Wright State University CORE Scholar
Browse all Theses and Dissertations Theses and Dissertations
2020
Influence of light va ailability on tree growth, defense, and emerald ash borer (Agrlius planipennis) success in white fringetree (Chionanthus virginicus) and black ash (Fraxinus nigra)
Michael S. Friedman Wright State University
Follow this and additional works at: https://corescholar.libraries.wright.edu/etd_all
Part of the Biology Commons
Repository Citation Friedman, Michael S., "Influence of light va ailability on tree growth, defense, and emerald ash borer (Agrlius planipennis) success in white fringetree (Chionanthus virginicus) and black ash (Fraxinus nigra)" (2020). Browse all Theses and Dissertations. 2310. https://corescholar.libraries.wright.edu/etd_all/2310
This Thesis is brought to you for free and open access by the Theses and Dissertations at CORE Scholar. It has been accepted for inclusion in Browse all Theses and Dissertations by an authorized administrator of CORE Scholar. For more information, please contact [email protected]. INFLUENCE OF LIGHT AVAILABILITY ON TREE GROWTH, DEFENSE, AND EMERALD ASH BORER (AGRILUS PLANIPENNIS) SUCCESS IN WHITE FRINGETREE (CHIONANTHUS VIRGINICUS) AND BLACK ASH (FRAXINUS NIGRA)
A Thesis submitted in partial fulfillment of the requirements for the degree of Master of Science
by
MICHAEL S. FRIEDMAN B.S., Massachusetts College of Liberal Arts, 2017
2020 Wright State University
WRIGHT STATE UNIVERSITY GRADUATE SCHOOL March 25, 2020
I HEREBY RECOMMEND THAT THE THESIS PREPARED UNDER MY SUPERVISION BY Michael S. Friedman ENTITLED “Influence of light availability on tree growth, defense mechanisms, and emerald ash borer (Agrilus planipennis) success in white fringetree (Chionanthus virginicus) and black ash (Fraxinus nigra)” BE ACCEPTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE.
______Don Cipollini, Ph.D. Thesis Director
______David Goldstein, Ph.D. Chair, Department of Biological Sciences Committee on Final Examination:
______Don Cipollini, Ph.D.
______Megan Rúa, Ph.D.
______John O. Stireman III, Ph.D.
______Barry Milligan, Ph.D. Interim Dean of the Graduate School
ABSTRACT
Friedman, Michael S. M.S. Department of Biological Sciences, Wright State University, 2020. Influence of light availability on tree growth, defense mechanisms, and emerald ash borer (Agrilus planipennis) success in white fringetree (Chionanthus virginicus) and black ash (Fraxinus nigra).
White fringetree is a host for the invasive emerald ash borer (EAB) despite being lower quality than black ash. Observations suggest that host trees grown in full sun are more resistant to EAB than those grown in shade, however chemical defense mechanisms and the impact of environmental stress have not been assessed. We quantified constitutive and induced defenses and other characteristics white fringetree and black ash phloem tissue grown under differential light conditions, and these traits were related to EAB larval performance. White fringetree had significantly lower constitutive and induced activities of defense associated enzymes and lignin but higher phenolic, non-structural carbohydrate, and oleuropein concentrations compared to black ash. Light limitation did not impact measured defense traits or EAB larval performance, although it did impact growth and photosynthetic efficiency. Our results suggest that phenolic profiles, metabolite abundance, and growth traits are important in mediating white fringetree susceptibility to EAB infestation.
iii TABLE OF CONTENTS
Page
LIST OF FIGURES...... vi
LIST OF TABLES...... vii
ACKNOWLEDGEMENTS...... viii
INTRODUCTION...... 1
Emerald ash borer-host tree system...... 6
Ash species and EAB...... 9
White fringetree and EAB...... 12
SPECIFIC AIMS AND HYPOTHESES...... 15
METHODS AND MATERIALS...... 17
Experimental design and location...... 17
Growth and photosynthetic efficiency...... 18
Methyl jasmonate treatment...... 19
EAB larval performance...... 20
Protein extraction and activity assays...... 20
Total phenolic and lignin content...... 22
Non-structural carbohydrate content...... 24
Oleuropein concentration...... 24
Statistical analyses...... 26
iv RESULTS...... 27
Growth and photosynthetic efficiency...... 27
Enzyme activity assays...... 28
Total phenolic and lignin content...... 29
Non-structural carbohydrate content...... 30
Oleuropein concentration...... 30
Principal component analyses...... 30
EAB larval performance...... 31
DISCUSSION...... 32
LITERATURE CITED...... 59
v
LIST OF FIGURES
Figure Page
1. Terminal bud growth throughout the study, presented by species and light treatment....43
2. Total growth rate, presented by species and light treatment...... 44
3. Initial terminal bud growth by species...... 45
4. Photosynthetic efficiency by light treatment...... 46
5. Protein concentration by species and MeJA treatment...... 47
6. Peroxidase, β-glucosidase, and chitinase activities...... 48
7. Polyphenol oxidase and trypsin inhibitor activities...... 50
8. Lignin, total phenolic, and non-structural carbohydrate concentrations...... 51
9. Oleuropein concentration by species and MeJA treatment...... 53
10. PCA 1: Control v. MeJA treated black ash...... 54
11. PCA 2: control v. MeJA treated white fringetree...... 55
12. PCA 3: control black ash v. control white fringetree...... 56
13. PCA 4: MeJA treated black ash v. MeJA treated white fringetree...... 57
14: Emerald ash borer larval gallery width by species...... 58
vi LIST OF TABLES
Table Page
1. Impact of species, methyl jasmonate, and light treatment on defense-associated enzyme activities, three-factor ANOVA results...... 42
vii ACKNOWLEDGEMENTS
I would like to thank my advisor and mentor, Dr. Don Cipollini, for taking me on as a student, for all of the laboratory training necessary to complete this study, and for the professional opportunities I have had over the past two years. I also need to include members of the Cipollini and Stireman labs including Emily Ellison, Dr. Donnie
Peterson, Alicia Goffe, and Juan Manuel Perilla Lopez for assistance with lab and field work when needed. Thank you to my committee members Dr. Megan Rúa and Dr. John
Stireman III for always being available to answer my questions. And a huge thanks to all the undergraduate and graduate students, staff, and faculty members of the Biological
Sciences Department that have made my time at Wright State University incredible.
Lastly, to my family and friends: thank you for all of the love and support you have given me. And especially to my grandmother, Sarah J. Frankland Ed.D., who is my biggest inspiration to never stop learning: thank you.
This research was possible due to funding from the USDA APHIS Cooperative
Agreement and the Graduate Student Assembly of Wright State University.
viii INTRODUCTION
Plants are known to possess many different mechanisms that are utilized as defenses against herbivory. Biochemical defenses, in particular, include a wide array of secondary metabolites and proteins which can reduce the digestibility of plant tissues, increase plant tissue toxicity, or cause oxidative stress to herbivores through the synthesis of reactive oxygen species (Bowles, 1990; Fürstenberg-Hägg et al., 2013; War et al.,
2012).
Studies on plant defense responses have addressed the impact of abiotic stressors, such as resource limitation, on the presence and effectiveness of particular defense mechanisms. Light, the main driver of carbon allocation for plants via photosynthesis, is an essential abiotic resource necessary for plant growth, development, and metabolism. It has been suggested that low light environments (shade), and thus carbon limitation, may constrain the synthesis of carbon-based secondary metabolites and limit the activity of well-known defensive proteins (Agrell et al., 2000; Calder et al., 2011; Folgarait &
Davidson, 1994; Hemming & Lindroth, 1999; Nabeshima et al., 2001; Yang et al., 2008), which may lead to increased feeding by herbivorous insect species (Raffa et al., 1998;
Suárez-Vidal et al., 2017; Yang et al., 2008). This relationship has previously been explained by the carbon-nutrient balance hypothesis which posits that in low light environments, where carbon is limited, plants will allocate resources to growth rather than carbon-based secondary metabolites (Bryant et al. 1983). However, this hypothesis has been widely challenged and criticized in recent decades as it cannot explain the subtle
1 variation among systems of plant defense (Hamilton et al., 2001; Stamp, 2003).
Regardless, plenty of studies have investigated the relationship between light limitation and plant defense; here, I provide a few selected examples.
Peroxidases (POXs) and Polyphenol oxidases (PPOs)
Peroxidases and polyphenol oxidases are two large classes of enzymes with a diversity of functions within the context of plant-insect interactions. Both POXs and
PPOs may potentially act as direct toxins (Duffey & Stout, 1996) or may readily oxidize phenolic metabolites in the presence of hydrogen peroxide (H2O2) and O2, respectively, forming electrophilic quinones that can permanently bind to and damage proteins, thus lowering the nutritional quality of the plant tissue for herbivorous insects (Constabel &
Barbehenn, 2008; Felton et al. 1994). In plant cells POXs are also involved in important processes such as lignin and suberin formation which can also act as a defensive response to herbivory by physically strengthening cell walls (Chen, 2008). Different isozymes of
POX are also known to both detoxify and produce reactive oxygen species (ROS) such as hydrogen peroxide, superoxide, hydroxyl radicals, and singlet oxygen (Schaffer and
Bronnikova 2012) which can be antinutritive, or toxic to herbivores (War et al. 2012).
The activities of POXs and PPOs in cotton (Gossypium hirsutum L.) leaves have been shown to significantly increase following infestation of cotton leaf worm
(Spodoptera litrua F.) (Usha Rani and Pratyusha 2013). Further, POX activities have been shown to be induced in resistant wheat (Triticum aestivum L.) varieties following infestation of the Russian wheat aphid (Diuraphis noxia Mordvilko) in comparison to susceptible wheat varieties, indicating their role in defense (van der Westhuizen et al.,
2 1998). POX activity has also been shown to increase in response to defoliation treatments
(Roitto et al. 2003) low light conditions (Zhang et al., 2010), and the interaction of low light conditions combined with herbivory treatments (Mooney et al., 2009). Conversely,
Cipollini (2005) showed there was no difference in POX activities between light exposed and laterally shaded Arabadopsis thaliana, and Shao et al. (2014) showed the highest levels of POX activities in a 50% irradiance treatment as compared to treatments of 30,
20, and 5% irradiance, although after 20 days this activity was reduced drastically.
However, it is worthwhile to note that the lateral shade treatment utilized by Cipollini
(2005) likely did not significantly alter total light exposure. Treatments of 50, 60, and
80% shading reduced the activity of PPO in lettuce (Lactuca sataiva L.) leaves when compared to lettuce grown in full sun conditions (Chutichudet and Chutichudet 2011).
Aside from these, no other studies that I am aware of have investigated the impact of light limitation on PPO activities. Based on a search of primary literature, the impact of light exposure and shade on POX and PPO activities is idiosyncratic, and likely cannot be generalized across species and ecological systems.
β-glucosidase (βG)
Unlike POXs and PPOs, β-glucosidases are not oxidative enzymes but hydrolytic enzymes that catalyze the breakdown of glycosides by cleaving off a sugar group and leaving an altered aglycone molecule (War et al. 2012). In plant cells, βG and its glycosidic substrates are isolated from one another and do not interact. However, mechanical damage caused by herbivory is likely to rupture cells, allowing βG to contact suitable substrates. Studies show that this two-step process, where the aglycone is often
3 more toxic than the original glycoside as a whole, is an important defense against herbivory for trees in the Oleaceae (Konno et al., 1999; Spadafora et al., 2008). The β- glucosidase isozyme specific to oleuropein, a unique secoiridoid glycoside, catalyzes a reaction that transforms oleuropein into a toxic and unstable alkylating agent that can crosslink and denature proteins necessary for herbivore growth (Konno et al. 1999).
Oleuropein is known to be found in the tissues of white fringetree (Gülçin et al., 2008), and therefore it is likely that this two-step defense mechanism is important with regard to insect resistance in this tree.
While the differential responses of βG activity to abiotic stressors has not been widely investigated, a few studies have highlighted potential trends. The canopy position of olive fruits was shown to affect the activity of βG at different stages of maturity; immature fruits on the outside of the canopy had higher βG activities, while at a mature stage fruits on the inside of the canopy had higher βG activities (Bartolini et al., 2014). In addition, expression levels of the olive GLU gene, which codes for the synthesis of β- glucosidase, were measured in olive branches incubated at normal light or dark conditions and results suggest that darkness lead to decreased expression levels of the olive GLU gene (Velázquez-Palmero et al. 2017). Future studies exploring if and how differential levels of light exposure may alter the activity of βG as well as the concentration and abundance of suitable substrates in other members of the Oleaceae, such as ash or white fringetree, may help understand intraspecific patterns in resistance to the emerald ash borer and other pests.
4 Chitinase (CHI) – Chitinases are enzymes most commonly associated with a plant’s resistance to pathogens. However, they have also been shown to aid in resistance to insect pests by breaking down chitin, a major component of arthropod exoskeleton and peritrophic matrix. Lawrence & Novak (2006) showed that the expression of poplar CHI is induced by gypsy moth (Lymantria dispar L.) attack, and further that the expression of poplar CHI in tomato (Solanum lycopersicum L.) plants inhibited Colorado potato beetle
(Leptinotarsa decemlineata Say) development. When expressed in tobacco, a chitinase from tobacco hornworm also inhibited the development of tobacco budworm. However, a bean chitinase expressed in potatoes had no inhibiting or negative effects on the aphid
Lacanobia oleracea L. (Gatehouse et al. 1997) and only a slight impact on female fecundity (Gatehouse et al. 1996). While studies have shown that CHIs are likely an important enzyme in plant defense responses, it remains unclear to what extent CHI is utilized as a protection against herbivory. Further, no studies have investigated the impact of stress on the activity of this enzyme in plant tissues.
Trypsin inhibitors (TI) – Trypsin inhibitors are proteins classified as serine protease inhibitors that bind to the receptors of proteases necessary for the digestion and breakdown of plant proteins by herbivores (Koiwa et al., 1997). TIs are known to be inducible by wounding, insect attacks, and chemical elicitation in both leaves and stems
(bark and vascular tissues) of trembling aspen (Populus tremuloides Michx) (Haruta et al., 2001) and have been suggested as a potential defense against the red oak borer
(Enaphalodes rufulus Haldeman) (Crook et al., 2009). Along with proteinaceous trypsin
5 inhibitors phenolic based trypsin inhibiting activity has been reported in species of
Fraxinus (Rigsby et al., 2016).
In one study, leaves of amaranth (Amaranthus hypochondriacus L.) grown under full sun displayed significantly higher activity of trypsin inhibitors than leaves from individuals grown in partial shade (Nagamatsu López et al., 2004). Similarly, cabbage
(Brassica oleracea L.) plants grown at slightly higher concentrations of light (+60 µmol photosynthetic photon flux density m-2 s-1) had significantly higher TI activity than those grown under a lower concentration of light (Broadway and Missurelli 1990). However, it is worth noting that this study did not have a shaded treatment, but simply a reduced light intensity treatment. In contrast to these studies, results from Cipollini (2005), where activity of TIs was higher in laterally shaded A. thaliana, suggest the opposite influence of shading.
EMERALD ASH BORER – HOST TREE SYSTEM
One class of herbivorous insects that are particularly sensitive to variations in host defense are woodboring beetles of the genus Agrilus (Buprestidae). These beetles, which feed on xylem and phloem tissues of trees, are known to be secondary pests of stressed or dying trees in their native ranges. North American examples include the bronze birch borer (Agrilus anxius Gory) and the two-lined chestnut borer (Agrilus bilineatus Weber), which experience periodic outbreaks due to host declines, often after an extended period of drought or following infestation by another insect pest (Haack et al. 1983, Muilenburg and Herms 2012). Additionally, other species such the emerald ash borer (Agrilus planipennis Fairemaire; EAB) and the gold spotted oak borer (Agrilus auroguttatus
6 Schaeffer) have been unintentionally introduced to new hosts on which they have become invasive primary pests subsequently causing extensive mortality and thus economic and ecological damage (Haavik et al. 2013, Klooster et al. 2018).
Interspecific variation in resistance and susceptibility of hosts to these wood- boring beetles is often attributed to differences in chemical defense mechanisms, would healing abilities, and non-structural carbohydrate reserves (Dunn et al. 1987, 1990,
Muilenburg et al. 2011, Villari et al. 2016). However, despite these hypotheses, no clear patterns in resistance to these beetles are known, and overall investigation into defense mechanisms of hardwood trees susceptible to Agrilus species is limited.
Emerald ash borer is a wood-boring beetle native to eastern Asia that was accidentally introduced to North America sometime in the late 1980’s or early 1990’s, and was discovered in the Detroit, Michigan area in 2002. Since then, EAB has spread unimpeded across the continent causing extensive mortality to native North American species of ash trees (Fraxinus spp.; Herms and McCullough 2014). In its larval stage,
EAB consumes the phloem tissue under the outer bark of its host tree, slowly leaving serpentine galleries behind as they develop into adults. As these galleries multiply, the removal of phloem tissue restricts nutrient transport and effectively girdles the host tree, leading to its eventual death. In its introduced range, EAB has caused unprecedented damage to black ash (Fraxinus nigra Marshall), white ash (Fraxinus americana L.), green ash (Fraxinus pennsylvanica Marshall), and to a lesser extent blue ash (Fraxinus quadrangulata Michaux), and pumpkin ash (Fraxinus profunda (Bush) Bush) (Villari et al., 2016). In addition, other North American Fraxinus species outside of EAB’s current introduced range may also be susceptible to attack (Herms 2015).
7 While the susceptibility of many North American ash species to EAB has been known for close to a decade, the susceptibility of trees outside of the Fraxinus genus was not realized until EAB larvae were discovered developing on white fringetree
(Chionanthus virginicus L.) (Cipollini, 2015). Dendrochronological techniques have revealed that EAB has likely been using white fringetree as a host since 2011 in southwest Ohio, if not earlier (Peterson & Cipollini, 2017; Thiemann et al., 2016). White fringetree is a small, multistemmed tree in the family Oleaceae that is closely related to ash (Jensen et al., 2002; Wallander & Albert, 2000), which is native to the southeastern
United States. Outside of its native range white fringetree is commonly planted as an ornamental tree along the East Coast of the United States and in Midwestern states such as Ohio and Indiana (Cipollini & Rigsby, 2015). It has recently been proposed that this host expansion is a result of ecological fitting (Cipollini & Peterson, 2018), a process by which a novel association is formed as a result of a novel host possessing similar traits as those of an ancestral or primary host. The close phylogenetic relationship of white fringetree and ash (Wallander and Albert 2000) suggests that these species may be physiologically similar, allowing EAB to easily locate and utilize it as a host.
In addition, observations have suggested that ash trees infested by EAB survive longer when growing in full sun conditions compared to those growing in shaded environments (Don Cipollini, personal communication). These observations suggest that low light has a limiting effect on the synthesis of secondary metabolites and activity of defense-associated enzymes and a negative impact on other mechanisms of resistance in ash trees.
8 ASH SPECIES AND EAB
In its larval stage, EAB is a wood-boring insect that consumes the phloem tissues beneath the bark of its host trees. Thus, quantifying differences in the profiles of secondary metabolites and activities of defense-associated enzymes is a strong method for comparing interspecific variations in resistance to EAB (Kehr 2006).
Cipollini et al. (2011) quantified activities of peroxidases (POX), polyphenol oxidases (PPO), and trypsin inhibitors (TI), as well as total protein content, the browning rate of phloem extracts, concentrations of total phenolic compounds and lignin, and phenolic profiles of Manchurian ash (F. mandshurica Ruprecht; the ancestral host of
EAB), and two North American host trees: black ash and green ash. Lignin levels and activity of PPO were not significantly different among species. They found that
Manchurian ash has high levels of protein, low TI activity, high rate of phloem extract browning, and intermediate intensities of POX activity and phenolic concentrations compared to North American species. White ash, on the other hand, displayed low rates of phloem extract browning and low activity of POX and phenolic compounds. Lastly, green ash showed similarly high activities POX and TI, high phenolic concentrations, and intermediate intensities of phloem extract browning. High performance liquid chromatography detected nine phenolic compounds unique to Manchurian ash, and principal components analysis based on phenolic profiles showed a clear distinction between Manchurian ash and North American species. While no clear patters in enzyme activities were reported with respect to EAB susceptibility, distinct differences in phenolic profiles suggest that secondary metabolites are important components of evolved defenses against EAB.
9 In order to uncover more precise variation in defense mechanisms between ash species Rigsby et al. (2016) quantified activities of defense-associated enzymes in phloem sampled from Manchurian and black ash before and after exogenous application of methyl jasmonate (MeJA). MeJA originates from jasmonic acid (JA), an important hormone in signal transduction pathways of plant defense responses which in this esterized volatile form is commonly utilized to simulate insect attack on trees and induce defense responses (Erbilgin et al., 2006). Constitutive activity levels, prior to MeJA application, of lipoxygenase (LOX), chitinase (CHI), ß-glucosidase (ßG), POX, and PPO were higher in Manchurian ash than black ash. However, MeJA application increased the activities of LOX in both species and the activity of phenolic based trypsin inhibitors in
Manchurian ash (Rigsby et al. 2016). These results support those of a previous study which also found no effect of MeJA on POX, PPO, or CHI activities but did find a strong induction of TIs, lignin, and the concentration of verbascoside, a phenolic glycoside, in
North American ash species. (Whitehill et al. 2014). These studies further highlight certain traits which may be important in determining interspecific variation in defense mechanisms to EAB.
To further investigate differences in ash resistance to EAB, and the impact of physiological stress on defense responses, larval performance, secondary metabolites
(phenolic profiles), and defense-associated enzyme activities were quantified in girdled and non-gridled Manchurian ash (Rigsby et al. 2018). Larval performance was significantly higher (2.3 times) in girdled compared to non-girdled trees. Girdling caused total starch and protein quantities to decline, increased levels of protein damage, and had no effect on phenolic profiles. However, girdling increased the activity of cinnamyl
10 alcohol dehydrogenase (CAD), CHI, and POX. These results suggest that although stress has a significant influence on larval development the activity of enzymes such as CAD,
CHI, and POX do not contribute to the reduced resistance of wounded Manchurian ash to
EAB, and that stress-induced mechanisms of resistance may be different than those governing constitutive variation in resistance between different ash species (Rigsby et al.
2016).
Environmental stresses such as light limitation and its impact on host tree defenses was tested in leaves of ash trees but not in the phloem (Chen and Poland 2009).
Light limitation had a strong negative impact on phenolic compounds and chymotrypsin inhibitors in young leaves, although no impacts were detected for mature leaves.
Additionally, trypsin inhibitors were more active in young leaves. Overall, adult EAB survived longer when feeding on mature leaves, although no impact of light limitation was detected in this study (Chen and Poland 2009). These results suggest that a stress such as low light may have stronger impacts on the chemistry of actively growing and differentiating tissues rather than already mature tissues. This study attempts to place these results in the context of the carbon-nutrient balance hypothesis which states that under carbon stress (low light) the amount of carbon-based secondary metabolites will decrease and the amount of nitrogen-based secondary metabolites will increase, and that trees grown in the sun will exhibit higher levels of carbon-based secondary metabolites
(Bryant et al., 1983). The finding of increased content of phenolic compounds (carbon- based) in young leaves grown in the sun is consistent with the carbon-nutrient balance hypothesis. However, the dramatic increase of chymotrypsin inhibitors (nitrogen-based) was inconsistent with the hypothesis, which has previously garnered substantial criticism
11 (Hamilton et al., 2001). Similar to Rigsby et al. (2018), Chen and Poland (2009) presents evidence that stress has a significant influence on ash tree defenses, and also highlights a few key mechanisms such as enzymes and phenolic metabolites that may be responsible for interspecific variations in resistance and susceptibility to EAB. However, this study is limited as it only investigated the effect of light and girdling stress on leaf chemistry and
EAB adult performance. Future studies should utilize a similar design to assess the impact of light limitation on host phloem defense responses and EAB larval success, as this is the stage of development which causes the most damage. Considering the carbon- nutrient balance hypotheses, as well as other hypotheses aimed at the balance of plant growth and defense, may be useful in understanding the results of a study like this.
WHITE FRINGETREE AND EAB
Since being discovered as a novel host of EAB in North America, scientific inquiry into the susceptibility of white fringetree has been limited. Larval success has been tested on white fringetree and two closely related species, Chinese fringetree
(Chionanthus retusus Lindley & Paxton) and devilwood (Osmanthus americanus Benth.
& Hook.f.) in field-grown trees and found evidence of infestation on white fringetrees
(32%) but no infestation on Chinese fringetree or devilwood. In a subsequent 40-day bioassay of larval success on cut stems, 97% of EAB individuals survived the bioassay on white fringetree and 100% of individuals survived on green ash, suggesting both hosts provide adequate nutrition for survival; however, the mass of larvae reared on white fringetree was significantly lower (Cipollini and Rigsby 2015). This result suggests that although the larvae can survive on white fringetree, they may incur a nutritional deficit or
12 face higher defense response which prevents them from thriving to the extent they do on
North American ash species. These conclusions support the notion that white fringetree, while of lesser quality than ash trees, is a suitable host for EAB development, although the mechanisms influencing this interspecific variation have not been determined.
Water availability was manipulated to investigate the influence of water stress on
EAB larval development in white fringetree and white ash (Rutledge and Arango-Velez
2017). The authors hypothesized that EAB would develop faster and grow larger in water deficient trees, as is the case in their coevolved host, Manchurian ash (Chakraborty et al.
2014). Their results showed that the overall rate of larval establishment (larvae found/larvae hatched) and survival was significantly lower on white fringetree (45%) than white ash (75%) when data from all sampling dates were combined, and survival rates at each sampling date respectively were significantly lower on white fringetree.
These results correspond with those reported by Cipollini and Rigsby (2015), suggesting that although white fringetree is a suitable host for EAB, it is likely of lower quality than ash species. Additionally, water treatment did not influence survival rates of EAB larvae at 21 days or 36 days, but at 61 days survival rates were significantly lower on water deficient than well-watered trees (Rutledge and Arango-Velez 2017). This is in contrast to results from previous studies which showed increased larval survival on ash species in response to decreased water availability (Chakraborty et al., 2014, Showalter et al.,
2018). Furthermore, callus presence, a measure of wound healing which may impede herbivore survival, was inversely associated with larval development but was not affected by water treatments and thus does not explain the response of larvae to a water deficit treatment. The authors suggest that the observed decrease in larval survival could
13 possibly be explained by decreased levels of carbon and nitrogen (due to environmental stress), and thus the building blocks necessary for the synthesis of secondary metabolites and defense associated enzymes, although there is no direct evidence presented to support this hypothesis. Future studies of this type would benefit from including an analysis of defense mechanisms, such as secondary metabolites and defense-associated enzymes, in both stressed and not stressed trees.
Along with these findings, two other studies found that the parasitoid Tetrastichus planipennisi (Hymenoptera: Eulophidae) located and parasitized EAB larvae significantly less (and in one study not at all) reared in white fringetree as compared to larvae reared in
North American ash bolts (Hoban et al. 2018, Olson and Rieske 2019). Notably, the latter study also found the cambial tissue of white fringetree to have significantly higher C content and greater density than white or blue ash, indicating interspecific differences in phloem chemistry. However, efforts are needed to elucidate more about potential defense mechanisms of white fringetree in comparison to ash species in order to better understand the future of EAB spread in North America. In light of the relatively close phylogenetic relationship of white fringetree and ash species (Wallander and Albert 2000, Jensen et al.
2002), it would be beneficial to quantify defense mechanisms implicated as potentially relevant in ash species as a starting point.
Nothing is currently known about the defense mechanisms employed by white fringetree in response to emerald ash borer infestation, which remains a significant ecological threat across eastern North America. The objective of this study is to provide the first insight into both constitutive and induced defense mechanisms of white fringetree in comparison to those of a highly susceptible ash species, black ash.
14 Additionally, in this study I examine and quantify the effects of limited light, a potential abiotic stress, on the ability of both species to mount induced defenses. Further, I relate activity levels of defense-associated enzymes and content of other measured compounds to rates of tree growth and photosynthetic efficiency in order to more completely understand the physiological impacts of light limitation. Lastly, I relate the potential defenses quantified here to rates of EAB larval success, growth, and development in the phloem tissue of the subject host trees. This study will contribute to the currently small body of literature regarding hardwood bark defense mechanisms in response to herbivory by wood borers. As EAB continues to spread unimpeded, an understanding of white fringetree defense mechanisms could be valuable for protecting this tree in its native and planted range across North America. Furthermore, due to increasing levels of globalization, the introduction of other non-native insects to novel environments is a phenomenon likely to continue, making any information regarding plant-insect interactions, such as mechanisms of plant defense, necessary for chemical ecologists and conservationists of the twenty-first century.
SPECIFIC AIMS AND HYPOTHESES
Aim 1: Quantify impacts of low light on constitutive and induced activities of phloem defense-associated enzymes
I hypothesized that the activities of defense-associated enzymes would be significantly higher in the phloem of white fringetree than in black ash, highlighting their role in defense against EAB, similar to what was found with Manchurian ash (Rigsby et al. 2016). Further, black ash would have higher concentrations of protein and non-
15 structural carbohydrates, while white fringetree would have higher levels of total phenolics, lignin, and oleuropein. With respect to the impact of abiotic stress here, I hypothesized that under low light conditions defense mechanisms would be weakened in both species due to resource limitation, although overall measures of defense would still be higher in white fringetree when compared to black ash. Additionally, I hypothesized that MeJA would increase the activity of defense-associated enzymes and secondary metabolites in all trees, although this increase would be more apparent in white fringetrees than black ash. Results from this investigation were designed to elucidate two main points of interest: 1) interspecific variation in resistance mediated by the activity of defense-associated enzymes, and 2) variation in this resistance as modulated by abiotic stress (here, light limitation), on both an intra- and interspecific basis.
Aim 2: Quantify the impacts of low light and defense induction on tree growth rate and photosynthetic efficiency
I hypothesized that white fringetree and black ash would have comparable mean rates of growth based on the variables measured. However, within species I hypothesized that trees grown under low light conditions would have significantly decreased growth rates when compared to trees grown in normal light. In terms of photosynthetic efficiency, I hypothesized that trees grown in low light conditions would have decreased levels of photosynthetic efficiency and that MeJA application would have no effect.
Aim 3: Quantify the impacts of low light and induced defenses on EAB larval establishment, growth, and development
16 I hypothesized that EAB larvae would have greater overall success when reared on black ash as compared to larvae reared on white fringetree, which has been shown in previous studies (Cipollini and Rigsby 2015, Rutledge and Arango-Velez 2017). I also hypothesized that in both tree species, larvae reared on trees grown in the limited light environment would have greater overall success based on our hypothesis that light limitation will decrease our host trees’ defense capabilities. This larval assay will provide important knowledge regarding the suitability of white fringetree as a novel host for EAB in comparison to a well-known susceptible species. Furthermore, it will allow us to compare the effects of abiotic stress and pre-infestation induced defenses on larval development between both tree species, a comparison which has not been made before.
METHODS AND MATERIALS
EXPERIMENTAL DESIGN AND LOCATION
This study took place on the campus of Wright State University in Fairborn, Ohio and consisted of 30 individual potted white fringetrees and 32 individual potted black ash that were arranged in a small plot on the northeastern side of campus (39.791207, -
84.049423) and surrounded with a six foot mesh fence to deter mammalian herbivores.
All 62 trees were re-potted in early Spring after acquisition in 7-gallon nursery containers using PRO-MIX BX potting medium with mycorrhizae (Premier Tech Horticulture,
Quakertown, PA) after washing roots of all previous potting medium. One quarter cup of
Osomocote slow release 14-14-14 fertilizer (the Scotts Miracle-Gro Company,
Marysville, OH) was added to each tree after being repotted. Black ash trees were originally sourced from Bailey Nurseries (St. Paul, Minnesota) and donated to Wright
17 State University in 2015 by The Ohio State University. White fringetrees were sourced from Bailey Nurseries through Siebenthaler Garden Center (Beavercreek, Ohio). Mean stem diameters measured 10 cm above the soil for black ash were 2.26 cm (± 0.33) and
2.90 cm (± 0.70) for white fringetrees. The study utilized a generalized randomized block design with eight blocks containing six to eight trees (difference due to uneven number of individuals between species), approximately four black ash and four white fringetrees.
Half of the blocks were designated as “shade” and the other half designated as “full light”. Blocks designated as “shade” were covered by a shade house constructed using a polyvinyl chloride pipe frame fitted with one layer of 80% black knitted shade cloth from
Greenhouse Megastore (Danville, Illinois) on all sides. The shade treatment was constructed in a way that mimics light level available in a forest understory. Within blocks, two individuals of each species were assigned to either a control treatment or
MeJA treatment. Shade houses and trees were arranged prior to their buds breaking in spring 2019. After the emergence of buds from all individuals, the trees were grown in this controlled arrangement for 90 days, receiving water as needed when soil became dry.
No precautions were taken to limit exposure to rain over the course of the experiment.
GROWTH AND PHOTOSYNTHETIC EFFICIENCY
Prior to the appearance of leaves, all terminal buds of each tree were marked with flagging tape. Total terminal bud growth – herein referred to as stem growth – was measured throughout the course of the experiment every two weeks using digital calipers.
All black ash trees had one terminal bud to measure, while white fringetrees had between one and six (due to their multistemmed growth habit), so the growth of all stems was
18 summed. In addition, both black ash and white fringetree are determinant trees, meaning their leaves grow only at the beginning of the growing season and not continuously throughout. Therefore, I calculated the initial rate of growth as:
(initial stem length – final stem length) / initial stem length using the first measurement taken as initial stem length, and the measurement taken 17 days later as final stem length. Using an OS-30 chlorophyll fluorometer (Opti-Sciences;
Hudson, New Hampshire), fluorescence of two leaves on each individual tree was measured 30, 60, and 95 days after the appearance of leaves, respectively. The latter measurement, at 95 days, was post MeJA application. Measurement of maximum photosynthetic efficiency of PSII is reported as FVM where FV represents variable fluorescence and FM represents maximal fluorescence. The average FVM for each tree was calculated at each sampling event.
METHYL JASMONATE TREATMENT
After 90 days of growth, induced defense responses were elicited through the application of a 100 mM solution of MeJA in deionized water and 0.01% Tween 20, as utilized by Whitehill et al. (2014) and modified by Rigsby et al. (2016). This solution was applied to the entire bark surface of a single branch on each tree with a foam paintbrush.
As an experimental control, a solution of deionized water and 0.01% Tween 20 (without
MeJA) was applied in the same way to all trees not treated with MeJA. Five days after
MeJA application, approximately 2 g of outer bark and phloem tissue was harvested from each of the trees, immediately placed on dry ice, and transferred back to the lab where it was stored at -20° C until extraction (Whitehill et al. 2014).
19
EAB LARVAL PERFORMANCE
After phloem samples and measurements of growth have been collected each individual tree was inoculated with five EAB eggs previously oviposited on coffee filters supplied by the USDA-APHIS-PPQ EAB Biocontrol Rearing Facility in Brighton,
Michigan. Cut pieces of filter with one egg were secured to the trunk of each tree with parafilm and left for 40-days while larvae developed in the phloem. Trees remained in their respective low and high light environments throughout the larval assay. After seven days hatch rate was assessed by visually inspecting the eggs. After 40-days, the whole tree (black ash) or the infested stem (white fringetree) was harvested and stripped of bark to inspect for larvae and their feeding galleries in the phloem tissue. Establishment rate, or the rate that neonate larvae that were able to begin feeding and successfully bore into the vascular tissue was determined by locating the start of each gallery. Gallery width at the widest point and larval head dimensions were measured using digital calipers to estimate instar stage, as done in Cappaert et al. (2005), and the establishment rates, overall survivorship, and mass of each larvae was recorded.
PROTEIN EXTRACTION AND ENZYME ACTIVITY ASSAYS
Phloem tissue was ground finely in liquid nitrogen with a mortar and pestle, after which proteins were extracted as according to Cipollini et al. (2011) with modified protocols outlined by Rigsby et al. (2016). Briefly, ground tissues were extracted in a
0.01 M sodium phosphate buffer with 5% polyvinylpolypyrrolidone (pH 7.0) at a ratio of
0.2 g tissue per ml of buffer. The resulting homogenate was vortexed for five seconds and
20 centrifuged at 12,000 rpm for 12 minutes at 4° C. After centrifugation, the supernatant was transferred to a clean tube and frozen at -20° C for storage.
Total protein content and enzyme activity assays were quantified spectrophotometrically using a SPECTRA MAX 190 Microplate Reader (Molecular
Devices; Sunnyvale, California). Total protein content in extracts was quantified as in
Bradford (1976) by comparing absorbance at 595 nm to a standard curve created with bovine serum albumin. POX activities were quantified as in Rigsby et al. (2016) by analyzing the oxidation rate of guaiacol, a common natural phenolic, as well as the rate of oxidative polymerization of oleuropein, a secoiridoid glycoside found in the Oleaceae family, for three minutes at 485 nm. 20 μl of protein extract were added to 200 μl of a 23 mM guaiacol solution with 0.375% H2O2 and 200 μl of a 3 mM oleuropein solution with
0.375% H2O2, respectively. Similarly, the activity of PPOs was quantified by analyzing the oxidation rate of both caffeic acid and catechol, natural phenolics which act as an intermediate compound in lignin biosynthesis and metabolites involved with wound response, respectively. For both assays, 30 μl of protein extract were added to 150 μl of each respective substrate solution and the change in absorbance was monitored for three minutes at 470 nm, as performed in Cipollini et al. (2004) and Cipollini et al. (2011).
Both POX and PPO activities were expressed as enzyme activity units per minute per milligram protein. βG activities were quantified following a modified procedure using 4-
Nitrophenyl β-D-glucopyranoside (pNBG), a synthetic glucoside, as a substrate (Konno et al. 1999, Romero-Segura et al. 2009, Velázquez-Palmero et al. 2017). 20 μl of protein extract were incubated with 20 μl of 11 mM pNBG for two hours at 25° C, after which 40
μl of 1 M of Na2CO3 were added to stop the reaction. The absorbance of the reaction
21 mixture was read at 405 nm and compared to a standard curve to quantify the concentration of p-nitrophenol released from the reaction. βG activities were expressed as the amount of enzyme necessary to release one umol of p-nitrophenol per minute per milligram protein. Activity of general CHIs was quantified using a suspension of chitin azure (Pedraza-Reyes and Lopez-Romero 1991, Rigsby et al. 2016) as a substrate, and measuring the absorbance at 575 nm. A suspension of 200 μl of protein extract and 200
μl of chitin azure was prepared at a ratio of 0.002 g per ml buffer and was gently shaken for 60 minutes at 30° C. Tubes were centrifuged at 10,000 rpm for 10 minutes, and the absorbance of the supernatant at 575 nm was compared to standard solution of chitin azure and sodium phosphate buffer. CHI activities were expressed as the amount of enzyme that released an amount of Remazol Brilliant Violet 5R Dye from hydrolysis of the chitin azure substrate equivalent to an increase in absorbance of 0.001.
The activity of protein based trypsin inhibitors was quantified by measuring the radial diffusion of soluble proteins in agar gels containing bovine trypsin as outlined by
Jongsma et al. (1993) and modified by Cipollini & Bergelson (2000). Results from this assay were compared to a standard curve prepared with soybean trypsin inhibitor (STI) through the same radial diffusion technique and results are expressed as μg of trypsin inhibitors per milligram STI equivalent.
TOTAL PHENOLIC AND LIGNIN CONTENT
Total phenolic concentration of samples was quantified using a modified procedure performed by Cipollini et al. (2011) and Bonello and Pearce (1993). HPLC grade methanol was used to extract soluble phenolics from 100 mg samples twice for 24
22 hours in the dark at 4 ° C. After centrifugation for five minutes at 13,4000 x g the two supernatants from each sample were pooled. New 2 ml Eppendorf tubes were filled with
150 μl methanol, 1 ml of d.d. water, and 5 μl of methanol extract, after which 75 μl of
Folin-Ciocalteu reagent were added to mixture. After three minutes 75 μl of 1 NaHCO3 were added, mixtures were vortexed for five seconds, incubated at 25° C for one hour, and the absorbance of samples was observed at 725 nm. Absorbance readings were compared to a standard curve prepared with gallic acid and expressed as mg phenolics per gram fresh weight.
Lignin concentration was quantified using the acetyl bromide method, where a reaction mixture of acetyl bromide is used to solubilize normally insoluble lignin
(Moreira-Vilar et al. 2014). A dry sample of 0.3 g phloem tissue was ground to a fine powder, homogenized in 7 ml of 50 mM sodium phosphate buffer (pH 7.0), and centrifuged at 2,000 x g for 5 minutes. The resulting pellet was washed and centrifuged consecutively twice with 7 ml of sodium phosphate buffer, twice with 7 ml of 1% Triton
X-100 in sodium phosphate buffer, twice with 1 M NaCl in sodium phosphate buffer, twice with 7 ml d.d. water, and twice with 5 ml of acetone to isolate the cell wall and remove any proteins that may increase absorbance of the sample. After washing, the remaining pellet was dried at 60° C for 24 hours. The next day, 20 mg of the pellet was placed into a glass tube with screw cap, and 0.5 ml of a 25% acetyl bromide in glacial acetic acid solution was added. Tubes were capped and placed in an incubator for 30 minutes at 70° C. After 30 minutes, the tubes were quickly cooled in an ice bath, and 0.9 ml of 2 M NaOH, 0.1 ml of 5 M hydroxylamine-HCl, and 6 ml of glacial acetic acid were added to each tube. Tubes were centrifuged at 1,400 x g for 5 minutes, and the
23 absorbance at 280 nm of 250 μl of the supernatant was read in duplicate. Lignin concentration is reported as mg of acetyl bromide soluble (ABS) lignin per gram tissue.
NON-STRUCTURAL CARBOHYDRATE CONTENT
Total non-structural carbohydrates were extracted by incubating 20 mg of ground plant tissue in 1 ml of 0.2 N H2SO4 for one hour in a boiling water bath. After one hour, samples were cooled, centrifuged at 10,000 rpm for 10 minutes, and the supernatant was transferred to a new tube. In a 13 x 100 mm glass culture tube, 15 μl of sample solution were added to 385 μl of d.d. water, to which 400 μl of a 5% phenol solution and 2 ml of concentrated H2SO4 were added. Solutions were incubated at room temperature for 10 minutes, vortexed for 5 seconds, and incubated at room temperature for 30 more minutes.
After incubation, the absorbance was read at 490 nm in a Genesys 10 UV Scanning
Spectrophotometer (Thermo Scientific; Waltham, Massachusetts) and compared to a standard curve of D-glucose. The concentration of non-structural carbohydrates is reported as μg per ml of extract.
OLEUROPEIN CONCENTRATION
Powdered tissue was extracted in 500 μl 100% HPLC-grade methanol containing
500 μg per ml butylated hydroxyanisole (BHA) as internal standard. Extraction solvent was added, tubes vortexed, and homogenates sonicated in an ice bath for 5 min before centrifugation at 10,000 g (5 min). Supernatants were transferred to fresh tubes, the procedure was repeated, and supernatants pooled. Tubes containing extract were centrifuged at 21,000 g (5 min) and 100 μl aliquots were transferred to autosampler vials.
24 A four-point standard curve containing 1000, 100, 10, and 0 μg ml-1 oleuropein was additionally prepared in extraction solvent.
The quantification of phenolics was performed essentially as described by Eyles et al. (2007) and Whitehill et al. (2012) using a Shimadzu LC-2030C 3D Plus
Prominence-i high performance liquid chromatograph-photodiode array detector (HPLC-
PDA). A Nucleosil C18 (250 x 4.6 mm; 5 µm particle size) column equipped with a 5 mm pre-column of the same stationary phase was used. The binary solvent system was 2%
(v:v) acetic acid (mobile phase A) and 2% (v:v) acetic acid in methanol (mobile phase B) at a flow rate of 1 ml min-1. The PDA was set to scan 190-400 nm, the column oven was set to 50°C, the autosampler was set to 4°C, and 10 μl extract were injected into the
HPLC. The elution program was set as follows (percentages refer to proportion of solvent
B): 5–15% (0–15 min); 15–30% (15–35 min); 30–40% (35–40 min); 40–60% (40–50 min); 60–90% (50–55); 100% (55–60 min); 5% (60–65 min) (Eyles et al. 2007; Whitehill et al. 2012).
The i-PeakFinder algorithm in LabSolutions software (Shimadzu) was used to integrate all peaks from 41.00-56.00 minutes with a minimum peak area of 10,000. Peak identity of oleuropein (RT = 43.58 min, λmax = 280 nm) was confirmed in samples based on retention time and UV spectral matching to the external standard and quantified based on regressions (R2 > 0.99) of the four-point standard curve. Oleuropein peak areas were quantified at 280 nm and expressed as mg oleuropein per gram fresh weight.
25 STATISTICAL ANALYSES
All analyses were performed in R version 3.5.2 (R Core Team 2020). Outliers were identified and removed using the ‘outliers’ package (Komsta 2011), removed from the dataset, and replaced with “NA”. To meet the assumptions of normality, the distribution of residuals for all response variables were transformed as necessary. In particular, POX activities, PPO activity using catechol, CHI activity, and total protein concentration were log10 transformed while PPO activity using caffeic acid as a substrate,
ßG, and TI activities were square root transformed. The effects of species, MeJA treatment, light treatment, and their interactive effects on enzyme activities, total phenolic, lignin, non-structural carbohydrate, and oleuropein contents, along with EAB larval gallery width were modeled using a linear model approach. Models were tested using three-factor ANOVA, where statistically significant differences between groups were assessed at an alpha of 0.05. Differences in photosynthetic efficiency as effected by species and light treatment were assessed at days 30 and 60 using a two-factor analysis of variance. At day 95, the effects of species, light treatment, and MeJA application on photosynthetic efficiency were tested in a three-factor analysis of variance. Total stem growth was only measured throughout the first 90 days of the experiment, prior to MeJA application, and therefore the effects of light treatment and species on this variable were assessed in a two-factor analysis of variance using the same approach. Effects of species and light treatment on initial growth rate (between the first two measurements) were assessed using a two-factor analysis of variance as well. The effects of species, MeJA treatment, light treatment and their interactions on hatch and establishment rates of EAB eggs and larvae, respectively, were tested with a logistic regression approach and Chi-
26 squared tests. Using the ‘emmeans’ package, Tukey-HSD post-hoc analyses were conducted when our models showed that multifactor interactions had significant effects on the dependent variables (Lenth 2019).
Principal components analysis was used to visualize the main factors driving interspecific differences in defense induction. This was accomplished by four separate analyses comparing levels of defense grouped as: between control and MeJA in white fringetree (1) and black ash (2) separately, comparing control levels between species (3), and comparing levels after MeJA treatment between species (4). To account for any missing data due to experimental errors, transcriptional errors, or outlier removal (less than 5% of the total data) multivariate imputation by chained equations, via the ‘mice’ package, was used to impute data using a random forest algorithm (van Buuren and
Groothuis-Oudshoorn 2011). Data including enzyme activities, protein, non-structural carbohydrate, phenolic, lignin, and oleuropein content of trees were used in all four principal component analyses. All data were standardized to mean of 0 and standard deviation of 1. The difference between MeJA and control treatments or species was highlighted in the biplot using ellipses representing 95% confidence intervals. Statistical differences between groupings were analyzed by analysis of similarities using the
ANOSIM function in the ‘vegan’ package (Oksanen et al. 2019).
RESULTS
GROWTH AND PHOTOSYNTHETIC EFFICIENCY
Results from two-factor ANOVA using showed that both species and light treatment interactively impacted total stem growth (F1,56 = 6.62; P = 0.01). Specifically,
27 total growth of white fringetrees in the full light treatment was 74.6% greater than white fringetrees in the shade treatment whereas black ash grown in the shade treatment was
23.5% greater than black as in the full light treatment (Figure 1, 2). Similarly, initial growth rate was impacted by species and light treatment (F1,56 = 5.74; P = 0.02). Initial growth rate of white fringetree grown in the full light treatment was 33.3% greater than white fringetree grown in the shade, and initial growth rate of black ash grown in the shade treatment was 42.9% greater than black ash grown in the sun (Figure 3).
Photosynthetic efficiency was significantly different between light treatments at
30 (F1,46 = 47.30; P < 0.001), 60 (F1,51 = 94.41; P < 0.001), and 95 (F1,50 = 27.15; P <
0.001) days of growth but there was no significant effect of species, MeJA treatment, or the interaction terms in our analyses. Briefly, photosynthetic efficiency, FVM, at 30 days was 52.0% higher for shade grown trees than for sun trees, at 60 days was 35.0% higher for shade trees than for sun trees, and at 95 days was 33.3% times higher for shade trees than for sun trees (Figure 3).
ENZYME ACTIVITY ASSAYS
Total protein concentrations in phloem samples were 150.0% higher in white fringetree than in black ash and were 53.4% higher in control trees than in trees treated with MeJA (Table 1; Figure 4). The activity of POX measured using guaiacol as a substrate was 29.3% higher in black ash than in white fringetree and was 23.1% higher in
MeJA treated trees than in control trees (Table 1; Figure 5A, B). The activity of POX measured using oleuropein as a substrate was not significantly impacted by any of the treatments. βG activity using pNBG as a substrate was 119.3% higher in black ash than in
28 white fringetree and was 42.1% higher in MeJA treated trees than in control trees (Table
1; Figure 5C, D). CHI activity measured using chitin azure as a substrate was 61.2% higher in black ash than in white fringetree and 32.5% higher in MeJA treated trees than in control trees (Table 1; Figure 5E, F). The interactive effect of species and MeJA was found to significantly impact the activity of PPO when caffeic acid was used as a substrate. Specifically, there was no difference between control and MeJA treated white fringetrees. However, PPO activity was 346.0% higher in MeJA treated than control black ash (Table 1, Figure 6A). The activity of PPO using catechol as a substrate was only significantly impacted by species, where activity was 1,526.1% higher in black ash than in white fringetree, where it was almost nonexistent (Table 1, Figure 6B). Finally, the activity of protein-based TIs was 64.5% higher in trees grown in the shade treatment than in full sun. Other than TI, no other measured defense associated enzyme was influenced by light treatment (Table 1; Figure 6C).
TOTAL PHENOLIC AND LIGNIN CONTENT
The interactive effect of species and light treatment was found to have a significant impact on total phenolic content (F1,52 = 5.31; P = 0.025). Pairwise post-hoc analysis revealed that species was the main factor driving differences in our model, where overall total phenolic content of white fringetree was 9.3% higher than that of black ash
(F1,52 = 38.93; P < 0.001) (Figure 7B). Mean lignin content was 31.0% higher on average in black ash than in white fringetree (F1,44 = 42.99; P < 0.001), and there was no significant effect of light or MeJA treatments (Figure 7A).
29 NON-STRUCTURAL CARBOHYDRATE CONTENT
On average, the content of non-structural carbohydrates was 36.1% higher in white fringetree than in black ash (F1,52 = 30.95; P < 0.001). No other factors or interactions in our model had any effect on non-structural carbohydrate content (Figure
7C).
OLEUROPEIN CONCENTRATION
Average oleuropein concentration was significantly impacted by both species and
MeJA treatment (F1,52 = 8.79; P = 0.005). Specifically, oleuropein content in control white fringetrees was 7.18 times higher than in MeJA treated white fringetrees, 11.72 times higher than control black ash, and 25.60 times higher than MeJA treated black ash
(Figure 8).
PRINCIPAL COMPONENT ANALYSES
Principal component analyses were used to visualize the differences in levels of constitutive and induced defenses in both species, separately, as well as differences between species at both of these levels of defense. In our first PCA which compared black ash defenses in both control (constitutive) or MeJA treated (induced) trees, PC1 accounted for 45.8% of the variation while PC2 accounted for 12.8% of the variation, in sum accounting for 58.6% of total variation (Figure 9). Overall weak differences were found between these groups (R = 0.081; P = 0.043). Our second PCA which compared white fringetree defenses in both control and MeJA treated trees displayed weak differences between groups (R = 0.368; P = 0.001), with PC1 accounting for 37.7% and
30 PC2 accounting for 13.1% of the variation, in sum 50.8% (Figure 10). The third PCA comparing constitutive levels of defense (control, untreated) between black ash and white fringetree in our study displayed a moderate and significant difference between groupings
(R = 0.462; P = 0.001) with PC1 accounting for 46.7% and PC2 accounting for 18.1% of the variation, in total the first two dimensions accounting for 64.8% (Figure 11). Our fourth PCA comparing induced levels of defense (MeJA treated trees) displayed moderate differences between species (R = 0.347; P = 0.001). PC1 accounted for 45.3% of variation, PC2 accounted for 12.6% of variation, and together both PCs accounted for
57.9% of total variation in the analysis (Figure 12).
EAB LARVAL PERFORMANCE
Hatch rate of EAB eggs on all trees in this study was 63.0% (n = 287). Light treatment had a significant effect on hatch rate (P < 0.001). Specifically, hatch rate on trees grown in full sun conditions was 0.74 ± 0.22, while hatch rate on trees grown in the shade treatment was 0.51 ± 0.29. Establishment rate, or the rate of neonate larvae that were able to begin feeding and successfully bore into the vascular tissue, was significantly higher on trees grown in the full sun (0.86 ± 0.25) than on trees grown in the shade (0.85 ± 0.23) (P < 0.01). Weight of larvae was not compared between species as I only retrieved three surviving individuals from all white fringetrees in the study.
However, of those three, average weight was 0.0047 g ± 0.003. Weight of larvae collected from black ash trees in all treatments was not significantly affected by MeJA or light treatments, and on average was 0.007 g ± 0.005, which is notably higher than the few collected from white fringetrees. Species had a significant effect on average larval
31 gallery width; on average, larval galleries were 30.4% wider on black ash than on white fringetree (F1,52 = 4.81; P = 0.03) (Figure 13).
DISCUSSION
Previous studies have shown that although EAB can successfully utilize white fringetree as a host, larvae survive at a lower rate and develop slower than if reared on
North American ash trees (Cipollini and Rigsby 2015, Rutledge and Arango-Velez 2017).
Despite these findings, no studies to date have investigated the particular mechanisms mediating this interspecific difference in host suitability, or more specifically any defense responses that white fringetree may possess that North American ash species do not.
Certain defense-associated enzymes including peroxidases, polyphenol oxidases, chitinases, and β-glucosidases were found to be more active in the phloem tissues of the ancestral host of emerald ash borer, Manchurian ash, as compared to the far more susceptible North American black ash, indicating that some level of resistance may be conferred through these enzymatic pathways (Rigsby et al. 2016). Considering the close relationship between the genera Fraxinus and Chionanthus (Wallander and Albert 2000,
Jensen et al. 2002), it is logical that similar defense mechanisms may be present in these genera. In this study, one of my main objectives was to quantify differences in activities of a suite of defense-associated enzymes and other defense related traits in the phloem of white fringetree and black ash in order to determine if they contribute to the observed differences in host suitability between trees.
Surprisingly, I found that activities of POX, PPO, βG, and CHI were all significantly higher in protein extracts from black ash than white fringetree. In addition,
32 treatment of the trees in our study with MeJA induced a defense response that significantly increased the activities of POX (using guaiacol as a substrate), PPO (using caffeic acid as a substrate), βG, and CHI in both species. Prior to this study, I hypothesized that the functional roles of these enzymes would likely confer some resistance to EAB in the form of a defense response in the white fringetree, comparable to responses observed in Manchurian ash. These results do not support this hypothesis and indicate that these enzymes alone are not responsible for the observed variation in host suitability observed between white fringetree and native North American ash species as they are more active in the phloem of black ash, which is much more susceptible to larval EAB. However, despite having lower activities of the measured enzymes, I found total protein content to be higher in white fringetree extracts than in black ash, and lower on average in MeJA treated trees than control trees. Increased levels of soluble protein may be a result of higher concentrations and activities of vigorous wound-healing
(Cipollini 2015) and other defense associated enzymes in the tissues of white fringetree that were not measured in the present study. Wound healing ability of white fringetree should be measured and compared to highly susceptible North American ash species, as this indicates white fringetree has a higher tolerance to EAB infestation in addition to the possibility of being better defended. Further, isoforms of the enzyme groups measured may be specific to substrates naturally prevalent in phloem tissue white fringetree that different from those used here in activity assays. Lastly, and most importantly, enzyme activity that is dependent on more toxic and abundant phenolic profiles (substrate profiles) in white fringetree may compensate for lower observed activities of enzymes like POX, PPO, and βG, and may still be highly active in this species’ defense response.
33 However, because CHI acts on substrates not produced by the plant itself (chitin), our study indicates that this functional enzyme is not important in white fringetree resistance to EAB.
Lignin has been indicated as an important defense against herbivory by physically toughening plant tissues and making them harder to consume (War et al. 2012). However, the role of this ubiquitous polymer as an inducible defense against EAB is inconclusive
(Cipollini et al. 2011, Whitehill et al. 2012) although lignin may play a role in lower nutritional qualities of resistant Manchurian ash (Whitehill et al. 2014). Here, I found the concentration of acetyl bromide soluble lignin to be higher in black ash than in white fringetree and I observed no difference between trees treated with MeJA compared to untreated trees. These interspecific differences in lignin concentration in the present study indicate that increased lignin concentrations may not be important in determining the suitability of white fringetree as a host, which does not support my original hypothesis.
Despite this, total phenolic concentrations were significantly higher in white fringetrees than black ash suggesting that secondary metabolites play an important role in defense against wood boring insects. In the past, the role of specific classes of phenolic compounds including hydroxycoumarins, lignans, and phenylethanoids in defense against
EAB has been both supported (Eyles et al. 2007) and unsupported (Whitehill et al. 2012) by data. However, these studies both indicated that Manchurian ash, the highly resistant evolutionary host for EAB, contains higher levels of pinoresinol dihexoside than susceptible green and white ash. In addition, phenolics such as oleuropein, verbascoside, and pinoresinol were found in high concentrations of white fringetree phloem tissue (D.
Cipollini, unpublished data). While verbascoside was tested for biological relevance to
34 emerald ash borer larvae in feeding assays and was shown to decrease EAB survival
(Whitehill et al. 2014), other implicated metabolites have not undergone the same investigations. The secoiridoid glycoside oleuropein is characteristic of species in the
Oleaceae family (Jensen et al. 2002) and specifically found in higher concentrations in
Manchurian ash than North American species (Cipollini et al. 2011, Whitehill et al.
2012). Oleuropein has been shown to be a strong protein crosslinker after it is hydrolyzed into its respective aglycone by βG isozymes (Konno et al. 1999). In the present study although I found βG activities to be higher in black ash, I found the concentration of oleuropein to be significantly higher in control white fringetrees than fringetrees treated with MeJA and black ash in either treatment. Even with lower enzyme activities, an increased availability of substrate material is likely sufficient for this defense mechanism to be effective against herbivores such as EAB. Further, along with being hydrolyzed by
βG into its toxic aglycone, oleuropein, a polyphenolic metabolite, can also be readily oxidized by POX and PPO into orthoquinones making its importance in defense apparent.
The role these enzyme groups and oleuropein is consistent with what I observed in this study. I found that MeJA application increased enzyme activity, and after MeJA treatment I observed a significantly lower amount of oleuropein in white fringetree. The decrease of this metabolite is likely due to metabolism by these enzymes.
In addition to protein concentrations, enzymatic activity, and oleuropein content, non-structural carbohydrate concentrations were higher in white fringetree than black ash. While the role of non-structural carbohydrates in defense is not entirely clear and has not been the focus of any studies utilizing white fringetree, they may play an important role as building blocks for secondary metabolites that are biological relevant in antibiosis
35 while also being an important marker for examining the balance of defense mechanisms and growth. The multi-stemmed growth habit of white fringetree, and thus its increased amount of photosynthetic tissue (i.e. leaves) compared to black ash, may allow for amplified carbon acquisition and translocation to non-photosynthetic tissue stores throughout the growing season in the form of non-structural carbohydrates. Populus tremuloides with higher NSC/N ratios have shown to have greater levels of foliar phenolic glycosides that are biologically relevant the following year (Najar et al. 2014). I did not measure N content in the phloem of our study species, although exploring this relationship would likely help explain the influence of resource limiting stress on the defense ability of novel hosts.
Another main objective of this study was to test whether light limitation can impact constitutive defenses or constrain the trees ability to induce defenses necessary to respond to EAB infestation. This hypothesis was based on anecdotal evidence that host trees grown in areas of full sun seem to suffer less die-back due to EAB and potentially are less suitable hosts for the emerald ash borer than conspecifics growing in shaded conditions where light is a limited resource (D. Cipollini, personal communication). In the present study, the only defense traits that were impacted by light availability were the activity of trypsin inhibitors and total phenolic content. However, post-hoc pairwise comparisons revealed no statistically significant differences when grouped by species and light treatment. The activity of trypsin inhibitors was significantly lower in trees grown in full sun than in shade and was not affected by MeJA treatment or species. However, the conclusions that light availability had little to no impact on defense traits is also
36 consistent with results from our EAB larval assay, where light availability also had no observable impact on larval performance in either species of tree.
Although our light treatment did not have any strong effects on the activity of potential defense-related enzymes or traits, it did have a noticeable effect on photosynthetic efficiency of either tree species. Photosynthetic efficiency as measured by chlorophyll fluorescence indicates the amount of light energy that is released from the leaf as fluorescence under a dark-adapted state where no photosynthesis is occurring and under a high light state where the maximum amount of reaction centers at photosystem II are open and able to function in the passing of electrons to quinone carriers. This measurement of photosynthetic efficiency is often used to measure the degree of photoinhibition of a plant, which can lead to oxidative stress caused by an excess of reactive oxygen species thought to damage proteins such as the D1 protein that functions in PSII repair (Nishiyama et al. 2006). While I originally hypothesized that our low light treatment would have a negative effect on photosynthetic efficiency, I found that these plants had fluorescence measurements closer to what is considered normal, near 0.83 –
0.85 (Favaretto et al. 2011). Conversely, our plants growing in ambient light conditions, had significantly lower levels of photosynthetic efficiencies, indicating a higher level of energy being emitted as fluorescence rather than being captured and used in photosynthesis. It is possible that trees grown in higher light levels suffered from photo- oxidative stress caused by excess light, however, antioxidant enzymes known to be directly involved in the management of reactive oxygen species including superoxide dismutase, catalase, and ascorbate peroxidase were not specifically measured in this study. Alternatively, trees grown in the sun likely have enough light available, and
37 therefore do not need to maintain as high an efficiency of PSII as trees grown in the shaded conditions, where their use of light needs to maintain maximum efficiency.
Despite having an influence on the photosynthetic efficiency of the trees in our study, light treatment did not significantly impact either measurement of terminal bud growth throughout the growing season or in the first 20 days when most of the growth occurred. This lack of effect, along with the decreased fluorescence of trees grown in full sun, may be an indication that these species rely more on stored carbon assimilated the previous year than newly acquired carbon for meristematic growth. If the trees in our study relied on newly assimilated carbon for new growth, I would assume those grown in full sun suffering from oxidative stress would not be able to assimilate as much carbon as their shade grown neighbors, and thus have a decreased amount of new growth. Our measurements of bud growth also follow the measured pattern of non-structural carbohydrate concentration, both of which were higher in samples from white fringetree than black ash. This pattern may indicate a different strategy of carbon use and allocation between species that may have an impact on defense as well, however this needs to be explored more explicitly as this was not an objective of our study. Additionally, we found the results of this study do not agree with the assumptions of the carbon-nutrient balance hypothesis often used to explain the effect of carbon limitation on plant defenses.
The lack of impacts from light limitation in this study may be in part due to the fact that this experiment occurred through one growing season only. Chronic light limitation over multiple growing seasons may have a much stronger effect on both growth and defense traits. A multiyear study would likely provide a better representation of wild trees growing in shaded conditions where levels of light are relatively static year
38 to year. Decreased total phenolic concentrations coupled with an increase in herbivory was observed in light limited Salix dasyclados Wimm. when compared to high light control plants (Larsson et al. 1986), and a recent study found foliar total phenolic content of Piper betle L. to be lowest under a 50% shade treatment compared to control plants
(Muttaleb et al. 2018). Both of these studies show a consistent effect of light limitation on secondary metabolism in foliar tissues, where an effect is likely larger and more apparent than in a single layer of newly developed phloem surrounded by tissues from previous years, as in the present study.
Emerald ash borer egg hatch rate was affected by the interaction of light and
MeJA treatment. High light treatment seemed to increase hatch rate which could be due to physical differences such as increased temperature. Interestingly, this pattern was also more dramatic in trees treated with MeJA. Establishment of neonate larvae was affected by light treatment, where on trees grown in high light there were more successfully established larvae than in shaded trees. This may indicate that in our study high light acted as an environmental stress to the tree, subtly decreasing defense abilities against larvae as they enter the vascular tissue. However, the potential stress from high light in our experiment did not significantly affect larval weight (tested with larvae from black ash only) or larval gallery width. MeJA also had no effect on either of these two metrics of insect success, although gallery width was significantly higher on black ash than white fringetree which is consistent with other studies that indicate the latter to be a poorer host.
One possible source of error that may have led to a lack of significant differences in larval weight and gallery width between MeJA treatment may have been the small size of the trees used in our experiment. Young trees less with a diameter at breast height of less
39 than 3 cm have been shown to be seldomly infested in EAB’s native range (Wang et al.
2010). Because the trees used in this study were smaller than trees the emerald ash borer infests naturally, an upper limit of growth for the larvae may have led to non-detectable differences in development between groups.
Overall, although I did not detect any impacts of the light treatment investigated in this study on defense induction in black ash or white fringetree, I did find quantifiable differences in defense-associated traits observed in extracts made from the phloem tissues of each species. I found white fringetree to have lower activities of defense related enzymes, but higher levels of total phenolics, potential substrates to the measured enzymes, and nonstructural carbohydrates in compared to black ash. These observed variables, coupled with an EAB larval performance assay where significantly decreased performance was recorded on white fringetree, indicate that despite lower activities these enzymes are likely conferring resistance by acting on phenolic substrates such as oleuropein and directly increasing defensive function. In addition, this species’ strategy for carbon allocation may potentially be important in EAB resistance and tolerance.
Future, more targeted studies, of the white fringetree secondary metabolome in comparison to other trees susceptible to EAB infestation coupled with larval and adult feeding trials would be beneficial in determining what is specifically responsible for the observed interspecific variations in host suitability. Additionally, as emerald ash borer continues to spread further in the southeastern region of the United States, studies investigating geographical variation in susceptibility and resistance of white fringetree might inform strategies for conservation if they continue to be targeted by this invasive beetle. Lastly, long term experiments exposing these trees to different types of chronic
40 stress via resource limitation may better reveal how stress mediates susceptibility to herbivory and infestation. Continued research on alternate hosts of the emerald ash borer is vital as the beetle continues to spread and pose a threat to novel tree species in North
America and worldwide. An increased number of introductions of non-native and potentially invasive wood-boring insects to novel hosts is a side effect of an increasingly connected world, which threatens a huge number of ecologically, economically, and societally important plant species. Thus, any information that can help us understand the roles of plant defense against arthropod herbivores is important
41
Protein POX POX PPO PPO ßG CHI TI Factor Caffeic Chitin Guaiacol Oleuropein Catechol pNBG Trypsin Acid Azure Df 1, 50 1, 49 1, 44 1, 45 1, 45 1, 45 1, 49 1, 48
F = 15.95 F = 5.28 F = 1.83 F = 9.63 F = 13.95 F = 15.70 F = 22.84 F = 0.67 Species P = < 0.001 P = 0.03 P = 0.18 P = 0.003 P = < 0.001 P = < 0.001 P = < 0.001 P = 0.42
F = 4.18 F = 6.82 F = 1.51 F = 6.25 F = 2.74 F = 4.08 F = 9.87 F = 3.54 MeJA P = 0.05 P = 0.01 P = 0.23 P = 0.02 P = 0.11 P = 0.05 P = 0.003 P = 0.07
F = 0.40 F = 0.17 F = 0.09 F = 1.70 F = 0.78 F = 0.08 F = 0.92 F = 4.50 Light P = 0.53 P = 0.68 P = 0.77 P = 0.20 P = 0.38 P = 0.78 P = 0.34 P = 0.04
F = 0.15 F = 0.18 F = 0.93 F = 4.65 F = 2.74 F = 0.74 F = 1.55 F = 0.74 Species x MeJA P = 0.70 P = 0.67 P = 0.34 P = 0.04 P = 0.11 P = 0.40 P = 0.22 P = 0.39
F = 0.43 F = 0.03 F = 0.91 F = 2.00 F = 0.80 F = 2.35 F = 0.12 F = 1.23 Species x Light P = 0.52 P = 0.87 P = 0.35 P = 0.16 P = 0.38 P = 0.13 P = 0.73 P = 0.27 F = 0.56 F = 0.99 F = 1.03 F = 0.21 F = 0.004 F = 0.16 F = 0.01 F = 0.37 MeJA x Light P = 0.46 P = 0.32 P = 0.32 P = 0.65 P = 0.95 P = 0.69 P = 0.92 P = 0.54
Species x MeJA F = 0.13 F = 0.42 F = 0.26 F = 0.39 F = 0.01 F = 0.68 F = 0.35 F = 1.80 x Light P = 0.71 P = 0.52 P = 0.61 P = 0.54 P = 0.94 P = 0.41 P = 0.56 P = 0.19
Table 1. Results from three-factor analysis of variance testing the single factor and interactive effects of species, methyl jasmonate application, and light treatment on total protein concentrations and activities of POX, PPO, ßG, CHI, and TI extracted from the phloem tissues of white fringetree and black ash.
42
Figure 1. Total average terminal bud growth over time separated by species and light treatments of each tree from the start of the experiment until harvest at the end of the
EAB larval assay, error bars indicate standard deviation from the mean.
43
Figure 2. Total growth rate (mm) for white fringetree and black ash grown in sun and shade conditions. Different letters indicate significant differences between groups.
44
Figure 3. Average initial terminal bud growth rate through the first 17 days of the experiment for white fringetree and black ash. Different letters indicate significant differences between groups. No significant differences were found between light treatments.
45 ● 0.8 )
m ● ● F v F (
y 0.7 c n
e Light i c i f
f ● Shade E
c ● i Sun t e 0.6 ● h t n
y ● s o t o h P 0.5 ●
2019−05−26 2019−06−25 2019−07−30 Date
Figure 4. Average photosynthetic efficiency (chlorophyll fluorescence; Fvm) of trees exposed to full sun and shade (low light) treatments. Fvm was measured at three points throughout the course of the experiment. No differences were found between white fringetree and black ash, and MeJA treatment had no influence on the third measurement of Fvm.
46
Figure 5. Average protein concentrations of phloem extracts shown for (A) white fringetree and black ash, and (B) methyl jasmonate and control treatments pooled across the two species. Different letters indicate significant differences between groups. No significant differences were found between light treatments.
47
Figure 6. Figures A, C, and E show average enzyme activities for black ash and white fringetree. Figures B, D, and F show average enzyme activities pooled between species and separated by control and MeJA treatments. (A) Activities of peroxidase with guaiacol as a substrate by species and (B) between control and MeJA treated trees. (C) Activities of β-glucosidase using pNPG as a substrate by species and (D) between control and
48 MeJA treated trees. (E) Activities of chitinase using chitin azure as a substrate by species and (F) and between control and MeJA treated trees. Values for POX and CHI activities were log10 transformed and βG activities were square root transformed. Different letters indicate significant differences between groups.
49 1
- C. virginicus F. nigra g a m
1 - 150 − A n i m
U
y t i b v 100 − i t c ● a
) d i c
a 50 −
c i
e b b ● f f a c ( 0 − O
P Control MeJA Control MeJA P Treatment a 1
- ● g B ● m
●
1 40 − - n i ● m
U 30 − y t i v i t c a 20 − ) l o h c e t 10 − a b c (
O ●
P 0 − P C. virginicus F. nigra Species
1 ● - g C m a
t 20 − n e l a v i
u 15 − q e b I T S
10 − g u
) n i
s 5 − p y r t (
I
T 0 − Shade Sun Light
Figure 7. (A) The interactive effect of species (white fringetree and black ash) and MeJA treatment on PPO activities measured using caffeic acid as a substrate. (B) Activities of
PPO using catechol as a substrate separated by species. (C) Activities of trypsin inhibitors shown separated by light treatment. Values for PPO activity using caffeic acid as a substrate and trypsin inhibitors were square root transformed, values for PPO using catechol as a substrate were log10 transformed. Different letters indicate significant differences between groups.
50
Figure 8. (A) Acetyl-bromide soluble (ABS) lignin concentrations and (C) and nonstructural carbohydrate concentration as they differ between white fringetree and black ash. Average total phenolic concentrations are displayed for both white fringetree and black ash in sun and shade treatments because the principal ANOVA indicated the
51 interaction effect between these factors to be significant. However, our post hoc analysis showed the main differences to be driven primarily by species. Different letters indicate significant differences between groups. For all three of these traits there was no significant difference between MeJA and control or light treatments.
52
Figure 9. Average concentrations of oleuropein in control and MeJA treated white fringetree and black ash. Different letters indicate significant differences between groups.
No significant difference was found between light treatments.
53 8
4
carbohydrates ● ● lignin ti ● bg ● oleouropein● ● pox_gua ppo_cat Groups ● phenolics ppo_caf ● ●● ● 0 protein ● ● Control chi ● pox_ole ● MeJA ●
Dim2 (12.8%) ● ● ●
−4
−8 −8 −4 0 4 8 Dim1 (45.8%)
Figure 10. Principal component analysis displaying differences between control and
MeJA treated black ash (R = 0.081; P = 0.043). Arrows indicate variable loadings.
Ellipses indicate 95% confidence intervals.
54 8
4
ppo_caf
● pox_ole ● ● Groups ● ● ● protein 0 bg ● ● ●● Control phenolics● pox_gua ● MeJA oleouropein●
Dim2 (13.1%) chi lignin ● ti
ppo_cat ● carbohydrates●
−4
−8 −8 −4 0 4 8 Dim1 (37.7%)
Figure 11. Principal component analysis displaying differences between control and
MeJA treated white fringetree (R = 0.368; P = 0.001). Arrows indicate variable loadings.
Ellipses indicate 95% confidence intervals.
55 5
lignin protein● Groups ● ● ● ppo_caf 0 ● ●● C. virginicus ● ● ppo_cat chi F. nigra ●● phenolics ● pox_gua bg Dim2 (18.1%) ● ● pox_ole oleouropein● ti ● carbohydrates ●
−5
−5 0 5 Dim1 (46.7%)
Figure 12. Principal component analysis displaying differences between black ash and white fringetree in the control (no MeJA treatment) groups (R = 0.462; P = 0.001).
Arrows indicate variable loadings. Ellipses indicate 95% confidence intervals.
56 5
lignin
protein Groups ● ppo_cat ●● C. virginicus 0 ● ● ●● bg chi ● ●● carbohydrates●● pox_ole ppo_caf F. nigra ● Dim2 (12.6%) phenolics● ti pox_gua oleouropein
●
−5
−5 0 5 Dim1 (45.3%)
Figure 13. Principal component analysis displaying differences between methyl jasmonate treated black ash and white fringetree (R = 0.347; P = 0.001). Arrows indicate variable loadings. Ellipses indicate 95% confidence intervals.
57
Figure 14. Average emerald ash borer larval gallery width shown between white fringetree and black ash. Different letters indicate significant differences between groups.
No significant difference was found between MeJA and control or light treatments.
58 LITERATURE CITED
Agrell, J., E. P. McDonald, and R. L. Lindroth. 2000. Effects of CO2 and light on tree
phytochemistry and insect performance. Oikos. 88: 259–272.
Bartolini, S., A. Leccese, and L. Andreini. 2014. Influence of canopy fruit location on
morphological, histochemical and biochemical changes in two oil olive cultivars.
Plant Biosyst. - Int. J. Deal. Asp. Plant Biol. 148: 1221–1230.
Bonello, P., and R. B. Pearce. 1993. Biochemical defence responses in primary roots of
Scots pine challenged in vitro with Cylindrocarpon destructans. Plant Pathol. 42:
203–211.
Bowles, D. J. 1990. Defense-Related Proteins in Higher Plants. Annu. Rev. Biochem. 59:
873–907.
Bradford, M. M. 1976. A Rapid and Sensitive Method for the Quantitation of Microgram
Quantities of Protein Utilizing the Principle of Protein-Dye Binding. Anal.
Biochem. 72: 248–254.
Broadway, R. M., and D. L. Missurelli. 1990. Regulatory mechanisms of tryptic
inhibitory activity in cabbage plants. Phytochemistry. 29: 3721–3725.
Bryant, J. P., F. S. Chapin, and D. R. Klein. 1983. Carbon/Nutrient Balance of Boreal
Plants in Relation to Vertebrate Herbivory. Oikos. 40: 357–368. van Buuren, S., and K. Groothuis-Oudshoorn. 2011. mice: Multivariate Imputation by
Chained Equations in R. J. Stat. Softw. 45: 1–67.
59 Calder, W. J., K. J. Horn, and S. B. St. Clair. 2011. Conifer expansion reduces the
competitive ability and herbivore defense of aspen by modifying light
environment and soil chemistry. Tree Physiol. 31: 582–591.
Cappaert, D., D. G. McCullough, T. M. Poland, and N. W. Siegert. 2005. Emerald ash
borer in North America: a research and regulatory challenge. Am. Entomol. 513
152-165. 51.
Chakraborty, S., J. G. A. Whitehill, A. L. Hill, S. O. Opiyo, D. Cipollini, D. A. Herms,
and P. Bonello. 2014. Effects of water availability on emerald ash borer larval
performance and phloem phenolics of Manchurian and black ash. Plant Cell
Environ. 37: 1009–1021.
Chen, M.-S. 2008. Inducible direct plant defense against insect herbivores: A review.
Insect Sci. 15: 101–114.
Chen, Y., and T. M. Poland. 2009. Interactive influence of leaf age, light intensity, and
girdling on green ash foliar chemistry and emerald ash borer development. J.
Chem. Ecol. 35: 806–815.
Chutichudet, B., and P. Chutichudet. 2011. Shading Application on Controlling the
Activity of Polyphenol Oxidase and Leaf Browning of “Grand Rapids” Lettuce.
Int. J. Agric. Res. 6: 400–409.
Cipollini, D. 2005. Interactive Effects of Lateral Shading and Jasmonic Acid on
Morphology, Phenology, Seed Production, and Defense Traits in Arabidopsis
thaliana. Int. J. Plant Sci. 166: 955–959.
Cipollini, D. 2015. White Fringetree as a Novel Larval Host for Emerald Ash Borer. J.
Econ. Entomol. 108: 370–375.
59 Cipollini, D., S. Enright, M. B. Traw, and J. Bergelson. 2004. Salicylic acid inhibits
jasmonic acid-induced resistance of Arabidopsis thaliana to Spodoptera exigua.
Mol. Ecol. 13: 1643–1653.
Cipollini, D. F., and J. Bergelson. 2000. Environmental and Developmental Regulation of
Trypsin Inhibitor Activity in Brassica napus. J. Chem. Ecol. 26: 1411–1422.
Cipollini, D., and D. L. Peterson. 2018. The potential for host switching via ecological
fitting in the emerald ash borer-host plant system. Oecologia. 187: 507–519.
Cipollini, D., and C. M. Rigsby. 2015. Incidence of Infestation and Larval Success of
Emerald Ash Borer ( Agrilus planipennis ) on White Fringetree ( Chionanthus
virginicus ), Chinese Fringetree ( Chionanthus retusus ), and Devilwood (
Osmanthus americanus ). Environ. Entomol. 44: 1375–1383.
Cipollini, D., Q. Wang, J. G. A. Whitehill, J. R. Powell, P. Bonello, and D. A. Herms.
2011. Distinguishing defensive characteristics in the phloem of ash species
resistant and susceptible to emerald ash borer. J. Chem. Ecol. 37: 450–459.
Constabel, C. P., and R. Barbehenn. 2008. Chapter 12 Defensive Roles of Polyphenol
Oxidase in Plants.
Crook, D. J., S. Prabhakar, and B. Oppert. 2009. Protein digestion in larvae of the red oak
borer Enaphalodes rufulus. Physiol. Entomol. 34: 152–157.
Duffey, S. S., and M. J. Stout. 1996. Antinutritive and toxic components of plant defense
against insects. Arch. Insect Biochem. Physiol. 32: 3–37.
Dunn, J. P., T. W. Kimmerer, and D. A. Potter. 1987. Winter starch reserves of white oak
as a predictor of attack by the twolined chestnut borer, Agrilus bilineatus (Weber)
(Coleoptera: Buprestidae). Oecologia. 74: 352–355.
60 Dunn, J. P., D. A. Potter, and T. W. Kimmerer. 1990. Carbohydrate reserves, radial
growth, and mechanisms of resistance of oak trees to phloem-boring insects.
Oecologia. 83: 458–468.
Erbilgin, N., P. Krokene, E. Christiansen, G. Zeneli, and J. Gershenzon. 2006. Exogenous
application of methyl jasmonate elicits defenses in Norway spruce (Picea abies)
and reduces host colonization by the bark beetle Ips typographus. Oecologia. 148:
426–436.
Eyles, A., W. Jones, K. Riedl, D. Cipollini, S. Schwartz, K. Chan, D. A. Herms, and P.
Bonello. 2007. Comparative Phloem Chemistry of Manchurian (Fraxinus
mandshurica) and Two North American Ash Species (Fraxinus americana and
Fraxinus pennsylvanica). J. Chem. Ecol. 33: 1430–1448.
Favaretto, V. F., C. A. Martinez, H. H. Soriani, and R. P. M. Furriel. 2011. Differential
responses of antioxidant enzymes in pioneer and late-successional tropical tree
species grown under sun and shade conditions. Environ. Exp. Bot. 70: 20–28.
Felton, G. W., C. B. Summers, and A. J. Mueller. 1994. Oxidative responses in soybean
foliage to herbivory by bean leaf beetle and three-cornered alfalfa hopper. J.
Chem. Ecol. 20: 639–650.
Folgarait, P. J., and D. W. Davidson. 1994. Antiherbivore Defenses of Myrmecophytic
Cecropia under Different Light Regimes. Oikos. 71: 305–320.
Fürstenberg-Hägg, J., M. Zagrobelny, and S. Bak. 2013. Plant Defense against Insect
Herbivores. Int. J. Mol. Sci. 14: 10242–10297.
Gatehouse, A. M. R., G. M. Davison, C. A. Newell, A. Merryweather, W. D. O.
Hamilton, E. P. J. Burgess, R. J. C. Gilbert, and J. A. Gatehouse. 1997.
61 Transgenic potato plants with enhanced resistance to the tomato moth, Lacanobia
oleracea: growth room trials. Mol. Breed. 3: 49–63.
Gatehouse, A. M. R., R. E. Down, K. S. Powell, N. Sauvion, Y. Rahbé, C. A. Newell, A.
Merryweather, W. D. O. Hamilton, and J. A. Gatehouse. 1996. Transgenic potato
plants with enhanced resistance to the peach-potato aphid Myzus persicae.
Entomol. Exp. Appl. 79: 295–307.
Gülçin, İ., R. Elias, A. Gepdiremen, K. Taoubi, and E. Köksal. 2008. Antioxidant
secoiridoids from fringe tree (Chionanthus virginicus L.). Wood Sci. Technol. 43:
195.
Haack, R. A., D. M. Benjamin, and K. D. Haack. 1983. Buprestidae, Cerambycidae, and
Scolytidae Associated with Successive Stages of Agrilus Bilineatus (Coleoptera:
Buprestidae) Infestation of Oaks in Wisconsin. 16: 10.
Haavik, L. J., T. W. Coleman, M. L. Flint, R. C. Venette, and S. J. Seybold. 2013.
Agrilus auroguttatus (Coleoptera: Buprestidae) seasonal development within
Quercus agrifolia (Fagales: Fagaceae) in southern California. Ann. Entomol. Soc.
Am. 1062 189-197. 106: 189–197.
Hamilton, J. G., A. R. Zangerl, E. H. DeLucia, and M. R. Berenbaum. 2001. The carbon–
nutrient balance hypothesis: its rise and fall. Ecol. Lett. 4: 86–95.
Haruta, M., I. T. Major, M. E. Christopher, J. J. Patton, and C. P. Constabel. 2001. A
Kunitz trypsin inhibitor gene family from trembling aspen (Populus tremuloides
Michx.): cloning, functional expression, and induction by wounding and
herbivory. Plant Mol. Biol. 46: 347–359.
62 Hemming, J. D. C., and R. L. Lindroth. 1999. Effects of Light and Nutrient Availability
on Aspen: Growth, Phytochemistry, and Insect Performance. J. Chem. Ecol. 25:
1687–1714.
Herms, D., A. 2015. Host range and host resistance. In: Van Driesche RG, Reardon R,
eds. Biol. Control Emerald Ash Borer - Tech. Bull. FHTET 2014-09 USDA For.
Serv. 65–73.
Herms, D., A., and D. McCullough G. 2014. Emerald Ash Borer Invasion of North
America: History, Biology, Ecology, Impacts, and Management. Annu. Rev.
Entomol. 59: 13–30.
Hoban, J., N., J. Duan J., and P. Shrewsbury A. 2018. Host utilization and fitness of the
larval parasitoid Tetrastichus planipennisi are influenced by emerald ash borer’s
food plants: Implications for biological control. Biol. Control. 127: 85–93.
Jensen, S. R., H. Franzyk, and E. Wallander. 2002. Chemotaxonomy of the Oleaceae:
iridoids as taxonomic markers. Phytochemistry. 60: 213–231.
Jongsma, M. A., P. L. Bakker, and W. J. Stiekema. 1993. Quantitative Determination of
Serine Proteinase Inhibitor Activity Using a Radial Diffusion Assay. Anal.
Biochem. 212: 79–84.
Kehr, J. 2006. Phloem sap proteins: their identities and potential roles in the interaction
between plants and phloem-feeding insects. J. Exp. Bot. 57: 767–774.
Klooster, W. S., K. J. K. Gandhi, L. C. Long, K. I. Perry, K. B. Rice, and D. A. Herms.
2018. Ecological Impacts of Emerald Ash Borer in Forests at the Epicenter of the
Invasion in North America. Forests. 9: 250.
63 Koiwa, H., R. A. Bressan, and P. M. Hasegawa. 1997. Regulation of protease inhibitors
and plant defense. Trends Plant Sci. 2: 379–384.
Komsta, L. 2011. outliers: Tests for outliers.
Konno, K., C. Hirayama, H. Yasui, and M. Nakamura. 1999. Enzymatic activation of
oleuropein: A protein crosslinker used as a chemical defense in the privet tree.
Proc. Natl. Acad. Sci. 96: 9159–9164.
Larsson, S., A. Wirén, L. Lundgren, and T. Ericsson. 1986. Effects of Light and Nutrient
Stress on Leaf Phenolic Chemistry in Salix dasyclados and Susceptibility to
Galerucella lineola (Coleoptera). Oikos. 47: 205–210.
Lawrence, S. D., and N. G. Novak. 2006. Expression of Poplar Chitinase in Tomato
Leads to Inhibitionof Development in Colorado Potato Beetle. Biotechnol. Lett.
28: 593–599.
Lenth, R. 2019. emmeans: Estimated Marginal Means, aka Least-Squared Means.
Mooney, E. H., E. J. Tiedeken, N. Z. Muth, and R. A. Niesenbaum. 2009. Differential
induced response to generalist and specialist herbivores by Lindera benzoin
(Lauraceae) in sun and shade. Oikos. 118: 1181–1189.
Moreira-Vilar, F. C., R. de C. Siqueira-Soares, A. Finger-Teixeira, D. M. de Oliveira, A.
P. Ferro, G. J. da Rocha, M. de L. L. Ferrarese, W. D. dos Santos, and O.
Ferrarese-Filho. 2014. The Acetyl Bromide Method Is Faster, Simpler and
Presents Best Recovery of Lignin in Different Herbaceous Tissues than Klason
and Thioglycolic Acid Methods. PLOS ONE. 9: e110000.
64 Muilenburg, V. L., and D. A. Herms. 2012. A Review of Bronze Birch Borer
(Coleoptera: Buprestidae) Life History, Ecology, and Management. Environ.
Entomol. 41: 1372–1385.
Muilenburg, V. L., P. L. Phelan, P. Bonello, and D. A. Herms. 2011. Inter- and Intra-
Specific Variation in Stem Phloem Phenolics of Paper Birch (Betula papyrifera)
and European White Birch (Betula pendula). J. Chem. Ecol. 37: 1193–1202.
Muttaleb, Q., A. Tl, H. Sa, R. Aa, T. S, A. Oa, and A. Da. 2018. The Role of Shade and
Nitrogen on Physiological Traits and Secondary Metabolites of Piper betle L. J.
Hortic. 05.
Nabeshima, E., M. Murakami, and T. Hiura. 2001. Effects of Herbivory and Light
Conditions on Induced Defense in
crispula. J. Plant Res. 114: 403–409.
Nagamatsu López, Y., A. Blanco Labra, J. Délano Frier, and E. Pimienta Barrios. 2004.
Light intensity and activity of trypsin inhibitors in amaranth leaves and seeds.
Rev. Fitotec. Mex. 27.
Najar, A., S. M. Landhäusser, J. G. A. Whitehill, P. Bonello, and N. Erbilgin. 2014.
Reserves Accumulated in Non-Photosynthetic Organs during the Previous
Growing Season Drive Plant Defenses and Growth in Aspen in the Subsequent
Growing Season. J. Chem. Ecol. 40: 21–30.
Nishiyama, Y., S. I. Allakhverdiev, and N. Murata. 2006. A new paradigm for the action
of reactive oxygen species in the photoinhibition of photosystem II. Biochim.
Biophys. Acta BBA - Bioenerg. 1757: 742–749.
65 Oksanen, J., F. G. Blanchet, M. Friendly, R. Kindt, P. Legendre, D. McGlinn, P. R.
Minchin, R. B. O’Hara, G. L. Simpson, P. Solymos, M. H. H. Stevens, E. Szoecs,
and H. Wagner. 2019. vegan: Community Ecology Package.
Olson, D. G., and L. K. Rieske. 2019. Host range expansion may provide enemy free
space for the highly invasive emerald ash borer. Biol. Invasions. 21: 625–635.
Pedraza-Reyes, M., and E. Lopez-Romero. 1991. Chitinase activity in germinating cells
ofMucor rouxii. Antonie Van Leeuwenhoek. 59: 183–189.
Peterson, D. L., and D. Cipollini. 2017. Distribution, Predictors, and Impacts of Emerald
Ash Borer (Agrilus planipennis) (Coleoptera: Buprestidae) Infestation of White
Fringetree (Chionanthus virginicus). Environ. Entomol. 46: 50–57.
R Core Team. 2020. R: A language and environment for statistical computing.
Raffa, K. F., S. C. Krause, and P. B. Reich. 1998. Long-Term Effects of Defoliation on
Red Pine Suitability to Insects Feeding on Diverse Plant Tissues. Ecology. 79:
2352–2364.
Rigsby, C. M., D. A. Herms, P. Bonello, and D. Cipollini. 2016. Higher Activities of
Defense-Associated Enzymes may Contribute to Greater Resistance of
Manchurian Ash to Emerald Ash Borer Than A closely Related and Susceptible
Congener. J. Chem. Ecol. 42: 782–792.
Rigsby, C. M., C. Villari, D. L. Peterson, D. A. Herms, P. Bonello, and D. Cipollini.
2018. Girdling increases survival and growth of emerald ash borer larvae on
Manchurian ash. Agric. For. Entomol. 1–6.
Roitto, M., A. Markkola, R. Julkunen-Tiitto, T. Sarjala, P. Rautio, K. Kuikka, and J.
Tuomi. 2003. Defoliation-Induced Responses in Peroxidases, Phenolics, and
66 Polyamines in Scots Pine (Pinus sylvestris L.) Needles. J. Chem. Ecol. 29: 1905–
1918.
Romero-Segura, C., C. Sanz, and A. G. Perez. 2009. Purification and Characterization of
an Olive Fruit β-Glucosidase Involved in the Biosynthesis of Virgin Olive Oil
Phenolics. J. Agric. Food Chem. 57: 7983–7988.
Rutledge, C. E., and A. Arango-Velez. 2017. Larval Survival and Growth of Emerald
Ash Borer (Coleoptera: Buprestidae) on White Ash and White Fringetree Saplings
Under Well-Watered and Water-Deficit Conditions. Environ. Entomol. 46: 243–
250.
Schaffer, W. M., and T. V. Bronnikova. 2012. Peroxidase-ROS interactions. Nonlinear
Dyn. 68: 413–430.
Shao, Q., H. Wang, H. Guo, A. Zhou, Y. Huang, Y. Sun, and M. Li. 2014. Effects of
Shade Treatments on Photosynthetic Characteristics, Chloroplast Ultrastructure,
and Physiology of Anoectochilus roxburghii. PLOS ONE. 9: e85996.
Showalter, D. N., C. Villari, D. A. Herms, and P. Bonello. 2018. Drought stress increased
survival and development of emerald ash borer larvae on coevolved Manchurian
ash and implicates phloem-based traits in resistance. Agric. For. Entomol. 20:
170–179.
Spadafora, A., S. Mazzuca, F. F. Chiappetta, A. Parise, E. Perri, and A. M. Innocenti.
2008. Oleuropein-Specific-β-Glucosidase Activity Marks the Early Response of
Olive Fruits (Olea europaea) to Mimed Insect Attack. Agric. Sci. China. 7: 703–
712.
67 Stamp, N. 2003. Out of the Quagmire of Plant Defense Hypotheses. Q. Rev. Biol. 78: 23–
55.
Suárez-Vidal, E., X. López-Goldar, L. Sampedro, and R. Zas. 2017. Effect of Light
Availability on the Interaction between Maritime Pine and the Pine Weevil: Light
Drives Insect Feeding Behavior But Also the Defensive Capabilities of the Host.
Front. Plant Sci. 8.
Thiemann, D., V. Lopez, A. M. Ray, and D. Cipollini. 2016. The History of Attack and
Success of Emerald Ash Borer (Coleoptera: Buprestidae) on White Fringetree in
Southwestern Ohio. Environ. Entomol. 45: 961–966.
Usha Rani, P., and S. Pratyusha. 2013. Defensive role of Gossypium hirsutum L. anti-
oxidative enzymes and phenolic acids in response to Spodoptera litura F. feeding.
J. Asia-Pac. Entomol. 16: 131–136.
Velázquez-Palmero, D., C. Romero-Segura, R. García-Rodríguez, M. L. Hernández, F. E.
Vaistij, I. A. Graham, A. G. Pérez, and J. M. Martínez-Rivas. 2017. An
Oleuropein β-Glucosidase from Olive Fruit Is Involved in Determining the
Phenolic Composition of Virgin Olive Oil. Front. Plant Sci. 8.
Villari, C., D. A. Herms, J. G. A. Whitehill, D. Cipollini, and P. Bonello. 2016. Progress
and gaps in understanding mechanisms of ash tree resistance to emerald ash borer,
a model for wood-boring insects that kill angiosperms. New Phytol. 209: 63–79.
Wallander, E., and V. A. Albert. 2000. Phylogeny and classification of Oleaceae based on
rps16 and trnL-F sequence data. Am. J. Bot. 87: 1827–1841.
68 Wang, X.-Y., Z.-Q. Yang, J. R. Gould, Y.-N. Zhang, G.-J. Liu, and E.-S. Liu. 2010. The
biology and ecology of the emerald ash borer, Agrilus planipennis, in China. J.
Insect Sci. 10.
War, A. R., M. G. Paulraj, T. Ahmad, A. A. Buhroo, B. Hussain, S. Ignacimuthu, and H.
C. Sharma. 2012. Mechanisms of plant defense against insect herbivores. Plant
Signal. Behav. 7: 1306–1320. van der Westhuizen, A. J., X.-M. Qian, and A.-M. Botha. 1998. Differential induction of
apoplastic peroxidase and chitinase activities in susceptible and resistant wheat
cultivars by Russian wheat aphid infestation. Plant Cell Rep. 18: 132–137.
Whitehill, J. G. A., S. O. Opiyo, J. L. Koch, D. A. Herms, D. F. Cipollini, and P. Bonello.
2012. Interspecific Comparison of Constitutive Ash Phloem Phenolic Chemistry
Reveals Compounds Unique to Manchurian Ash, a Species Resistant to Emerald
Ash Borer. J. Chem. Ecol. 38: 499–511.
Whitehill, J. G. A., C. Rigsby, D. Cipollini, D. A. Herms, and P. Bonello. 2014.
Decreased emergence of emerald ash borer from ash treated with methyl
jasmonate is associated with induction of general defense traits and the toxic
phenolic compound verbascoside. Oecologia. 176: 1047–1059.
Yang, Y., C. Han, Q. Liu, B. Lin, and J. Wang. 2008. Effect of drought and low light on
growth and enzymatic antioxidant system of Picea asperata seedlings. Acta
Physiol. Plant. 30: 433–440.
Zhang, M., T. Cao, L. Ni, P. Xie, and Z. Li. 2010. Carbon, nitrogen and antioxidant
enzyme responses of Potamogeton crispus to both low light and high nutrient
stresses. Environ. Exp. Bot. 68: 44–50.
69