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Effects of ozone on host-parasite relations Resh, Howard Martin 1975

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EFFECTS OF OZONE ON HOST-PARASITE RELATIONS by HOWARD MARTIN RESH B.S.A., The University of British Columbia A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in the Department of PLANT SCIENCE We accept this thesis as conforming, to the required standard THE UNIVERSITY OF BRITISH COLUMBIA Apri l , 1975 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make i t 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 British Columbia Vancouver, B.C. V6T 1W5 Canada Date May 23, 1975 i i ABSTRACT Investigations of the effects of the air pollutant ozone on the growth of bush bean {Phaseolus vulgaris L. cv Pure Gold Wax) were undertaken to determine their nature and the modifications in growth responses caused by interactions of the pollutant with con-current development of two plant pathogens, rust (Uromyaes phaseoli [Rebent.3 Wint.) and halo blight (Pseudomonas phaseolioola [Burkh.] Dowson). Two dosages of ozone (0.04 to 0.05 ppm = "low" and 0.09 to 0.10 ppm = "high"), administered daily during the photoperiod for 6 or 12 days, were used in most experiments. Neither dosage is sufficient to cause symptoms of acute injury. When compared with growth in f i l tered, ozone-free air, treatment, "low" ozone frequently resulted in enhanced growth of the host plant. "High" ozone treatment invariably resulted in impaired growth and the appearance of chronic injury symptoms after 10-12 days of treatment. A reflectance spectro-photometry method was developed to permit quantitative assessment of such injury. Growth responses to ozone in the presence of rust infection varied with infection level. In "high" ozone, the reduced dry weight gains of uninfected plants were reversed by heavy infection, leading to significant increases in total dry weight of infected leaves. Lower levels of infection, however, had l i t t le effect in modifying the nature of the growth responses of bean to ozone. The effects of i i i heavy infection in counteracting ozone-induced growth suppression appear to be due to the ability of the fungus to maintain vigorous growth in the presence of ozone, rather than to an effect oh host susceptibility to ozone injury. Halo blight infection offered no protection against ozone stress, but on the contrary, increased the overall retardation of plant growth. The presence of rust infection offered l i t t le protection against ozone-induced premature senescence except in the "green islands" immediately surrounding the rust pustules. Higher levels of cytokinins have been associated with green islands, and both kinetin and 6-benzyladenine were found to offer some protection against ozone-induced, accelerated senescence, but not against injury to the leaf palisade cel ls , as revealed by reflectance measurements. Exposures of the two host-parasite systems to ozone levels used in this study revealed interactions rather than additive effects. "High" ozone in the presence or absence of the pathogen impaired plant growth with l i t t le protection offered by the pathogen to the host against the pollutant. Fungal development (before sporulation) was retarded by ozone but greater pustule frequency and the formation of secondary pustules maintained inoculum potential under ozone stress. iv TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS iv LIST OF TABLES v i i i LIST OF FIGURES x ACKNOWLEDGEMENTS x i i i LIST OF ABBREVIATIONS xiv INTRODUCTION 1 LITERATURE REVIEW 5 1. Effects of Ozone on Higher Plants (Hosts) 5 A. Natural Occurrence of Ozone and Plant Sensitivity 5 B. Symptomatology 9 C. Effect on Growth of Host 10 D. Mechanisms 13 E. Environmental Factors Influencing Response . . . . 18 2. Effects of Ozone on Pathogens 26 A. Effects on Growth 26 B. Environmental Factors Influencing Response . . . . 30 3. Effects of Ozone on Plant Pathogen Interactions . . . . 30 A. Effects on Growth 30 B. Mechanisms 34 C. Genetic and Ecological Effects (Adaptation) . . . . 36 MATERIALS AND METHODS 41 V Page 1. Plant Materials 41 2. Exposure Chambers 41 3. Quantitative Measurement of Chronic Injury 45 A. Introduction 45 B. Visual Assessment 46 C. Chlorophyll Extraction 46 D. Reflectance Measurements 49 E. Comparison of Chlorophyll Extraction and Reflectance Procedures 50 4. Experimental Designs 57 A. Introduction 57 B. Bean - Bean Rust Experiments 57 (a) Experiments of Timing on Inoculation and Fumigation 57 (b) Experiments on Levels of Ozone 60 C. Bean - Halo Blight Experiments 61 D. Bean - Siderochrome Experiments 62 E. Bean - Cytokinin Experiments 63 5. Growth Analysis of Host Plants 64 A. Introduction 64 B. Bean - Bean Rust Experiments (A,C,D,E,F,G,J,K) . . 65 C. Bean - Halo Blight Experiments (L and 0) 67 D. Cytokinin Experiment (N) 68 6. Inoculation 68 A. Bean Rust [Uromyoes phaseoli) 68 B. Bean Halo Blight (Pseudomonas phaseolioola) . . . . 69 vi Page 7. Measurement of Rust Infection 69 RESULTS AND DISCUSSION 71 1. Preamble 71 2. Ozone Dosage and Uptake . 72 3. Main Effects on Bean Growth 75 A. Effects of Ozone 75 (a) Ozone dosage 75 (b) Timing of fumigation 77 B. Effects of Rust Infection 79 (a) Intensity of infection 79 (b) Timing of inoculation 81 C. Effects of Halo Blight Infection 83 4. Interactions Between Ozone and Rust on Bean Growth 85 A. Richards' Diagrams (Factor Diagrams) 85 B. Effects of Level of Infection and Level of Ozone on Bean Growth 87 C. Effects of Timing of Fumigation on Bean Growth Response to Ozone 107 D. Effects of Ozone on Pathogenesis 128 5. Interactions Between Ozone and.Halo Blight 135 A. Effects of Infection and Level of Ozone on Bean Growth 135 B. Effects of Infection and Timing of Ozone on Bean Growth 144 C. Effects of Ozone on Pathogenesis T44 vii Page 6. Effects of Cytokinins and Siderochromes 145 A. Effects of Ozone on Cytokinin Responses and Cytokinin on Ozone Responses 145 B. Effects of Rhodotorulic Acid on Ozone Response . . . 160 7. General Discussion 161 A. Effects of Ozone on Bean Growth 161 (a) Low ozone levels 161 (b) High ozone levels 164 B. Effects of Pathogenesis on Bean Growth 165 C. Effects of Pathogenesis on Ozone Damage 167 D. Effects of Ozone on Pathogenesis 169 E. Relationship of Pathogenesis to Kinetin Treatment and Ozone Response 169 SUMMARY 176 LITERATURE CITED 182 APPENDIX 198 vii i LIST OF TABLES Table Page I. Absorption of ozone by chambers, plants and soil expressed in ozone levels observed (ppm) at the exit ports of the chambers 74 II. Effects of ozone dosage on bean growth parameters -a summary table of differences from controls 76 III. Effects of ozone fumigation timing on bean growth parameters - a summary table of differences from controls 79 IV. Effects of intensity of rust infection on bean growth parameters - a summary table of differences from controls 80 V. Effects of timing of rust infection on bean growth parameters - a summary table of differences from controls 82 VI. Effects of halo blight infection on bean growth parameters - a summary table of differences from controls 84 VII. Interaction effects of ozone at a given timing of inoculation - a summary table of differences from unfumigated controls 112 VIII. Interaction effects of timing of inoculation at a given timing of ozone - a summary table of differences from unfumigated controls 122 IX. Number and size of primary pustules produced in presence (+) and absence (-) of ozone 129 ix Table Page X. Effects of ozone on cytokinin responses - a summary table of differences from unfumigated controls . . . . 146 XI. Effects of cytokinin treatment on bean stem and root growth response to low levels of ozone 154 XII. Effects of ozone and cytokinin treatment on numbers and weights of root nodules 159 XIII. Dry weights of parts of 22-day-old bean plants grown in filtered air or filtered air containing ozone (0.03 ± 0.01 ppm) for 12 days 162 X LIST OF FIGURES Figure Page 1. Atmospheric nitrogen dioxide photolytic cycle 6 2. Interaction of hydrocarbons with atmospheric nitrogen dioxide photolytic cycle 7 3. Ozone fumigation chambers 42 4. Chronic ozone injury to bean primary leaves 47-5. Reflectance spectra of upper surfaces of bean leaves after 12 days in filtered air or 0.05 ppm ozone . . . . 51 6. Comparison of reflectance and chlorophyll content of bean leaves subjected to treatments 1, 5, 7, 8, 9, 10 53 7. Comparison of reflectance and chlorophyll content of bean leaves subjected to treatments 1, 2, 3, 4, 5, 6'. 55 8. Summary of treatments in experimental runs 59 9. Effect of level of ozone in the presence or absence of medium rust infection on primary leaf dry weight 88 10. Effect of level of ozone and level of rust infection on primary leaf injury (percent reflectance) 90 11. Effect of level of ozone and light rust infection on primary leaf injury (percent reflectance) 93 12. Effect of level of ozone and level of rust infection on primary leaf area 95 xi Figure Page 13. Effect of level of ozone and level of rust infection on primary leaf area and dry weight 97 14. Effect of level of ozone and level of rust infection on primary leaf dry weight 99 15. Photographs of chronic ozone injury adjacent to pustules 102 16. Effect of level of ozone and level of rust in-fection on trifoliate dry weight 104 17. Effect of level of ozone and level of rust in-fection on stem dry weight 108 18. Effect of level of ozone and level of rust in-fection on root dry weight 110 19. Effect of timing of inoculation in the presence or absence of continuing ozone on primary leaf injury (percent reflectance) 113 20. Effect of ozone timing in the presence or absence of early or late light inoculation on primary leaf injury (percent reflectance) 116 21. Effect of ozone timing in the presence or absence of early or late light inoculation on primary leaf area 118 22. Effect of ozone timing in the presence or absence of early or late light inoculation on primary leaf dry weight 120 23. Photographs of premature senescence in the presence of ozone and rust infection 123 xii Figure Page 24. Effect of timing of ozone in the presence or absence of late or early light rust infection on primary leaf senescence 126 25. Photographs of reduced pustule size and development of secondary pustules under ozone treatment 130 26. Photographs of development of symptoms of chronic ozone injury with accompanying retardation of pustule development 132 27. Effect of level of ozone in the presence or absence of early bacterial infection on primary leaf area 136 28. Effect of level of ozone in the presence or absence of early bacterial infection on primary and t r i -foliate leaf dry weights 138 29. Effect of level of ozone in the presence or absence of early bacterial infection on root dry weight . . . 140 30. Effect of ozone, benzyladenine (BA) and kinetin (K) on leaf reflectance (550 nm) after 26 days of ozone treatment 147 31. Effect of ozone and cytokinins on primary leaf area after 42 days of ozone treatment 149 32. Effect of ozone and cytokinins on primary leaf dry weight after 42 days of ozone treatment 151 33. Effect of ozone and cytokinins on primary leaf senescence 156 xi i i ACKNOWLEDGEMENTS I wish to thank Dr. V.C. Runeckles, Chairman, Department of Plant Science, The University of British Columbia, under whose super-vision this thesis was carried out, for his guidance, criticism and assistance in the preparation of this manuscript. I would also like to thank the members of my graduate committee for their interest in my research and the reviewing of this thesis. I am grateful for financial support from the Leonard S. Klinck Fellowship and for equipment and supplies provided by the National Research Council through a grant to Dr. V.C. Runeckles. I wish also to thank Mrs. M. El l is for typing the thesis. Finally, I wish to thank my loving wife Elvira for her moral support and assistance in collecting data. I dedicate this manuscript to Elvira. LIST OF ABBREVIATIONS x Adenosine triphosphate 6 - benzyl adenine Carbon dioxide Fluoride Ki neti n Leaf area ratio Nitrogen Nicotinamide adenine dinucleotide Reduced nicotinamide adenine dinucleotide Nicotinamide adenine dinucleotide phosphate Reduced nicotinamide adenine dinucleotide phosphate Net assimilation rate Nitric oxide Nitrogen dioxide Ozone Phosphorus Peroxyacetyl nitrate Parts per million Polyvinyl chloride Rhodotorulic acid Relative growth rate Sulfhydryl Sulphur dioxide Tobacco mosaic virus 1 INTRODUCTION In recent years researchers have become aware of the increasing presence of air pollutants in our environment. These air pollutants include gases such as ozone (0,), nitrogen oxides (NO ), peroxyacetyl nitrate (PAN), and other oxidants commonly found in photochemical smog, sulphur dioxide ( S O 2 ) , fluoride (F), ethylene, methane, and other hydrocarbons. They may also include heavy metals, such as lead, and particulate matter. Chambers (1968) defined air pollutants as sub-stances added to the atmosphere in sufficient quantity to produce a measureable effect on man or other animals, vegetation, or materials. He classified air pollutants into two general groups; (i) those emitted directly from identifiable sources, for example, by combustion of fossil fuels and emission from industrial processes, and (i i) those produced in the air by interaction among two or more primary pollutants, or by reaction with normal atmospheric constituents, with or without photoactivation. The latter group is exemplified by ozone which is formed by photochemical reactions of the products of combustion of petroleum fuels and forms an important constituent of the system known as photochemical oxidant or photochemical smog. Literature published on the effects of air pollutants on vegetation can be divided into two general categories: (i) that dealing with real-world situations and the relationship of naturally occurring field injury to measure pollutant levels (work of this nature usually emphasizes characteristic symptoms of the pollutant 2 injury on various plant species), and (ii) that dealing with simula-tions of field injury, usually carried out in greenhouses, fumigation chambers or growth chambers under varying degrees of environmental control. A prime objective of such studies has been to learn more about the nature of the injury, and the conditions favouring its occurrence. Such studies have frequently been extended to the cellular and biochemical level in search of explanations of mechanism. In situations in which living organisms are subjected to treat-ment with toxic agents, two types of reaction can occur. Reactions are termed "acute" when they result from one or two comparatively massive doses of the toxin, and "chronic" when they result from repetitive or continuous exposures to low doses, which individually are insufficient to cause visible injury. Air pollution effects on plants can thus be classified as acute or chronic. The bulk of the published work has related to specific single plant species and single pollutants, at dosages sufficient to cause symptoms of acute injury, with emphasis being placed on symptomato-logy. In particular, very few studies have been made on ecological effects, and the effects of air pollutants on interacting biological systems. Much of the published work carried out in the past in fumi-gation chambers or growth chambers is of limited relevance because of the use of unrealistically high levels of pollutants. These results frequently bear l i t t le relation to real-world situations in that they examine the effects of massive acute doses of pollutant, 3 often far in excess of those reported to occur even in highly polluted air. Particularly rare are studies of the effects of doses in the. sub-acute or chronic injury range. For these reasons the present studies have focused upon the effects of low levels of the air pollutant ozone on interacting biological systems involving host-parasite relations. It is partly in the knowledge of such effects that meaningful criteria can be established with respect to standards of air quality. Ozone was chosen since i t is the major pollutant formed by the action of ultraviolet light energy on the waste products of petroleum fuel combustion and i t also occurs naturally as one of the components of the upper atmosphere. Effects of air pollutants on host-parasite relations must be considered by examining the effects on both the host and the parasite. The severity of parasite-induced diseases may be altered indirectly through host predisposition or directly through action on the parasite. Any change in the host or parasite will upset the host-parasite balance and may, as a result, favour the host (greater sensitivity of the pathogen or increased resistance of the host) or favour the parasite (greater susceptibility of the host to infection). Thus, all aspects of both the host and parasite affected by air pollutants must be carefully examined, evaluated, and compared. In this way, the interaction of air pollutants on the host-parasite relationship can be defined in terms of the physiological and bio-chemical processes altered by the pollutants. 4 Protective effects of infection against ozone injury have been reported. Yarwood and Middleton (1954) reported that rust fungi infection prevented smog injury to leaves of bush bean and sun-flower. Kerr and Reinert (1968) observed an absence of ozone injury in "halo" areas adjacent to sites of bacterial infection of bean leaves while ozone flecking appeared over most of the surface of the leaflets exposed to ozone. Heagle and Key (1973) found that significantly less ozone injury occurred in mesophyll cells in the substomatal areas of Vuooinia graminis Pers. f. sp. tritioi Erikss. & Henn. inoculated wheat leaves than in noninoculated leaves. Such interaction effects of pathogenesis and host response to ozone have been observed in relatively few cases and warrant further examination. The overall objectives of the present study have therefore been to determine the effects of low (subacute) doses of ozone on bush bean (Phaseolus vulgaris L., cv Pure Gold Wax) in the presence and absence of two disease-producing organisms; the rust fungus, Uromyees phaseoli (Rebent.) Wint and the bacterium causing halo blight, Pseudomonas phaseolicola_ (Burkh.) Dowson and to determine the effects of ozone on pathogenesis, and the effects of pathogen growth on the host response to ozone. 5 LITERATURE REVIEW 1. Effects of Ozone on Higher Plants (Hosts) A. Natural Occurrence of Ozone and Plant Sensitivity The photochemical formation of ozone in the atmosphere is summarized in Figures 1 and 2. In the absence of hydrocarbon vapour (Figure 1) the cyclic process generates modest amounts of ozone, much of which is consumed in reacting with nitric oxide (NO). In the presence of hydrocarbon vapour (Figure 2), on the other hand, greater quantities of ozone are generated together with compounds such as peroxyacetylnitrate (PAN). The background atmospheric concentration of ozone in surface air at sea level has been estimated to be about 0.01 - 0.03 ppm (Tebbens, 1968). The maximum-content of ozone detected as a contam-inant of outdoor urban air in Los Angeles, is 0.99 ppm, measured dur-ing a smog episode in 1956 (Tebbens, 1968). Daily peaks commonly reach 0.15 to 0.38 ppm in the summer and f a l l , and 0.05 to 0.10 ppm in the winter in the Los Angeles region. Ozone has reached 0.50 ppm in San Francisco, 0.22 ppm in Salt Lake City, 0.16 ppm in Washington, D.C. and over 0.15 ppm in nearly every large city in which measure-ments have been made (Treshow, 1970). In Vancouver, at the University of British Columbia, background levels have been recorded during the summer and fall months at 0.02 to 0.03 ppm with maxima reaching 0.07 ppm. NITROGEN SUNLIGHT OXYGEN ATOM (0) + OXYGEN (o2) Atmospheric nitrogen dioxide photolytic cycle (after U.S. Department of Health, Education, and Welfare Publication No. AP-63). NITROGEN DIOXIDE + SUNLIGHT ENERGY Interaction of hydrocarbons with atmospheric nitrogen dioxide photolytic cycle (after U.S. Department of Health, Education, and Welfare Publication No. AP-63). 8 Diurnal fluctuations of ozone concentrations produce a typical pattern; low concentration at night, increasing to a maximum around noon, and practically disappearing at sunset following a gradual de-crease during the afternoon. Ozone concentrations of less than 0.10 ppm are damaging to many plant species. Plant sensitivity to ozone varies among species and even varieties of the same species. As ozone concentration increases all plant species show susceptibility to i t and reveal distinct symptoms of injury. Several workers have classified various herbac-eous (Hill et al_., 1961) and woody (Treshow, 1970) plants into three groups of relative sensitivity: sensitive, intermediate and resistant. Sensitive species were injured when exposed to ozone below 0.30 ppm for four hours; intermediate species were injured at 0.40 ppm for four hours; and resistant species were damaged at 0.53 - 0.56 ppm for four hours. These sensitivity categories are based on high levels of ozone exposure for short intervals of time and therefore give rise to symptoms of acute injury. Since these high acute levels tend to occur sporadically in nature, the defined categories may not apply to lower acute and chronic exposure levels of longer duration. Nonetheless, even low ozone concentrations between 0.05 and 0.12 ppm for 2 - 4 hours can injure the most sensitive species. For example, certain varieties of tobacco, such as Bel-W3 have been injured by ozone concentrations as low as 0.05 ppm for 4 hours (Hill et a l . , 1970). Eastern white pine has been injured by 0.07 ppm for 4 hours (Costonis and Sinclair, 1969) and sensitive varieties of alfalfa, spinach, clover, oats, radish, sweet corn, and bean have been 9 injured by 0.10 to 0.12 ppm for 2 hours (Hill et a l . , 1970). Responses of plants to ozone exposures causing acute injury (high levels of short duration), however, may be very different from those causing chronic injury (low levels of long duration). Hence care must be taken in correlating mechanisms involved in acute injury with those expected from chronic injury. Even within the range of ozone concentrations causing acute injury , Heck et_al. (1966) showed that the relationships of concentration, time and injury were complex and non-linear. B. Symptomatology Ozone may cause lesions of four different general types. Usually one type of injury is present, but several types may occur in the same or different areas. Pigmented lesions are the most common symptoms on many deciduous trees and shrubs, and some herbaceous species. This localized pigmentation of the cells results in small dot-like lesions (Ledbetter_et_ a j . , 1959). Palisade cells are most prone to ozone injury and on leaves with palisade parenchyma, primary lesions are often limited to small groups of palisade cells. The injury is observable primarily on the upper leaf surface. Bleaching in the form of small, unpigmented necrotic spots or more general upper-surface bleaching is a common type of injury on many herbaceous and woody species (Ledbetter et_ a l . , 1959). This type of symptom is usually the result of more severe injury under acute levels of ozone exposure. In this case the palisade cells 10 and upper epidermal cells collapse and become bleached. Bifacial necrosis occurs when all of the tissue through the thickness of leaf is kil led, drawing the upper and lower surfaces together to form a thin, papery lesion. A water-soaked appearance often develops prior to the drying and bleaching. This type of symptom is associated with exposure to high levels of ozone, especially when relatively sensitive plants are exposed to such ozone levels. Chlorosis is a later developing symptom and occurs particularly when primary lesions are limited to small groups of palisade cells and the injury is observable primarily on the upper leaf surface. Such chlorosis occurs in the tissues surrounding the primary lesions and may extend throughout the leaf. C. Effect on Growth of Host Reports of plant growth responses to ozone include general growth suppression, reduced shoot and flower weight, reduced flower number, slower rate of flowering (Adedipe et a j . , 1972), reduced leaf area (Evans, 1973; Feder, 1970), reduced fresh weight of tops and roots and reduced nodulation (Tingey and Blum, 1973; Manning et a l . , 1971), reduced plant yield (Tingey et aJL , 1971; Heagle et a l . , 1972), reduced pollen germination (Mumford et al,., 1972), and varietal sensitivity to ozone (Ormrod et j i l . , 1971; Cameron and Taylor, 1973; Feder, 1970; Adedipe et ^1 . , 1972). Cultivars of geranium and carnation exhibit a reduction of side branching, a retardation of floral initiation, and a decrease in 11 floral productivity when exposed daily for 5 - 7 hours to 0.10 ppm ozone for 1 - 3 months (Feder, 1970). These plants also exhibited a reduction in leaf size, an increase in internode length, a progressive destruction of leaf tissue and eventual defoliation in the case of geranium. Chronic ozone exposures (0.10 - 0.12 ppm) increased fresh weight but not size of flowers of petunia and geranium (Craker and Feder, 1972). Bract size decreased in poinsettia under the same ozone treatments. When soybean varieties were exposed to ozone concentrations exceeding 0.09 ppm for 6 hours per day, 5 days per week, for a 3-week period, the fresh weight of both tops and roots was reduced and a reduction in nodulation was observed (Reinert et al_., 1971). Manning et^  aj_. (1971) reported that Pinto beans in carbon-filtered air-developed nodules, whereas those exposed to 0.10 to 0.15 ppm ozone for 28 days did not. They suggested an indirect effect of ozone on nodulation by a modification of plant metabolism and/or metabolite translocation. The growth and yield of radish plants exposed to low concen-trations of ozone (0.05 ppm for 40 hours per week for 5 weeks) were significantly reduced (Tingey et_ al_. 1971). They also found a reduction in leaf and root fresh weight and attributed the reduction in root fresh weight to an indirect effect of ozone through the impairment of leaf metabolism and a resultant reduction of photo-synthate available for translocation. Heagle et aj_. (1972) reported that yields of sweet corn were significantly lower for plants exposed to chronic ozone levels. The average percentage ear f i l l , kernels 12 per plant, and kernel dry weight were also reduced by ozone treatments, but the differences were not statistically significant. Germination of corn, petunia, and tobacco pollen was reduced following ozonation (Feder, 1970). Heagle et al_. (1972) suggested that in some cultivars of corn, reproductive processes necessary for successful seed set are more sensitive to low-level ozone exposures than are vegetative growth processes. Cultivar differences in sensitivity to ozone have been reported for a wide variety of crop plants, including cereals (Sechler and Davis, 1964), onion (Engle et_ a]_., 1965), begonia (Leone and Brennan, 1969) and petunia (Feder et al_., 1969). While many workers have focused their attention on acute injury effects with emphasis on symptomatology, the significant agri-cultural losses due to chronic ambient pollutant levels are due mainly to growth suppression, reduction in photosynthetic activity, premature senescence, and the resultant losses in productivity. Feder (1970) claims that chronic exposures to low dosages of pollutants exert a more general effect upon plant growth and physiology, while short-term exposures exert more specific effects upon only a few leaves of a single age or maturity level. Plants as a whole can survive acute doses once the pollutant is removed or i f i t does not reach toxic levels again, leaving only a few injured leaves as a legacy of exposure. The chronically exposed plant does not recover as long as it is being exposed to levels as low as 0.10 ppm and i f the pollutant is removed from the plant's environment, recovery will depend 13 upon the growth pattern of the plant, and the time of exposure during the growth cycle. Since the real-world situation is frequently one of chronic exposure to pollutants over the entire growing season, this fact should be taken into consideration when attempting to define the impact of air pollution upon agricultural productivity. On the other hand, acute doses of pollutant which cause rapid injury can cause extremely high losses of crops in terms of market value, not because of any appreciable effect on yield, but because subsequent injury renders the crop unsaleable because of appearance, or impairs storage l i fe because of tissue breakdown. D. Mechanisms Much work has been conducted to determine the physiological and biochemical effects of ozone damage. Ozone damage was demonstrated to be characterized by the breakdown of the plasmalemma and other cellular membranes (Thomson et al_., 1966). Such changes in permeabil-ity have been attributed to the action of ozone on SH-groups and the breaking of -SH dependent hydrogen bonds within the plasmalemma (Wedding and Erickson, 1955). Increased respiration rate and decreased photosynthetic rate have been reported after ozone exposure of plants (Macdowall, 1965a; Dugger and Palmer, 1969). Hill and Litt lefield (1969) reported that stomatal closure, reduced apparent photosynthesis, and reduced transpiration occurred simultaneously during ozone fumigation, indi-cating that suppression of transpiration and apparent photosynthesis probably resulted from stomatal closure. 14 Hanson and Stewart (1970) found that photochemical oxidants somehow block or retard certain steps of the soluble carbohydrate translocation process. Cotton exposed to ozone at 0.7 to 0.8 ppm for 1 hour showed maximum susceptibility when the leaves contained a minimum amount of soluble carbohydrates and amino acids (Ting and Mukerji, 1971). Previous work showed that ozone damage was associated with low sugar content (Dugger et a]_., 1962). It was also demonstrated that an exogeneous supply of soluble sugar prevented ozone damage. On this basis Ting and Mukerji (1971) concluded that low concentrations of soluble components (sugars and amino acids) were directly related to the susceptibility of cotton leaves to ozone. Evidence has been presented which suggests the possibility of a correlation between high concentrations of ATP and SH- groups within a leaf and its resistance to ozone damage (Siegel, 1962; Tomlinson and Rich, 1968).- Todd (1958) has presented evidence that ozone can inactivate enzymes because of its ability to oxidize the SH-group. Mudd (1965) stated that ozone destroys NADH and NADPH. In studies by Tomlinson and Rich (1967) on bean, tobacco, beet, corn, barley, and rye plants fumigated with 1 ppm of ozone for 1 hour they found in all plants that there was a net increase in various amino acids and concluded that protein synthesis was probably decreased. Macdowall (1965b) working on tobacco found that a leaf did not "fleck" until a week or so after its grand period of growth was over. The leaf's susceptibility to ozone was thus associated with a metabolic change toward senescence, which was indicated by a decline in protein that immediately followed the cessation of leaf expansion. 15 Tomlinson and Rich (1969, 1970) presented evidence that fatty acid content may be a factor in determining ozone-resistance, and that fatty acid content decreases in leaves exposed to ozone. They related this to decreased SH- content in ozone-exposed leaves, and to the fact that SH-groups are essential for fatty acid synthesis. Howell (1970) reported that the diphenol, caffeic acid, was present in injured leaves at twice the concentration detected in healthy plant leaves. Air pollutants thus affect phenolic metabolism in plants as do plant pathogens and other physiological stress factors. Studies on the influence of ozone on the growth and ribosomal RNA content of Pinto bean demonstrated that ozone at high concentrations (0.35 ppm ozone for 20 - 40 minutes) specifically decreased the popu-lation of chloroplast ribosomes, but not that of cytoplasmic ribosomes (Chang, 1971; 1972). A reduction in ribosomal RNA was therefore attributed to a loss of chloroplast ribosomes. Ozone decreased the polysome population of chloroplast ribosomes per unit fresh weight of leaves. Chang (1971) suggested that the polysome level of chloro-plast ribosomes (which decreased well before the stage of natural senescence) could account for the initiation of ozone-induced senesence, since this reduction would depress the level of protein synthesis below that of the control. Ozone, therefore, may initiate premature senesence by decreasing the polysome level of chloroplast ribosomes after the plants are exposed to this oxidant. Pinto beans exposed to 0.05 ppm ozone exhibited premature senescence after 3 days of fumigation (Engle and Gabelman, 1967). Additional observations of premature senescence caused by oxidant 16 injury have been reported (Menser et al_., 1966; Manning et a l . , 1971). It has been suggested that ozone-damaged cells could produce ethylene in situ and thus cause early maturity of the surrounding cells (Rich, 1964). More recently ozone injuries to plants in the form of early senescence, premature leaf drop (Taylor, 1968), in-creased fruit respiration (Todd, 1956), and increased swelling of mitochondria (Lee, 1968), have been suggested to be responses to ethylene (Craker, 1971a). Studies with tomato, tobacco, and bean plants treated with 0.25 ppm ozone for 2 hours revealed that they had increased rates of ethylene production. Ethylene production from oxidant-treated plants appeared to be related to the extent of injury. Plants which received higher concentrations or longer periods of ozone fumigation gave off more ethylene. Similarly, in a comparison of tomato varieties, the more susceptible variety produced more ethylene. These results would be expected i f ethylene formation is induced by ozone injury to the ce l l . More severe plant injury is a result of more cells being damaged by the pollutant and thus would induce more cells to evolve ethylene. These results indicate that some responses of plant tissue to ozone fumigation may be due to an enhanced production of ethylene as a secondary consequence of injury (Craker, 1971a). The literature on the control of air pollution damage to plants by chemical means identifies many compounds, including fungicides (Middleton, 1956; Ordin et al_., 1962; Pell issier, 1971) vitamins (Menser, 1967; Walker, 1961; 1967), the stomatal regulators, phenyl-17 mercuric acetate and the monomethyl ester of decenylsuccinic acid (Rich, 1964), antitranspirants (Gale and Hogan, 1966) and others (Heck, 1968; Heggestad and Heck, 1971; Kendrick et al_., 1962). None of these chemicals has gained extensive use in agriculture, largely because of cost and the need for frequent and thorough applications. Fletcher and associates (1972) treated bean plants with abscisic acid 4 hours prior to exposure to Qg at 0.20 to 0.40 ppm for 3 hours. They found that abscisic acid protected bean leaves from 0g injury and related this protection to the effect of abscisic acid in causing stomatal closure. In radish plants, Adedipe and Ormrod (1972) found that ozone at 0.25 ppm for 4 hours had no effect on leaf weights of plants pre-treated with 30 mg/litre of benzyladenine (BA), gibberellic acid (GA) or indoleacetic acid (IAA), but decreased the leaf weight of control plants and of plants treated with (2-chloroethyl) phosphonic acid (ethephon). In terms of radish root weight, however, ozone had no effect only in plants treated with BA. BA was found to be the most active of the growth regulators tested, in the protection of plants from oxidant damage, both in terms of ozone-induced growth suppression and decrease in chlorophyll content of the leaf. Lee (1966) had earlier obtained, in tobacco, increased ozone susceptibility with kinetin (K) pre-treatment. The apparent conflict could be due to the use of detached leaves or to differential species response, as suggested by Adedipe et ah (1973). 18 Tomlinson and Rich (1973) reported that benzimidazole, ben-zyl adenine and kinetin retarded senescence of bean leaves, inhibited ozone injury and inhibited loss of, free sterol in whole tissue and chloroplasts of leaves exposed to an ozone dosage causing acute injury (0.50 ppm for 1 hour). They suggested that ozone increased the conversion of free sterol to sterol glycosides. Plants treated with the anti-senescence compounds did not lose free sterol from their membranes nor did they show symptoms of cell leakage during ozonation. They presented this action on free sterol as evidence for the correla-tion between free sterol content and the development of ozone injury, which indicated that these anti-senecescence compounds inhibited ozone injury by inhibiting sudden changes in the structure and function of membranes. The systemic fungicide benomyl has recently been shown to be effective in suppressing chronic 0^  injury on beans (Manning and Vardaro, 1973a; Manning et aj_., 1973b) and on poinsettia (Manning et^  aj_;, 1973a). No suggestions of mechanisms were made. It was not determined whether protection was due to a change in the physiology of the plant induced by the treatment or by direct inactivation of 0g by the chemical. E. Environmental Factors Influencing Response Response of plants to ozone is dependent on various environmental factors (Heck, 1968; Ting and Dugger, 1968). Factors such as nutrition, light, relative humidity and temperature, as well as the stage of 19 plant growth may determine the response to a given ozone dosage, Some environmental factors may indirectly influence plant response to ozone by altering plant sensitivity. For example, moisture stress can close stomata thereby reducing plant sensitivity to ozone. Conversely, ozone injury to Bel-W3 tobacco and Pinto bean plants was reported to increase with humidity (Heggestad and Heck, 1971; Macdowal1, 1965b). A good correlation was found between plant injury and increased stomatal aperture as humidity increased (Otto and Daines, 1969). There is evidence that Og itself may induce stomatal closure, reducing the amount of Og entering the leaf and contributing to the resistance to Og injury of certain varieties. In tobacco, stomatal closure in Og tolerant cultivars has been reported (Dugger and Ting, 1970; Rich and Turner, 1968) and has been implicated in the apparent tolerance of young and old leaves (Menser et al_., 1963). Although some movement of the air pollutant may occur through the cuticle, the principal movement occurs through the stomata, with the result that the guard cells regulate, to a large extent, what goes in and out of the leaf. They are the f i rst cells to experience an effect by the air pollutant diffusing in from the atmosphere and react in several ways. They may lose turgor, temporarily closing the stomata, thus offering the other cells inside the leaf some degree of protection or they may close permanently i f they are damaged and cannot regain turgor, causing a reduction in plant growth since COg intake for photosynthesis is reduced. Stomatal closure is the only known mechanism of resistance to Og injury among plants of genetic variability in Og tolerance. It is speculated that the sensitivity of the membranes of the guard cells to Og is controlled by a dominant gene system in tolerant onions (Engle and Gabelman, 1966). In the presence of Og, the membranes of the guard cells in resistant plants lose their permeability and leak, thereby closing the stomata while those of susceptible plants are less sensitive to ozone. With regard to stage of plant growth, i t was found that Og injury to tobacco plants occurred a week or so after their "grand period of growth" (Macdowall, 1965b). The leaf's susceptibility to Og was thus associated with metabolic change toward senescence, which was indicated by a decline in protein synthesis that immediately followed the expansion of the leaf. Similarly, the most susceptible stage of cotton leaves to Og injury was reported at about 75 percent full expansion (Ting and Dugger, 1968). Recent studies showed that soybean trifoliate leaves were most sensitive to Og injury four to seven days before they reached maximum fresh weight or leaf expansion (Tingey ejt al_., 1973a). In soybean, this stage of leaf development is characterized by rapid leaf expansion, the formation of inter-cellular spaces.and the cessation of cell division in the palisade layers. During this period, low stomatal resistance permits maximum gas exchange between palisade cells and the atmosphere. Tingey et al_. (1973a) suggested that, as the leaf continues to mature, the mesophyll cells develop a cutin layer, which could reduce gas exchange and thereby render the older cells more tolerant to ozone. Nutrition also plays an important role in plant response to ozone. Tobacco plants grown under excess nitrogen produced leaves of increased susceptibility to ozone damage as a result of their succulent growth (Macdowall, 1965b). Similarly, the dry weight of radish plants was reduced more at high than at low nitrogen levels, following Og exposure (Ormrod et a h , 1973). Nutritional studies with Lemna and tobacco showed a direct relationship between total nitrogen and susceptibility of the plant to Og injury (Craker, 1971b; Menser and Hodges, 1967). Low nitrogen supply was found to decrease severity and incidence of oxidant damage to field plants (Middleton et al_., 1958). Work with beans led Adedipe et_ aj_. (1971) to suggest that sulphur is also an important nutritional factor influencing susceptibility of plants to Og, in that low sulphur level resulted in greater injury. They explained their findings in terms of the levels of sulphur necessary for the production of compounds containing sulphydryl groups which have a direct relationship to the tolerance of plants to air pollutants (Tomlinson and Rich, 1968). Brewer et £l_., (1961) reported interactions of phosphorus with nitrogen and potassium influencing plant susceptibility to oxidant injury. Higher phosphate or potassium levels increased susceptibility of Lemna to Og when other nutrients were maintained at levels for optimum growth, as had also been found for other species by Brewer et al_. (1961). Craker (1971b) suggested that nutritional status induced different physiological ages of leaf tissues which could make them more or less susceptible to ozone. 22 Leone e^ t al_. (1966) reported that tobacco plants receiving an optimal supply of nitrogen were more susceptible to Og injury than those receiving a deficient supply, probably because of excess carbohydrates in nitrogen-deficient plants. A similar accumulation of carbohydrates under phosphorus deficiency was reported on tomatoes (Leone and Brennan, 1970). They suggested that an available carbo-hydrate reserve as induced by withholding either nitrogen or phosphorus serves to enable the plant to survive a variety of adverse conditions such as Og stress. A further possible explanation for the effect of phosphorus deficiency on Og response was that on stomatal opening. Wallace and Frolich (1965) reported that stomata failed to open on old leaves of plants from which phosphorus was withheld. Porometer readings have corroborated these findings (Leone and Brennan, 1970). The susceptibility of tobacco plants to Og was increased by low day temperatures and by high night temperatures (Macdowall, 1965b). In contrast, Heck et al_. (1965) found that low temperatures for one to several days prior to exposure reduced sensitivity in tobacco and Pinto bean. They attributed this increased sensitivity to Og at higher than optimal temperatures to a light and temperature inter-action. An interaction effect of temperature on radish response to Og under several levels of nitrogen and phosphorus has recently been reported by Ormrod et al_. (1973) who found that Og treatments resulted in decreased dry weight of low- and high-N plants at both temperature regimes (two day/night temperature regimes of 20/15 and 30/25°C) and of low- and high-P plants only at the lower temperature. 23 Sensitivity at time of Og exposure increases with increasing light intensity (Heck, 1968). Photoperiod controls many aspects of plant development and, not surprisingly, therefore, also exerts a marked effect on the sensitivity of plants to air pollutants (Brandt and Heck, 1968). Shortened photoperiods apparently increase the susceptibility of plants to Og damage (Macdowall, 1965b; Heck et a l . , 1965). High carbon dioxide levels, at the time of exposure, reduce sensitivity presumably by causing the closing of stomata (Heck, 1968). Air velocity affects the severity of plant injury by Og, but i t is greatly influenced by plant species (Heagle et al_., 1971). When tomato and cucumber plants were exposed to identical concentrations of either Og or SO2, but at different flow rates, plants exposed at higher flow rates exhibited more severe toxicity symptoms, especially at the lower concentrations (0.3 ppm SO2 and 0.18 ppm Og) of pollutants (Brennan and Leone, 1968). Tobacco was often injured more at high air velocity, but the differences were usually not significant. Bean was more severely injured at high than at low velocity, and the results were often statistically significant (Heagle et al_., 1971). They explained these results in terms of disruption of the air boundary layer of the leaf surface at higher velocities which would sustain higher pollutant levels in the boundary layer and therefore permit greater uptake and subsequent injury. Most of the work described concerns acute injury resulting from high Og exposure for short duration. These doses ranged from 0.10 ppm to 1.0 ppm for 1 hour to 12 hours with the most common 24 dosages being between 0.30 and 0.50 ppm for 2 to 4 hours. Such exposures cause dramatic injury often with collapse of the entire.leaf tissue. Thus, very significant effects of other environmental factors on plant response to Og can be expected. In contrast, l i t t le information is available on the effects of such environmental factors on plant response to low levels of oxidants or ozone. However, in general, i t appears that the more vigorous the plant, the greater its susceptibility to phytotoxic air pollutants (Heck et al_., 1965). Finally, i t should be stressed that almost all of these effects of environmental factors on plant response to ozone or oxidants ignored one other factor, namely, the effect of previous exposures. Heck and Dunning (1967) found that plants were more sensitive when given a specific high dose of Og in 1 hour than when this same dose was divided into two half-hour exposures, with variable time periods between exposures. This reduction in injury was attributed to stomatal closure after the init ial exposure; to partial recovery to incipient injury; or to.the ability of the tissue to remove a certain amount of the Og with partial recovery of the removal system before the second exposure. On the other hand, Macdowall (1965b) reported that a low dose of Og acted synergistically with a second consecutive low dose. However, he also found that high doses acted antagonistically against high doses that immediately followed, and he surmised that, after an init ial high Og dose, less surviving susceptible tissue was available for injury by a following exposure. In addition, stomata were less open in freshly damaged areas of leaves than in comparable 25 but healthy areas. Both his low doses of Og (about 0.25 ppm) and his high doses (about 0.50 ppm) were in fact sufficient to cause acute injury. More significant perhaps are recent observations of Runeckles and Rosen (1974) on the effects of exposure to sub-acute ozone levels prior to acute doses. Their work on the pre-treatment of bean and mint plants with daily exposures to low levels of Og (0.02 - 0.08 ppm) for several days, followed by exposure to higher levels of 0o (0.15 - 0.25 ppm) for one or two days shows that the in the case of bean pretreatment markedly reduces susceptibility/to the subsequent high levels of ozone. In the case of mint, on the other hand, the effects of pretreatment and treatment were found to be synergistic. Thus, environmental stresses, including low levels of air pollutants, can influence the physiological resistance of plants to subsequent pollutant exposures. The effects of oxidants on plants can be altered through synergistic or antagonistic responses with other pollutants in the atmosphere. Working with the sensitive Bel-W3 variety of tobacco, Menser and Heggestad (1966) found that a 4-hour exposure to 0.05 ppm of 0g and 0.25 ppm of S0£ caused severe injury while the same levels of the pollutants were not independently injurious to the tobacco. Heck (1968) reported a synergistic action between N02 and S02 on Bel-W3 tobacco. Tingey et al_. (1971) found that radish exposed to 0.05 ppm 0g and 0.05 ppm SO2 for 40 hours per week for 5 weeks had significantly reduced plant fresh weight, leaf fresh weight, root fresh weight and dry weight. These effects were additive or less than additive. Tingey e_t al_. (1973b) reported that growth reductions 26 in soybean resulting from a mixture of 0.05 ppm Og plus 0.05 ppm SO2 were greater than the additive reductions of the single gases. Such pollutant interactions can greatly influence the relation between pollutant concentration and plant injury. 2. Effects of Ozone on Pathogens A. Effects on Growth The inhibition of the growth of fungal pathogens by air pollu-tants is suggested from observations that mildew fungi (Hibben and Walker, 1966; Koch, 1935),blister rust cankers (Linzon, 1958), black spot of roses (Saunders, 1966), and various other foliar pathogens (Scheffer and Hedgcock, 1955) are less apparent on plants in areas of high air pollution. Not only could pathogens at the infection site be inhibited by air pollutants, but the viability of air- and soi l -borne propagules might also be reduced before they reach the host. Ozone has been reported to inhibit fungal growth on refrig-erated foods (Smock and Watson, 1941; Watson, 1942), and on bread and leather; to reduce sporulation of Venicillium spp. on citrus fruits (Harding, 1968); to damage conidiophores of Alternaria solani (E l l . & Mart.) Jones & Grout.and to stimulate germination of spores s t i l l attached to conidiophores (Rich and Tomlinson, 1968); to suppress aerial hyphae on agar (Klotz, 1936; Kuss, 1950; Watson, 1942); to prevent germina-tion and growth of several fungi in liquid and solid culture (Klotz, 1936; Watson, 1942); to increase sporulation of Alternaria and to prevent germination of spores of Mycosphaerella citrullina (C.O.Sm.) Gross. (Richards, 1949); and to stimulate sporulation of several fungi with accompanying reduction in germination of spores ozonated while attached to sporophores (Kuss, 1950). In studies with fungi using low ozone concentrations (0.10 ppm), spore germination was reduced in some species (Hibben, 1966) while in others i t was stimulated (Hibben and Stotzky, 1969). Ozone stimulated spore production in Alternaria oleraoea Milbr. (Treshow et al_., 1969). Hibben and Stotzky (1969) concluded that 0 3 must contact the spores in order to be toxic, because a direct relation-ship was apparent between Og concentration and time of exposure. The mechanism of Og injury to germinating spores, which they proposed, was an effect on membrane integrity, perhaps similar to the increase in membrane permeability reported to occur in green plants (Dugger et a h , 1966; Mudd and McManus, 1964; Tomlinson and Rich, 1967) primarily as a result of oxidation by 0g of l ipid fractions necessary for the synthesis of long-chain fatty acids. Inhibition of spore germination was speculated to be due to inactivation by 0g of certain essential enzymes as a result of oxidation of SH-groups. On the other hand, Rich and Tomlinson (1968) found that 0g stimulated the germination of Alternaria spores attached to conidiophores, possibly by either inactivating germination inhibitors or altering cell wall permeability. They suggested that certain correlations 28 existed between the morphology of spores and their susceptibility to Og. Spores most sensitive to Og were, in general, relatively small and hyaline, whereas the most resistant spores were large, pigmented and multicellular. Spores with relatively thick walls tended to have some degree of protection from Og injury. However, once germination had taken place, the internal components of the fungal cell with its thin wall were not protected from Og injury. The suppression of aerial hyphae (Treshow et al_., 1969) and the occasional stimulation of sporulation of colonies maintained in sublethal doses of Og confirmed that this pollutant, even when not fungicidal, could affect the developing thallus. These growth abnormalities, which were also observed when colonies from mycelial rather than from spore inoculations were maintained in Og, are apparently characteristic reactions of ozonated fungi (Klotz, 1936; Kuss, 1950; Richards, 1949; Watson, 1942). Recent work with barley and powdery mildew, Erysiphe gramin-is DC. ex Merat hordei E. Marchall, exposed to low levels of Og showed a significant reduction in percentage infection by conidia exposed to Og, but germination of conidia was not significantly reduced by the same exposures which inhibited infection (Heagle and Strickland, 1972). This differential sensitivity of fungi to Og with respect to spore germination demonstrates the potential effect which air pollutants such as Og could have on competition among fungi in their ability to colonize hosts. In the presence of Og, the fungi most sensitive to 0^ , could be out-competed by the less ozone-sensitive 29 species in infecting host tissues. This advantage of ozone-stimulated or less ozone-sensitive species over the more ozone-sensitive species could be of major ecological significance. Under Og fumigation, a stimulation of spore production would greatly increase inoculum potential, accelerate the disease cycle and help to ensure survival. Very l i t t le work has been done on the effects of air pollu-tants on plant pathogenic bacteria. However, considerable work has been carried out on the use of Og, not as an air pollutant, but as an oxidizing agent in sewage-disposal plants for treatment of sewage to ki l l bacteria, and in the preservation of meat during the tenderizing process. Scott and Lesher (1963) studied the effect of Og on survival and permeability of Escherichia coli B. They postulated that the primary attack of Og was on the cell wall or membrane of the bacteria, probably by reaction with the double bonds of l ipids; the leakage or lysis of cells depended on the extent of that reaction. In the case of viruses pathogenic to plants, again relatively l i t t le work has been carried out. Treshow et al_. (1967) began the f i rst work of this nature when they found that fluoride stimulated TMV-induced lesions on bean plants. Tests conducted on Pinto bean leaves by Brennan and Leone (1970) indicated that there was a time-dependent stimulating action of 0 ? on virus activity. 30 B. Environmental Factors Influencing Response Environmental factors which can seriously affect the initiation and development of infectious plant diseases are temperature, moisture, light, and soil reaction. A comprehensive review of environmental predisposition on plant diseases has been presented by Yarwood (1959a). 3. Effects of Ozone on Plant-Pathogen Interactions A. Effects on Growth The extent to which a pathogen parasitizes a host plant may be partially dependent upon the physiological state of the plant; the conditions under which the plant grows ultimately affect its susceptibility to a pathogen and its development. This concept is termed predisposition, and was defined by Yarwood (1959a) as the tendency of nongenetic conditions, acting before infection, to affect the susceptibility of plants to disease. Natural variations in temperature, humidity, light, mineral content, and air pollutants all exert their influence on plant growth, development and physiology which in turn influence a plant's susceptibility to pathogenic invasion. The host-parasite relation is generally a highly specific interaction involving numerous physiological, ecological, and physiochemical factors operating concurrently. When an additional environmental stress, such as air pollution, is superimposed on this relation, there are potentially three alternative results: 31 (1) the pollutant may render the parasite more infective, either by predisposing the host to enhanced infection or by increasing the activity of the parasite; (2) the pollutant may render the parasite less infective, either by direct action on the parasite or by increas-ing the resistance of the host; (3) the pollutant may not affect the relationship. Studies on host-parasite-pollutant interactions have yielded a variety of results. In some cases, the parasite reduced the phyto-toxic effect of the pollutant on the host plant; that i s , the plant appeared more resistant to the pollutant after infection. For example, Yarwood and Middleton (1954) reported that infection with rust fungi prevented smog injury to leaves of bush bean and sunflower. However, i t is not known against which of the components of smog the protective effect was directed, since an undefined mixture of gasoline vapour and ozone was used. The response of bean to acute doses of Og as related to infection by Pseudomonas phaseolicola was described by Kerr and Reinert (1968). Ozone flecking appeared over most of the surface of the leaflets exposed to Og, but did not occur in the chlorotic or "halo" areas adjacent to the sites of bacterial infection. The authors proposed that the decreased occurrence of flecking in the chlorotic areas indicated less sensitivity of the diseased tissue to Og than of the healthy tissue. Brennan and Leone (1969) studied the suppression of Og toxicity symptoms in virus-infected tobacco. On the day following 32 each exposure to Og, typical symptoms of Og toxicity always developed on the virus-free plants, but never on those infected with TMV. Recently Heagle and Key (1973) studied the effect of Puecinia gvaminis f. sp. tvitioi on Og injury in wheat. Exposure of the inoculated plants to 0.30 ppm 0g for 5 hours revealed that signif i -cantly less 0g injury occurred in mesophyll cells in the substomatal areas of inoculated wheat leaves than in noninoculated leaves. The mesophyll cells under stomata with appressoria attached were rarely injured. Mesophyll cells under stomata without appressoria attached were also protected in areas of the leaf that were inoculated. Non-inoculated areas were not protected. Similar protection around parasitic infections has also been reported on peanut infected with peanut leaf spot fungus, Cercospora arachidicola Hori.(Heagle, 1973), on broad bean infected with Botrytis cinevea Pers. (Magdycz and Manning, 1973), on l i lac infected-with Micvosphaeva alni (Wallr.) Salm. (Heagle, 1973) and on pinto bean infected with TMV ( Yarwood, 1959b). Pollutants may also predispose plants to infection. An increase in disease severity may be expected when air pollutants weaken plants, making them more susceptible to infections by weak parasites. For example, 0g injury to leaves of potato (Manning et a l . , 1969) and geranium (Manning et al_., 1970) increased the suscepti-bil i ty of the plants to infection by the fungus, Botrytis cinevea. Air pollutants may also stimulate parasite activity and proliferation in the host. Tobacco leaves infected with TMV exhibited increased viral replication upon exposure to 0.30 ppm 0g for 6 hours (Brennan and Leone, 1970). Conversely, pollutants may be toxic to a parasite, thereby reducing or eliminating infection. Microorganisms and plant diseases have been reported to be less abundant around certain industries and urban centres than at some distance. Koch (1935) was among the f i rst to report an air pollutant modifying a disease pattern. Over an interval of eight years he observed the complete absence of oak mildew around certain factories while the disease was otherwise widespread. Absence of the fungus was attributed to sulphur oxides. More recently, Linzon (1958) suggested that blister rust cankers appeared on fewer white pine trees near the nickel smelters at Sudbury, Ontario, than in more distant sampling areas. Closest to the smelter, blister rust was almost entirely absent. Exposures of several species of oats to low concentrations of Og (0.10 ppm for 6 hours for 10 days) after infection with crown rust fungus reduced the growth of uredia of the pathogen and caused a decrease in spore production (Heagle, 1970). Ozone decreased disease of gladiolus flowers inoculated with Botvytis gladiolovum Timmerm. (Magie, 1960; 1963) and disease of chrysanthemum petals infected with Botvytis sp. (Magie, 1963). Invasion of geranium flowers by B. cinevea was restricted in the presence of ozone (Manning et al_., 1970). Invasion and infection of poinsettia bracts by B. cinevea were not affected by ozone, and bracts were not injured by ozone after inoculation (Manning et al_. 1972). Heagle and Key (1973) studied the effects of low levels of 0g on various phases of uredial development of the wheat stem rust fungus. They found that 0^  exposures 24 to 48 hours before inoculation injured the plants and reduced penetration and infection. When plants were inoculated immediately after exposure and before injury developed, no reduction in penetration and infection occurred. Germination and infection of untreated wheat plants by spores produced on Og -treated plants was not impaired. No effects on infection or on rust morpho-logy were observed when inoculated plants were exposed immediately after incubation, during penetration. They stated that this was further evidence of a lack of direct action by Og on the fungus. They speculated that the lack of Og effects at the early stages of develop-ment may have been the result of the fact that the infection struc-tures arising from appressoria are formed within the substomatal cavity of the host plant where they may be partially protected from exposure to ozone. B. Mechanisms Numerous mechanisms have been proposed for the reduced phyto-toxic effect of the pollutant on the host plant in the presence of the parasite. Yarwood and Middleton (1954) attributed the protective effect of rust fungi on beans and sunflower against smog injury not to stomatal closure or high content of pantothenic acid in the rust-infected tissue, but to some undefined substance which diffused beyond the limits of the rust mycelium. Kerr and Reinert (1968) in their study of halo blight of bean suggested that decreased Og sensitivity in the halo areas was related to differences in the stomatal function between diseased and healthy areas in the leaf and that there was a metabolite in the chlorotic area that functioned to reduce sensitivity to ozone. Brennan and Leone (1969) speculated that TMV in tobacco reduced Og toxicity by hastening the maturation of the tobacco plants. They pointed out that many other biochemical changes are induced in the host tissue as a result of virus infection, such as profound changes in phosphorus metabolism, organic acid metabolism, phenol metabolism, and the elaboration of growth sub-stances. Thus, any one of these factors may account for the resistance of TMV-infected tissue to Og damage. Heagle and Key (1973) in work on the effect of Puocinia gvaminis f. sp. tvitioi on Og injury in wheat suggested that a diffusible substance could be produced by germinating spores and infection structures which results in protection of localized areas of wheat leaf tissue from Og injury. When cytokinins are applied to leaves, they stimulate metabol-ism, delay senescence, and maintain protein and chlorophyll synthesis (Galston and Davies, 1970). Metabolism is similarly stimulated in leaf tissues infected with certain pathogens, such as the rust fungi, in the green islands surrounding sites of infection. Atkin and Neilands (1972) tested representative siderochromes (polyhydroxamates) produced by many higher fungi, for example rhodotorulic acid from yeast-like heterobasidiomycetes and ferrioxamines from actinomycetes and some bacteria, and found that all were potent inducers of green islands in detached leaves. They state that i t has not been proved that cytokinins are responsible for naturally occurring green islands, and that i t is doubtful that the fungal metabolites that induce green 36 islands in vivo could be acting exclusively as cytokinins. Higher cytokinin activities, on the other hand, have been reported in tissues of rust-infected bean (Kiraly et al_., 1966). Accumulation of nutrients and prevention of premature senescence caused by rust-infected tissue were simulated by application of benzyladenine (BA). Craker (1971a) observed that ozone itself can induce ethylene synthesis as can some plant-pathogenic bacteria (Goodman et, al_., 1967). Such ethylene synthesis accounted for premature senescence. Cytokin-ins are known to play a role in ethylene effects. Adedipe and Ormrod (1972) found that BA protected plants against ozone-induced growth suppression and chlorophyll loss. The cytokinins delay senescence, in part, at least, through maintenance of the synthesis of RNA and protein. Galston and Davies (1970) proposed that auxin coming from the leaf blade retards abscission, but once senescence has started, such auxin may promote abscission. They attribute this response to the formation of ethylene, which in turn stimulates the synthesis of new enzymes, such as cellulase, promoting the dissolution of cell walls in the abscission zone. C. Genetic and Ecological Effects (Adaptation) In a recent review, Babich and Stotsky (1972) claim that adaptations by organisms to air pollutants are of two types: geno-topic and phenotypic. Genotypic adaptation has been demonstrated in the onion, Allium oepa L. as mentioned previously (Engle and Gabelman, 1966). Substantial evidence for a genetic basis for resistance to 0 -^induced weather fleck in tobacco has been found although specific genes have not been identified (Sand, 1960; Povilait is, 1967). Phenotypic adaptability has been suggested in the response of some fungal species to Og fumigation. Normally, agar cultures of colonies of these fungi develop aerial hyphae that extend upward. During exposure to Og, however, the hyphae grow oppressed to and into the agar, but, during subsequent incubation in carbon-filtered air , the new hyphae regain their aerial growth habit (Hibben and Stotzky, 1969). Babich and Stotzky (1972) point out that the ability of an organism or a population of organisms to adapt to adverse atmospheric conditions is of major"importance when considering the long-range ecological impact of air pollution. They ask the questions: Will air pollution gradually eliminate individuals with lesser capability to adapt? Will this, in turn, affect the ecological succession of an environment? They speculate that significant alteration of the host-parasite relations by air pollutants may eventually lead to a gradual elimination of the more sensitive individuals in a species and, conversely, that decreased plant injury by phytotoxic pollutants and decreased infectivity by plant pathogens may result in an increase in the total primary productivity of the ecosystem. But they admit that the final outcome from these host-parasite-pollutant interactions on an ecosystem are diff icult to predict. The fact that considerable genetic variability in pollutant sensitivity exists within varieties of any given species implies that there is a potential for genotypic adaptation. As air pollutants become more prevalent in our environment, natural selection will favour less sensitive strains of plants. Thus, tolerant strains or varieties will out-compete the less tolerant ones. Similarly, selection for pollutant-resistant microorganism strains will occur. In this way, host-parasite relations may be altered in the presence of air pollutants. Pollution may select hosts more resistant to infection or parasites less capable of infecting or conversely pollutants may select hosts less resistant to infection or parasites more capable of infecting them. Babich and Stotzky (1972) emphasize that pollutants may also affect the balance of nature by eliciting positive responses in organisms. For example, stimulation by pollu-tants of fungal spore germination might result in increased numbers of pathogens. Finally, in reviewing the literature on the effects of low levels of ozone on plants i t can be noted that many plants show increased growth responses to Og when exposed to concentrations in the range 0.02 - 0.05 ppm for various numbers of hours per day. Plant height and fresh weight were found by Heagle et al_. (1972) to be stimulated in certain varieties of corn {Zea mays L.) and lateral bud elongation of Pinto bean plants {Phaseolus vulgaris) was enhanced by Og (Engle and Gabelman, 1967), although in neither case were the effects statistically significant. A similar stimulation of growth of tomato by Og has been reported (Neil et al_., 1973). Several weeds and understorey herbs from aspen communities in Utah showed signif i -cantly increased plant weights at 0.05 and 0.15 ppm ozone (Harward and Treshow, 1971). Thompson and Taylor (1969) found that the weights of prunings of lemon and orange trees in California were slightly greater in a "low ozone air" chamber than in one with filtered air. At the physiological level, Barnes (1972) has reported small increases in photosynthesis in seedlings of four Plnus species when exposed to 0.05 ppm ozone. At the reproductive level, Harward and Treshow (1971) found that two species of Polygonum in their study of aspen understorey produced significantly more and heavier seed in 0.05 - 0.15 ppm ozone. Flower fresh weight but not flower size increased significantly from 0.02 to 0.12 ppm 0g in petunia and geranium, as was the case with poinsettia bracts (Craker and Feder, 1972). Additional studies in the present investigation have supple-mented the evidence for apparent stimulations of plant growth by low ozone concentrations when compared with growth in ozone-free air. (Bennett et al_., 1974). In all of these cases apparent stimulations in plant growth by low ozone levels appeared when compared with growth in carbon-filtered air. Bennett et al_..(1974) have chosen to suggest that this situation reflects the fact that plants adapt to low levels of ozone and that they are then at a disadvantage when grown in carbon-filtered air. In summary, most of the literature deals with ozone exposures causing acute injury and the effects of such levels on individual plant species. Many speculations on possible mechanisms of oxidant injury at the physiological and biochemical levels have been proposed. However, l i t t le work has been done on the long-term chronic injury response of plants to oxidants. This lack of knowledge about such long-term responses is particularly evident in terms of interactions of pollutants with biological systems such as those involving host-parasite relations. 41 MATERIALS AND METHODS 1. Plant Materials Bush bean {Phaseolus vulgaris L.) cultivar Pure Gold Wax was used in all experiments. Seed was sown in pasteurized soil in 5-inch (12.7 cm) plastic pots. The seedlings were generally grown for about 10 to 12 days under greenhouse conditions with supplementary art i f icial lighting (cool white fluorescent) to pro-vide an intensity of 10.7 klx and a 14-hour daily photoperiod, before being transferred to the fumigation chambers. 2. Exposure Chambers The bean plants were treated in seven plexiglass fumigation chambers (Figure 3). The chambers were constructed of 1/4-inch (0.635 cm) clear plexiglass fitted on to a plywood base. Each exposure chamber measured 13.5 inches (34.4 3 cm) wide by 18 inches (45.8 cm) high by 48 inches(122 cm) long, with a volume of 6.75 cu ft (193 cm ). The height of the chambers was sufficient to contain the mature bush bean plants. Carbon-filtered air or filtered air to which Og was added was regulated by gate valves to an air speed of 3 mph (4.83 km/hr) at the 1-inch (2.54 cm) diameter inlet ports, equivalent to a flow 3 of 1.44 cfm (41.2 cm /min) through the chambers giving an average air change every 4.7 minutes. The air within each chamber was well 42 Figure 3 Ozone fumigation chambers. 43 mixed by means of a 4-inch (10.2 cm) diameter fan located at the exit end of each chamber. Air from the chambers was exhausted via a 3-inch (7.62 cm) PVC plastic pipe manifold system to the outside. Ozone was generated by passing tank oxygen over a bank of 6 ultraviolet lamps (General Electric G4S11). The flow of 0 3 into each chamber was regulated by use of a micrometer valve and flow meter between an ozone-distribution manifold and the 1-inch filtered-air inlet pipe to each chamber. This design allowed injection of 0^  at concentrations from 0.00 to 0.20 ppm for any chamber for any desired length of exposure time. Ozone levels were monitored con-tinuously by means of a Mast Model 724 Ozone Meter and Mast Model 725-3CS Strip Chart Recorder. Each chamber was monitored sequentially by means of a timer-operated teflon rotary sampling valve (Chromatronix Inc., Model R60V6A) to which the air sampling hoses from each chamber were connected. Each chamber was monitored for approximately 5 minutes at a time to enable steady readings to be obtained. All investigations described in this thesis employed levels of ozone below those which cause acute injury. Low levels are defined in these studies as ozone concentrations from 0.03 to 0.05 ppm, whereas, high levels are from 0.08 to 0.10 ppm. The daily duration of exposure varied from 8 hours in most experiments to 12 hours in others. Eight pots could be placed in each chamber, allowing a minimum of 8 replicates per treatment i f only one plant was sown in each pot. The .pots were placed in plexiglass trays which were flooded once per day from a central irrigation reservoir located under the exposure 44 chamber table. The irrigation trays were covered with 1/4-inch (0.635 cm) plexiglass. Each cover had eight 5-inch (12.7 cm) diameter holes, large enough to contain the 5-inch (12.7 cm) plastic pots, the lips of which formed a seal when seated in the trays. This subirrigation system minimized any contact of water surface with the chamber air mixture so that Og would not be lost at the air-water interface. In addition, the system was fully automated from a 24-hour time clock so that the chambers did not have to be disturbed during experimental runs in order to water the plants. A 14-hour daily photoperiod under illumination of 10.7 klx was maintained throughout all experiments by means of a mixture of cool white fluorescent and tungsten lamps. Temperatures ranged from 15.5 to 18.5°C (night) and from 21 to 24°C (day). Humidity varied between 50 and 60 percent RH except during the 10-hour dark period after inoculation, during which time it was kept at saturation to encourage uniform spore germination. An automatic misting system regulated by time clocks was installed in the exposure chambers in an attempt to maintain high RH during pathogen incubation periods. However, results were not consistent and the system was not used in experimental tr ials. High humidities following inoculation were achieved by placing polethylene bags over individual plants (see Section 6). 3. Quantitative Measurement of Chronic Injury 45 A. Introduction Ozone injury to plants is usually measured by visual rating (Dass and Weaver, 1968; Manning et^  al_., 1973c) or by a visual estimate of percentage leaf injury (Ting and Mukerji, 1971; Craker, 1971a} Tingey et_ al_., 1973b). These visual ratings are widely used with acute injury in which differences of injury severity are fairly dis-tinct. But with chronic injury differences are very subtle, since they tend to involve different degrees of mild chlorosis spread over the sensitive upper leaf surface. Visual ratings are subjective and therefore ratings given by one researcher can differ greatly from those of another. In addition, the same researcher may unintention-ally judge the same severity of injury at different points on the scale on different occasions. Some workers (Fletcher et al_., 1972) have used chlorophyll extraction as a quantitative measure of Og injury. While chlorophyll extraction is a good indicator of acute injury and premature senescence caused by Og, i t does not differentiate among small differences in chlorophyll content due to chronic injury by ozone. Since low ozone levels (<0.10 ppm) damage only the upper palisade cell layer, chloro-phyll extraction of the entire leaf tissue is unable to detect these small differences. On the other hand, reflectance spectrophotometry can be used to focus specifically on the upper (adaxial) epidermis and palisade mesophyll chlorophyll content by measuring the reflec-tance of this surface. 46 B. Visual Assessment The difficulty in visually assessing small differences in chlorophyll content due to chronic ozone injury is demonstrated in Figure 4. Chronic injury often appears as a mild chlorosis extend-ing throughout the upper leaf surface. Small differences in the degree of chlorosis are diff icult to allocate to different values on a visual rating scale. In addition, since most of the leaf tissue is uniformly injured, i t is laborious to attempt a percentage injury rating of such damage. Accordingly, no data are presented on attempts at visual rating of such injury. C. Chlorophyll Extraction Initial attempts at estimating sub-acute injury were made by estimations of extracted chlorophyll. Chlorophyll was extracted from 8 leaf discs (1 disc per plant) for each treatment. Leaf discs of several sizes were used in the extraction procedures of various experimental runs in an effort to reduce experimental error. The procedure for the extraction and measurement of pigments was based on that of Bruinsma (1963). The leaf discs were ground using a mortar and pestle with washed sea sand and cold 80 percent aqueous acetone (reagent grade, ACS) as a solvent. The mixture was then centrifuged at 11,500 - 12,000 RPM for 20 minutes. The supernatant was decanted into a 100 ml volumetric flask. The residue in the centrifuge tubes was washed with additional cold 80 percent aqueous acetone and treated as before. The supernatants were combined and made to 47 Figure 4 Chronic ozone injury to bean leaves (22-day-old plants). (A) Control-filtered air (See Figure 8, Treatment 1). (B) Late ozone treatment (See Figure 8, Treatment 3). (C) Continuing ozone treatment (See Figure 8, Treatment 5). 100 ml. Concentrations of total chlorophyll were determined by measuring the absorbance of the extracts at 652 nm using a Beckman DU spectrophotometer. Pigment concentrations were then calculated using the following equations (Bruinsma, 1963): Total Chi (mg/1) = 27.8 0D g 5 2 or Total Chi (mg/dm2) = (27.8 0D f i „ ) V d x 1000 x A where : 2 Chi = concentration in mg/1 or mg/dm leaf area ODg,^ = optical density at 652 nm d = length of light path in cm V = final volume of extract in ml 2 A = total leaf area of material used in dm . The results of such chlorophyll extractions are discussed below in Section E. D . Reflectance Measurements Reflectance spectrophotometry was used to measure the amount of incident light reflected from the upper surface of leaf discs. Measurements were obtained using a diffuse reflectance integrating sphere attachment installed in a Perkin-Elmer Coleman Model 124 double beam spectrophotometer. One-inch (2.54 cm) diameter leaf discs were used. The instrument was adjusted to 100 percent reflec-tance using gypsum standards. Reflectance was routinely measured at the peak response in the 550 nm wavelength region (Figure 5). In addition, complete reflectance spectra between 500 and 700 nm were recorded from time to time, where leaves showed signs of senescence. This reflectance spectrophotometry method has been described in detail by Runeckles and Resh (In Press). E. Comparison of Chlorophyll Extraction and Reflectance  Procedures Numerous experimental runs using various treatment combinations were conducted employing only chlorophyll extraction as a measure of ozone damage. The means of such treatments are compared with means obtained for similar treatments by reflectance measurements during subsequent experimental runs in the Richards' diagrams, Figures 6 and 7 (see p. 85 for description of Richards' diagrams). Reflectance measurements indicate a significant increase in ozone damage by continuing ozone exposure regardless of the presence or absence of rust infection. Chlorophyll extraction does not discern such ozone damage under these treatments. The standard errors of the chloro-phyll extraction data are so great that the large visual increases in ozone damage resulting from continuing ozone exposure go undetected. On the other hand, the small standard errors of the reflectance measurements enable it to distinguish the small amounts of ozone damage that occurred under late ozone exposure (comparisons 1 - 3) and small changes in leaf colour due to the presence of rust pustules 51 Figure 5 Reflectance spectra of upper surfaces of primary leaves of bean plants after 12 days of growth in filtered air or filtered air containing 0.05 ppm ozone. Treatment 1: filtered air control; 5: daily ozone; 9: daily ozone for the f i rst 6 days. Curves 5a and 5b show the reflec-tance of leaves in advanced stages of senescence induced by exposures to 0.1 ppm. • ' I ' l l — T 1 - ! — ' — I 1 1 1 I I I I 1 ' | ' 1 r 53 Figure 6 Richards' (factor) diagrams of reflectance (550 nm) and chlorophyll content (mg/dm ) of primary leaves of bean plants treated according to the regimes 1, 5, 7, 8, 9, 10 described in Figure 8. Solid lines connect treatments with increasing exposures to 0.05 ppm ozone (1, 8: none; 7, 9: early; 5, 10: continuing). Broken lines connect treatments which compare the effect of infection with rust fungus. 54 55 Figure 7 Richards' (factor) diagrams of reflectance (550 nm) and chlorophyll content (mg/dm ) of primary leaves of bean plants treated according to the regimes 1, 2, 3, 4, 5, 6 described in Figure 8. Solid lines connect treatments with increasing exposures to 0.05 ppm ozone (1, 2: none; 3, 4: late; 5, 6: continuing). Broken lines connect treatments which compare the effect of infection with rust fungus . O —• KJ 00 O -I 1 -y£ reflectance to ^ 1 1 to o T " /A chlorophyll (mg^dm 2) CJ CO -U-O 00 O K3 I 1 1 1 1 1 T-en 57 (comparisons 1 - 2) as shown in Figure 7. Thus, while chlorophyll extraction can detect large differences in tissue damage and senescence, reflectance can detect the smaller differences of damage usually associated with chronic ozone injury. 4. Experimental Designs A. Introduction The number of treatments that could be used during any experi-mental run was limited to seven since there were only seven exposure chambers. For this reason, the incomplete factorial arrangement of treatments was broken down into two separate complete factorial designs, one for each experimental run, as outlined below. The objectives of the treatments were: to compare early with late inoculation; to compare early, continuing, or late ozone; and to compare any of these with their appropriate controls. A second set of experiments was conducted to compare the effects of continuing exposure to three levels of Og with appropriate controls (± inoculation). B. Bean - Bean Rust Experiments (a) Experiments on timing of inoculation and fumigation Plants were grown in the greenhouse until 10 days old before placing them in the exposure chambers. Early inoculation refers to inoculation after the photoperiod of day 0, immediately upon placing the plants in the chambers; late inoculation treatments were carried 58 out after the photoperiod of day 6. Early ozone exposure began on day 1 and continued through day 6 while late ozone exposure began on day 7 and continued through day 12. Continuing ozone exposure began on day 1 and terminated on day 12. Ozone levels of 0.08 - 0.09 ppm for 12 hours duration per day were used. Two experimental runs had to be used to accommodate these treatments as outlined in Figure 8. These treatments were used in the f i rst seven experiments (Experiments A, B, C, D, E, F, and G) and may be summarized as follows: BASIC INCOMPLETE FACTORIAL DESIGN Ozone I nocul a t iorT^^^ None Late Early Continuing None 1 3 9 5 Early 2 4 - 6 Late 8 - 7 10 (Numbers refer to individual treatment combinations ; these numbers are used to identify treatment combinations in the text and figures). Experiment runs were divided as follows: RUN 1 Ozone I n o c u 1 a t i orT~~~-\^ None Late Continuing None 1 3 5 Early 2 4 6 With treatment 7 added in the seventh chamber. Treatment Run I Treatment Run H No. No. 1 — 1 _ 2 t _ 8 * 3 -wnH 9 ------••-•-»-U *• — — • 7 5 BnHHBi^H 5 nHHOBBHI 7 MHH-BB^  _~ ^ c i z i r r r r i ^ 0 6 12 days 0 6 12 da ^ inoculation m ozone exposure Figure 8 Summary of treatments in experimental runs . 60 RUN 2 ^ ^ ^ . ^ ^ Ozone I n o c u 1 a t i o n""""^---^ None Early Continuing None 1 9 5 Late 8 7 10 With treatment 4 added in the seventh chamber. Each treatment contained 8 replicates. Each replicate con-sisted of one plant per pot. The data from each run were analyzed statistically by analysis of variance. The level of significance used was P 0.05. Duncan's New Multiple Range Tests were used to compare treatment means. Since experimental runs were isolated by time, treatment means of one run were not compared statistically with those of another run. (b) Experiments on levels of ozone Two experiments (Experiments J and K) were conducted in order to compare the effects of continuing exposure to three levels of ozone with appropriate controls: Continuing I n oc u 1 a t i o n None Low High None 1 5a 5b Early 2 6a 6b (Treatment numbers correspond to those used in earlier experiments). 61 These studies consisted of 6 treatments and 8 replicates per treatment. Three 0 3 levels (0.0, 0.03 - 0.04 and 0.09 ppm) of continuing exposure for 12 days and 8 hours per day were combined with two inoculation levels (none and early). Three sample periods were used in harvesting the plants; day 0 (10-day-old plants), day 6 (16-day-old plants) and day 12 (22-day-old plants). Six seeds were sown per pot and after 8 days the pots were thinned to leave the 3 most uniform plants. During each harvest one plant per pot was removed at random to give 8 replicates per treatment at each sampling period. Experiment J involved a heavy infection by the fungus (about 350 pustules per 1-inch [2.54 cm] leaf disc) while Experiment K involved medium infection (50 - 75 pustules per disc). The data from each run were analyzed statistically as described above. C. Bean - Halo Blight Experiments A single experiment (Experiment L) was performed to determine the effect of three levels of ozone (0.0, 0.03 - 0.04 and 0.09 ppm) for 8 hours per day on host-bacterium relations. This study consisted of 6 treatments as follows: ^ ^ ~ \ 0 z o n e Continuing Inoculation None Low High None 1 5a 5b Early 2 6a 6b 62 A second experiment (Experiment 0) was carried out to deter-mine the effect of timing of fumigation. The treatments were as follows: — O z o n e InoculatioiT\^^ None Late None 1 3 Early 2 4 In both Experiments L and 0, each treatment was replicated 8 times (8 pots with 1 plant per pot). A single harvest was made after 12 days of Og exposure (22-day-old plants) in Experiment L and after 8 days of Og exposure (31-day-old plants) in Experiment 0. The data were analysed as before. D. Bean - Siderochrome Experiments Two experiments (Experiments H and I) were conducted to deter-mine whether or not the siderochrome, rhodotorulic acid, might offer protection against Og injury. Its effects were compared with those of the cytokinin, 6-benzyladenine, against controls, in the presence of filtered air throughout or in continuing or late ozone. The three chemical treatments were: 1. 0.1%, Tween-20 (polyoxyethylene sorbitan monooleate) - a surfactant (control). 63 2. 0.1% Tween-20 + 344 ppm rhodotorulic/acid (RA). 3. 0.1% Tween-20 + 10 ppm 6-benzyladenine (BA). Late ozone exposure began on day 7 and continued through day 12. Continuing ozone exposure began on day 1 and ifinished on day 12. Ozone level was maintained at 0.09 ppm for 12 hours per day. Each of the chemical treatments was applied once to one-half of one primary leaf of each plant to give 8 replicates per treatment. Reflectance measurements and photographs were taken at the end of 12 days of treat-ment. The reflectance data for each experimental run were analysed as described before. E. Bean-- Cytokinin Experiments The effects of cytokinins on the response of primary leaves to 0g exposure were studied in Experiments M and N. Three levels of continuing ozone exposure (0.0, 0.03 - 0.04 and 0.09 ppm) for 8 hours per day were used. In experiment M, the cytokinins 6-benzyl adenine (BA) and kinetin (6-furfuryladenine)(K) were applied to the top and bottom surfaces of one primary leaf per plant while the other was wetted with water. These cytokinins were applied at a concentration of 30 mg/1 (ppm) once at day 1 (12-day-old plants) before 0g exposure began. Each treatment contained 8 replicates (each replicate was one primary leaf on one plant). The time for the primary leaves to senesce was recorded over a period from 21 to 42 days of daily 0g exposure. Senescence was defined as at least 75 percent of the leaf turned yellow or bleached. 64 Experiment N was done similarly, but with the cytokinins applied once a day for 8 consecutive days to the upper surface only of one primary leaf per plant. The second primary leaf of each plant acted as a control. All data for each experimental run were tested statistically as before. 5. Growth Analysis of Host Plants A. Introduction Air pollutants, like other environmental factors, can limit plant growth. The severity of any environmental stress imparted on a plant will be reflected in the degree of restriction in the growth of the plant. In the past much work has been done in measuring the effects of environmental factors such as light, temperature, mineral nutrition, and moisture on plant growth by use of growth analysis techniques (Blackman, 1961). Since air pollution is another environmental factor affecting plant growth it was felt its effects could also be studied by the use of growth analysis techniques. Similarly, parasites which use the metabolites of their hosts alter or limit the growth of the host plant and these effects on the latter can also be determined by growth analysis, but such measurements have been virtually ignored in the literature (Manners and Myers, 1973). 65 B. Bean - Bean Rust Experiments (A,C,D,E,F,G,J,K) The following growth measurements were made in various of the above experiments: 1. Plant height - - measured every second day of treatment beginning at day 0. 2. Internode length - - primary leaf to f i rst trifoliate and f i rst to second trifoliate internodes were measured at harvest period 3 (12 days of 0^  exposure). 3. Primary leaf fresh weighty M e a s u m J a t e a c n n a r v e s t p e r i o d „ n • i ^ j . , , 1 (0,6,12 days of 0o exposure), 4. Primary leaf dry weight ) v y ' J 3 K 5. Stem fresh weight | [ Measured at each period. 6. Stem dry weight ) 7. Primary leaf area — measured at each period. 8. Reflectance - - measured at each harvest period. 9. Root fresh weightj M e a s u r e d at the f i rst and third .10. Root dry weight i harvest periods. 11. First trifoliate middle leaflet fresh weight i Measured at n o r-- , , *_f n * j- . j j i i £i 4. J • U4. I harvest period 3. 12. First trifoliate middle leaflet dry weight ) ^ Dry weights were taken after oven drying of plant material for 48 hours at 100 - 105°C. Leaf area was calculated by use of a Keuffel and Esser Company compensating polar planimeter. The raw data were later transformed and the following relation-ships were examined: 66 1. L A R ~ fresh weight basisj > for the three harvest periods, 2. L A R - - dry weight basis ) where: L A R - t o t a l l e a f a r e a ' total plant weight 3. Total shoot fresh weight j t for harvests 1 and 3. 4. Total shoot dry weight ) 5. Root/shoot ratio - - fresh weight basis. J- for harvests 1 and 3, 6. Root/shoot ratio - - dry weight basis ' • where: root/shoot = ^ j ? 1 , ™ * w.f3gK 7. Total plant fresh weight 8. Total plant dry weight ' total shoot weight for the three harvests, 9. Total plant leaf area } f o r t h e t h r e e harvests. iqht^ 10. Total plant dry weight/total plant fresh weig  11. Leaf ratio - - fresh weight basis. I for the three harvests, 12. Leaf ratio - - dry weight basis ' , i x 4.- total leaf weight where: leaf ratio = t o t a 1 p 1 a n t w e i | jht • 13. Stem ratio — fresh weight basis \ for the three harvests, 14. Stem ratio - - dry weight basis ' total stem weight where: stem ratio = total plant weight ' 15. Root ratio - - fresh weight basis 16. Root ratio - - dry weight bas i . I for harvest periods 1 and 3, is ' , . . . total root weight where: root ratio = ^ total plant weight 67 These variables were measured in the heavy infection and medium infection experiments J and K. In earlier experiments only reflect-ance and leaf area were always measured, while primary leaf fresh weight and dry weight were measured in a few of the earlier experiments. In the Results and Discussion section i t has been necessary to reduce the amount of data presented to manageable proportions and consequently not all the original and none of the transformed data are presented. Few statistical differences were found among LAR, leaf, stem and root ratios, and hence relative growth rates (RGR) and net assimilation rates (NAR) were not calculated. Thus, the information presented in this thesis is really growth data and growth effects but not the type of analysis used by Blackman (1961). C. Bean - Halo Blight Experiments (L and 0) Similar measurements were made after 12 days to those described above for bean - bean rust experiments, except that f i rst trifoliate leaf areas and weights were not separated into leaflet components, measurements were made on the second and third trifoliates and counts were made of root nodules. Leaf areas were determined by use of a Hayashi Denko Co. Ltd. Automatic Area Meter Model AAM-5. The raw data were later transformed and the following relation-ships were examined: 1. total plant fresh weight 2. total plant dry weight 3. total plant leaf area 68 4. LAR - fresh weight basis 5. LAR - dry weight basis 6. total trifoliate leaf area 7. total trifoliate leaf fresh weight 8. total trifoliate leaf dry weight. D. Cytokinin Experiment (N) Measurements of primary leaf area and the fresh and dry weights of primary leaves, stems and roots were made after 12 days and up to 42 days of treatment. For those plants whose primary leaves had not senesced after 12 days of ozone treatment, exposure was continued up to a maximum of 42 days. At that time those plants whose primary leaves had not senesced were harvested and the measurements taken. 6. Inoculation A. Bean Rust {Uromyces phaseoli) Primary leaves were inoculated by spraying both surfaces to saturation with a "standard" uredospore suspension before placement in the fumigation chambers. Light infection experiments (A to G) used spore suspensions of 0.4 to 0.6 g spores in 200 ml disti l led water for inoculation. The medium infection experiment (K) was inoculated with 0.08 g/200 ml and the heavy infection experiment (J) was inoculated with a 0.30 g/200 ml spore suspension. The differ-ence in the degree of infection between Experiments A to G and Experiments J and K resulted from increased viability of the younger 69 spores used in the latter experiments. During the 10-hour incubation dark period, the relative humidity was kept close to saturation to encourage uniform spore germination by covering the plants with poly-ethylene bags which were removed at the start of the normal photo-period. B. Bean Halo Blight [Pseudomonas phaseolicola) Primary leaves of 10-day-old bean plants were inoculated by spraying to saturation both surfaces with a dense, opaque suspension made by washing 5 - 6 petri dishes of bacterial colonies with sterilized disti l led water. Since the trifoliate leaves were unfolding (about 1/2-3/4 inch [1-2 cm] in length) at the time of inoculation, they were also saturated with the inoculum suspension. The sprayed plants were covered with polyethylene bags to retain high moisture conditions and incubated for 20 hours in the dark before commencing 0 3 exposure in the fumigation chambers. The bacteria were propagated on King's medium B agar (Ktng et al_., 1954) in petri dishes for 36hbucs' before preparation of the inoculum suspension. 7. Measurement of Rust Infection At the end of 12 days of treatment the number and size of primary pustules were recorded. Pustules were counted on four randomly selected 1-inch (2.54 cm) diameter discs from each primary leaf of each of 8 plants per treatment. Pustule size was measured by use of a micrometer eyepiece (15 scale divisions = 1 mm). Twenty pustules were measured at random on each primary leaf of each plant. 70 The maximum diameter of the pustules was recorded. Photographs were taken through a microscope to record the size differences of pustules and the occurrence of secondary pustules. 71 RESULTS AND DISCUSSION 1. Preamble Only a selection of the data collected for the variables described in the Materials and Methods section is presented in the body of this thesis, namely: the percent reflectance, area and dry weight of primary leaves, trifoliate leaf dry weight, stem dry weight and root dry weight. Many of the other variables measured showed similar results to those selected for presentation. For example, in general, fresh weights and dry weights were closely related. It is believed, however, that dry weights are better indi-cators of plant growth responses than fresh weights, since fresh weights often depend on the turgidity of the plant at the time of harvesting and are subject to errors as a result of water loss on harvest. In addition, when studying effects of a pathogen or air pollutant upon a plant, attention must be focused on those parts of the plant which are most responsive to such treatments. For example, inoculation of primary leaves of a plant will result in infection largely of those leaves, not of the stem, roots, or trifoliates. Although such infection will indirectly affect the entire growth of the plant the greatest influences are on the actual area infected. Similarly, in the case of Og, the greatest effect is on plant parts exposed for the longest period of time and those parts having the largest surface area: volume ratio such as the leaves. 72 The experiments conducted in this research will not be dis-cussed in chronological order, but rather in order of subject matter. The results of various experiments have been collected and are pre-sented in the following sequence of subject areas: main effects of Og or pathogens on bean growth; interactions between Og and rust; interactions between Og and Pseudomonas; and effects of cytokinins and siderochromes on response to ozone. Most of the results will be presented in tabular form or as Richards' (factor) diagrams (see. p.. 84) of mean values. Primary data and statistical analyses are included in appendices. 2. Ozone Dosage and Uptake In the literature, particular Og exposure levels are cited in various studies, using levels which induce either acute or chronic responses. However, data are rarely presented to indicate where in the experimental system the pollutant levels were monitored and how the levels reported relate to the effective dosage received by plants. The Og concentration entering any given exposure chamber containing plants and soil or other media is always higher than the concentration leaving the chambers, since some Og is absorbed by the plant material, the rooting medium, and the chamber walls, while some breakdown of Og occurs in the atmosphere of the chambers (Runeckles, 1974). This situation can be expressed in terms of the equation: [ 0 3 ] IN = C 03 ]0UT + L"°3]S + [ 0 3 ] P + C ° 3 ] C D where [0 3 3 s , [0 3 ] p and [0 3 ]Q D represent uptake by s o i l , plants and chamber (including decomposition) respectively. In these experiments 0 3 levels were monitored at the exit ends of the chambers. However, for several days prior to any experiment, the 0 3 levels were recorded with the chambers empty, to permit adjustments to be made to get the desired level of ozone represented by [0 3 ] j N - [0 31QD- Once these levels were stabilized, the potted plants were placed in the chambers. Immediately, a significant drop in 0 3 level could be seen when stable conditions were reached, as the result of absorption by the soil and the plants. Measurements of ozone removed by soil have been reported to be a function of the physical and chemical characteristics of the soil and a function of the resistance of the air boundary layer near the soil surface (Turner et al_., 1973). In this research, exit 0 3 levels without plants were recorded at 0.09 to 0.10 ppm, while the levels fel l to 0.03 to 0.04 ppm when plants were placed in the chambers. The relationship between exit levels with and without plants and soil is shown by the data of Table I. Soil and plant material each absorbed approximately equal amounts of 0 3 at low levels, but at higher levels the plant material appeared to absorb greater amounts, but this increase could not be demonstrated statistically. In terms of the levels of 0 3 taken up by the plants, the effective dosages can be derived from the data of Table I. Thus, at the start of the experiments, low level exposures were obtained TABLE I Absorption of ozone by chamber, plants and soil expressed in ozone levels observed (ppm) at the exit ports of the chambers-Empty Chambers (a) Chambers + Soil + Pots (b) Chambers + Potted Soil + Plants (c) Absorbed by Soil [0 3 ] S (d = !a-b) Absorbed by Plants [0 3 ] p (e = b-c) 0.05.1- 0.06 0.03 - 0.04 0.01 0.02 0.02 - 0.03 0.09 - 0.10 0.06 - 0.07 0.03 - 0.04 0.03 0.03 .0.13 - 0.14 0.09 - 0.10 0.04 - 0.05 0.04 0.05 0.20 0.15 0.06 - 0.07 0.05 0.08 - 0.09 The data of the f i rst three columns are for [C^IQUT' observed with the empty chambers, with so i l , and with soil and plants respectively. For the empty chambers (a): L-0 3] 0 U T = t°3 ]IN " L"03]CD For the chambers containing soil in pots (b): ^ 0 U T = L-03] I N - [0 3 ] C D , - [0 3 ] s ; For the chambers containing soil and,plants (c): L^OUT = C 03 ] IN " t°3^CD " ^ S " L"°3]P > where [0 3]Q D, [0 33 s and [0 3 ] P represent concentration losses due to absorbtion by the chamber materials (plus decomposition) by the soil and by the plants, respectively. 75 with inputs to the empty chambers of approximately 0.10 ppm, leading to uptake by the plants of 0.03 to 0.04 ppm and an exit 0g concentration of 0.03 to 0.04 ppm (Table I). For higher level exposures, uptake by the plant of 0.08 to 0.09 ppm and exit levels of 0.06 to 0.07 ppm were achieved by inputs to the empty chambers of approximately 0.20 ppm. During the experiments, growth of the plants increased their effective area for absorbing 0g and the input levels were increased accordingly to maintain a steady exit concentration. Throughout these experiments, therefore, the 0g levels quoted will refer to the effective dosages received by the plants, in terms of the concentrations absorbed by the plant materials, rather than to the concentrations in the atmosphere to which they were exposed. 3. Main Effects on Bean Growth A. Effects of Ozone (a) Ozone dosage The effects of 0g on bean growth were studied in all experiments since each experimental design included untreated controls in filtered air as well as plants receiving 0g. Specific comparisons of dosage level were made in Experiments J , K and L (See Materials and Methods, pp.59-61). In these experiments, plants were exposed daily from 10 to 22 days after sowing, to low levels at an effective dosage of 0.04 -0.05 ppm 0g, or to higher levels at 0.09 - 0.10 ppm. Table II presents a summary of the results for the three experi-ments, expressed in terms of differences from control plants maintained in filtered air. TABLE II Effects of ozone dosage on bean growth parameters -a summary table of differences from controls. Ozone Levels Primary Leaves Trifoliate Dry Weight (g) Stem Dry Weight (g) Root Dry Weight (g) % Reflectance 2 Area (cm ) Dry Weight (g) Experi-ment J K J : K L J K L J K L J K L J K L Low High (+) (-) + + (-) (+) (-) (-) (+) (-) +* - (-)** + (+) (-) + (+) (+) (+) (-) " + Significant increase at the 5 percent level. Significant decrease at the 5 percent level. () Non-significant effect at the 5 percent level. * A significant increase in dry weight accompanied by a significant decrease in primary leaf fresh weight. An almost significant decrease in dry weight accompanied by a significant decrease in fresh weight. Note: All significant effects are at the 5 percent level using a Duncan's New Multiple Range Test. Individual mean values are presented in the Appendix (Experiments J , K, L). 77. While low Og levels (0.02 to 0.04 ppm) produced apparent growth stimulations these trends in most cases were not statistically significant. However, the frequency of such observations in the present studies together with comparable observations of others have led to the suggestion (Bennett et al_., 1974) that plants exhibit a degree of adaptation to air pollutants as a result of exposure to "natural" pollutant levels during their evolutionary past. In turn, this suggestion has led to questioning the validity of the experimental use of f i l tered, pollutant-free air as a control atmosphere in air pollution studies. High Og levels significantly decreased all plant variables meas-, ured except percent reflectance which increased significantly, indicating a significant loss of chlorophyll in the primary leaves. This result was expected since Og at 0.09 ppm causes pronouced bleaching of the upper epidermis of leaves with the breakdown of the palisade cells (Ledbetter ejt ah , 1959). Significant reductions in primary leaf area and dry weight, and root dry weight have also been reported for "Pinto" bean (Manning et al_., 1973b), and soybean (Tingey et al_., 1973b). The growth reductions caused by high 0g levels (0.09 to 0.10 ppm) resulted from injury to the photosynthetic tissue leading to reduced photosynthetic leaf area. This reduction in photosynthetic capacity of the plant under 0g stress would lead to decreased translocation of photo-synthates throughout the plant and to depressed overall growth. (b) Timing of fumigation In these experiments (A, C, D, E, F, G, H, I, 0: see Materials and Methods, pp. 56 to 59 , 61 to 62 ) the bean plants received effective dosages of 0.09 to 0.10 ppm 0 3 for 12 hours per day. Table III presents a summary of results expressed in terms of differences from control plants maintained in filtered air. Early Og caused significant increases in percent reflectance of primary leaves and significant decreases in primary leaf area and dry weight in most experiments. Late Og produced greater significant increases in primary leaf percent reflectance than did early ozone. This would be expected since late Og exposure occurred during the time of primary leaf full expansion and metabolic change towards maturity when leaf susceptibility to Og injury was greatest (Tingey et a l . , 1973a; Ting and Mukerji, 1971). This increased percent reflectance resulted not only from increased Og injury, but also from premature senescence. Although a significant decrease in leaf area was observed in one experiment, no significant decrease in dry weight was recorded. Late Og might not be expected to cause a great reduc-tion in primary leaf dry weight since most of the structure of the leaf would have been laid down before Og exposure caused any apprec-iable tissue damage. Continuing Og caused significant increases in percent reflec-tance and significant decreases in area and dry weight of primary leaves in most experiments. Since Og injury was present on these leaves before their grand period of growth photosynthetic area would have been reduced sufficiently to impair growth. 79 TABLE III Effects of ozone fumigation timing on bean growth parameters -a summary table of differences from controls . Ozone Timing P R I M A R Y L E A V E S % Reflectance p Area (cm ) Dry Weight Experiment C D E F G H I 0 A C D E F 0 A C D E F 0 Early (-) + + - (-) - (-)(-) -Late + + + + + (-) .( + ) (+) Continuing + + + + + + + - (-) - - - (-) - (-) - -Note: See Table II for explanation of the symbols . Individual mean values are presented in the Appendix (Experiments A, C, D, E, F, G, H, I, 0). B. Effects of Rust Infection (a) Intensity of infection Three infection levels of bean rust were used; light (less than 50 pustules per 1-inch [2.54 cm] diameter leaf disc), medium (50 to 75 pustules per disc); and heavy (about 350 pustules per disc) (Experiments D, F, G, J , K; see Materials and Methods, pp. 5 6 to 60 )• Light early infection in the absence of 0^  in one trial significantly increased percent reflectance (Table IV). Primary leaf area was decreased significantly in one case. No significant effect TABLE IV Effects of intensity of rust infection on bean growth parameters -a summary table of differences from controls. Infection Level Primary Leaves Trifoliate Stem Root % Reflectance Area Dry Weight Dry Weight Dry Weight Dry Weight Experiment D F; G J K D F J K (cm2) D F J K (g) J K (g) J K (g) J K (g) Light (+)(+) + - (-) (+)(-) Medium ( + ) - (-) (-) -Heavy + - + - - -Note: See Table II for explanation of the symbols . Individual mean values are presented in the Appendix (Experiments D, F, G, J , K). 81 on primary leaf dry weight was recorded. With medium infection, primary leaf area, and stem and root dry weights were significantly reduced. Heavy infection created a significant increase in percent reflectance and dry weight of primary leaves and significant decreases in primary leaf area, and trifoliate leaf, stem and root dry weights. The significant increase in dry weight of primary leaves under heavy infection is attributed partly to the weight of the large number of sporulating rust pustules. This heavy infection caused premature senescence which was the reason for a significant increase in percent reflectance. (b) Timing of inoculation Timing of inoculation was either early (10 days after sowing), or late (16 days after sowing). Only the primary leaves were inoculated. The light infection level was used in these experiments. (Experiments A, C, D, E, F, G; see Materials and Methods, pp. 56 to 60). Early inoculation caused a significant increase in percent reflectance in one run (Table V). Primary leaf area decreased significantly in one case. Since all treatments were of light infection levels, large significant effects were not anticipated,as was pointed out in the previous section on trials dealing with infection intensity. Late inoculation produced no significant effects on percent reflectance or growth. The only symptoms of infection after 6 days were immature, white sori. At this early stage of rust development TABLE V Effect of timing of rust infection on bean growth parameters -a summary table of differences from controls. Inoculation Timing P R I M A R Y L E A V E S % Reflectance Area (cm ) Dry Weight (g) Experiment C D E F G A C D E F A C D E F Early (+) (+) + (-) (+) (-) Late (-) (-) (+) (+) (+) (-) (-) (+) (+) Note: See Table II for explanation of the symbols. Individual mean values are presented in the Appendix (Experiments A, C, D, E, F, G) . 00 83 there was no evidence of a significant reduction in chlorophyll con-tent, as measured by reflectance. It has been reported that although there is no decline in photosynthetic activity per unit of chlorophyll (Allen, 1942), the chlorophyll content in the leaves is reduced by infection, so that the overall photosynthetic activity of the leaves is reduced and like-wise plant growth . The greater the intensity of infection the greater is the stress on the plant. Similarly, the earlier the infection occurs on the leaves, the greater its effect on that part of the plant. A reduction in photosynthesis usually accompanies chlorophyll loss in the late stages of infection (Allen, 1954; Livne, 1964b). C. Effects of Halo Blight Infection All treatments involved early inoculation (10-day-old plants). (Experiments L, 0; see Materials and Methods, pp. 60-61). The presence of bacteria caused a significant decrease in primary leaf area and dry weight, tr i fol iate, stem and root dry weights (Table VI). Experiment 0 was harvested 31 days after sowing and Experiment L, 22 days after sowing. Pseudomonas phaseolicola produced on susceptible leaves the characteristic symptoms of necrotic lesions with chlorotic haloes. At the time of inoculation the trifoliate leaves were already unfolding and were thus also saturated with the inoculum suspension. Some of the tr i fol iates, as a result, became infected to produce large "haloes." The expansion of these 84 haloes to large irregular chlorotic to necrotic areas caused signif i -cant decreases in leaf area. Such reductions in leaf area impaired photosynthetic efficiency, which in turn led to overall decreases in plant growth. Chlorotic areas around the haloes have been attri-buted to a toxin ("halo-inducing toxin") produced by the bacterium (Waitz and Schwartz, 1956) which would further contribute to the observed plant growth reductions. TABLE VI Effects of halo blight infection on bean growth parameters -a summary table of differences from controls. Inoculation Primary Leaves Tri fol -iate, . Stem Root % Reflectance Area (cm2) Dry Wt. (g) : Dry Wt. (g) Dry Wt. . ( g ) Dry Wt. (g) Experiment 0 L 0 L 0 L 0 L 0 L 0 Early (+) - - - - (-) - (-) -Note: See Table II for explanation of the symbols. Individual mean values are presented in the Appendix (Experiments L and 0). 85 4. Interactions between Ozone and Rust on Bean Growth A. Richards' Diagrams (Factor Diagrams) In much of the experimental work described in this thesis, interactive effects between treatments predominate. Accordingly, to make the nature of such interactions and their quantitative value more apparent, extensive use has been made of the diagramatic representation of the results of factorial experiments devised by Richards (1941). In such diagrams, the abscissae normally represent successive increments in the level of a single factor or the introduction of additional factors. For example, in a 2 x 3 factorial design: ^ \ L e v e l Level Factor 2 1 2 3 F a c 1 A B C t 0 r 2 D E. F 1 the abscissa for a Richards' diagram runs from zero to 3, with treat-ment A corresponding to zero, treatments B and D corresponding to 1, C and E to 2 and F to 3, i .e . , each factor moves one increment on the abscissa corresponding to the next level of that factor in the experi-mental design. Such diagrams have a linear abscissa, but this does not prevent their use for factors which vary non-1inearly. 86 The particular usefulness of the Richards' diagram is its ability to illustrate the form and magnitude of interactions. Thus, where there are no interactions between two factors, the diagram takes the form of a parallelogram or parallelograms, with each factor acting independently. Where an interaction occurs, this is immediately revealed by the absence of parallelism. For example: D -*E-0 1 2 3 Factors (Solid lines connect treatments involving levels of Factor 1; broken lines connect treatments involving levels of Factor 2). Here there is no interaction between the effects of Factor 1 at the higher levels of Factor 2 (parallelogram B,C F E). At lower levels of Factor 2, however, the interaction is revealed by the shape of quadri-lateral A B E D . Furthermore, the nature of the interaction of Factor 2 with Factor 1 is negative, since (E - D) < (B - A); the size of the interaction can be measured in terms of the distance between the midpoints of the two diagonals of the quadrilateral, which is the difference between the means of the diagonals in the corresponding two-way table of data. 8 7 For example, in Experiment L, where three harvests were made, typical results such as those depicted in Figure 9 were obtained. The inclusion of time as a factor in this diagram reveals that there were few interactions with time, as shown by the parallelism of those combinations of factors involving subsequent harvests. This was found to be generally true and, since effects revealed at 16 days were magnified at 22 days, only the latter data will be presented for discussion. B. Effects of Level of Infection and Level of Ozone on  Bean Growth The results of specific treatments in Experiments G, J and K (See Materials and Methods, pp. 56 to 60 and Appendix) are presented in Figure 10 and show that at low O3, significant increases in percent reflectance (Og damage) of the primary leaves occurred in the presence of either medium or heavy infection (6a). This increased injury did not occur in the absence of the fungus (5a). With both medium and heavy infection at low ozone levels the dominant stress on the plant was due to the fungus (6a) rather than to the low ozone (5a). On the other hand, under high Og exposure, the dominant stress was due to the pollutant (5a-5b), with non-significant effects of the pathogen (5b-6b). Infection reduced the effect of high 0 3 (5a 5b 6b 6a is not a parallelogram) indicating an interaction between infection and Og stresses. While medium infection reduced the effect of high Og, (6a-6b vs 5a-5b) this reduction was . much greater under heavy infec-tion. This is demonstrated by a smaller but s t i l l significant high 88 Figure 9 Effect of level of ozone in the presence or absence of medium rust infection on primary leaf dry weight. Experiment K. Factor diagrams of primary leaf dry weight (g) after 6 days and 12 days (16-day-old and 22-day-old plants respectively) of treatment. Solid lines connect treatments with increasing ozone levels. Broken lines connect treatments which compare the effect of infection with rust fungus. Dotted lines connect the same treat-ments over the time period from 6 days to 12 days of treatment. Treatments Day 10-Day 16 Day 17-Day 22 1 filtered air. filtered air. 2 filtered air -inoculated on day 10. filtered air. 5a daily low Og. daily low 0g. 5b daily high Og. daily high 0g. 6a daily low O3 -inoculated on day 10. daily low 0g. 6b daily high O3 -inoculated on day 10. daily high 0g. Significantly different at the 5 percent level using a Duncan's New Multiple Range Test. o CN CO 00 CN CN o CN ( 6 ) ; M Xjp Figure 10 Effect of level of ozone and level of rust infection on primary leaf injury (percent reflectance). Experiments J and K. Factor diagrams of reflectance of primary leaves of bean plants after 12 days (22-day-old plants) of treatment Solid lines connect treatments with increasing ozone levels. Broken lines connect treatments which compare the effect of infection with rust fungus. Treatments Day 10-Day 22 1 filtered air. 2 filtered air-inoculated on day 10. 5a daily low Og. 5b daily high Og. 6a daily low O3 -inoculated on day 10. 6b daily high Og -inoculated on day 10. Significantly different at the 5 percent level using Duncan's New Multiple Range Test. Medium infection level (50-75 pustules per 1-inch leaf disc). Experiment K. Heavy infection level (about 350 pustules per disc). Experiment J 92 Og response with medium infection compared to the non-significant response with heavy infection (6a-6b). These same trends were recorded for light infection (Figure 11). High Og caused a significant rise in percent reflectance both in the presence (6b) and absence (5b) of the fungus, but in the presence of light infection the magnitude of Og injury was reduced (2-6b vs 1 -5b). Figure 12 reveals that primary leaf area in low Og was signif i -cantly reduced by the presence of medium and heavy infection (5a-6a). Thus, the pathogenic stress was greater than the pollutant stress on this aspect of plant growth under.low Og fumigation. In the presence of high Og, primary leaf area was significantly depressed under all levels of infection (6b) including light infection (Figure 13). While light and medium infection did not significantly influence the high Og response, heavy infection significantly increased i t (5a-5b.vs 6a-6b) (Figure 12). This contrasts with the protective effect of heavy infection on injury by high Og revealed by reflectance measurements. Primary leaf dry weight under low Og was significantly increased in the presence or absence of medium infection (Figure 14) without interaction (l-5a, 2-6a). However, heavy infection had no significant effect on low Og response (Figure 14). Hence, with medium infection the pollutant stress predominated (l-5a, 2-6a), while with heavy infection, the pathogen stress exerted the major effect (1-2, 5a-6a). Light and medium infection (6b) (Figures 13-14) did not modify the high Og stress (5b) which greatly reduced primary leaf dry weight, but heavy infection significantly increased primary leaf dry weight response to high 0^  exposure (5b-6b) (Figure 14). Figure 11 Effect of level of ozone and light rust infection on primary leaf injury (percent reflectance). Experiment G. Factor diagram of reflectance of primary leaves of bean plants after 12 days (22-day-old plants) of treatment. Solid lines connect treatments with increasing ozone levels. Broken lines connect treatments which compare the effect of infection with rust fungus (< 50 pustules per 1-inch leaf disc). Treatments Day 10-Day 22 1 filtered air. 2 filtered air -inoculated on day 10. 5b daily high Og. 6b daily high O3 -inoculated on day 10. Note: See Figure 10 legend for explanation of symbols. 26r <D O S 24 -20r 18h 16 k 14r 12h 10 Figure 12 Effect of level of ozone and level of rust infection on primary leaf area. Experiments J and K. Factor diagrams of area (cm ) of primary leaves of bean plants after 12 days (22-day-old plants) of treatment. Treatments Day 10-Day 22 1 filtered air. 2 filtered air -inoculated on day 10. 5a daily low Og. 5b daily high Og. 6a daily low O3 -inoculated on day 10. 6b daily high O3 -inoculated on day 10. Note: See Figure 10 legend for explanation of symbols. Figure 13 Effect of level of ozone and level of rust infection on primary leaf area and dry weight. Experiments D and F. Factor diagrams of area (cm ) and dry weight (g) of primary leaves of bean plants with light infection level (< 50 pustules per 1-inch leaf disc) after 12 days (22-day-old plants) of treatment. Treatments Day 10-Day 22 1 filtered air. 2 filtered air -inoculated on day 10. 5b daily high Og. 6b daily high 0 3 -inoculated on day 10. Note: See Figure 10 legend for explanation of symbols. 00 CN CN O CN (6) } M Xjp CN o CO o ( OI3) B8JB o o o o o CN Figure 14 Effect of level of ozone and level of rust infection on primary leaf dry weight. Experiments J and K. Factor diagrams of dry weight (g) of primary leaves of bean plants after 12 days (22-day-old plants) of treatment. Treatments Day 10-Day 22 1 filtered air. 2 filtered air -inoculated on day 10. 5a daily low Og. 5b daily high Og. 6a daily low O3 -inoculated on day 10. 6b daily high O3 -inoculated on day 10. Note: See Figure 10 legend for explanation of symbols. 100 ( 6 ) - J M / j p 101 The significant increase in primary leaf dry weight under high Og exposure in the presence of heavy infection could be attributed to several factors. The presence of a large number of rust pustules with their green islands resulted in the retention of a relatively large portion of the leaf free of premature senescence caused by the Og fumigation. While ozone injury was observed within the green islands, it was most severe around their peripheries as shown in Figure 15. These green islands would act as sinks in which metabolites would have accumulated, while the metabolites in ozone-induced senescent areas would have been translocated to other parts of the plant or have been lost in areas of Og damage. Rust infection has been reported to induce abnormal phloem transport of nutrients in bean plants (Livne, 1964a; Pozsar and Kiraly, 1964). The translocation of labeled sub-stances out of uninfected secondary leaves to infected primary leaves increased considerably, but mobilization of nutrients out of infected leaves was almost fully inhibited. Wang (1961) has shown, in the case of bean rust, that these green islands are photosynthetically active and that starch tends to accumulate in them to a greater extent than in surrounding areas. Hansen and Stewart (1970) reported that starch retention was observed in leaves showing no visible Og damage but not in those leaves with visible damage. Thus, the pro-tective effect of fungal infection against Og injury may be due to starch accumulation in the green islands and immediately adjacent tissue. Infection of plants is characterized by an increase in respiration as well as an increase in weight of the infected tissue, 1 m m Figure 15 Chronic ozone injury adjacent to pustules. Early inoculation, late ozone (see Figure 8 , Treatment 4). 103 which has been attributed to an increased synthesis of carbohydrate (Goodman et^  al_., 1967). In rust-infected bean leaves this was shown to be due to enhanced dark fixation of COg at the infection sites (Zaki and Mirocha, 1965). In filtered air and in low Og, trifoliate leaf weights were significantly reduced by infection of the primary leaves with medium or heavy inocula (Figure 16). This loss of weight was proportionately greater under heavy than under medium infection and eliminated a significant low Og stimulation in the absence of infection (5a). Similarly, under high Og treatment trifoliate leaf dry weights were significantly decreased in the presence of heavy infection but not with medium infection (6b). The lack of an interaction effect with medium infection is demonstrated by the near-parallelogram (5a 5b 6b 6a), although the effect of infection was significant in low ozone (5a-6a), but failed to reach significance in high ozone (5b-6b). Since the trifoliate leaves were not inoculated with the fungus, few, i f any, pustules developed on them. Therefore, any effects of infection on these leaves have to be indirect effects of primary leaf infection on the entire plant. Where heavy rust infection reduced Og injury to primary leaves (Figure 10), the accumu-lation of photosynthates would have been greater in these infected leaves than in the non-infected ones which suffered from greater Og injury. Although some workers claim that mobilization of nutrients out of infected leaves is inhibited (Livne, 1964a; Pozar and Kiraly, 1964), some of these metabolites may have been translocated to the 104 Figure 16 Effect of level of ozone and level of rust infection on trifoliate dry weight. Experiments J and K. Factor diagrams of dry weight (g) of trifoliate leaves of bean plants after 12 days (22-day-old plants) of treatment. Treatments Day 10-Day 22 1 filtered air. 2 filtered air -inoculated on day 10. 5a daily low O 3 . 5b daily high Og. 6a daily low O 3 -inoculated on day 10. 6b daily high O 3 -inoculated on day 10. Note: See Figure 10 legend for explanation of symbols. 105 ( 6 ) - J M X j p 106 trifoliates, especially during the earlier stages of infection before the appearance of green islands and sporulation, which could account for the decrease in Og response of trifoliate leaf dry weight of plants whose primary leaves were heavily infected. An alternative explanation for this decreased response of heavily infected plants to high Og is that the fungus may produce a kinetin-like substance (Kiraly et al_., 1966; Pozsar and Kiraly, 1966) which could be translocated to other plant parts and thus offer pro-tection against Og damage (Adedipe and Ormrod, 1972; Adedipe et a l . , 1973). Under low Og exposure the effect of heavy infection counter-acted the low Og stimulation (Figure 16), probably as a result of stimulated fungal growth at the expense of translocation to the trifoliates. The effect of heavy fungal infection on the primary leaf dry weight was to increase i t at all levels of Og exposure, while its effect on trifoliate dry weight was to decrease it at all levels of Og. These results agree with other reports that rust infection induces translocation of substances out of uninfected secondary leaves to infected primary leaves and inhibits mobilization of nutrients out of infected leaves (Livne, 1964a; Pozsar and Kiraly, 1964). The same effect was evident under medium infection, but to a smaller degree. The fewer number of infection sites produced non-significant effects on primary leaf dry weight and decreased trifoliate dry weight, but much less than in the case of heavy infection. Under medium infec-tion, no interactions occurred between the fungus and 0-, response at 107 all levels of 0 3 exposure. The effects of both low and high 0 3 levels were similar on dry weights of primary and trifoliate leaves with a low 0 3 stimulation and a marked high 0 3 depression. Stem dry weight (Figure 17) and root dry weight (Figure 18) responded similarly to trifoliate dry weight. These growth components were significantly reduced under low 0 3 exposure and medium or heavy infection (6a) while, in the absence of infection (5a), there was l i t t le response to low 03- In this case the dominant stress was that of infection. In high 0 3 , the plant response was neutralized by heavy infection, while medium infection reduced the magnitude of the decreases in weight caused by ozone. Thus, with medium infection, the effect of ozone predominated, but with heavy infection, the reductions in weight were largely the result of the presence of the fungus. C. Effects of Timing of Inoculation and Timing of Fumigation  on Bean Growth Response to Ozone Experiments (A, C, D, E, F, G; see Materials and Methods, pp. 57,59), performed to test the effects of timing of inoculation on 0 3 response, showed few significant effects since infections were light (Table VII). The high 0 3 stress was, in general, greater than any infection stress. The results of Experiment G are typical (Figure 19) and show that late inoculation (10) had no significant effect on injury caused by continuing ozone treatment. However, early inoculation (6b) reduced this injury significantly (5b-6b), although there was also a signif i -108 Figure 17 Effect of level of ozone and level of rust infection on stem dry weight. Experiments J and K. Factor diagrams of dry weight (g) of stems of bean plants after 12 days (22-day-old plants) of treatment. Treatments Day 10-Day 22 1 filtered air. 2 filtered air -inoculated on day 10. 5a daily low Og. 5b daily high Og. 6a daily low O3 -inoculated on day 10. 6b daily high O3 -inoculated on day 10. Note: See Figure 10 legend for explanation of symbols. (6) } M Xjp 110 Figure 18 Effect of level of ozone and level of rust infection on root dry weight. Experiments J and K. Factor diagrams of dry weight (g) of roots of bean plants lifter 12 days (22-day-old plants) of treatment. Treatments Day 10-Day 22 1 filtered air. 2 filtered air -inoculated on day 10. 5a daily low Og. 5b daily high Og. 6a daily low O3 -inoculated on day 10. 6b daily high O3 -inoculated on day 10. Note: See Figure 10 legend for explanation of symbols. (6) - J M Ajp TABLE VII Interaction effects of timing of inoculation at a given timing of ozone (all high ozone and light inocu-lation) - a summary table of differences from unfumigated controls. Inoculation Timing Ozone Timing P R I M A R Y L E A V E S % Reflectance 2 Area (cm ) Dry Weight (g) Experiment C D E F G A C D E F A C D E F Late Early (+) (-) + (+)(+) (+) (-)(-) (+) Early Late (+) - (-). Early Continuing - - (-) (-) (-) (-) Late (-) (+)(+) (+) (+)(+) .(+) Note: See Table II for explanation of the symbols. Individual mean values are presented in the Appendix (Experiments A, C, D, E, F, G). 113 Figure 19 Effect of timing of inoculation in the presence or absence of continuing ozone on primary leaf injury (percent reflectance). Experiment G. Factor diagram of percent reflectance of primary leaves of bean plants with light infection (< 50 pustules per 1-inch leaf disc) after 12 days (22-day-old plants) of treatment. Treatments Day 10-Day 16 Day 17-Day 22 1 filtered air. filtered air. 2 filtered air-inoculated on day 10. filtered air. 5b daily high Og, daily high Og. 6b daily high O3 inoculated on day 10. daily high Og. 8 filtered air. filtered air -inoculated on day 17. 10 daily high Og. daily high O3 inoculated on day 17. Note: See Figure 10 legend for explanation of symbols. 114 115 cant increase in injury caused by ozone in spite of the presence of the fungus (2-6b). Furthermore, established fungal infection successfully prevented an increase in reflectance caused by late fumigation (Figure 20, quadrilateral 1 2 4 3), but was not able to prevent a decrease in leaf area (Figure 21) although this was not accompanied by any significant effect on leaf dry weight (Figure 22). There were few significant effects of timing of inoculation on primary leaf area or dry weight (Table VII). Timing of ozone fumigations, on the other hand, significantly influenced reflectance and leaf area and dry weight (Table VIII). Figure 20 shows that late ozone treatment resulted in approximately half the injury caused by continuing treatment (1, 3, 5b) in the absence of rust, while the presence of the fungus successfully countered the effect of late fumigation (2, 4, 6b). When initiation of ozone fumigation was delayed (late 0^  treatments, Figure 23B) i t permitted the fungus to develop more rapidly than when ozone treatment began at the time of inoculation (Figure 23C). As a consequence, in such treatments the green-island effect was more apparent than in treatments in which ozone was administered from the outset. In contrast, early ozone appeared to have somewhat less effect on injury and may have facilitated fungal development, since late inoculation caused a significant increase in reflectance (Figure 20) (9-7). This supports earlier work by Manning et al_. (1969; 1970) in which Og injury to leaves of potato and geranium increased the susceptibility of the plants to infection by the fungus Botrytis cinerea. The effects of 116 Figure 20 Effect of ozone timing in the presence or absence of early (A) or late (B) light inoculation on primary leaf injury (percent reflectance). Experiment G. Factor diagrams of percent reflectance of primary leaves of bean plants after 12 days (22-day-old plants) of treatment. Treatments Day 10-Day 16 Day 17-Day 22 1 filtered air. filtered air. 2 filtered air -inoculated on day 10. filtered air. 3 filtered air. daily high 0g. 4 filtered air -inoculated on day 10. daily high Og. 5b daily high Og. daily high Og. 6b daily high O3 inoculated on day 10. daily high O3. 7 daily high Og filtered air -inoculated on day 17. 8 filtered air. filtered air -inoculated on day 17. 9 daily high Og filtered air. 10 daily high Og daily high O3 inoculated on day 17. Note: See Figure 10 legend for explanation of symbols. 117 Figure 21. Effect of ozone timing in the presence of absence of early (A) (Experiment D) or Late (B) (Experiment E) light inoculation on primary leaf area. Experiments D and E. Factor diagrams of area (cm ) of primary leaves of bean plants after 12 days (22-day-old plants) of treatment. Treatments Day 10-Day 16 Day 17-Day 22 1 filtered air. filtered air. 2 filtered air -inoculated on day 10. filtered air. 3 filtered air. daily high 0g. 4 filtered air -inoculated on day 10. daily high 0g. 5b daily high Og. daily high 0g. 6b daily high O3 inoculated on day 10. daily high 0g. 7 daily high Og. filtered air -inoculated on day 17. 8 filtered air. filtered air -inoculated on day 17. 9 daily high Og. filtered air. 10 daily high Og. daily high O3 inoculated on day 17. Note: See Figure 10 legend for explanation of symbols. 119 Figure 22 Effect of ozone timing in the presence or absence of early (A) (Experiment D) or late (B) (Experiment E) light inoculation on primary leaf dry weight. Experiments D and E. Factor diagrams of dry weight (g) of primary leaves of bean plants after 12 days (22-day-old plants) of treatment. Treatments Day 10-Day 16 Day 17-Day 22 1 filtered air. filtered air. 2 filtered air -inoculated on day 10. filtered air. 3 filtered air. daily high 0g. 4 filtered air - daily high 0,. inoculated on day 10. 5b daily high Og. daily high 0g. 6b daily high O3 - daily high 0 V inoculated on day 10. 7 daily high 0^ ,. filtered air -inoculated on day 17. 8 filtered air. filtered air -inoculated on day 17. 9 daily high Og. filtered air. 10 daily high 0Q. daily high 0g -inoculated on day 17. Note: See Figure 10 legend for explanation of symbols. o CN 00 CN (6 ) ' } M Xjp TABLE VIII Interaction effects of timing of ozone at a given timing of inoculation -a summary table of differences from unfumigated controls-P R I M A R Y L E A V E S Ozone Timing Inoculation Timing % Reflectance Area (cm ) Dry Weight (g) Experiment C D E F G A C D E F A C D E F Early Late ( + ) + + - (-)(-) (-) Late Early + ( + ) - + Early + + + - (-) Continuing Late + + + - - (+)(-) H Note: See Table II for explanation of the symbols-Individual mean values are presented in the Appendix (Experiments A, C, D, E, F, G). 123 Figure 23 Premature senescence in presence of ozone and rust infection. (A) Early inoculation control (see Figure 8, Treatment 2). (B) Early inoculation, late ozone (see Figure 8, Treatment 4). (C) Early inocu-lation, continuing ozone (see Figure 8, Treatment 6). I I 1 c m 125 late and continuing ozone on reflectance are mirrored in the effects on leaf area (Figure 21). In contrast, early fumigation (1-9) resulted in proportionately greater reductions of leaf area and dry weight than continuing exposures (9-5b) (Figures 21 and 22). That is , the reductions due to early fumigation were greater than that part of the continuing exposure corresponding to late fumigation (1-9) > (9-5b). Reflectance'measurements were greatest under late ozone expos-ure when leaf susceptibility to ozone injury was greatest, but growth effects were greatest under early ozone exposure at the time of greatest rate of plant growth. During this period of rapid growth any environmental stress such as ozone would impair plant development. Short duration of stress could be overcome by compensatory growth (Leopold, 1964) upon its release. However, prolonged stress, such as in the case of early ozone exposure which continued throughout the grand period of primary leaf growth, would impair the final develop-ment since compensatory growth would not occur. The concentration and timing of ozone exposure determined the degree of ozone-induced senescence. Exposures to 0.10 ppm ozone greatly accelerated premature senescence (3, 5b) (Figure 24) which occurred generally within 10 to 12 days after commencement of fumi-gation (20-22-day-old plants), while control plants receiving no ozone treatment did not senesce even when 30-40 days old. Plants exposed to early 0g (Figure 24, Treatment 9) showed no premature senescence of primary leaves while those exposed to late 0g (Treatment 3) and continuing 0^  (Treatment 5b) revealed enhanced senescence. 126 Figure 24 Effect of timing of ozone in the presence or absence of early (A) (Experiments D and G) or late (B) (Experiments C, E and G) light rust infection on primary leaf senescence-Experiments C, D, E and G. Factor diagrams of percent of primary leaves senesced of bean plants after 12 days (22-day-old plants) of treatment. See Figure 10 legend for explanation of symbols. Senes-cence was defined as at least 75 percent of the leaf turned yellow or bleached . Treatments Day 10-Day 16 Day 17-Day 22 1 filtered air . f i 1tered ai r . 2 filtered air-inoculated on day 10. filtered a i r . 3 filtered a i r . daily high 0g • 4 filtered air-inoculated on day 10 . daily high 0g • 5b daily high Og daily high Og • 6b daily high 0g-inoculated on day 10. daily high Og • 7 daily high Og • filtered air • inoculated on day 17. 8 filtered air . filtered air • inoculated on day 17 . 9 daily high Og filtered a i r . 10 daily high Og • daily high Og inoculated on day 17 . 128 These findings support reports that the leaf's susceptibility to ozone is associated with a metabolic change toward maturity (Macdowall, 1965b; Ting and Mukerji, 1971; Tomlinson and Rich, 1968). The most susceptible period is a week or so after the leaf's grand period of growth. Late fungal inoculation had l i t t le effect on ozone-induced premature senescence as shown by the near parallelograms 9 5b 10 7 and 18 7 9 of Figure 24. Early fungal infection acted synergistically with late Og fumigation in giving rise to accelerated senescence (quadrilateral 12 4 3) but reduced the senescence induced by continuing Og as shown by quadrilateral 3 4 6b 5b. Apparently, senescence is accelerated by the presence of well-established rust infection and this is enhanced by late Og fumigation. The relative decrease in enhancement of rust-induced senescence which occurred when continuing fumigations were used probably reflects an impairment in the development of the fungus. Thus, in terms of senescence, the presence of established infection prior to fumigation leads to accelerated senescence, while development of the fungus and concurrent fumigation leads to a reduction in enhanced senescence. D. Effects of Ozone on Pathogenesis Ozone modified the development of primary pustules by increas-ing their number but decreasing their size, as shown in Table IX and Figure 25. The observed reduction in pustule size confirms observations made on crown rust on oats (Heagle, 1970), while the increased number TABLE IX Number and size of primary pustules produced in presence (+) and absence (-) of ozone3. Treatment Days Mean no. of Pustules0 Treatment Mean Square Mean Diameter of Pustules0 Treatment Mean Square 12 9.9 18.7 532.5d 12 3.3 6.8 98.9l 10.5 6.2 150.6C 16 7.4 6.4 4.51 17 3.2 5.5 34.6' 9.9 6.2 54.5l With an amount of 0.1 ± 0.01 ppm ozone present during 14-h diurnal photoperiod . ^Pustules counted on four randomly selected 16-mm-diameter disks from each primary leaf of each of eight plants per treatment. cFifteen units = 1 mm. Twenty pustules measured at random on each primary leaf of each plant. Signif icant at P = 0.01 . 130 Figure 25 Reduced pustule size and development of secondary pustules under ozone treatment. (A) Control, (B) Ozone treated. 131 of primary pustules must, in some way, reflect either enhanced uredos-pore germination or penetration of the host, or a reduction in the abortion of incipient infections. Heagle (1970) was unable to detect any effect of ozone on spore germination, appressoria formation, or penetration in the case of crown rust. The reduction in pustule size which can be clearly seen on visual inspection of infected leaves may be partly due to a retardation in the rate of fungal development before sporulation. The data in Table IX show that, once mature, the pustules do not change in size between 12 and 17 days of ozone treatment. Concomitant with pustule development was the development of symptoms of chronic injury to those plants fumigated with ozone. As these symptoms progressed, so the development of the rust was retarded (Figure 26). This was especially true of the intercostal lamina, which was more severely injured by ozone and on which the greatest retardation of pustules was observed, while, closer to the veins, injury was less and pustules developed more rapidly. Ozone treatment induces numerous changes in the metabolism of plant leaves, as well as causing visible injury. For example, changes in the levels of carbohydrates, l ipids, organic acids, ribonucleic acid fractions, and other constituents have been reported for various species, tissues and fumigation conditions (Dugger and Ting, 1970; Chang, 1971). The impaired development of the fungus could thus be caused by the disturbance of the normal metabolic processes for the host. Alternatively, ozone could affect the growth of the fungus per se. Few obligately parasitic fungi have to date 132 Figure 26 Development of symptoms of chronic ozone injury with accompanying retardation of pustule development. (A) Control, late inoculation (see Figure 8, Treatment 8). (B) Continuing ozone, late inoculation (see Figure 8, Treatment 10). 133 i i 1 c m 134 been successfully cultured axenically. Hence i t has not been possible to determine the degree of susceptibility of Uromyces phaseoli to ozone directly. Effects of high doses of ozone on fungal spore germin-ation and germ tube development ranging from no effect to marked in-hibition have been reported (Hibben and Stotzky, 1969). In some cases ozone caused enhanced sporulation (Treshow et^  al_., 1969). It was observed that the later stages of rust development on bean leaves subjected to ozone were additionally modified. Not only were the pustules reduced in size but there was considerable develop-ment of secondary pustules at the margins of the green islands surround-ing each primary pustule, as illustrated in Figure 25. Such secondary pustule development is frequently found in rust-infected plants subjected to stress such as nitrogen deficiency. As a consequence the effects observed with ozone may reflect action on the host rather than on the fungus itself . While no measurements of total uredospore yield have been made, i t appears that the net consequence of reduced pustule size, greater pustule frequency and secondary pustule development is the maintenance of inoculum potential by the fungus in the face of impairment of the normal metabolism of the host. A similar conclusion was drawn by Treshow et aj_. (1969) from observations of increased sporulation of Alternavia oleraoeae in culture or agar. 135 5. Interactions Between Ozone and Halo Blight A. Effects of Infection and Level of Ozone on Bean Growth Results of Experiments L and 0 (see Materials and Methods, pp. 60-61 and Appendix) indicate that bacterial infection in the pres-ence of low or high Og levels significantly reduced primary leaf area (Figure 27) and the dry weight of primary and trifoliate leaves (Figure 28) . The effects on stem dry weights resembled those on trifoliate leaf weights. No interaction between infection and Og response was found in primary leaf area, and trifoliate dry weight, as shown by the presence of the parallelograms 5a 6a 6b 5b . The absence of parallelograms in the primary leaf dry weight and root dry weight diagrams (Figures 28-29) indicates interactions between infection and Og response. The response of primary leaf dry weight to high Og was significantly enhanced by the presence of infection (6b). This was a synergistic effect between Og and infection in reducing primary leaf dry weight. The response of root dry weight to high Og (Figure 29) was not significantly enhanced by the presence of infection (6b), therefore an additive effect was present (statistically quadrilateral 5a 6a 6b 5b was not significantly different from a parallelogram). Likewise, the relationship between infection and Og response in primary leaf area, tr i fol iate, and stem dry weights were additive. In no case did a protective effect of infection against Og injury reveal itself in the growth studies. Therefore, such protection, i f i t existed, was of very small magnitude. The stresses imparted on plant growth components by Oo and infection separately created a significant 136 Figure 27 Effect of level of ozone in the presence or absence of early bacterial infection on primary leaf area. Experiments L and 0. Factor diagrams of area (cm ) of primary leaves of bean plants after 12 days (22-day-old plants - Experiment L) and after 21 days (31-day-old plants - Experiment 0) of treatment. See Figure 10 legend for explanation of symbols. Treatments Day 10-Day 16 Day 17-Day 22 Experiment L 1 filtered air . filtered air . 2 filtered air -inoculated on day 10. filtered air. 5a daily low Og. daily low 0g. 5b daily high 0g daily high 0g. 6a daily low 0g -inoculated on day 10. daily low 0g . 6b daily high 0g inoculated on day 10. daily high Og. Experiment 0 Day 10-Day 23 Day 24-Day 31 1 filtered air . filtered air . 2 • filtered air -inoculated on day 10. filtered air . 3 filtered a i r . daily high Og. 4 filtered air -inoculated on day 10 . daily high Og. 137 138 Figure 28 Effect of level of ozone in the presence or absence of early bacterial infection on primary and trifoliate leaf dry weights. Experiment L. Factor diagrams of dry weight (g) of primary (P) and trifoliate (T) leaves of bean plants after 12 days (22-day-old plants) of treatment. See Figure 10 legend for explanation of symbols. Treatments Day 10-Day 22 1 filtered a i r . 2 filtered air -inoculated on day 10. 5a daily low Og . 5b daily high Og. 6a daily low Og -inoculated on day 10. 6b daily high Og -inoculated on day 10. o o CO in CN o CN (6) - J M " Ajp Figure 29 Effect of level of ozone in the presence or absence of early bacterial infection on root dry weight. Experiment L. Factor diagram of dry weight (g) of roots of bean plants after 12 days (22-day-old plants) of treatment. See Figure 10 legend for explanation of symbols. Treatments Day 10-Day 22 1 filtered air-2 filtered air -inoculated on day 10. 5a daily low Og. 5b daily high O .^ 6a daily low O3-inoculated on day 10. 6b daily high O 3 -inoculated on day 10 . 141 ,05 142 reduction in each component measured. In some cases the magnitude of the Og stress exceeded that of the infection stress and vice versa as has already been pointed out (pp. 87 -92). There were trends for low Og in the presence of infection (6a) to give rise to insignificant increases in all parameters except for a significant increase in root dry weight (Figures 27-29). Such non-significant effects, however, could be due to experimental error, or to an interaction between the low Og and bacterial stresses within the plant. Previous evidence for a reduction in protein synthesis by both Og (Dass and Weaver, 1968; Tomlinson and Rich, 1968) and bacterial toxins (Goodman et aj_., 1967) is contrary to the present findings, which suggests that these results are due to experimental error. However, the above workers at all times were using very high levels of Og from 0.50 to 1.0 ppm for several hours duration. Such levels of Og would certainly have detrimental effects on plant growth. (Under low chronic levels of 0.03 to 0.05 ppm 0g the effects may be different when growth stimulation occurs.) High 0g and early inoculation (6b) significantly decreased all growth measurements. Such highly significant reductions in plant growth could be explained by large reductions in protein synthesis brought on by both 0g and bacterial stresses. Under both levels of 0g, bacterial infection reduced signif i -cantly all plant measurements except root dry weight. Since primary and trifoliate leaves were both infected with the bacterium, direct effects of the bacterium on growth would be 143 expected. In contrast to rust infection, which stimulates photosyn-. thesis, bacterial infection causes chlorosis suggestive of a direct effect upon chloroplasts, resulting in a reduction in the rate of photosynthesis (Goodman et al_., 1967). Some evidence exists that bacterial infection, like fungal infection, stimulates respiration (Goodman £t al_., 1967). While there is evidence that fungal infection induces abnormal phloem transport of nutrients (Livne 1964a; Pozsar and Kiraly, 1964), none exists in the case of bacterial infection. However, bacterial infection has been reported to block the xylem of the host tissue and to produce toxins which suppress plant growth (Goodman et al_., 1967). Reductions in primary leaf area and dry weight, tr i fol iate, root and stem dry weights caused by bacterial infection could be attributed to decreased photosynthesis, increased respiration and the presence of toxin(s). With the exception of primary leaf dry weight, growth measurements reported in these studies showed that bacterial and ozone stresses were additive with no protective effect of the bacterium against ozone response as was observed with earlier experi-ments using fungi. Since primary leaves were more heavily infected than trifol iates, a stress threshold could have been reached at which the bacterium predisposed them to more severe 0^  injury. These growth reductions in the presence of both the pathogen and ozone should not be confused with earlier reported protective effects of the pathogen against superficial ozone injury (flecking) of the upper palisade tissue immediately adjacent infection sites (Kerr and Reinert, 1968). The results of these studies are in agreement with those of Kerr and Reinert, (1968) in that, while ozone flecking appeared over most of the surface of the bean leaves, it did not occur in the "halo" areas adjacent to the sites of bacterial infection. B. Effects of Infection and Timing of Ozone on Bean Growth Established infection did not affect the response to late Og treatment (Figure 27), although late Og itself significantly decreased trifoliate and root dry weights in the presence of infection (Appendix, Experiment 0). Primary leaf area was significantly reduced in the presence of infection and late Og, but-the effect'was additive as "shown by parallelogram 1 2 4 3 of Figure 27. While late Og and early infection produced significant decreases in trifoliate and root dry weights only, continuing Og and early infection caused significant decreases in primary leaf area and dry weight and stem dry weight. These results can be explained by the fact that late Og exposure began after the primary leaves had fully expanded and since the harvesting of the plants was done 6 days later, Og injury would not have been severe enough at that time to have reduced leaf area or dry weight. C. Effects of Ozone on Pathogenesis In these investigations, direct effects of Og on the bacterium were not examined since the number and sizes of lesions were not con-sistent within any treatment. The sizes of lesions varied from a few millimeters to about 15-20 millimeters in diameter. Some inoculated leaves produced only 1 or 2 lesions while others developed numerous lesions scattered uniformly or non-uniformly over the entire lamina. 6. Effects of Cytokinins and Siderochromes 145 A. Effects of Ozone on Cytokinin Responses and Cytokinin  on Ozone Responses Increase in spectral reflectance at 550 nm, used as a measure of chronic injury, occurred as a result of daily exposure for 26 days to 0.10 ppm ozone, but not when the ozone concentration was reduced to 0.05 ppm, as shown in Table X and Figure 30 (Treatments 1, 5 & 9; 3, 7 & 11). Treatment for 8 days with BA and K significantly reduced the increase in reflectance caused by 0.10 ppm (Figure 30; 9-10 and 11-12 for BA and K respectively) without affecting the reflectance either of the controls (1-2 and 3-4) or those leaves treated with 0.05 ppm ozone (5-6 and 7-8). Conversely, low 0g had no significant effect on cytokinin response (indicated by parallelograms 2 6 5 1 and 4 8 7 3), but high 0g stress overcame any protective effect of the cytokinins (indicated by quadrilaterals 10 6 5 9 and 12 8 7 11). The effects of treatment on areas and dry weights of primary leaves are shown in Figures 31 and 32 for experiments involving 8 repeated cytokinin applications. Comparable data were obtained from an experiment with a single cytokinin application (Table X). After 42 days of ozone treatment, application of BA had resulted in signif i -cant increases in leaf area (1-2, 5-6, 9-10), which were, in turn, significantly and progressively reduced by ozone (2-6, 6-10)(Figure 31). In the case of kinetin treatment, ozone again reduced leaf area, but 146 TABLE X Effects of ozone on cytokinin responses - a summary table of differences from unfumigated controls. Ozone Level Chemical Primary Leaves Stem Root % Reflectance Area (cm2) Dry Weight (g) Dry Weight (g) Dry Weight (g) Experiment M N M N M N N N Low None BA K + + (+) (+) (+) (-) (-) + (-) (-) (-) (+) (-) -(+) (-) (-) High None BA K + + + + + + -(-) -* * Note: See Table II for explanation of the symbols. Individual mean values are presented in the Appendix (Experiments M and N) . * These effects are not available since one of each pair of primary leaves per plant was treated with BA or K . 147 Figure 30 Effects of ozone, benzyl adenine (BA) and kinetin (K) on primary leaf reflectance (550 nm) after 26 days of ozone treatment. Treatments numbered according to the table: Treatment Ozone Cytokinin Treatment Ozone Cytokinin Numbers ppm Numbers ppm 1 0 0 3 0 0 2 0 BA 4 0 K 5 0.05 0 7 0.05 0 6 0.05 BA 8 0.05 K 9 0.10 0 11 0.10 0 10 0.10 BA 12 0.10 K Comparisons marked V are significantly different at P0.05. Solid lines connect ozone treatments. Broken lines connect cytokinin treatments . 149 Figure 31 Effects of ozone and cytokinins on primary leaf area after 42 days of ozone treatment. Details as for Figure 30 . 150 151 Figure 32 Effects of ozone and cytokinins on primary leaf dry weight after 42 days of ozone treatment. Details as for Figure 30. 1 CO 153 the level of cytokinin applied was insufficient to cause significant increases in area although the trends of the data are similar to those for BA treatment. In neither case was there any appreciable inter-action between ozone and cytokinin treatments, as indicated by the parallelism between treatment effects. Primary leaf dry weight data after 42 days of ozone treatment show significant effects of ozone dosage and cytokinin treatment as illustrated in Figure 32. Again, there were no appreciable treatment interactions. The experimental design used involving comparisons of treat-ments between members of pairs of primary leaves did not permit examin-ation of the direct result of cytokinin treatment on the effects of ozone on stem or root growth since one of each pair of primary leaves per plant was treated with BA or K. However, comparisons of the dry weights of the stems or roots show significant effects of ozone when one of the primary leaves of a pair had been treated with BA or K (Table XI). Both BA and K caused increased leaf growth in the presence or absence of ozone. BA,in particul ar,,more than counteracted the effects of 0.10 ppm ozone in reducing leaf area and dry weight (Figures 31-32), but such counteraction did not occur with regard to the stem and root weight (Table XI). In spite of the evidence for immobility of exogeneous Gytokinins (Kiraly, El Hammady and Pozsar, 1967), this work indicated that treatment of one leaf of a pair with either BA or K resulted in some stimulation of the growth of the other leaf. This supports the contention of limited mobility of cytokinins (Fletcher, 1969). The mean, primary leaf areas of other plants treated concurrently with those reported in 154 TABLE XI Effects of cytokinin treatment on bean stem and root growth response to low levels of ozone9. Ozone ppm Treatment'3 Stem Dry Weight (g) Root Dry Weight (g) 0 BA 0.97a 1.16a 0 K 0.94a 1.09a 0.05 BA 0.78b l . l l 9 0.05 K 0.70b 0.91 b 0.10 BA 0.51C 0.48C 0.10 K 0.50° 0.50C aMeans not followed by the same letter are significantly different at P0.05 (Duncan's New Multiple Range Test). b0ne member of each primary leaf pairs treated with BA or K (30 ug/ml). 155 Figure 31 but with no kinin applied to either primary leaf were 2 respectively 83.8 and 100.9 cm for the filtered air and 0.05 ppm ozone treatments. Their leaf dry weights were 0.406 and 0.460 g respectively (cf. Figure 32) (see Appendix, Experiment N). With both types of measurements, therefore, the untreated leaf of a treated plant yielded significantly higher values than the leaves of an untreated plant. Measurements of the upper surfaces of chronically injured bean leaves showed an increase in the spectral reflectance peak at 550 nm which is related to the destruction of chlorophyll in the palisade mesophyll (see Materials and Methods pp. 45-57). As leaves senesce, whether naturally or more rapidly as a result of ozone treatment, reflectance increases generally throughout the range 550-700 nm. In the present experiments, the increases in reflectance resulting from ozone treatment (Figure 30) were invariably confined to. the 550 nm region, wherever BA or K had been applied, while untreated leaves yielded spectra with increased reflectance between 550 and 700 nm. These measurements confirmed the visual impression that ozone treat-ment resulted in a progressive bleaching of cytokinin-treated leaves, without appreciable yellowing. In contrast, the untreated leaves became chlorotic, although at a slower rate than those on untreated plants. The effects of cytokinins on ozone-induced senescence are presented in Figure 33. Both levels of ozone caused increases in premature senescence in the presence or absence of the cytokinins. While K offered l i t t le protection against ozone-induced senescence, BA reduced it slightly (1-2, 5-6). Treatment 10 was recorded as 156 Figure 33 Effect of ozone and cytokinins on primary leaf senescence. Experiment M. Factor diagrams of percent of primary leaves senesced of bean plants after 12 days (22-day-old plants) of treatment. See Figure 27 legend for explanation of symbols. Senescence was defined as at least 75 percent of the leaf turned yellow or bleached. 158 showing that all primary leaves had senesced, but, in fact, these leaves had not yellowed but were bleached by the high ozone level. They were s t i l l firmly attached to the plant in this condition. Root nodule numbers, fresh weights and dry weights were also decreased in the presence of ozone (Table XII). While only the reduc-tion in nodule number in BA-treated plants was significant at the low ozone level, all reductions were significant under high ozone exposure. Cytokinin treatments did not prevent reductions due to ozone. Under zero and 0.05 ppm ozone levels, BA-treated plants had greater nodule numbers, of greater fresh and dry weights than K-treated plants. However, these differences were not significant at the P0.05 level. Since no absolute control (no cytokinin, no ozone treatment) was included in the experiment, no conclusions can be drawn regarding the effect of cytokinins in the absence of ozone on these growth measurements. The reduction in nodule number, fresh and dry weights may have resulted from the penetration of ozone into the soil or from the modification of plant metabolism and/or metabolite translocation within the plant. Because of the high reactivity of ozone on coarse and moist surfaces, such as s o i l , i t is unlikely that the ozone penetrated the root medium to affect nodulation directly. Turner et aJL (1973) reported that soil is an important sink for atmospheric ozone (also see Results and Discussion pp. 72 -75) and that the degree of uptake by the soil of ozone depends on such factors as the air boundary layer near the soil surface, and the soi l 's physical and chemical properties. 159 TABLE XII Effects of ozone and cytokinin treatment on numbers and weights of root nodules3 . Ozone ppm Treatment*3 Nodule Number Nodule Fresh Weight (g) Nodule Dry Weight (g) 0 BA 106 a 0.47 a 0.053 a 0 K 70 ab 0.30 ab 0.036 ab 0.05 BA 53 be 0.29 ab 0.033 abc 0.05 K 29 be 0.16 b 0.016 be 0.10 BA 2 c 0.01 b 0.001 c 0.10 K 4 c 0.01 b 0.001 c aMeans not followed by the same letter are significantly different at P 0.05 (Duncan's New Multiple Range Test). One member of each primary leaf pairs treated with BA or K (30 ug/ml). The weights and number of all root nodules 1 mm or greater in diameter were recorded after plants were exposed to ozone for 42 days. 160 The observed significant reduction in nodule growth due to high ozone and the non-significant reduction due to low ozone demon-strate the role of greater ozone stress on the shoot in inducing a greater reduction in root development. Reductions in nodule growth by ozone treatment of 0.75 ppm for 1 hour have been reported in soy-beans (Tingey and Blum, 1973). Such acute levels of ozone would severely injure the plant shoot and thus reduce photosynthetic productivity. Reductions of chlorophyll content as described earlier (see Materials and Methods, pp. 45 - 57 ), suggest that the photosynthetic capacity of the plants was in the present study also reduced and that there was less photosynthate for translocation to the root. Earlier reports suggested that ozone reduces root growth indirectly by an effect on translocation of metabolites (Tingey et al_., 1971). The reported effects of ozone on photosynthesis and root growth support the concept that the reduction in nodulation resulted from reduced photosynthate available for trans-location to the root needed for its growth and nodule formation. B. Effects of Rhodotorulic Acid on Ozone Response Treatment with rhodotorulic acid prior to ozone exposure was found to have no effect on ozone-induced injury (see Appendix, Experiments H and I). In particular, no significant protective effect against ozone could be demonstrated. However, i t should be pointed out that Atkin and Neilands (1972) were only able to demonstrate green island formation in detached leaves. In agreement with their observations, no green islands were observed in attached, treated primary leaves in the present study. Whether the absence of effect is the result of differences in trans-location between detached and attached leaves is not known. 161 7. General Discussion A. Effects of Ozone on Bean Growth (a) Low ozone levels Most of the work described in the literature on the effects of air pollution on plants has been conducted at levels infrequently found in nature. Such studies with levels of from 0.25 to 1.0 ppm have been undertaken to obtain information as to the nature of, and casual relationships with, injurious effects on plant growth. Never-theless, in some reports increases in various growth parameters were noted in the presence of low levels of pollutants such as fluoride (Treshow et al_., 1967; Treshow and Harner, 1968; Thompson and Taylor, 1969; Hitchcock et^  al_., 1971), sulphur dioxide (Bell and Clough, 1973), and ozone (Heagle el: al_., 1972; Engle and Gabelman, 1967; Harward and Treshow, 1971; Thompson and Taylor, 1969; Ormrod, 1973). In several experiments conducted in this research additional supporting evidence was obtained in favour of "apparent" growth stimu-lations in the presence of low levels of ozone (0.02 to 0.04 ppm) over filtered-air controls (Table XIII). In contrast to work on barley and smartweed (Bennett et al_., 1974), apparent stimulation of bean growth in terms of dry weights was modest, but this might be expected since the bean plants were only 22 days old and were exposed 162 TABLE XIII Dry weights (g) of parts of 22-day-old bean plants grown in filtered air or filtered air containing ozone (0.03 ± 0 . 0 1 ppm) for 12 days. First Trifoliate Leaves Stems Primary Leaves Middle Leaflet Other Leaflets Treatment Run 1 Run 2 . Run 1 Run 2 Run 1 Run 2 Run 1 Run 2 Filtered Air 0.52* 0.58 0.32 0.30* 0.098* 0.083 0.178 0.158 Ozone 0.58* .0.61 0.32 0.36* 0.113* 0.093 0.201 0.171 Percent Increase in Ozone 11.4 4.8 0 18.2 14.8 11.4 13.0 7.9 Significant at P 0.05. 163 to ozone for only the final 12 days. On considering specific compon-ents of the growth of bean (Table XIII) there were consistent differences observed between the controls and treatments, with statistically significant increases observed in stem, primary leaf and middle leaflet of the f i rst trifoliate leaf dry weights. In cases where the observa-tion from a second experiment failed to show statistically significant differences at P 0.05, the data nevertheless show consistent trends, in many cases approaching the 5 percent level of probability. There were also consistent trends towards stimulated root dry weight, inter-node length and middle trifoliate leaflet area, although in no cases did the differences reach P 0.05. The studies described above involv-ing cytokinin treatments also provided examples of apparent stimulations of plant growth by low ozone dosages (see Results and Discussion, p. 155). These apparent stimulations in growth in relation to that in filtered air could be attributed to adaptation of plants to air pollution stress. Low levels of pollution are nothing new, since during the past, pollution has resulted from volcanism, electrical storms, f i re , and from natural photochemical oxidant reactions. In particular, low ozone levels comparable to those used in the present studies to give rise to growth stimulations are commonplace ambient concentrations(Tebbens,1968) and may therefore more truly reflect the norm than does filtered air (Bennett et al_., 1974). Thus, i f plants evolved under such conditions, they might be expected to have adapted to such pollution. 164 (b) High ozone levels High Og levels (0.10 to 0.50 ppm) have been reported to reduce significantly growth parameters such as primary leaf area and dry weight, trifoliate leaf dry weight, stem and root dry weights in poinsettia, Pinto bean (Manning et al_., 1973a; 1973c; 1973b), and soybean (Tingey et a l . , 1973b). In the research of this thesis, 0.09-0.10 ppm 0g significantly reduced the following plant parameters at the P 0.05 level of probability: 1. primary leaf area 2. primary leaf fresh weight 3. primary leaf dry weight 4. stem fresh weight 5. stem dry weight 6. root fresh weight 7. root dry weight 8. plant height 9. trifoliate fresh weight 10. trifoliate dry weight 11. trifoliate area 12. total plant fresh weight 13. total plant dry weight 14. total plant leaf area. In addition, a significant increase in plant injury (0g damage to upper surface of the primary leaves) was recorded through a signif i -cant increase of percent reflectance of leaf discs at 550 nm. 165 The observed growth reductions in 0.09-0.10 ppm 0^  may have resulted from biochemical interference in the process of photosynthesis leading to a reduction of effective photosynthetic leaf area. The degree of 0^  injury and the leaf area ratio of the plant at a given point in time are determining factors as to the degree of general plant growth reduction which might occur. These factors are influenced by the age of the plant at the time of commencement of 0^  injury and the length of exposure period. The greatest reduction of leaf area occurred under continuing 0 3 exposure from early leaf stage until maturity. The reduction in photosynthesis under 0 3 stress would thus become a growth-limiting factor. Reduced translocation of photosynthate to the other parts of the plant and especially to the roots would result in reduction in their growth. Roots receive only the excess photosynthate that is not required for top growth and under conditions of assimilate deficiency shoot growth has priority over root growth (Ward!aw, 1968). In turn, the reduction in root size decreases the absorption of water and nutrients and affects photosynthetic rate through reduction of "sink" size (Humphries and Thorne, 1964). B. Effects of Pathogenesis on Bean Growth Few studies on growth effects of pathogens have been reported. The effects of yellow rust of wheat {Puccinia striiformis Westend.) and a cereal smut {Ustilago nuda [Jens.] Rostr.) on wheat growth and develop-ment were studied by Manners and Myers (1973). The effect on the host depended on which part of the plant was infected and on the duration of infection. Full rust infection caused the greatest reductions in growth 166 and yield of wheat. The proportional decrease in root dry weight was larger than that of any other part of the plant. The maturation of the plant as a whole was retarded by infection. Smut infection produced comparable effects to those produced by rust infection. Harrison and Isaac (1968) did growth analysis studies on wilt diseases of potatoes caused by infection with Vevticillium albo-atrum Reinke & Berth, and V. dahliae Kleb. They found that 6 weeks after infection while no obvious pathological symptoms had appeared, there were significant reductions in stem height and leaf area. When plotted against plant dry weight, leaf area ratio of the infected plants was lower than that of the controls from the fifth week after infection onward, because infection caused a proportionately larger decline in specific leaf area, where: total leaf area specific leaf area = total leaf weight Harrison (1968) attributed this to early maturation of the foliage, the infected plants having the specific leaf area characteristic of control plants of considerably larger size. Significant suppression of bean growth by rust and halo blight were found in this research. However, while sufficient data were gathered on growth of plant parts, insufficient data were obtained on the overall plant growth to enable detailed growth analyses similar to those of the above work to be carried out. 167 C. Effects of Pathogenesis on Ozone Damage Parasitic disease may affect the development of ozone injury. With a variety of plants and parasites a localized decrease of ozone injury has been observed. The effect is often similar whether the parasite is a fungus, a bacterium or a virus (Yarwood and Middleton, 1954; Heagle and Key, 1973; Kerr and Reinert, 1968; Brennan and Leone, 1969). These experiments show no consistent protective effect at light or medium infection levels of bean rust (Uromyees phaseoli). At heavy infection level (350 + pustules per 1-inch leaf disc) chronic ozone injury symptoms to the lamina tissue were reduced as indicated by a reduction in spectral reflectance measurements. However, the injury which s t i l l occurred in the lamina tissue, did so immediately adjacent to pustules (Figure 15) within the green islands as well as at some distance from the pustules. When initiation of ozone fumi-gation was delayed (late 0^  treatments, Figure 23B) i t permitted the fungus to develop more rapidly than when ozone treatment began at the time of inoculation (Figure 23C). As a consequence, in such treat-ments the green-island effect was more apparent than in treatments in which ozone was administered from the outset. In addition, it was observed in cases of delayed fumigation that, while ozone injury occurred within the green islands, it was most severe around their peripheries, as shown in Figure 15. Since the tissues immediately surrounding the green islands become chlorotic and senescent more rapidly than more distant tissue, while 168 the islands themselves senesce more slowly, the relative severity of ozone injury to these areas is in keeping with the known relationship between susceptibility to ozone at acute levels and tissue maturity (Macdowall, 1965b). A protective effect against ozone occurs with leaves in which the rust has become well established (early inoculation) and the infection level is relatively high.on a unit leaf area basis (heavy infection). Thus, i t appears at best to be a transitory phenomenon, occuring late in the development of the pustules with their associated green islands. Furthermore, the fungus shows l i t t le ability to retard the onset of premature senescence of the leaf as a whole, caused by ozone. In several experiments, it was consistently observed that leaves treated with ozone senesced at the same rate, whether inoculated or not (Figure 23C vs. Figure 4C). As a consequence, these results suggest that the previously reported protective effect of rust infection against smog injury (Yarwood and Middleton, 1954), does not occur at low levels, particu-larly when ozone treatment begins at the time of inoculation, unless the intensity of infection is very great. Furthermore, the fungus is unable to delay premature senescence and death of the host tissue caused by ozone. The studies with Pseudomonas support earlier work of Kerr and Reinert (1968) in that, while 0^  flecking appeared over most of the surface of the bean leaves, i t did not occur in the chlorotic or "halo" areas adjacent to the sites of bacterial infection. 169 D. Effects of Ozone on Pathogenesis In the present studies, exposure to ozone was found to retard pustule growth of bean rust but pustule number was increased. In addition, secondary pustules were found at the margins of the green islands surrounding each primary pustule. The greater pustule fre-quency and secondary pustule formation probably compensates for the smaller pustules to maintain inoculum potential under ozone stress. E. Relationship of Pathogenesis to Kinetin Treatment and  Ozone Response These experiments confirm the retardation of leaf senescence by application of exogeneous cytokinins (Fletcher, 1969) and indicate that such applications offer some protection to the leaf against the effects of ozone. Both benzyladenine (BA) and kinetin (K) decreased injury caused by repeated exposures to 0.10 ppm ozone, although neither treat-ment was able to provide complete protection. Tomlinson and Rich (1973) reported similar partial protection of pinto bean leaves and suggested that i t occurred as a result of effects of the exogenous cytokinins in maintaining the integrity of membranes, especially those of the chloro-plast, since degree of protection was closely related to prevention of chlorophyll degradation and the retardation of senescence. These results shed no new light on such mechanisms. However, they show that, even though typical senescence is retarded, there is progressive loss of chlorophyll which has far-reaching effects on the plant's development. Thus, in spite of increases in primary leaf 170 area and dry weight in cytokinin-treated leaves, whether or not they were fumigated with ozone, the growth of the rest of the plant (stem and roots) was severely affected by ozone (Table XI). However, the situation is not clear-cut, since both 0.05 and 0.10 ppm ozone reduced stem and root growth, but only 0.10 ppm ozone caused significant loss of chlorophyll. Both the loss of assimilatory capacity caused by the latter and the relative reduction in leaf size and weight appear to contribute to the overall effect of 0.10 ppm ozone. In contrast, the reduction in stem and root weight of plants exposed to 0.05 ppm may reflect merely the decreased leaf area, since there were no indications of loss of chlorophyll. Such results could be explained by a direct effect of ozone on translocation, but this is not supported by any direct evidence in the present study. Indeed, Adedipe and Fletcher (1971) showed that, in intact plants, cytokinin-treated leaves or areas of leaves do not act as sinks, but rather that the retardation of senescence which they exhibit is associated with metabolic self-sustenance and a high retention of the products of photosynthesis. Thus, in the present study, i t appears that transport to the roots and stems was reduced in part by a direct effect of cytokinin treatment, and in part indirectly by reduction of photosynthetic capacity, in the case of 0.10 ppm ozone treatment. No measurements of protein or RNA contents were made in the present study, but the facts that leaf growth occurred as a result of cytokinin application, concurrently with loss of chlorophyll as a result of ozone treatment, indicate a situation different from natural senescence. Recently, Adedipe et aj_. (1973) have suggested that 171 senescence induced in tobacco by ozone dosages leading to acute injury differs from natural senescence in that it is manifested by loss of chlorophyll without loss of protein or RNA. They suggested that ozone affects, more directly, the morphological and physiological condition of the chloroplast, than the protein-synthesizing system. While the present evidence clearly shows that cytokinin treat-ment of bean leaves may offer them a degree of protection against 0.10 ppm ozone (Figures 29 and 32) such protection is at the expense of the rest of the plant. Furthermore, ozone at the sub-acute level of 0.05 ppm also causes a reduction in root and stem growth even when leaf growth is stimulated by cytokinins, with no apparent loss of chlorophyll. Overall, these results are in agreement with those of Tomlinson and Rich (1973) and extend their hypothesis to lower dosages. They also indicate that cytokinins are able to confer limited protection against loss of chlorophyll caused by repeated sub-acute fumigations and hence their presence at elevated levels in the green islands surrounding rust pustules could account for the reduced sensitivity to ozone of those tissues (Resh and Runeckles, 1973). Kiraly, Pozsar and El Hammady (1966) reported that in diseased tissues of bean and broad bean infected with Uromyoes phaseoli and u. fabae (Grev.) de Bary respectively, there are higher cytokinin activities. According to these workers accumulation of nutrients in the infected tissue as well as senescence of the uninfected leaf tissues near the sites of infection can be simulated by benzyladenine applied to the leaves. When half-leaves were infected by Uromyoes phaseoliy or treated with 30 mg/1 of BA the opposite half-leaves senesced while the 172 treated halves retained juvenility as in the control leaves receiving no treatment. Some plant-pathogenic bacteria can induce ethylene synthesis (Goodman et al_., 1967). Since ethylene can account for premature senescence (Galston and Davies, 1970), the action of cyto-kinins in preventing senescence may be in part due to effects on ethylene synthesis. The observation of Craker (1971a) that ozone itself can induce ethylene synthesis adds another factor to the complexities of the possible interactions between ozone and infection as they influence senescence. Ethylene is known to hasten senescence but i t is not known whether i t acts on permeability or protein synthesis. Perme-ability could be selectively increased by ethylene, and this might then lead to an increased synthesis of degradative enzymes such as nucleases, peroxidases, and proteases involved in senescence (Galston and Davies, 1970). There is considerable evidence that ethylene is derived from methionine under the action of the enzyme peroxidase (Yang, 1969). If peroxidase is involved in ethylene synthesis, and the enzyme is among those induced by ethylene, then the induction of peroxidase by ethylene could be the means for promoting the production of ethylene in adjacent tissues. Ozone has been reported to increase peroxidase activity (Curtis and Howell, 1971; Dass and Weaver, 1968). Thus the role of ozone in ethylene production might be as follows: 173 Ozone Damage Ethylene Production A Leakage from one cellular compartment to another • Increased -Membrane Permeability Release of degradative enzymes (nucleases, proteases, peroxidases) Increased Ethylene Production Senescence If these relationships are true for bean rust, and i f ozone increases peroxidase activity (Curtis and Howell, 1971; Dass and Weaver, 1968), then ozone exposure combined with rust infection should enhance the onset of premature senescence of the leaf beyond that which would occur i f either were present alone. On the other hand, i f bean rust decreases peroxidase activity then ozone injury and premature senescence would be reduced in the presence of the rust. The results of the present research suggest that rust does l i t t le to retard accelerated ozone-induced senescence (Figure 24). Late infection had no effect on ozone-induced senescence since pustules with their associated green islands had not developed before premature senescence occurred. However, even in the presence of fully developed pustules (early infection) the onset of ozone-induced senescence was not retarded. In contrast, early rust infection in the presence of late or continuing 0 3 caused a significant increase in senescence (Figure 24). Late and continuing ozone exposure alone caused signif i -cant increases in premature senescence. An interaction between rust infection and ozone treatment in promoting premature senescence is 174 suggested by the fact that infection alone had less effect on senescence. Difficulty arises in that, when dealing with an entire leaf, ozone-induced premature senescence is fairly uniform, while in the presence of established fungus, peripheral areas may senesce more rapidly but the green island effect cancels this out leaving no net difference from ozone alone. If the mode of action of cytokinins in delaying senescence was to decrease peroxidase activity they could also be responsible for delayed senescence by fungal infection. Green islands surrounded by chlorotic halos, such as are produced by certain rust fungi, have an increased respiration and increased dry weight per unit area, when compared with normal tissue (Atkins and Neilands, 1972). Assays indicate that simple extracts of spores from bean rust and of certain fungi contain cytokinins (Bushnell, 1966; Johnson et al_., 1966), nevertheless, some workers (Atkins and Neilands, 1972) doubt that the fungal metabolites that induce green islands in  vivo could be exclusively cytokinins. On the other hand, they reported that siderochromes, such as rhodotorulic acid, from yeast-like hetero-basidiomycetes are potent inducers of green islands. They hypothes-ized that siderochromes in green islands affect the function and the metabolism of fungal cytokinins and endogenous plant hormones, or that they directly affect the breakdown of chlorophyll. They further suggested that sidrochromes could poison sensitive enzymes such as indoleacetic acid oxidase and others involved with metabolism of plant hormones or of chlorophyll. As mentioned earlier (see p. 164), some 0Q injury was observed 175 within the green islands but not nearly as much as around their peri-pheries. Furthermore, the rust pustules had. to be mature and the green islands wel1-developed before even this limited amount of pro-tective effect became apparent. Diffusible metabolites of the fungus, which are not necessarily cytokinins, yet produce similar effects in retarding senescence, have been suggested to be responsible for protective effects against acute smog injury adjacent to rust pustules in bean leaves (Yarwood and Middleton, 1954). The present studies are in keeping with such a hypothesis, since cytokinin treatment was found to offer limited protection against ozone, corresponding to the limited protection observed in the green islands. The present studies do not permit any elaboration of the actual mechanisms involved in the modifications to natural senescence induced by either ozone treatment or pathogenesis. However, the general picture which emerges is that while ozone levels below those which cause acute injury induce rapid senescence, the presence of established infection may accelerate this effect beyond the boundaries of the green islands, but retards i t within their boundaries, suggesting a role for cytokinins. When infection is becoming established concurrently with ozone treatment, green island development is impaired and therefore the acceleration of overall senescence by ozone predominates. The role of ethylene may thus be to accelerate senescence in uninfected leaves and to induce more rapid maturation of the tissues invaded by the rust fungus, thereby decreasing the ability to induce green island formation, by counteracting the tendency for a retardation of senescence in such green islands. In turn, this could modify the normal developmental pattern of the fungus. 176 SUMMARY The purposes of this research were to investigate the inter-action effects of ozone and host-parasite relations using the systems bean-bean rust and bean-halo blight infections, to determine whether pathogenesis protects the host from chronic injury at low ozone levels, to determine the effects of such low ozone levels on pathogenesis, to measure chronic ozone effects on bean growth and their modification by infection, to study effects of ozone on cytokinin responses and vice versa, and to observe the effects of ozone and/or pathogenesis on premature senescence. The main results are summarized below: 1. The use of chlorophyll extraction as a quantitative measure of chronic ozone injury was found to be inadequate. Such a technique can be useful in measur-ing acute ozone injury or premature senescence, but not for measuring chronic ozone injury which occurs only in the upper palisade layer of the leaves. 2. A method of spectral reflectance was developed to measure chronic ozone injury of leaves and was shown to be capable of consistently detecting small changes in ozone injury which could not be differentiated by the use of chlorophyll extraction. 177 3. The dosage of an air pollutant must be defined for given experimental conditions. In a fumigation chamber ozone is absorbed by the chamber walls, the so i l , and the plants. Such losses, other than through absorption by plants, must be considered and the pollutant input adjusted to give the desired concentration to the plant itself . 4. Apparent growth stimulation in the presence of low levels of ozone (0.02 to 0.04 ppm) in contrast to growth in filtered air was observed frequently and is attributed to adaptation of plants to air pollution stress. 5. Ozone levels as high as 0.10 ppm caused general chlorosis of the upper surface of primary leaves after 12 days of 14 hours exposure per day. 6. 0.10 ppm ozone caused premature senescence of primary leaves after 12 days of exposure (22-day-old plants). It is speculated that this premature senescence is a result of increased ethylene production incited by increased peroxidase activity. 7. The number of root nodules, nodule fresh weight and nodule dry weight per plant were significantly reduced by 0.10 ppm ozone. Root growth (fresh and dry weights) was also reduced. Modification of plant metabolism and/or metabolite translocation is suggested to cause the reductions in growth of nodules and roots. 178 8. Primary leaf area, fresh weight, dry weight, stem fresh weight, dry weight, plant height, trifoliate area, fresh weight, dry weight, total plant leaf area, total plant fresh weight and dry weight were all significantly reduced by 0.10 ppm ozone. These effects on plant growth are attributed primarily to reduced rates of photosynthesis in the leaves, result-ing in part from the destruction of chlorophyll, leading in turn to a restriction of leaf development and reduced translocation of metabolites. 9. There was some protective effect of rust, Uromyoes phaseolij on bean against chronic ozone injury when the intensity of infection was high. 10. Some chronic injury symptoms occurred within the green islands surrounding the rust pustules, but injury was more severe around their peripheries. 11. The fungus showed no ability to retard the onset of ozone-induced premature senescence of the leaf as a whole. 12. The intensity of rust infection is important in con-sidering the effects of rust or ozone injury of the host. It is believed that an interplay of stresses between these two factors determines to what degree plant injury will be due to one or other of them. 179 For example, under heavy infection (greater than 350 pustules per 1-inch disc) and 0.10 ppm ozone the pre-dominant stress factor which determines symptom expression is infection and thus i t appears to prevent some of the ozone injury from occurring. On the other hand, under light (less than 50 pustules per 1-inch disc) and medium infection (50-75 pustules per 1-inch disc) \ 0.10 ppm ozone is the dominant stress causing injury with infection offering no protection. 13. Ozone modified the development of primary pustules by increasing their number but decreasing their size. The reduction in pustule size may be partly due to retarda-tion in the rate of fungal development before sporulation. The more severe the ozone injury the greater the retard-ation of pustule development. It is suggested that these effects on fungal development are caused by dis-turbance of the host metabolism by ozone injury. 14. Later stages of rust development on bean leaves subjected to ozone were additionally modified through the develop-ment of secondary pustules at the margins of the green islands surrounding each primary pustule. 15. The greater pustule frequency and secondary pustule formation probably compensated for the smaller pustules to maintain inoculum potential under ozone stress. 180 16. In the halo blight studies on bean i t was observed that, while ozone flecking appeared over most of the surfaces of the exposed leaves it did not occur in the chlorotic or "halo" areas adjacent to the sites of bacterial infection. 17. Bacterial infection gave no protective effect against ozone stress, but on the contrary, increased the over-all retardation of plant growth. 18. Since higher cytokinin levels have been reported to be associated with the green islands surrounding rust pustules, the effects of cytokinin treatment on leaf response to ozone were studied. Exposure to 0.10 ppm ozone caused significant leaf injury and significantly reduced leaf area, dry weight, stem dry weight and root dry weight, even following a single application of a cytokinin (kinetin or benzyl adenine) to the leaves. 19. With eight applications of cytokinins (one application of 30 yg/ml per day for 8 consecutive days) ozone injury occurred but there were no significant effects on plant growth after daily exposures to 0.10 ppm ozone for 42 days. Both kinetin and benzyl adenine greatly increased primary leaf area, fresh weight, dry weight, stem fresh weight and dry weight and root fresh and dry weights under 0.10 ppm ozone treatments. 181 20. In all cases, both kinetin and benzyl adenine offered some protection against ozone injury to plant growth. Benzyladenine provided the greatest protection. This protection is attributed to increased photosynthate associated with increased leaf area. In summary, while low levels of ozone (0.02 to 0.04 ppm) caused apparent growth stimulation in bean,, {Phaseolus vulgaris), higher levels (0.10 ppm) caused significant reductions in plant growth due to decreased photosynthetic capacity of the plant. The protective effect of rust, Uromyces -phaseoli, on bean against chronic ozone injury when the intensity of infection was high can be explained in terms of an interplay of stresses between these two factors. The fungus showed no ability to retard the onset of premature senescence caused by ozone. Ozone modified the development of primary pustules by increasing their number but decreasing their size; however, the greater pustule frequency and secondary pustule formation probably compensated for the smaller pustules to maintain inoculum potential under ozone stress. 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Phytopathology 55_: 1303-1308. 198 A P P E N D I X Tables of Treatment Means of Various Growth Parameters for All Experimental Trials. 199 TABLE OF MEANS* EXPERIMENT: A Treatment Number Ozone Inoculation PRIMARY LEAVES Area (cm ) Dry Weight (g) 1 None None 113.07 abc 0.20 a 8 None Late 118.04 a 0.17 ab 9 Early None 83.20 ef 0.17 ab 7 Early Late 91.98 de 0.14 b 5 Contin- None 75.53 f 0.17 ab 10 Contin. Late 83.98 ef 0.18 ab Each mean is based on 8 observations. Means not followed by the same letters are significantly different from each other at the .05 level, using Duncan's New Multiple Range Test for comparisons. 200 TABLE OF MEANS EXPERIMENT: C Treatment Number Ozone Inoculation PRIMARY LEAVES % Reflectance p Area (cm ) Dry Weight (g) 1 None None 13.79 c 103.59 bed • .0L)20 a 8 None Late 13.39 c 114.55 ab 0.21 a 9 Early None 13.56 c 93.20 de. 0.18 ab 7 Early Late 14.11 c 102.68 cd 0.17 ab 5 Contin. None 21.65 a 91.85 de 0.15 b 10 Contin. Late 16.95 b 101.65 cd 0.17 ab Each mean is based on 8 observations. Means not followed by the same letters are significantly different from each other at the .05 level, using Duncan's New Multiple Range Test for comparisons. 201 TABLE OF MEANS EXPERIMENT: D Treatment Number PRIMARY LEAVES Ozone Inoculation % Reflectance Area(cm ) Dry Weight (g) 1 None None 14.16 d 113.91 a 0.17 ab 2 None Early 14.25 d 98.56 b 0.18 ab 3 Late None 18.65 c ' 101.20 b 0.20 a 4 Late Early 19.29 c 82.37 c 0.18 ab 5 Contin. None 23.71 a 63.34 d 0.15 b 6 Contin. Early 21.26 b 56.44 d 0.14 b Each mean is based on 8 observations. Means not followed by the same letters are significantly different from each other at the .05 level, using Duncan's New Multiple Range Test for comparisons. TABLE OF MEANS* EXPERIMENT: E Treatment Number Ozone Inoculation PRIMARY LEAVES % Reflectance 2 Area (cm ) Dry Weight (g) 1. None None 14.28 de 96.62 a 0.18 a 8 None Late 13.78 e 86.88 a 0.19 a 9 Early None 16.81 c 58.50 b 0.13 b 7 Early Late 16.16 cd 71.27 b 0.15 ab 5 Contin. None 24.69 a 58.11 b 0.12 b 10 Contin. Late 21.52 b 60.44 b 0.15 ab Each mean is based on 8 observations. Means not followed by the same letters are significantly different from each other at the .05 level, using Duncan's New Multiple Range Test for .comparisons. 203 TABLE.OF MEANS* EXPERIMENT: F Treatment Number PRIMARY LEAVES Ozone Inoculation % Reflectance Area (cm ) Dry Weight (g) 1 None None 14.66 b 99.91 a 0.21 a 2 None Early 14.94 b 97.30 a 0.20 a 5 Contin. None 21.32 a 62.18 b 0.16 b 6 Contin. Early 20.98 a 57.60 b 0.15b Each mean is based on 8 observations. Means not followed by the same letters are significantly different from each other at the .05 level, using Duncan's New Multiple Range Test for comparisons. 204 TABLE OF MEANS* EXPERIMENT: G Treatment Number Ozone Inoculation Primary Leaf % Reflectance 1 None None 11.81 d 8 None Late 12.56 d 9 Early None 14.94 c 7 Early Late 17.25 b 5 Contin. None 25.12 a 10 Conti n. Late 23.94 a 1 None None 11.81 e 2 None Early 16.50 d 3 Late None 18.75 c 4 Late Early 16.75 d 5 Contin. None 25.12 a 6 Contin. Early 21.44 b Each mean is based on 8 observations. Means not followed by the same letters are significantly different from each other at the .05 level, using Duncan's New Multiple Range Test for .-.comparisons. 205 TABLE OF MEANS EXPERIMENT : H Ozone Chemical Primary Leaf % Reflectance None Tween-20 9.31 c None R.A. 9.25 c None B.A. 9.19 c None Fungus 8.69 c Late Tween-20 13.19 ab Late R.A. 16.12 a Late B.A. 13.81 ab Late Fungus 13.56 ab Conti nuing Tween-20 14.62 ab Continuing R.A. 14.25 ab Continuing B.A. 12.88 b Continuing Fungus 13.31 ab * Each mean is based on 8 observations. Means not followed by the same letters are significantly different from each other at the .05 level, using Duncan's New Multiple Range Test for comparisons. 206 TABLE OF MEANS EXPERIMENT : I Ozone Chemical Primary Leaf % Reflectance None Tween-20 13.62 c None R.A. 13.75 c None B.A. 13.25 c Late Tween-20 31.06 a Late R.A. 29.81 a Late B.A. 29.94 a Continuing Tween-20 21.62 b Continuing R.A. 22.81 b Continuing B.A. 21.62 b Each mean is based on 8 observations. Means not followed by the same letters are significantly different from each other at the .05 level, using Duncan's New Multiple Range Test for comparisons. TABLE OF MEANS* EXPERIMENT: J- l (HARVEST 1) Treatment Number Ozone Inoculation Primary Leaves Stem Dry Weight (g) Root Dry Weight (g) % Reflectance Area (cm )^ Dry weight (g) 1 None None 15.81 a 55.25 a 0.1087 a 0.0969 a 0.0244 a 2 None Heavy 15.44 a 44.31 a 0.0900 a 0.0937 a 0.0194 a 5a Low None 16.81 a 53.11 a 0.1012 a 0.0931 a 0.0319 a 6a Low Heavy 16.19 a 53.79 a 0.0850 a 0.0962 a 0.0250 a 5b High None . 15.75 a 48.94 a 0.0969 a 0.0950 a 0.0281 a 6b High Heavy 16.31 a 58.70 a 0.1131 a 0.1019 a 0.0294 a *Each mean is based on 8 observations, Means not followed by the same letters are significantly different from each other at the .05 level, using Duncan's New Multiple Range Test for comparisons, TABLE OF MEANS* EXPERIMENT: J^2 (HARVEST 2) Treatment Number Ozone Inoculation Primary Leaves Stem Dry Weight (g) % Reflectance Area (cm )^ Dry Weight (g) 1 None None 18.38 be 163.81 a 0.2700 be 0.1869 a 2 None Heavy 18.56 abc 133.33 b 0.2969 ab 0.1206 be 5a Low None 17.56 be 158.64 a 0.2862 ab 0.1931 a 6a Low Heavy 19.12 ab 112.56 b 0.3075 ab 0.0994 be 5b High None 18.69 abc 123.04 b 0.2400 c 0.1775 a 6b High Heavy 19.88 a 115.40 b 0.3275 a 0,1469 ab *Each mean is based on 8 observations, Means not followed by the same letters are significantly different from each other at the ,05 level, using Duncan's New Multiple Range Test for comparisons, TABLE OF MEANS EXPERIMENT: J-3 (HARVEST 3) Treatment Number Ozone Inoculation Primary Leaves Trifoliate Dry Weight (g) Stem Dry Weight (g) Root Dry Weight (g) % Reflectance p Area(cm ) Dry Weight (g) 1 None None 17.94 d 189.69 a 0.3200 cd 0.2756 b 0.5162 b 0.3187 a 2 None Heavy 21.38 c 157.38 b 0.3631 b 0.1388 d 0.2381 d 0.1819 be 5a Low None 18.06 d 175.12 ab 0.3187 cd 0.3137 a 0.5675 a 0.3531 a 6a Low Heavy 22.62 b 121.78 c 0.3444 be 0.1368 d 0.1925 def 0.1187 c 5b High None 23.44 ab 133.52 c 0.3000 de 0.2043 c 0.4650 c 0.2025 b 6b High Heavy 24.19 a 35.28 d 0.4087 a 0.1174 d 0.2312 de 0.1169 c Each mean is based on 8 observations. Means not followed by the same letters are significantly different from each other at the .05 level, using Duncan's New Multiple Range Test for comparisons. I N D o TABLE OF MEANS* EXPERIMENT: K-1 (HARVEST 1) Treatment Number Ozone Inoculation Primary Leaves Stem Dry Weight (g) Root Dry Weight (g) % Reflectance Area (cm )^ Dry Weight (g) 1 None None 15.44 a 36.50 a 0.0874 a 0.0931 a 0.0337 a 2 None Medium 15.69 a 31.90 a 0.0796 a 0.0737 a 0.0269 a 5a Low None 14.81 a 30.06 a 0.0824 a 0.0956 a 0.0387 a 6a Low Medium 15.00 a 33.21 a 0.0874 a 0.0831 a 0.0325 a 5b High None 14.94 a 34.45 a 0.0905 a 0.1025 a 0.0400 a 6b High Medium 15.00 a 33.43 a 0.0796 a 0.0981 a 0.0450 a *Each mean is based on 8 observations. Means not followed by the same letters are significantly different from each other at the .05 level, using Duncan's New Multiple Range Test for comparisons. TABLE OF MEANS* EXPERIMENT: K-2 (HARVEST 2) Treatment Number Ozone Inoculation Primary Leaves Stem Dry Weight (g) % Reflectance Area (cm )^ Dry Weight (g) 1 None ' None 16.62 a 155.85 a 0.2492 ab 0.1606 a 2 None Medium 16.44 a 125.28 be 0.2531 ab 0.1244 a 5a Low None 16.25 a 161.93 a 0.2715 a 0.1575 a 6a . Low Medium 16.81 a 138.96 ab 0.2662 a 0.1269 a . 5b High None 16.50 a 124.38 be 0.2369 ab 0.1531 a 6b High Medium 16.25 a 106.25 c 0.2137 b 0.1087 a *Each mean is based on 8 observations. Means not followed by the same letters are significantly different from each other at the .05 level, using Duncan's New Multiple Range Test for comparisons. TABLE OF MEANS EXPERIMENT: K-3 (HARVEST 3) Treatment Number Primary Leaves Trifoliate Stem Root Ozone Inoculation % Reflectance p Area(cm ) Dry Weight (g) Dry Weight (g) Dry Weight (g) Dry Weight (g) 1 None None 16.75 cd 189.57 a 0.3034 b 0.2412 ab • 0.5825 a 0.3102 a 2 None Medium 17.94 c 144.09 b 0.2994 b 0.1968 b 0.3525 c 0.2019 b 5a Low None 16.50 cd 191.44 a 0.3579 a 0.2631 a 0.6106 a 0.2944 a 6a Low Medium 20.62 b 164.60 b 0.3566 a 0.2044 b 0.4231 b 0.2100 b 5b High None 27.38a 94.91 d 0.2505 c 0.1524 c 0.3169 cd 0.1381 c 6b High Medium 26.88 a 112.10 cd 0.2444 c 0.1181 c 0.2756 d 0.1287 c * Each mean is based on 8 observations. Means not followed by the same letters are significantly different from each other at the .05 level, using Duncan's New Multiple Range Test for comparisons. TABLE OF MEANS EXPERIMENT: L Treatment Number Ozone Inoculation (Bacteria) Primary Leaves Trifoliate Dry Weight (g) Stem Dry Weight (g) Root Dry Weight (g) 2 Area (cm ) Dry Weight (g) 1 None None 191.72 a 0.2725 a 0.6612 a 0.4400 a 0.2637 a 2 None Early 144.10 b 0.1825 b 0.5087 be 0.3487 b 0.1700 c 5a Low None 177.62 a 0.2450 a 0.6425 a 0.4500 a 0.2212 b 6a Low Early 145.06 b 0.2000 b 0.5675 b 0.3675 b 0.2200 b 5b High None 138.38 b 0.2550 a 0.4675 c 0.3312 b 0.1212 d 6b High Early 97.94 c 0.1400 c 0.3600 d 0.2237 c 0.0900 d Each mean is based on 8 observations. Means not followed by the same letters are significantly different from each other at the .05 level, using Duncan's New Multiple Range Test for comparisons. oo 214 TABLE OF MEANS* EXPERIMENT: M Ozone Chemical Primary Leaves % Reflectance 2 Area (cm ) Dry Weight (g) None No B.A. 15.81 e 94.77 bed .0.2700 b None B.A. 13.81 e 121.12 a 0.4150 a None No Kinetin 15.31 e 96.51 be 0.2050 be None Kinetin 15.44 e 99.43 b 0.2162 be Low No B.A. 17.19 de 83.09 bede 0.1763 be Low B.A. 14.88 e 97.12 b 0.2825 b Low No Kinetin 20.38 cd 79.94 cde 0.1650 be Low Ki neti n 17.31 de 78.76 de 0.1450 c High No B.A. 26.88 a 73.82 e 0.1250 c High B.A. 22.75 be 85.14 bede 0.1487 c High No Kinetin 24.94 ab 73.83 e 0.1300 c High Kinetin 24.25 ab 72.55 e 0.1350 c * Each mean is based on 8 observations. Means not followed by the same letters are significantly different from each other at the .05 level, using Duncan's New Multiple Range Test for comparisons. TABLE OF MEANS EXPERIMENT: N Ozone Chemical Primary Leaves Stem Dry Weight (g) Root Dry Weight (g) % Reflectance Area (cm ) Dry Weight (g) None None 8.69 e 83.84 9 0.4056 g None No B.A. 9.81 e 129.84 cde 0.6200 ef } 0.9725 a 1.1625 a None B.A. 9.69 e 192.89 a 1.1525 a / None No Kinetin 9.06 e 131.32 cde 0.7563 cd \ 0.9388 a 1.0900 a None Kinetin 9.56 e 140.23 cd 0.8225 c / Low None 11.25 d 100.94 f 0.4600 g Low No B.A. 8.56 e 121.27 e 0.6125 ef } 0.7750 b 1.1125 a Low B.A. 8.62 e 174.56 b 0.9588 b Low No Kinetin 9.00 e 118.10 e 0.5313 f ) 0.6975 b 0.9050 b Low Kinetin 9.00 e 125.90 e 0.6138 ef / High No B.A. 22.44 a 86.68 f 0.3437 g I High B.A. 16.06 c 143.19 c 0.6875 de j 0.5075 c 0.4762, c High No Kinetin 18.94 b 94.83 f 0.3762 g ) High Kinetin 17.19 c 100.55 f 0.4100 g / 0.5013 c 0.5000 c * Each mean is based on 8 observations. Means not followed by the same letters are significantly different from each other at the .05 level, using Duncan's New Multiple Range Test for comparisons ro TABLE OF MEANS EXPERIMENT: 0 Treatment Number Ozone Inoculation (Bacteria) Primary Leaves Trifoliate Dry Weight (g) Stem Dry Weight (g) Root Dry Weight (g) % Reflectance Area (cm ) Dry Weight (g) 1 None None 14.25 b 220.99 a 0.3975 a 1.3975 a 0.7662 b 1.0937 a 2 None Early 20.31 ab 184.16 be 0.2975 b 1.2925 ab 1.1012 a 0.8887 b 3 Late None 22.44 a 212.00 ab 0.4075 a 1.0350 be 0.8825 ab 0.5462 c 4 Late Early 23.12 a 177.61 c 0.3275 ab 0.9062 c 1.0925 a 0.5375 c Each mean is based on 8 observations. Means not followed by the same letters are significantly different from each other at the .05 level, using Duncan's New Multiple Range Test for comparisons. e n 

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