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"thunk. . ....h.uu~...rh no.” u u. ‘ (”Jan - wfi“%LrOHMWVH.~.U-wafl . z... 1 ailihfinh... 1.. ollti. .. Iii v iv. g. at“. \0nu.%.on-t..7hhyk|"[t.¢ 7 Ifl.nmln.w‘nl‘lyl . V .IOL . . lol. flmo . NH. VWWLQ Vaulu. .s x ‘ ,IIHPI‘I‘O‘III.‘ ‘}-llb 5".” ill...- Illlfllillljillililliillilll'illlllliill 93 01581 2740 This is to certify that the thesis entitled Paper Birch and Sugar Maple Resistance to Insect Folivores as Influenced by Defoliation, Drought, and Fertilization presented by Beth Dankert has been accepted towards fulfillment of the requirements for M - S - degree in __t_Qmoln_gEn Y m/fl/g/Mm Major professor Date 18 NOV. 199i 0-7639 MS U is an Affirmative Action/Equal OppOrtuniry Institution LIBRARY Michigan State ‘ University PLACE It RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or More data duo. DATE DUE DATE DUE DATE DUE MSU loAnAffirmotivo AotioNEquol Opportunity intuition Wm: _L if *7 _ 7 PAPER BIRCH AND SUGAR MAPLE RESISTANCE TO INSECT FOLIVORES AS INFLUENCED BY DEFOLIATION, DROUGHT, AND FERTILIZATION By Beth Dankert A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Entomology and Program in Ecology and Evolutionary Biology 1996 ABSTRACT PAPER BIRCH AND SUGAR MAPLE RESISTANCE TO INSECT FOLIVORES AS INFLUENCED BY DEFOLIATION, DROUGHT, AND FERTILIZATION By Beth Dankert This study tested models of genotypic and phenotypic variation in tree resistance to insects, including life history theory, the carbon / nutrient balance and growth/ differentiation balance hypotheses, and models of defoliation- induced resistance. Effects of drought, soil fertility, and defoliation on paper birch and sugar maple resource acquisition and allocation, as well as their resistance to gypsy moth, forest tent caterpillar, and whitemarked tussock moth were consistent with life history theory. Effects of soil fertility and drought stress on tree growth, photosynthesis, foliar chemistry, and herbivore resistance were consistent with the growth/ differentiation balance but not the carbon / nutrient balance hypothesis. Defoliation effects on herbivore resistance were environmentally dependent but generally consistent with the carbon/ nutrient balance hypothesis. Forest tent caterpillar defoliation affected the performance of other species feeding on the same tree at the same time, later in the same year, and in the year following defoliation. For aiding and abetting aspiring chemical ecologists, this work is dedicated to Steve Malcolm. ACKNOWLEDGMENTS I wish to extend my sincerest thanks to the members of my committee for their contribution to the quality of the final product. Thanks to my major professor, Dr. Dan Herms, for the opportunity to work on a challenging and rewarding project. His careful revisions and restraint in never asking when the next chapter would be done are gratefully acknowledged. I am indebted to Dr. Ray Hammerschmidt for his seemingly infinite patience, understanding, and encouragement. He diligently and enthusiastically read and reread various drafts while maintaining unwavering faith in the quality of the final product. I thank Dr. Steve Malcolm for his support, advice, good cheer and confidence in my future academic career. He somehow manages to always be around when I need him. I would also like to thank Dr. Jim Miller for helpful advice during the research process and his revision of the manuscript. I owe a great debt of thanks to the graduate students who kept me sane throughout the writing process. Thanks first and foremost to Cheryl Frankfater, the best friend a stressed grad student could have; a pillar of support and a mountain of inspiration. I couldn’t have done it without you! I look forward to the day that the Dankert/Frankfater Society for the Advancement of Chemical Ecology is a reality. Many thanks go to the grad students of Entomology and Botany who enrolled in ENT 890: Chemical Ecology and the distinguished scientists who so graciously agreed to speak. I learned more from that experience than any other during my masters program. iv I am deeply indebted to Tim Work, Amy Roda, and Chris Steiner for dragging me to the bar when I really needed it. Thanks also to Amy Christensen for friendship and support. A huge thanks to the extremely patient Eileen Eliason for entering most of my references. I am very grateful to Mark Hammond and Lea Hunt for long distance support and for keeping me well supplied with chocolate throughout the writing process. Jennifer Miles provided capable and expert assistance during the trials and tribulations of the 1995 field season. With her efficient help, we were gracefully able to recover from the forces of evil (some by air, some by land) conspiring against us. I must also extend my appreciation to my parents who not only spent one long, hot summer Saturday helping me collect frass, but drove all the way to Midland to help me do it! Lastly, I express thanks to the College of Natural Science for the continuing fellowship which funded me during most of the writing process and to the Dow Gardens where this research was performed. TABLE OF CONTENTS LIST OF TABLES ............................................................................................................ viii LIST OF FIGURES ............................................................................................................. x CHAPTER 1 INTRODUCTION ............................................................................................................... 1 What is the ecological cost of secondary metabolism? ........................................... 1 Interspecific patterns of plant defense ..................................................................... 2 Induced resistance .................................................................................................... 2 Intraspecific patterns of plant defense ..................................................................... 3 The carbon/nutrient balance hypothesis .................................................................. 4 The growth/differentiation balance hypothesis ....................................................... 5 Integrating interspecific and intraspecific patterns of defense ................................ 8 Induced resistance as an active defense ................................................................... 9 Tree species tested: paper birch and sugar maple .................................................... 9 Insect species tested: forest tent caterpillar, gypsy moth, and whitemarked tussock moth .......................................................................................................... 10 Can DIR regualate population cycles? .................................................................. 13 Objectives .............................................................................................................. 13 CHAPTER 2 PHYSIOLOGICAL EFFECTS OF DEFOLIATION, SOIL MOISTURE, AND SOIL FERTILITY ON PAPER BIRCH AND SUGAR MAPLE ............................................... 15 Introduction ............................................................................................................ 15 Interspecific patterns of plant defense ....................................................... 15 Intraspecific patterns of plant defense ........... 16 Methods ................................................................................................................. 20 Experimental control plots ........................................................................ 20 Experimental treatments ............................................................................ 21 Plant physiological measurements ............................................................. 23 Statisitical analysis Results .................................................................................................................... 24 1994 ............................................................................................................ 25 1995 ............................................................................................................ 35 Discussion .............................................................................................................. 40 vi CHAPTER 3 EXPRESSION OF INDUCED RESISTANCE OF PAPER BIRCH AND SUGAR MAPLE TO OUTBREAK LEPIDOPTERA IS MEDIATED BY SOIL MOISTURE AND FERTILITY .............................................................................................................. 46 Introduction ............................................................................................................ 46 Methods .................................................................................................................. 51 1994 forest tent caterpillar/gypsy moth bioassay ....................................... 51 1995 gypsy moth bioassay ......................................................................... 52 Statistical analysis ...................................................................................... 54 Results .................................................................................................................... S4 1994 forest tent caterpillar/gypsy moth bioassay ....................................... 54 1995 gypsy moth bioassay ......................................................................... 63 Discussion .............................................................................................................. 72 1994 ............................................................................................................ 72 1995 ............................................................................................................ 73 CHAPTER 4 ‘ INTERSPECIFIC INTERACTIONS BETWEEN INSECT FOLIVORES MEDIATED BY HOST-PLANT RESISTANCE: EFFECTS OF EARLY SEASON DEFOLIATION ON A LATE SEASON FOLIVORE ................................................................................. 76 Introduction ............................................................................................................ 76 Methods ............................................................................................ 78 1994 ............................................................................................................ 78 1995 ............................................................................................................ 79 Statistical analysis ...................................................................................... 80 Results .................................................................................................................... 81 1994 ............................................................................................................ 81 1995 ............................................................................................................ 82 Discussion .............................................................................................................. 96 1994 ............................................................................................................ 96 1995 .......................................................................................................... 100 CHAPTER 5 CONCLUSIONS .............................................................................................................. 106 APPENDD( 1 Record of deposition of voucher specimens ................................ 109 APPENDD( 1.1 Voucher specimen data ................................................................ 110 LIST OF REFERENCES ................................................................................................. 1 l 1 vii LIST OF TABLES Table 1-1: Predictions of the Carbon/Nutrient Balance and Growth/Differentiation Balance Hypotheses for the effect of nutreint deficiency and drought on carbon/nutrient balance and growth differentiation balance, respectively ....................................................... 7 Table 2-1: Amount of water received by irrigated and non-irrigated trees during the 1994 and 1995 growing seasons ......................................................................... 22 Table 2-2: F -values and df for analysis of tree species (paper birch and sugar maple), defoliation, irrigation, fertilization effects and their interactions on trunk growth, trunk diameter, height growth, and total number of leaves of paper birch and sugar maple in 1994 ................................................................................................... 2 6 Table 2-3: The effects of forest tent caterpillar defoliation on trunk growth, trunk diameter, and height growth of paper birch and sugar maple in the year of defoliation (1994) and the year following defoliation (1995) ................................................................. 27 Table 2-4: F -values and df for ANOVA of tree species (paper birch and sugar maple), defoliation, irrigation, fertilization effects and their interactions on photosynthesis (A) and stomatal conductance (g) in 1994 and 1995 ...................................................... 32 Table 2-5: F -values and df from ANOVA of tree species (paper birch and sugar maple), defoliation, inigation, fertilization effects and their interactions on % foliar carbon, % foliar nitrogen, and C:N ratio in 1994 ................................................................... 33 Table 2-6: F -values and df for analysis of tree species (paper birch and sugar maple), defoliation, irrigation, fertilization effects and their interactions on trunk growth, trunk diameter, height growth and total number of leaves of paper birch and sugar maple in 1995 .................................................................................................. 3 8 Table 2-7: F -values and df from ANOVA of tree species (paper birch and sugar maple), defoliation, in'igation, fertilization effects and their interactions on % foliar carbon, % foliar nitrogen, and C:N ratio in 1995 ................................................................... 41 Table 2-8: The effects of forest tent caterpillar defoliation in the previous year on foliar carbon, nitrogen and C:N ratio in the year following defoliation ............................. 42 Table 3-1: Nutritional indices used in experiments with fourth instar gypsy moth to determine effects of host quality on growth and nutrition. Calculated on a dry weight basis .................................................................................................. 5 3 viii Table 3-2: First instar to two weeks. F -values and df from AN OVA of tree species (paper birch and sugar maple), defoliation, fertilization effects and their interactions on individual biomass of first instar forest tent caterpillar and gypsy moth reared in the lab for two weeks from eclosion ........................................................................................ 55 Table 3-3: First instar to two weeks. Chi-square values and df from categorical ANOVA of tree species (paper birch and sugar maple), defoliation, fertilization effects and their interactions on survival of first instar forest tent caterpillar and gypsy moth reared in the lab for two weeks from eclosion ....................................................................... 56 Table 3-4: F-values and df from ANCOVA of tree species, defoliation, inigation, fertilization effects and their interactions on nutritional indices of fourth instar gypsy moth ................................................................................................... 65 Table 3-5: F—values and df from ANCOVA of tree species (paper birch and sugar maple), defoliation, irrigation, fertilization effects and their interactions on development time (the number of days to reach pupation from the fourth instar) and pupal mass of gypsy moth ................................................................................................... 69 Table 4-1: F-values and df from ANCOVA on the effects of leaf type (immature birch, mature birch, or mature maple), defoliation (D), irrigation (D, fertilization (F) and their interactions on nutritional indices of fourth instar whitemarked tussock moth in 1995 ..... 83 Table 4-2: F-values and df from ANOVA on the effects of leaf type (immature birch, mature birch, or mature maple), defoliation (D), irrigation (I), fertilization (F) and their interactions on larval duration (number of days until pupation) and pupal mass of whitemarked tussock moth in 1995 ............................................................... 91 Table 4-3: F -values and df from a nested ANOVA on the effects of tree species (paper birch and sugar maple), defoliation, irrigation, fertilization and their interactions on development time (the number of days to reach pupation) and pupal mass of whitemarked tussock moth reared from eclosion to pupation in the field ..................................... 97 Table 4-4: Predictions of the Carbon/Nutrient Balance and Growth/Differentiation Balance Hypotheses for the effect of nutrient deficiency and drought on carbon/nutrient balance and growth differentiation balance, respectively and observed results ........................... 104 ix LIST OF FIGURES Figure 2-1: The effect of forest tent caterpillar defoliation and irrigation on the trunk growth (at 50 cm above ground) of paper birch (A) and sugar maple (B) in 1994 (i SE) ................................................................................................. 28 Figure 2-2: The effect of forest tent caterpillar defoliation and irrigation on the trunk diameter at 50 cm above ground of paper birch (A) and sugar maple (B) at the end of the 1994 growing season (iSE) ....................................................................... 29 Figure 23: The effect of forest tent caterpillar defoliation and irrigation on the height growth (cm) of paper birch (A) and sugar maple (B) during the 1994 growing season (:1: SE) ................................................................................................. 30 Figure 2-4: The effect of defoliation by forest tent caterpillar and irrigation on foliar carbon concentration of paper birch (A) and sugar maple (B) in June 1994 (i SE) ................. 34 Figure 2-5: The effect of forest tent caterpillar defoliation and fertilization on the amount of foliar nitrogen concentration of paper birch (A) and sugar maple (B) in June 1994 (:i: SE) ................................................................................................ 3 6 Figure 2-6: The effect of defoliation in the previous year, as affected by irrigation and fertilization on the trunk growth of paper birch and sugar maple (species combined) during the 1995 growing season (:t. SE) .................................................................. 39 Figure 3-1: The effects of soil moisture and soil fertility on the biomass of first instar forest tent caterpillar reared on paper birch (A) and sugar maple (B) in 1994 (t SE) ....... 58 Figure 3-2: The effects of defoliation and soil moisture on percent survival of first instar forest tent caterpillar reared on paper birch (A) and sugar maple (B) in 1994 (:1: SE) ....... 59 Figure 3-3: The effects of defoliation and soil moisture on the percent survival of first instar gypsy moth reared on paper birch (A) and sugar maple (B) in 1994 (i SE) .......... 61 Figure 3-4: The effects of soil moisture and soil fertility on the percent survival of first instar gypsy moth reared on defoliated (A) and non-defoliated (B) paper birch and sugar maple in 1994 (:tSE) ................................................................................ 62 Figure 3-5: The effects of soil moisture and soil fertility on the percent survival of first instar gypsy moth reared on foliage from paper birch (A) and sugar maple (B) in 1994 (i SE) ................................................................................................. 64 Figure 3-6: The effects of defoliation and fertilization on growth of fourth instar gypsy moth reared on paper birch and sugar maple (i SE) ............................................ 66 Figure 3-7: The effects of defoliation and fertilization on the amount of digested food converted to biomass for fourth instar gypsy moth reared on paper birch and sugar maple foliage (i SE) ........................................................................................ 68 Figure 3—8: The effects of defoliation and soil moisture on the development time (number of days required for larvae to reach pupation) of fourth instar gypsy moth reared on paper birch (A) or sugar maple foliage (B) (:1: SE) ..................................................... 70 Figure 3-9: The effects of defoliation and soil moisture on pupal mass of fourth instar gypsy moth reared on paper birch (A) and sugar maple foliage (B) (:t SE) .................. 71 Figure 4-1: The effects of leaf age and defoliation on the growth of fourth instar whitemarked tussock moth reared on mature or immature birch foliage (t SE) ............. 84 Figure 4-2: The effects of leaf age and defoliation on the amount of digested food converted to biomass by fourth instar whitemarked tussock moth reared on mature or immature birch foliage (1: SE) ..................................................................... 86 Figure 4~3z The effects of defoliation, soil moisture, and soil fertility on the amount of food digested by fourth instar whitemarked tussock moth reared on defoliated (A) or non- defoliated (B) birch foliage (1 SE) ................................................................ 87 Figure 4-4: The effects of leaf age and soil moisture on the amount of digested food converted to biomass by fourth instar whitemarked tussock moth reared on mature and immature birch foliage (:t SE) ..................................................................... 88 Figure 4-5: The effects of leaf age and fertilization on the amount of food digested by fourth instar whitemarked tussock feeding on mature or immature birch foliage (:1: SE) ................................................................................................. 90 Figure 4—6: The effects of leaf type and defoliation in the previous year on the pupal mass of whitemarked tussock moth (:t SE) ............................................................. 92 Figure 4—7: The effects of leaf type, defoliation in the previous year, and soil moisture on the development time (number of days from eclosion to pupation) or whitemarked tussock moth reared on mature birch (A) or mature maple (B) foliage (i SE) ......................... 93 Figure 4-8: The effects of defoliation, soil moisture, and soil fertility on the development time (number of days from eclosion to pupation) of whitemarked tussock moth reared on defoliated (A) or non-defoliated (B) birch foliage (i SE) ...................................... 94 Figure 4-9: The effects of defoliation, soil moisture, and soil fertility on the pupal mass of whitemarked tussock moth reared on defoliated (A) or non-defoliated (B) birch foliage (:tSE) ................................................................................................. 95 Figure 4-10: The effects of defoliation in the previous year and soil moisture on the pupal mass of whitemarked tussock moth reared on paper birch (A) or sugar maple (B) foliage in the field (:t SE) ...................................................................................... 98 Figure 4-11: The effects of defoliation in the previous year and soil moisture on the development time (number of days from eclosion to pupation) of whitemarked tussock moth reared on paper birch (A) or sugar maple (B) foliage in the field (1 SE) .............. 99 xi Chapter 1 INTRODUCTION Plants, by virtue of their immobility, have developed a range of chemical and physical defenses to avoid natural enemies. Among these are plant secondary metabolites, which, unlike primary metabolites, are not physiologically essential to the plant, and are not ubiquitous among all plant families. Different plant species have different repertoires of secondary metabolites. This variation is a product of evolved life histories and taxonomy (Ehrlich and Raven 1964, Gottleib 1989). While they serve other functions, plant secondary metabolites are in general believed to be defensive (Harbome 1994). The majority of theories developed to explain allocation of plant resources to secondary metabolism have come from studies of plant- herbivore interactions. IEU'Il l'lli 1 ”1.? An underlying premise of plant defense theory is that there is a negative correlation between growth and defense. Primary and secondary metabolites share common precursors and intermediates which may force trade-offs between resources allocated among growth, reproduction and defense (Herms and Mattson 1992). The negative correlation between growth and secondary metabolite production may mean that defense is costly. If secondary metabolism uses resources that could otherwise be used to increase growth or reproduction, then a cost is incurred. However, allocating 2 resources to defense is cost-effective if the resources saved from herbivory exceed the resources allocated to defense. However, if growth is limited by low resource availability then resources can be diverted to defense at no cost to growth (Bryant et a1. 1985, 1988, Tuomi et a1. 1988) I .E. E l | 1 E Interspecific life-history patterns are in part the evolutionary result of trade-offs in resource allocation (Bryant et a1. 1983, Coley et a1. 1985, Loehle 1988, Herms and Mattson 1992). In general, slow growing plants have higher levels of constitutive defense compounds and are less susceptible to herbivory than fast growing species (Bryant et al. 1983, Coley 1983, Coley et a1. 1985, Loehle 1988). Production of constitutive defenses is thought to be favored in slow-growing species because it is less costly to defend resources in these species than to replace resources lost to herbivory (Janzen 1974, Bryant et a1. 1983, Coley et al. 1985, Chapin 1989). Fast growing plants are less likely to divert large quantities of resources from growth to defense because foliage is more expendable and fast growth is needed to preempt competitors (Feeny 1976, Bryant et al. 1983, Herms and Mattson 1992). Rapid growth may also dilute the concentration of secondary metabolites, increasing the metabolic cost of maintaining high concentrations in fast growing plants (Gershenzon 1994). Lnduceiresiatance To reduce the cost of maintaining constitutive defenses, fast growing plants may employ defenses that are induced by herbivore attack (Edwards and Wratten 1983, Haukioja 1990). Defoliation by herbivores or infection by pathogens can induce changes in a plant that make it less susceptible to current and future attack. Defoliation-induced changes in plant chemistry may occur in hours to days following defoliation (rapid-induced changes), or 3 in the several years following defoliation (delayed-induced changes) (Haukioja and Niemela 1977, Valentine et al. 1983, Williams and Myers 1984). Delayed-induced resistance (DIR) decreases herbivore performance in the year after defoliation (Haukioja 1980, Haukioja 1982, Schultz and Baldwin 1982, Tuomi et al. 1984, Haukioja and Neuvonen 1985). This response may last up to three years after defoliation (Haukioja and Niemela 1979, Haukioja 1982). DIR has been proposed as a delayed density-dependent mechanism responsible for driving cycles of outbreak Lepidoptera (Haukioja 1980). The proposed mechanism is thus: as the population of herbivores increases, increased defoliation elicits a delayed-induced resistance response. Delayed- induced resistance decreases host-plant quality causing herbivore populations to decline. As populations decrease, delayed-induced resistance relaxes, foliage quality improves and with it herbivore performance. I 'f H E l | 1 l Intraspecific variation in secondary metabolism can be affected by a variety of biotic and abiotic factors. Soil fertility (Mihaliak and Lincoln 1985, Bryant et al. 1987, Price et al. 1989), light intensity (1N aterman et a1. 1984, Bryant et al. 1987, Price et al. 1989), soil moisture (Gershenzon 1984, Homer 1990), temperature (Appleton and van Staden 1989), herbivory (Schultz 1988, Haukioja 1990) and pathogen infection (Nicholson and Hammerschmidt, 1992, Hammerschmidt and Schultz 1996) all influence production of secondary metabolites. Two hypotheses commonly used to explain environmental effects on phenotypic variation in secondary metabolism are the carbon/ nutrient balance (CNB) hypothesis (Bryant et al. 1983, Tuomi et al. 1984, Tuomi et al. 1988) and the growth/ differentiation balance (GDB) hypothesis (Loomis 1932, Lorio 1986, Herms and Mattson 1992). Both hypotheses share the key premises that secondary metabolite production 4 diverts resources from growth and that growth is more sensitive to stress than photosynthesis. I] l , . l l l l . The CNB hypothesis proposes that foliar C/ N ratios control expression of secondary metabolism. Carbon-based secondary metabolite concentration (i.e. phenolics, terpenes) is predicted to be positively correlated with the C/ N ratio of a plant and nitrogen-based secondary metabolite concentration (i.e. alkaloids, cyanogenic glycosides) is predicted to be inversely correlated with the C / N ratio of a plant (Bryant et al. 1983). The CNB hypothesis was originally proposed to explain the effects of shade and fertilization on secondary metabolism (Bryant et al. 1983). Predictions of the CNB hypothesis for the effects of nutrient deficiency on secondary metabolism are derived from the premise that moderate nutrient deficiency limits the growth of plants more than photosynthesis. Thus, moderately nutrient deficient plants accumulate excess carbon which increases the C/ N ratio of the plant. These excess carbohydrates can be allocated to C-based defenses. In fertile soil, plants that assimilate nitrogen in excess of what is needed for growth may be able to allocate extra nitrogen to N-based defenses, and should have lower C/ N ratios. Hence, concentrations of C-based defenses are predicted to be directly correlated with the C/ N ratio of the plant, while N-based defenses are predicted to be inversely correlated with plant C / N ratio. The predictions of the CNB hypothesis for shade are based on the premise that shade limits carbon assimilation more than nutrient uptake, decreasing the C/ N ratio of a plant. Levels of C-based secondary metabolites decrease due to decreased availability of carbohydrates. Levels of N-based secondary metabolites increase as nitrogen is assimilated in excess of growth, 5 which is light limited. As light intensity increases plant C/ N ratio increases as photosynthesis increases. Levels of C-based secondary metabolites increase as available carbon increases. The CNB hypothesis has also been extended to explain the effects of delayed-induced resistance (Tuomi et al. 1984, Bryant et al. 1988). The premises are these: proportionally more of the nutrients of deciduous trees are contained in the foliage. Carbon is stored in woody tissues. Thus, when a tree is defoliated it can lose more nitrogen than carbon (Tuomi et al. 1984, Bryant et a1. 1988). This results in an increase in the C / N ratio of the tree, enabling it to allocate carbon to the production of C-based defenses such as phenolics and terpenes. Since only resources accumulated in excess of growth requirements are used in defense, there is no trade-off between growth and defense. The effects of defoliation on C/ N ratio can be tested by fertilization. Fertilizing defoliated trees should compensate for nitrogen lost to defoliation, and therefore alleviate a DIR response. The GDB hypothesis subsumes the CNB hypothesis, predicting that any environmental factor that slows growth more than photosynthesis, such as drought or low temperature (as well as nutrient deficiency), can cause a carbohydrate surplus, which increases resources available to support secondary metabolite production (Loomis 1932, Lorio 1986). The GDB hypothesis proposes secondary metabolism is constrained in growing cells and that there is a resource based trade-off between growth and secondary metabolism. Thus, increased growth decreases the amount of resources available for allocation to secondary metabolites or other differentiation processes. 6 There is support for both the CNB and the GDB hypotheses. The CNB hypothesis has successfully predicted the outcome of some studies manipulating fertilization and shading. Fertilization has been shown to increase nitrogen content, decrease C/ N ratio, and with it levels of carbon- based secondary metabolites and herbivore resistance (Mihaliak and Lincoln 1985, Bryant et al. 1987, Price et al. 1989). Shading has been shown to decrease carbon assimilation more than nutrient uptake, resulting in a lower C/ N ratio and lower concentrations of carbon-based secondary metabolites (Waterman et a1. 1984, Bryant et al. 1987, Mole et al. 1988). However, results of studies manipulating C02 concentrations and drought have not been consistent with the predictions of the CNB hypothesis (Herms and Mattson 1992, Baldwin 1994). Drought is predicted to decrease C/ N ratio, but has in some cases increased concentrations of C-based secondary metabolites (Gershenzon 1984, Homer‘1990). Elevated C02 is predicted to increase C / N ratio, but has had no effect on secondary metabolism in some studies (Fajer et al. 1989, Johnson and Lincoln 1991). Fertilization has in some cases ameliorated induced resistance (Bryant 1993), but has enhanced resistance in others (Haukioja et al. 1985). Consistent with the GDB hypothesis, studies have shown increased secondary metabolism in response to drought (Gershenzon 1984, Mattson and I-Iaack 1987). I Simultaneous testing of the effects of nutrient deficiency and drought may be a means of delineating between the predictions of the two hypotheses (Table 1-1). The GDB hypothesis predicts both drought and nutrient deficiency slow growth more than photosynthesis, resulting in increased levels of C-based defenses. The CNB hypothesis also predicts that nutrient stress will decrease secondary metabolism. However, since drought limits carbon acquisition more than nutrient uptake (Mattson and Haack 1987) and Table 1-1: Predictions of the carbon/nutrient balance and growth/differentiation balance hypotheses for the effect of nutrient deficiency and drought on carbon/nutrient balance and growth/differentiation balance, respectively. Change in Change in UN Balance G/D Balance Nutrient Deficiency T i Drought 1 l 8 thus should decrease the C/ N ratio of the plant, the CNB hypothesis predicts that drought will decrease C-based defenses. Since the GDB hypothesis predicts that nutrient deficiency and drought will have the same effect on growth and defense, but the CNB hypothesis predicts that nutrient deficiency and drought will have different effects on C/ N ratio and thus growth and defense, it should be possible to exclude one of these models through simultaneous testing of these factors. This thesis represents an attempt to do 50. The CNB hypothesis and the GDB hypothesis have frequently been invoked to explain phenotypic plasticity in secondary metabolism as an incidental response to environmental variation (Ayres 1993). However, environmental variation including intraspecific patterns of growth and differentiation in response to herbivory may be adaptive expressions of phenotypic plasticity, analogous to patterns of phenotypic plasticity in other life history traits (Herms and Mattson 1992). Herms and Mattson (1992) used the GDB hypothesis as a mechanism for integrating interspecific and intraspecific theories of plant defense. To minimize the cost of defense, faster growing individuals within a species may be able to express phenotypic plasticity in secondary metabolism, including induced responses to herbivores. When a plant is growing in high resource environments where there is intense competition for resources, rapid growth is favored over defense. In low resource environments where growth is slow and resource retention is favored, the cost of producing secondary metabolites is low and the same plant will produce high levels of constitutive defense. 9 I l l . . l E While there are studies supporting the view that induced defenses are an incidental by-product of nutrient deficiency, there are also discrepancies that point towards an adaptive theory of defense (see Herms and Mattson 1992 for a review of both views). Among these are several studies in which insect damage caused a greater induced response than artificial defoliation (Coley et al. 1985, Edwards and Wratten 1985, Haukioja and Neuvonen 1985, Neuvonen et al. 1987, Hartley and Lawton 1991). Furthermore, secondary compounds induced by herbivores can be similar or identical to those induced by bacterial and fungal pathogens or viruses, suggesting that induced responses may be an adaptive response, though not necessarily targeted at herbivores (Hartley and Lawton 1987, Hammerschmidt and Schultz 1996). I . 1. l . l 1 1 Environmental effeCts on resistance depend on the kind of defenses possessed by the plant. The research in this thesis examines environmental effects on the resource acquisition and allocation of two species of deciduous trees with contrasting life histories: paper birch (Betula papyrifera Marsh.) and sugar maple (Acer saccharum Marsh.) Paper birch is a fast growing, early successional species and sugar maple is a slower growing, late successional species. Betula spp. produce carbon-based secondary metabolites (phenolics, terpenoids, flavonoids and glycosides) but no known N-based secondary metabolites (Palo 1984, Reichardt et al. 1984, Sunnerheirn et al. 1988, Glasby 1991). The induction of phenolics in birch by herbivorous insects is a well established phenomenon (Haukioja 1990). Acer spp. produce both C-based secondary metabolites (flavonoids, tannins, and saponins) and N-based 10 secondary metabolites (alkaloids) (Miller and Feeny 1983, Barbosa and Krisch 1987). 1 an oa- - -c.. ,o ‘ t-1 «no-- :41- “1.! LI! 1c' '11: co. practicum Different insect species have evolved different mechanisms to overcome the defenses of their plant hosts. By manipulating how much they consume, the variety of plants in their diet, and which part of the plant they consume, insects may best exploit their hosts and thereby minimize the effect of defenses (Slansky 1992). Decreased host quality can affect survival directly and lower pupal weight resulting in decreased fecundity. Poor host quality can also increase chances of predation; through increased mobility, as insects search for a higher quality food source (Schultz 1983, Bergelson and Lawton 1988), or by lengthening larval period, thus increasing length of exposure to predators. This study looks at the resistance of paper birch and sugar maple to three phytophagous generalists: gypsy moth (Lymantria dispar L.), forest tent caterpillar (Malacosoma disstria Hiibner), and whitemarked tussock moth (Orygia leucostigma Smith). Forest tent caterpillar and whitemarked tussock moth are native insect species, sharing an evolutionary history with paper birch and sugar maple. Gypsy moth is an introduced species and does not share an evolutionary history with paper birch and sugar maple. Thus the two tree species are predicted to be less well defended against gypsy moth than forest tent caterpillar and whitemarked tussock moth. Lepidoptera maintain an alkaline midgut lumen of pH 8 to 12, presumably an evolutionary adaptation to feeding on high-tannin diets (Feeny 1970, Berenbaum 1980, Martin and Martin 1984, Martin et al. 1987). Although this high pH is a disadvantage in digesting phenolics, it may help 11 to minimize the impact of oxidative enzymes on plant protein (Felton and Duffey 1991) and the extraction of proteins and carbohydrates is greater under highly alkaline conditions (Appel 1994). Solubility of leaf nitrogen is also greater (Betschart and Kinsella 1973). Insects regulate both the pH and Eh (redox) of the midgut and consumed foliage is titrated to the alkalinity of the midgut (Schultz and Lechowicz 1986). The metabolic costs associated with maintaining these levels is dependent on pH and buffering capacity of the plant species consumed and if the expenditure is great enough may influence host preference (Appel 1994). Dow (1986) estimates that maintaining an alkaline pH may expend 10% of a caterpillar's ATP. Allelochemicals which reach the insect gut interact with other foliar nutrients as well as each other. In some cases it may be the combined effect of allelochemicals which causes decreased performance (Berenbaum 1985). Gypsy moth often consume multiple species of host plants in their diet (Stoyenoff et al. 1994). The number, quality, and sequence of dietary hosts can have a large effect on fecundity of gypsy moth (Barbosa et al. 1986, Stoyenoff et al. 1994). Manipulating combinations of host plants consumed (and thus host plant allelochemicals consumed) may be a means by which phytophagous Lepidoptera increase digestive success. Gypsy moth can also compensate for poor food quality and low efficiency of conversion by increasing consumption rates (Stoyenoff et al. 1994). In this study, sugar maple is used as a representative tree species possessing N-based constitutive defenses. Research on gypsy moth food preference indicates that alkaloid presence is a basis for host-plant rejection (Barbosa and Krisch 1987). Perhaps it is for this reason that the alkaloid- containing sugar maple is not a preferred host for gypsy moth. It is not 12 known what effect alkaloids have on forest tent caterpillar and whitemarked tussock moth. Gypsy moth raised on alkaloid diets have a slower developmental rate, lower larval weight, and higher mortality (Miller and Feeny 1983). This is believed to be due to an inability to metabolize alkaloids (Barbosa and Krischik 1987). Little is known however, about the mode of action or how alkaloids interact with insect gut fluids (Appel 1994). Defoliated paper birch and sugar maple are used to test for the effects of induced C-based defenses on outbreak Lepidoptera in this study. Covalent binding of phenolics in the gut of Lepidoptera inhibit digestion (Appel 1994) and may be responsible for decreased insect performance. In contrast to their performance on alkaloids, gypsy moth seem to be adapted to feeding on high levels of phenolics (Barbosa and Krischik 1987). However, when normal levels of phenolics escalate in response to defoliation as part of a delayed- induced resistance response, gypsy moth are negatively affected (Wallner and Walton 1979, Schultz and Baldwin 1982). Schultz and Baldwin (1982) found that gypsy moth grew more slowly, had smaller pupae, fewer eggs and were less vigorous when fed on previously defoliated red oak (Quercus rubrum). Phenolic glycosides also have a negative effect on survival, pupal mass, and development time of forest tent caterpillar (Lindroth and Bloomer 1991). It is not known whether induced levels of phenolics have a negative effect on whitemarked tussock moth. In one study, dietary tannic acid increased the relative growth rate of whitemarked tussock moth (Karowe 1989). Whitemarked tussock moth are believed to be tannin tolerant due to ultrafiltration of tannins by the peritrophic membrane (Barbehann and Martin 1992). 13 can [213 madam 1291211131191] 93:91:57 Gypsy moth and forest tent caterpillar are considered the two most important outbreak defoliators of deciduous trees in North America (Mattson et al. 1991). Whitemarked tussock moth populations may also reach outbreak levels (Johnson and Lyon 1991). Outbreaks of phytophagous insects commonly last for several years and then rapidly decline. Defoliation- induced changes in leaves have been implicated in the regulation of population dynamics of outbreak species (Haukioja 1980, Rhoades 1983). However, the role of delayed-induced resistance in this process has not been firmly established. Rather than a single controlling factor, populations of phytophagous insects may be regulated by a combination of induced phytochemicals, decreased nutritional quality, and natural enemies. Induced phytochemicals and viruses may interact ‘to control insect populations. Studies have shown that host plant chemistry can affect the susceptibility of gypsy moth to NPV (nuclear polyhedrosis virus) (Keating et al. 1988, Schultz et a1. 1990). Hunter and Schultz (1993) found that delayed-induced resistance decreased the susceptibility of gypsy moth to NPV. Q] . |' Studies testing tree responses to defoliation have produced conflicting results. The responses of trees to environmental stress are not well understood. These variable results may be due to differing effects of abiotic factors on interspecific and intraspecific variation in the acquisition and allocation of resources such as carbon and nitrogen. This thesis reports the results of a two-year controlled field study on the effects of a factorial combination of two levels of defoliation, fertilization, and irrigation on the physiology of paper birch and sugar maple and their 14 resistance to three species of outbreak Lepidoptera. The objectives of this study were: 1) to test the predictions of life history theory, by looking at the resource acquisition and allocation of two tree species (paper birch and sugar maple) with divergent life histories, 2) to test the predictions of the carbon / nutrient balance and growth / differentiation balance hypotheses, by manipulating soil fertility and soil moisture 3) to determine the effects of abiotic stress on rapid and delayed-induced resistance of birch and maple to outbreak Lepidoptera species, and 4) to determine the effects of changes in host-quality elicited by an early season-folivore on a late-season folivore. This study provides greater insight into evolutionary and environmental effects on the expression of plant defense. Rarely have the effects of abiotic and biotic stresses on resistance been tested in a controlled field experiment. It is hoped that through increased understanding of the relationship between insect defoliators and their plant hosts, we will be better able to predict insect outbreaks and develop effective pest management strategies based on host-plant resistance. Chapter 2 PHYSIOLOGICAL EFFECTS OF DEFOLIATION, SOIL MOISTURE, AND SOIL FERTILITY ON PAPER BIRCH AND SUGAR MAPLE INTRODUCTION Insect outbreaks have often been linked with plant stresses such as nutrient deficiency or drought (Mattson and Addy 1975). Stress may improve host-plant quality and facilitate outbreak conditions by weakening plant defenses (Rhoades 1983) or through enhancing the nutritional quality of foliage (White 1984). Defoliation-induced changes in leaf chemistry have been proposed as delayed-density dependent factors regulating cyclic populations of outbreak insects (Haukioja 1980). However, studies testing tree responses to defoliation have often produced conflicting results. These variable results may be due to differing effects of abiotic factors on interspecific and intraspecific variation in the acquisition and allocation of resources such as carbon and nitrogen. I .E. E l | l E Resource allocation patterns of a tree may be constrained by life history. Life history theory is based on the premise that resource allocation patterns have evolved to maximize fitness. Primary metabolites used for growth and secondary metabolites used in defense share common precursors and intermediates, forcing trade-offs between resources allocated among growth, reproduction and defense (Herms and Mattson 1992). Interspecific life-history 15 l6 patterns are the evolutionary result of these trade—offs (Bryant et al. 1983, Coley et al. 1985, Loehle 1988, Herms and Mattson 1992). In general, slow growing plants have higher levels of constitutive defense compounds and are less susceptible to herbivory than fast growing species (Bryant et al. 1983, Coley 1983, Coley et al. 1985, Loehle 1988). Production of constitutive defenses is thought to be favored in slow-growing species because it is less costly to defend resources than to replace resources lost to herbivory (J anzen 1974, Bryant et al. 1983, Coley et al. 1985, Chapin 1989). Fast growing plants are less likely to develop high concentrations of constitutive defenses than slow growing plants because foliage is more expendable and fast growth is needed to preempt competitors (Feeny 1976, Bryant et al. 1983, Herms and Mattson 1992). Faster growth may also dilute the concentration of foliar secondary metabolites, increasing the metabolic cost of maintaining high concentrations in fast growing plants (Gershenzon 1994). To reduce the cost of maintaining constitutive defenses, fast grong plants may employ defenses that are induced after herbivore attack (Edwards and Wratten 1983, Haukioja 1990). I .E. E l 1 E Phenotypic plasticity in secondary metabolism may be an adaptive response to variation in resource availability (Gershenzon 1984, Herms and Mattson 1992). Phenotypic plasticity might enable the plant to alter its phenotype to suit the environment (Grime et al. 1986, Weis 1992). Plant fitness may be maximized by maintaining an optimal level of constitutive defense, such that benefits derived from producing constitutive defenses outweigh the metabolic costs of their production (Tuomi et al. 1991). Plants could reduce the cost of maintaining constitutive defenses by allocating resources to defense only when they are needed. Fast growing plants are predicted to show greater phenotypic plasticity in growth and secondary metabolism than slow growing 1 7 plants (Grime 1979, Bryant et al. 1983, Grime et al. 1986), including more pronounced defoliation-induced responses (Mattson et al. 1991). Several hypotheses have been proposed to explain intraspecific phenotypic variation in plant defense in response to resource availability. Two hypotheses commonly used to explain environmental effects on phenotypic variation in secondary metabolite content are the carbon/ nutrient balance (CNB) hypothesis (Bryant et al. 1983, Tuomi et al. 1984, Tuomi et al. 1988) and the growth/ differentiation balance (GDB) hypothesis (Loomis 1932, Lorio 1986, Herms and Mattson 1992). Both hypotheses share the premise that secondary metabolite production diverts resources from growth (i.e. a negative correlation between growth and defense) and that growth is more sensitive to stress than photosynthesis. The negative correlation between growth and secondary metabolite production may mean that defense is costly. If secondary metabolism uses resources that could otherwise be used to increase growth or reproduction, then a cost is incurred. Allocating resources to defense is cost effective if the resources saved from herbivory exceed the resources allocated to defense. However, if growth is limited by low resource availability then resources can be diverted to defense at no cost to growth (Bryant et al. 1985, 1988, Tuomi et a1. 1988). The CNB hypothesis proposes that changes in secondary metabolite concentration may be an incidental response to environmental variation and that foliar carbon / nutrient ratio controls quantitative allocation to secondary metabolism. Concentrations of carbon-based secondary metabolites such as phenolics or terpenes are predicted to be positively correlated with the C/ N ratio of a plant, while nitrogen-based secondary metabolites, such as alkaloids or cyanogenic glycosides are predicted to be inversely correlated with the C/ N ratio of a plant (Bryant et al. 1983, Tuomi et al. 1984, Tuomi et al. 1988). 18 The GDB hypothesis predicts that any environmental factor which slows growth more than photosynthesis (such as drought or low temperature as well as soil fertility) can cause a carbohydrate surplus, which then can be diverted to production of secondary metabolites (Loomis 1932, Lorio 1986, Herms and Mattson 1992). Increased allocation of resources to growth decreases the amount of resources available for allocation to secondary metabolism or other differentiation processes. Thus, predictions of the CNB hypothesis are a subset of the predictions of the GDB hypothesis (Herms and Mattson 1992). This study examines environmental effects on the resource acquisition and allocation of two species of deciduous trees with contrasting life histories: paper birch (Betula papyrifera Marsh.) and sugar maple (Acer saccharum Marsh). Paper birch is a fast growing, early successional species and sugar maple is a slower growing, late successional species. Betula spp. produce C-based secondary metabolites (terpenoids and phenolics), but no known N-based secondary metabolites (Palo 1984, Reichardt 1984, Sunnerheirn et al. 1988, Glasby 1991). The induction of phenolics in birch by herbivorous insects is a well established phenomena (Haukioja 1990). Acer spp. produce both C-based secondary metabolites (flavonoids, tannins, and saponins) and N-based secondary metabolites (alkaloids) (Miller and Feeny 1983, Barbosa and Krischik 1987). The objective of this study was to simultaneously test the predictions (outlined below) of the CNB hypothesis, GDB hypothesis, and life history theory. I examined the effects of defoliation, fertilization and soil moisture on the growth, photosynthesis, stomatal conductance, and foliar C/ N ratio of paper birch and sugar maple over a two year period (1994-1995). Under high resource conditions paper birch is predicted to grow faster than sugar maple. Paper birch requires high levels of nutrients and water (Perala and Alrn 1990) and should be more sensitive to fertilization and drought l 9 treatments than sugar maple. Sugar maple is a late successional species, that is better adapted to lower resource environments than paper birch (Loehle 1988). Thus, sugar maple growth is predicted to be less sensitive to fertilization level and drought than paper birch. Because birch is a faster growing species than maple, it should be able to replace tissue lost to herbivory much faster than sugar maple. Thus, birch is predicted to be more tolerant to defoliation than sugar maple, and the effect of defoliation on the growth of birch will be less pronounced than the effect on maple. As a fast growing species, paper birch is also predicted to have a higher photosynthetic rate than sugar maple. Severe defoliation decreases the amount of photosynthetic leaf area, but has been observed to increase the photosynthetic rate in remaining leaves (Welter 1989). If paper birch can more quickly replace lost foliage, the negative effect of defoliation on total carbon assimilation should be more pronounced in sugar maple. Drought can decrease stomatal conductance and thus photosynthesis (Hsiao 1973, Farquhar and Sharkey 1982). If paper birch is more sensitive to drought, drought should have a larger effect on its gas exchange and growth. Fertilization can increase photosynthetic rate (Field and Mooney 1986). Since birch inhabit nutrient-rich environments, their growth should be more sensitive to fertilization. If nutrient deficiency limits growth more than photosynthesis, foliar C/ N ratio should increase in non-fertilized trees. Drought can decrease photosynthesis (Hsaio 1973, Farquhar and Sharkey 1982), and thus drought stressed trees should have lower foliar C/ N ratios than irrigated trees. Defoliated trees lose more foliar nitrogen than carbon are then predicted to have a higher C/ N ratio than non-defoliated trees (Tuomi et al. 1984, Bryant et al. 1988). Since maple are predicted to be more responsive to defoliation, the effect on C/ N ratio should be more pronounced in maple than in birch. 20 In order to distinguish between the predictions of the CNB and GDB hypotheses, I simultaneously tested the effects of nutrient deficiency and drought. From the GDB hypothesis it is possible to predict that both drought and nutrient deficiency will lower growth more than photosynthesis, resulting in high levels of C-based defenses. However, drought limits carbon acquisition more than nutrient uptake (Mattson and Haack 1987) and is predicted to increase C/ N ratio. Since the GDB hypothesis predicts that nutrient deficiency and drought will have the same effect on growth and defense, but the CNB hypothesis predicts that nutrient deficiency and drought will have different effects on C/ N ratio and thus growth and defense, it should be possible to delineate between the two hypotheses through simultaneous testing of these factors. METHODS Experimental Control Plots Experimental plots consisting of 96 cells arranged in 6 blocks, each containing 16 cells (2 rows of 8), were constructed at Dow Gardens in Midland, Michigan. Cells 4 m2 in area were constructed by trenching to a depth of 1 m. Cells were encased by a water impermeable liner (30 mil PVC) extending 10 cm above ground level and 1 m deep. These cells allowed soil fertility and moisture levels to be manipulated independently on a cell by cell basis. The experiment was designed as a randomized complete block consisting of 96 trees planted one to a cell. Four main effect treatments (species, defoliation, soil moisture and soil fertility), each with two levels, and their interactions were replicated once within each of the six blocks (2 x 2 x 2 x 2 factorial). Each block had uniform soil consistency. 21 Experimental Treatments Species One birch or one maple tree was planted in each cell (48 total of each species). Trees were selected for uniformity from a group of 250 half-sibs (same mother), grown together under the same conditions from seed. Each block contained eight trees of each species. Birch trees were 2-years-old and sugar maple were 5-years-old when they were transplanted in April 1992. Birch originated from Wisconsin and maple originated from Minnesota. D i l' |' Studies contrasting artificial defoliation (manual tearing or cutting) with natural defoliation by insects have shown that there is a difference in the level of resistance elicited (see Baldwin 1990 for a review of experimental use of mechanical damage v. true herbivory). For this reason, I used forest tent caterpillar larvae (Malacosoma disstria Hiibner) to naturally defoliate half of the trees in May-June, 1994. Ten egg masses were affixed to the branches of each tree at bud break. This treatment was estimated visually to result in 90% defoliation. The other half of the trees were left undefoliated. Control trees were protected from defoliators by sticky bands placed on the trunk to prevent caterpillar access, and were monitored daily for stray caterpillars which were promptly removed. Low levels of background herbivory occurred and were not controlled for. Induced responses to catastrophic defoliation are not likely to be biologically important if they can not be detected over background levels of herbivory. I . . In 1994, all trees in the study received 39.6 cm of natural precipitation during the field season (from bud break to leaf abscission) (Table 2-1). Half the trees received an additional 23 cm irrigation (62.4 cm of water, total) applied in increments of 3.8 cm throughout the growing season (May-August). Trees 22 Table 24: Amount of water received by irrigated and non-irrigated trees during the 1994 and 1995 growing seasons. 1994 1995 Non-irri gated (cm) Irrigated (cm) Drouglflm) Irrigated (cm) May 6.6 10.4 0.0 2.5 June 12.6 27.8 0.0 16.5 July 10.4 10.4 0.0 10.2 Must 10.0 13.8 0.0 10.0 Total 39.6 62.4 0.0 39.2 23 received 47.1 cm of natural precipitation between the end of the 1994 growing season in September and the beginning of the 1995 growing season in April. For the drought experiments in 1995, beneath-canopy tents constructed of black plastic sheeting (30 mil PVC) were used to exclude precipitation. The plastic sheeting was supported by wood frames at 70 cm above ground level. Absorbent precipitation barriers at the trunk / plastic interface intercepted stem flow. Tents were open at both ends to allow for air circulation. Beneath-canopy tents were deployed 16 May-25 May and removed after leaf abscission in the fall. Half the trees received 0 cm irrigation during this period; the other half received a total of 39.2 cm, spaced equally throughout the season (Table 2-1). Two levels of fertilization were applied: trees received either no fertilization (ambient soil fertility) or 18:5:9 N,P,K applied at the rate of 225 kg N, 62.5 kg P, 112.5 kg K/ ha/ yr. Half the annual amount of fertilizer was applied at bud break and the other half just after leaf abscission each year, starting in the fall of 1993. Soil in the experimental area is a relatively infertile sandy to sandy- loam mineral soil. Plant Physiological Measurements Iteefimfli Tree growth was measured each year as annual radial trunk growth, height growth, overall trunk diameter, and total number of leaves. Tree height and trunk diameter were measured at the beginning and end of the growing season. Trunk diameter was measured at 50 cm above ground level. Total number of leaves was determined by manually counting the leaves on each tree after the cessation of indeterminate growth in August. 24 W Photosynthesis and stomatal conductance were measured periodically throughout the season using a LICOR LI-6200 Portable Photosynthesis System (LICOR Inc., Lincoln, NE). Readings were taken on one leaf per tree for all 96 trees. To minimize variation in light intensity, measurements were taken on clear (cloudless) days and leaves in full sun were used. To standardize for leaf age, only mature leaves were used. Readings were taken four times during the 1994 field season (27 June, 12 July, 19 July and 15 August) and five times during the 1995 field season (12 June, 20 June, 11 July, 27 July and 15 August). E 1' C ,1 I . Leaves were sampled for C / N analysis in June of each year. Leaves were picked 8-9am EST to standardize for diurnal variation in chemistry. Twelve leaves were sampled per tree, collected randomly throughout the canopy. Leaves with noticeable insect damage were excluded. Collected leaves were placed in ziplock plastic bags and kept on ice for transit to the laboratory. Following collection the area of sampled leaves was measured using an image analyzer. Leaves were then dried at 60°C for 48 h. Dried leaves were first weighed and then ground to pass through a size 30 mesh screen using a Wiley mill. The carbon and nitrogen content of ca. 0.10 g samples was determined by a Carbo Erba CHN analyzer, model NA 1500, using the method described by Daun and DeClercq (1994). Statistical Analysis Gas exchange data were analyzed using multivariate repeated measures analysis of variance (RMANOVA) (SAS Institute, Inc. 1985). Trunk diameter was analyzed by analysis of covariance (ANCOVA) (SAS Institute, Inc. 1985) using 1992 trunk diameter as a covariate. The remainder of the data was analyzed using analysis of variance (ANOVA) (SAS Institute, Inc. 1985). 25 RESULTS 1994 1mm Paper birch grew faster than sugar maple (Table 2-2). In 1994, mean trunk growth was 12.5 i 0.4 mm for paper birch and 5.3 i 0.4 mm for sugar maple. At the end of the 1994 growing season, mean trunk diameter was 35.6 i- 1.6 mm and 19.8 i 1.6 mm for 4-year-old birch and 7-year-old maple, respectively. During 1994, height growth of birch increased 69% more than that of maple; birch increased an average of 78.2 i 2.6 cm in height during the season and maple height increased an average 46.3 i 2.6 cm during the season (Table 2.2). Growth of trees defoliated by forest tent caterpillar decreased dramatically in 1994 (Table 2-2, 2-3). The overall response to defoliation was greater in sugar maple than in paper birch. Defoliation decreased radial trunk growth of maple 76% and radial growth of birch 45%. Height growth of maple was decreased 47% by defoliation and height growth of birch was decreased 42%. Fertilization increased trunk and height growth of paper birch, but not sugar maple (Table 2-2). Mean trunk growth of fertilized birch was 13.9 :i: 0.6 mm as compared to 11.2 i 0.6 mm of trunk growth for unfertilized birch. Mean height growth was 88.1 i 3.7 cm for fertilized birch and 58.5 i 3.7 cm for unfertilized birch. Fertilization increased trunk diameter of both species 9% over non-fertilized trees (Table 2-2). There was no significant main effect of irrigation on growth in 1994 (Table 2-2). There was however, a significant species x defoliation x irrigation interaction on trunk growth , trunk diameter, and height growth (Table 2-2). Irrigation had no effect on trunk growth (Figure 2-1), trunk diameter (Figure 2-2), or height growth (Figure 2-3) of defoliated trees of either species. For non- defoliated trees however, the effect of irrigation differed by tree species. Non- 26 .83.. u _. .83.. u .2. 28.8.. n .2... .8868 u t: . 2. on an. ow a. Sufi E.— 56 cud 9.0 — Ed Q..— vod cod _ em; :.— and w—._ _ SN 56 wc. — ch — mm.N .. find a. 3.”. .. N04,. _ _N.N mod and c-d _ end ..2.. 5%: mm; .. 56 — EN “—6 a. 8.? _.. wfim _ MNA mm; 86 ad — on; >56 :6 Ed _ av. _ mod cm.— med _ and E .3 mad 2 .N _ .12.... 3.0— mo.m VN; ovd — ca; .....2.2.. wvdm 12.2.. 36m 3.2.2.. wodm— _ 3.22.. Stan 32.2.. Sims. 3.2.2.. hcdm .21.... NVéM— — 3.501— % 30,—. 539.0 2.de ~205an “EFF 5306 3:3,“. Km» 23— Eoeooc Co 329.. u xv ”82332.5(. _ xv cetm A87. 355 ~33 Sargon cease=Eumucezemtha=eu~=£5$£comm ceuafi—Eomuceuamt:1:23.800 counEEomaeeuawE—«8.00am conga—Eulagzazefin«museum actuate—«=23..809..quon :euan___tom..=e_§_£on :eua~___:om*mo_ooam 5:355". =2§=E$8§e£ 8.3.2.52385 cougar—78.00am .82..me 8320988.; .8230qu 3.8mm .232 E 0.92: 396 can :85 .23.. Co 8.60. Co .35.... .82 v5. .532» 232. c.2256 355 .56..» EB. :e meeuoflug he... 93 98:0 5235...... 6282.5 £632.83. .3.an 396 was :23 69.3 8.8a... ooh .«e £928.. .8 xv e5. 82?. k "a." 03:. 27 Table 2-3: The effects of forest tent caterpillar defoliation on trunk growth, trunk diameter, and height growth of paper birch and sugar maple in the year of defoliation (1994) and the year following defoliation (1995) (:t SE)”. Trunk Growth (mm) Trunk Diameter (mm) Height Growth (cm) Total Number of Leaves Trunk Growth (mm) Trunk Diameter (mm) Height Growth (cm) Total Number of Leaves 1994 Paper Birch Sugar Maple Defoliated Non-defoliated Defoliated Non-defoliated 8.9 :1: 0.6 16.2 :1: 0.6 2.0 i 0.6 8.5 :t 0.6 31511.8 39.821: 1.8 17011.9 22.721: 1.7 57.5 :i: 3.7 98.8 :i: 3.7 32.0 :1: 3.7 60.5 i 3.7 1194 :i: 162 2078 1:162 921 :1: 166 494 i 162 1995 Paper Birch Sugar Maple Defoliated Non-defoliated Defoliated Non-defoliated 15.3 21:07 17.0:t0.7 9.1 10.7 11.7 10.7 48.5 :1: 2.4 58.6 :1: 2.4 23.7 i 2.7 33.0 i 2.3 128.2 :1: 6.8 132.1 d: 6.8 31.4 :1: 6.9 82.9 :1: 6.9 3503 i444 6055 i444 829:1:455 1118 i455 1trunk growth = increase in trunk diameter (at 50 cm above ground) between beginning and end of growing season (May and August) 2trunk diameter (at 50 cm above ground), height growth, and total number of leaves were measured after the cessation of indeterminate growth in August 20 fl M i I Irrigated D Non-irrigated Trunk Growth (mm) '5 M r Defoliated Non-defoliated Paper Birch 20 — M 1 I Irrigated D Non-irrigated Trunk Growth (mm) 8 M 1 Defoliated Non-defoliated Sugar Maple Figure 2-1: The effect of forest tent caterpillar defoliation and irrigation on the trunk growth (at 50 cm above ground) of paper birch (A) and sugar maple (B) in 1994 (:1: SE). 50 Trunk Diameter (mm) M O 50 N O 1 Trunk Diameter (mm) 8 8 — O 1 8 8 H O 29 j_ Defoliated Paper Birch Non-defoliated I Irrigated U Non-irrigated Defoliated Non-defoliated Sugar Maple I Irrigated D Non-irrigated Figure 2-2: The effect of forest tent caterpillar defoliation and irrigation on the trunk diameter at 50cm above ground of paper birch (A) and sugar maple (B) at the end of the 1994 growing season (2: SE). 30 120 § co C 8 I Irrigated D Non-irrigated 8 Height Growth (cm) N C 1 O I Defoliated Non-defoliated Paper Birch 120 100~ 80a I Irrigated D Non-irrigated Height Growth (cm) 20- Defoliated Non-defoliated Sugar Maple Figure 2-3: The effect of forest tent caterpillar defoliation and irrigation on the height growth (cm) of paper birch (A) and sugar maple (B) during the 1994 growing season (:1: SE). 31 defoliated paper birch that were irrigated had less trunk and height growth and a larger trunk diameter than non-defoliated paper birch that were not irrigated. There was no effect of irrigation on non-defoliated sugar maple. Overall, defoliation in 1994 decreased numbers of paper birch leaves by 43% but increased the number of leaves on sugar maple by 46%, during the 1994 season (Table 2-2, 2-3). The increase in sugar maple leaves was due to increased production of immature leaves during reflush after defoliation. In August, defoliated maple trees had 85% more immature leaves but 84% fewer mature leaves than non-defoliated trees. GasEzsrhange Paper birch had a much higher photosynthetic rate than sugar maple (Table 2-4). In 1994, mean net photosynthesis for birch was 15.16 i 0.47 umol C02 m'zs’2 while mean net photosynthesis for maple was 6.79 :i: 0.46 umol COz m'zs‘2 . Paper birch also had a higher rate of stomatal conductance than sugar maple (Table 2-4). Mean stomatal conductance in 1994 was 0.47 :t 0.02 cm 3'1 for birch and 0.16 :i: 0.02 cm 5'1 for maple. There was no effect of defoliation, irrigation, or fertilization on photosynthesis or stomatal conductance in 1994 (Table 2-4). W Paper birch had higher levels of foliar carbon and nitrogen than sugar maple in 1994 and 1995 resulting in lower overall foliar C/ N ratios (Table 2-5). In 1994, mean percent nitrogen was 4.13 :I: 0.07% for birch and 2.79 i 0.07% for maple. Mean percent carbon was 49.88 i 0.25% for birch and 47.82 i 0.25% for maple. The effect of defoliation on foliar carbon content was dependent upon irrigation and tree species (significant species x defoliation x irrigation interaction, Table 2-5, Figure 2-4). Irrigation increased foliar carbon 32 .mcdwa u a Socodwn u use...“ .6280... .e 828.. n xv ”8338.59: 2 N. S S Seem Nod .dd dvd .md . =e..an...:u...a:e..ew.t.aco..a..e.on.«8.8qm add mm.~ d.d xmd . :e..a~...ton.a=o..am.t.acod....e..oa 3d Ed mnd bod . 22.3...tomaeo..mm.h.a8.8..m 8.. .. 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R8 2.. 9.... no. . =0.....8un...8.8..m mwd mod add . 5.32800 3.2.2.. 32n— ..2.2.2.. end: ..2.2.2.. hNfim . 8.00am on... 26 awe... 8. 5.5 $ a. va— igo. =.o..a. 2.0 .2... demos... 3.8 R. 63.30 8.8 fl. .8 88.8.3... .8... .33 308.0 5.28.5.8 58.8.... 62.3.83 .8.an an... .2... :8... 89.9 8.8... 8.. ..o <>OZ< Ea... xv v5. 839.. k 2m." 033—. 34 8 M O 8 Foliar Carbon (°/o) I Irrigated U Non-irrigated Defoliated Non-defoliated Paper Birch 8 M O 4 S 8 I Irrigated D Non-irrigated Foliar Carbon (%) 8 ~ 0 l Defoliated Non-defoliated Sugar Maple Figure 2-4: The effect of defoliation by forest tent caterpillar and irrigation on foliar carbon concentration of paper birch (A) and sugar maple (B) in June 1994 (:l: SE). 35 concentration of defoliated birch and decreased foliar carbon of non-defoliated birch. There was no difference between the carbon content of defoliated birch and non-defoliated birch that were not irrigated. There was also no effect of defoliation or irrigation on foliar carbon content of maple. The effect of defoliation on foliar nitrogen was dependent on fertilization and species (significant species x defoliation x fertilization interaction, Table 2—5, Figure 2-5). Fertilization increased foliar nitrogen of defoliated birch trees but had no effect on non-defoliated birch trees. However, a different pattern was seen for maple; fertilization increased the nitrogen content of non-defoliated trees but had no effect on defoliated trees. The combined result of the species x defoliation x irrigation interaction on foliar carbon and the species x defoliation x fertilization on foliar nitrogen was that defoliation had no main effect on the foliar C/ N ratio of birch trees (12.00 i 0.43 defoliated; 12.59 i 0.43 non-defoliated), but increased the foliar C/ N ratio of maple (18.28 :1: 0.44 defoliated, 16.90 i 0.43 non-defoliated) (Table 2-5). Fertilized trees had lower levels of foliar carbon (48.45 i 0.25%) than non- fertilized trees (49.24 i 0.25%). Maple that were fertilized had higher levels of foliar nitrogen (3.00 i 0.10%) than non-fertilized maple (2.58 i 0.10%), but fertilization had no effect on the nitrogen content of birch foliage. Thus, fertilization had no effect on the overall C/ N ratio of birch foliage (11.97 :t: 0.43 fertilized; 12.62 i 0.43 non-fertilized), but fertilized maple trees had a lower foliar C/ N ratio (16.04 :t 0.44) than unfertilized maple trees (19.14 i 0.43). 1995 W Defoliation in 1994 continued to have a negative effect on tree growth of both species in 1995. Trunk growth, trunk diameter, and height growth of both birch and maple defoliated in the previous year were lower than non-defoliated 36 5.0 1“ d l l” d l . Fertilized U Non-fertilized .N d 1 Foliar Nitrogen (%) fl O l 0.0 - Defoliated Non-defoliated Paper Birch 5.0 :5 d L 9’ d L I Fertilized D Non-fertilized P o L Foliar Nitrogen (%) ~ 0 L 0.0 - Defoliated Non-defoliated Sugar Maple Figure 2-5: The effect of forest tent caterpillar defoliation and fertilization on the amount of foliar nitrogen concentration of paper birch (A) and sugar maple (B) in June 1994 (:l: SE). 37 trees (Table 2-3, 2-6). This effect was most pronounced in the height growth of sugar maple. Previously defoliated sugar maple grew 67% (51.5 cm) less in height than non-defoliated sugar maple (Table 2-3). Paper birch were more sensitive to the 1995 drought treatment than sugar maple (Table 2-6). Trunk growth was 17.9 i 0.7 mm for irrigated birch, but only 14.3 i- 0.7 mm for drought stressed birch. Drought stress had no effect on trunk growth of maple or height growth of either species. Fertilization interacted significantly with defoliation and irrigation (Table 2-6, Figure 2—6). In trees that had been defoliated and fertilized, there was no effect of soil moisture on trunk growth. However, irrigation increased the trunk growth of defoliated trees that were not fertilized and drought decreased it. For non-defoliated trees, it was the fertilized trees that were sensitive to soil moisture level (with irrigation increasing trunk growth and drought stress decreasing it) and the non-fertilized trees that showed no response to drought or irrigation. Both paper birch and sugar maple defoliated the previous year had fewer leaves than undefoliated trees (Table 2-3, 2-6). Previously defoliated paper birch had 42% fewer leaves than that of non-defoliated paper birch and previously defoliated sugar maple had 26% fewer leaves than non-defoliated sugar maple (Table 2-3). W Mean net photosynthesis for birch was 14.21 :I: 0.52 mol C02 m'25'2 and mean net photosynthesis for maple was 7.13 :l: 0.53 mol C02 m'zs'2 (Table 2—4). Net photosynthesis of maple that had been defoliated in the previous year was 20% less than non-defoliated maple. There was no effect of defoliation on the photosynthesis of birch (Table 2-4). Irrigation and fertilization had no effect on the net photosynthesis of either species. Mean stomatal conductance in 1995 was 38 .38.. u .. 20.8. n .2. 28.0w. u 2.... 2.8.8. u 2.: . 3. an. 2. wk. ...... amfi med ONN 3.6 end . .o.o cod mwd ... ovfi . mm... 36 n. .c w . d . GN. NM. end Ed . 8.. .cd Ed 2.0 . C... bed S6 56 . mm. mm. No. —vd . 2d 26 wNN 8d . 92m ch NN.m mo. _ . FNN mm. and 36 _ .N.. Om. 6.6 ...... 0N.» _ and ... 8% Nod .. .hé . .. end ...... .md mad mvd . ...... add .....2.. mm... ........2.. mvém ...... 2d — 3.2.2.. 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E 910 « I Irrigated (D DDrought x C E l- 5 ~ 0 Fertilized Not fertilized Non-defoliated Figure 2-6: The effect of defoliation in the previous year. as affected by irrigation and fertilization on the trunk growth of paper birch and sugar maple (species combined) during the 1995 growing season (:1: SE). 40 0.41 i 0.02 cm 3‘1 for birch and 0.21 i 0.02 cm 5'1 for maple (Table 2-4). Defoliation, irrigation and fertilization had no effect on stomatal conductance. W In 1995, paper birch again had higher levels than maple of foliar nitrogen and carbon, resulting in lower C/ N ratios (Table 2-7). Mean percent nitrogen was 3.25 i 0.04% for birch and 2.42 i 0.04% for maple. Percent carbon was 48.80 :t 0.15% for birch and 46.95 i 0.15% for maple. Trees defoliated by forest tent caterpillar in 1994 had lower levels of foliar nitrogen (2.72 i 0.04%) than non-defoliated trees (2.94 i 0.04) (Table 2-7, 2-8). There was no effect of defoliation on carbon content of foliage, resulting in lower C/ N ratios for defoliated trees (18.16 :t 0.22%) than non-defoliated trees (16.66 i 0.22%) in 1995 (Table 2-7, 2-8). Fertilized trees had higher levels of foliar nitrogen (2.91 i 0.04%) than non- fertilized trees (2.75 :l: 0.75%), experienced no change in foliar carbon and thus had lower overall C/ N ratios (16.90 i 0.22% fertilized; 17.92 i 0.22% non- fertilized) (Table 2-7). Soil moisture level had no effect on foliar carbon, nitrogen, or C/ N ratio in 1995. DISCUSSION Results were consistent with predictions based on tree life history. Sugar maple had a slower growth rate and a lower photosynthetic rate than paper birch. Defoliation decreased the trunk growth, trunk diameter and height growth of both species across both seasons. However, birch recovered more quickly, and in the year after defoliation the effect on growth of sugar maple was larger than the effect on birch. Sugar maple had less capacity to compensate for growth lost to defoliation than paper birch and this effect carried into the year after defoliation, as evident by lower growth and photosynthetic rates. Maple were also less able to compensate for nitrogen lost to defoliation as indicated by 41 686.6%. u .22.... 66.6w. u 2.. 66600.. .6 80.60.. H xv ”86.3.8.3... 2. 2. m6 xv .66... 3.6 66.6 «.6 . 66.30..6.0......66..0w........66..0._6.0n....8.00..m ~66 6..6 6m. . 66:60:68"...66..0u.......66..0..6.0o .6. . 3.6 2.6 . 66..0~.....0n....66..6w.......8.00am 3.6 6. .6 n . .6 . 66.30.....0ms66..0..6.0n....8.00..m 66.6 3.6 66.6 . 66..0~...ton....66..6w.... 5.6 66.6 and . 66..6N.....0......66..6..6.0Q and 6.6 6N6 . 66600.....0n....m0.00..m ...... had. ...... find— a—d . :o..a~._...0n— 66.6 m . .6 m6.6 . 66..0w.......66..6..6.09..8.00..m NN.6 66.6 9.6 . 66..6w.......66..6..6.0n. .66 8.6 6... . . 66..6w..:....m0.006m mnd mm.m 66.6 . 66.86.... ...... 606 .6.— .66.. . Sea—6.5.8.0066 2.2.2.. N©.MN 0...: 2.6. 3.. _ 62.23.00 2...... ...... 2...... 3.... ......2. an... _ 8.8% no. ...... mcé «...: mwd m zoo—m 2.3. 2.6 308.2 ... 8.6.6 ... ... ma— .2 n66. 6.6.6.. 7.0 666 6066...: .6..6. .... 666.00 .6..6. .... 66 86.80.86. ..0... .060 800:0 6660......0. 66606.... .66..0..6.0.. A260... .6660 660 60.5 .0603 8.00... 00.. .6 .3072 .66.. xv 666 86.0? .. "Wu 036,—. 42 Table 2-8: The effects of forest tent caterpillar defoliation in the previous year on foliar carbon, nitrogen and CIN ratio in the year following defoliation (1995) (:t SE). 1995 Paper Birch Sugar Maple Defoliated Non-defoliated Defoliated Non-defoliated % Carbon 48.57 :t 0.21 49.04 :t 0.21 46.93 :t 0.22 46.98 :1: 0.22 % Nitrogen 3.17 :t 0.05 3.33 :t 0.05 2.28 i 0.05 2.56 :t 0.05 CIN Ratio 15.53 :t 0.30 14.81 :t 0.30 20.79 i 0.31 18.51 :1: 0.31 43 lower foliar nitrogen concentrations in the year after defoliation. In 1995, maple defoliated in the previous year had a lower photosynthetic rate, but there was no effect of defoliation on the photosynthetic rate of birch. Surprisingly, at the end of the 1994 season, defoliated maple trees had more leaves than non-defoliated trees. This was due to increased production of immature leaves during reflush. Levels of mature leaves for birch and maple were lower at the end of the season due to defoliation, as expected. Defoliation did not increase photosynthetic rate; it had no effect on the photosynthetic rate of either species in 1994. Consistent with life history predictions, growth of paper birch was less affected by defoliation, but more sensitive to the effects of irrigation and fertilization than sugar maple. The effects of fertilization on growth were inconsistent across the two seasons. Fertilization increased trunk growth and height growth of birch in 1994, but had no effect on maple. In 1995, fertilization had no effect on the growth of either species. Fertilization had no effect on the photosynthetic rate of birch or maple in either year. Irrigation treatment had no effect on the growth or photosynthesis of either species in 1994 perhaps because ambient rainfall was sufficient to prevent drought stress. As a result, rain was excluded from the trees in 1995. In 1995, drought decreased the trunk and height growth of birch. There was no effect of drought on photosynthesis. The observation that sugar maple were less susceptible to the effects of fertilization and irrigation may be due to a life history strategy that necessitates resource conservatism. A major premise of the carbon / nutrient balance and growth/ differentiation balance hypotheses that growth is more sensitive to stress than photosynthesis was confirmed. Defoliation limited growth more than photosynthesis due to a decrease in growth and no change in photosynthesis (rather than increased photosynthesis as observed in other studies (Welter 1989)). 44 The carbon/ nutrient balance hypothesis did not accurately predict delayed- defoliation effects on C/ N ratio. Foliar nitrogen levels decreased in response to defoliation, but photosynthesis did not increase (photosynthetic rate of maple actually decreased), resulting in a decreased C/ N ratio for defoliated trees in 1995. Herbivory generally causes an increase in photosynthetic rate (Welter 1989) but this effect may be more common in grasses than in other species (Rosenthal and Kotanen 1994). The effect of defoliation on foliar carbon concentration was mediated by irrigation in 1994. Irrigation increased foliar carbon concentration of defoliated birch and decreased foliar carbon content of non-defoliated birch. When trees were not irrigated, there was no difference in the foliar carbon of defoliated and non-defoliated trees. Thus, defoliated birch may be more likely to produce excess carbon which could be allocated to defense in wet, rather than dry years. Since foliar carbon increased in defoliated trees, but decreased in non-defoliated trees, the main effect of irrigation on C/ N ratio was not significant. The predictions of the CNB hypothesis for fertilization effects were supported in some cases but not others. Fertilization was predicted to decrease C/ N ratio. In 1994, fertilization decreased foliar carbon of birch and maple, but increased foliar nitrogen in maple only. Thus, fertilization decreased the C/ N ratio of maple only. In 1995, fertilization increased both the foliar nitrogen and foliar carbon of both species, resulting in a lower C/ N ratio for fertilized trees. Some of the intricacies of environmental effects on foliar carbon and nitrogen are lost by looking at C/ N ratios. In this study, C/ N ratio was not an accurate predictor of what was happening with foliar nitrogen concentration or foliar carbon individually. In 1994 for example, irrigation of defoliated birch caused an increase in foliar carbon and fertilization of defoliated birch caused an increase in foliar nitrogen. In concert, these effects canceled each other such that 45 The carbon / nutrient balance hypothesis did not accurately predict delayed- defoliation effects on C/ N ratio. Foliar nitrogen levels decreased in response to defoliation, but photosynthesis did not increase (photosynthetic rate of maple actually decreased), resulting in a decreased C/ N ratio for defoliated trees in 1995. Herbivory generally causes an increase in photosynthetic rate (Welter 1989) but this effect may be more common in grasses than in other species (Rosenthal and Kotanen 1994). The effect of defoliation on foliar carbon concentration was mediated by irrigation in 1994. Irrigation increased foliar carbon concentration of defoliated birch and decreased foliar carbon content of non-defoliated birch. When trees were not irrigated, there was no difference in the foliar carbon of defoliated and non-defoliated trees. Thus, defoliated birch may be more likely to produce excess carbon which could be allocated to defense in wet, rather than dry years. Since foliar carbon increased in defoliated trees, but decreased in non-defoliated trees, the main effect of irrigation on C/ N ratio was not significant. The predictions of the CNB hypothesis for fertilization effects were supported in some cases but not others. Fertilization was predicted to decrease C/ N ratio. In 1994, fertilization decreased foliar carbon of birch and maple, but increased foliar nitrogen in maple only. Thus, fertilization decreased the C/ N ratio of maple only. In 1995, fertilization increased both the foliar nitrogen and foliar carbon of both species, resulting in a lower C/ N ratio for fertilized trees. Some of the intricacies of environmental effects on foliar carbon and nitrogen are lost by looking at C/ N ratios. In this study, C/ N ratio was not an accurate predictor of what was happening with foliar nitrogen concentration or foliar carbon individually. In 1994 for example, irrigation of defoliated birch caused an increase in foliar carbon and fertilization of defoliated birch caused an increase in foliar nitrogen. In concert, these effects canceled each other such that Chapter 3 EXPRESSION OF INDUCED RESISTANCE OF PAPER BIRCH AND SUGAR MAPLE TO OUTBREAK LEPIDOPTERA IS MEDIATED BY SOIL MOISTURE AND FERTILITY INTRODUCTION Defoliation by herbivores has been shown often to induce resistance to future attack by decreasing host quality, but in some cases it has also been shown to induce susceptibility by improving host quality (Myers and Williams 1984, Fowler and Lawton 1985, Haukioja 1990, Karban and Niiho 1995). Defoliation-induced changes in plant chemistry may occur in hours to days following defoliation (rapid-induced changes), or in the several years following defoliation (delayed-induced changes) (Haukioja and Niemela 1977, Valentine et a1. 1983, Williams and Myers 1984). In general, there appears to be more variability in rapid than in delayed-induced changes in host quality in response to herbivory. Delayed-induced resistance occurs when in the year after defoliation trees exhibit increased herbivore resistance, which in some cases has been correlated with an increase in foliar phenolic content and decline in foliar nitrogen (Haukioja 1980, Haukioja 1982, Schultz and Baldwin 1982, Tuomi et a1. 1984, Haukioja 1985). This response may last up to three years after defoliation (Haukioja and Niemela 1979, Haukioja 1982). Delayed-induced resistance has been proposed as a delayed density-dependent mechanism 46 47 responsible for driving cycles of outbreak Lepidoptera (Haukioja, 1980). The proposed mechanism is thus: as the population of herbivores increases, increased defoliation elicits a delayed-induced resistance response. Delayed- induced resistance decreases host-plant quality and causes herbivore populations to decline. As the population decreases, delayed-induced resistance relaxes, foliage quality improves and with it herbivore performance. Phenotypic variation in secondary chemistry may be an adaptive response to environmental variation. Resistance-associated changes in plant chemistry may be an adaptive response to herbivory that enable the plant to alter its phenotype to suit the environment (Haukioja 1980). Plant fitness may be maximized by maintaining an optimal level of constitutive defense, such that benefits derived from producing constitutive defenses outweigh the metabolic costs of their production (Tuomi et al. 1991). Plants could reduce the cost of maintaining constitutive defenses by inducing defenses only when they are needed. Fast grong plants are predicted to show greater phenotypic plasticity in growth and secondary metabolism than slow growing plants (Crime 1979, Bryant et al. 1983, Grime et al. 1986), including more pronounced defoliation-induced responses (Mattson et al. 1991). Two hypotheses commonly used to explain environmental effects on phenotypic variation in secondary metabolite content are the carbon / nutrient balance hypothesis (Bryant et al. 1983, Tuomi et a1. 1984; 1988) and the growth / differentiation balance hypothesis (Loomis 1932, Lorio 1986, Herms and Mattson 1992). Both hypotheses share the premise that secondary metabolite production diverts resources from growth and that growth is more sensitive to stress than photosynthesis. 48 The carbon/ nutrient balance hypothesis proposes that foliar C/ N ratios control the expression of secondary metabolism. Concentration of carbon- based secondary metabolites, such as phenolics or terpenes, is positively correlated with the C/ N ratio of a plant and concentrations of nitrogen-based secondary metabolites, such as alkaloids, are inversely correlated with the C/ N ratio of a plant (Bryant et al. 1983). The CNB hypothesis has been invoked to explain defoliation-induced changes in tree resistance to herbivores (Tuomi et al. 1984, Bryant et al. 1988). Defoliation may affect secondary metabolism incidentally by altering the C/ N ratio of the plant. When a tree is defoliated it loses nitrogen and other nutrients contained in the leaves, leading to an increase in the C/ N ratio of the tree and thus increased concentrations of C-based defenses including phenolics. Fertilization of defoliated trees is predicted then to compensate for nitrogen lost to defoliation and alleviate the delayed-induced resistance response. The growth / differentiation balance hypothesis predicts that any environmental factor that slows growth more than net photosynthesis (such as drought or low temperature) can result in an increased supply of carbohydrates that can be used in the production of secondary metabolites (Loomis 1932, Lorio 1986, Herms and Mattson 1992). Increased growth decreases the amount of resources available for allocation to secondary metabolism or other differentiation processes. Thus, predictions of the carbon / nutrient balance hypothesis are a subset of the predictions of the growth / differentiation balance hypothesis (Herms and Mattson 1992). The growth / differentiation balance hypothesis predicts that trees experiencing drought and low nutrient levels will have high levels of C-based defenses. 49 These two hypotheses have been used to explain delayed—induced resistance as an incidental by-product of defoliation-induced nutrient deficiency (Tuomi et al. 1990). However, intraspecific patterns of growth and differentiation in response to herbivory may be adaptive expressions of phenotypic plasticity, analogous to patterns of phenotypic plasticity in life history patterns (Herms and Mattson 1992). In high resource environments where there is intense competition for resources, rapid growth is favored over defense. To minimize the cost of defense, faster growing plants may be able to express more phenotypic plasticity, manifested as induced responses. In low resource environments where resource retention is favored and growth is slow, the cost of producing secondary metabolites is low and high levels of constitutive defense are favored. While there are studies supporting the view that induced defenses are an incidental by-product of nutrient deficiency, there are also discrepancies that point towards an adaptive theory of defense (see Herms and Mattson 1992 for a review of both views). Among these are several studies in which insect damage caused a greater induced response than artificial defoliation (Coley et al. 1985, Edwards and Wratten 1985, Haukioja and Neuvonen 1985, Neuvonen et. a1 1987, Hartley and Lawton 1991). Compounds induced by herbivores can be similar or identical to those induced by bacterial and fungal pathogens or viruses, indicating induced responses may be an adaptive response, though not necessarily targeted at herbivores (Wink 1985, Hartley and Lawton 1987, Hammerschmidt and Schultz 1996). This study tests the effects of resource availability and defoliation on herbivore resistance in two tree species with contrasting life histories: paper birch (Betula papynfera Marsh.) and sugar maple (Acer saccharum Marsh.) Paper birch is a fast growing, early successional species and sugar maple is a 50 slower growing, late successional species. Fast growing plants are less likely to develop high concentrations of constitutive defenses than slow growing plants because foliage is more expendable and fast growth is needed to preempt competitors (Feeny 1976, Bryant et al. 1983, Loehle 1988). To reduce the cost of maintaining constitutive defenses, fast growing plants may employ induced defenses. Thus paper birch is predicted to have lower levels of constitutive defense and a higher level of delayed-induced resistance than sugar maple. Betula spp. produce C-based secondary metabolites (terpenoids and phenolics), but no known N-based secondary metabolites (Palo 1984, Reichardt et al. 1984, Sunnerheim et al. 1988, Glasby 1991). The induction of phenolics in birch by herbivorous insects is a well established phenomena. Acer spp. produce both C-based secondary metabolites (flavonoids, tannins, and saponins) and N-based secondary metabolites (alkaloids) (Miller and Feeny 1983, Barbosa and Krischik 1987). To test for herbivore resistance in this study, I used the outbreak defoliators gypsy moth (Lymantria dispar L.) and forest tent caterpillar (Malacosoma disstria Hiibner). Gypsy moth and forest tent caterpillar are considered the two most important outbreak defoliators of deciduous trees in North America (Mattson et al. 1991). Gypsy moth was accidentally introduced to the United States in the late 1860’s and appeared in Michigan in the 1950’s (USDA 1981). Forest tent caterpillar is native to Michigan. To simultaneously test the predictions of the above hypotheses of plant defense, the effects of forest tent caterpillar defoliation, soil moisture, and soil fertility on paper birch and sugar maple resistance to gypsy moth and forest tent caterpillar were tested. In 1994, treatment effects on rapid-induced responses to gypsy moth and forest tent caterpillar were tested for. In 1995, 51 delayed-induced responses were tested for, using gypsy moth. I predicted that induced resistance would be more pronounced in birch than maple, relative to background constitutive defense, and that fertilization of defoliated trees would counteract an induced response. Constitutive levels of defense are predicted to increase under drought conditions and decrease under high nutrient conditions. Since drought limits growth more than photosynthesis and does not directly alter C / N levels, it was used as a means of delineating between the predictions of the carbon/ nutrient balance and growth / differentiation balance hypotheses. METHODS The experimental design, treatments, and site were described in Chapter 2. Additional methods are described below. 1994 forest tent caterpillar/gypsy moth assay The forest tent caterpillar / gypsy moth bioassay was initiated in May, and run concurrently with defoliation of experimental trees by forest tent caterpillar in the field. Forest tent caterpillar and gypsy moth eggs were obtained from the Canadian Department of Forestry, Insect Production Laboratory, Sault Ste. Marie, Ontario. Insects were reared in petri dishes (8 cm x 2.5 cm) containing moist plaster of Paris with activated charcoal to remove impurities. Larvae were reared at 25°C on a 18:6 h light:dark photoperiod. The plaster was moistened to maintain humidity and leaf turgidity. Ten first instar forest tent caterpillar or five first instar gypsy moth were reared from egg hatch for two weeks (10 May-23 May) on detached foliage from one of the 96 trees. Insects received foliage from the same tree throughout the experiment. At the end of two weeks, surviving larvae were weighed as a group and the average mass of each individual was calculated. 52 1995 gypsy moth bioassay Newly molted fourth instar gypsy moth reared on artificial diet were obtained from the Canadian Department of Forestry, Insect Production Laboratory, Sault Ste. Marie, Ontario on 21 June 1995. The bioassay was initiated the next day. This coincided with the phenology of natural gypsy moth populations in the region. Larvae were reared at ca. 25°C under natural day / night photoperiods in petri dishes as described above. Insects were reared from fourth instar until pupation on detached foliage, one insect per tree. Each insect received foliage from the same tree throughout the experiment. Foliage was replaced every other day and supplied in excess of demand. Only fully-expanded mature foliage was used. E I] . | E 1' Detailed analysis of insect nutrition was performed during the fourth instar. Leaf area consumed, frass production and weight gained by larvae were quantified on a dry weight basis in a 48 h feeding assay. From these data growth (mg), consumption (mg), food digested (mg), and the amount of digested food converted to biomass (mg) were calculated (Table 3-1). Due to concern over statistical problems with analyzing ratios, nutritional data were analyzed not with nutritional indices, but with analysis of covariance (Packard and Boardman 1988, Raubenheimer and Simpson 1992). Treatment effects on growth were tested by analyzing final size with initial size as a covariate. Consumption was analyzed as the amount of food consumed, using initial size as a covariate. Treatment effects on the amount of food digested were determined by analyzing the amount of food assimilated using the amount of food consumed as a covariate (Doucet and Ball 1994). The ability of larvae to convert digested food to biomass was tested by analyzing 53 Table 3-1: Nutritional indices used in experiments with fourth instar gypsy moth to determine effects of host quality on growth and nutrition. Calculated on a dry weight basis. Nutritional Index (mg) Dependent Variable Quantified Covariate Growth final size initial size Consumption amount of food consumed1 initial size Food Digested assimilation 2 consumption Digested Food Converted to Biomass biomass increment increase 3 assimilation 1 amount of food consumed (mg) = foodi - fOOdf 2 assimilation (mg of food digested) = consumption - frass 3 biomass increment (mg) = final insect mass (Wf) - initial insect mass (Wi) 54 increase in larval biomass using the amount of food assimilated as a covariate. E ll . | . Following the fourth instar feeding assay, larvae were reared on foliage from the same trees until pupation. Pupal weight and development time (number of days from the beginning of the fourth instar to pupation) were measured. Statistical analysis The 1994 forest tent caterpillar / gypsy moth data were analyzed by analysis of variance (ANOVA) using SAS PROC GLM (SAS Institute, Inc. 1985) for individual biomass and SAS CATMOD (SAS Institute, Inc. 1985) for analysis of categorical survival data. Nutritional and pupal mass data from 1995 were analyzed by analysis of covariance (ANCOVA) using SAS PROC GLM (SAS Institute, Inc. 1985). Pupal mass was analyzed with initial weight as a covariate. Male and female pupal mass were analyzed both separately and together, with males converted to female equivalents. There was no significant difference between results for each sex, so combined results are presented. Development time was analyzed by ANOVA; initial weight was not significant as a covariate. RESULTS 1994 forest tent caterpillar/gypsy moth assay Forest tent caterpillar reared on birch foliage were twice as heavy as (0.08 :1: 0.01 mg) as forest tent caterpillar larvae reared on maple (0.04 i 0.01 mg) (Table 3.2). However, forest tent caterpillar reared on birch had a lower survival (51.2 i 4.7%) than forest tent caterpillar reared on maple (69.3 i: 4.7% survival) (Table 3-3). Gypsy moth performed equally well on both species. 55 .586 we... ......86841. fl......w..u..._.. ”8.6 w." . N .E6600.. .6 80.w06 u xv “066..0.>0.66< _ 6.. m6 xv .6..m. 666 66.6 . 6666......0n...66.32:...66..0..6.0D_._8.00..m .66 6m. . . 66..6~.....0......66..6w.......66..0..6.0n. 66.. .. vow . 66:60.....0....66..6w....u8.006m .. 36 66.6 . 66..0N.....0.._..66..6w.... .66 .66 . 66.806.604.462...26.09.00.003 .. .66 Ram . 66..6~.....0n.466..6._6.0n. ~66 ~06 . 66..6~.....0n....8.006m .66 ..66 . 66:60:68“. 6.6 6.... . 66..0w.......66..0._6.0n.«8.0066 666 .66 . 66..0m.......66..6..6.0n. 666 .m6 . 66.83.648.003 2.6 «N6 . 66.36.... 6m.m ...... . . 66..6._6.0D...8.00..m mN6 66.6 . 66633.00 .2. in... mm... . 8.8% 866.65 636.366. 0.86.6.6 6.66.366. xv 66..0..6> .6 00.66m 6.6.). $9.0 8:58.60 .60 ... .88... «66.8.8 .66.. 8.003 63. .6. 60. 0... 6. 60.60. 6.6:. .363 660 3.66.0.8 .60. .8.6. .206. .0... .6 086.6... 6.66.366. 66 066606.26. ..0... 666 0.60:0 66..0~._...0. .66..6..6.06 A286. .666... 660 60.... .0603 8.00.... 00.. .6 <>OZ< 6.6.. xv 660 86.6? ... .3003 63. 6. .306. .0... ”Na 036,—. 56 .586 Wm H 6.2.2.. 38.36 H ...: .....OWQ u ...... .mod Wu H a N 666600.. .6 80.w06 n xv “666.630.6616. _ 06 6.8.6 .66 ... 6.6 . 66666..6.0n....66666.......6666..6.0n.48.00% ...... 6m6. 66.. . 66.666.606.466636...666666.00 ... Ed mm... . 66..66..6.0n...6666w....*8.00..m 3.6 is... 6...m~ . 66666..6.0...*6666w..... $6 69m . 66..66..6.0n...6666..6.0n.68.00% 66.~ 666 . 66666..6.0n.46666..6.0n. .66 3...... 6m6~ . 66666..6.0..._..8.00..m ...... 66... 6.... . 666666.60". 1...... 66.6. 1...... 2.8 . 60..66.......6666._0.06_..60.006m :2. 8.0. mm... _ 8660662266606 ...... m6... ...6 . 6666w.6....8.00..m ...... mm... 6.... . 66666.... ... m6.n 6m.~ . 6666680048603 m6.~ m66 . 666666.00 86 3.2.2.. vwfim . 8.606% .6>.>.6m .6>.>.=m Nv 6666..6> .6 00.66m .662 >360 .6......0.6U .60 ... .8.6n. . N666.6200 6.6.. 0.0.003 63. .6. 66. 6. 60.60. .666. >663 666 .6......0.60 .60. .8.6. 6.66. .6... .6 .93.... 66 666606.86. ..0... 666 6.08.0 666666.60. 666666.06 8.66.6 .6666 666 60.... .066... 8.00.... 00.. .6 <>OZ< .60..6w0.60 6.6.. xv 666 8...? 0.663.60 8.003 63. 6. .636. .6.... "Wm 0.66... 57 Fertilization improved the host quality of maple, but decreased the host quality of birch. Fertilization decreased the survival of forest tent caterpillar larvae reared on birch by 10% (46.1 i 6.7% survival on fertilized birch, 56.3 :t 6.7% survival on non-fertilized birch), but increased larval survival on maple by 20% (fertilized maple 79.2 i 6.7%, non-fertilized maple 59.4 i 6.7%) (Table 3-3). The effects of fertilization and irrigation on the survival and mass of forest tent caterpillar were interactive. When fertilized trees were irrigated forest tent caterpillar survival decreased (53.76 i 6.66% survival on fertilized trees, 62.96 i 6.66% survival on non-fertilized trees), but when fertilized trees were not irrigated, survival increased (71.48 i 6.66% fertilized, 52.73 i 6.66% non-fertilized) (Table 3-3). The effects of fertilization and irrigation on forest tent caterpillar mass differed by tree species; there was a significant species x irrigation x fertilization effect (Table 3—2, Figure 3—1). There was no effect of irrigation or fertilization on the mass of forest tent caterpillar reared on birch foliage. When maple trees were irrigated, fertilization increased the mass of forest tent caterpillar larvae. There was no effect of fertilization on mass of larvae reared on non-irrigated maple. There was a significant species x defoliation x irrigation effect on forest tent caterpillar survival (Table 3-3, Figure 3-2). Irrigation enhanced rapid- induced resistance in birch, but induced susceptibility in defoliated maple. When birch were defoliated, irrigation decreased the survival of forest tent caterpillar larvae, but when birch were not defoliated, irrigation increased the survival of forest tent caterpillar larvae. For maple the opposite was observed. Irrigation increased the survival of forest tent caterpillar reared on defoliated maple, but decreased the survival of forest tent caterpillar reared on non-defoliated maple. 58 0.12 ’6» E. 0 (B E ..l 0.08 . R 3 32 > I ‘g I lrngated 3'5 D Non-irrigated to 0.04 4 (I) 6 E .9 m 0.00 - Fertilized Non-fertilized Paper Birch 0.12 0.08 ~ I Irrigated D Non-irrigated 0.04 - Biomass of Individual Larvae (mg) 0.00 - Fertilized Non-fertilized Sugar Maple Figure 3-1: The effects of soil moisture and soil fertility on the biomass of first instar forest tent caterpillar reared on paper birch (A) and sugar maple (B) in 1994 (:t SE). 59 100 804 8 Survival (%) I Irrigated D Non-irrigated ‘5 20~ Defoliated Non-defoliated Paper Birch B 100 801 Survival (%) I Irrigated D Non-irrigated 20* Defoliated Non-defoliated Sugar Maple Figure 3-2: The effects of defoliation and soil moisture on percent survival of first instar forest tent caterpillar reared on paper birch (A) and sugar maple (B) in 1994 (:t SE). 60 As with forest tent caterpillar, there was a species x defoliation x irrigation effect on gypsy moth survival (Table 3-3, Figure 3-3). However, the effect on gypsy moth was different than the effect on forest tent caterpillar. Irrigation enhanced the constitutive resistance of birch to gypsy moth. Gypsy moth larvae reared on foliage from non-defoliated birch had a much lower survival rate when the trees were irrigated than when the trees were not irrigated. Irrigation had no effect on rapid-induced resistance of birch or maple, or the constitutive resistance of maple to gypsy moth. There was also a significant defoliation x irrigation x fertilization effect on gypsy moth survival (Table 3-3, Figure 3-4). Constitutive resistance of unfertilized trees was higher when trees were irrigated than when they were not irrigated. There was no effect of irrigation on the constitutive resistance of fertilized trees or induced resistance (irrespective of fertilization treatment) to gypsy moth. Fertilization decreased constitutive resistance of trees to gypsy moth. Larvae were bigger on fertilized trees that were not defoliated (0.13 :t 0.01 mg fertilized, 0.11 i 0.01 non-fertilized) (Table 3-2). However, fertilization enhanced rapid-induced resistance to gypsy moth, decreasing size of gypsy moth reared on fertilized trees that were defoliated (0.10 :l: 0.01 mg fertilized, 0.13 :l: 0.01 mg non-fertilized). The effect of irrigation on gypsy moth biomass was also mediated by fertilization. Fertilization enhanced the resistance of irrigated trees but had no effect on the resistance of trees that were not irrigated. Gypsy moth larvae reared on foliage from irrigated trees were smaller when trees were fertilized (0.11 :l: 0.01 mg) than when they were not fertilized (0.13 i 0.01 mg). There was no effect of fertilization on the size of larvae reared on foliage from trees that were not irrigated. 61 100+ 80* Survival (%) 8 I Irrigated D Non-irrigated 4o _ 20 ~ 0 _ Defoliated Non-defoliated Paper Birch 100 — 80 4 Survival (%) 8 I Irrigated D Non-irrigated 4o _ 20 — o Defoliated Non-defoliated Sugar Maple Figure 3-3: The effects of defoliation and soil moisture on the percent survival of first instar gypsy moth reared on paper birch (A) and sugar maple (B) in 1994 (:I: SE). 62 100 « 80 - $3 .“2’ 60 4 g I Irrigated (I) D Non-irrigated 40 - 20 ~ 0 _. Fertilized Non-fertilized Defoliated L I Irrigated D Non-irrigated Survival (%) Fertilized Non-fertilized Non—defoliated FIgure 3-4: The effects of soil moisture and soil fertility on the percent survival of first instar gypsy moth reared on defoliated (A) and non-defoliated (B) paper birch and sugar maple in 1994 (t SE). 63 There was a significant species x irrigation x fertilization effect on gypsy moth survival (Table 3—3, Figure 3-5). When birch were fertilized, irrigation decreased the survival of gypsy moth larvae. However, irrigation had no effect on larvae reared on unfertilized birch. Irrigation had no effect on maple reared insects whether trees were fertilized or not. 1995 gypsy moth bioassay F ' t ' a a Tree species had a significant effect on growth, consumption and amount of digested food converted to biomass, but not amount of food digested (Table 3-4). In the year after defoliation, birch was a better host for fourth instar gypsy moth than maple (both birch and maple were of equal quality for first instars in 1994). Growth of gypsy moth larvae was greater on birch (44.13 i 0.74 mg) than maple (37.59 i 0.75 mg), although consumption of birch (136.79 i 6.42 mg) was less than maple (157.88 i 6.41 mg). The amount of digested food converted to biomass was greater on birch (18.25 i 0.71 mg) than maple (10.28 :I: 0.72 mg). Digestibility of birch and maple foliage was the same. Delayed-induced resistance decreased foliage consumption by gypsy moth larvae (134.59 i 6.48 mg on defoliated trees; 160.08 i 6.33 mg on non- defoliated trees). Delayed-induced resistance had no significant effect on the amount of food digested. The effect of delayed-induced resistance on growth and the amount of digested food converted to biomass was mediated by fertilization. Fertilization ameliorated the effects of delayed-induced resistance on growth of gypsy moth larvae on both paper birch and sugar maple (Table 3-4, Figure 3—6). Larvae reared on defoliated trees that were fertilized grew the same amount as larvae reared on trees that had not been defoliated in the 64 Survival (%) 8 I Irrigated D Non-irrigated 100~ . 801 40. 20- 0- Fertilized Non-fertilized Paper Birch 100 « 80 ~ 8 TS to. :Z: I Irrigated a) D Non-irrigated 40 - 20 « O .I Fertilized Non-fertilized Sugar Maple Figure 3-5: The effects of soil moisture and soil fertility on the percent survival of first instar gypsy moth reared on foliage from paper birch (A) and sugar maple (B) in 1994 (1 SE). 65 .58... man 2.2.2. :8... Wu" .23 30>»: 56%." .2." .mod wan ... N 68600.. .6 80.606 u xv ”666666.063... _ ... ... n. 2. 6.2.6 66*... ....NR 666 2.2.2.. .w.wm «*6... 3.66. . 26.66.50 8.0 8.0 2.0 2.0 . 8666660....8.606....8666606.62866 mm... ~66 5.6 .... . 60666660622666...606660.06 66... No.6 3.. .66 . 60666660628662.6286 66.6 66.6 . ..6 8.6 . 60666...60u.*6o..666.. a... 0.... 9.... .0... _ 8666.60.83.80...8.8.6 2.0 .. .6... a... ...... 66.. _ 562.660.286.806 666 . .6 66.6 666 . 66666.....0n._..8.00..m a... 6... 2.0 8.. . 863.660. 8.. 06.0 6.... 66.. _ 8.6066206666682866 66.6 5.6 .6.. 66.6 . 60666.5...6066..6.06 66.6 6... 2.6 66.6 . 666.666.78.866 606 6.6 3.6 66.6 . 66666.... 666 66.. ~66 36 . 66666806788066 66.6 8.6 ...... 6: ... S... . 606660.06 8.6 3...... 00.5 .. 66.6 :2. 66.2 . 3.866 566.68% 66.6.0660 6666656660 536.0 xv 6666..6> .6 00.666 .252: 3.6.0 .62. 5.8. .o .86... 6866.2. 66 666606.86. ..0... 666 m.00..0 666666.60. 66666.... 666666.06 2.0.00... 00.. .6 <>OUZ< 6.6.. xv 666 86.6? .6. 316 0.66... 66 50 40 .4 a g 30 -« g I Fertilized E a .. o Non-fertilized (‘5 20 ~ 10 T 0 Defoliated Non-defoliated Figure 3-6: The effects of defoliation and fertilization on growth of fourth instar gypsy moth reared on paper birch and sugar maple foliage (:l: SE). 67 previous year. Larvae reared on defoliated trees that were not fertilized grew 11.17% less over the 48 h period than larvae reared on defoliated trees that were fertilized. Fertilization also ameliorated the effects of delayed-induced resistance on the amount of digested food converted to biomass (Table 3-4, Figure 3-7). Fertilization had no effect on conversion of foliage from non- defoliated trees. Conversion of foliage from defoliated trees that were fertilized was not significantly different than conversion of foliage from non- defoliated trees. Conversion of foliage from defoliated trees that were not fertilized however, was 22.40% less than the conversion of defoliated, fertilized trees. Irrigation had no effect on the growth, consumption, digestibility, or amount of digested food converted to biomass of fourth instar gypsy moth feeding on birch and maple. r 'n t r a ’ n Gypsy moth larvae reared on birch took 21.8 :t 0.4 d to pupate from the fourth instar as compared to 20.6 i 0.4 d for larvae reared on maple (Table 3- 5). Pupal mass of insects reared on paper birch was 38.9% larger than that of insects reared on sugar maple (Table 3-5). There was a significant species x defoliation x irrigation effect on development time (Table 3-5, Figure 3-8) and pupal mass (Table 3-5, Figure 3- 9). Drought stress increased the constitutive defenses of birch, decreasing larval development time and pupal mass, but there was no effect of soil moisture on induced defenses. Pupae reared on non-defoliated drought stressed birch weighed 13.5% less than pupae on irrigated trees. Drought stress had the opposite effect on constitutive resistance of maple to gypsy moth. Again, drought stress had no effect on the induced-resistance of previously defoliated maple, but constitutive resistance of non-defoliated 68 N C 15~ I Fertilized D Non-fertilized Digested food converted to biomass (mg) '5' C l Defoliated Non-defoliated Figure 3-7: The effects of defoliation and fertilization on the amount of digested food converted to biomass for fourth instar gypsy moth reared on paper birch and sugar maple foliage (:t SE). 69 Table 3-5: F -values and df from ANCOVA of tree species (paper birch and sugar maple), defoliation, irrigation, fertilization effects and their interactions on development time (the number of days to reach pupation from the fourth instar) and pupal mass of gypsy moth”. Source of Variation df Development Time Pupal Mass Species 1 4.27 * 18.98 **** Defoliation 1 0.86 0. 10 Species*Defoliation l 5.13 * 0.24 Irrigation 1 0.00 0.14 Species*lrrigation 1 2.06 0.52 Defoliation*ln'igation 1 0.09 0.55 Species*Defoliation*Irrigation l 5.99 * 4.52 * Fertilization 1 0.00 3.88 Species*Fertilization 1 1.23 0.85 Defoliation*Fertilization l 0.20 3.31 Species*Defoliation*Fertilization 1 0.27 0.05 Irfigation*Fertilization l 0.33 0.56 Species*lnigation*Fertilization l 0.05 0.01 Defoliation*lrrigation*Fertilization 1 3.19 0.54 Species*Defoliation*lrrigation*Fertilization 1 0.91 0.13 Covariate l ....... 17.45 **** Error df 73 72 ' Abbreviations: df = degrees of freedom. 2 * =pS 0.05; **** =pS 0.0001. 7O b.) C N C I Irrigated D Drought Development Time (days) _ C 4 0 I Defoliated Non-defoliated Paper Birch 30 I Irrigated D Drought Development Time (days) 207 10- ‘ o- Defoliated Non-defoliated Sugar Maple Figure 3-8: The effects of defoliation and soil moisture on the development time (number of days required for larvae to reach pupation) of fourth instar gypsy moth reared on paper birch (A) or sugar maple foliage (B) (1: SE). 2000 71 1500 ~ 1000‘ Pupal Mass (mg) 500- I Irrigated D Drought Defoliated Non-defoliated Paper Birch h M 8 1 Pupal Mass (mg) § 500~ I Irrigated D Drought Defoliated Non-defoliated Sugar Maple Figure 3-9: The effects of defoliation and soil moisture on pupal mass of fourth instar gypsy moth reared on paper birch (A) and sugar maple foliage (B) (:t SE). 72 maple decreased, as evidenced by longer time required to pupate and no effect on pupal mass. Thus drought had disparate effects on the constitutive resistance of birch and maple and had no effect on induced-resistance of birch or maple. DISCUSSION 1994 In 1994, defoliation, irrigation, and fertilization had a larger effect on the survival of first instars than on larval growth. This suggests that early- instars that are able to survive stress-induced changes in host quality are able to compensate for the effects of decreased host-quality on their growth. First instar forest tent caterpillar were more sensitive to treatment effects than were gypsy moth. Maple was a better host for forest tent caterpillar than birch. The mean biomass of forest tent caterpillar larvae reared on birch was half of the biomass of larvae reared on maple, and survival was 18% less for forest tent caterpillar larvae reared on birch foliage than larvae reared on maple foliage. Defoliation had no effect on the biomass of early instar forest tent caterpillar, but did have an effect on survival. The effect of defoliation on survival was mediated by irrigation and was species specific. Defoliated birch expressed rapid-induced resistance to forest tent caterpillar when irrigated (lower forest tent caterpillar survival), but defoliated birch that were not irrigated did not have higher resistance (higher forest tent caterpillar survival). Defoliated trees that were irrigated had elevated levels of foliar carbon (Chapter 2) which may have provided enhanced substrate for secondary metabolism. This suggests that for birch, rapid induced resistance may be more pronounced in wet years than in dry years. Irrigation had the 73 opposite effect on defoliated sugar maple; irrigated maple didn’t express rapid- induced resistance, but non-irrigated maple did. Fertilization increased the quality of maple foliage for forest tent caterpillar, but decreased the quality of birch foliage, perhaps because fertilization increased foliar nitrogen content of sugar maple, but had no effect on the foliar nitrogen content of birch (Chapter 2). Forest tent caterpillar growth was higher on foliage from fertilized maple but only if trees were irrigated. Irrigation may have enhanced the nitrogen uptake of maple, improving foliage quality. Insects perform best on foliage that has high concentrations of nitrogen (Mattson 1980, Scriber and Slansky 1981) and adequate water (Scriber and Slansky 1981). As host plants for early instar gypsy moth, birch and maple were of equal quality. Growth and survival were equal on both tree species. Drought stress decreased the constitutive resistance of birch as evidenced by decreased gypsy moth survival but had no effect on sugar maple. Soil moisture also played a role in determining the effect of fertilization on gypsy moth size. When fertilized trees were irrigated gypsy moth mass decreased, but there was no effect of fertilization on gypsy moth when trees were not irrigated. Fertilization ameliorated constitutive defenses of birch and maple to gypsy moth, but enhanced induced defenses, resulting in lower gypsy moth survival. This is in contrast to a study by Hunter and Schultz (1993) in which fertilization mitigated a rapid-induced resistance response in one species of oak (Quercus prinus). 1995 Defoliation in 1994 caused delayed-induced resistance in paper birch and sugar maple in 1995. Delayed-induced resistance decreased growth, foliage consumption, and the amount of digested food converted to biomass 74 by gypsy moth larvae. Development time (number of days to pupation) increased for larvae reared on defoliated birch, but not on maple. There was no effect of delayed-induced resistance on pupal mass of gypsy moth. Birch was a better host for gypsy moth than sugar maple. Sugar maple maintain constitutive defenses that make them more resistant to gypsy moth. Insects reared on birch grew more, even though they consumed less foliage, and were able to convert more digested foliage to biomass. Pupae of insects reared on birch were also larger than the pupae of insects reared on maple. Gypsy moth may be unable to metabolize alkaloids present in sugar maple foliage (Barbosa and Krisch 1987). Consistent with predictions of the carbon/ nutrient balance hypothesis (Tuomi et al. 1984, 1990), delayed-induced resistance was ameliorated by fertilization for several of the variables tested. Larval growth on fertilized, defoliated trees did not differ from those of insects fed on non-defoliated controls. Fertilization also ameliorated the negative effect of defoliation on the amount of digested food converted to biomass. These results are consistent with those of Bryant et al. (1993) who found that fertilization reduced delayed-induced resistance to spear-marked black moth (Rheumaptera hastata) in manually defoliated Alaska paper birch (B. resinifera), presumably by inhibiting tannin induction. This supports the hypothesis that delayed-induced resistance is a result of defoliation-induced nutrient deficiency. However, despite the ameliorative effects of fertilization on resistance to insects, fertilization did not in actuality ameliorate the effects of defoliation on C / N ratio (Chapter 2). Drought stress has been proposed to facilitate insect outbreaks by elevating plant nutrient levels (Rhoades 1983, White 1984) and decreasing secondary defenses (Rhoades 1983). In this study however, larvae reared on 75 non-defoliated paper birch experiencing drought stress had a shorter development time and a smaller pupal weight than defoliated trees under drought conditions, suggesting that drought stressed foliage was of poorer host-quality. Drought lengthened development time of insects fed on non- defoliated sugar maple but had no effect on pupal mass. This may be the result of drought enhanced constitutive defenses. This study demonstrates that the expression and magnitude of delayed- induced resistance can be influenced by abiotic factors such as nutrient and water regimes. Several recent studies have demonstrated that nutrient availability has an effect on the expression of induced resistance (Bryant et a1. 1993, Hunter and Schultz 1993). The results of this study show that soil moisture is also an important factor determining variation in induction responses. The effects of soil moisture on the expression of defoliation- induced resistance have not previously been investigated, and deserve closer examination. Through further study it will be possible to evaluate which stresses have the largest impact on host quality. More studies are needed to understand how these multiple stresses interact and if the responses observed are consistent. This should enhance our understanding of how phytochemical changes in host quality mediate insect interactions and is critical to our understanding of how host quality influences the population dynamics of outbreak Lepidoptera. Chapter 4 INTERSPECIFIC INTERACTIONS BETWEEN INSECT FOLIVORES MEDIATED BY HOST-PLANT RESISTANCE: EFFECTS OF EARLY SEASON DEFOLIATION ON A LATE SEASON FOLIVORE ARE DEPENDENT ON RESOURCE AVAILABILITY INTRODUCTION Induced responses of trees to herbivory can slow growth and / or decrease feeding by herbivorous insects and may be an active defense against them. In order to be effective against a species of attacking insect, an induced response should be rapid, yet sustained long enough to affect the population of that insect. However, the longer a response is sustained, the more likely it is to affect other herbivore species. In the case of trees, resistance may be rapid, occurring in the season of defoliation, or delayed, occurring in the season (or seasons) following a defoliation event. Delayed-induced resistance in trees may play a role in the regulation of outbreak Lepidoptera having multi-year population cycles. By altering the nutritional quality of foliage for up to several years, a defoliating insect has the potential to affect other species, perhaps across guilds. Interspecific competition is generally not believed to play a large role in the structuring of insect communities (Hairston et al. 1960, Lawton and Strong 1981, Strong et al. 1984). Several studies have shown that competition via herbivory-induced changes in host quality may be an exception (West 1985, Faeth 1986, Harrison and Karban 1986, N euvonen et al. 1988, English-Loeb et a1. 76 77 1993, Felton et al. 1994, Hougen-Eitzman and Karban 1995). Chemical or physical changes in host-plant quality induced by herbivory have the potential to mediate interspecific interactions through rapid-induced changes when the two species feed concurrently or through delayed-induced changes when species are temporally separated. Herbivory-induced responses may also affect insect survival by influencing attack by natural enemies (Price et al. 1980, Faeth 1986, Bergelson and Lawton 1988, Schultz 1988). Damage may be a cue helping natural enemies to locate insects (Faeth 1986, Takabayashi and Dicke 1996). Decreased host quality may increase the mobility (Schultz 1983) or prolong the development of insects (Clancy and Price 1987, Haggstrom and Larsson 1995) exposing them to greater risk of predation. Within a season, defoliation may improve (as in Williams and Myers 1984) or decrease host-quality for insects. Complete defoliation by one species may result in production of higher quality reflush leaves for another species (Faeth 1987). Higher water content and lower toughness of reflush leaves may render them of a higher quality than the alternative mature leaves regardless of induced phytochemicals (Faeth 1992). The observed variation in resistance studies may be due to the timing, magnitude, or type of defoliation. Abiotic factors such as soil fertility or soil moisture may influence the magnitude of resistance responses. Late season insects accustomed to the seasonal deterioration of leaf quality may be better adapted to low leaf quality and less susceptible to induced changes than early season insects (Hanhimake 1989). The purpose of this study was to determine the effects of severe forest tent caterpillar defoliation (Malacosoma disstria Hiibner) on the performance of whitemarked tussock moth Orygia leucostigma (J .E. Smith). Forest tent caterpillar is an important defoliator of deciduous trees throughout the United States and Canada and has 8-11 year population cycles (Myers 1993). Whitemarked tussock 78 moth is a generalist feeder, found throughout much of North America (Johnson and Lyon 1991). They have 1-3 generations per year depending on latitude (Drooz 1985). Both forest tent caterpillar and whitemarked tussock moth are generalist feeders with overlapping host-plant and geographic distributions and could potentially compete directly and indirectly for resources. This study spanned two years. Paper birch and sugar maple were severely defoliated (90%) by forest tent caterpillar in May 1994. In the year of defoliation I tested the quality of the reflush foliage of paper birch for whitemarked tussock moth. Birch has long and short shoots which may respond to defoliation differently (Coleman and Jones 1991). In the year following defoliation I tested delayed-induced resistance to whitemarked tussock moth of birch and maple that had been defoliated during the previous year. To determine the effect of abiotic factors on the expression of induced resistance, I manipulated levels of soil moisture and soil fertility. METHODS The experimental design, treatments and site were as described in Chapter 2 unless otherwise noted as below. 1994 W106): The 1994 tussock moth experiment used a subset of 8 defoliated and 8 non-defoliated birch from the 24 experimental birch trees that were not fertilized. Insects were reared on one of three foliage treatments: immature leaves from non-defoliated birch, mature leaves from non-defoliated birch, or reflush leaves from defoliated birch. Level of irrigation was assigned randomly among the defoliated treatments and the effects of irrigation were not tested. Insects were reared in petri dishes (8 cm x 2.5 cm) containing moist plaster of Paris with activated charcoal to remove impurities. Insects were reared from 79 eclosion to pupation on detached foliage from each tree, four insects per tree. Eggs were obtained from the Canadian Department of Forestry, Insect Production Laboratory, Sault Ste. Marie, Ontario. Each insect received foliage from the same tree throughout the experiment. Larvae were reared at 25°C on a 18:6 h light:dark photoperiod. The plaster was moistened to maintain humidity and leaf turgidity. Foliage was replaced every other day and supplied in excess of demand. Assays were initiated in late July and timed to synchronize with the natural hatch of second-generation white-marked tussock moth in Midland county, Michigan. 21131110131512 Leaves for analysis of carbon / nitrogen content were sampled in August 1994. Leaves were picked 8-9am EST to standardize for diurnal variation in chemistry. Four samples of three leaves each were collected per foliage class (immature leaves from non-defoliated birch, mature leaves from non-defoliated birch, or reflush leaves from defoliated birch) from throughout the canopy. Sampled leaves were placed in ziplock plastic bags and kept on ice for transport to the laboratory. Leaves were dried, ground, and analyzed for C/ N content as described in Chapter 2. 1995 All 96 of the experimental trees were used for the 1995 assays. Eggs for the lab and field assays were obtained from the Canadian Department of Forestry, Insect Production Laboratory, Sault Ste. Marie, Ontario. 1.2211011910198656)! The laboratory assay was initiated on 18 July 1995. Larvae were reared at 25°C on a 18:6 h lightdark photoperiod in petri dishes as described above. Each larva was fed detached foliage from one of three classes: mature sugar maple, immature birch, and mature birch. Paper birch produces two distinct classes of 80 foliage; ”early leaves” which are formed in the bud the year before their emergence and ”late leaves”, the primordia of which are initiated during the same growing season in which they emerge. Since a late season defoliator would have the option of feeding on either immature or mature foliage, I tested the effects of feeding on each. Foliage was replaced every other day and supplied in excess of demand. Eight insects were reared in each petri dish until the fourth instar, at which time a detailed analysis of insect nutrition was performed. When half the larvae reached the fourth instar one fourth instar of intermediate size was selected for a 48 h feeding assay and the rest were discarded. At the end of the 48 h period, leaf area and dry mass consumed, frass production and weight gain during the 48 h period were quantified. Following the 48 h assay, larvae were fed fresh foliage three times a week until pupation. Pupal weights and the number of days from eclosion to pupation (development time) were recorded. 12W): The field assay was initiated on 20 July and ran concurrently with the laboratory assay. Five first instar larvae were reared until pupation outdoors in fine mesh bags, one bag per tree. Large branches with abundant foliage were selected. Insects were collected and weighed as pupae. Number of days required to reach the pupal stage from egg hatch was recorded. Statistical analysis Data were'analyzed using SAS (SAS Institute, Inc. 1985). The reflush experiment was analyzed as a nested ANOVA with insect nested within tree using SAS PROC GLM. Foliar C/ N content was also analyzed as a nested ANOVA with leaf class nested within tree. Tree was the unit of replication for both nested designs. The 48 h feeding assay was analyzed by analysis of covariance (ANCOVA) using covariates as described in Chapter 2 (Table 2—1). 81 Initial weight was not significant as a covariate for development time or pupal mass of lab or field insects and data were analyzed by ANOVA. RESULTS 1994 Wear Leaf type had a significant effect on development time (df=2, 34; F=41.0, =0.0001) and pupal mass (df=2, 30; F=4.6, p=0.0415) of males, but had no significant effect on the development time or pupal mass of females. Males reared on reflush foliage of previously defoliated trees had a longer development time and a smaller pupal weight than males reared on immature or mature birch foliage from non-defoliated birch. Male tussock moths reared on reflush leaves took longer to pupate (26.8 i 0.5 d) than males reared on mature (22.4 i 0.4 d) or immature (21.7 i 0.4 d) foliage. The pupal mass of males reared on immature leaves (0.181 i 0.004 mg) was larger than the pupal mass of males reared on reflush (0.163 :I: 0.005 mg) and mature (0.176 i 0.005 mg) foliage. gamma Reflush leaves were closer to immature leaves in foliar carbon and nitrogen content than to mature leaves. Mature foliage had a lower nitrogen content (2.64 i: 0.04%) than immature (3.33 i 0.04%) or reflush (3.15 i 0.04%) leaves (df=2,84; F=69.18; p=0.0001). Carbon content of mature foliage was also lower (49.59 :I: 0.13%) than that of immature (50.22 :t. 0.13%) or reflush (50.32 i 0.13%) foliage (df=2,84; F=9.24; p=0.0066). Thus immature and reflush leaves had lower overall foliar C/ N ratios than mature leaves. 82 1995 MW Leaf type There was no difference in growth between larvae reared on immature or mature paper birch leaves. Consumption and digestibility of immature birch leaves were higher than mature birch leaves, but the amount of digested food converted to biomass was the same for both leaf classes. Larvae consumed 30% more immature birch foliage than mature birch foliage and digested 26% more immature birch foliage than mature birch foliage. Growth of larvae was less on maple foliage than on birch foliage of either age. Larvae reared on mature mature maple grew 11.92 :I: 0.55 mg as compared to 15.97 i 0.54 mg of growth for larvae reared on mature birch foliage. Larvae reared on mature birch foliage consumed 76% more than insects reared on mature sugar maple foliage. The amount of digested mature maple foliage converted to biomass (4.96 i 0.37 mg) was lower than the amount of digested mature birch foliage converted to biomass (7.14 i 0.35 mg). There was no difference in digestibility between mature birch foliage and mature maple foliage. Defoliation Larvae reared on birch leaves were more sensitive to the effects of defoliation than larvae reared on maple leaves. Defoliation in the previous year induced susceptibility to whitemarked tussock moth in 1995. Larvae reared on mature leaves of birch defoliated in the previous year grew more than larvae reared on mature leaves of non-defoliated paper birch (leaf type x defoliation interaction, Table 4-1, Figure 4-1). There was no effect of defoliation on the consumption, conversion of digested food to biomass, or digestibility of mature birch foliage. When feeding on immature foliage, defoliation decreased the 83 .268de H .....i... £8.an— H ..:.:.. ..odwn .I.. .3. H936 fl ... . 6.6602. .6 600.606 0 x6 ”66666.>0.66< . 2. 6.. 2. R. .... E. F 6.. .....i... 0.56 .....i... 2.6m mnfi ......e... N96. :0... .wdme .. 06.? an...” .....i... afig . 66.. 66.6 6.66 6... NON .. N66 666 .. ~66 . 6.." 66.6 N66 .86 ..i 36 mm.” 36 66.6 . 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N . .m .N.. . 666 2.... 666. 1...... 6666 2...... 6.66 .. $6 666 ... 66.». mm... . 66686.6 66.6.3660 666666.660 4.36.6 $686.6 66.6.3660 6666664660 6.36.6 9.6.656 60.... .> 9.6.66.6 0.66.2 6.36586 .6.... ... 6:262. .666 6. 8:6 0.65.60 666.8... .63 nun—«Q ......25 .63 6.6.2.... .63 76.0.5. .63 l..— "TC 7.0 6.3.6.63 6.2.... .63 6.25 .60.. 6 . 6 0...... .60.. .2n66. 6. 6.6.6 0.06666. 63666.86...» 6666. 5.66. .6 600.66. 6.666.666 66 666606.86. ..0... 666 ...... 66666660. .8 66666.... ..e 666666.06 A2666. 0.6.66. .6 .60.... 0.6.6... .60.... 0.6.6.66... 06.0 .60. .6 6.00..0 0.6 66 <>OUZ< 6.6.. \6 666 86.6? k ”.... 036,—. 84 20.0 15.0 « 2'" . 10 0 I Defoliated g - DNon-defoliated 2 (D 5.0 ~ 0.0 — Mature Birch Immature Birch Figure 4-1: The effects of leaf age and defoliation on the growth of fourth instar whitemarked tussock moth reared on mature or immature birch foliage (1: SE). 85 ability of larvae to convert digested food to biomass (Table 4—1, Figure 4-2). However defoliation did not effect the growth of larvae reared on immature foliage, their consumption, or conversion of immature foliage to biomass. There was a significant defoliation x irrigation x fertilization effect on the ability of larvae to digest birch foliage (Table 4—1, Figure 4-3). Defoliation increased the digestibility of foliage from unfertilized trees that were drought stressed. Defoliation in the previous year had no effect on the growth of larvae reared on maple or on the amount of digested food converted to biomass, consumption, or digestibility of maple foliage by larvae. Irrigation Irrigation increased the amount of mature birch foliage converted to biomass by larvae, but had no effect on the conversion of maple foliage to biomass (Table 4-1, Figure 44). Soil moisture level had no effect on the growth of larvae reared on birch, or the consumption or digestibility of birch foliage by larvae. There was also no effect of soil moisture on the growth of larvae reared on maple or on the amount of digested food converted to biomass, consumption or digestibility of maple foliage. Fertilization Fertilization increased the growth of larvae reared on birch foliage but not those reared on maple (Table 4-1). Larvae reared on fertilized birch grew 18.11 :1: 0.58 mg, while larvae reared on non-fertilized birch leaves grew 16.00 i 0.58 mg. There was no effect of fertilization on the consumption of either birch or maple foliage. The amount of digested food converted to biomass was higher for insects reared on mature foliage from fertilized birch and maple (6.77 i 0.32 mg) than on mature foliage from non-fertilized trees (5.38 i 0.33 mg). For birch, the effect of fertilization on amount of digested food converted to biomass was more pronounced for immature than for mature leaves (Table 4-1). Larvae converted 86 15.0 a 15. 8 «310.0 . E .2 m . o I Defoliated 8 D Non-defoliated E 5.0 4 d) > C O o 0.0 - Mature Birch Immature Birch Figure 4-2: The effects of leaf age and defoliation on the amount of digested food converted to biomass by fourth instar whitemarked tussock moth reared on mature or immature birch foliage (:t SE). 87 70.0 T6 .3 O O l g I Fertilized DNot fertilized U) P C Food Digested (mg) .8 0 fl .° 6 P 6 I Irrigated Drought Defoliated 70.0 60.0 - 50.0 4 40.0 - , I Fertilized (I: Not fertilized 30.0 - 20.0 - Food Digested (mg) 10.0 4 0.0 - Irrigated Drought Non-defoliated Figure 4-3: The effects of defoliation, soil moisture, and soil fertility on the amount of food digested by fourth instar white marked tussock moth reared on defoliated (A) or non-defoliated (B) birch foliage (1: SE). 88 15.0 10.0 - I Irrigated D Drought Conversion to Biomass (mg) U: 6 0.0 - Mature Maple Mature Birch Figure 4-4: The effects of leaf age and soil moisture on the amount of digested food converted to biomass by fourth instar whitemarked tussock moth reared on mature and immature birch foliage (3: SE). 89 10.12 i 0.63 mg of immature birch foliage to biomass and 8.77 i 0.66 mg of mature foliage to biomass when trees were fertilized as compared to 7.20 i 0.69 mg and 7.42 i 0.67 mg of immature and mature foliage respectively when trees were not fertilized. Fertilization decreased the amount of immature birch foliage digested (Table 4-1, Figure 4-5), but not the amount of mature birch or maple foliage digested. Thus, although immature foliage from fertilized trees was more difficult for larvae to digest, they were better able to convert it to biomass. Larvae reared on mature maple leaves took slightly longer to pupate (27.2 i 0.5 d) than larvae reared on mature birch leaves (25.8 i 0.5 d) (Table 4-2). Pupal weight of insects reared on mature maple was also smaller (336.31 i 9.95 mg) than pupal weight of insects reared on mature birch (397.56 i 9.43 mg) (Table 4- 2). Defoliation had no effect on the pupal weight of insects reared on sugar maple (Figure 4-6), but pupae of insects reared on defoliated birch were smaller than the pupae of insects reared on non-defoliated birch (Table 4-2). There was a significant leaf type x defoliation x irrigation effect on the development time of insects reared on mature birch and maple leaves (Table 4-2, Figure 4-7). Irrigation increased the development time of larvae reared on mature birch leaves from trees that were defoliated in the previous year and the development time of larvae reared on maple leaves from trees that were not defoliated. There was a significant defoliation x irrigation x fertilization effect on development time (Table 4-2, Figure 4-8) and pupal weight (Table 4-2, Figure 4-9) of larvae reared on paper birch. Fertilization decreased development time and increased pupal weight of larvae on defoliated birch that were drought stressed. Fertilization also decreased the development time and increased the pupal weight of larvae reared on non-defoliated birch that were irrigated. 90 U. 9 o S c I Fertilized 30.0 ~ DNot fertilized 20.0 a Food digested (mg) 10.0 ~ 0.0 - Mature Birch Immature Birch Figure 4-5: The effects of leaf age and fertilization on the amount of food digested by fourth instar whitemarked tussock feeding on mature or immature birch foliage (1 SE). 91 .8868 u 1...... fichn n .. N .5338 we 88mg u xv umcoufi>8£< _ 2. K 8 2 8 Saw a: .. as. Ed 38 _ 8:38»... 83 :3 Ed .. 3n .. awe _ "Ea Ed 2.” m3 3a _ 9.788983 one on; .3 mac _ $925.83 8d .. 0.2. N: 8... _ 79.25 .8,— 93 m3 :3 a3 _ "E ”3 m3 *3 85 _ "re So as :4 3o _ re 85 9: 85 e3 _ 8.25 :3 3c 2; a; 2: _ 725 83 .. a? a: mg 8... _ 9.25. as: Ed So :3 SN 8 ... one men 22 n3 _ _ Ed m3 ”3 S..— . o ...: 3.2 .. NB. 8.... 8o _ 25 83 mas—2 395 858:6 3.53 "“32 395 838.6 Eta..— he ...—me: 8:32 .> :85 8:32 :85 83855 .> :85 8:32 .232 E 59: 383.3 coo—38223 .8 88: 395 can 30:83 5:: $96 mo 83:55 .5383 its. :6 8036825 :05 98 An: 838.33.. .6 :23me .2: sous—£3 A242: 822: .8 .283 88.9.: .283 8:388: 25 .32 go 38:6 65 co <>OZ< Ea: xv new 839». k "Na. 93am. 92 500.0 400.0 J 300.0 . I Defoliated D Non-defoliated Ts’ O Pupal Mass (mg) 2‘ O 0.0 Mature Birch Mature Maple Figure 4-6: The effects of leaf type and defoliation in the previous year on the pupal mass of whitemarked tussock moth (:t SE). 93 35.0 30.0 ~ 25.0 ~ 20.0 - I Irrigated D Drought 15.0 ‘ 10.0 - Development Time (days) 5.0 - 0.0 - Defoliated Non-defoliated Mature Birch 35.0 30.0 ~ 25.0 - 20.0 - I Irrigated 15.0 ~ D Drought 10.0 . Development Time (days) 5.0 . 0.0 - Defoliated Non-defoliated Mature Maple Figure 4-7: The effects of leaf type. defoliation in the previous year, and soil moisture on the development time (number of days from eclosion to pupation) of whitemarked tussock moth reared on mature birch (A) or mature maple (B) foliage (:t SE). 94 35.0 30.0 a 25.0 - 20.0 - I Fertilized DNot fertilized 15.0 a 10.0 - Development Time (days) 5.0 J 0.0 - Irrigated Drought Defoliated 35.0 30.0 . 25.0 . 20.0 . I Fertilized DNot fertilized 15.0 4 10.0 - Development Time (days) 5.0 . 0.0 - Irrigated Drought Non-defoliated Figure 4-8: The effects of defoliation. soil moisture, and soil fertility on the development time (number of days from eclosion to pupation) of white marked tussock moth reared on defoliated (A) or non-defoliated (B) birch foliage (:l: SE). 95 500.0 400.0 « ’6; .5, a) 300.0 4 (I) g I Fertilized 7;; 200.0 + DNot fertilized l I 100.0 — 0.0 - Irrigated Drought Defoliated 500.0 400.0 — ’6: E. a) 300.0 ~ (I) g [I Fertilized § 200.0 . DNot fertilized a. 100.0 4 0.0 — Irrigated Drought Non-defoliated Figure 4-9: The effects of defoliation. soil moisture. and soil fertility on the pupal mass of whitemarked tussock moth reared on defoliated (A) or non-defoliated (B) birch foliage (t SE). 96 Heldassax Due to high predation by stink bugs, only 122 of 470 whitemarked tussock moth larvae survived to pupation in the field: 52 females and 70 males. Species had a large effect on the pupal weight of both sexes (Table 4-3). Larvae reared on paper birch had a much higher pupal weight than larvae reared on sugar maple. Mean pupal weight of females was 493.3 1 15.88 mg for larvae reared on birch and 368.0 1 13.06 mg for larvae reared on maple. For males mean pupal weight was 179.70 i 5.00 mg for larvae reared on birch and 142.65 :I: 6.07 mg for larvae reared on maple. There was a significant species x defoliation x irrigation effect on pupal weight (Table 4-3, Figure 4-10) and development time (Table 4-3, Figure 4-11). Though combined results for males and females are presented, males were more sensitive to experimental treatments than females. There was no effect of delayed-induced resistance on the pupal mass of larvae reared on irrigated sugar maple foliage. However, larvae reared on foliage from drought stressed maple had lower pupal weights when trees had been defoliated, than on non-defoliated trees. There was no interactive effect of defoliation and irrigation on the pupal mass of larvae reared on birch foliage. Irrigation increased the negative effect of constitutive defenses of paper birch on development time of larvae, but decreased the constitutive defenses of sugar maple on development time. There was no effect of irrigation on delayed-induced resistance of either birch or maple. DISCUSSION 1994 During 1994 (the season of defoliation), male whitemarked tussock moth larvae were sensitive to the effects of defoliation, but females were not. Reflush leaves from defoliated trees were poor quality hosts for males. Males reared on reflush foliage had a longer development time and a smaller pupal weight than 97 ~m cod nNd — 2.6 whfi — ... mad Gd _ 86 2.0 _ 86 XV.— _ g; hvd — mad amd _ E .6 had _ ... mad and _ mam cad _ mod sh.— _ vod med u th wmd ~ cvd mmd _ 3:1. 0%: mN.m — mm“: Han—a Duh—«H. aflOEQO—D>OQ \wu 38.8.01: nBowen... . 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Defoliated Non-defoliated Paper Birch 600.0 500.0 . 400.0 1 I Irrigated D Drought 300.0 4 Pupal Mass (mg) 200.0 - 100.0 4 0.0 - Defoliated Non-defoliated Sugar Maple Figure 4-10: The effects of defoliation in the previous year and soil moisture on the pupal mass of whitemarked tussock moth reared on paper birch (A) or sugar maple (B) foliage in the field (:l: SE). 99 50.0 E 40.0- as E m , .E 30.0 - '— E I Irrigated E 200 . DDrought 2 d) > d) o 10.0 — 0.0 - Defoliated Non-defoliated Paper Birch 50.0 ’5’; 40.0— m 3 . 0 .§ 30.0 i '— E I Irrigated g 20.0 ~ DDrought 2 m > 0 o 10.0 - | 0.0 - Defoliated Non-defoliated Sugar Maple Figure 4-11: The effects of defoliation in the previous year and soil moisture on the development time (number of days from eclosion to pupation) of whitemarked tussock moth reared on paper birch (A) or sugar maple (B) foliage in the field (:I: SE). 100 larvae reared on immature or mature birch foliage. Thus, defoliation earlier in the season decreased host-quality later in the season. Other studies have also found birch regrowth foliage to be less palatable to herbivores (Haukioja and Niemela 1979, Bryant et a1. 1981, 1987). Immature leaves of non-defoliated birch were the highest quality hosts for male whitemarked tussock moth; males had the largest pupal weights and shortest development time when reared on immature leaves. Female tussock moth larvae were not affected by defoliation, indicating that they may be more efficient at compensating for changes in host quality. If fecundity is closely correlated with pupal mass of whitemarked tussock moth (as with other Lepidopteran species), it may be more important for females to regulate pupal mass than males. Reflush leaves were closer in foliar carbon and nitrogen content to immature leaves than mature leaves. Although insects found feeding on reflush leaves most comparable to feeding on mature leaves, the levels of foliar nitrogen and carbon of reflush foliage were not significantly different than carbon and nitrogen levels of immature foliage. Mature leaves were intermediate hosts, but had lower levels of foliar nitrogen than immature and reflush leaves. Thus the resistance of reflush foliage to whitemarked tussock moth males was not correlated with any change in foliar C/ N ratio. 1995 In the year after defoliation, birch was still a better host for whitemarked tussock moth than maple. Larvae reared on maple consumed less foliage than larvae reared on birch and were not able to convert as much foliage to biomass, resulting in less overall growth. Larvae reared on mature and immature birch foliage grew the same amount, but larvae had to consume and digest more immature birch foliage to achieve the same amount of growth. As late season 101 feeders, whitemarked tussock moth may be more well adapted to feeding on mature foliage. Larvae also took longer to develop to pupation on maple foliage than on birch foliage, but there was no difference in pupal size of insects reared on birch or maple. In contrast to the delayed-induced resistance of birch and maple to gypsy moth earlier in the season (Chapter 3), defoliation induced susceptibility of birch and resistance of maple to whitemarked tussock moth. Defoliation improved the quality of mature birch leaves for whitemarked tussock moth. Larvae reared on mature birch leaves from previously defoliated trees grew more than insects reared on mature birch leaves from non-defoliated trees. However, there was no corresponding effect of defoliation on the consumption, amount of digested food converted to biomass or digestibility of mature birch leaves for these insects. Defoliation decreased the amount of immature foliage converted to biomass by larvae, but had no effect on the overall growth of larvae or the consumption or digestibility of foliage. Larvae reared on sugar maple had lower pupal weights than larvae reared on non-defoliated sugar maple. Little is known about the feeding preferences of whitemarked tussock moth that could explain these variable responses. Whitemarked tussock moth are a tannin tolerant species (Karowe 1989, Barbehenn and Martin 1992). If resistance to gypsy moth earlier in the season was carbon-based, this may explain the positive effects on whitemarked tussock moth. The effects of delayed-induced resistance may also disappear later in the season (Hanhimaki 1989). Level of birch phenolics tapers off during the season, while level of maple phenolics remains constant (Schultz et al. 1982). Differential responses of gypsy moth and whitemarked tussock moth to feeding on birch could be due to a relaxation of resistance in birch during the season, or simply to different feeding preferences between the two species. 102 As in Chapter 3, extrinsic factors influenced plant resistance to insects. Soil moisture had no effect on the growth of larvae reared on birch or maple, but did effect the ability of larvae to convert birch foliage to biomass. Irrigation increased the conversion of birch foliage to biomass by insects and drought decreased the conversion of birch foliage to biomass. Irrigation increased the induced defenses of birch and the constitutive defenses of maple against whitemarked tussock moth, increasing development time. Fertilization increased the amount of maple foliage that larvae converted to biomass, but had no overall effect on growth. Fertilization did increase the growth of insects on birch, but had a larger effect on larvae reared on immature birch foliage than mature birch foliage. Larvae reared on mature leaves from fertilized birch converted more digested food to biomass and grew more than larvae reared on unfertilized trees. Fertilization decreased the amount of immature foliage that was digested, but also increased conversion of digested foliage to biomass resulting in a larger overall increase in growth. The effects of fertilization on the development time of larvae reared on birch were mediated by a defoliation x irrigation interaction. Fertilization increased induced defenses of drought stressed birch, and the constitutive defenses of irrigated birch. Field results were comparable to those obtained in the lab. Insects reared on paper birch in the field had bigger pupae than inSects reared on sugar maple. The effects of defoliation on pupal weight and larval duration were mediated by irrigation. Drought increased the induced resistance of maple, decreasing pupal weight, but had no effect on the induced resistance of birch. Irrigation increased the constitutive defenses of birch, resulting in longer development time, but decreased the constitutive defenses of maple, increasing development time of larvae on irrigated trees. Irrigation had no effect the development time of larvae reared on defoliated trees. 103 This study demonstrates that an early season defoliator can have an impact on host-quality for late season folivores in the year of and the year following defoliation. In the season of defoliation, reflush leaves were poorer hosts for whitemarked tussock moth. In the year following defoliation, the defoliated trees were better hosts for whitemarked tussock moth than non- defoliated trees. This indicates that defoliation-induced resistance by one species may have unpredictable effects on other species. Immature birch leaves were consistently better hosts across both years. This underscores the importance that leaf age may have on resistance. Leaf age is a particularly important factor in birch, because long shoots contain leaves of all ages. This is yet another factor creating considerable variability in results of plant resistance studies. This study attempted to delineate between the predictions of the carbon / nutrient balance and growth / differentiation balance hypotheses through simultaneous testing of nutrient deficiency and drought. The growth/ differentiation balance hypothesis predicts that both nutrient deficiency and drought will decrease growth and increase secondary metabolism (resulting in a decrease in growth / differentiation balance). The carbon / nutrient balance hypothesis predicts that drought will decrease C/ N ratio and thus secondary metabolism. The negative effect of drought on gypsy moth (Chapter3) and whitemarked tussock moth provides support for the growth/ differentiation balance hypothesis (Table 4-4). Since drought decreased tree growth (Chapter 2) and insect performance (Chapters 3 and 4) (presuming this decrease in performance is correlated with an increase in secondary metabolism), the growth / differentiation balance is supported. Nutrient deficiency did increase 104 Table 4-4: Predictions of the carbon/nutrient balance and growth/differentiation balance hypotheses for the effect of nutrient deficiency and drought on carbon/nutrient balance and growth/differentiation balance, respectively and observed results. Change in CIN Balance Change in G/D Balance Predicted Observed Predicted Observed Nutrient Deficiency T T l l Drought i no change i l 105 C/ N ratio, but drought had no effect on C/ N ratio (Chapter 2). Thus, the predictions of the carbon / nutrient balance hypothesis were unsubstantiated in this case. Chapter 5 CONCLUSIONS Results were consistent with predictions based on tree life history. Paper birch had higher growth and photosynthetic rates than sugar maple. Sugar maple had less capacity to tolerate defoliation, and this effect carried over into the following year; sugar maple defoliated in 1994 had lower growth, photosynthetic and foliar nitrogen rates in 1995. Growth of paper birch was less affected by defoliation, but more sensitive to the effects of irrigation and fertilization. Fertilization increased paper birch growth in 1994, but had no effect on sugar maple. Drought decreased the growth of birch in 1995, but had no effect on sugar maple. Sugar maple had higher levels of constitutive defense against gypsy moth and whitemarked tussock moth than paper birch. Defoliation by forest tent caterpillar in 1994 caused delayed-induced resistance to gypsy moth in 1995. Delayed-induced resistance decreased growth, foliage consumption, and the amount of digested food converted to biomass by gypsy moth larvae. Development time increased for larvae reared on defoliated birch, but not maple. There was no effect of delayed-induced resistance on pupal mass of gypsy moth. 106 107 The carbon / nutrient balance hypothesis did not accurately predict delayed-induced resistance effects on C/ N ratio. Defoliation was predicted to increase C / N ratio. Foliar nitrogen levels decreased in response to defoliation, but photosynthesis did not increase, resulting in a decreased C/ N ratio for defoliated trees. However, defoliation effects on herbivore resistance were generally consistent with the carbon/ nutrient balance hypothesis. Delayed-induced resistance to gypsy moth was ameliorated by fertilization. The expression and magnitude of induced resistance can be influenced by abiotic factors such as nutrient and water regimes. The role of soil moisture in the expression of rapid and delayed-induced resistance has been previously unexamined and appears to be important. The effect of irrigation on foliar carbon of defoliated birch suggests that defoliated birch may be more likely to produce excess carbon which could be allocated to defense in wet, rather than dry years. Irrigation mediated the effect of defoliation on host quality in both lab and field assays. Drought stress has been proposed to facilitate insect outbreaks by improving host quality. However in this study, drought stressed foliage was a poorer quality host for gypsy moth. Gypsy moth larvae reared on non- defoliated paper birch experiencing drought stress had a shorter development time and a smaller pupal weight than defoliated trees under drought conditions, suggesting that drought stressed foliage was of poorer host-quality. Drought lengthened development time of gypsy moth reared on non- defoliated sugar maple, but had no effect on pupal mass. 108 Effects of soil fertility and drought stress on tree growth, photosynthesis, foliar chemistry, and herbivore resistance were consistent with the growth / differentiation balance but not the carbon / nutrient balance hypothesis. The carbon / nutrient balance hypothesis did not accurately predict the effects of drought on resistance to insects, but the growth / differentiation balance hypothesis did. As predicted by the growth / differentiation balance hypothesis, drought decreased growth and increased secondary metabolism (as determined by resistance to insects). The carbon/ nutrient balance hypothesis predicts that drought will decrease C/ N ratio and secondary metabolism. Drought had no effect on C/ N ratio and increased resistance to insects. Thus, the predictions of the carbon / nutrient balance hypothesis were unsubstantiated. Forest tent caterpillar defoliation affected the performance of other species feeding on the same tree at the same time, later in the same year, and in the year following defoliation. Defoliation by forest tent caterpillar early in 1994 decreased quality of foliage for gypsy moth and forest tent caterpillar feeding concurrently with defoliation and whitemarked tussock moth feeding later in the same year. In 1995, the previous year’s defoliation induced resistance to gypsy moth feeding early in the season and susceptibility of birch and resistance of maple to whitemarked tussock moth feeding later in the season. APPENDICES APPENDIX 1 109 APPENDIX 1 Record of Deposition of Voucher Specimens* The specimens listed on the following sheet(s) have been deposited in the named museum(s) as samples of those species or other taxa which were used in this research. voucher recognition labels bearing the voucher No. have been attached or included in fluid-preserved specimens. Voucher No.: 19964 Title of thesis or dissertation (or other research projects): Paper Birch and Sugar Maple Resistance to Insect Folivores as Influenced by Defoliation, Drought, and Fertilization Museum(s) where deposited and abbreviations for table on following sheets: Entomology Museum, Michigan State University (MSU) Other mseums: Investigator's Name (a) (typed) Beth Dankert Date .11/18/96 *Reference: Yoshimoto, C. M. 1978. Voucher Specimens for Entomology in Nbrth America. Bull. Entomol. Soc. Amer. 24:141-42. Deposit as follows: Original: Include as Appendix 1 in ribbon copy of thesis or dissertation. Copies: Included as Appendix 1 in copies of thesis or dissertation. Museum(s) files. Research project files. This form is available from and the Voucher No. is assigned by the Curator, Michigan State University Entomology Museum. APPENDIX 1.1 110 APPENDIX 1.1 Vbucher Specimen Data Pages of Page i mama .83qu 223*: muse WNW\ SQQ\ w \mm\&\ 1 N .Eflmmfiz Guam 3333.5 33m ea 23: 0.5 5 388.. you ecosauoom vuumwa o>ona onu vo>auuem .oz nonuao> Himmma Avonhuv onuamz u.uousw«umo>=m Amuanaoooa ma masons Heaowuwvvo easy mama sass m mmmm mm .uomm afinmpco .mwnmz .mam paamm .nmh cospoauona pommCH am: a >pumonou mo .pnmo cmfiumcmo spasm memfipmooama mfimxpo mama ocaw om mnmpmcH cannon mm .uoom ownmpco .mwnmz .oum uaamm .nmm cowuuauonn pommCH am: e >nummuou mo .pamo cmflnmcmo nmcnaz mfiuummfiu meomoumamz mama mean am mumumCH cannon mm .uomm onempco .mhumz .mpm panmm .nmh cowpoanoua pommCH :mz v snummnou mo .pamo cmfiumcmu .4 gunman chances»; 400+ wouamonov was com: no wouuuHHou condo nonuo no mououam m e r r m m e .m n newswuoam now must Henna mssmmmmme uuw.«4..u A in P n".a E "we nonesz LIST OF REFERENCES LIST OF REFERENCES Appel, H. M. 1994. 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