual estimates to actual or absolute necrotic injury, a method was developed employing an Automatic Area Meter (Type AAM-5, Hayashi 34 Denko Ltd.), a photoelectric beam planimeter. For analysis, the primary leaves on bean plants and those leaves greater than 4 cm in length on radish plants were removed 2 days following exposure. The outlines of the leaves and the necrotic areas were then traced on transparent acetate sheets, using a fine-tip black ink felt marking pen. The outlines of the necrotic areas were filled in in black. The acetate leaf silhouette was then cut out and both the silhouette and leaf were passed through the planimeter. Absolute injured area (from the acetate tracing) was calculated as a proportion or percentage of total leaf area (from the leaf). Eight measurements, comprising two primary leaves each, were obtained in each bean treatment and 15 measurements, each comprised of all leaves greater than 4 cm in length were made in each radish treatment. Visual injury assessment data were also obtained for the same primary leaves in Treatment 5 of Experiments 1 and 2 with beans and in all treatments of Experiments 1 and 2 with radishes. I l l RESULTS AND DISCUSSION Treatment 1 in all experiments constituted a charcoal-filtered con-trol; hence, there was no ozone injury. Table 4 summarizes absolute injury data in beans. The absolute injury level in Treatment 5 .was high, with a grand mean of 78.3% for the combined data of the bean experiments. Results of visual injury assessment in Treatment 5 were collected by this author and are presented in Table 5. There is an apparent over-estimation of plant injury with the visual rating system. Table 6 summarizes absolute injury data in radishes. 35 Table 4. % Absolute Injury (Beans) Treatment Two Three Four Five Experiment One Two One Two One Two One Two 1* 6.5 11.3 36.7 34.8 15.8 21.7 84.8 85.3 2 8.1 6.0 23.6 22.9 16.6 15.9 84.7 91.5 3 12.1 5.4 18.0 41.8 18.1 22.0 86.1 79.0 4 8.6 3.6 27.7 37.7 32.7 28.9 79.9 82.1 5 4.8 6.5 20.5 26.1 36.9 17.7 83.5 83.1 6 5.1 41.5 32.9 61.3 28.6 15.5 75.5 83.3 7 10.7 7.8 70.0 52.8 24.3 36.2 70.2 76.3 8 • 45.3 7.7 53.8 19.8 19.7 34.4 54.7 53.1 Mean 12.6 11.2 35.4 37.2 24.1 24.0 77.4 79.2 S.D. ±13.4 ±12.4 ±17.9 ±14.5 +7.9 ±8.1 ±10.6 ±11.4 * Each replicate represents pooled measurements for 2 primary leaves of a single plant. Table 7 presents visual injury assessment data for both experiments and all treatments with radishes. In contrast to the visual assessment of injury to bean leaves, in the case of radish injury, visual assessments covered a wide range, comparable to the range of absolute areas. Table 8 summarizes injury levels in beans exposed in Treatment 5 and in radishes exposed to all the treatment regimes. There is a consistent over-estimation of injury by the visual 36 Table 5. % Visual Injury (Beans) Treatment Five Experiment One Two 1 95 95* 2 95 95 3 95 95 4 90 95 5 95 95 6 95 90 7 95 90 8 95 85 Mean 94.4 92.5 S.D. ±1.7 ±3.7 Individual visual injury data are related one-for-one with absolute data in this treatment. technique when compared with the absolute injury assessment. In order to compare visual injury assessment and absolute injury assess-ment, the over-estimation of the visual assessments for each of the four treatments in radishes and the single treatment with beans is also reported in Table 8. Both visual and absolute injury assessment in Treatments 2 and 37 Table 6. % Absolute Injury (Radish) Treatment Two Three Four Five Experiment One Two One Two One Two One Two 1* 9.3 36.4 65.8 64.4 6.3 31.9 43.1 72.6 2 10.3 7.3 49.2 51.7 6.9 61.1 39.3 37.0 3 13.7 0- 21.3 22.0 0 60.4 52.5 26.9 4 6.2 58.6 9.9 20.8 66.0 16.5 61.1 84.4 5 46.9 8.3 11.7 16.7 39.0 0 18.8 79.3 6 47.0 6.8 9.6 54.7 53.0 0 66.0 39.1 7 14.9 49.5 68.7 49.4 20,4 20.0 51.1 36.6 8 16.2 34 j 44.7 25.4 6.8 13.1 53.7 18.8 9 17.9 0 34.0 48.2 34.2 27.8 57.0 13.8 10 5.6 46.8 68.0 30.7 4.7 29.1 0 72.5 11 5.5 15.5 28.9 25.0 0 23.6 16.7 48.0 12 0 11.3 41.9 30.6 6.9 12.9 16.0 72.9 13 20.8 4.4 38.4 54.4 24.6 16.9 72.1 51.2 14 0 8.7 32.6 66.3 37.9 7.9 42.1 24.9 15 41.4 0 40.7 90.1 25.3 6.5 57.6 20.8 Mean 17.0 19.2 37.7 43.4 22.1 21.8 43.1 45.3 S.D. ±15.7 ±20.1 ±19.8 ±21.0 ±20.3 ±18.5 ±21.1 ±24.4 Each repl icate represents pooled measurements for a l l leaves greater than 4 cm in length on a s ingle p lant . 38 Table 7. % Visual Injury (Radishes) Treatment Two Three Four Five Experiment One Two One Two One Two One Two 1 10 45 90 95 5 35 85 95* 2 5 5 80 40 10 80 35 80 3 15 0 25 15 0 50 80 65 4 25 60 5 20 60 25 95 80 5 20 50 5 20 40 0 30 95 6 5 45 5 75 45 0 85 25 7 20 50 90 55 15 20 90 45 8 20 30 60 25 10 15 95 25 9 15 0 25 50 15 40 80 10 10 5 70 90 35 5 60 0 90 11 5 25 20 40 0 35 30 40 12 0 5 40 35 5 30 10 85 13 65 10 40 50 35 30 100 25 14 0 5 25 60 55 5 55 30 15 65 0 50 95 40 5 95 30 Mean 18.3 26.7 43.3 47.3 22.7 28.'3 64.3 54.7 S.D. ±20.4 ±24.7 ±31.8 ±25.3 ±20.8 ±22.8 ±34.3 ±30.4 * Individual visual injury data are related one-for-one with absolute data in all radish treatments. 39 Table 8. % Injury* % Over-Treatment Visual Absolute Estimation 1 - Control 0 0 0 2 - Radish 22.5 + 22.4 18.1 + 17.9 15.0" 3 - Radish 45.3 + 28.5 40.5 + 20.4 10.5 4 - Radish 25.7 + 21.8 21.9 + 19.4 14.7 5 - Radish 59.5 + 52.3 44.2 + 22.7 25.7 5 - Bean 93.4 + 2.7 78.3 + 11.0 19.3 Data of Experiments 1 and 2 are pooled. 4 are comparable. In addition, the percent over-estimation is similar in these two treatments. Percent over-estimation is lowest in Treatment 3 and highest in Treatment 5. Based upon percent over-estimation calculations, a pattern emerges in which the visual rating system may conceivably be more sensitive or responsive in the "lower" and "middle" ranges of injury and less so at the "upper" end of the range. Previous workers (Todd and Arnold, 1961) have noted a relative insensitivity of the visual rating system at the upper end of the range. Over-estimation is compounded by the inherent subjectivity of the technique. It is apparently more difficult to quantify injury visually when the injury lesions are dense and closely compacted as opposed to when a moderate necrotic stippling occurs. The evidence, 40 however, is insufficient to unequivocally confirm decreased visual assess-ment sensitivity at the upper extreme of the injury spectrum. A relationship between percent visual and percent absolute injury is observed. There is a distinct increase in percent absolute injury with an increase in percent visual injury. The relationship between the two variables is not one of dependence, however. Figures 1 and 2 describe a simple linear correlation between percent absolute and percent visual injury in the two radish experiments. The positive correlation coefficients in both experiments with radishes are highly significant and indicative of a close association between the two assessment techniques. The-'absolute injury assessment technique described depends upon measure-ment of the total area of lesions,on a leaf of known area. Correlation data suggest that the absolute technique can serve as a suitable periodic check upon the visual rating system-.- Observations in beans suggest, and those in radishes confirm, a consistent over-estimation with the visual technique that becomes most pronounced near the upper end of the injury range. The absolute injury determination approach described in this section is employed in the calculation of extent of injury subsequent to plant exposure to variable ozone regimes (Chapter 5). 41 80 E X P E R I M E N T ONE RADISH r = 0- 8 9 5 >-or ZD 70 60 8 B 50 o Ui m < 40 o • 8 U J o cc L U C L 30 20 i 10 -4 ~! 1 1 1 1 ! ! 1— 10 20 30 40 50 60 70 8 0 P E R C E N T V I S U A L I N J U R Y 90 Figure 1. Scatter diagram of injury levels in Raphanus sativus. Each of 4 ozone treatments is represented by 15 points. 42 8 0 i E X P E R I M E N T T W O R A D I S H r = 0 - 8 0 8 7 0 o o >-rr 6 0 H 5 0 O to 4 0 GO < H :z U J o or U l CL 3 0 2 0 i 10 ~i e « 10 i 2 0 I 3 0 ~ I — 4 0 5 0 T 6 0 I 7 0 P E R C E N T V I S U A L I N J U R Y "1— 8 0 9 0 Figure 2. Scatter diagram of injury levels in Raphanus sativus. Each of 4 ozone treatments is represented by 15 points. 43 CHAPTER 5  VARIABLE OZONE DOSE APPLICATION I INTRODUCTION Virtually all air pollutant investigations in controlled environments have utilized "steady state" gaseous fOimigations. In contrast, ambient air pollutant levels are rarely static but fluctuate as a consequence of changes in the rates of emission from pollutant sources, and of changes in environmental factors such as wind, turbulence, temperature, topography, and pollutant stability. While fluctuations in levels of pollutants such as SO^ emitted from point sources may be expected as a result of atmospheric turbulence, the same is true of "secondary" pollutants such as ozone which are formed within the atmosphere from precursors. Thus, Sagert and Tennis (1975) have reported the fluctuations in ozone levels experienced in the city of Vancouver and at a station 100 km to the east in the Fraser Valley, as shown in Figure 3. This illustrates both the normal diurnal rise in ozone concentration during daylight hours typical of regions with high vehicle density, and the way in which these elevated concentrations can persist and move within an air mass, away from the site of formation. In this example, the delayed appear-ance of high ozone levels at the inland site is a function of distance and the normal on-shore breeze. The figure also shows hour to hour fluctuations which follow distinctly different patterns throughout the day. For example, the data for the first day show a steadily increasing concentration (Figure 3, solid lines) at one site, reaching a maximum at sunset. On the second 44 T I M E ( H O U R S ) Figure 3. Comparison of ozone measurement in the Lower Mainland Air Basin, Vancouver, B.C. Source: Sagert and Tennis, 1975. 45 day, the levels rise rapidly at the same site and, with some minor fluctu-ations, remain essentially constant until sunset. On the third day, on the other hand, there is a rapid rise following sunrise, with a peak at midday, followed by a steady decline. In terms of dosage, ,the integrals between the hours of 0600 and 2200 for these three days are respectively 25.3, 41.0, and 51.5 pphm-h (Figure 3, solid lines). These data, therefore, also illustrate how dosage may vary appreciably even though the same peak concentration may be reached (first day vs. second day), and how comparable dosages may occur (second day vs. third day) with a two-fold difference in peak concentration. ; The ozone dosages in Figure 3 are relatively modest and would not in themselves cause injury to the most susceptible plants. However, they reveal the variability in the pattern of concentrations which may occur within a given dosage period, and which may equally well occur at concen-trations above the injury threshold. Numerous workers have stressed the greater importance of peak concen-trations rather than duration of exposure in determining plant response (Guderian, ,1960; Heck, 1966; Webster, 1967). Van Haut (1961), for example, showed that when radish was subjected to treatments of 3, 6, 9, and 12 mg -3 -3 m SO^  for 12, 6, 4, and 3 hours respectively (36 mg m h dosages through-out), the following leaf injury occurred: 2, 20, 40, and 76 percent of leaf area. In Van Haut's studies, however, the duration of the exposure varied inversely with the concentration. Few studies have been made of situations in which the total duration of the exposure to a pollutant has been kept constant, together with the dosage, but in which the concentrations 46 contributing to the dosage have varied. Those studies which have been conducted have taken the form of fumigations for brief periods with dif-ferent concentrations interspersed with periods of varying duration in which no pollutant is administered. Zahn (1961) again using SO^ , conducted experimental trials on spinach. All plants were fumigated with 1.5 ppm SG^  for 3 hours. Exposure was for 3 consecutive hours or 3 separate hourly exposures with one, two, or three SC^-free hours between exposures. Injury was assessed visually on a scale from 1 (slight injury) to 5 (total injury). An injury coefficient of 3.3 was obtained when plants were exposed to 3 consecutive hours of SG^  treatment. Injury was reduced to 0.5 i f the longer (3 hour) SO^ -free periods were interspersed between the shorter (1 hour) SO2 exposures. In subsequent studies, he noted that recovery due to a break in pollutant exposure is contingent upon that period exceeding a minimum time. However, he observed that the duration of elevated concen- . trations was of more importance than the pause. For example, doubling the duration of the concentration could not be compensated for by doubling the recovery time. Little information .is available pertinent to plant response under fluctuating ozone regimes. Heck and Dunning (1967) have noted that tobacco and pinto beanare more sensitive to a specified dose given in one hour than to the same dose divided into two k-hour exposures. A partial recovery may be attributed to either a stomatal closure during the "recuperation" interval or a diffusion of the internal ozone during that period. Menser (1962), however, demonstrated the reverse in four tobacco varieties. Subsequently, Menser et al_. (1965) undertook a similar investigation to that of Zahn (1961), in which three varieties of tobacco were subjected to identical 47 ozone doses in which the durations of exposure were inversely proportional to the concentrations used. Three different dosages were applied, namely 0.15, 0.30, and 0.60 ppm-h, and in each case, three concentrations were employed for the appropriate durations: 0.30, 0.20, and 0.10 ppm. In every comparison, the treatment which involved the highest concentration resulted in the greatest injury. In contrast to these studies of dissected doses, a few investigators have recognized that the interruptions or "recuperation" intervals under field conditions are unlikely to be periods during which the level of the pollutant in question drops to zero, but rather that the level will merely drop to one below the threshold for acute injury. Zahn (1970) and Runeckles and Rosen (1974) observed that,.depending upon the species studied, sub-acute doses could either increase or decrease the magnitude of the acute injury response to higher concentrations. Zahn's work with SOg suggested that cereals and larch were protected by sub-acute dosages, while dicotyle-dons such as alfalfa and rapeseed tended to show injury enhancement. This latter finding confirmed earlier observations made on spinach (Zahn, 1963). Runeckles and Rosen, working with ozone similarly showed that different species responded in opposite ways to sub-acute pretreatments, with mint leaves demonstrating an additive effect and bean primary leaves showing a reduction in injury. Subsequently, they were able to demonstrate that the nature of the response of bean leaves was dependent upon the state of maturity of the leaves, and that the protective effect only occurred in "middle-aged" leaves (Runeckles and Rosen,.1977). Heagle and Heck (1974) reported an increased susceptibility of the highly susceptible tobacco cultivars Bel-W3 to acute injury from natural oxidants (mostly ozone) in field situations, 48 following exposures to sub-acute concentrations. The primary objective in these earlier studies had been to acquire an understanding of the ways in which plants respond to various dosages, regardless of the variations in response attributable to other environ-mental, edaphic, and biotic factors. However, only recently has an experi-mental system been devised which can simulate the normal variability in pollutant concentrations over extended periods (McLaughlin ejt al_., 1976). The system is dependent upon rapid flushing of air within the experimental chamber, with the pollutant concentration (in this case SC^ ) being intro-duced with thorough mixing at rates determined by pre-set exposure regimes based upon records of ambient concentrations. To date, however, no data obtained with the system have been reported. Nevertheless, the system appears capable of generating useful information, since it is designed to simulate field conditions. It should also be noted that the system uses wind speeds of sufficient magnitude as to avoid one of the serious criticisms of much of the experimentation with exposure chambers that has been carried out to date on the effects of air pollutants on plants. Ashendon and Mansfield (1977) have stated the criticism in the fol-lowing words: "If laboratory experiments are to provide a realistic indication of the sensitivity of plants to SO,, in the air, it is clear that attempts must be made in future work to ensure that fumigations are not carried out in conditions of air movement that are a great deal less than those prevailing out-of-doors....Where plants have been exposed to pollutants in virtually s t i l l air (in chambers), the high boundary layer resistance could be expected to reduce the rate of entry into leaves and result in an over-estimate of the concentration required to cause damage. 49 This simple fact has not been recognized by many experimenters in the past, so that there is considerable doubt about the validity of their estimates of the threshold S02 concentrations required to induce injury to plants". While Ashendon and Mansfield's remarks focus on S02, the criticism is equally valid with regard to much of the published work on ozone. The criticism is of particular significance in relation to studies of the rates of uptake of pollutants, aimed either at reaching an under-standing of pollutant flux and hence of the actual or "effective" doses received by plant tissues (Runeckles, 1974), or at developing predictive models of injury. The role of stomata in regulating the uptake of pollutants is well established and dates back to the work of Loftfield (1921). However, stomata themselves vary in their response to pollutants, although in most cases pollutants cause stomatal closure (Mansfield and Majernick, .1970). Stomatal diffusive resistance is an important component of the models of pollutant uptake developed by Bennett et al_. (1974) and more recently by 0'Del 1 ejt al_. (1977). The latter workers also point out, independently of Ashendon and Mansfield (1977), the importance of aerodynamic or boundary layer resistance to pollutant uptake at wind speeds less than lm. s ^ . At greater wind speeds and with pollutant gases of high solubility in water, stomatal resistance approximates the total resistance to uptake. Hence, under such conditions, stomatal resistance can be the major factor affecting the effective dosage of a pollutant within the leaf. Work on pollutant uptake and dose-response relationships has to date been largely independent, although workers such as Guderian and Stratmann 50 (1960) have incorporated "resistance factors" into threshold response models. The work described in the present chapter was therefore undertaken to obtain a clearer understanding of the injury caused by different patterns of exposure to ozone in which the total duration and dosage was essentially constant and to develop a predictive model of acute injury in which ambient dosage, peak concentration, and the effects of treatment on stomatal resistance are all involved in determining the effective dosage to which the leaf tissues respond. II MATERIALS AND METHODS Phaseolus vulgaris (Pure Gold Wax) and Raphanus sativus (Cherry Belle) seed was sown in a manner identical to that described in Chapter 3. Experi-mental chamber conditions (12 hour photoperiod; temperatures: 17°C night, 22°C day; RH 75-80%), ozone generation and measurement, and plant age were the same as in Chapter 4. Continuous Stomatal Measurement Two experiments were conducted initially, one with beans and one with radishes. Plants were maintained in charcoal-filtered air throughout except during the times at which ozone was added to the filtered air stream. Each experiment was composed of five treatments, as shown in Table 9. Total ozone doses in all bean treatments were 2.8 ppm-hrs and 1.4 ppm-hrs in radishes. The ozone concentration was adjusted to the appropriate level at the beginning of each hour. Treatments 3, 4 and 5 in both plant species were characterized by variation in the time of occurrence of the 51 Table 9. Ozone Treatments Administered to Bean and Radish Plants. Ozone Treatments Commenced at the End of the Second Hour of the Photoperiod Hour of Treatment* Treatment Number 1 2 3 4 5 6 7 Remarks Beans: 1 0 0 0 0 0 0 0 filtered air throughout 2 0.4 0.4 0.4 0.4 0.4 0.4 0.4 ppm ozone; "steady state" 3 0.1 0.3 0.6 0.8 0.6 0.3 0.1 ppm ozone; "middle peak" 4 0.1 0.2 0.-3 0.4 0.5 0.6 0.7 ppm ozone; "late peak" 5 0.7 0.6 0,5 0.4- 0.3 0.2 0.1 ppm ozone; "early peak". 3A 0.15 0.35 0.55 0.70 0.55 0.35 0.15 ppm ozone; "middle peak" 4A 0.05 0.15 0.30 0.40 0.50 0.60 0.80 ppm ozone; "late peak" 5A 0.80 0.60 0.50 0.40 0.30 0.15 0.05 ppm ozone; "early peak" Radish: 1 0 0 0 0 0 0 0 filtered air throughout 2 0.2 0.2' 0.2 0.2 0.2 0.2 0.2 ppm ozone; "steady state" 3 0.05 0.15 0.25 0.50 0.25 0.15 0.05 ppm ozone; "middle peak" 4 0.05 0.10 0.15 0.20 0.25 0.30 0.35 ppm ozone; "late peak" 5 0.35 0.30 0.25 0.20 0.15 0.10 0.05 ppm ozone; "early peak" Ozone fumigation extended from hours 2 through 8 in the photoperiod. 52 peak concentration, in the middle, late, or early part of the exposure period. Bean treatments 3A, 4A and 5A provided a full complement of treatments for that crop species with two .peak concentrations - 0.7 ppm and 0.8 ppm. The legends in figures presented in this chapter describe the variable exposure regimes as bell curve (middle peak), step up (late peak) and step down (early peak) to assist in visualizing conditions prior to and following the occurrence of peak ozone concentrations. Two days following exposure, ozone injury was assessed using both visual injury assessment {% LAN) and the absolute assessment technique described in Chapter 4. Ten replicate stomatal resistance measurements were made starting approximately 20 minutes after the beginning of each hour, and lasting approximately 10 to 15 minutes. The same two primary leaves for each of five plants were measured in every hour in the 12 hour photoperiod. Sto-matal resistance was measured with a diffusion resistance porometer (Model LI-60, Lambda Instrument Corporation, Lincoln, Nebraska) fitted with a horizontal sensor cup (Model LI-20S). All measurements were made on the abaxial leaf surface. The treatments were conducted on progressive days in the same growth chamber using plants of the same age sown at daily intervals. Hence, each experiment progressed through 5 days. In this way time was afforded for the continuous collection of stomatal data. In order to make the stomatal measurements without opening the door of the chamber and thus causing a significant reduction in ozone concentration, a "glove-box" approach was used. A plexiglass sheet sealed across the door opening, contained two 53 15 cm diameter ports around which were sealed 45 cm long polyethylene "sleeves". The two ports allowed access to the chamber with minimal dis-turbance to the required ozone concentration. Periodic checks confirmed an ozone concentration decline of no greater than 5-8% during the sampling interval. To avoid disturbance to the sealed plexiglass window, plants were introduced through the top of the chamber. A completely random design was assumed in all experiments. Stomatal data were subjected to an analysis of variance (ANOVA). If the F calculated was significant, the ANOVA was followed by the Student-Newman-Kuels (SNK) multiple range test. Growth of Radish Two experiments were conducted in which total plant, shoot, and root dry weights of radish were obtained following exposure to Treatments 1, 2, 3, 4 and 5 (Table 9). There were 14 replicates in the first experiment, each replicate consisting of 2 radish plants. In the second experiment, 14 replicates each consisted of 8 radish plants. The plants were harvested seven days after the termination of the ozone exposures during which time they were maintained in the growth chamber in filtered air. Plants were oven-dried at 60°C for 24 hours prior to weighing. In these experiments, single stomatal resistance measurements were made on 28 plants in each treatment, 24 hours after the completion of the treatments. 54 III RESULTS AND DISCUSSION Plant Injury and Exposure Components Injury levels are summarized in Table 10. Table 10. Ozone Injury with Various Constant Dosage Regimes Treatment C * max t + max % Absolute Number (ppm) (hr) Injury Beans: 1 0 0 0 2 0.4 1-7 15.4 3 0.8 4 36.2 4 0.7 7 24.1 5 0.7 1 78.3 3A 0.7 4 32.1 4A 0.8 7 28.6 5A 0.8 1 84.5 Radish: 1 0 0 0 2 0.2 1-7 18.1 3 0.5 4 40.5 4 0.35 7 21 .9 5 0.35 1 44.2 *Peak ozone concentration +Hour in the exposure period in which the peak concentration occurred 55 Percent injury varied with a corresponding variation in &max (peak ozone concentration). If C is reduced from 0.8 ppm to 0.7 ppm as in max the middle peak treatment (3 and 3A), percent injury is also reduced. If C , is increased from 0.7 ppm to 0.8 ppm as in the late and early max peak treatments (4 and 4A; 5 and 5A), percent injury is also increased. The magnitude of C is an important factor in dictating the extent of 3 max ^ 3 plant injury and it becomes apparent that the time at which that peak occurs is also important in determining the subsequent extent of injury. Injury levels with the steady state exposure are less in both beans and radishes than those observed in the other treatments. Three of the five treatments used in this study were characterized by hour to hour changes in ozone concentration. Figures 4 and 5 (A s in time) indicate similarities in stomatal response in beans and radishes. Analyses of variance in both species detected a significant difference among treatments. The Student-Newman-Kuels multiple range test confirmed differences between specified pairs of means (n = 10) commencing in the fourth hour after the start of treatment of beans and in the fifth hour of treatment in the case of radishes. In both species the single signifi-cantly different pair is the control and the early peak treatments. Significant differences between pairs of means become more prevalent in the latter half of the photoperiod in both species. The control and late peak treatments do not differ significantly until the eleventh hour of the photoperiod for either species. The stomatal resistance of the control remained virtually unchanged. The steady state, middle peak, and late peak were all characterized by a steady increase in stomatal closure, the greatest rate of increase 56 B E A N o o UJ to UJ o z < co CO UJ < < o y-C O N T R O L S T E A D Y S T A T E B E L L C U R V E S T E P UP S T E P D O W N 4 0 3 0 2 0 10 2 3 T I r 4 M E —I 1 5 6 ( H O U R S ) 8 10 II — 12 Figure 4. Stomatal resistance through a 12 hour photoperiod with beans in the steady s tate , middle peak (bel l curve), late peak (step up), and early peak (step down) exposures. Ozone treatment occurred i n hours 2 through 8 of the photoperiod. S.E. = 3.29. 57 o U J to R A D I S H C O N T R O L '."I"""'""" S T E A D Y S T A T E B E L L C U R V E S T E P U P S T E P D O W N 4 0 I LO O < re < z o I -3 0 2 0 I 0 --•s^  „ — — — -0 I T I M E ( H O U R S ) Figure 5. Stomatal resistance through a 12 hour photoperiod with radish in the steady state, middle peak (bell curve), late peak (step up), and early peak (step down) exposures. Ozone treatment occurred in hours 2 through 8 of the-photoperiod. S.E. = 2.91. 58 occurring in the early peak treatment. Table 11 summarizes stomatal resistance and absolute injury data in the two plant species for Experiments 1 and 2. In spite of the fact that stomates are the primary ports of pollutant entry, the mean absolute injury and stomatal resistance data appear to show li t t l e relation to each other. While lower stomatal resistances are to be expected to lead to higher injury levels, the data show no such relationship for either species. Since there is no obvious relationship between subsequent injury and mean stomatal resistance during the exposure to ozone, i t appears that the treatment exposure regime itself must be important in dictating the extent of plant injury. In particular, the peak concentration and the time of its occurrence might be expected to play an important role in determining injury, since there is a lag in stomatal response to treatment. As a result, peak concentrations occurring before the stomatal resistance has increased markedly might be expected to result in the greatest effec-tive dosages being administered. Figures 6 and 7 illustrate comparisons between the ozone treatment patterns and stomatal resistance over time for the two species. Figures 6a and 7a show an increase in stomatal resistance throughout the course of the steady state treatment period and that the rate of increase itself increases with time. This constant treat-ment results in a greater closure rate in beans than in radishes. Figure 6b shows that the stomatal resistance in the middle peak treatment follows essentially the same curve, and that at the time of the peak concentration, the stomatal resistance was only 13.9 sec cm" ^. As shown in Figure 6c, the stomatal resistance was somewhat higher, 19.4 sec cm"'', at the time of peak concentration in the late peak treatment. In addition, the 59 Table 11. Treatment % Injury Mean Stomatal*, Resistance (sec cm ) Beans: 1 0 11.8 2 15.39 21.8 3 36.24 20.8 4 24.06 17.9 5 78.32 25.7 Radish: 1 0 10.9 2 18.11 17.4 3 40.53 18.9 4 21.99 14.7 5 44.20 23,9 * Measurements are grand means for each treatment. There are 12 hours per treatment with 10 measurements per hour. general form of the curve for stomatal resistance is here very different from that in either Treatments 2 and 3, and undergoes a decline in the rate of increased stomatal resistance. Perhaps this decline is indicative of a periodic stomatal response independent of the shape of dose application. Figure 6d shows that in the case of the early peak treatment, the peak 60 BEAN co UJ cc O Z O N E C O N C E N T R A T I O N r o LO CO ~ 4 0 LU O z < O- 8 3 0 0-6 o N o o o z o m < < o I-2 0 I 0 0 4 0-2 7} > O z D T) T T 1 2 3 4 O Z O N E F U M I G A T I O N T I M E T 5 1 6 ( H O U R S ) 7 Figure 6a. Stomatal resistance (r ) during the steady state ozone fumigation in bean (Phaseolus vulgaris). 61 Figure 6b. Stomatal resistance (r ) during the middle peak ozone fumigation in bean (Phaseolus vulgaris). 62 Figure 6c. Stomatal resistance (r ) during the late peak ozone fumigation in bean (Phaseolus vulgaris). 63 Figure 6d. Stomatal resistance (r ) during the early peak ozone fumigation in bean (Phaseolus vulgaris). 64 occurred at the time of the lowest stomatal resistance, 13.3 sec cm 1. Here, stomatal resistance increased rapidly during the early stages of the treatment. It should also be pointed out that since the stomatal measurements were made 20-30 minutes after the start of the ozone treat-ment, the mean value for the critical stomatal resistance in Figure 6d may represent a partial closure, as suggested by Figure 4. A comparison of the subsequent injury levels and the values of stomatal resistance at the time of the peak ozone concentration is shown in Table 12. Table 12. Stomatal Resistance at C m n v and Subsequent Injury Levels. r (at C ) sec cm-1 Injury (%) Beans: s x max' 1 -2 10.2'-.28.4 15.4 3 13.9 36.2 4 19.4 24.1 5 13.3 78.3 Radish: 1 2 9.9 - 20.9 18.1 3 13.0 40.5 4 14.3 22.0 5 12.7 44.2 65 Radish data presented in Figure 7 tend to substantiate the obser-vations made with beans. Treatments in which stomatal resistances were relatively low at the time of peak concentration resulted in higher injury levels than those in which stomatal resistance was relatively high at the time of peak concentration. While there is a general inverse relation between injury and r g at Snax' t'1is c*oes n o t t e ^ t'ie w n 0^ e s t o ry» since in Treatment 2 there was no peak concentration. Although injury was least in this treatment for both species, it was nevertheless significant. While i t could be argued that the stomata in Treatment 2 were responding to a "peak" in the first hour of exposure, i t is more likely that the injury here is primarily a function of cumulative dosage. Because the ozone treatments described in this study represent a continuum as opposed to singular, discrete fumigations, cumulative dosage must be considered in conjunction with magnitude and time of occurrence of peak concentration in determining stomatal response and subsequent plant injury. The relative differences in stomatal resistance (Table 12) are insufficient to account for the wide range in injury levels among the treatments. In addition, previous researchers (Zahn, 1961; Runeckles and Rosen, 1977) have noted the importance of events prior to or following exposure (or peak concentration) in determining subsequent plant injury. Whether stomatal closure affords a plant protection will therefore depend upon cumulative dosage through a fumigation period as well as peak concen-tration characteristics. Hour to hour dose accumulation varies according to the pattern of ozone application. Table 13 shows the hourly variation in dose accumulation in the five treatments with beans and radishes. 66 o o Ul R A D I S H O Z O N E C O N C E N T R A T I O N r 4 0 0 4' U J o z <t H to to U J cc < < o w 3 0 2 0 I 0 0 3 0 2 0 T —~l I r -I 2 3 4 O Z O N E F U M I G A T I O N 1 5 T I M E "1 F ( H O U R S ) Figure 7a. Stomatal resistance (r ) during the steady state ozone fumigation in radish (Raphanus sativus). 67 O Z O N E F U M I G A T I O N T I M E ( H O U R S ) Fig. 7b. Stomatal resistance (r ) during the middle peak ozone fumigation in radish (Raphanus sativus). 68 Fig. 7c. Stomatal resistance (rs>) during the late peak ozone fumigation in radish (Raphanus sativus). 69 Fig. 7d. Stomatal resistance (r ) during the early peak ozone fumigation in radish (Raphanus sativus). 70 Table 13. Cumulative Ozone Dose Beans Radish Treatment: 2 Hour: Treatment: 1 0.4 ppm-hrs 2 Hour: 1 0.2 2 0.8 2 0.4 3 1.2 3 0.6 4 1.6 4 0.8 5 2.0 5 1.0 6 2.4 6 1.2 7 2.8 7 1.4 1 0.1 3 1 0.05 2 0.4 2 0.20 3 1.0 3 0.45 4 1.8 4 0.95 5 2.4 5 1.20 6 2.7 6 1.35 7 2.8 7 1.40 1 0.1 4 1 0.05 2 0.3 2 0.15 3 0.6 3 0.30 4 1.0 4 0.50 5 1.5 5 0.75 6 2.1 6 1.05 7 2.8 7 1.40 1 0.7 5 1 0.35 2 1.3 2 0.65 3 1.8 3 0.90 4 2.2 4 1.10 5 2.5 5 1.25 6 2.7 6 1.35 7 2.8 7 1.40 ppm-hrs 71 As dose accumulation varies in time with the individual treatments, stomata respond differentially. Such differences are noted in the com-parison of the steady state (Figure 8) with the variable exposure regimes (Figures 9, 10 and 11). It must be emphasized that the extension of lines to the left-hand vertical axis, to form a series of seven planes in each figure, is merely a graphical convenience while the individual points of interest rise vertically from a position described by time and the associ-ated cumulative dose. Comparison of the steady state and middle peak exposures (Figures 8 and 9) indicates that early in the treatment period cumulative dose the middle peak/lags behind the steady state in cumulative ozone dose. Stomatal resistance in the first four hours is similar in the two treatments (Figure 4). In the fourth hour of treatment, the dosage begdns to accumulate more rapidly in the middle peak treatment while the stomatal resistance remains lower than in the steady state. Injury levels were higher in the middle peak than in the steady state treatment with the lag in stomatal resistance and the higher cumulative dose likely contributors to this enhanced injury. Comparison of the steady state and late peak treatments (Figures 8 and 10) confirms a lag in cumulative ozone dose in the late peak through all treatment hours. In the last three exposure hours, the stomatal resis-tance in the late peak treatment lags behind that of the steady state. The slightly higher injury in the late peak is determined, in part, by this lag in stomatal resistance in the latter hours of the exposure. In the early peak (Figure 11) the cumulative dose is greater in all hours than the steady state. Stomatal resistance in the early peak is also greater in all hours than the steady state, yet the injury level is also 72 Fig. 8. Changes in r s in beans with increases in cumulative dose in time (steady state exposure). 73 Fig. 9. Changes (middle in r in beans with increases in cumulative dose in time s peak or bell curve exposure). 74 Fig. 10. Changes in r g in (late or step up beans with increases to peak exposure). in cumulative dose in time 75 11. Changes in r g in beans with increases in (early or step down from peak exposure). cumulative dose in time 76 considerably greater. Evidently, the extent of plant injury is dependent on other factors as well as the pattern in which dose accumulates. Because there is no obvious relation between cumulative dose, stomatal resistance, and extent of injury in the early peak exposure, consider-ation should once again be given to the role of magnitude and time of occurrence of peak concentration in dictating plant injury. The initial high level exposure in the early peak treatment may be sufficient to induce extensive acute leaf injury despite an apparent compensatory stomatal closure in the latter part of the exposure. A similar series of plots was developed for radishes (Figures 12, 13, 14 and 15°) in which stomatal response patterns were also similar to beans. Comparison^  of the steady state and middle peak exposures (Figures 12 and 13) reveals that although the trend in cumulative dose and stomatal resistance is similar to that observed in beans, there occurs a greater stomatal resistance in hour seven of the middle peak exposure. Perhaps this is an attempted compensation response to the ozone stress; if so, i t is apparently too late as the injury level is more than twice as high in the middle peak as in the steady state (40.5% vs. 18.1%). Both the late peak and early peak dose accumulation and stomatal response in radishes are similar to that observed in beans. Stomatal responses to variable cumulative ozone doses in time are reproducible with two plant species. v Dose Response MacDowall et al. (1964) devised a model for approximating ozone dose 77 R A D I S H Fig. 12? Changes in r g in radish with increases in cumulative dose in time (steady state exposure). 78 •13. Changes in r g in radish with increases in cumulative dose in time (middle peak or bell curve exposure). 79 R A D I S H S T E P UP Fig. 14. Changes in r g in radish with increases in cumulative dose in time (late or step up to peak exposure). 80 Fig. 15. Changes in r g in radish with increases in cumulative dose in time (early or step down from peak exposure). 81 affecting tobacco leaf tissue in which dose (time x concentration) was modified by an empirical evapotranspiration factor. This concept was adjusted to accommodate the continuous exposure emphasis in this study. An approximation of ozone flux (gaseous flow into leaves) is obtained by multiplying stomatal conductance (reciprocal of resistance) by the ozone concentration in a specified hour. Summation of these values describes the total flux through a specified hour; £(l/rs X C). Figure 16 illustrates patterns of ozone flux in beans throughout the ozone fumigation. Although an analysis of variance failed to detect a significant difference among the exposure profiles, the treatment means are variable in any given hour. The early peak treatment was characterized by a greater ozone flux than the other three treatments through the first three hours of ozone fumigation. The rate of increase in the last three hours of exposure declined in the early peak. This may be indicative of stomatal closure following saturation of the sub-stomatal cavity (Koritz and Went, 1953). Plants exposed in the steady state have a lower ozone flux than the early peak to hour five. At that point the steady state curve intercepts that of the early peak and in hours six and seven i t ranges slightly higher than the early peak curve. The middle peak curve describes a low ozone flux in the first three hours of treatment with only plants in the late peak exhibiting a lower flux. The middle peak flux rises above that of all other treatments in the last four hours of exposure. Throughout the fumigation, the late peak treatment is characterized by a consistently reduced flux. Figure 17 shows.ozone flux patterns in radishes. With minor deviations, the flux patterns in radish are roughly the same as those observed in beans. 82 B E A N e o S T E A D Y S T A T E B E L L C U R V E S T E P UP S T E P D O W N X Z3 U . o N O 0-20 0 1 6 0 1 2 0 0 8 0 0 4 II 2 O Z O N E i i 3 4 F U M I G A T I O N 6 7 T I M E ( H O U R S ) Fig. 16. ': Ozone flux defined as stomatal conductance 0/r s) multiplied by ozone concentration (C) summed through a specified hour (z(l/r s x C)) in bean. Fig. 17. Ozone flux defined as stomatal conductance O/r ) multiplied by ozone concentration (C) summed through a specified hour (z(l/r s x C)) in radish. 84 The extent of plant leaf injury following air pollutant exposure is apparently attributable in part to the pattern of flux throughout an exposure period. The conventional steady state exposure with no variation in ozone concentration is characterized by a consistent increase in ozone flux that is similar to that observed in the early peak exposure. That the early peak injury level (in beans, 78.3%) was so much greater than that of the steady state (in bean, 15.4%) must be attributable to the initial ozone dose and, hence, flux. Plant response in the first hour.or less of exposure to pollutant stress is apparently critical in determining the extent of injury at the termination of an exposure. A noteworthy pattern emerges in the first three to four hours of exposure; 1) the early peak treatment exhibits the highest hour to hour flux and eventually the highest injury, 2) the middle peak exposure exhibits a "middle range" ozone flux and the injury is also "middle range", and 3) the late peak treatment exhibits the lowest hour to hour ozone flux and the lowest injury. Interpretation of plant response to the steady state exposure is difficult perhaps because it is rarely representative of ambient con-ditions. It is conceivable that this exposure may be unique in that it affords the plant valuable adjustment time to an unchanging air pollutant concentration. It is reasonable to assume that if air pollutant stresses are of the same magnitude (equivalent doses), adjustment to a consistent stress will be more efficient than that to a changing stress. Single stomatal resistance measurements made 24 hours after the termin-ation of ozone exposure in beans indicated that in none of the treatments was there a decline in stomatal resistance to a level comparable to that 85 observed in control plants (Table 14). Table 14. Single Stomatal Resistance Measurements in Beans 24 hrs After  the Termination of Ozone Exposure (n = 28). Treatment s 1 10.2 ±8.15 S.D. 2 22.9 ± 5.35 3 26.4 ±4.70 4 19.6 ±5.59 5 18.9 ± 4.89 Although stomatal pores provide the primary ports for gas exchange between leaf tissue and the atmosphere (Mansfield and Majernik, 1970), they may not be the critical factor in determining the extent of plant injury following exposure. Rather, magnitude and time of occurrence of peak concentration, pattern of dose accumulation, and air pollutant flux when considered concurrently, may clarify plant response in a manner that defies explanation solely by examination of stomatal resistances. Growth of Radish The two experiments involved the collection of total plant, shoot, and r-oot dry weight data seven days after the termination of the ozone exposures in radishes. Figures 18 and 19 indicate similar trends in dry 86 R A D I S H 16 j E X P E R I M E N T O N E T O T A L P L A N T S H O O T 1-4 I R O O T < CC CD 12 1 0 V-X o LU > -r r o 0-8 0 6 0 4 * * * * -r \ \ 0-2 ~i — r r* 1 — - — - r — 1— 1 r~ 10 2 0 3 0 4 0 5 0 6 0 7 0 8 0 P E R C E N T I N J U R Y Fig. 18. Patterns of dry weight change in radish with increasing injury in Experiment 1. Injury levels: Treatment 1 - 0%, 2 - 18.1%, 3 -40.5%, 4 - 21.9%, and 5 - 44.2%. 87 4-8 H 4-2 R A D I S H E X P E R I M E N T TWO T O T A L S H O O T ROO T P L A N T < c c o 3-6 3 0 2 4 H x (3 UJ >-CC Q 1-2 / / / / X \ \ \ 0 6 H I 20 10 PER l 30 CENT I 40 "T 5 0 - I 6 0 70 8 0 INJURY Fig. 19. Patterns of dry weight change in radish with increasing injury in Experiment 2. Injury levels: Treatment .1 - 0%, 2 - 18.1%, 3 -40.5%, 4 - 21.9%, and 5 -.44.2%. 88 weight in the two replicate experiments with increasing injury. There were variations in all three growth parameters with the different exposure regimes. With regard to total plant weight, control, steady state, and middle peak plants were not significantly different from one another while the late peak dry weight was significantly greater than the control. The early peak was significantly less than the three other ozone exposures, but not less than the control. Shoot dry weight increased slightly in all ozone treatments with the late peak,.middle peak, and early peak significantly greater than the control. It is noteworthy that in the early peak there was an increase in shoot dry weight whereas the overall plant dry weight was reduced. The root dry weight curve tended to mirror that of the total plant. Once again, control plants were not significantly different from the steady state nor the middle peak. The late peak was significantly greater than the control while the early peak was significantly less. The two experiments confirmed increases in total plant, shoot and root dry weight in response to variable ozone exposures with only the early peak response of the total plant and roots consistent with the growth reductions observed by Tingey (1974). A number of tentative hypo-theses can be suggested to account for experimental increases in dry weight; stomates may open wider following exposure permitting more C0£ to be incorporated into photosynthate, irreversible guard cell injury could lock stomates in an open or partially open position, or induction processes such as phototranslocation (Hartt, 1965) may be altered by the pollutant exposure. Regardless of the suitability of any hypothetical proposal, a 89 fundamental difficulty in data interpretation lies in the fact that only a single harvest was made in both experiments. Plants were 21 days old at the time of treatment,.and 28 days old at harvest. The seven day interval coincides roughly with the maturation period for the spring variety, Cherry Belle. It is conceivable that rapid growth in this period masked the actual plant response to the gaseous pollutant. Although the growth increases noted here are not an artefact, i t becomes apparent that additional work is necessary to elicit a complete explanation of them. Injury Prediction When stomatal conductance is multiplied by the peak concentration in an exposure, a measure-,of flux at the time of C m a x is obtained. Fluxes calculated in this way are-.shown in Figures 20a and 20b with a decline from 0.058 cm sec"^ ppm to 0.035 cm sec-^ ppm in the middle peak exposure when C is reduced from 0.8 ppm to 0.7 ppm. Figures 21a and max 21b illustrate ozone flux patterns when the late and early peak .C „ is max elevated to 0.8 ppm while the middle peak C „ is either 0.7 ppm or 0.8 max ppm. By .themselves, Figures 20 and 21 serve only to illustrate the impor-tance of C in determining an hourly flux, max Consideration of no single exposure component is sufficient in charac-terizing the factors that dictate plant injury. A functional prediction model should incorporate ambient dosage, peak concentration, and those factors regulating stomatal diffusive resistance. Models described in the literature have emphasized gaseous deposition rates at leaf surfaces 90 007' B E A N • S T E A D Y S T A T E o B E L L C U R V E * S T E P UP • S T E P DOWN o 006 - 005 x 3 004 0 03 ui z O £} 0 02 1 < 00I 1 UJ 10 —i 1— 20 30 P E R C E N T 4 0 5 0 I N J U R Y 6 0 7 0 " T — 8 0 Fig. 20a. Ozone flux at the time of peak concentration. Peak concentrations: 0.4 ppm (steady state), 0.8 ppm (bell curve), 0.7 ppm (step up), and 0.7 ppm (step down). 91 p 0 0 7 0 0 6 B E A N e S T E A D Y S T A T E B E L L C U R V E S T E P UP S T E P D O W N W 0 0 5 X r> 0 0 4 UJ z o M O 0 0 3 0 0 2 < UJ CL 0 0 1 — I ~ I 0 I 2 0 P E R I— 30 C E N T i r~ 4 0 5 0 I N J U R Y 6 0 ~~\— 7 0 \— 8 0 Fig. 20b. Ozone flux at the time of peak concentration. Peak concentrations: 0.4 ppm (steady state), 0.7 ppm (bell curve), 0.8 ppm (step up), and 0.8 ppm (step down). 92 0 0 7 1 o x 0 06 B E A N * S T E A D Y S T A T E ° B E L L C U R V E 4 S T E P UP • S T E P DOWN 0 0 5 ' x 0 0 4 u. 0 0 3 £ 0 0 2 ' < 0 : 0 I 0 2 0 ' E R 3 0 C E N T I— 4 0 — I — 5 0 T 6 0 7 0 T 8 O I N J U R Y Fig. 2la. Ozone flux at the time of peak concentration. Peak concentrations: 0.4 ppm (steady state), 0.7 ppm (bell curve), 0.8 ppm (step up), and 0.8 ppm (step down). 93 0 0 7 1 B E A N e S T E A D Y S T A T E ° B E L L C U R V E A S T E P UP • S T E P DOWN o * 0 0 6 00 5 x r ) _J u. UJ z o K l O 004 0 0 3 002 < U l a 001 —I -1 1 1— 10 2 0 3 0 40 P E R C E N T I N J U R Y 5 0 60 7 0 8 0 Fig. 21b. Ozone flux at the time of peak concentration. Peak concentrations: 0.4 ppm (steady state), 0.8 ppm (bell curve), 0.8 ppm (step up), and 0.8 ppm (step down). 94 (Thorne and Hanson, 1972; Slinn, 1977), threshold concentrations (O'Gara, 1922; Zahn, 1963), and prediction of a concentration required to elicit a previously determined level of leaf tissue injury (Larsen and Heck, 1976). Few authors have incorporated injury as an unknown, however. It is suggested that injury prediction encompasses the following terms: I = ( D o s e) x (cmax) m^ax where: I is injury, Dose is the "cumulative" dose or flux through a fumi-gation period. As previously defined, this is the stomatail. conductance multiplied by the ozone concentration which is then summed through the total fumigation period; ^(l/r x C). C m a x is the peak concentration attained during the exposure period and t is the hour in which that peak con-centration occurred. The "cumulative" dose is a necessary component of the model since the effective dosage is dependent not only upon the concen-tration at the leaf surface, but also upon the ability of the leaf to accept the pollutant gas. Regardless of the potential flux the atmosphere can deliver to the plant, i f the leaf sink cannot accept the pollutant gas, the process is rate-limited elsewhere. Because rate limitation probably occurs with the diffusion of gas through plant cell membranes and since access to cell membranes is via the stomates, .stomatal conductance is important in determining the flux delivered to cell membranes. Injury data described earlier indicate that injury is directly related to "cumulative" dose and C „ and inversely related to t . As the C v max max max shifts later into the exposure period, the injury lessens. Figures 22 and 23 describe predicted injury changes with corresponding I 0 2 0 P E R 1 3 0 C E N T I — 4 0 —r—• 5 0 ~!— 6 0 — r — 7 0 8 0 I N J U R Y Fig. 22. Injury prediction in bean. I = injury, Dose = "cumulative" fl or dose, C = peak ozone concentration, and T = time of max max occurrence of peak concentration. 96 P E R C E N T | K J U R Y Fig. 23. Injury prediction in radish. I = injury, Dose = "cumulative" flux or dose, C „ = peak ozone concentration, and T = time of max max occurrence of peak concentration. 97 changes in absolute injury. Two comments are appropriate: the predicted injury levels occur in the "correct" ascending order in both plant species and the magnitude of change among the treatments is roughly that of the absolute assessment technique. The injury prediction model should also incorporate additional exposure components as the following modification of the first model suggests: 1 = < D o s e ) x C C m a x ) x (flux) x (C r a n g e) x (T) x (x) t max where: flux is the air pollutant flow (1/r x C ) in the hour of peak s max concentration, C is the variation in the concentration through the range 3 exposure interval, J is the total exposure time, and C- is the mean ozone, concentration. Because C r a ng g, T, and C- are usually the same in all treatments of a controlled, variable application experiment, these factors, are incorporated into a constant, K. Hence, j = K (Dose) x (C m a x) x (flux) t "~ max In Figure 24, the three variable exposures peaked at 0.7 ppm with the dashed line indicating the influence of elevating the concentration to 0.8 ppm in the middle peak exposure. Alternatively, Figure 25 illustrates the predicted injury pattern when all three variable exposure regimes peaked at 0.8 ppm while the dashed line indicates the predicted reduction in injury when the middle peak ozone concentration was 0.7 ppm. Both the absolute assessment technique and injury prediction model were capable of discerning 98 Fig. 24. Injury prediction in bean. Components of the model are defined in the text. 99 > -c c 3 o UJ H O Q UJ CC a 0 016 i o 0 0 1 4 6 0 0 1 2 0- 010 0 0 0 8 ' 0 0 0 6 0 0 0 4 ' 0 0 0 2 1 0 B E A N a o S T E A D Y S T A T E B E L L C U R V E S T E P UP S T E P DOWN 0-8 ppm 0^ PEAK 0 7 ppm 0 3 PE AK P E R C E N T 4 0 5 0 I N J U R Y 6 0 ~ l — 70 8 0 Fig. 25. Injury prediction in bean. Components of the model are defined in the text. 100 differences in injury with two peak concentrations whereas the visual assessment had failed to detect a difference. The important role of peak concentration is illustrated in Figure 26 with radishes in which both absolute and predicted injury in the early peak and the middle peak are comparable. Perhaps the peak concentration, 0.50 ppm in the middle peak and 0.35 ppm in the early peak, contributes to similar injury levels in the two exposures. The algebraic expression derived in this study is adequate in the prediction of gaseous air pollutant injury to vegetation in that it incor-porates those exposure components deemed important in eliciting injury responses. 101 Fig. 26. Injury prediction in radish. Components of the model are defined in the text. 102 CONCLUSION Preliminary investigations with ozone and greenhouse whitefly in this study suggested an interaction between the two that resulted in an enhanced visual injury level in bean leaf tissue. A potential synergism may be due to increased entry of pollutant gas into the leaf through tissues damaged by the insect. Alternatively, the combined action of insects and ozone may stimulate ethylene production by plant tissue. Similar experiments utilizing reflectance spectrophotometry in injury assessment failed to confirm an interaction and, in fact, indicated a greater injury level in beans exposed to ozone alone as opposed to exposure to the combination of ozone and white-fly. Although i t is conceivable that subtle differences in the environment of the growth chamber may contribute to variations in injury observed with the two techniques, it is more likely that differences inherent in the assessment techniques themselves are at least partially responsible for the inability to substantiate an air pollutant-insect interaction. In the light of the foregoing, emphasis was shifted from an examination of the interaction of abiotic and biotic factors to the fundamental problem of accurately quantifying leaf tissue injury following air pollutant fumigations. An injury assessment technique was developed that involves determination of the proportion of actual injured leaf tissue area to total leaf area. The assessment procedure is not suggested as a replacement for visual rating systems, but rather as a useful periodic check upon the visual system. It was concluded that the visual system tends to over-estimate injury as determined by the silhouette tracing procedure, with an increased insensitivity in the visual system near the upper extreme of the 103 injury spectrum. The silhouette tracing procedure, in addition to serving in aligning visual data with the true injury levels in plant tissue, may also be useful in the training of laboratory personnel so as to increase the reliability of interrelating data from different laboratories. Utilizing the silhouette tracing procedure, significant differences in the extent of leaf tissue injury were observed in beans and radish when the plants were subjected to various constant dosage exposures of ozone in which single peak concentrations were of different magnitude and occurred at different times during the exposure. Injury trends were reproducible with the two crop species with the steady state injury the lowest, the late peak injury slightly higher than the steady state, the middle peak injury higher than the steady state and late peak, and the early peak injury higher than in all other exposures. Magnitude and time of occurrence of peak concentration are important determinants of the extent of injury in plant tissue following ozone exposure. Because plants are influenced by events prior to and following a peak con-centration, at least two additional exposure characteristics, cumulative dose and pollutant flux, must also be considered as contributors to the ultimate injury status of the plant. These exposure components provided sufficient information to develop a simple workable model that can be used in the prediction of injury. Injury is directly related to dose (defined as £(l/'r x C)), maximum pol-lutant concentration (C ), and pollutant flux; but inversely related max to the time of occurrence of the peak concentration. Because the dose term relies upon stomatal diffusive resistance data, the model provides for an approximation of actual effective ozone dosage. When the model is 103A implemented, means of the various treatment exposures are aligned in the "correct" ascending order of injury with roughly the appropriate magnitude of difference among treatments. The f i r s t attempts to describe a i r pol lutant injury in terms of a simple product of pol lutant concentration (above maximum level continuously to lerable without v i s i b l e injury) and time have been supplanted by more recent models that consider the potential higher t o x i c i t y of short exposures of high concentrations in contrast to lower levels of longer duration. Few models, however, have recognized the c r i t i c a l importance of determining the actual concentration impinging upon a l l reactive s i tes within the sub-stomatal cav i ty . On the contrary, most researchers have been content to report pol lutant concentrations at chamber i n l e t or out let ports or at a r b i t r a r y locations within the chamber. In a d d i t i o n , ambient f l u c t u a t i o n s , most notably in magnitude and time of occurrence of peak concentrations, have rare ly been simulated in growth chamber studies. This thesis i s not meant as a d e f i n i t i v e statement of plant response to ambient conditions employing growth chamber fumigation studies. Rather, i t i s intended as recognition of some of the exposure components important in d i c t a t i n g the extent of plant t issue i n j u r y . The model, although useful i n the characterizat ion of plant i n j u r y , does not elucidate the r e l a t i v e importance of the various exposure components in determining plant i n j u r y , nor does i t consider mult iple peak phenomena, In a d d i t i o n , i t i s well docu-mented that the chronic exposures are often as prevalent in ambient s i tuat ions as the acute concentrations to which plants were exposed in t h i s study. 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I N S E C T S _ I 1 0 0 19 0 0 1 1 0 0 1 1 0 0 Proposed treatments designed to elicit a mechanical and/or chemical effect in plants (for example, ethylene production) by insects in the presence of ozone. 


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