Abstract
Street- and park-planted ash (Fraxinus spp.) trees infested with emerald ash borer (Agrilus planipennis Fairmaire) ranging in size from 30 to 55 cm (11.8 to 21.7 in) dbh and 10 to 20 m (32.8 to 65.6 feet) in height were measured over two sites. The first group was treated with an emamectin benzoate stem injection at 10 ml/2.54 cm dbh (0.4 g ai) in June 2014, and the second group was left as an untreated control. Chlorophyll concentration and fluorescence was measured to assess plant fitness and vitality over three summers. Trees treated with emamectin benzoate showed improvements in chlorophyll concentration and plant fitness and vitality over the course of the study with peak improvement occurring in the second year. The untreated control trees showed continued signs of decline in each year of the study. This work demonstrates the utility of chlorophyll fluorescence for detecting plant stress related to forest health threats and could potentially inform managers on both short-term and long-term management options.
- Agrilus planipennis
- Ash
- Emerald Ash Borer
- Chemical Control
- Chlorophyll Fluorescence
- Plant Fitness
- Plant Vitality
The emerald ash borer Agrilus planipennis Fairmaire (Coleoptera: Buprestidae) is native to East Asia and Russia (Haack et al. 2002). This insect was first discovered in North America in 2002 when widespread ash (Fraxinus spp.) mortality was reported in southeast Michigan and Windsor, Ontario (Haack et al. 2002). Newly infested ash trees do not typically exhibit any external or visible symptoms (McCullough et al. 2009; Poland and McCullough 2006), making early detection difficult. Epicormic shoots, bark deformities, canopy dieback, yellow foliage, and blonding from woodpecker feeding are all external symptoms of an A. planipennis infestation that do not typically occur until trees are heavily infested. Ash yellows, phytoplasmas, are also present in the northeastern United States and can complicate early detection of A. planipennis. Healthy stands of ash, once infested by A. planipennis, can suffer greater than 99% mortality within six years of initial infestation (Knight et al. 2013), but this presumably follows a longer lag period (Klooster et al. 2014).
Adult A. planipennis emergence begins in the spring through characteristic D-shaped emergence holes starting in mid-May with a peak in June (Cappaert et al. 2005). Newly emerged adults feed on ash foliage for approximately one week before mating, followed by oviposition where females can lay 50 to 90 eggs (Bauer et al. 2004). Larvae undergo four instars and create serpentine galleries while feeding in cambial region on phloem and outer xylem (Cappaert et al. 2005). As larval densities increase, water transport is directly impacted through girdling. Girdled tissue ultimately results in a decrease in the photosynthetic electron transport rate and increases photoprotective energy dissipation (Urban and Alphonsout 2007).
As trees experience increased stress (drought, resource competition, extreme temperatures, nutrient deficits, insect and disease attack), photosynthesis is reduced, and plants shift towards photoprotection mechanisms to safely dissipate excess energy (Peñuelas and Munné-Bosch 2005). Chlorophyll fluorescence is a measure of photosynthetic efficiency, illustrating relationships between both structure and function of Photosystem II (PSII), Reaction Center (RC), and core complexes (Rosenqvist and van Kooten 2003). Chlorophyll fluorescence is a useful tool to measure the physiological status of plant performance (fitness and vitality) under a large range of conditions (Baker and Rosenqvist 2004; Johnstone et al. 2014), including the effects of insect herbivory (Tang et al. 2006; Zangerl et al. 2002). Photochemical change due to stress is measured through a unique group of fluorescence parameters derived from chlorophyll a fluorescence kinetics. The maximum quantum yield of PSII photochemistry (FV/FM), where FV is the maximum capacity for photochemical quenching and FM is the maximum chlorophyll fluorescence, can be used to measure solar energy conversion to fixed carbon and serve as a strong indicator of overall plant fitness (Strasser et al. 1995). The average FV/FM value for healthy plants is > 0.8 (Björkman and Demmig 1987) with decreasing values indicating a reduction in maximum quantum efficiency associated with increased stress (Maxwell and Johnson 2000). Plant vitality is characterized by the Performance Index (PIabs) (Strasser et al. 2000) to best explain the functionality of both photosystems I and II while providing quantitative data on plant performance under stress (Strasser et al. 2004).
Millions of ash trees have been killed in urban, rural, and forested settings in North America since 2002, with management costs potentially reaching $10.7 billion across 25 states in the eastern United States (Kovacs et al. 2010). Management options have been proposed for A. planipennis that range from doing nothing to retaining ash trees through insecticide application (Vannatta et al. 2012). There are currently several insecticide products registered to combat A. planipennis infestations, with emamectin benzoate being the most effective option for slowing A. planipennis population growth (McCullough and Mercader 2012). Annual trunk injections have been demonstrated to be efficacious against A. planipennis (Cappaert et al. 2005; Herms et al. 2009; Smitley et al. 2010b). Single, one-time injections have also shown success against A. planipennis for two to four years post-injection, showing promise for reducing annual treatments and injection rates and protecting more trees (Smitley et al. 2010a; Smitley et al. 2015).
Several factors must be considered during the decision process to treat and retain or remove ash trees in A. planipennis infested areas such as tree size, health, location, and value. Measuring the health and potential recovery of a tree over time can serve as a valuable tool in quantifying treatment efficacy and future management decisions. The goal of this study was to gain a better understanding of the physiological response (decline or recovery) of the infested tree using foliar chlorophyll content and fluoresesnce of A. planipennis infested trees treated with emamectin benzoate or un-treated and measured for three years.
MATERIALS AND METHODS
Two sites in Concord, New Hampshire were selected for this study. Measurements were collected once every July over the course of three summers from 2014 to 2016. Both sites were identified as A. planipennis infested during a 2013 delimitation survey conducted by the New Hampshire Division of Forests and Lands Forest Health Program and all trees selected for the study had D-shaped exit holes characteristic of adult A. planipennis emergence along the upper stem and crown base. The first site was a dog park (DP) with open-grown green ash (Fraxinus pennsylvanica Marshall), all showing early signs of decline due to A. planipennis. The second site was a city street (CS) with smaller diameter white ash (Fraxinus americana L.) in a more advanced state of decline, with characteristic blonding of the bark from woodpeckers and galleries visible in the mid-upper bole. Both sites were selected by the New Hampshire Division of Forests and Lands Forest Health Program as an opportunity to create side-to-side visual comparison of chemically treated and untreated infested trees to increase community awareness.
Ten green ash trees were selected for chemical treatment (Chem) at the DP site and paired with ten un-treated green ash trees (Control). Five white ash trees were selected for treatment at the CS site and paired with five un-treated white ash trees. Diameter at breast height (dbh) (cm) and height (m) was measured for each tree. TREE-äge® (emamectin benzoate, Syngenta Crop Protection, Inc.), a systemic insecticide currently registered in all states where A. planipennis has been detected, was used at both sites. TREE-äge® was applied once at 10 ml/2.54 cm dbh (0.4 g ai) on June 4, 2014 at both sites. The Arborjet Tree IV system was used and TREE-äge® was then diluted 1:1 with water and placed into a pressurized 3.16 kg/cm2 bottle connected to four injection needles. The injection sites were evenly spaced around the base of the trunks at a height of 20 to 40 cm (7.8 to 15.7 in) above the soil surface. Four holes were then drilled into the sapwood at each of the four injection sites and a plastic septum (Arborjet #4 plug) was inserted. The injection needles were then placed in the septa and the injection was completed. The injection process has been found to not compromise structural integrity, lead to infection, or negatively impact tree health (Doccola et al. 2011).
Chlorophyll Content and Fluorescence
Healthy and unhealthy vegetation demonstrate differences in pigment and moisture content. Healthy cells are more capable of quenching and utilizing light energy towards photosynthetic processes than dead or damaged cells. An atLEAF chlorophyll meter (FT Green LLC, Wilmington, Delaware, U.S.A.) was used to measure relative chlorophyll concentration using wavelengths of 660 and 940 nm. Five samples were taken for each individual tree and averaged. Each sample was a leaf collected from the sunlit portion of the canopy. All collection days took place under relatively clear, blue-sky conditions. All atLEAF values were then converted to SPAD units (Zhu et al. 2012) to calculate total chlorophyll concentrations (mg/cm2) (Richardson et al. 2002). A Handy PEA fluorimeter (Hansatech Instruments, Norfolk, UK) using a high light emitting device (LED) light source with rapid measurement capabilities was used to capture the polyphasic rise in chlorophyll fluorescence (OJIP curve) known as the Kautsky effect (Kautsky and Hirsch 1931). Sampling was conducted once per year every July. All foliage was collected from sunlit branches and processed on-site. Five samples were taken per tree and averaged. All samples were immediately placed into a dark adaptation clip for thirty minutes and then subjected to a saturating red actinic light intensity of 1500 μMol m−2 s−1 for one second. The PSII reaction centers of leaves transferred from darkness to light are progressively closed. This creates an immediate increase in yield of chlorophyll fluorescence, primarily in the first second of illumination, before fluorescence levels begin to decrease again. All samples were grouped by treatment (Chem and Control) and measured for vitality (PIabs) and fitness (FV/FM). The PIabs parameter is calculated as follows:
Where F0 is the fluorescence intensity at 50 μs, FM is the maximal fluorescence intensity, VJ is the relative variable fluorescence at 2 ms calculated as VJ – (FJ – F0) / (FM – F0), and M0 represents the initial slope of fluorescence kinetics, which can be derived from the equation: M0 = 4x(F300 μs – F0) / (FM – F0). The maximum quantum efficiency of PS II photochemistry (Fv/Fm) is calculated as follows:
Chlorophyll concentration, plant vitality (PIabs), and plant fitness (Fv/Fm) values were analyzed using a univariate repeated measures Analysis of Variance (ANOVA) using PROC GLM (SAS Institute, 2004). Tukey’s HSD (α = 0.05) was used for pairwise comparisons among fluorescence means within and between treatments. Each location was analyzed separately.
RESULTS
Average dbh for the DP site was 53.9 cm (± 5.1) for chemically treated trees and 48.1 cm (± 5.5) for control trees, respectively. Average heights for the DP site were 15.6 m (± 0.9) for the chemically treated trees and 17.1 m (± 0.8) for the control trees, respectively. Average dbh for the CS site were 34.7 cm (± 5.1) for chemically treated trees and 31.6 cm (± 2.2) for control trees, respectively. Average heights for the CS site were 12.9 m (± 0.8) for the chemically treated trees and 13.2 m (± 0.9) for the control trees, respectively.
Results from both DP and CS locations indicate continued health improvement over time from the emamectin benzoate treated trees in year two and three of this study. Figure 1 shows the chlorophyll a fluorescence OJIP transient curves of chemically treated and control trees at the DP and CS sites during the summer (2014–2016).
Chlorophyll a fluorescence OJIP curves from green ash at the Dog Park site (a-c) and white ash at the City Street site (d-f) in 2014, 2015, and 2016, respectively.
Insecticide-treated trees show improvement in 2015 and continued improvement in 2016. There was a significant interaction between treatment and time for chlorophyll content at the Dog Park (F1, 2 = 46.3, P < 0.001) and City Street (F1, 2 = 15.9, P < 0.001) sites. There was no significant difference between mean chlorophyll concentrations in 2014. However, mean chlorophyll concentration was significantly greater in chemically treated trees at both sites in 2015 and 2016 (Figure 2).
Mean chlorophyll concentration (mg/cm2) over time for green ash at the Dog Park (a) and white ash at the City Street sites (b), respectively. An asterisk indicates significant differences (P < 0.05) between chemically treated trees and untreated control trees.
There was a significant interaction between treatment and time for plant vitality (PIabs) at the Dog Park (F1, 2 = 51.5, P < 0.001) and City Street (F1, 2 = 158.1, P < 0.001) sites. There was no significant difference between mean plant vitality in 2014. However, mean plant vitality was significantly greater in chemically treated trees at both sites in 2015 and 2016 (Figure 3). There was a significant interaction between treatment and time for plant fitness (FV/FM) at the Dog Park (F1, 2 = 16.7, P < 0.001) and City Street (F1, 2 = 9.9, P < 0.001) sites. There was no significant difference between mean plant fitness in 2014. However, mean plant fitness was significantly greater in chemically treated trees at both sites in 2015 and 2016 (Figure 4).
Mean plant vitality measured as Performance Index (PIabs) over time for green ash at the Dog Park (a) and white ash at the City Street sites (b), respectively. An asterisk indicates significant differences (P < 0.05) between chemically treated trees and untreated control trees.
Mean plant fitness measured as maximum quantum yield of PS II photochemistry (FV/FM) over time for green ash at the Dog Park (a) and white ash at the City Street sites (b), respectively. An asterisk indicates significant differences (P < 0.05) between chemically treated trees and untreated control trees.
DISCUSSION
Trees treated with emamectin benzoate, TREE-äge®, showed positive physiological response in the form of increased chlorophyll concentration and improvements in both vitality and fitness at both sites during this study with the peak of the response occurring in the second year, indicating the untreated control trees were physiologically more stressed due to continued larval attack by A. planipennis. Chlorophyll fluorescence continued to decline in the control trees while the emamectin benzoate–treated trees showed greater improvement during year two with values showing signs of decline again in year three. Smitley et al. (2010a) found emamectin benzoate–treated trees were free of larvae even when the trees were surrounded by heavily infested trees. Plant vitality, measured as the Performance Index (PIabs), and fitness, measured as the maximum quantum yield of PSII photochemistry (FV/FM), are useful tools for measuring solar energy conversion to fixed carbon while measuring plant performance under stress (Dodds et al. 2016; Strasser et al. 2000; Strasser et al. 2004).
Leaf photosynthetic capacity and the hydraulic properties of tree stems have been demonstrated to be related (Brodribb and Field 2000; Brodribb et al. 2007) and directly used for pest-related stress detection (Christen et al. 2007; Dodds et al. 2016). Increased larval densities of A. planipennis negatively impact water transport of the infested tree, ultimately reducing the leaf photosynthetic capacity resulting in direct reduction in plant vitality and fitness. Chlorophyll fluorescence is therefore an excellent non-destructive tool to measure the physiological state of photosynthetic tissues (Maxwell and Johnson 2000) and could provide valuable health information in preparation for potential infestations as well as informing long-term management of an ash resource.
Previous work has shown that a single stem injection of emamectin benzoate at a 0.4 g ai/2.54 cm dbh rate reduces A. planipennis larvae up to almost 100% for three years (Smitley et al. 2010a) with biennial treatments being optimal (Flower et al. 2015). Our work shows that trees previously infested with A. planipennis also show physiological signs of improvement, suggesting at least short-term recovery from attack is also possible through continued applications every three years.
ACKNOWLEDGMENTS
We greatly appreciate the assistance of the New Hampshire Division of Forests and Lands Forest Health Group for treating the trees and allowing us to measure them over the course of this study. We are also grateful to Andy Graves (USFS) for providing helpful comments to improve this manuscript.
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