INTEGRATED BIOLOGICAL CONTROL OF WOOLLY APPLE APHID IN WASHINGTON
STATE
By
LESSANDO MOREIRA GONTIJO
A dissertation submitted in partial fulfillment of the requirements for the degree of
DOCTOR OF PHILOSOPHY
WASHINGTON STATE UNIVERSITY Department of Entomology December 2011
To the Faculty of Washington State University: The members of the Committee appointed to examine the dissertation of LESSANDO MOREIRA GONTIJO find it satisfactory and recommend that it be accepted.
______Elizabeth H. Beers, Ph.D., Chair ______William E. Snyder, Ph.D. ______Jeb P. Owen, Ph.D. ______John P. Reganold, Ph.D.
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ACKNOWLEDGEMENTS At first, I would like to thank my major advisor Dr. Elizabeth Beers for the opportunity to work on this interesting project and for all the guidance provided throughout my graduate studies. I also want to thank Dr. William Snyder for being my co-advisor as well as campus advisor while I was in Pullman doing my course work, and for providing me with invaluable suggestions when planning some of my experiments. Likewise, I want to thank all the other committee members Dr. Jeb Owen and Dr. John Reganold, for their always insightful and wise comments on my work. Special thanks to Dr. Vincent Jones who provided me with invaluable suggestions on some of my experimental designs.
I would like to show my appreciation to all our technicians Peter Smytheman, Bruce
Greenfield and Randy Talley who helped me with the setup and data collection of some experiments. Along with that, I want to thank all the summer helpers, especially David
Gutierrez, for the assistance they provided me with.
I want to express a special thanks to Dr. Tom Unruh, who although was not part of my committee, was always willing to dialogue and provide me with information relevant to my project.
I also thank Dr. James Johnson at the Dept. of Plant, Soil and Entomological Sciences –
University of Idaho for identification of coccinellid and lacewing specimens.
I am also in debt with Andrew Kahn, an IPM consultant, who helped me finding orchards with woolly apple aphid parasitoids. Thanks also to Jerry Tangren in Wenatchee for helping me with computer problems and such.
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My sincere thanks to all the colleagues and staff members from the WSU department of
Entomology as well as from the Tree Fruit Research and Extension Center who definitely helped in one way or another to make my Ph.D. journey more enjoyable and a little less troublesome.
At last, I must express my gratitude to my greatest source of support and inspiration which is my family: my father and hero Jésus Moreira (in memoriam), my mother Maria
Moreira, my wife Angela and my brother Cristiano.
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INTEGRATED BIOLOGICAL CONTROL OF WOOLLY APPLE APHID IN WASHINGTON
STATE
by Lessando Moreira Gontijo, Ph.D. Washington State University December 2011
Chair: Elizabeth H. Beers
Abstract
Woolly apple aphid Eriosoma lanigerum Hausmann is a secondary pest of apples whose outbreaks have occurred more often since about 2000. The increase in outbreaks appears to be associated with changes in pesticide programs and disruption of biological control. Because of the banning of azinphos-methyl and restriction posed on other organophosphates, growers are turning more to biological control as an alternative tactic to control woolly apple aphid. This aphid has been documented to have a wide host of natural enemies around the world including syrphids, coccinellids, chrysopids, predatory hemipterans, earwigs and the endoparasitoid
Aphelinus mali (Haudeman). A survey I conducted in 2008 in central Washington confirmed that syrphids, chrysopids, coccinellids and A. mali are the most common natural enemies of woolly apple aphid occurring in Washington. Conservation of woolly apple aphid natural enemies in the orchard was also studied in 2008, 2010 and 2011. Sweet alyssum Lobularia maritima (L.)
Desvaux was the flowering plant that attracted significantly more predatory syrphids into the orchards. A faster response by natural enemies to woolly apple infestation was observed on plots planted with sweet alyssum. In addition, a movement of natural enemies between sweet alyssum and tree canopy was confirmed by an imunomarking technique. Exclusion cage studies
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conducted in 2010 and 2011 showed that predators together with A. mali can efficiently control woolly apple aphid in orchards under a soft pesticide program. In addition, syrphids did not seem to disrupt parasitism of A. mali, but instead showed an additive effect when combined. The lethal and sublethal effect of orchard pesticides on A. mali were also examined in 2009, 2010 and 2011.
Spinetoram, spinosad, carbaryl, organophosphates and neonicotinoids showed a high acute toxicity to A. mali killing more than 90% at full rate. Chlorantraniliprole, lambda-cyhalothrin, novaluron, cyantraniliprole, spirotetramat, sulfur and the mixture of Zinc/Manganese + copper hydroxide killed less than 60% of the parasitoids in the acute bioassays at both rates. Only cyantraniliprole, spinetoram and lambda-cyhalothrin showed significant sublethal effects on A. mali. The sublethal effect of cyantraniliprole was due to induction of low fecundity in A. mali, whereas for spinetoram and lambda-cyhalothrin the effect was due to adult A. mali and host mortality. All the studies suggest that biological control of woolly apple aphid has the potential to work in the field. Nevertheless, conservation measures like augmenting alternative food sources for natural enemies and spraying selective pesticides should be adopted.
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TABLE OF CONTENTES
Page
ACKNOWLEDGEMENTS…………………………………………………………… iii ABSTRACT…………………………………………………………………………… v LIST OF TABLES……………………………………………………………………... ix LIST OF FIGURES……………………………………………………………………. xi CHAPTER ONE – INTRODUCTION AND LITERATURE REVIEW……………… 1 Introduction and literature review……………………………………………………... 1 References……………………………………………………………………………… 7 CHAPTER TWO – SURVEY OF WOOLLY APPLE APHID NATURAL ENEMIES IN WASHINGTON STATE…………………………………………………………... 13 Abstract………………………………………………………………………………… 13 Introduction……………………………………………………………………………. 14 Materials and Methods………………………………………………………………… 15 Results and Discussion………………………………………………………………… 17 References……………………………………………………………………………… 23 CHAPTER THREE – EFFECTS OF FLOWERING PLANTS ON ATTRACTION OF PREDATORY SYRPHIDS AND WOOLLY APPLE APHID SUPPRESSION…. 35 Abstract………………………………………………………………………………… 35 Introduction……………………………………………………………………………. 36 Materials and Methods………………………………………………………………… 38 Results and Discussion………………………………………………………………… 45 References…………………………………………………………………………….. 53 CHAPTER FOUR – EFFECTS OF PREDATORS AND PARASITOID ON THE CONTROL OF WOOLLY APPLE APHID…………………………………………... 72 Abstract……………………………………………………………………………….. 72 Introduction…………………………………………………………………………… 73 Materials and Methods………………………………………………………………… 74 Results and Discussion………………………………………………………………… 78
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References…………………………………………………………………………….. 83 CHAPTER FIVE – IMPACT OF SYRPHIDS ON THE PARASITISM OF WOOLLY APPLE APHID BY APHELINUS MALI………………………………… 102 Abstract………………………………………………………………………………… 102 Introduction……………………………………………………………………………. 103 Materials and Methods………………………………………………………………… 105 Results and Discussion………………………………………………………………… 110 References……………………………………………………………………………… 114 CHAPTER SIX – ACUTE AND CHRONIC EFFECTS OF ORCHARD PESTICIDES ON APHELINUS MALI, A PARASITOID OF WOOLLY APPLE APHID………………………………………………………………………………… 122 Abstract……………………………………………………………………………….. 122 Introduction…………………………………………………………………………… 123 Materials and Methods………………………………………………………………… 124 Results and Discussion………………………………………………………………… 127 References……………………………………………………………………………… 131 APPENDICES……………………………………………………………………...... 141 Appendix 4.1: Does Aphelinus mali go through the organdy mesh used in the exclusion cage experiments?...... 142 Appendix 4.2: Estimation of total tree leaf surface area …………………………….. 144 Appendix 4.3: Estimation of woolly apple aphid numbers based on colony size ……. 145 Appendix 6.1: Toxicity of orchard pesticides to woolly apple aphid…………………. 147
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LIST OF TABLES
CHAPTER TWO Table 1. Surveyed predators identified to species……………………………………… 28 Table 2. Percentage parasitism of woolly apple aphid by Aphelinus mali ± SE………….. 28 Table 3. Predators collected for posterior gut content analyses using DNA technique (2011)…………………………………………………………………………………… 28 CHAPTER THREE Table 1. Mean number of adult syrphids (± SE) attracted by different flowering species…………………………………………………………………………………… 58 Table 2: Plant morphological and phenological characteristics ………………………... 58 Table 3. Syrphids collected during the experiment and identified to species ………….. 59 Table 4. Mean number of woolly apple aphid (± SE) at different dates………………... 59 Table 5. Mean number of woolly apple aphids (± SE) on transect trees………………... 59 Table 6. Numbers of natural enemies associated with woolly apple aphid colonies on potted trees at the time of evaluation……………………………………………………. 60 Table 7. Syrphids collected from sweet alyssum plots with sweep net for gut dissection and species identification………………………………………………………………. 60 Table 8. Total ambient natural enemies sampled via beating tray, sweep net, card board band and 2-minute direct count ………………………………………………… 61 Table 9. Percentage of natural enemies from sweet alyssum, trees and distant traps that tested positive for the protein marker…………………………………………………… 62 CHAPTER FOUR Table 1. Treatment and treatment*time interaction effects on woolly apple aphid densities through time…………………………………………………………… 86 Table 2. Mean number of woolly apple aphids per tree (+ SE) at different dates and cage treatments………………………………………………………………………….. 87 Table 3. Insecticides sprayed on orchards during the exclusion cage experiments……. 88 Table 4. Number of natural enemies observed during the exclusion cage experiments in block 5 (TFRC)…...... 89 Table 5. Number of natural enemies observed during exclusion cage experiments in block 1 (Smith tract)…………………………………………………………….. 90 Table 6. Number of natural enemies observed during the exclusion cage experiments in block 11 (Columbia view)……………………………………………………. 91
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Table 7. Temperature measurements collected inside cages by pendant data loggers during each experiment………………………………………………………………… 92 CHAPTER 6 Table 1. Pesticide rates used in the bioassays (maximum label rate: 1.0x)……………... 136 Table 2: Mortality of Aphelinus mali at 24 and 48 hours after pesticide treatment……. 137 Table 3: Mortality of Aphelinus mali at 24 and 48 hours after pesticide treatment (continuation)……………………………………………………………………………. 138 Table 4. Population growth parameters measured for Aphelinus mali during sublethal bioassays………………………………………………………………………………… 139
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LIST OF FIGURES
CHAPTER TWO
Fig. 1. Orchards visited during the 2008 survey (yellow marker = conventional orchard, green marker = organic orchard)……………………………………………… 30
Fig. 2. Woolly apple aphid predator composition and abundance at different times of the year (2008)…………………………………………………………………………. 31
Fig. 3. Total mean number of predators collected per orchard type during the survey (+ SE) (2008) (Data pooled for predators across time)……………………………………. 32
Fig. 4. Mean number of woolly apple aphid colonies counted in a 5-minute walk per orchard type at different times (+ SE) (2008)……….………………………………….. 33
Fig. 5. Woolly apple aphid aerial colony densities in 20 orchards in central Washington, 2008; (C), conventional orchards; (O), organic orchards………………… 34
CHAPTER THREE
Fig. 1. Flowering plant species constituting the treatments in the syrphid/flower attraction study…………………………………………………………………………. 64
Fig. 2. Experimental plots having either grass or sweet alyssum flowers……………… 65
Fig. 3. Mean number of adult syrphids (+ SE) attracted to different plant species during a 2-minute observation…………………………………………………………………. 66
Fig. 4. Mean number of adult syrphids and honeybees (+ SE) attracted to different plant species (data pooled over all observation dates). Bars with different capital letters (syrphids) or lower case (honeybees) differ statistically at P < 0.05 (PROC GLM)…………………………………………………………………………………….. 67
Fig. 5. Mean number of woolly apple aphids (+ SE)/ tree at different dates. Last evaluation date of experiment 1 (27 Sep) coincides with the deployment of infested trees in experiment 2……………………………………………………………………. 68
Fig. 6. Number of woolly apple aphids on potted trees at 10 and 20 m away from experimental plots (transect study). ……………………………………………………. 69
Fig. 7. Mean total number of natural enemies (+ SE)/ plot at different dates………….. 70
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Fig. 8. Mean number of sweet alyssum flowers (+ SE)/ square foot (left Y axis), and 71 percentage coverage (+ SE) of plots by sweet alyssum plants (right Y axis)……………
CHAPTER FOUR
Fig. 1. Experimental cages: Treatment 1 is intended to exclude everything, treatment 2 is intended to exclude predators but no the parasitoid A. mali, and treatment 3 is intended to allow everything (predators + parasitoid A. mali)………………………… 94
Fig. 2. Organdy meshes used on experimental cages. A: mesh used in control cages (trt 1), B: mesh used in parasitoid cages in 2010 (trt 2), C: mesh used in parasitoid cage in 2011 (trt 2). Parasitoid = 1 mm in length………………………………………………. 94
Fig. 3. Mean number of woolly apple aphid per tree (+ SE) at different dates and cage treatments. Cage treatments: ● = WAA only, ○ = WAA + A. mali, and ▼ = WAA + A. mali + predators………………………………………………………………………… 95
Fig. 4. Mean number of woolly apple aphid colonies (+ SE) observed on orchard trees during a 5-minute walk conducted during each evaluation date. ● = Block 11 (C.V.) 2011, and ○ = Block 1 (S.T.) 2011. There was not woolly apple aphid on the orchard trees of block 5 (T.F.) in 2010…………………………………………………………. 96
Fig. 5. Percentage parasitism of woolly apple aphid by A. mali per tree (+ SE) at different dates. Cage treatments: ■ = WAA only, □ = WAA + A. mali, and ■ = WAA + A. mali + predators…………………………………………………………………… 97
Fig. 6. Percentage parasitism of woolly apple aphid by A. mali (+ SE) observed on orchard trees (10 colonies/ evaluation date). ■ = Block 1 (S.T.), and ■ = Block 11 (C.V.)…………………………………………………………………………………… 98
Fig. 7. Size of woolly apple aphid colonies: Mean number of aphids/ colony (+SE). Cage treatments: ■ = WAA only, □ = WAA + A. mali, and ■ = WAA + A. mali + predators………………………………………………………………………………… 99
Fig. 8. Regression between number of woolly apple aphid/ colony and proportion of mummies (N = 1231 aphid colonies)…………………………………………………… 100
Fig. 9. Total tree leaf surface area (+ SE) (cm2). ■ = first day, and ■ = last day of experiment. Bars followed by the same letters do not differ statistically at p<0.05 (ANOVA - PROC GLM)………………………………………………………………. 101
CHAPTER FIVE
Fig. 1. A: Cage experiment setup in greenhouse, 2009. B: Sleeve cage used in the field experiment in 2011……………………………………………………………………… 119
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Fig. 2. Final number of woolly apple aphid per tree under different treatments. A: parasitoid and syrphid tested separately. B: parasitoid and syrphid tested separately and combined. Bars followed by the same letters do not differ significantly at P <0.05, Anova (PROC GLM)…………………………………………………………………… 119
Fig. 3. A: Mean number of woolly apple aphids per branch (+ SE) at the beginning and at the end of the experiment. B: Mean number of mummies/ branch (+ SE), and mean percentage parasitism of woolly apple aphid by Aphelinus mali (+ SE). ■ = WAA + parasitoid + syrphid, ■ = WAA + parasitoid. Bars followed by the same letters do not differ significantly at P <0.05, T-test (PROC TTEST)…………………...... 120
Fig. 4. A: Mean number of woolly apple aphids and mummies per twig (+ SE) at the beginning and at the end of the experiment. B: Mean number of mummies/Petri dish (+ SE). ■ = initial number of WAA, □ = final number of WAA, ■ = initial number of mummies, ■ = final number of mummies……………………………………………... 120
Fig. 5. A: Number of Aphelinus mali making a choice between plant material with aphids and void choice. B: Number of Aphelinus mali choosing odors sources with and without predator cues. * = significant difference between treatments at P <0.05, two- sided binomial test (PROC FREQ)……………………………………………………… 121
CHAPTER 6
Fig. 1. Example of experiment set up for the sublethal bioassays: Young potted tree with woolly apple aphid colonies at the top exposed to a female A. mali enclosed by a glass cylinder cage………………………………………………………………………………………………. 140 Fig. 2. Estimated population growth of A. mali based on stage-structured matrix model from Poptools. ● = control, ○ = pesticide………………………………………………. 141
APPENDIX 4.1
Fig. 4.1.1. Percentage of female Aphelinus mali that visited the aphid colonies enclosed by organdy sleeves in a 24-hour period………………………………………………… 143 APPENDIX 4.2
Fig. 4.2.1. Regression between apple leaf surface area and leaf mid rib length. 2 Regression equation: Y= 0.48 * X – 0.51 * X + 2.29…………………………………… 144 APPENDIX 4.3
Fig. 4.3.1. Regression between surface area covered by woolly apple aphid colony and aphid numbers. Regression equation: Y = 110.27 * X + 276.11………………………… 146
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APPENDIX 6.1
Fig. 6.1.1. Percentage mortality of woolly apple aphid at 48 hours after pesticide treatment. A: experiment 1 conducted in 2008, B: experiment 2 conducted in 2011……………………………………………………………………………………… 148
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DEDICATION
I dedicate this dissertation to my father Jésus Moreira (in memoriam) and my mother Maria
Moreira, whose unconditional love and support played a major role in this achievement.
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CHAPTER ONE
INTRODUCTION AND LITERATURE REVIEW
Washington State has been leading the vanguard of apple production in the United States since the 1920‟s. Today, approximately 192,000 bearing acres (WSU Extension 2010) of apples are cultivated primarily in the semi-arid, irrigated central part of the state, and the tree fruit industry is the economic foundation of many communities in Washington. Among many of the agricultural problems that pose obstacles to apple production, arthropod pests are one of the most devastating. Apples are attacked by a wide range of both direct and indirect arthropod pests
(Beers et al. 1993). One indirect pest that has received more attention lately is the woolly apple aphid Eriosoma lanigerum Hausmann (Hemiptera: Aphididae) which has been occurring at high levels more frequently since about 2000. The problem appears to be associated with changes in pesticide programs as wells as disruption of biological control.
Woolly apple aphid is a small purplish aphid, with adults measuring approximately 1-2 mm in length. Its life cycle is composed of four nymphal instars and an adult stage. The first instar nymphs, called crawlers, are the most motile forms whereas the remaining instars and the adult stage usually become “sessile” after they start feeding (Asante et al. 1991). This aphid species is native to North America and originally was a holocyclic, heteroecious aphid that used the American elm tree Ulmus americana L. as the winter host and one or more different woody summer hosts (Crataegus or Sorbus). After the introduction of cultivated apples in Eastern North
America by European settlers this aphid started to use apples Malus domestica (Borkh) as its summer host, and in areas where elm trees did not occur the apples became the aphid host throughout the year (Sandanayaka and Bus 2005). In North America woolly apple aphid can reproduce by both sexual (elm) and asexual (apple) means (Patch 1913, Sandanayaka and Bus
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2005). On elm trees, they overwinter in the egg stage, which hatch in the spring and produce fundatrice females, a morph found only on the primary host. The fundatrices will give birth to a few generations from which alate spring migrant forms will eventually appear, and thereafter move to secondary hosts.
Woolly apple aphid feeding causes the formation of hypertrophic galls on the roots as well as on the aerial parts of the apple trees (Brown et al. 1991). The galls can restrict sap flow and frequently rupture plant tissues providing further feeding sites and allowing the invasion of fungal diseases (Weber and Brown 1988). In addition, heavy infestations may also reduce tree vigor, damage buds, reduce productivity, and lower fruit quality by direct feeding in the calyx and fruit core (Essig 1942, Bertus 1986, Brown and Schmitt 1990, Brown et al. 1995). This aphid also produces waxy filament over its body and secretes honeydew, which are both considered a nuisance for apple pickers during the harvest time. In addition, woolly apple aphid may overwinter as adult females on both edaphic and aerial parts of the plant, however; larger populations are found to overwinter on the root level (edaphic region). The populations overwintering underground will keep reproducing at a low rate, whereas the ones overwintering on the aerial parts will be dormant and more susceptible to extreme cold temperatures (Thwaite and Bower 1983). Asante et al. (1993) observed woolly apple aphid to have a highly aggregated spatial distribution within orchards as well as a density dependent type of dispersal. In infested trees, first instar nymphs (crawlers) are found to disperse from parent colonies to form new colonies in other parts of the tree canopy (Asante et al. 1993). In the spring, crawlers produced by overwintering females migrate from the roots to the plant canopy thereby serving as a source of infestation for every new season (Damavandian and Pringle 2007, Beers et al. 2010). Woolly
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apple aphid can have approximately 10-14 generations each season depending on the climatic conditions (Schoene and Underhill 1935).
The management of woolly apple aphid is characterized by the use of resistant rootstock, chemical control, biological control, or by the integration of these three tactics. As the roots are often attacked by woolly apple aphid, and also shelter overwintering populations, it is of paramount importance to develop rootstocks that are resistant to this aphid species. Various resistant rootstocks have been developed in the past from the variety “Northern Spy” at the East
Malling Research Station in the United Kingdom, including the Malling-Merton series that has frequently been used as rootstock for scions of commercial varieties (Walker 1985). However, biotypes of woolly apple aphid feeding on some of these rootstocks in the past have already been reported to occur in some locations like South Africa (Giliomee et al. 1968), South Australia
(Self 1966) and North Carolina – USA (Rock and Zeiger 1974). More recently, the apple rootstock breeding program based in Geneva New York has developed various new resistant genotypes (Johnson et al. 2001). A study by Beers et al. (2007) has shown that the new Geneva rootstocks present a higher degree of resistance to woolly apple aphid in Washington State than the Malling-Merton rootstocks. Although these rootstocks would help mitigate the aphid impact on the root system, the effect of rootstock on the development of aerial colonies is still unknown.
Chemical control of woolly apple aphid in the 1980s was based solely on the use of the organochlorine (endosulfan) and organophosphate (diazinon, dimethoate, methyl parathion and azinphosmethyl) insecticides (Walker 1985). Endosulfan and diazinon are still being used for woolly apple aphid control in Washington State and are highly effective. Other important broad- spectrum organophosphates that have been used to control woolly apple aphid in Chile are chlorpyrifos and methidathion (Gonzales 2006). However, the organophosphate azyphosmethyl
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is slated to be removed from the US market in 2012 based on the Food Quality Protection Act established in 1996 (Jones et al. 2009), and restrictions are being posed on other organophosphates. In addition to organophosphates, the neonicotinoids imidacloprid and thiametoxam have also been used to control woolly apple aphid with good results (Gonzales
2006). Another important form of chemical control is the use of mineral oil, which has detrimental effects on woolly apple aphid (Fernandez et al. 2005). Chemical control is primarily aimed at aerial colonies because of the relative difficulty in controlling subterranean colonies.
For example, Brown et al. (1992) tested a systemic aphicide to control subterranean colonies, but did not obtain satisfactory aphid control. Nonetheless, one of the main problems with pesticide use is the disruption of biological control (Shaw and Walker 1996, Nicholas 2000), which may result in future reliance on this method.
Known predators of woolly apple aphid include syrphids, coccinellids, lacewings, predatory hemipterans and earwigs (Walker 1985, Asante 1997, Mueller et al. 1988, 1998; Short and Bergh 2004). It is also attacked by the specialist endoparasitoid Aphelinus mali Haldeman
(Mueller et al. 1992, Brown and Schmitt 1994, Asante 1997). Less frequently, woolly apple aphid has also been observed to be infected by the fungal pathogen Verticillium lecanii (Zimm)
(Asante 1997) and the nematode Steinernema carpocapsae (Weiser) which attacks edaphic aphid colonies (Brown et al. 1992).
Syrphids are one of the most common predators of woolly apple aphid in the US, where both specialist and generalist species occur in apple orchards (Carroll and Hoyt 1984, Short and
Bergh 2004). Two of the most abundant species observed in eastern Washington are Eupeodes americanus Wiedeman and Eupeodes fumipennis Thomson (unpublished data). The chrysopids are the second most common predators of wooly apple aphid encountered in this region, with
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Chrysopa nigricornis Burm being by far the most common species (James 2003). Coccinellids are also very common predators, and have been documented to significantly reduce woolly apple aphid infestation in the early summer in Europe (Kozár et al. 1979). One of the most common coccinellid species encountered feeding on woolly apple aphid in Washington is Coccinella trasversogutatta Fald (Walker 1985). Other predators observed to occur, although in lower frequency, are mirids Deraeocoris brevis Uhler (Walker 1985) and earwigs Forficula auricularia
L. (Mueller et al. 1988, Nicholas 2000). The latter is also considered a pest of apples, but under correct management it can be an important predator of woolly apple aphid (Nicholas 2000).
Most of the woolly apple aphid natural enemies documented to date are generalists. One of the most acclaimed specialist natural enemies has been the endoparasitoid Aphelinus mali, which was once thought to be the panacea for woolly apple aphid problems. This parasitoid has been credited with the successful control of woolly apple aphid in a number of regions around the world (Howard 1929, McLeod 1954, DeBach 1964, Brown and Schmitt 1994, Shaw and
Walker 1996) whereas in other regions it has not been able to provide enough control to maintain the aphid population at acceptable levels. One reason for this disparity in the success of control may be due to differences in the regional climatic conditions (Mols and Boers 2001).
Specifically, low temperatures in many regions of the world where woolly apple aphid occurs have been observed to have a detrimental effect on the development of A. mali (Walker 1985,
Asante and Danthanarayana 1993, Nicholas 2000).
Aphelinus mali is native to North America and has been introduced in many regions of the world in attempt to control woolly apple aphid (Lundie 1924, Howard 1929). Aphelinus mali is a solitary, specialized arrhenotokous endoparasitoid (Lundie 1924) that parasitizes mainly aerial wooly apple aphid colonies (Lundie 1924). This parasitoid appears to prefer third instar
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woolly apple aphid; aphids parasitized at an earlier stage tend to produce male biased adults, whereas the opposite is true for later instars (Mueller 1992). The adult females may live up to 47 days whereas the larval + pupal stage lasts up to 12 days, depending on temperature (Lundie
1924, Bonnemaison 1965). The female parasitoid lays as many as 140 eggs during its life span, and there could be up to 15 generations a year depending on the region (Lundie 1924,
Bonnemaison 1965).
Another important specialist natural enemy is the predatory syrphid Heringia calcarata
Loew. This species has been observed attacking woolly apple aphid in the eastern US (Short and
Bergh 2004, Bergh and Short 2008). An additional attribute of H. calcarata is its ability to attack superficial woolly apple aphid root colonies, which generalist species cannot (Short and Bergh
2004).
The existence of such a diverse group of natural enemies, including both generalists and specialists, increases the opportunities for implementing a stable biological control program for woolly apple aphid. The biological control approach is a preferable alternative at the current time because of the loss and restriction on organophosphates, coupled with increasing public demand for more sustainable pest control management strategies. Thus, conducting studies to determine which natural enemies have the greatest impact and to inform conservation measures towards them is essential.
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88: 127–133.
8
Carroll, D. P., and S. C. Hoyt. 1984. Natural enemies and their effects on apple aphid, Aphis
pomi DeGeer (Homoptera: Aphididae), colonies on young apple trees in central
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Damavandian, M. R., and K. L. Pringle. 2007. The field biology of subterranean populations
of the woolly apple aphid, Eriosoma lanigerum (Hausmann) (Hemiptera: Aphididae), in
South African apple orchards. African Entomol. 15: 287-294.
DeBach, P. 1964. Success, trends and future possibilities, pp. 673-713. In: Biological Control of
insect pests and weeds. P. DeBach (Eds), Chapman and Hall, London.
Essig, E. O. 1942. Woolly apple aphid infesting cores. J. Econ. Entomol. 35: 281.
Fernandez, D. E., E. H. Beers, J. F. Brunner, M. D. Doerr, and, J. E. Dunley. 2005. Effects
of seasonal mineral oil application on the pest and natural enemy complexes of apple. J.
Econ. Entomol. 98 (5): 1630-1640.
Gonzales, R. H. 2006. Alternative proposals of chemical and hormonal control of the codling
moth. Revista Fruticola (April Edition).
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Merton rootstocks susceptible to woolly apple aphid, Eriosoma lanigerum in the Western
Cape. S. Afr. J. Agric. Sci. 11: 183-186.
Howard, L. O. 1929. Aphelinus mali and its travels. Ann. Entomol. Soc. Am. 22: 311-368.
James, D. G. 2003. Field evaluation of herbivore-induced plant volatiles as attractants for
beneficial insects: methyl salicylate and the green lacewing, Chrysopa nigricornis. J.
Chem. Ecol. 29:1601–1609.
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Orchard trial performance of elite Geneva Series rootstocks. Acta Hort. 557: 63-67.
9
Jones, V. P ., T. R. Unruh, D. R. Horton, N. J. Mills, J.F. Brunner, E. H. Beers, and P. W.
Shearer. 2009. Tree fruit IPM programs in the western United States: the challenge of
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Kozár, F., L. Szalay-Marzo, A. Meszleny, G. Lovei, and S. Szabo. 1979. Data to the
population and host susceptibility of apple woolly aphid, Eriosoma lanigerum Hausm.
(Hom. Aphidoidea). Novonyvedelem 15: 542-550.
Lundie, A. E. 1924. A biological study of Aphelinus mali Hald., a parasite of the woolly apple
aphid, Eriosoma lanigerum Hausm. NY Agric. Exp. Stn. Ithaca Mem. 79:1-27.
McLeod, J. H. 1954. Statuses of some introduced parasites and their hosts in British Columbia.
Proceedings of the Entomological Society of British Columbia 50: 19-27.
Mols, P. J. M., and J. M. Boers. 2001. Comparison of a Canadian and a Dutch strain of the
parasitoid Aphelinus mali (Hald) (Hym., Aphelinidae) for control of woolly apple aphid
Eriosoma lanigerum (Haussmann) (Hom., Aphididae) in the Netherlands : a simulation
approach. J. Appl. Entomol. 125: 255-262.
Mueller, T. F., L.H.M. Blommers, and P. J. M. Mols. 1988. Earwig (Forficula auricularia)
predation on wooly apple aphid, Eriosoma lanigerum. Entomol. Exp. Appl. 47. 145-152.
Mueller, T. F., L.H.M. Blommers, and P. J. M. Mols. 1992. Woolly apple aphid (Eriosoma
lanigerum Hausm.,Hom., Aphidae) parasitism by Aphelinus mali Hal.(Hym.,
Aphelinidae) in relation to host stage and host colony size, shape and location. J. Appl.
Entomol. 114,143-154.
10
Mueller, T. F., L.H.M. Blommers, and P. J. M. Mols. 1998. Earwig (Forficula auricularia)
predation on the woolly apple aphid, Eriosoma lanigerum. Entomol. Exp. Appl. 47:145-
152.
Nicholas, A. H. 2000. The pest status and management of woolly aphid in an Australian apple
orchard IPM program. Ph.D. dissertation, University of Western Sydney Hawkesbury,
Sydney, Australia.
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Patch, E. M. 1916. Elm leaf rosette and woolly aphid of the apple. Bull. 256, Maine Agricultural
Experiment Station.
Rock, G. C., and D. C. Zeiger. 1974. Woolly apple aphid infests Malling and Malling-Merton
rootstocks in propagation beds in North Carolina. J. Econ. Entomol. 67: 137-138.
Sandanayaka, W.R.M., and V.G.M. Bus. 2005. Evidence of sexual reproduction of woolly
apple aphid, Eriosoma lanigerum, in New Zealand. J. Insect Sci. 5:27.
Schoene, W. J., and G. W. Underhill. 1935. Life history and migration of the woolly apple
aphids. Virginia Agric. Exp. Tech. Bull. 57:1-31.
Self, B. G. 1966. Progress in research. Rep. E. Malling Res. Sta. for 1965: 1201-1206.
Shaw, P. W., and J.T.S. Walker. 1996. Biological control of woolly apple aphid by Aphelinus
mali in an integrated fruit production programme in Nelson. Proc. 49th N.Z. Plant
Protection Conf. 59-63.
11
Short, B. D., and J. C. Bergh. 2004. Feeding and egg distribution studies of Heringia calcarata
(Diptera:Syrphidae), a specialized predator of woolly apple aphid (Homoptera :
Eriosomatidae) in Virginia apple orchards. J. Econ. Entomol. 97:813-819.
Thwaite, W. G., and C. C. Bower. 1983. Woolly Aphid. Agfact H4. AE.3. NSW Agriculture,
Orange, Australia.
Walker, J.T.S. 1985. The influence of temperature and natural enemies on population
development of woolly apple aphid, Eriosoma lanigerum (Hausmann). Ph.D. Thesis,
Washington State University, Pullman.
Weber, D. C., and M. W. Brown. 1988. Impact of woolly aphid (Homoptera: Aphididae) on the
growth of potted apple trees. J. Econ. Entomol. 81: 1170–1177.
WSU Extension. 2010. Apples in Washington. Retrieved on September 8th, 2011 from:
http://county.wsu.edu/chelan-douglas/agriculture/treefruit/Pages/Apples_in_Washington_State
12
CHAPTER TWO
SURVEY OF WOOLLY APPLE APHID NATURAL ENEMIES IN WASHINGTON
STATE
ABSTRACT
Woolly apple aphid, Eriosoma lanigerum (Hausmann), has become a pest of increasing importance in Washington apple orchards for the past few years. The increase in aphid outbreaks appears to be associated with changes in pesticide programs and disruption of biological control. We sampled woolly apple aphid colonies in Washington apple orchards for natural enemies of this pest from April through October 2008. The most common predator species encountered were syrphids (Syrphus opinator Osten Sacken and Eupeodes americanus
Wiedemann), lacewings (Chrysopa nigricornis Burmeister), and coccinellids (Coccinella transversoguttata Brown and Hippodamia convergens Guérin-Méneville). The specialist syrphid
Heringia calcarata Loew was found for the first time occurring in Washington apple orchards.
The only parasitoid found in the aerial colonies was Aphelinus mali Haldeman. Aerial colonies of woolly apple aphid appear to reach the highest peaks around the months of July and August.
Identification of important natural enemies provides a better basis for conservation biological control of this pest.
Key words: Eriosoma lanigerum, natural enemies, biological control, survey, apple.
13
INTRODUCTION
Woolly apple aphid, Eriosoma lanigerum (Hausmann), has been a pest of apple orchards in Washington State for over 100 years, but during the past few years there has been an increase in the incidence and severity of outbreaks (Beers unpublished data). This increase is likely to be associated, to some extent, with changes in pesticide programs and disruption of biological control.
Wooly apple aphid is native to eastern North America and originally was a holocyclic, heteroecious aphid species which used the American elm tree, Ulmus americana L., as the winter host and one of several woody plants (Crataegus or Sorbus) as summer hosts. After the introduction of cultivated apples, Malus domestica (Borkhausen), by European settlers this aphid started to use apple as its summer host. This pest was subsequently spread to apple-growing areas around the world; and in areas where U. americana was absent, it adapted to survive on apple throughout the year. Sexual reproduction occurs on the winter host (Patch 1913), but reproduction on apple is generally regarded to be asexual only (Sandanayaka and Bus 2005).
Woolly apple aphid feeding causes hypertrophic gall formation on the roots and aerial parts of the trees (Brown et al. 1991), which can restrict sap flow and rupture plant tissues, thereby providing further feeding sites and allowing the invasion of fungal diseases (Weber and
Brown 1988). In addition, the wax and honeydew produced by the colonies are a nuisance to pickers at harvest time, and aphids found on packed apples are a quarantine concern in countries like China and Taiwan which are major importers of Washington apples (Warner 2006).
Nevertheless, there is a good potential for biological control of this aphid. Woolly apple aphid is known to be attacked by a large natural enemy complex, including various
14
aphidophagous predators such as ladybird beetles, lacewings, syrphids, earwigs and predatory bugs (Walker 1985, Asante 1997, Mueller et al. 1988, Short and Bergh 2004). In addition, this aphid is also attacked by a specialized hymenopterous endoparasitoid, Aphelinus mali Haldeman
(Mueller et al. 1992, Brown and Schmitt 1994, Asante 1997). Aphelinus mali has been introduced into many regions throughout the word in attempt to control woolly apple aphid and has shown satisfactory results in many of those regions (McLeod 1954, DeBach 1964, Brown and Schmitt 1994).
The objective of this survey was to identify the key natural enemies of woolly apple aphid occurring in central Washington apple orchards, providing a better foundation for implementing an integrated biological control program for this pest.
MATERIALS AND METHODS
The natural enemies of woolly apple aphid were surveyed in Washington State in 2008, from Bridgeport in the north to the Tri-Cities in the south. The orchards sampled had a previous history of woolly apple infestation. Only predators physically associated with the woolly apple aphid colonies were collected during the survey. A total of 20 orchards were sampled including
13 conventional and 7 organic orchards. The orchards were located in the following counties:
Okanogan (3), Douglas (2), Grant (5), Yakima (3) Franklin (5), and Chelan (2) (Fig. 1). Orchards were sampled weekly or biweekly from April through October. The sample unit consisted of a
10-cm section of shoot containing one, or more aphid colonies. Colony size varied from a few to hundreds of aphids. Aphid colonies were first inspected in situ for motile predators, which, if present, were collected for species identification. After visual inspection, each sample unit was removed from the tree and placed in a self-sealing plastic bag. Up to fifty sampling units were collected per visit using this method. The adult predators found were killed and pinned, and the
15
larvae were reared to the adult stage by feeding them woolly apple aphids collected from an untreated apple orchard located in the Tree Fruit Research & Extension Center in Wenatchee,
WA. In addition, the size of the woolly apple aphid population was estimated during each visit by counting the number of aerial colonies observed during a five-minute walk within each orchard.
The percentage parasitism by A. mali was also estimated in a subsample of up to 15 colonies per orchard and visit by counting the number of mummies (intact and exited) and the total number of aphids. The percentage of colonies parasitized (i.e., with one or more mummies) was also calculated. Aphids with no visible external signs of parasitism were not included in these estimates.
An additional 405 predators were collected in 2011 for a posterior gut content analysis using DNA technique (King et al. 2008). All predators were collected from three WSU orchards infested with woolly apple aphid from July through September. Collections were made mainly by picking predators manually and placing them into individual 2 ml plastic vials, and then in an ice chest. In addition, pitfall traps and card board bands were also used to collect terrestrial predators. All specimens collected were brought to the laboratory and preserved at – 80 0C.
16
RESULTS AND DISCUSSION
Predators
Syrphids, chrysopids and coccinellids were the most common predator groups encountered attacking woolly apple aphid, with syrphids being the most abundant (Fig. 2).
Syrphids were more abundant during the month of July coinciding with the mid-season peak in aphid densities (Fig. 2). The syrphid species identified from the samples included Eupeodes americanus Wiedeman and Heringia calcarata Loew (Table 1), with the former being a generalist predator while the latter a specialist. Heringia calcurata has been reported previously to feed on woolly apple aphid in the US (Short and Bergh 2004). However, up to now, H. calcarata had only been found in apple orchards in the eastern US (Short and Bergh 2004, Bergh and Short 2008). To my knowledge, this is the first documented occurrence of H. calcarata in
Washington, or for that matter, the western US. This specialist syrphid is one of the few natural enemies, besides the nematode Steinernema carpocapsae Weiser (Brown et al. 1992), known to attack the root aphid colonies below the soil surface (Short and Bergh 2004), thus making it an important biological control agent for this pest. The fact that both generalist and specialist predatory syrphids occur in the orchards may be advantageous in the control of woolly apple aphid. Predators with different diet breadth occurring at different time periods may provide better and continuous prey control. For example, generalist predators such as E. americanus can provide early season woolly apple aphid control, before the aphid density increases and H. calcarata comes into the system (Bergh and Short 2008).
Chrysopids were the second most abundant predator and occurred slightly later in the season than the syrphids (July-September) (Fig. 2). The most common chrysopid species
17
identified included Chrysopa nigricornis, Hemerobius sp. and Chrysoperla plorabunda Fitch, with the former being by far the most common species in this survey (Table 1). Because of their commercial availability and resistance to some insecticides, green lacewings are among one of the most commonly released predators for augmentative biological control in orchards and other agroecosystems (Aldrich 1999). Chrysopa nigricornis is a large lacewing species that occurs throughout the US, especially in the Pacific Northwest (James 2003a), and is an important generalist predator that feeds on aphids and other soft-bodied arthropods (Tauber et al. 2000).
Many lacewings, C. nigricornis in particular, have been a target of experiments testing their attraction to pheromone and other semiochemical-based attractants. For instance, Zhang et al.
(2006) observed the pheromone iridodial (male-produced pheromone) to be attractive to all lacewing species that attack woolly apple aphid, especially C. nigricornis. Lacewings have also been reported to be attracted to the semiochemical methyl salicylate (James 2003b, 2006). The use of these attractants in apple orchards may help control woolly apple aphids by bringing lacewing species into the orchards.
The coccinellids were the third most common predator and tended to be more numerous in July and August (Fig. 2). The coccinellid species identified included Coccinella transversoguttata, Hippodamia convergens, Coccinella septempunctata (L.), Adalia bipunctata
(L.), Harmonia axyridis Pallas (n= 2) and Hippodamia tredecimpunctata (L.), with the former two being the most abundant species observed (Table 1). All of these species are generalist predators that feed on aphids and other soft-bodied arthropods; however, only C. transversoguttata, H. convergens (Walker 1985) and A. bipunctata (Aslan and Karaca 2005) had been reported previously to feed on woolly apple aphid. Harmonia axyridis and C. septempunctata are exotic species, and are known to have the potential to displace native species
18
(Burgio et al. 2002, Alyokhin and Gary 2004, Evans 2004, Snyder and Evans 2006). Their introduction into the US dates back as early as 1916 for H. axyridis (Koch and Galvan 2008) and early in the 1970s for C. septempunctata (Angalet et al. 1979).
Other predator groups encountered attacking woolly apple aphid, although in lower numbers, were nabis (Hemiptera:Nabidae), Deraeocoris sp. (Hemiptera:Miridae), spiders
(Araneae) and earwigs (Dermaptera).
Predators were more abundant during the months of June, July and August (Fig. 2). This abundance appeared to coincide with the time of highest woolly apple aphid densities in the orchards (Fig. 4 and 5). In addition, predators tended to be more abundant on organic orchards compared to conventional (Fig. 3). Nevertheless, the low number of predators at the end of season coupled with optimal temperatures for aphid population growth (13-25 OC, September and October) may contribute to an increase in aphid population towards the end of autumn
(Beers et al. 2010). This late-season population increase may occur because of a higher aphid reproductive rate (Asante et al. 1991) and/ or because of aphid release from natural enemy pressure (Walker 1985).
The density of woolly apple aphid colonies tended to be higher in conventional orchards
(Fig. 4), which may have been induced by pesticides disrupting biological control. Nonetheless, the aerial aphid colony seasonal patterns fell into three general groups: a late-season peak (3 orchards, Fig. 5A), a mid-season peak (6 orchards, Fig. 5B), and a mid- and late-season peak (11 ochards, Fig. 5C). One orchard in the late season group (a research orchard that had been deliberately perturbed with pesticides) had a very high population from July through October, peaking at about 200 colonies per 5 minute search. This orchard is an example of how a prior
19
history of infestation, together with disruption of biological control can produce a severe outbreak of this pest. Because we did not track pesticide use in the surveyed orchards, it is difficult to determine what effect they might have had on the population timing and severity. In general, growers rarely apply pesticides after late August because of preharvest intervals, or harvest operations that are in progress.
The predator composition encountered during this survey is similar to the predator complex encountered around the world (Asante 1997). However, that literature review did not document chrysopids as predator of woolly apple aphid in the US before that time.
In a previous study conducted in Wenatchee, WA by (Walker 1985), the main predator groups observed attacking woolly apple aphid were coccinellids (C. transversoguttata, H. convergens, Coccinella novemnotata Herbst), chrysopids (C.nigricornis and Chrysopa coloradensis Banks) and predatory bugs (Deraeocoris brevis Uhler). Walker‟s (1985) findings, generally agree with the results of my study, with the exception that only one syrphid species
Eupeodes (Metasyrphus) fumipennis Thompson was observed in his study. In addition, his study also found Deraeocoris sp. to be one of the three most important predators, while it occurred in very low frequencies in our studies. The difference may be ascribed to the limited nature of
Walker‟s observations (single orchard, two years) versus the regional survey we conducted; however, it is also possible that there might have been a genuine shift in the predator composition in response to changing pesticide programs through time. While syrphids are known to be predators of woolly apple aphid (Asante 1997), there is relatively little information on the species present in the western US.
20
Like any sampling method, the one employed in this study had both strengths and weaknesses. Because the predators collected were in the aphid colonies, there is a reasonable degree of confidence that they were preying on woolly apple aphid. However, these samples were taken only during the day, so nocturnal predators such as earwigs and spiders may have been underrepresented. The more mobile predators (e.g., lacewings and coccinellids) may also have been underrepresented in relation to the syrphid larvae, which are relatively sessile once settled in a colony. The predatory bugs are highly mobile in both adult and nymphal stages, and even with the precaution of observing the colony before removing it, they may have escaped detection. In addition, there may be a bias in the species reported if there was differential survival in laboratory rearing of larvae specimens collected during the survey; the overall survival of the various groups was 10% (syrphids), 42% (chrysopids) and 100% (coccinellids).
Nonetheless, the more concentrated collection conducted in 2011 should help to confirm the predator status of the specimens collected in 2008 via gut content analysis. The predators collected in 2011 were spiders (74), coccinellids (134), chrysopids (59), syrphids (48),
Deraeocoris sp (40), nabis (31), earwigs (15), ground beetles (2) and Staphylinidae (2) (Table 3).
During the time of collection, 214 predators were physically associated with the aphid colonies whereas the remaining 191 were not (Table 3). In addition, 313 predators were collected directly by hand, 78 by pitfall traps, and 14 by cardboard bands.
Aphelinus mali.
All the mummies observed were from woolly apple aphid colonies collected on the aerial parts of the trees. To our knowledge, A. mali has never been documented to attack edaphic
21
colonies and data collected in previous years, while not exhaustive, confirm this (Cockfield and
Beers unpublished). Up to now, the only natural enemies known to attack woolly apple aphid in the roots are the syrphid H. calcarata (Short and Bergh 2004) and the nematode S. carpocapsae
Weiser (Brown et al. 1992).
Aphelinus mali was the only parasitoid found parasitizing woolly apple aphid during this survey. I observed an overall parasitism rate by A. mali of approximately 30% in July, August and September, whereas in October this parasitism went down to about 10% (Table 2). However; the percentage parasitism was lower on organic orchards when compared to conventional (Table
2). This may reflect the lower aphid densities observed in the organic orchards (Fig. 4). Although this parasitism rate may not translate into complete suppression of the aphid population, I believe that the combination of parasitism and predation could significantly help to reduce woolly apple aphid in the field.
Studies such as this provide a better and more quantitative basis on which to base future integrated control strategies. Determining which natural enemies are most abundant is a first step; however, their relative impact on prey/host densities needs to be determined. Another factor in integrated control in a highly pesticide-managed system such as tree fruits is determining the nontarget impact of insecticides used in western orchards. Lastly, the nontarget effect must be linked with the phenology of the various groups to minimize any detrimental effects, and enhance opportunities for biological control.
22
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26
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(Diptera: Syrphidae), a specialized predator of woolly apple aphid (Homoptera :
Eriosomatidae) in Virginia apple orchards. J. Econ. Entomol. 97:813-819.
Snyder, W. E., and E. W. Evans. 2006. Ecological effects of invasive arthropod generalist
predators. Annu. Rev. Ecol. Evol. Syst. 37:95–122.
Tauber, M. J., C. A. Tauber, K. M. Daane, and K. S. Hagen. 2000. Commercialization of
predators: Recent lessons from green lacewings (Neuroptera: Chrysopidae: Chrysoperla).
Am. Entomol. 46:26–38.
Zhang, Q. H., R. G. Schneidmiller, D. R. Hoover, K. Young, D. O. Welshons, A.
Margaryan, J. R. Aldrich, and K. R. Chauhan. 2006. Male-produced pheromone of
the green lacewing, Chrysopa nigricornis. J. Chem. Ecol. 32: 2163–2176.
Walker, J. T. S. 1985. The influence of temperature and natural enemies on population
development of woolly apple aphid, Eriosoma lanigerum (Hausmann). Ph.D.
Dissertation, Washington State University, Pullman.
Warner, G. 2006. Woolly apple aphid treated as quarantine pest. Good Fruit Grower 57 (7): 10-
11.
Weber, D. C., and M. W. Brown, 1988. Impact of woolly aphid (Homoptera: Aphididae) on the
growth of potted apple trees. J. Econ. Entomol. 81: 1170–1177.
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Table 1. Surveyed predators identified to species
Predator Species N % of total Predator type Eupeodes americanus Wiedeman 13 92.85% Generalist Syrphids Heringia calcarata Loew 1 7.15% Specialist Chrysopa nigricornis Burmeister 24 88.88% Generalist Chrysopids Chrysoperla plorabunda Fitch 2 7.42% Generalist Hemerobius sp 1 3.70% Generalist Adalia bipunctata (L.) 2 8.70% Generalist Coccinella septempunctata (L.) 2 8.70% Generalist Coccinella transversoguttata Brown 8 34.80% Generalist Coccinellids Harmonia axyridis Pallas 2 8.70% Generalist Hippodamia convergens Guerin-Meneville 8 34.80% Generalist Hippodamia tredecimpunctata (L.) 1 4.30% Generalist
Table 2. Percentage parasitism of woolly apple aphid by Aphelinus mali ± SE
Month Organic Conventional Combined July 12.92 ± 5.46 32.36 ± 4.54 34.15 ± 1.22 August 19.64 ± 6.85 18.70 ± 5.41 32.15 ± 1.85 September 13.00 ± 5.27 19.22 ± 3.96 30.47 ± 1.94 October 2.94 ± 0.82 15.24 ± 3.13 10.83 ± 0.78
Table 3. Predators collected for posterior gut content analyses using DNA technique (2011)
Specimen from waa colonies from elsewhere Total Spider 1 73 74 Coccinellid larvae 67 6 73 Coccinellid adult 28 33 61 Chrysopid larvae 52 2 54 Syrphid larvae 48 0 48* Deraeocoris sp 18 22 40 Nabis 0 31 31 Earwig 0 15 15 Chrysopid adult 0 5 5 Ground beetle 0 2 2 Staphylinidae 0 2 2 Total 214 191 405 *68 more syrphid larvae were collected, but were used for the intraguild predation study.
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Figure Legends
Fig. 1. Orchards visited during the 2008 survey (yellow marker = conventional orchard, green marker = organic orchard).
Fig. 2. Woolly apple aphid predator composition and abundance at different times of the year (2008).
Fig. 3. Total mean number of predators collected per orchard type during the survey (+ SE) (2008) (Data pooled for predators across time).
Fig. 4. Mean number of woolly apple aphid colonies counted in a 5-minute search per orchard type at different times (+ SE) (2008)
Fig. 5. Woolly apple aphid aerial colony densities in 20 orchards in central Washington, 2008; (C), conventional orchards; (O), organic orchards.
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Fig. 1. Orchards sampled during the 2008 survey (yellow marker = conventional orchard, green marker = organic orchard).
30
120
Syrphids Chrysopids 100 Coccinellids Other
80
60
40
Number of predators
20
0 April May June July Aug Sept Oct
Fig. 2. Woolly apple aphid predator composition and abundance at different times of the year (2008).
31
25
20
15
10
5
Total number of predators/ orchard + SE
0 Organic Conventional
Fig. 3. Total mean number of predators collected per orchard type during the survey (+ SE) (2008) (Data pooled for predators across time).
32
40
30 Organic Conventional
20
10
0
Mean number of WAA colonies + SE
April May June July Aug Sept Oct
Fig. 4. Mean number of woolly apple aphid colonies counted in a 5-minute search per orchard type at different times (+ SE) (2008)
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12 250 DNR (C) A PRO (O) 10 ST1 (C) 200
8 150
6
100 4
50 2
WAA colonies/ 5-minute search 0 0
WAA colonies/ 5-minute search (ST1)
80 B ARR (C) MCD (C) S56 (C) 60 S00 (C) SCY (C) SPH (O)
40
20
WAA colonies/ 5-minute search 0
140 BEL (O) C CAC (C) 120 CRA (C) DTZ (O) FER (C) 100 MTV (C) PMA (O) 80 RRY (O) RFL (O) SMT (C) 60 S01 (C)
40
20
WAA colonies/ 5-minute search
0
Apr May Jun Jul Aug Sep Oct Nov
Fig. 5. Woolly apple aphid aerial colony densities in 20 orchards in central Washington, 2008; (C), conventional orchards; (O), organic orchards.
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CHAPTER THEE
EFFECTS OF FLOWERING PLANTS ON ATTRACTION OF PREDATORY
SYRPHIDS AND WOOLLY APPLE APHID SUPPRESSION
ABSTRACT
Woolly apple aphid Eriosoma lanigerum has become a pest of increasing importance in
Washington apple orchards since about 2000. A preliminary survey of natural enemies has indicated that syrphids (Diptera: Syrphidae) are one of the most common predators found in woolly apple aphid colonies in Washington State. One approach to enhance biological control is the conservation of natural enemies. This may be achieved by altering crop systems to provide necessary resources for beneficial insects. Adult female syrphids are known to rely on the ingestion of nectar for energy and pollen for gametogenesis. Thus, engineering the orchard ecosystem to include flowering plants that provide these resources to adult syrphids should enhance biological control. In this 3-year work I investigated the effects of sweet alyssum
Lobularia maritima on the attraction of syrphids, and suppression of woolly apple aphid as well as the natural enemy movement between cover crop and apple trees. Sweet alyssum was highly attractive to syrphids. A faster response to woolly apple aphid infestation was observed on sweet alyssum plots, thus providing further evidence of its potential to indirectly contribute to conservation biological control of woolly apple aphid. In addition, syrphids and other predators were found to move between the flowers and the canopy of the trees. Syrphids were found to fly up to 200 m away from the floral source, thus indicating that this cover crop does not need to be planted in the entire orchard floor.
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Key words: woolly apple aphid, conservation biological control, insectary plants, cover crop, syrphids.
INTRODUCTION
Conservation of natural enemies is an important approach in enhancing biological control. This may be achieved, in part, by altering crop systems to provide necessary resources for beneficial insects. The provision of alternative food resource through cover crops can help to increase the abundance of predators and parasitoids in many agroecosystems (Kruess and
Tscharntke 1994, Landis et al. 2000, Tscharntke et al. 2005, Ambrosino et al. 2006). However, an increase in the abundance of natural enemies may not always translate into improved pest control if those natural enemies do not target the pest (Baggen and Gurr 1998). In addition to providing alternative food (i.e., pollen and nectar), some plants can also provide beneficial insects with overwintering shelter, mating and refuge sites (Tscharntke et al. 2005). Various studies have documented the provision of flowering plants to enhance biological control of pests within particular crop systems (Gurr et al. 2004, Berndt et al. 2006, Ponti et al. 2007). The use of floral resources is of paramount importance for predators such as syrphids, whose adult females rely on their nectar for energy and their pollen for gametogenesis (Haslett 1989). The larval stage of predatory syrphids feeds on many aphid species, including woolly apple aphid Eriosoma lanigerum Hausmann, which is a pest of increasing concern in apple orchards in Washington.
In a recent survey of this pest‟s natural enemies in Washington, syrphids were found to comprise more than half of all predators encountered; therefore, making it a reasonable choice of biological control agent to be preserved in orchards. Conventional orchards in central
Washington rarely have an abundance of flowering plants; row middles are a mix of grasses and broadleaf weeds, while strips beneath the trees are treated with herbicide making flowering
36
plants even more scarce. Organic orchards are more likely to have a cover crop as part of their agroecosystem; however, grass sod and tillage strips are still very common. Thus, an opportunity exists to attract and retain adult syrphids in these orchards by adding a consistent and abundant source of alternative food.
Although the provision of floral resources has shown to be important for the development and establishment of syrphids, there are other factors that should be taken into account when deciding whether or not a candidate plant makes a suitable cover crop. Cover crops must be able to grow and develop under normal orchard management practices. They must not be a resource for pests of the desired crop, or unduly compete with crops for nutrients, light or water (Bone et al. 2009). To reduce management costs, cover crops should preferentially be perennial, or if annual, be able to reseed itself.
A wide variety of flowering plant species used as cover crops are known to attract syrphids, including the annual species sweet alyssum, Lobularia maritima (L.) Desvaux.
(Ambrosino et al. 2006), coriander, Coriandrum sativa L. (Lovei et al. 1993, Ambrosino et al.
2006), buckwheat, Fagopyrum esculentum Moench (Kloen and Altieri 1990, Ambrosino et al.
2006), phacelia, Phacelia tanacetifolia Benth (Kloen and Altieri 1990, Hickman and Wratten,
1996), cosmos, Cosmos sulphureus Cav. (Sadeghi 2008), marigold, Calendula officinalis L.
(Colley and Luna 2000, Sadeghi 2008) mustard, Brassica juncea L. (Kloen and Altieri 1990,
Lovei et al. 1993), and perennial species: yarrow, Achillea millefolium L. (Kloen and Altieri
1990, Lovei et al. 1993), fennel, Foeniculum vulgare Miller (Kloen and Altieri 1990, Lovei et al.
1993), and Korean licorice mint, Agastache rugosa Fischer & Meyer (Colley and Luna, 2000).
I hypothesized that sowing flowering plants between the rows of apple trees could attract predatory syrphids that would feed on woolly apple aphid. Thus, the main objectives of this
37
study were: 1) evaluate plant species for attractiveness to syrphids and agronomic suitability in central Washington; 2) determine the indirect effect of flowering plants on suppression of woolly apple aphid; and 3) evaluate the movement of syrphids and other natural enemies between sweet alyssum and apple trees.
MATERIAL AND METHODS
Screening of flowering plants. This experiment was conducted in September 2008 in an open field that had formerly been planted with apples located on the grounds of the Tree Fruit
Research & Extension Center in Wenatchee, WA. The field was surrounded by apple orchards to the north and west, a cherry orchard to the south, and buildings to the east. A large tract of unmanaged ground with plants native to the shrub-steppe habitat adjoined the apple orchard area on the west.
The treatments consisted of six different flowering plant species: marigold, Calendula officinalis; buckwheat, Fagopyrum esculentum; cosmos, Cosmos sulphureus; mustard, Brassica juncea;, zinnia, Zinnia hybrida; and sweet Alyssum, Lobularia maritima (Figure 1). All plants tested were annuals in the interior fruit-growing districts of central Washington. In addition to their documented attraction to syrphids, the plants were chosen based on their potential for easy management in the orchard floor (compact growth), which requires multiple operations during the growing season (mowing, weeding, irrigation). The plants were grown from seed in the greenhouse in 10-inch pulp pots with Miracle-Gro Promixing soil (Marysville, OH), and then transported to the field 10 days after germination (mid-August). The existing in-ground irrigation system (impact sprinklers on 0.60 m risers) was used to provide moisture. Plants were irrigated twice per week.
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The experiment was a completely randomized design with repeated measures, having six treatments and four replicates. Each replicate consisted of approximately 30 pots placed in a triple row (9-11 pots/row) in a 1.2 m wide rotovated strip in the center of the former drive rows of the former orchard. Plots/replicates measured approximately 1 x 3 m and were separated by empty spaces 10.5 m in the east-west direction, and 15 m in the north-south direction.
Flower attractiveness was measured by recording the feeding-visit frequency of adult syrphids observed in 2-min observation period per replicate plot. After the 2-min observation period, adult syrphids were captured with an aerial net during an additional 1-2 minute period.
These adults were killed and pinned for later species identification. Evaluations were made on 5 dates with a 7-day interval during the entire month of September 2008. All evaluations were made between 10:00 am and 12:00 pm. The temperature for those time slots varied from 23 to
28 C, and the conditions were sunny for the 1st, 3rd, and 4th sample dates, and partly cloudy for the 2nd and 5th sample dates.
Data analyses. The effect of treatments throughout time was assessed by repeated measures analyses of variance (PROC MIXED) (SAS 2008). Covariance structures for the mixed model repeated measures were constructed, and the ANTE(1) covariance structure was determined as the best-fit model for the data using the Baysian Information Criterion (BIC).
Pairwise comparisons using LSMeans (PROC GLM) (SAS 2008) were conducted separately on each date because of treatment by date interaction observed in the previous analysis.
Effect of sweet alyssum on woolly apple aphid suppression. This experiment was conducted in 2010 in a 4-year old unsprayed WSU orchard (mix of the cultivars „Gala‟,
39
„Ambrosia‟, „Golden Delicious‟, and „Jonagold‟). The block was bordered by pears on north, apples on the west, cherries on south and a basalt cliff with a narrow strip of native habitat on the east. The orchard floor was covered mostly by grass between tree rows. There was also an irrigation system of micro sprinklers running along the tree rows (south-north lengthwise). The orchard was watered 2-3 times a week. The experimental plots were about 15 x 3.60 m sections of the orchard floor in the tree-row middles (the 1-m herbicide strip beneath the trees was not used). Plots were spaced 120 m apart (north-south, down rows) and 40 m apart (east-west, across rows).
Experiment 1. There were two treatments for the experiment: 1) grass plot (control) which was mowed regularly (Fig. 2A) sweet alyssum plot (Fig. 2B). There were three replicates for each treatment arranged in a completely randomized design. Each plot had four infested potted apple trees, two in each tree row near the plot‟s north-south center about 5 m apart.
About 4 months prior to the experiment, dormant trees were transplanted into 8-inch pots with the same soil media described above, and allowed to leaf out in the greenhouse. The trees were Auvil Early Fuji (cultivar 216) grafted on rootstock M-9 RN-29 (Van Well nursery inc.
Wenatchee, WA). A month before the beginning of the experiment each tree was infested with a
2-cm apple twig containing about 50-70 woolly apple aphids obtained from a greenhouse colony.
At the time of field deployment, the trees were ca. 1.2 m tall.
The plots randomly assigned to receive the sweet alyssum treatment were tilled with a tractor-drawn rotovator a few weeks before sowing the seeds. To ensure high plant density and high germination rate, the seeds (American Meadows, Williston, VT) were sown by hand (ca.
150 g/plot). After sowing, the top 1-2 cm of soil was carefully turned over using a rake. The
40
germination was lower on one plot with sandy soil, which required two additional seedings.
Sweet alyssum plots were kept free of weeds prior to the beginning of the experiment by weekly manual removal.
Two days before placing the infested trees in the field, the number and size of woolly apple aphid colonies were adjusted (2 colonies of 1 cm2/ tree) with the aid of a brush so that each tree would have approximately the same initial aphid numbers.
Data on woolly apple aphid and its natural enemies were collected weekly for three weeks after the deployment of infested trees. Colony size was used to estimate woolly apple aphid densities (Appendix 4.3), and a non-destructive visual search for predators on the infested potted trees was conducted at the same time. The orchard floor (control and sweet alyssum) was first examined visually for adult syrphids hovering near the grass or flowers, and number of adults recorded during a two-minute period. A small subsample (one or two adult syrphids) was netted for identification and gut content analysis (see below); otherwise, this sample was non- destructive in order to minimize interference with biological control. After that, grass and sweet alyssum plots were sampled with a sweep net (3 sweeps/plot), and the natural enemies collected were recorded and released. In addition, natural enemies were sampled on four of the field- planted apple trees bordering the plots with a beating tray (1 tap per each of the four trees).
Earwigs were sampled on field-planted trees on the plot border using a 10 cm roll of cardboard tied to the tree trunk 10 cm above the soil level. The cardboard traps were collected and replaced on each sample date.
The subsample of adult syrphids and lacewings collected weekly were preserved in 70% alcohol. The specimens were dissected on the same day of collection, their gut contents placed
41
on a glass slide, and stained with safranin (Wratten et al. 1995). The slides were examined under a compound microscope at 4 100 to determine the presence of sweet alyssum pollen.
In addition, a transect study was deployed in the orchard during the same period to investigate a potential gradient in woolly apple aphid predation/parasitism as a function of distance from the sweet alyssum source. The transect was composed of two infested trees spaced out about 10 m apart and placed at the four cardinal directions around each experimental plot.
The transect trees were grown and infested in the same way described above. The trees were adjusted to an approximately equal aphid density immediately prior to deployment, and a single aphid density estimate was performed at the end of the experiment to determine the effect of the treatments.
The number of sweet alyssum flowers per square foot and percentage plot coverage by the ground cover were also estimated weekly. A data logger (Onset Comp., Pocasset, MA) was installed in the middle of the orchard to monitor temperature.
Experiment 2. The experimental treatments and procedures were the same as described above. The experimental plots were also the same. The only difference was the number of subsamples (infested potted trees placed in each replicate). Only two infested trees were used in each replicate, and no transect trees were deployed at this time.
Data Analysis. The effect of treatments on woolly apple aphid densities through time was assessed by repeated measures analyses of variance (PROC MIXED) in SAS v.9.2 (SAS 2008).
Covariance structures for the mixed model repeated measures were constructed, and the CS covariance structure was determined as the best-fit model for the data (analyzed separately or combined) using the Baysian Information Criterion (BIC). First evaluation dates (infested tree
42
deployment date) were dropped from the analyses since they were the same values across treatments and experiments. In addition, pairwise comparisons were conducted using LSMeans
(PROC MIXED) (SAS 2008). The same analysis was conducted to assess the treatment effect on the density of natural enemies through time. A correlation between the natural enemy and woolly apple aphid density within treatments was also carried out (PROC CORR) (SAS 2008).
Movement of natural enemies between sweet alyssum and apple trees. This experiment was conducted from June 27 to August 30, 2011 in the same WSU orchard described above. There was not a natural infestation of woolly apple aphid in the orchard. Sweet alyssum seeds were sown as described above in three plots measuring 15 x 3.6 m each, and spaced 40 m apart (east-west, across tree rows) following the same alignment. Plots were irrigated 1–2 times a week via micro-sprinkler irrigation system (after natural enemy collection). Four potted trees infested with woolly apple aphids (two on each side) were placed on each sweet alyssum plot as in the 2010 experiment (potted trees were heavily infested, at least 6000 aphids each). The potted trees were always replaced when the aphid numbers were very low (below 1000). The potted trees were „Fuji September Wonder‟/ EMLA 26 (C&O Nursery, Wenatchee, WA). The field- planted trees were not infested with woolly apple aphid during the course of the experiment, thus the infested potted trees were the only nearby source of this aphid species.
Egg protein (liquid egg white; All Whites®, Crystal Farms, Minnetonka, MN) was used as a marker to assess the movement of predators and parasitoids between sweet alyssum and apple trees. The sweet alyssum plots were sprayed with a 20% solution of egg whites diluted in water. Water softener (tetrasodium ethylenediaminetetraacetic [EDTA]; The Herbarie at Stone
Hill Farm Inc., Prosperity, SC) at 16 g per 10 liters of solution was also added to reduce water hardness. The egg white plus EDTA solution was applied once a week using a 16-liter backpack
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sprayer (Smith Sprayers®, Utica, NY). Immediately after each spray, white and yellow sticky traps were hung at a height of 1-1.5 m on the canopies of infested potted trees and uninfested field-grown trees. Each side of the sweet alyssum plots had a 2 x 2 permutation of sticky trap and tree type (yellow and white sticky traps on both infested potted trees and unifested field-grown trees). One card board band was also placed at base of an infested potted tree and uninfested field-grown tree (one on each side of the alyssum plot). In addition, two white sticky traps parallel to each other and 10 m apart were hung individually on the canopy of filed-grown trees as described above at different distances away from the experimental area on the four cardinal directions (50 m on east, 100 and 200 m on west, 100 m on south, 100 and 200 m on north). The natural enemy collection focused more on beneficial insects that are known to feed on or parasitize woolly apple aphid. Natural enemies were collected 24 hours after each spray and trap deployment. Natural enemies were collected from the deployed sticky traps, and also directly from tree canopies (2 infested and 2 uninfested trees from each plot) and sweet alyssum flowers by shaking their limbs and flowers respectively, onto a tray covered by waxed paper coated with adhesive (Tanglefoot®; Grand Rapids, MI). All Specimes caught on sticly traps and directly from tree canopy were processed before sweet alyssum samples to reduce the risk of contamination. Samples of sweet alyssum flowers and leaves were also collected at each time to confirm the presence of egg protein on the cover crop. Natural enemies caught on the sticky traps and tray were removed with the aid of toothpicks (one for each specimen to avoid contamination) and individually transferred to 1.5 ml microtubes. The microtubes were quickly placed into an ice chest for transport to the laboratory. Samples were stored at -4 oC until they were tested for the egg protein. At the assay, specimens were removed from storage and washed individually with 100 μl of 1X PBS (buffer) and then vortexed and centrifuged. The buffer wash
44
was thereafter subjected to indirect ELISA (Jones et al. 2006) for identifying the egg protein marker.
RESULTS AND DISCUSSION
Screening of flowering plants. There was a significant effect of flower species on syrphid attraction (F = 54.40; df = 5; P < 0.0001) as well as an interaction between flower species and time (F = 2.62; df = 24; P = 0.023). The number of syrphids observed in the 2-min observation periods was consistently higher in the sweet alyssum plots (Table 1, Figs. 3 and 4).
The attractiveness of sweet alyssum may have been due in part to its early and prolific flowering habit, but it also appeared to be intrinsically attractive. The amount of floral resources (flower density) has been shown to affect the overall abundance of foraging syrphid flies (Sutherland et al. 2001). Mustard and buckwheat were also attractive, but less so than sweet alyssum. The reduction in the attraction of buckwheat from the third evaluation date (Fig. 3) may be due to a decline in its flower production. The results of this study are similar to the findings of Colley and
Luna (2000) and Ambrosino (2006), which indicated that sweet alyssum and buckwheat to be fairly attractive to syrphids. Marigold, zinnia and cosmos started to flower much later than the other flower species (Table 2). Marigold was however intermediate in attractiveness, while zinnia and cosmos attracted relatively few syrphids. Sadeghi (2008) observed cosmos to have a high attraction to syrphids whereas marigold a very low attraction. However, the climatic conditions and syrphid species observed in his study were different from those found in central
Washington. Differences in locations regarding landscape composition and climate can affect the number and species composition of syrphids attracted to the flowering plants (Colley and Luna
2000). In addition, flower color can also influence the choice. For example, Cowgill (1989) observed in his experiment that yellow and white flowers are more attractive than flowers with
45
other colors. Lastly, because feeding preference and species composition may also vary throughout the season, the attractiveness of the tested plants could have been different if evaluated during longer or different periods.
The tallest plants were buckwheat and mustard, whereas sweet alyssum was the shortest
(Table 2). Sweet alyssum, in addition to its ability to attract adult syrphids, also appears to have favorable plant growth habits that facilitate horticultural management in the orchards (i.e., driving between tree rows whe spraying). Sweet alyssum forms a low-growing mat of plants with a high flower density and is able to reseed itself. Although the duration of this test was fairly short, sweet alyssum produced flowers very quickly from seed, with continuous flowering through frost. The early flowering habit of sweet alyssum in this test may be partly responsible for the higher total numbers of syrphids observed. Mustard plants were considerably taller than sweet alyssum, with an upright growth. One interesting characteristic of mustard was its high attractiveness to honeybees (Fig. 4). This attractiveness could have positive or negative effects, depending on the circumstances. If any pesticides toxic to honeybees were applied in the orchard during the bloom period, bee mortality could be substantial. In addition, the honeybees may compete with the syrphids for resources on mustard, or vice versa. During the experimental evaluation adult syrphids were observed to spend great amount of time trying to dislodge and chasing off honeybees from the flowers. Some syrphid species are known to be territorial
(Wellington and Fitzpatrick 1981, Fitzpatrick 1981), which may explain this observation.
Nonetheless, there was no correlation (Pearson, r = 0.14 and P = 0.43) between the number of syrphids and honeybees occurring in the mustard plots. This could indicate that honeybees do not affect syrphid recruitment.
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Adult syrphids collected for identification were captured roughly in proportion to their feeding-visit frequency on the various plants. A total of 53 adults were captured, killed and mounted for identification to species. Most of the species identified were pollinators Eristalis arbustorum L. (Table 3), which were found feeding on all tested plants with the exception of zinnia. Predatory syrphids were only present among the specimens collected from sweet alyssum and mustard (Table 3). The predatory syrphid Eupeodes americanus Wiedemann was observed on sweet alyssum and Scaeva pyrastri L. on mustard, whereas Syrphus opinator Osten-
Sacken was observed on both. Given the short time of this experiment (about one month), it is possible that we may have missed the peaks for the predatory syrphids we collected, or even completely missed different species that may occur earlier in the season. Nevertheless, E. americanus and S. opinator larvae had already been observed feeding on woolly apple aphid in the field (Chapter 2), which indicates the potential for sweet alyssum to attract predatory syrphids into infested orchards, which could result in enhanced biological control.
Effect of sweet alyssum on woolly apple aphid suppression.
Woolly apple aphid densities. There was a significant treatment effect in experiment 1 (F
= 17.01, df = 1, P = 0.05) as well as a significant date effect (F = 40.83, df = 2, P < 0.001); the interaction of the two main effects was not significant (F = 1.21, df = 2, P = 0.35). Likewise, in experiment 2 there was a significant treatment (F = 44.79, df = 1, P = 0.02) and date effect (F =
11.20, df = 3, P < 0.001), but no significant interaction (F = 3.03, df = 3, P < 0.07). These results remained essentially the same when the data from both experiments were combined. The treatment effect was generally due to a better woolly apple aphid control in the sweet alyssum plots.
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Although aphid densities were similar at the end of both experiments, a faster response to aphid infestation and subsequent faster aphid reduction was observed in the first week in the sweet alyssum plots compared to control (Table 4, Fig. 5). In experiment 1, woolly apple aphid numbers were reduced to nearly zero in four weeks on both treatments. In experiment 2, the woolly apple aphids were reduced to about half of the initial numbers in 5 weeks (Table 4, Fig.
5). The slower aphid reduction observed in experiment 2 may be due to the lower temperatures prevailing in October (20/10 C max/min) versus September (25/14 C max/min), which may have slowed predation or parasitism rates. Aphid suppression was mostly due to predation since the parasitism rate by A. mali was negligible. In experiment 1, parasitism was zero on all dates.
In experiment 2, the parasitism was zero on the first two dates and below 1% on the last three dates in both the control and sweet alyssum plots. Low parasitism may be due to absence of a natural aphid infestation of aphids in the orchard prior to the experiment which may lead to low numerical response, or because of intraguild predation which has been observed in many instances for other parasitoids (Wheeler et al. 1968, Colfer and Rosenheim 2001).
Transect Study. The transect study indicates a decline in biological control (increase in woolly apple aphid numbers) with increasing distance from both the control and sweet alyssum plots (Table 5, Fig. 6). However, because we have only two woolly apple aphid counts on the transect trees (at the beginning and at the end of the experiment) it is not possible to determine if the same weekly pattern occurred as in the plot subsample trees. Also, there may have not been enough spatial separation of the plots for relatively mobile predators.
Total natural enemies. More natural enemies were found to occur in plots planted with sweet alyssum compared to controls (Fig. 7). There was a significant treatment effect on the total number of natural enemies occurring in the plots (F = 126.01, df = 1, P = 0.0004), but there was
48
neither a significant date (F = 0.59, df = 3, P < 0.632) nor treatment*date interaction effect (F =
1.23, df = 3, P = 0.34). In addition, there was no significant correlation between the total natural enemy and woolly apple aphid densities in neither the sweet alyssum (Pearson, r = -0.296, P =
0.191) nor the control treatments (Pearson, r = -0.172, P = 0.454) meaning that there may be a particular species responsible for most of the aphid control. However, correlations for separate natural enemy taxon were not done because of the low numbers.
Natural enemies on potted trees. Substantially more natural enemies were found in experiment 1 than in experiment 2 (Table 6), an indication that aphids may be less subject to predation as temperatures and photoperiods decline in the fall. This is a possible explanation for late-season outbreaks of woolly apple aphids, which have a relatively low optimal developmental temperature, and thus can maintain a high rate of reproduction during cool temperature (Asante et al. 1991). The numbers of active stages of natural enemies encountered during sampling were relatively low. The low numbers of syrphid larvae associated with the aphid colonies make it difficult to attribute the aphid control to syrphids alone, even though this control was most likely due to predation rather than parasitism. Lacewing eggs were occasionally encountered during experiment 1, but no larvae were found. Higher numbers of lacewing adults were found in the sweet alyssum plots, but it is difficult to interpret the value of this given that adults of many species are not predaceous. Low numbers found in this direct observation may be due in part to the short time frame in which these are made (a few minutes during the 7-day period), and may not reflect the total effect of predation during that period. This is especially problematic with the more active predators, which either leave/drop off the tree or seek sheltered spots when disturbed.
49
Syrphid species complex and abundance. Seven species of syrphid adults were collected in or near the sweet alyssum plots (Table 7). These species were (from most to least abundant)
Syrphus opinator Osten-Sacken (35), Eupeodes fumipennis Thomson (7), Scaeva pyrastri L. (3),
Eupeodes volucris Osten-Sacken (2), Syrphus ribesii L. (2), E. americanus Wiedeman (1), and unidentified Eristalinae (pollinator) (1). Of the predatory species found, all except S. pyrastri and E. volucris have been observed to feed on woolly apple aphid colonies in previous studies
(Walker 1985; Chapter 2), and the latter two are known predators of green apple aphid Aphis pomi (Carroll and Hoyt 1984).
Adult syrphids were found exclusively in the sweet alyssum plots (Table 8), with a peak of approximately 12 specimens per 2-min observation in mid-October. If the 28 October species collection can be used as an indicator, most of the adults attracted were species that are known to prey on woolly apple aphid. This is positive evidence that sweet alyssum attracts useful syrphid species, even though the time frame of these experiments may have been too short to demonstrate effective biological control by their larvae. In addition, the adults are highly mobile, and could have oviposited on aphid colonies in both treatments (some syrphid larvae were also found on the potted trees in the control plots). The spatial scale of the effect is yet undetermined; and thus, a study addressing the predator movement (i.e., distance traveled from floral resource) within the orchards is warranted. Some adult syrphid species have been documented to fly up to
200 m away from a floral source when no physical barriers are present (Wratten et al. 2003).
Six out of eight adult syrphids collected throughout the experiments were identified positive for sweet alyssum pollen in their gut, confirming that they were using the flowers as a food source. In contrast, all the eight adult lacewings collected at the same time as the syrphids tested negative for sweet alyssum pollen. However, it is difficult to assure whether or not the
50
lacewings fed on the pollen because they were not collected directly from the flowers, and the pollen may have degraded some time after feeding.
Ambient natural enemy densities. More natural enemies in general were found in the ground cover (sweep nets) than on field-planted apple trees (beating trays) (Table 8). Spiders were the most abundant predator found in either sample type. Lacewing adults were moderately abundant in tray samples, but not in sweep nets. Adults of Aphelinus mali were relatively rare in both sample types. This orchard had no previous history of woolly apple aphid infestation, thus
A. mali may have not had enough time to become established during the two months of the study. Earwigs were moderately abundant in cardboard bands during experiment 1 (September), but almost absent during experiment 2 (October). The low earwig densities in October may reflect a movement to overwintering shelters as winter approached (Horton et al. 2002).
It is unclear whether or not sweet alyssum directly attracted natural enemies other than syrphids. It is possible that some were attracted to other phytophagous insects inhabiting the sweet alyssum plots. For example, Harwood and Obrycki (2007) found that linyphiid spiders tend to aggregate in prey-rich micro-sites.
In this study, sweet alyssum also attracted the tarnished plant bug Lygus sp., which could restrict the adoption of this cover crop in places where this pest is of major concern. Under this scenario, phenology studies of Lygus sp. with sweet alyssum would be warranted. This would help to devise sowing time that prevents the blossom from coinciding with the time of this pest peak.
Flower density. Sweet alyssum establishment on sandy soil was slower than on heavier- textured soils, although it did eventually become established and flowered on both soil types.
51
The average number of flowers per square foot was about 1,500 and 1,100 at beginning and at the end of the experiment respectively (Fig. 8). The plant coverage of the orchard floor was averaged 80-90% throughout the two experiments. Despite occasional tractor traffic over the plots, the plants were still vigorous and blooming through the beginning of November.
In summary, this experiment provides further indirect evidence for the potential of sweet alyssum plantings to enhance biological control of woolly apple aphid. Although predatory syrphids were the primary target species, this flowering plant was able to attract other beneficial arthropods. However, it was not possible to identify which natural enemies contributed the most to aphid suppression. In addition, the spatial and temporal scales of these experiments limit the comprehensive interpretation of the results. Thus, further research is needed to demonstrate the utility of sweet alyssum in a commercial setting, and provide a closer causative relationship between the use of flowering plants and biological control of woolly apple aphid.
Movement of natural enemies between sweet alyssum and apple trees. The three main predator groups of woolly apple aphid (syrphids, lacewings and coccinellids) and the parasitoid A. mali were found to visit the canopy of the trees after visiting the sweet alyssum
(Table 9). Some syrphids were caught on sticky traps up to 200 m away from the experimental area (Table 9). This is additional confirmation that syrphids are attracted to sweet alyssum, and that it could be planted in discontinuous strips in the orchard, which might ultimately lead to saving money on seed cost and sowing. In addition, the fact that A. mali and other predators also visit the flowers increases the benefits of sweet alyssum as an insectary plant. Spiders and earwigs were also found to move between sweet alyssum and trees. However, it is unknown whether these predators were attracted by the floral resource or only happened to walk over it.
Deraeocoris sp. was the only predator that did not test positive for the marker; however, it was
52
caught in very low numbers (Table 9). Nabis and Geocoridae were the only predators that were only caught on sweet alyssum (Table 9).
This three-year work shows that sweet alyssum has the potential to attract beneficial insects into the orchards, which might have a negative impact on woolly apple aphid population.
This is an important first step for developing a conservation biological control program for this pest.
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57
Table 1. Mean number of adult syrphids (± SE) attracted by different flowering species
Plant 1 Sep 6 Sep 13 Sep 20 Sep 27 Sep mustard 2.75 ± 0.85 b 2.25 ± 0.75 ab 2.28 ± 0.85 b 2.00 ± 0.70 b 3.25 ± 0.47 b buckwheat 4.00 ± 0.70 a 3.50 ± 1.55 a 1.50 ± 0.50 b 0.25 ± 0.24 c 1.50 ± 0.28 c cosmos * * * 0.50 ± 0.28 c 1.00 ± 0.40 c zinnia * * 0.75 ± 0.47 b 0.00 ± 0.00 c 0.50 ± 0.28 c sweet alyssum 5.25 ± 1.03 a 4.50 ± 0.86 a 5.75 ± 0.62 a 5.00 ± 0.57 a 8.75 ± 0.47 a marigold 0.75 ± 0.47 c 0.75 ± 0.75 b 1.00 ± 0.47 b 0.75 ± 0.48 bc 3.50 ± 0.45 b * Plants had not flowered yet.
Means followed by different letters within date differ significantly at P < 0.05 (Proc GLM ANOVA).
Table 2: Plant morphological and phenological characteristics
Plant Germination Flowering time Plant height Flower color Flower size Flower shape Mustard 3 20 76.00 ± 5.08 yellow 6.33 ± 0.33 cruciform Buckwheat 3 18 66.00 ± 7.50 white 2.66 ± 0.33 bowl-shaped Cosmos 6 54 29.60 ± 1.11 yellow 4.66 ± 0.33 bowl-shaped Zinnia 5 40 24.54 ± 3.68 reddish 45.66 ± 2.33 flat Sweet alyssum 3 21 16.91 ± 0.84 white 4.66 ± 0.33 saucer-shaped Marigold 5 34 29.62 ± 2.89 yellow and reddish 57.33 ± 1.45 flat Germination and flowering time = days after sow; plant height = cm; flower size = diameter across the corolla (mm).
58
Table 3. Syrphids collected during the experiment and identified to species
Plant Syrphid Species N % of total type Eristalis arbustorum L. 10 66.66% pollinator Scaeva pyrastri L. 1 6.66% predator Sphaerophoria philanthus Meigen 1 6.66% pollinator Mustard Syrphus opinator Osten Sacken 1 6.66% predator Parasyrphus relictus Zetterstedt 1 6.66% pollinator Eristalis hirta Loew 1 6.66% pollinator Eristalis arbustorum L. 17 73.90% pollinator Eristalis hirta Loew 1 4.35% pollinator Sweet alyssum Syrphus opinator Osten Sacken 2 8.70% predator Eupeodes americanus Wiedemann 2 8.70% predator Syritta pipiens L. 1 4.35% pollinator Buckwheat Eristalis arbustorum L. 11 100% pollinator Cosmos Eristalis arbustorum L. 1 100% pollinator Zinnia * Marigold Eristalis arbustorum L. 2 100% pollinator No specimen was captured for species identification.
Table 4. Mean number of woolly apple aphid (± SE) at different dates
Experiment 1 Experiment 2 Date control sweet alyssum Date control sweet alyssum 13 Sep 1304.00 ± 108.00 a 886.38 ± 131.87 b 4 Oct 1004.88 ± 190.23 a 572.26 ± 208.26 b 20 Sep 280.00 ± 135.88 a 90.16 ± 56.00 a 11 Oct 930.35 ± 197.19 a 667.80 ± 157.42 a 27 Sep 24.10 ± 13.46 a 5.25 ± 2.53 a 18 Oct 999.80 ± 209.33 a 467.05 ± 129.00 b 25 Oct 402.69 ± 118.48 a 347.67 ± 109.97 a Means followed by different letters within dates at each experiment differ significantly at P < 0.05 (LSMeans-PROC MIXED).
Table 5. Mean number of woolly apple aphids (± SE) on transect trees.
Mean distance from exp. plots Control (grass) Sweet alyssum 10 m 14.83 ± 8.24 17.17 ± 12.67 20 m 76.41 ± 32.45 33.41 ± 15.65
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Table 6. Numbers of natural enemies associated with woolly apple aphid colonies on potted trees at the time of evaluation
Treatments Experiment 1 Experiment 2 Control 6 13 20 27 Total 27 4 11 18 25 Total Sep Sep Sep Sepz Sepz Oct Oct Oct Oct Syrphid larvae 0 0 1 0 1 0 0 0 1 3 4 Lacewing adult 0 1 1 0 2 0 0 0 0 1 1 Lacewing eggs 0 97 0 0 97 0 0 0 0 0 0 Coccinellid adult 0 0 1 0 1 0 0 0 0 0 0 Deraeocoris sp 0 0 1 0 1 0 0 0 0 0 0 Aphelinus mali adult 0 0 0 0 0 0 0 0 0 0 1 Spiders 0 0 0 0 0 0 0 0 0 0 0 Sweet alyssum Syrphid larvae 0 0 3 0 3 0 0 0 0 0 0 Lacewing adult 0 6 0 0 6 0 0 0 0 0 0 Lacewing eggs 0 31 0 0 31 0 0 0 0 0 0 Coccinellid adult 0 0 0 0 0 0 0 0 0 0 0 Deraeocoris sp 0 0 0 0 0 0 0 0 0 0 0 Aphelinus mali adult 0 0 0 0 0 0 0 0 0 0 0 Spiders 0 1 0 0 1 0 0 0 0 0 0 Z Last date of experiment 1 overlaps the first date of experiment 2
Table 7. Syrphids collected from sweet alyssum plots with sweep net for gut dissection and species identification
Syrphid Species 13 Sepy 20 Sepy 27 Sepy 11 Octy 25 Oct 28 Oct Total Eupeodes americanus 1 (f) 0 0 0 0 0 1 Eupeodes fumipennis 0 0 0 0 1 (f) 3 (f), 3(m) 7 Eupeodes volucris 0 0 1 (f) 1 (f) 0 0 2 Eristalinae 0 0 0 0 0 1 (f) 1 Scaeva pyrastri 0 0 0 0 1 (m) 1 (f), 1 (m) 3 Syrphus ribesii 0 0 0 0 0 2 (f) 2 Syrphus opinator 0 1 (m) 1 (f) 1 (f) 9 (f), 1 (m) 17 (f), 5 (m) 35 f = female, m = male; ySpecimens collected for gut dissection
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Table 8. Total ambient natural enemies sampled via beating tray, sweep net, card board band and 2-minute direct count
Experiment 1 Experiment 2 control sweet alyssum control sweet alyssum Specimens Sep.6 Sep.13 Sep.20 Sep.27* Sep.6 Sep.13 Sep.20 Sep.27* Oct.4 Oct.11 Oct.18 Oct.25 Oct.4 Oct.11 Oct.18 Oct.25 Natural enemies/ 4 trays Spider 5 1 0 2 4 2 2 7 4 2 1 2 2 1 3 1 Deraeocoris sp. 1 1 0 0 1 0 0 0 1 0 0 0 0 0 0 0 Lacewing ad. 0 0 2 0 0 1 2 0 0 1 0 0 1 0 5 0 Aphelinus .mali 0 0 0 1 0 2 0 0 0 0 0 0 1 0 0 0 Minute pirate bug 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 Pred. hemipteran 0 0 0 0 0 0 0 0 1 0 0 0 1 0 0 0 Coccinellid ad. 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 Total 6 2 2 3 5 5 4 8 6 4 1 2 5 1 8 1 Total by exp & trt 13 22 13 15 Natural enemies/ 3 sweeps y Spider 0 0 1 0 0 7 20 28 0 1 0 0 13 16 17 17 Pred. hemipteran 0 0 0 0 0 1 0 1 1 0 0 0 0 1 1 0 Big-eyed bug 0 0 0 1 0 0 0 0 0 0 0 0 2 1 0 1 61 Minute pirate bug 0 0 0 0 0 0 0 2 0 0 0 0 1 1 2 2 Syrphid ad. 0 0 0 0 0 0 0 1 0 0 0 0 1 0 2 0 Nabis 0 0 0 0 0 0 0 0 0 0 0 0 2 1 1 0 Aphelinus .mali 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 Total 0 0 1 1 0 8 20 32 1 1 0 0 19 20 24 20 Total by exp & trt 2 60 2 83 Earwigs/ 4 bands@ 13 2 13 2 2 1 4 3 0 0 0 0 1 0 0 0 Total by exp & trt 30 10 0 1 Adult syrphids/2- 0 0 0 0 0 7 13 11 0 0 0 0 13 36 14 22 minute count Total by exp & trt 0 31 0 85 Grand total/date 19 4 16 6 7 21 41 54 7 5 1 2 38 57 46 43 Grand total exp & trt 45 123 15 184 * Last date of experiment 1 overlaps the first date of experiment 2.
@ earwigs were sampled via card board bands.
sweep net sampling and 2-minute count of adult syrphids started on September 13th .
Table 9. Percentage of natural enemies from sweet alyssum, apple trees and distant traps that tested positive for the protein marker
Sweet alyssum Infested trees Uninfested 50 m* 100 m* 200 m* Total Natural enemies N % positive N % positive N % positive N % positive N % positive N % positive N % positive Syrphidae 1 100.0 19 47.4 23 30.4 0 0.0 8 50.0 5 40.0 56 41.1 Chrysopidae 0 0.0 59 42.4 16 62.5 0 0.0 3 0.0 1 0.0 79 44.3 Chrysopidae larvae 0 0.0 8 50.0 0 0.0 0 0.0 0 0.0 0 0.0 8 50.0 Coccinellidae 0 0.0 9 33.3 3 33.3 0 0.0 1 0.0 0 0.0 13 28.6 Coccinellidae larvae 0 0.0 2 100.0 0 0.0 0 0.0 0 0.0 0 0.0 2 100.0 Deraeocoris sp. 0 0.0 2 0.0 1 0.0 0 0.0 0 0.0 0 0.0 3 0.0 Nabis sp. 11 81.8 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 11 81.8 Anthocoridae 9 77.7 2 50.0 0 0.0 0 0.0 0 0.0 0 0.0 11 72.7 Geocoridae 3 33.3 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 3 33.3
62 Forficulidae 0 0.0 7 42.8 18 33.3 0 0.0 0 0.0 0 0.0 25 36.0
Spiders 20 95.0 4 50.0 4 75.0 0 0.0 0 0.0 0 0.0 28 85.7 Aphelinus mali 0 0.0 26 50.0 17 70.5 2 50.0 1 100.0 0 0.0 46 58.7 Total 44 84.1 138 47.32 82 47.6 2 50.0 13 38.5 6 33.3 285 50.9 *White sticky traps at different distances away from experimental area.
Figure Legends
Fig. 1. Flowering plant species constituting the treatments in the syrphid/flower attraction study.
Fig. 2. Experimental plots having either grass or sweet alyssum flowers.
Fig. 3. Mean number of adult syrphids (+ SE) attracted to different plant species during a 2- minute observation.
Fig. 4. Mean number of adult syrphids and honeybees (+ SE) attracted to different plant species
(data pooled over all observation dates). Bars with different capital letters (syrphids) or lower case (honeybees) differ statistically at P < 0.05 (PROC GLM).
Fig. 5. Mean number of woolly apple aphids (+ SE)/ tree at different dates. Last evaluation date of experiment 1 (27 Sep) coincides with the deployment of infested trees in experiment 2.
Fig. 6. Number of woolly apple aphids on potted trees at 10 and 20 m away from experimental plots (transect study).
Fig. 7. Mean total number of natural enemies (+ SE)/ plot at different dates.
Fig. 8. Mean number of sweet alyssum flowers (+ SE)/ square foot (left Y axis), and percentage coverage (+ SE) of plots by sweet alyssum plants (right Y axis).
63
Figure 1A. Mustard Figure 1B. Buckwheat
Figure 1C. Cosmos Figure 1D. Zinnia
Figure 1E. Sweet alyssum Figure 1F. Marigold
Fig.1. Flowering plant species constituting the treatments in the syrphid/flower attraction study.
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Fig. 2A. Grass row middles (control) (Trt. 1). Fig. 2C. Sweet alyssum row middles (Trt. 2)
Fig. 2. Experimental plots having either grass or sweet alyssum flowers.
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10 mustard buckwheat cosmos zinnia sweet alyssum Marigold 8
6
4
2
Number of syrphids/ 2 minutes + SE 0
1 Sept. 6 Sept. 13 Sept. 20 Sept. 27 Sept.
Fig. 3. Mean number of adult syrphids (+ SE) attracted to different plant species during a 2- minute observation.
66
10
syrphids a honey bees 8
A
6
4 B B b B Mean number of syrphids + SE 2 C b C b b b 0
mustard cosmos zinnia buckwheat marigold sweet alysson
Fig. 4. Mean number of adult syrphids and honeybees (+ SE) attracted to different plant species
(data pooled over all observation dates). Bars with different capital letters (syrphids) or lower case (honeybees) differ statistically at P < 0.05 (PROC GLM).
67
1800 Exp. 1 Exp. 2 1600 Control 1400 Sweet alyssum
1200
1000
800
600
400
Number of WAA/ tree + SE 200
0
6 Sept 13 Sept 20 Sept 27 Sept 4 Oct 11 Oct 18 Oct 25 Oct
Fig. 5. Mean number of woolly apple aphids (+ SE)/ tree on different dates. Last evaluation date of experiment 1 (27 Sep) coincides with the deployment of infested trees in experiment 2.
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20 20 waa 40 waa 60 waa 80 waa 100 waa 120 waa 140 waa
Distance fromDistance (m) plots
10
1 2 3
20 control plots
Distance fromDistance (m) plots
10 1 2 3 sweet alyssum plots Fig. 6. Number of woolly apple aphids on potted trees at 10 and 20 m away from experimental plots (transect study).
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30 Exp. 1 Exp. 2
25 Control Sweet Alyssum
20
15
10
5
0
Total number of natural enemies/ plot + SE
6 Sept 13 Sept 20 Sept 27 Sept 4 Oct 11 Oct 18 Oct 25 Oct
Fig. 7. Mean total number of natural enemies (+ SE)/ plot at different dates.
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2500 120 % visual cover of plots number of flowers 100 2000
80
1500
60
1000 40
Number of flowers + SE
500 20 % coverage of orchard floor + SE
0
6 Sept 13 Sept 20 Sept 27 Sept 4 Oct 11 Oct 18 Oct 25 Oct
Fig. 8. Mean number of sweet alyssum flowers (+ SE)/ square foot (left Y axis), and percentage coverage (+ SE) of plots by sweet alyssum plants (right Y axis).
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CHAPTER FOUR
EFFECTS OF PREDATORS AND PARASITOID ON THE CONTROL OF WOOLLY
APPLE APHID
ABSTRACT
Outbreaks of woolly apple aphid Eriosoma lanigerum have become more frequent in
Washington State orchards since about 2000. Although there are only few insecticides known to effectively control woolly apple aphid, various natural enemies have been documented to prey on or parasitize woolly apple aphid. These include predators such as syrphids, coccinellids, chrysopids, earwigs, hemipterans, and the specialist parasitoid Aphelinus mali. However; little is known about their actual impact on woolly apple aphid population in the field. In this study, exclusion cage experiments were conducted in 2010 and 2011 to investigate the effect of the parasitoid A. mali alone and in combination with predators on woolly apple aphid suppression.
Results showed that predators together with A. mali can keep woolly apple aphid at very low densities in the field. Aphelinus mali alone was not able to suppress woolly apple aphid, but was able to slow down the growth of aphid population. There was a negative correlation between the size of woolly apple aphid colonies and the proportion of aphids parasitized by A. mali. The data suggest that biological control of woolly apple aphid work well in orchards where natural enemies are not disrupted.
Key words: biological control, predators, Aphelinus mali, woolly apple aphid, exclusion cage.
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INTRODUCTION
Understanding the role of existing natural enemy community in arthropod pest population dynamics is of paramount importance for developing effective pest management strategies. Thus, studying the impact of natural enemies on pest population is a critical step in implementing a biological program for agricultural pests. Woolly apple aphid is a secondary pest of apples whose outbreaks can affect fruit quality (Brown and Schmitt 1990, Brown et al. 1995) and harvest operations because of all the wax and honeydew produced. Outbreaks of woolly apple aphid is believed to be associated with disruption of natural enemies by broad spectrum pesticides (Nicholas 2000). Nonetheless, there is a good potential for implementing a biological control program of this pest in orchards that use selective pesticides. Woolly apple aphid is known to be preyed upon by various predators like coccinellids, chrysopids, syrphids, earwigs and predatory hemipterans (Walker 1985, Asante 1997, Mueller et al. 1998, Short and Bergh
2004). In addition, a specialized endoparasitoid, Aphelinus mali Haldeman is known to parasitize woolly apple aphid (Mueller et al. 1992, Brown and Schmitt 1994, Asante 1997).
However, little is known about their impact on woolly apple aphid population in the orchards.
Exclusion cage experiments have been used previously in other studies to assess the impact of natural enemies on other aphid species (Fox et al. 2004, Rutledge et al. 2004). Despite some of the caveats, studies like this allow a better understanding of the relative contribution of different natural enemy group in controlling the aphid population in the field. Understanding the role of natural enemy guilds is an important step in prioritizing conservation and augmentation measures.
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Preliminary results by Walker (1985) (also using exclusion cages) indicated that A. mali alone was not capable of suppressing woolly apple aphid population growth in Washington State.
While his results are plausible, several issues could be raised regarding the procedures used: 1) in the control cages parasitoid exclusion was accomplished by spraying fenvalerate (pyrethroid), 2) the parasitoid-alone treatment was a closed system (no ambient parasitoids entered the cages), and 3) trees of adjacent blocks were regularly sprayed with azinphosmethyl to control codling moth, and these sprays disrupt biological control of woolly apple aphid (Nicholas 2005). Thus, given these facts and the possibility that there may also have been a genuine shift in the predator complex in response to changing pesticide programs through time, a new assessment of the natural enemy impact on woolly apple aphid in Washington is warranted.
This study examines the hypothesis that the parasitoid A. mali alone is unable to suppress field populations of woolly apple aphid in central Washington. Therefore, the main objective of this work was to assess the effects of predators and parasitoid (separately and in combination) on the suppression of woolly apple aphid in orchards.
MATERIALS AND METHODS
Field exclusion cage experiments. Six exclusion cage experiments were conducted in three
WSU orchards (two sequential experiments in each orchard) in 2010 and 2011. The first two experiments (A and B) were conducted respectively in June-August and September-October
2010. This was conducted on an unsprayed 40-year-old apple orchard (mix of „Golden
Delicious‟ and „Delicious‟) located in the Tree Fruit Research and Extension Center in
Wenatchee, WA (Block 5). The other four experiments were conducted in June-July and August-
September 2011 (C and D in block 1; E and F in block 11). The orchard Smith tract block 1 was
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located at the Columbia River valley near Orondo, WA, and was planted with 21-year-old „Fuji‟ apples. The other orchard was located at the Columbia View WSU farm which was planted with
33-year-old „Delicious‟ apples (block 11). The orchards row middles were a grass sod, with a 1- m herbicide strip beneath the trees. The field planted trees were irrigated with under-tree impact sprinklers. Supplemental water was provided weekly for potted trees in the experimental cages because the pots could not hold enough water for the week.
The experimental treatments were: 1) control (woolly apple aphid only), 2) woolly apple aphid + parasitoid A. mali and 3) woolly apple aphid + A. mali + predators (Fig. 1). There were five to six replicates for each treatment which were arranged in a completely randomized design within each orchard. The experimental unit consisted of a potted tree infested with woolly apple aphid enclosed in an organdy (screen) or sham cage. The cages were constructed of a PVC pipe frame (1.60 1 1 m) and were covered with screening material (BioQuip, Rancho Domingues
CA). Two mesh sizes were used, the first (70/inch) excluded all natural enemies (treatment 1) and the second (24 x 20/inch) excluded predators, but not parasitoid (treatment 2). The third cage type was an open PVC frame with no fabric, allowing access by both predators and parasitoids (treatment 3) (Fig. 1). In 2011, fabric with a slightly larger mesh (18 13/inch) was used for treatment 2 (JoAnn Fabrics, Wenatchee, WA) (Fig. 2). An independent test was conducted in the laboratory to assure that parasitoids would go through the organdy mesh in treatment 2 (Appendix 4.1). The cages in treatment 1 and 2 were all enclosed with organdy
(screening) on all six sides, with one side closed with hook and loop tape (Velcro inc.,
Manchester, NH) allowing access to the tree inside. Each cage was placed between trees in the orchard tree rows and spaced approximately 10-20 6-12 m apart in the experimental setup. At least two tree rows were left as a buffer zone on all sides of the orchards.
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Before each experiment, young dormant ¾” apple trees „Fuji September Wonder‟/
EMLA 26 (2010 experiments) and „Fuji Top Export‟ / NIC 29 (2011 experiments) (C&O
Nursery, Wenatchee, WA) were transplanted into a 10-inch round plastic pot and pruned to about
1.0-1.2 m tall with three initial branches. After leafing out (2-3 weeks after transplanting), each tree was infested with approximately 30 first-instars of woolly apple aphid (experiment A), or two 1-2 cm twigs with an aphid colony (experiments B, C, D, E and F) collected from greenhouse colonies. In experiment A, the initial number of woolly apple aphid colonies was counted on each tree a month after infestation, and then randomly assigned to a cage treatment.
In all other experiments the aphid colony size and number were adjusted for each tree (two 1 1 cm colonies/tree).
To assure similar tree canopy structure across treatments, total leaf surface area was also estimated before deploying trees in the field. This also allows for an indirect measure of aphid effect on trees by comparing initial and final leaf surface area at the different cage treatments.
Total leaf surface area was estimated based on an equation obtained from a regression between leaf mid rib length and surface area (Appendix 4.2).
Data on woolly apple aphid and its natural enemies were collected weekly for six-seven weeks for each experiment. The number and size of woolly apple aphid colonies were recorded as well as the natural enemy composition (from potted trees and field-grown trees) and parasitism rate of aphids. Because of the difficulty in counting the precise number of aphids in large colonies, a regression equation between the colony surface area and aphid numbers was developed in order to estimate the aphid density on experimental trees (Appendix 4.3). In 2011, woolly apple aphid colony density was also estimated on the field-grown trees by counting the number of colonies found during a five-minute search. Predators were sampled via four methods:
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1) Direct count of predators physically associated with aphid colonies on potted trees in treatment 3 (this was the only sampling method used in experiment A). 2) Beating tray on field- grown trees (Doerr et al. 2005), where every week each branch of ten different trees were carefully inspected for motile natural enemies and then tapped into the tray, and the natural enemies were counted and released. 3) Card board bands (Horton et al. 2002) were attached to ten randomly selected field-grown trees; the earwigs found in them were counted and replaced with new bands weekly. 4) White sticky traps (9 15 cm) were hung weekly on ten field-grown trees (at least 8 m away from experimental cages) to trap predators and A. mali (2011 experiments only). Parasitism rate by A. mali was evaluated on both experimental and orchard trees. The percentage parasitism rate on experimental trees was estimated based on the total number of woolly apple aphid and intact mummies per tree on each date. Exited mummies were not included in this estimate, because they accumulate over time, leading to an inflated parasitism estimate. Predators eventually found in the control and parasitoid-only cages during the evaluations were recorded and removed.
The temperature was measured inside cages every hour using pendant data loggers (two per cage type) (Hobo Pendant Loggers, Onset Comp., Pocasset, MA). However, in experiment
A the temperature measurement was carried out only post hoc. In all experiments data loggers were suspended from tree branch at a height of about 0.5 m.
Data analyses. Cage treatment effect on woolly apple aphid density throughout time was assessed by repeated measures analyses of variance (PROC MIXED) (SAS 2008). The data from the first evaluation date was not included in the analysis when the initial number of aphids was the same across treatments (exps B, C, D, E and F). Data were analyzed by experiment and also combined by year. Covariance structures for the mixed model repeated measures were
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constructed for each experiment. ANTE (1) covariance structure was determined as the best-fit model for the second experiment in 2011 in Smith tract block 1 (experiment D), whereas
UNSTRUCTURE was determined as the best-fit model for all other experiments and combined data. Both covariance structures were chosen based on the Akaike Information Criterion (AIC).
In addition, pairwise comparisons were conducted using LSMeans (PROC MIXED) (SAS 2008).
Separate ANOVAS (PROC GLM) (SAS 2008) were conducted for each date within each experiment when there was a treatment and time interaction in the PROC MIXED. Pearson correlation was done between size of woolly apple aphid colonies (number of aphids/colony) and proportion of intact mummies/colony (aphid colonies with zero mummies were not included).
Differences in total tree leaf surface area at the beginning and at the end of experiments were assessed by separate ANOVAS (PROC GLM) (SAS 2008). Lastly, ANOVAS were also carried out to assess temperature difference among cage treatments (PROC ANOVA) (SAS 2008).
RESULTS AND DISCUSSION
There was a significant treatment effect in all experiments except experiment E (Table 1).
Likewise, there was a significant interaction between treatment and time for all experiments, except experiment E (Table 1). There was also a treatment effect for the combined data in both years as well as treatment and time interaction (Table 1). There was no block effect when pooling the data (considering the time of the experiment as a “temporal block”) (2010, F = 0.25,
P = 0.622, df = 1; 2011, F = 0.70, P = 0.405, df = 1), meaning that the results were consistent across experiments.
The open cage treatment (WAA + A. mali + predators) had a significant impact on the suppression of woolly apple aphid compared to the control (WAA only) and parasitoid cages
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(WAA + A. mali). The woolly apple aphid density was reduced to zero, or near zero in the open cages of all experiments (Table 2, Fig. 3A-D, F), except in experiment E (Table 2 and Fig. 3E).
This exception in aphid control may be due to disruption of biological control caused by pesticides sprayed in June and July in block 11 (Table 3). Overall, fewer natural enemies were observed in experiment E, compared to the other experiments that used equivalent predator sampling methods (Table 6). The organophosphate diazinon was only sprayed on the selected orchard trees (two rates with four replicates of three trees each, away from the cages). However, this insecticide is known to be highly toxic to natural enemies of woolly apple aphid (Shaw and
Walker 1996, Nicholas 2000), and any small section of the orchard sprayed with it could have some detrimental effect on biological control agents. In addition, delegate (spinetoram) was sprayed on the entire orchard on 11 and 27 June (Table 3). This is a new pesticide introduced in
2008 to control codling moth Cydia pomonella L. and has been shown to be toxic to the parasitoid A. mali (chapter 6). In addition, Biddinger et al. (2011) observed a reduction of natural enemies in orchards in Pennsylvania sprayed with spinetoram, which correlated with high incidence of woolly apple aphid. Likewise, the woolly apple aphid population on the orchard trees of block 11 also peaked about the time of the sprays, and thereafter plummeted to nearly zero (Fig. 4). This may indicate that woolly apple aphid is an induced pest, and thus more problematic in orchards that are sprayed with non-selective pesticides. In fact, outbreaks of woolly apple aphid have been associated with the use of broad-spectrum pesticides that kill natural enemies (Penam and Chapman 1980, Nicholas 2000). Low woolly apple aphid densities on the orchard trees of block 1 (Fig. 4) did not seem to hamper natural enemy recruitment (Table
5), and hence, biological control was efficient in the open cages of experiments C and D (Fig.3).
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The aphid control in the parasitoid cage treatment (WAA + A. mali) was not as effective as in the open cage treatment (Fig. 3). In most of the dates it was not significantly different from the control cages (Table 2). However, while A. mali was not able to completely control woolly apple aphid, parasitism appeared to consistently slow aphid population growth. Parasitism rate by A. mali in the parasitoid-only cages was lower or equal to the parasitism in the open cages on most of the dates for the first half of the summer in all experiments (Fig. 5A, 5C and 5E). In contrast, parasitism was higher during the second half of the summer in the parasitoid-only cages compared to the open cages (Fig. 5B, 5D and 5F). Likewise, parasitism was also higher on the second half of the summer on the orchard trees of block 1 and 11 (Fig. 6). This trend may be explained by two factors: predators eliminating aphid colonies in the open cages, and occurrence of cooler temperatures in the second half of the summer that favor A. mali reproduction. Walker
(1985) observed a reproductive decline of A. mali under temperatures above 32o C, which can cause parasitized aphids to die. In fact, a higher parasitoid density was observed during the second half of the summer in blocks 1 and 11 (Table 5 and 6). A possible explanation for higher levels of parasitism in the first half of the summer in the open cages compared to parasitoid-only cages (Fig. 5) is the presence of smaller aphid colonies (Fig. 7) which facilitate parasitism by A. mali (Mueller et al. 1992). Similarly, the smaller aphid colonies on the orchard trees (average 25 aphids, range 5 - 93) used to estimate the parasitism may help explain why parasitism on orchard trees was higher compared to the parasitoid-only cages. This is also supported by a negative correlation found between number of aphids per colony and proportion of mummies per colony
(Pearson, r = - 0.5066, P = <0.0001) (Fig.8).
The most common natural enemies found in the orchards were earwigs, Aphelinus mali,
Deraeocoris sp., syrphids, coccinellids and chrysopids (Tables 4, 5 and 6). All natural enemies
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found in these experiments have also been reported in other studies regarding woolly apple aphid control (Walker 1985, Asante 1997, Mueller et al. 1988, 1998, Short and Bergh 2004, Nicholas et al.2005). However, to my knowledge, little mention of earwigs as biocontrol agents of woolly apple aphid is made in US literature, although it is considered a key predator in some regions of
Europe (Mueller et al. 1988). The number of natural enemies observed on the potted trees was much lower compared to field trees (Tables 4, 5 and 6). This low number is likely a result of the direct count that is done on a very short period of time as opposed to the traps that stay in the orchard for a week. White sticky trap and card board band were the most efficient sampling methods, trapping most of the natural enemies (Tables 5 and 6). However, caution is advised when ranking natural enemies by order of occurrence/importance, since the sampling method used may influence the proportion of predator taxon collected. Predator and parasitoid densities tended to be similar in the first half of the summer, but the latter was higher on second half of the summer (Tables 5 and 6). A much lower number of natural enemies were recorded in 2010 because only one or two sampling methods were used (Table 4).
A few predators were found in cages that were intended to exclude them. Spiders were the most common predator found (18), followed by nabis (2), Deraeocoris (1), and coccinellids
(1). All were immediately removed from the cages after being encountered.
There was no difference in the total tree leaf surface area between treatments at the beginning and end of all experiments (Fig. 9). This indicates that tree size or morphology was not a factor in aphid population growth or foraging success by natural enemies. In addition, similar leaf surface area among treatments at the end of the experiment suggests that aphid feeding did not negatively affect plant growth during the course of the experiment.
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Data loggers revealed no significant difference in temperature inside the different cage treatments (Table 7). This indicates that temperature was not a factor for differences in the size of aphid population inside the cages.
The results suggest that biological control has the potential to effectively control woolly apple aphid, if not disrupted by non-selective pesticides. Even though there was not a predator- alone treatment, it appears that predators were the main cause of aphid mortality. It is especially clear in the second half of the summer of all experiments where parasitism is near zero in the open cages. This indicates that either predators are eliminating the aphid colonies before parasitism takes place or are preying on parasitized aphids too. Our findings are similar to those of Walker (1985), who also concluded that predation was the main cause of woolly apple aphid reduction in the field in eastern Washington. Conversely, in areas of the world that do not have wide range of generalist aphidophagous predators, the parasitoid A. mali is felt to play an important role in biological control of woolly apple aphid (Shaw and Walker 1996, Nicholas
2005).
Our results indicate that generalist predators should be the primary target of conservation programs aimed at promoting biological control of woolly apple aphid; implementation of such programs are likely to promote supplemental control by A. mali, which can contribute to the overall success and stability of control.
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Table 1. Treatment and treatment*time interaction effects on woolly apple aphid densities through time.
Treatment effect Treatment*time interaction effect Experiment P F d.f. P F d.f. A 0.0020 9.32 2 0.0010 6.60 2 B <0.0001 46.09 2 <0.0001 14.71 2 C <0.0001 20.38 2 0.0009 8.46 2 D 0.0002 19.25 2 <0.0001 12.98 2 E 0.1600 2.16 2 0.0980 7.35 2 F 0.0100 7.25 2 0.0090 5.11 2 Combined (A-B)* <0.0001 24.59 2 <0.0001 10.78 12 Combined (C-D)Z <0.0001 37.72 2 <0.0001 11.14 8 *Pooled data for 2010 experiments; ZPooled data for 2011 experiments.
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Table 2. Mean number of woolly apple aphids per tree (+ SE) at different dates and cage treatments.
Block 5 (T.F.) – Experiment 1 (2010)* Date WAA only A. mali only A. mali + predators June 18 470.04 ± 146.12 a 318.80 ± 63.48 a 405.17 ± 104.61 a June 28 1041.12 ± 471.84 a 464.04 ± 234.89 a 245.17 ± 222.12 a July 05 2398.80 ± 884.05a 1274.14 ± 393.16 a 232.70 ± 203.92 b July 12 4518.35 ± 1796.67 a 2677.62 ± 617.55 a 257.76 ± 231.03 b July 19 9399.25 ± 2922.77 a 5825.85 ± 1977.56 a 221.44 ± 202.49 b July 26 13779.65 ± 3503.98 a 8245.45 ± 1714.18 a 186.03 ± 166.92 b August 2 15279.9 ± 2454.37 a 8845.11 ± 1467.84 b 70.70 ± 60.34 c Block 5 (T.F.) – experiment 2 (2010)* September 7 772.76.04 ± 0.00 a 772.76.04 ± 0.00 a 772.76.04 ± 0.00 a September 15 2447.76 ± 358.96 a 2003.68 ± 100.44 a 2003.47 ± 402.09 a September 22 3393.25 ± 522.30a 2913.42 ± 336.92 a 2448.23 ± 402.09 a September 29 4337.69 ± 653.40 a 3252.72 ± 534.84 a 414.00 ± 235.65 b October 6 7251.69 ± 421.50 a 3907.09 ± 446.54 b 3.00 ± 1.43 c October 13 10034.18 ± 1118.19 a 5550.79 ± 859.52 b 0.00 ± 0.00 c October 20 9435.83 ± 1061.74 a 6986.47 ± 1017.87 b 0.00 ± 0.00 c Block 1 (S.T.) – Experiment 1(2011)* 22 June 772.76 ± 0.00 a 772.76 ± 0.00 a 772.76 ± 0.00 a 29 June 1316.04 ± 175.70 a 811.05 ± 146.78 b 681.18 ± 96.67 b 6 July 3561.89 ± 633.16 a 2029.98 ± 478.00 b 644.45 ± 218.39 b 13 July 6074.74 ± 914.12 a 2453.92 ± 568.15 b 375.50 ± 134.38 c 20 July 7177.32 ± 1555.96 a 2888.83 ± 897.64 b 286.83 ± 110.83 b 27 July 13315.97 ± 2663.23 a 5156.58 ± 1315.25 b 159.80 ± 49.72 c Block 1 (S.T.) – Experiment 2 (2011)* 3 August 772.76 ± 0.00 a 772.76 ± 0.00 a 772.76 ± 0.00 a 10 August 984.33 ± 162.64 a 1066.46 ± 125.70 a 397.23 ± 206.26 b 17 August 3056.76 ± 639.17 a 2135.52 ± 472.59 a 8.20 ± 8.20 b 24 August 7017.60 ± 1592.86 a 2922.79 ± 500.03 b 0.00 ± 0.00 c 31 August 9576.82 ± 2300.63a 2935.30 ± 628.88 b 0.00 ± 0.00 c 7 September 9760.07 ± 1397.77 a 3922.962 ± 454.20 b 0.00 ± 0.00 c Block 11 (C.V.) – Experiment 1(2011)Z 24 June 772.76 ± 0.00 a 772.76 ± 0.00 a 772.76 ± 0.00 a 1 July 1072.23 ± 77.63 a 898.28 ± 85.59 a 952.99 ± 106.83 a 8 July 2695.84 ± 686.81 a 1442.29 ± 358.32 a 709.50 ± 269.23 a 15 July 4368.98 ± 1273.32 a 2978.68 ± 578.74 a 1811.67± 612.87 a 25 July 4166.81 ± 718.52 a 4083.31 ± 1142.49 a 2526.44 ± 818.28 a 29 July 5586.29 ± 991.38 a 3772.07 ± 766.86 a 2355.48 ± 805.85 a Block 11 (C.V.) – Experiment 2 (2011)* 5 August 772.76 ± 0.00 a 772.76 ± 0.00 a 772.76 ± 0.00 a 12 August 1037.01 ± 176.18 a 1199.06 ± 269.57 a 434.18 ± 169.73 a 19 August 2628.40 ± 435.42 a 2625.41 ± 440.46 a 551.23 ± 374.22 b 26 August 4721.35± 766.88 a 1837.66 ± 718.45 b 4.00 ± 4.00 c 2 September 4307.96 ± 1078.14 a 2715.17 ± 1201.94 a 0.00 ± 0.00 b 9 September 5863.38 ± 1869.82 a 4338.56 ± 1596.47 a 0.00 ± 0.00 b *Separate Anovas (PROC GLM) were conducted for each date due to treatment and time interaction. Means followed by different letters within dates differ significantly at P<0.05 (LSMeans PROC GLM or PROC MIXEDZ)
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Table 3. Insecticides sprayed on orchards during the exclusion cage experiments
Block 5 (T.F) (2010) Block 1 (S.T.) (2011) Block 11 (C.V.) (2011) Unsprayed Altacor on June 14th, 2011 Delegate on June 11th, 2011 Altacor on June 28th, 2011 Delegate on June 27th, 2011 Intrepid on July 18th, 2011 *Ultor on July 18th, 2011 *Warrior II on July 18th, 2011 *Intrepid on July 18th, 2011 *Sulfaxaflor on July 18th, 2011 *Diazinon (rate 1) on July 20th, 2011 *Diazinon (rate 2) on July 20th, 2011 *Ultor on August 1st , 2011 * Rimon on August 1st , 2011 * Altacor on August 1st , 2011 T.F.: Tree Fruit Research and Extension Center orchard, S.T.: Smith Tract orchard, C.V.: Columbia View orchard
* Sprayed only on 4 replicates of 3 trees each (each replicate was at least 7 m away from cages)
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Table 4. Number of natural enemies observed during the exclusion cage experiments in block 5 (TFRC)
Natural enemies observed on the orchard trees Natural Experiment 1 Experiment 2 Enemies 28 June 5 July 12 July 19 July 26 July 2 Aug Total 7 Sept 15 Sept 22 Sept 29 Sept 6 Oct 13 Oct 20 Oct Total Syrphid lv ...... 0 0 0 0 0 0 0 0 Syrphid ad ...... 0 0 1 2 0 0 0 3 Chrysopid lv ...... 0 0 0 0 0 0 0 0 Chrysopid ad ...... 0 0 0 0 0 0 0 0 Coccinellid lv ...... 0 0 0 0 0 0 0 0 Coccinellid ad ...... 0 0 0 0 0 0 0 0 Deraeocoris sp ...... 0 0 0 0 0 0 0 0 Earwig ...... 49 15 25 26 8 0 0 123 Spider ...... 0 2 4 0 0 2 0 8 Nabis ...... 0 0 0 0 0 0 0 0 Aphelinus mali ...... 0 0 2 10 0 0 0 12
89 Predator total ...... 49 17 30 38 8 2 0 144
Total ...... 49 17 32 51 15 2 0 156 Percent caught by different sampling method (%) Beating tray ...... 0.00 11.80 21.88 49.02 46.67 100.00 0.00 21.16 Cardboard ...... 100 88.20 78.12 50.98 53.33 0.00 0.00 78.84 band Natural enemies observed on potted trees during the experiment evaluation (direct count only) Syrphid lv 0 0 0 0 0 0 0 0 0 10 11 2 0 0 23 Syrphid ad 0 0 0 0 0 0 0 0 0 1 1 1 0 0 3 Chrysopid lv 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Chrysopid ad 2 0 0 0 1 0 3 0 0 0 0 0 0 0 0 Coccinellid lv 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Coccinellid ad 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Deraeocoris sp 1 0 0 0 1 1 3 0 0 0 0 0 0 0 0 Earwig 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Spider 0 0 0 0 0 0 0 0 0 0 1 4 0 0 5 Nabis 0 0 0 1 2 1 4 0 0 0 0 0 0 0 0 Aphelinus mali 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Predator total 3 0 0 1 4 2 10 0 0 11 13 7 0 0 31 Total 3 0 0 1 4 2 10 0 0 11 13 7 0 0 31
Table 5. Number of natural enemies observed during exclusion cage experiments in block 1 (Smith tract)
Natural enemies observed on the orchard trees Natural Experiment 1 Experiment 2 Enemies 22 June 29 June 6 July 13 July 20 July 27 July Total 3 Aug 10 Aug 17 Aug 24 Aug 31 Sept 7 Sept Total Syrphid lv 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Syrphid ad 0 0 0 0 1 0 1 0 0 1 1 2 2 6 Chrysopid lv 0 0 0 0 0 0 0 0 0 0 0 0 0 8 Chrysopid ad 0 0 0 0 0 0 0 0 0 1 0 0 0 1 Coccinellid lv 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Coccinellid ad 5 0 1 0 0 2 8 1 0 0 0 0 0 1 Deraeocoris sp 0 0 0 0 2 1 3 0 0 0 1 0 0 2 Earwig 1 96 77 105 58 66 403 50 59 36 63 54 36 298 Spider 1 3 6 3 1 0 14 2 0 1 3 3 8 17 Nabis 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Aphelinus mali 0 159 119 39 35 61 413 29 115 158 202 138 176 818
90 Predator total 7 99 84 108 60 69 428 53 59 39 68 59 46 333
Total 7 259 213 147 98 133 841 82 174 197 270 197 222 1142 Percent caught by different sampling method (%) Beating tray 100 0.38 2.95 2.04 1.04 0.76 2.25 1.22 33.15 1.01 0.37 0.50 2.70 0.96 Cardboard band 0.00 37.20 37.94 71.42 59.37 50.76 47.68 60.97 0.00 18.27 23.33 27.41 15.76 25.89 Sticky trap 0.00 62.40 59.11 26.53 39.58 48.46 50.05 37.80 66.85 80.71 76.30 72.08 81.53 73.14 Natural enemies observed on potted trees during the experiment evaluation (direct count only) Syrphid lv 0 0 6 0 0 0 6 0 0 0 0 0 0 0 Syrphid ad 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Chrysopid lv 0 0 0 0 0 0 0 0 8 0 0 0 0 8 Chrysopid ad 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Coccinellid lv 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Coccinellid ad 0 0 1 0 0 0 1 0 0 0 0 0 0 0 Deraeocoris sp 0 0 0 0 1 0 1 0 1 0 0 0 0 0 Earwig 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Spider 0 0 1 0 0 0 1 0 0 0 0 0 0 0 Nabis 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Aphelinus mali 0 1 2 0 1 3 7 0 0 0 0 0 0 0 Predator total 0 0 8 0 1 0 9 0 9 0 0 0 0 9 Total 0 1 10 0 2 3 16 0 9 0 0 0 0 9
Table 6. Number of natural enemies observed during the exclusion cage experiments in block 11 (Columbia view)
Natural enemies observed on the orchard trees Natural Experiment 1 Experiment 2 Enemies 24 June 1 July 8 July 15 July 25 July 29 July Total 5 Aug 12 Aug 19 Aug 26 Aug 2 Sept 9 Sept Total Syrphid lv 0 0 0 0 0 0 0 0 1 0 0 0 0 1 Syrphid ad 3 1 0 0 0 3 7 12 12 1 2 6 5 38 Chrysopid lv 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Chrysopid ad 0 0 0 0 0 4 4 0 0 0 1 1 1 3 Coccinellid lv 1 0 0 0 0 0 1 2 1 0 0 0 0 3 Coccinellid ad 0 1 0 0 1 4 6 5 0 2 0 0 1 8 Deraeocoris sp 1 0 8 15 38 8 69 109 80 19 5 14 8 235 Earwig 1 6 12 17 15 8 59 6 10 17 24 15 6 78 Spider 7 5 0 3 2 0 17 2 1 2 0 5 1 11 Nabis 0 0 0 0 0 0 0 0 0 0 1 1 0 2 Aphelinus mali 6 8 29 5 21 40 109 88 115 340 159 129 125 956
91 Predator total 13 13 20 35 56 27 164 136 95 41 33 42 22 369
Total 19 21 49 40 77 67 273 224 210 381 192 171 147 1325 Percent caught by different sampling method (%) Beating tray 31.57 4.76 4.08 7.50 6.32 2.98 6.90 3.30 2.72 0.78 1.56 5.26 0.00 2.11 Cardboard band 5.26 28.57 24.48 42.50 18.98 11.94 21.47 2.83 4.54 4.46 12.50 8.77 4.09 5.89 Sticky trap 63.15 66.66 71.42 50.00 74.68 85.07 71.63 93.86 92.72 94.75 85.93 85.96 95.91 91.98 Natural enemies observed on potted trees during the experiment evaluation (direct count only) Syrphid lv 0 0 1 0 0 0 1 0 0 0 0 0 0 0 Syrphid ad 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Chrysopid lv 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Chrysopid ad 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Coccinellid lv 0 0 0 0 0 0 0 0 0 1 0 0 0 1 Coccinellid ad 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Deraeocoris sp 0 1 0 0 1 0 2 0 1 0 0 0 0 1 Earwig 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Spider 0 0 0 0 0 1 1 0 2 0 0 0 0 2 Nabis 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Aphelinus mali 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Predator total 0 1 1 0 1 1 4 0 3 1 0 0 0 4 Total 0 1 1 0 1 1 4 0 3 1 0 0 0 4
Table 7. Temperature measurements collected inside cages by pendant data loggers during each experiment
Temperatures 0C Block/ Orchard Date trt 1 trt 2 trt 3 19 Aug – 21 Aug, 2010* 38.46 ± 1.41 a - 37.56 ± 1.41 a Block 5 (T.F) 7 Sept – 20 Oct, 2010 14.76 ± 1.72 a 14.81 ± 1.72 a 14.53 ± 1.73 a 22 June – 27 July, 2011 20.75 ± 1.41 a 20.80 ± 1.41 a 19.80 ± 1.40 a Block 1 (S.T.) 3 Aug – 7 Sept, 2011 22.05 ± 1.41 a 22.00 ± 1.41 a 21.87 ± 1.41 a 24 June – 29 July, 2011 23.00 ± 1.41 a 21.25 ± 1.41 a 21.75 ± 1.41 a Block 11 (C.V.) 5 Aug – 9 Sept, 2011 23.15 ± 1.41 a 21.45 ± 1.42 a 23.20 ± 1.41 a *Post hoc test
Mean temperatures followed by the same letter within date do not differ statistically at p<0.05 (PROC ANOVA).
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Figure legends
Fig. 1. Experimental cages: Treatment 1 is intended to exclude everything, treatment 2 is intended to exclude predators but no the parasitoid A. mali, and treatment 3 is intended to allow everything (predators + parasitoid A. mali).
Fig. 2. Organdy meshes used on experimental cages. A: mesh used in control cages (trt 1), B: mesh used in parasitoid cages in 2010 (trt 2), C: mesh used in parasitoid cage in 2011 (trt 2).
Parasitoid = 1 mm in length.
Fig. 3. Mean number of woolly apple aphid per tree (+ SE) at different dates and cage treatments. Cage treatments: ● = WAA only, ○ = WAA + A. mali, and ▼ = WAA + A. mali + predators.
Fig. 4. Mean number of woolly apple aphid colonies (+ SE) observed on orchard trees during a
5-minute walk conducted during each evaluation date. ● = Block 11 (C.V.) 2011, and ○ = Block
1 (S.T.) 2011. There was not woolly apple aphid on the orchard trees of block 5 (T.F.) in 2010.
Fig. 5. Percentage parasitism of woolly apple aphid by A. mali per tree (+ SE) at different dates.
Cage treatments: ■ = WAA only, □ = WAA + A. mali, and ■ = WAA + A. mali + predators.
Fig. 6. Percentage parasitism of woolly apple aphid by A. mali (+ SE) observed on orchard trees
(10 colonies/ evaluation date). ■ = Block 1 (S.T.), and ■ = Block 11 (C.V.).
Fig. 7. Size of woolly apple aphid colonies: Mean number of aphids/ colony (+SE). Cage treatments: ■ = WAA only, □ = WAA + A. mali, and ■ = WAA + A. mali + predators.
Fig. 8. Regression between number of woolly apple aphid/ colony and proportion of mummies
(N = 1231 aphid colonies).
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Fig. 9. Total tree leaf surface area (+ SE) (cm2). ■ = first day, and ■ = last day of experiment. Bars followed by the same letters do not differ statistically at p<0.05 (ANOVA - PROC GLM).
trt 1= (WAA only) trt 2= (WAA + A. mali) trt 3= (WAA + A. mali + predators)
Fig. 1. Experimental cages: Treatment 1 is intended to exclude everything, treatment 2 is intended to exclude predators but not the parasitoid A. mali, and treatment 3 is intended to allow everything (predators + parasitoid A. mali).
A B C
Fig. 2. Organdy meshes used on experimental cages. A: mesh used in control cages (trt 1), B: mesh used in parasitoid cages in 2010 (trt 2), C: mesh used in parasitoid cage in 2011 (trt 2).
Parasitoid = 1 mm in length.
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20000 12000
18000 A - Block 5 (T.F.), 2010 B - Block 5 (T.F.), 2010 10000 16000
14000 8000 12000
10000 6000
8000 4000 6000
4000 2000
No. of WAA/ tree + SE 2000 No. of WAA/ plant + SE 0 0
5 July 2 Aug 6 Oct 18 June 28 June 12 July 19 July 26 July 7 Sept 15 Sept 22 Sept 29 Sept 13 Oct 20 Oct
18000 14000
16000 C - Block 1 (S.T.), 2011 12000 D - Block 1 (S.T.), 2011 14000 10000 12000
10000 8000
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No. of WAA/ tree + SE 2000 No. of WAA/ tree + SE
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7000 10000
E - Block 11 (C.V.), 2011 F - Block 11 (C.V.), 2011 6000 8000
5000 6000
4000 4000 3000
2000 2000
No. of WAA/ tree + SE
No of WAA/ plant + SE 1000 0
0
1 July 7 July 24 June 15 July 25 July 29 July 5 Aug 12 Aug 19 Aug 26 Aug 2 Sept 9 Sept
Fig. 3. Mean number of woolly apple aphid per tree (+ SE) at different dates and cage treatments. Cage treatments: ● = WAA only, ○ = WAA + A. mali, and ▼ = WAA + A. mali + predators.
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400 400 A B 300 300
200 200
100 100
0 0
No of WAA colonies/ 5 minute + SE
No of WAA colonies/ 5 minute + SE
6 - 8 July 3-5 Aug 1-2 Sept 7-9 Sept 22 -24 June 13 - 15 July20 - 25 July27 - 29 July 10-12 Aug 17-19 Aug 24-26 Aug 29 June- 1 July
Fig. 4. Mean number of woolly apple aphid colonies (+ SE) observed on orchard trees during a
5-minute search conducted during each evaluation date. A = first half of summer; B = second half of summer. ● = Block 11 (C.V.) 2011, and ○ = Block 1 (S.T.) 2011. There was not woolly apple aphid on orchard trees of block 5 (T.F.) in 2010.
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6 6
A - Blck 5 (T.F.), 2010 B - Block 5 (T.F.), 2010 5 5
4 4
3 3
2 2
% parasitism of WAA + SE 1
% parasitism of WAA + SE 1
0 0
5 July 2 Aug 6 Oct 18 June 28 June 12 July 19 July 26 July 7 Sept 15 Sept 22 Sept 29 Sept 13 Oct 20 Oct
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C - Block 1 (S.T.), 2011 D - Block 1 (S.T.), 2011 30 30
25
20 20
15
10 10
% parasitism of WAA + SE 5 % parasitism of WAA + SE
0 0
6 July 22 June 29 June 13 July 20 July 27 July 8 Aug 10 Aug 17 Aug 24 Aug 31 Aug 7 Sept
0.6 18
E- Block 11 (C.V.), 2011 16 F -Block 11 (C.V.), 2011 0.5 14
0.4 12
10 0.3 8
0.2 6
4
% parasitism of WAA + SE 0.1
% parasitism of WAA + SE 2
0.0 0
1 July 8 July 24 June 15 July 25 July 29 July 5 Aug 12 Aug 19 Aug 26 Aug 2 Sept 9 Sept
Fig. 5. Percentage parasitism of woolly apple aphid by A. mali per tree (+ SE) at different dates.
Cage treatments: ■ = WAA only, □ = WAA + A. mali, and ■ = WAA + A. mali + predators. 97
100 100 A B
80 80
60 60
40 40
20 20
Percetage parasitism of WAA + SE
Percetage parasitism of WAA + SE
0 0
6 - 8 July 3-5 Aug 1-2 Sept 7-9 Sept 13 - 15 July 20 -25 July 27 - 29 July 10-12 Aug 17-19 Aug 24-26 Aug
Fig. 6. Percentage parasitism of woolly apple aphid by A. mali (+ SE) observed on orchard trees
(10 colonies/ evaluation date). ■ = Block 1 (S.T.), and ■ = Block 11 (C.V.).
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800 800
A - Block 5 (T.F.), 2010 B - Block 5 (T.F.), 2010
600 600
400 400
200 200
No. of WAA/ colony + SE
No. of WAA/ colony + SE
0 0
5 July 2 Aug 6 Oct 18 June 28 June 12 July 19 July 26 July 7 Sept 15 Sept 22 Sept 29 Sept 13 Oct 20 Oct
600 600
C - Block 1 (S.T.), 2011 D - Block 1 (S.T.), 2011 500 500
400 400
300 300
200 200
No. of WAA/ colony + SE 100 No. of WAA/ colony + SE 100
0 0
6 July 22 June 29 June 13 July 20 July 27 July 3 Aug 10 Aug 17 Aug 24 Aug 31 Aug 7 Sept
600 600
E - Block 11 (C.V.), 2011 F - Block 11 (C.V.), 2011 500 500
400 400
300 300
200 200
No. of WAA/ colony + SE No. of WAA / colony + SE 100 100
0 0
1 July 8 July 24 June 15 July 25 July 29 July 5 Aug 12 Aug 19 Aug 26 Aug 2 Sept 9 Sept
Fig.7. Size of woolly apple aphid colonies: Mean number of aphids/ colony (+SE). Cage treatments: ■ = WAA only, □ = WAA + A. mali, and ■ = WAA + A. mali + predators.
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1.0
2 0.8 R = 0.57 P < 0.0001
0.6
0.4
0.2
Proportion ofProportion WAA colony mummies/ 0.0
0 500 1000 1500 2000 2500 No. of WAA/ colony
Fig. 8. Regression between number of woolly apple aphid/ colony and proportion of mummies/ colony (N = 1231 colonies).
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14000 14000 A - Block 5 (T.F.), 2010 B - Block 5 (T.F.), 2010 12000
) / tree 12000 ) / tree
2 A A A 2 10000 10000 a a 8000 8000 a A a A a A a 6000 6000
4000 4000
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Total leaf surface area (cm Total leaf surface area (cm 0 0
WAA only WAA + A. mali WAA only WAA + A. mali WAA + A. mali + predators WAA + A. mali + predators
14000 14000 C - Block 1 (S.T.), 2011 C -Block 1(S.T.), 2011 12000 12000 A A A
)/ tree + SE )/ tree + SE 10000
2 2 A 10000 a a A A 8000 8000 a a a a 6000 6000
4000 4000
2000 2000
0 0
Total leaf surface area (cm Total leaf surface area (cm WAA only WAA + A. mali WAA only WAA + A. mali WAA + A. mali + predators WAA + A. mali + predators
14000 14000 E - Block 11 (C.V.), 2011 F - Block 11 (C.V.), 2011 12000 12000 A A A
)/ tree + SE )/ tree + SE 10000 2 10000 2 a A a A a A a 8000 8000 a a 6000 6000
4000 4000
2000 2000
0 0
Total leaf surface area (cm Total leaf surface area (cm WAA only WAA + A. mali WAA only WAA + A. mali WAA + A. mali + predators WAA + A. mali + predators
Fig. 9. Total tree leaf surface area (+ SE) (cm2). ■ = first day, and ■ = last day of experiment. Bars followed by the same letters do not differ statistically at P<0.05 (ANOVA - PROC GLM). 101
CHAPTER FIVE
IMPACT OF SYRPHIDS ON THE PARASITISM OF WOOLLY APPLE APHID BY
APHELINUS MALI
ABSTRACT
Predatory syrphids and Aphelinus mali are important natural enemies of woolly apple aphid. Because generalist predators such as syrphids have broader feeding habits it is possible that sometimes they may reduce the overall effectivieness of biological control by feeding on specialist natural enemies like A. mali. This parasitoid has been credited with the successful control of woolly apple aphid in many countries around the world. Nevertheless, some countries did not achieve the same success from importation of A. mali. However, no one has yet investigated the possibility of intraguild predation being the contributing or responsible factor for the inefficiency of A. mali in certain regions. In this work I investigated the effects of the interaction between A. mali and predatory syrphids on suppression of woolly apple aphid. I conducted a series of greenhouse, laboratory and field experiments where I tested the efficiency of both natural enemies separately and in combination, syrphid preference for mummified versus unparasitized aphids, avoidance by A. mali of aphid colonies with predator cues, and effect of syrphids on parasitism of woolly apple aphid. Syrphids and A. mali were able to significantly reduce the population of woolly apple aphid, either separately or combined. Syrphids did not seem to disrupt parasitism of woolly apple aphid, and did not feed on mummified aphids. A. mali did not avoid aphid colonies with syrphids. Thus, it appears that syrphids do no compromise the parasitism of woolly apple aphid by A. mali, and could enhance biological control in areas where they co-occur.
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Key words: syrphid, Aphelinus mali, intraguild predation, woolly apple aphid, biological control.
INTRODUCTION
Natural enemy biodiversity has been shown to enhance biological control through increase of resource (prey/host) consumption/ parasitism (Loreau and Hector 2001). This increase in pest control may be achieved either by natural enemy complementarity or sampling effect. Complementarity occurs via either resource partitioning or facilitation, whereas sampling effect refers to the presence of key species with very high consumption or parasitism rate
(Loreau and Hector 2001). Nonetheless, these dynamics are affected by the specificity of natural enemies with regards to their resource. Thus, natural enemies of arthropods are broadly divided into specialists and generalists. Specialists feed on or parasitize relatively few pest species, whereas generalists have a broader prey/host range. Because of the close relationship between specialists and their resource, a higher numerical response and more effective pest control is expected from this group of natural enemies (Murdoch 1994, Turchin et al. 1999). Generalists, on the other hand, feed on many different species and thus respond more slowly to the fluctuations of pest density, which can compromise pest control (Murdoch 1985). In addition, the broader feeding habits of generalists can lead to reduced effectiveness of biological control via intraguild predation (Rosenheim et al. 1993, Polis and Strong 1996, Snyder and Ives 2001). This phenomenon occurs when generalists feed on other generalist or specialist natural enemies with which they share some common resource. However, specialist natural enemies with their narrower prey/host range are more likely to be attacked by generalist predators (Lucas et al.
1998). Despite all these limitations, generalists have shown to be effective biological control agents in many agroecosystems (Fagan et al. 1998, Snyder and Wise 2001).
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In this work I investigated the effects of the interaction between the specialist parasitoid
Aphelinus mali (Haudeman) and generalist predatory syrphids on suppression of woolly apple aphid. Aphelinus mali is an endoparasitoid native to north America known to parasitize only woolly apple aphid. This parasitoid has been credited with the successful control of woolly apple aphid in many countries around the world (DeBach 1964). Nevertheless, some countries did not achieve the same success from importation of A. mali. One possible reason cited for the disparity in the control success are differences in the climatic conditions (Mols and Boers 2001).
However, intraguild predation has not been explored as a contributing factor to reduced efficiency of A. mali in certain regions. In previous exclusion cage studies (Walker 1985,
Chapter 4) in central Washington, A. mali was not able to provide high levels of control of woolly apple aphid in the field. In addition, parasitism was very low in open cages where aphid colonies were exposed to both predators and parasitoids (Chapter 4), suggesting that intraguild predation of A. mali could be happening.
Syrphids are important predators of woolly apple aphid (Asante 1997, Bergh and Short
2008) and are often found feeding in aphid colonies in Washington orchards (Walker 1985,
Carroll and Hoyt 1984, Chapter 2). Generalist and specialist species have been found in
Washington, with the former being by far the most abundant. There are two facts that make syrphid larvae potential intraguild predators of A. mali. First, they are generalist aphidophagous predators that share a common resource with A. mali; and second, unlike the more mobile larvae of coccinellids and lacewings they move very infrequently between colonies, thus leaving them little choice of prey when settled in colonies with many parasitized aphids.
I hypothesized that syrphid larvae and A. mali independently have significant impact on woolly apple aphid suppression, but that aphid parasitism is negatively affected when syrphid
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and A. mali co-occur. The objectives of this work were: 1) measure the effect of syrphid larvae and A. mali on suppression of woolly apple aphid separately and combined, 2) assess whether or not syrphid larvae are able to distinguish between unparasitized aphids and mummified aphids, and 3) test whether or not A. mali avoids host patch with predator cues (kairomones).
MATERIALS AND METHODS
Greenhouse cage experiments. Two experiments were conducted in the greenhouse at the Tree
Fruit Research and Extension Center (TFREC) in Wenatchee, WA. The first experiment was conducted from 19 August to 9 September 2009. This experiment had a completely randomized design with three treatments and five replicates: 1 = four adult female parasitoids Aphelinus mali
+ woolly apple aphid, 2 = four second-third instar syrphid larvae + woolly apple aphid and 3 = control (woolly apple aphid only). Each experimental unit was comprised of an 80-cm tall potted apple tree infested with woolly apple aphid (cultivar 'Morning Mist Fuji‟/ M9-337). The trees were planted into 10- inch round pots using Promixing soil (Miracle-Gro Promixing, Marysville,
OH) and placed in the greenhouse to leaf out. Thirty four days before predators and parasitoids were released each tree was infested with 20 first instars (crawlers) of woolly apple aphid. After infestation each tree was enclosed by a circular plastic sheeting cage (92 x 40 cm) having a fine mesh organdy fabric on top and a 15 x 25 cm mesh ventilation port on the side (Fig. 1A). The parasitoid mummies and syrphid larvae were collected from woolly apple aphid colonies found in WSU research orchards. Prior to release, parasitoid mummies were held at indoor ambient temperature to emerge; and syrphid larvae were kept on apple twigs containing woolly apple aphids. Adult parasitoids were sexed 24 hours after emergence; and thereafter, the females and the syrphid larvae were randomly assigned and released into their respective treatment cages.
The environmental conditions for the greenhouse during the course of the experiment were
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temperature of 23 ± 0.15 ○C, a relative humidity of 59.74 ± 0.30% and photoperiod 16:8 (L:D).
During the experiment the trees were watered every three days with tap water. The experiment was terminated 21 days after the predator/parasitoid release. No aphid or natural enemy count was done before the end of the experiment. Adult syrphids encountered in the cages at the end of the experiment were saved for later species identification. The trees were cut out into smaller parts (10-15 cm) and the woolly apple aphids were carefully brushed into a 120-ml plastic cup containing 70% alcohol. Aphids were counted by pouring them into gridded Petri dishes and counting them under binocular microscope.
The second experiment was conducted from 20 October to 4 November 2009. This experiment had a substitutive design which controls for confounding effects of total natural enemy density and potential sampling effects (Huston 1997). The strengths and weaknesses of this design have been discussed before (Connolly 1988, Griffen 2006). There were four treatments with three-four replicates each: 1 = eight adult female parasitoids A. mali + woolly apple aphid, 2 = eight second – third instar syrphid larvae + woolly apple aphid, 3 = four adult female parasitoids + four second – third instar syrphid larvae + woolly apple aphid, and 4 = control (woolly apple aphid only). The experiment was terminated 15 days after predator/parasitoid release. The remaining procedures were the same as described above in experiment 1.
Field experiment. This experiment was conducted in August, 2011 in the WSU Columbia View farm (block 11). The syrphid larvae and parasitoids used in the experiment were collected in the same orchard. Syrphid larvae were collected two days before the experiment and kept on a woolly apple aphid diet until two hours prior to the start of the experiment. Only second and third instar larvae similar in appearance were used to increase the probablity of using specimens
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of the same species. Aphelinus mali were collected as mummies attached to apple twigs two- four days before their use in the experiment. The parasitoid mummies were carefully brushed off of the twigs and placed into empty 120 ml-plastic cups having the top covered with fine organza.
These cups were placed in ambient temperature in the lab for adult emergence. Syrphids and A. mali were collected from the field because it is unfeasible to establish and maintain a lab colony.
Two treatments with nine-ten replicates were used for this experiment. 1 = woolly apple aphid colony with unparasitized and recently parasitized aphids (control), and 2 = woolly apple aphid colony with unparasitized and recently parasitized aphids + single second -third instar syrphid larva. Each replicate consisted of the distal third of a „Delicious‟ apple branch with aphids enclosed by 30 x 15 cm organdy sleeve cage (Fig. 1B). Prior to the experiment, young woolly apple aphid colonies without visible signs of parasitism were selected from different trees, and enclosed by the organdy sleeve for six days to assure that the aphids were not previously parasitized. Just prior to the parasitoid release, the number of aphids was counted in each cage, and five 24-hour old female A. mali were released and allowed to parasitize for 24 hours. Three days after parasitoid removal, a single syrphid larva was added to half of the cages and allowed to feed for 24 hours. Syrphid larvae were then removed and reared to the adult stage for species identification. Six days after syrphid removal the branches with cages were cut and brought to the lab where number of aphids and mummies were counted under a binocular microscope.
Laboratory experiments. Two experiments were conducted in August 2011 to test whether or not syrphid larvae can feed on mummified aphids. The treatments in the first experiment were: 1) apple twig with unparasitized and mummified aphids (control), and 2, apple twig with unparasitized and mummified aphids + single second - third instar larva of syrphid.
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There were 8-13 replicates. Each experimental unit consisted of a plastic sheeting cylinder cage
(7 x 20 cm) having both ends covered by organza fabric, and containing inside the 17-cm apple twig infested with unparasitized and mummified aphids. The base of the apple twigs were immersed inside a glass bottle (4 x 8 cm) with water and sealed with parafilm to avoid water evaporation and syrphid larva falling into it. Syrphid larvae were collected as described in the previous experiment. Before the start of the experiment the number of unparasitized and mummified aphids on each twig was counted. After that, 2-hour starved syrphid larva was placed individually on twigs of half of the cages and allowed to feed for 24 hours. Thereafter, the syrphids were removed and the number of aphids and mummies were counted.
Two treatments were used in this second experiment: 1) ten mummified woolly apple aphids (control) and 2) ten mummified woolly apple aphids plus a single second - third instar larva of syrphid. There were twelve replicates, consisting of a 35 x 10 mm Petri dish containing the mummified aphids. Syrphid larvae were starved for two hours, then introduced into treatment the arenas and allowed to feed for 24 hours. Both experiments were conducted in the laboratory at 24 ± 10 C, R.H. of 40 ± 5%, with fluorescent lights set at a photoperiod of 16:8 L:D. Syrphid larvae in both experiments were reared to the adult stage for species identification.
Olfactometer experiments. The effects of predator cue (kairomones) on A. mali‟s choice of host patch were examined by Y-tube olfactomer tests. Syrphid larvae and parasitoids were collected as described above. Two experiments with five runs (ten parasitoids/run) each were conducted. In experiment 1 each female parasitoid had a choice between the odors of apple twigs infested with woolly apple aphid plus ten second - third instar syrphid larvae, and apple twigs infested with woolly apple aphid only. In experiment 2, the choices were apple twigs infested with woolly apple aphid and a void source (empty jar). In the treatments that required
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plant and aphid odor, four apple twigs measuring eight cm long infested with 200-400 woolly apple aphids each were used in each run. Some of the syrphid larvae were used twice in different runs. Each odor source treatment was placed inside a 945 ml glass jar (Kerr, Daleville, IN) and tightly closed 6-12 hours prior to the beginning of the experiment. The jars were independently connected to the arms of the Y-tube and the air flow adjusted to 45 ml/minute/arm before each run. One run was completed when ten 24-48 hour old female parasitoids successfully made a choice of odor in the Y-tube. A choice was scored only when the female touched the very end of one of the Y-tube arms. Female parasitoid was individually released at the base of the Y-tube with the aid of a camel brush, and was given 20 minutes to make a choice. All female parasitoids were naïve, with no previous oviposition experience or contact with plants. In order to remove any spatial bias, the position of the Y-tube arms were switched after five females had made a choice in each run. The experiments were conducted in a laboratory evenly illuminated by fluorescent lights, and with ambient temperature of 23 ○C.
Data Analyses. Statistical tests were performed using SAS software (SAS 2008). Differences in the final number of woolly apple aphids per tree among treatments were tested by running
ANOVA (PROC GLM) on the greenhouse cage experiment data. T-tests were performed with
PROC TTEST to test for significant differences between treatments in the field experiment
(initial and final number of woolly apple aphids/ branch, total number of mummies/branch and percentage parasitism by A. mali). A two-sided binomial test (PROC FREQ) was performed on the olfactometer data to test for differences in odor choice made by A. mali.
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RESULTS AND DISCUSSION
Greenhouse cage experiments. In both greenhouse experiments, the syrphid larva and parasitoid alone treatments were able to reduce woolly apple aphid numbers in the caged trees.
However, there was not a significant difference between the syrphid and parasitoid treatments
(Figs. 2A and 2B). In addition, there was not a clear effect of the combination treatment (syrphid larva plus parasitoid) on aphid suppression (Fig. 2B). However, if a marginal effect of P = 0.06 is considered for the combination treatment, then all natural enemy treatments in experiment two would show to equally reduce woolly apple aphid numbers when compared to the control. Thus, the treatment combination of syrphids and parasitoids would show an additive effect on aphid control. This outcome might be attributed to similar functional response of both natural enemies and differences in their plant space use. For instance, Chang (1996) found that combination of aphid predators that usually foraged and fed on different places within the plant had additive effects on aphid suppression. This may also explain why no signs of intraguild predation were observed in this study.
All the adult syrphids retrieved at the end of experiment one belonged to the species
Syrphus opinator (n = 6), whereas in experiment two they belonged to Eupeodes americanus (n
= 4) and Eupeodes fumipennis (n = 5). Although the syrphid species used in the two experiments were different, the results obtained were essentially the same. This may be so because of using larvae of similar instar, and potentially similar functional response.
Thus, the results reveal the potential of both syrphids and parasitoids, alone or in combination, to reduce woolly apple aphid density. However, due to the fact that cage
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experiments tend to simplify the system this assessment of syrphid and parasitoid effectiveness should be also conducted in the field in order to corroborate the results here observed.
Field experiment. There was no significant difference in the final number of woolly apple aphids between treatments (Fig. 3A). This outcome may be attributed to facilitation, where syrphid larvae might prefer injured/recently dead aphids attacked by A. mali. Lundie (1924) observed higher mortality of woolly apple aphids exposed to A. mali in small enclosure, because the parasitoid can parasitize the same aphid many times leading to debilitation or death.
Likewise, there was no significant difference in the numbers of mummies, or the percentage parasitism between the two treatments (Fig. 3B). This may indicate that syrphid larvae do not disrupt the parasitism of woolly apple aphid, but apparently, they do not enhance overall mortality in the presence of syrphids. A possible reason for this result is that syrphid larvae might be able to discern between parasitized and unparasitized aphids, and thus, prefer the latter.
In fact predatory syrphids are less likely to prey on parasitized aphids when compared to other generalist predators such as coccinellids (Brodeur and Rosenheim 2000). Another possible reason is that syrphid larvae and A. mali females could attack different instars of woolly apple aphid. For example, Ninomiya (1957) found syrphid larvae of Episyrphus balteatus to prefer only early instars of aphids.
The results indicate that syrphids do not interfere with woolly apple aphid parasitism.
Thus, the co-occurrence of these natural enemies in the orchards could benefit biological control of woolly apple aphid. However, the basis that underlies the “coexistence” between these natural enemies should be further investigated in order to better understand the ecological aspects of this interaction.
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Laboratory experiments. Syrphid larvae rejected mummified aphids in both lab experiments (Figs. 4A and B). When given a choice, syrphids only fed on unparasitized aphids, whereas in the no-choice test (only mummies) they did not feed at all. These results show that syrphid larvae are able to discern between unparasitized and mummified aphids, and prefer the former. In addition, it proves that intraguild predation does not occur when woolly apple aphids are in an advanced mummified stage. In fact, it has been documented that syrphid larvae of many species are not able to feed on mummified aphids because they can not open the mummy shelter
(Kindlmann and Ruzicka 1992, Meyhöfer and Klug 2002). It is also possible that some species could be inheritably pre-determined to avoid feeding on mummies based on the choice of ovipositing site made by the female syrphids. For example, Pineda et al. (2007) observed adults of Episyrphus balteatus to avoid ovipositing on aphid colonies that were parasitized by Aphidius colemani Viereck. The results also suggest that if A. mali larvae reach the mummy stage before syrphid eggs hatch there could be a complementary control of woolly apple aphid on the colonies co-occupied by these natural enemies.
Olfactometer experiments. All the syrphid larvae used in the 2011 experiments and reared successfully to the adult stage were Eupeodes americanus, (field experiment, n = 6; lab experiment, n = 6; olfactometer test, n = 10). The success rate of rearing the syrphid larvae to the adult stage was about 31% (22 out of 71). It shows that collecting larvae with identical morphology increases the chances of obtaining specimens from same species.
Each A. mali took an average of 8.5 minutes to make a choice after released in the Y- tube. Females of A. mali preferred the odors of woolly apple aphid colonies as opposed to a void source (Fig. 5A). This indicates that the olfactometer test worked and A. mali did not respond at random when given a choice of interest.
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Adult females of A. mali showed no preference when given a choice between aphid colonies with and without syrphid larvae (Fig. 5B). This suggests that A. mali females do not avoid ovipositing on aphid colonies that have syrphid larvae. This may happen because some syrphid species can not feed on parasitized aphids, and moreover, there would be a cost and risk involved when searching for the “perfect” aphid patch with no predator (Rosenheim and Mangel
1994). Foraging for predator-free patches could be even more costly for a parasitoid like A. mali which is not a strong flier. I addition, A. mali is known to parasitize more aphids at the margins of the colonies (Mueller et al. 1992) whereas syrphid larvae are frequently found in the center of the colonies. This could, therefore, facilitate their coexistence.
In conclusion, the results suggest that intraguild predation of A. mali by syrphid larvae of
E. americanus is unlikely to happen, or if it happens, it might not disrupt the biological control of woolly apple aphid. On the contrary, these two natural enemies together could perform a complementary control of woolly apple aphid, and thus, enhance biological control. However, other potential intraguild predators such as coccinellids, chryspids and other syrphid species should be also investigated in order to understand why parasitism sometimes is low in the field.
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Figure legends
Fig. 1. A: Cage experiment setup in greenhouse, 2009. B: Sleeve cage used in the field experiment in 2011.
Fig. 2. Final number of woolly apple aphid per tree under different treatments. A: parasitoid and syrphid tested separately. B: parasitoid and syrphid tested separately and combined. Bars followed by the same letters do not differ significantly at P <0.05, Anova (PROC GLM).
Fig. 3. A: Mean number of woolly apple aphids per branch (+ SE) at the beginning and at the end of the experiment. B: Mean number of mummies/ branch (+ SE), and mean percentage parasitism of woolly apple aphid by Aphelinus mali (+ SE). ■ = WAA + parasitoid + syrphid, ■
= WAA + parasitoid. Bars followed by the same letters do not differ significantly at P <0.05, T- test (PROC TTEST).
Fig. 4. A: Mean number of woolly apple aphids and mummies per twig (+ SE) at the beginning and at the end of the experiment. B: Mean number of mummies/Petri dish (+ SE). ■ = initial number of WAA, □ = final number of WAA, ■ = initial number of mummies, ■ = final number of mummies.
Fig. 5. A: Number of Aphelinus mali making a choice between plant material with aphids and void choice. B: Number of Aphelinus mali choosing odors sources with and without predator cues. * = significant difference between treatments at P <0.05, two-sided binomial test (PROC
FREQ).
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A B
Fig. 1. A: Cage experiment setup in greenhouse, 2009. B: Sleeve cage used in the field experiment in 2011.
20000 A 14000 A B
12000 A 15000 B 10000 AB
B 8000 B B 10000 6000
4000 5000
Number of WAA/ tree ± SE
Number of WAA/ tree + SE 2000
0 0
control (WAA only) syrphid + WAA parasitoid + WAA control (WAA only)parasitoid + WAApredator + WAA predator + parasitoid + WAA
Fig. 2. Final number of woolly apple aphid per tree under different treatments. A: parasitoid and syrphid tested separately. B: parasitoid and syrphid tested separately and combined. Bars followed by the same letters do not differ significantly at P <0.05, Anova (PROC GLM).
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500 40
A a B
400 A A 30
300
20 A 200 a a A a 10 100
Number of WAA/ branch + SE
0 0
No. of parasitismmummies percentage and (%) + SE Initial Final mummies parasitism
Fig. 3. A: Mean number of woolly apple aphids per branch (+ SE) at the beginning and at the end of the experiment. B: Mean number of mummies/ branch (+ SE), and mean percentage parasitism of woolly apple aphid by Aphelinus mali (+ SE). ■ = WAA + parasitoid + syrphid, ■
= WAA + parasitoid. Bars followed by the same letters do not differ significantly at P <0.05, T- test (PROC TTEST).
100 10
A B
80 8
60 6
40 4
No. of mummies + SE 20 2
No. of WAA and mummies + SE
0 0 syrphid no syrphid syrphid no syrphid
Fig. 4. A: Mean number of woolly apple aphids and mummies per twig (+ SE) at the beginning and at the end of the experiment. B: Mean number of mummies/Petri dish (+ SE). ■ = initial
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number of WAA, □ = final number of WAA, ■ = initial number of mummies, ■ = final number of mummies.
40 35 A B 30
30 25 NS A. mali 20
A. mali 20 * 15
Number of 10
Number of of 10
5
0 0 WAA + plant empty jar WAA + plant + syrphids WAA + plant
Fig. 5. A: Number of Aphelinus mali making a choice between plant material with aphids and void choice. B: Number of Aphelinus mali choosing odors sources with and without predator cues. * = significant difference between treatments at P <0.05, two-sided binomial test (PROC
FREQ).
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CHAPTER SIX
ACUTE AND CHRONIC EFFECTS OF ORCHARD PESTICIDES ON APHELINUS
MALI, A PARASITOID OF WOOLLY APPLE APHID
ABSTRACT
Aphelinus mali (Haudeman) is an important endoparasitoid of woolly apple aphid. This parasitoid is native to North America and has been introduced in many apple growing regions of the world. One of the main problems faced by natural enemies like A. mali is the use of harmful pesticides which can cause direct mortality or affect their reproduction and development, and therefore, disrupt biological control. In this study I investigated the acute and chronic effects of orchard pesticides on A. mali. For the acute bioassays two rates were tested, 1.0x = maximum label rate and 0.1x = 10% of the maximum label rate. For the chronic tests only one rate was tested, which was the higher of the two rates used in the acute test that killed 75% or less of the parasitoids. Spinetoram, spinosad, carbaryl, organophosphates and neonicotinoids were acutely toxic to A. mali, killing more than 90% at 1.0x rate. Chlorantraniliprole, lambda-cyhalothrin, novaluron, cyantraniliprole, sulfur, spirotetramat and zinc + manganese hydroxide killed less than 60% of the parasitoids in the acute bioassays at both rates. Only cyantraniliprole, spinetoram and lambda-cyhalothrin showed significant sublethal effects on A. mali. The sublethal effect of cyantraniliprole was due to induction of low fecundity in A. mali, whereas for spinetoram and lambda-cyhalothrin the effect was due to adult A. mali and host mortality.
Key words: pesticides, non-target effect, Aphelinus mali, biological control, woolly apple aphid.
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INTRODUCTION
Woolly apple aphid is an indirect pest of apples that causes serious problems in high densities (Bertus 1986, Brown and Schmitt 1990, Brown et al. 1995). Biological control is becoming an important component of IPM of woolly apple aphid, especially now with increasing restrictions on endosulfan and diazinon, two of the older pesticides that are still effective on woolly apple aphid (Bush et al 2011). Some of the most common natural enemies found attacking woolly apple aphid are syrphids, coccinellids, chrysopids, earwigs, predatory hemipterans and the parasitoid Aphelinus mali (Walker 1985, Asante 1997, Mueller et al. 1988,
1992; Short and Bergh 2004). This specialist endoparasitoid has received credit for the successful control of woolly apple aphid in many countries around the world (DeBach 1964).
Aphelinus mali is native to North America and has been introduced in many world regions in attempt to control woolly apple aphid (Lundie 1924, Howard 1929). This parasitoid is very important in parasitizing aerial aphid colonies, and thereby reducing woolly apple aphid density in the field. However, it has not been obeserved to parasitize edaphic aphid colonies.
One of the main problems faced by natural enemies like A. mali is the use of harmful pesticides which can cause direct mortality or affect their reproduction and development, and therefore, disrupt biological control. Shaw and Walker (1996) correlated higher incidence of woolly apple aphid with low densities of A. mali in orchards sprayed with non-selective insecticides. Laboratory studies have also shown broad spectrum pesticides to be acutely toxic to
A. mali (Cohen et al. 1996, Bradley et al 1997, Heunis and Pringle 2003).
Studies of non-target effect of pesticides conducted in laboratory are more common than field because of limitations posed by biotic and abiotic factors in the latter. However, in laboratory studies the natural enemies are usually exposed to only one or two routes of exposure
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(James 2004, Tillman and Mullinix 2004), whereas in the field they would be exposed to at least three different routes (Banken and Stark 1998). For this reason, A. mali was subjected to the topical, oral and residual routes of exposure during the sublethal studies.
Some of the newer orchard insecticides (i.e., spinetoram, chlorantraniliprole, novaluron) are expected to have a higher pest specificity, lower mammalian toxicity and lower potential for ground water contamination, hence; being labeled as “reduced risk” insecticides (Jones et al.
2009). However, little is known about their non-target effect on various natural enemies that occur in the orchard agroecosystem. This study evaluates the toxicity of fungicides, newer insecticides and more conventional insecticides (i.e., organophosphates). My hypothesis is that, unlike the fungicides the insecticides will show acute but not chronic toxicity to A. mali. Thus, the objective of this study was to evaluate the acute and sublethal effects of orchard pesticides on
A. mali.
MATERIALS AND METHODS
Acute toxicity bioassays. This series of bioassays were conducted at the Tree Fruit
Research and Extension Center in Wenatchee, WA. The bioassays were comprised of three treatments and approximately 30 replicates each: 1) control (distilled water), 2) 10% of the pesticide maximum label rate (0.1x), and 3) pesticide maximum label rate (1.0x). Both insecticide and fungicide were tested (Table 1). For the insecticides, the concentrations were based on the rate recommended for codling moth control Cydia pomonella L (Table 1). Each experimental unit consisted of an adult parasitoid Aphelinus mali (either male or female) enclosed in a plastic Petri dish (35 x 15 mm) with small orifice (≈ 5 mm in diameter) on the top covered by micropore tape. The experiment had a completely randomized design. The parasitoids were collected as mummies attached to apple twigs from WSU orchards. After
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collection, the parasitoid mummies were carefully brushed off of the twigs and placed into 120- ml plastic cups covered with fine organza. These cups were placed in a environment controlled room for the adult parasitoid emergence (25 ±1○C, 40 ± 5% R.H. and 16:8 L:D photoperiod).
Adult parasitoids 24 hours after emergence were placed individually into a plastic Petri dish (35 x 15 mm) with filter paper at the bottom. Before treatment, each Petri dish containing the parasitoid was placed inside the freezer at -5○C for about 0.6 – 1.0 minute in order to immobilize them. While immobilized the parasitoid was sprayed individually in a laboratory sprayer (Potter
Precision Spray Tower, Rickmansworth, England) with 2 ml of the respective pesticide treatment. This process was repeated for all treatments in the following order: control, 0.1x rate and 1.0x rate. Following the spray the parasitoids were individually transferred to a clean Petri dishes containing a small cotton ball (5 mm diameter) imbibed with 50% commercial honey/water solution, and then placed in an environment controlled room (25 ±1○C, 40 ± 5%
R.H. and 16:8 L:D photoperiod). The mortality was evaluated at 24 and 48 hours after the treatment. The data were analyzed as categorical with binary response (dead or alive) using
PROC GENMOD (Stokes et al. 2000) (SAS 2008).
Chronic toxicity bioassays. These bioassays had two treatments with approximately 30 replicates each. The treatments were: 1) control (distilled water) and 2) 1.0x rate used in the acute mortality test. The experiment was conducted in a completely randomized design in an environment controlled room with environmental conditions described above. Each experimental unit was composed of a 24-hour old mated adult female parasitoid caged on apex a potted apple tree infested with woolly apple aphid. Each female parasitoid was caged on a potted „Fuji‟ apple tree with one to three woolly apple aphid colonies (100-300 aphids total) at the apex. The colonies were enclosed by a glass cylinder cage (45 60 mm) (Wheaton, Millville, NJ) that had
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an organdy sleeve attached to the bottom to secure it to the stem, and the top glued with organdy
(Fig. 1). The trees were infested with approximately 10-15 first instar woolly apple aphids at 40 days prior to the beginning of the experiment. The parasitoids were collected and prepared as described in the acute mortality tests.
Each female parasitoid was exposed to the pesticide treatment via three routes of exposure for four days (≈1/3 of their adult longevity). The routes of exposure were: topical, residual and oral (all applied about the same time). Topical exposure was applied with a laboratory sprayer as described above. For the residual exposure, the inside of each glass cylinder was sprayed under the potter tower using 2 ml of treatment solution. The organdy sleeves for the cages as well as the potted trees and aphid colonies were also sprayed with the respective treatment solution using a180 ml-aerosol spray bottle (Nalgene Inc., Rochester, NY).
For the oral exposure, a small cotton ball drenched in honey-water solution (50%) was pinned to each tree close to the aphid colonies and sprayed at the same time of the tree and aphid colonies with the aerosol spray bottle. Approximately two to four hours after topical treatment, they were transferred individually to the treated glass cylinder cage on the tree.The toxicity of the pesticides were also tested on woolly apple aphids in independent tests (Appendix 6.1)
Every day during a four-day period the mortality of the parasitoids was evaluated, and at the end of the fourth day all remaining female parasitoids were removed from the cages. Thirteen and 16 days after the spray; the number of intact and exited mummies was counted, and the F1 sex ratio was assessed. The sex was based on their size (Mueller et al. 1992) and response to cold
(expose parasitoids to -5 0C for 3-4 minutes) causing the males to withdraw their genitalia.
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The data obtained were used to parameterize a stage-structured matrix model software
(PopTools version 3.2.3, Hood 2010). This software allows an estimate of r, the rate of increase, and a projection of recovery of treated and control populations.
RESULTS AND DISCUSSION
Acute toxicity. Spinetoram, spinosad, azynphos-methyl, carbaryl and chlorpyrifos presented the highest acute toxicity to A. mali (100% mortality at 1.0x rate) (Table 2 and 3).
Spinetoram and spinosad are insecticides that act on allosteric activators of the nicotinic acetylcholine receptors. Insecticides from the spinosad group have been reported to have negative effects on other small prasitoids, like the leaf miner parasitoids Neochrysocharis formosa and Ganaspidium nigrimanus (Moreno 2009). Azynphos-methyl, carbaryl and chlorpyrifos act similarly on the insect nervous system by inhibiting cholinesterase enzymes; therefore, a similar response from A. mali to these chemicals is expected. These three insecticides have also been documented to be toxic to adults of A. mali (Cohen et al. 1996,
Shawn and Walker 1996, Nicholas 2000). However, no negative effect has been observed on the mummy stage (Cohen et al. 1996, Bradley et al 1997, Heunis and Pringle 2003).
Acetaprimid and thiacloprid also showed high toxicity to A. mali killing about 83 and
92% respectively at 1.0x rate. These are neonicotinoid-based insecticides that act on the insect‟s nervous system by binding to postsynaptic nicotinic acetylcholine receptors. Acetaprimid and thiacloprid have been observed to also have detrimental effects on parasitoids like Leptomastix dactylopi, a natural enemy of the citrus mealybug (Cloyd and Dickinson 2006), and
Trichogramma cacoeciae (Schuld and Schmuck 2000). Likewise, another neonicotinoid imidacloprid has also shown to be highly toxic to A. mali in Israel (Cohen et al. 1996).
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Lambda-cyhalothrin showed an intermediate toxicity killing about 60% of the parasitoids at 1.0x rate. Lambda-cyhalothrin is a pyrethroid type of insecticide that acts on the stomach and may function as a sodium channel modulator. This insecticide has also shown to be toxic to leaf miner parasitoids Neochrysocharis formosa and Ganaspidium nigrimanus (Moreno 2009). In addition, lambda-cyhalothrin has also shown sublethal effects on the aphid parasitoid Aphidius ervi by inducing low fecundity (Desneux 2004).
The remaining insecticides chlorantroniprole, cyantraniprole, novaluron and spirotetramat killed less than 50% of the parasitoids at 1.0x rate (Table 2 and 3), thereby showing a better selectivity to A. mali. Chlorantroniprole and cyantraniprole are diamide insecticides that activate ryonodine receptos causing impaired muscle regulation; whereas novaluron is an insect growth regulator (IGR) that affects the development of immature insects. The lower toxicity of these three insecticides may be explained by the fact that their main target is immature lepidopterans. Spirotetramat is a systemic insecticide that targets sucking pests such as aphids, whiteflies and scales, which may help explain its lower toxicity to A. mali.
The fungicides sulfur and Zinc/Manganese + copper hydroxide killed respectively 10 and
3.12% of the parasitoids at 1.0x rate (Table 2). In fact, most of the fungicides tested to date against A. mali have shown no negative effect (Bradley et al.1997, Heunis and pringle 2003,
Rawat et al. 1988). However, unlike in my study; sulfur was documented by Cohen et al. (1996) to be moderately toxic to A. mali. This discrepancy between our results regarding sulfur may be due to the use of different A. mali strains, and/or the fact that he used sprayed leaf discs which can increase the contact period between the fungicide and parasitoid. Nonetheless, in order to assure the selectivity of these insecticides and fungicides that did not show high acute toxicity, a sub lethal study should be conducted. For example, novaluron had a small impact on A. mali in
128
the acute mortality test (Table 2), however, this insecticide has been observed elsewhere to reduce the emergence rates of Trichogramma parasitoids (Bastos et al. 2006).
In general, considering only the acute toxicity, it appears that in fact the fungicides and more recently developed insecticides such as chlorantroniprole, cyantraniprole, novaluron and spirotetramat have a smaller impact on A. mali. However, the sublethal effects of these pesticides should be investigated in order to assure their safety to A. mali. And those insecticides that showed high acute toxicity should be avoided in situations where biological control of woolly apple aphid is being implemented.
Chronic toxicity. Cyantraniliprole, spinetoram and lambda-cyhalothrin were the only pesticides tested that showed significant negative sublethal effect on A. mali population growth (Table 4 and Fig. 2). All the remaining insecticides and fungicides tested did not show significant difference from the control treatments (Table 4 and Fig. 2). Cyantraniliprole caused a reduction of more than 50% in the A. mali fecundity, which ultimately led to a low net reproductive rate
(R○) and rate of increase (r), thereby affecting population growth (Table 4). Cyantraniliprole has been also observed to have detrimental effects on the parasitoid of walnuts Trioxys pallidus
(Halliday) in California (Mills unpublished data). Cyantraniliprole and chlorantraniliprole belong to the class of diamide insecticides, and have similar mode of action. While both showed similir acute toxicity to A. mali, chlorantraniliprole did not have significant sublethal impact.
Teixeira et al. (2009) tested the sublethal effects of chlorantraniliprole on rhagoletis fruit fly and found that although egg oviposition was delayed in treated flies, there were not significant sublethal effects on either number of eggs laid or hatched. The sublethal effect of spinetoram was mainly due to acute mortality of adult parasitoids, whereas for lambda-cyhalothrin was due to mortality of adult parasitoids and woolly apple aphid. The mortality of woolly apple aphid was
129
used as an estimate of parasitoid egg/larva mortality. The remaining of the insecticides and fungicides tested showed to be relatively safe to A. mali, and therefore compatible with biological control and IPM of woolly apple aphid.
The sex ratio for the F1 offspring was slightly female biased (≈ 60%) on all bioassays.
This outcome is in accordance with a study done by Mueller et al. (1992). The fecundity observed was also in accordance with Lundie‟s studies (Lundie 1924). Nonetheless, a potential problem when measuring fecundity in a closed system (small cages), is that A. mali tend to overparasitize some aphids which can lead to death and consequently lower fecundity estimate
(Lundie 1924).
In conclusion, the fungicides and some of the newer insecticides showed to be relatively safe to A. mali, and could therefore be an important addition to biological control and IPM programs of woolly apple aphid. However, most of the more conventional insecticides
(organophosphates, neonicotinoids and spinosad) showed to be highly toxic to A. mali and should be avoided in places where woolly apple aphid outbreaks are a major problem.
Furthermore, in order to assure a wider basis for the knowledge of natural enemy conservation, the toxicity of all these pesticides should be also tested for other important natural enemies of woolly apple aphid.
130
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Table 1. Pesticide rates used in the bioassays (maximum label rate: 1.0x)
Per acre rate Pesticides A.I. name % A.I. Formula g fm/100gal g fm/ L g A.I./100gal ppm A.I./ L max label (oz) Delegate Spinetoram* 25% WG 7 198.45 0.52 49.61 131.07 Altacor Chlorantraniliprole* 35% WDG 4.5 127.57 0.33 44.65 117.97 Kocide Copper hydroxide 53.8% DF 64 1814.37 4.79 976.13 2578.95 Manzate Zinc + Manganese 75% DG 28.8 816.47 2.15 612.35 1617.84 Kumulus Sulfur 80% DF 320 9071.87 23.96 7257.50 19174.37 Entrust Spinosad 80% W 3 85.05 0.22 68.04 179.76 Guthion Azinphos-Methyl 50% WP 48 1360.78 3.59 680.39 1797.60 Assail Acetamiprid 70% WP 3.4 96.39 0.25 67.47 178.26 Per acre rate ml lb A.I. /gal Formula ml fm/ L lb A.I./100gal ppm A.I./ L max label (fl oz) fm/100gal Cyazypyr Cyantraniliprole* 0.83 lbs A.I./gal SE 20.5 606.02 1.60 0.13 160.17
136 Warrior II Lambda-cyhalothrin* 2.08 lbs A.I./gal CS 2.56 75.68 0.20 0.041 49.85 Rimon Novaluron* 0.83 lbs A.I./gal EC 50 1478.10 3.90 0.32 388.54
Sevin Carbaryl 4 lbs A.I./gal F 96 2837.95 7.50 3.00 3595.19 Ultor Spirotetramat* 1.25 lbs A.I./gal L 14 413.87 1.09 0.13 163.84 Lorsban Chlorpyrifos 4 lbs A.I./gal EC 64 189.17 4.99 2.00 2396.79 Calypso Thiacloprid 4 lbs A.I./gal F 8 236.50 0.62 0.25 299.60 * Newer pesticides (recently released in the market)
0.1x rate concentration was obtained by mixing 10 ml of 1.0x rate solution with 90 ml of water.
A.I. = active ingredient, oz =ounces, fl oz =fluid ounces, g = grams, ml = milliliters, fm = formula, lb = pound, gal = gallon, ppm = parts per million.
Table 2: Mortality of Aphelinus mali at 24 and 48 hours after pesticide treatment.
Pesticide time after spray control% 0.1xrate % 1.0xrate% χ2 d.f. Sig. p-value (hrs) (n) (n) (n) 24 6.66 a 90.00 b 100.00 b 81.71 2 * <.0001 Delegate 48 6.60 a 100.00 b 100.00 b 96.90 2 * <.0001 (Spinetoram) No. of parasitoids N = 30 N = 30 N = 30
24 6.6 a 10.00 a 20.00 a 2.61 2 n.s. = 0.270 Altacor 48 13.33 a 33.33ab 43.33 b 6.66 2 * = 0.028 (Chlorantraniliprole) No. of parasitoids N = 30 N = 30 N = 30
24 6.66a 13.33ab 33.33b 7.79 2 * <0.020 Warrior II 48 13.33a 40.00b 60.00b 15.01 2 * 0.0005 (Lambda-cyhalothrin) No. of parasitoids N = 30 N = 30 N = 30 137
24 0.00a 13.33b 10.00b 6.13 2 n.s. = 0.046 Rimon 48 6.66a 23.33ab 33.33b 7.29 2 n.s. = 0.026 (Novaluron) No. of parasitoids N = 30 N = 30 N = 30
24 0.00a 6.66a 6.66a 3.34 2 n.s. = 0.188 Cyazypyr 48 0.00a 30.00b 40.00b 27.51 2 * = <.0001 (Cyantraniliprole) No. of parasitoids N = 30 N = 30 N = 30
24 0.00a 0.00a 6.66a 4.44 2 n.s. = 0.108 Kumulus 48 0.00a 0.00a 10.00b 6.73 2 * = 0.034 (Sulfur) No. of parasitoids N = 30 N = 29 N = 30
Manzate + Kocide 24 0.00a 0.00a 3.12a 2.18 2 n.s. = 0.336 (Zinc/Manganese + 48 3.12a 6.66a 3.12a 0.59 2 n.s. = 0.745 copper hydroxide) No. of parasitoids N = 32 N = 30 N = 32 * - treatments differ significantly at P<0.05 within each date (Pearson Chi-Square)
n.s - there is not significant difference among treatments at P<0.05(Pearson Chi-Square)
Means followed by different letters within each date differ significantly at p<0.05 (pair wise comparison using Likelihood ratio tests).
Table 3: Mortality of Aphelinus mali at 24 and 48 hours after pesticide treatment (continuation)
Pesticide time after spray control% 0.1xrate % 1.0xrate% χ2 d.f. Sig. p-value (hrs) (n) (n) 24 0.00a 100.00b 96.66b 107.14 2 * <.0001 Entrust 48 6.66a 100.00b 100.00b 96.90 2 * <.0001 (Spinosad) No. of parasitoids N = 30 N = 30 N = 30
24 0.00a 76.66b 100.00c 89.31 2 * <.0001 Guthion 48 6.66a 76.66b 100.00c 72.99 2 * <.0001 (Azinphos-Methyl) No. of parasitoids N = 30 N = 30 N = 30
24 0.00a 96.66b 100.00b 107.14 2 * <.0001 Sevin 48 6.66a 96.66b 100.00b 89.67 2 * <.0001 (Carbaryl) No. of parasitoids N = 30 N = 30 N = 30
138 24 0.00a 0.00a 3.33a 2.17 2 n.s. = 0.337 Ultor 48 6.66a 7.14a 10.00a 0.26 2 n.s. = 0.879 (Spirotetramat) No. of parasitoids N = 30 N = 28 N = 30
24 0.00a 93.33b 100.00b 102.45 2 * = <.0001 Lorsban 48 0.00a 96.66b 100.00b 107.14 2 * = <.0001 (Chlorpyrifos) No. of parasitoids N = 30 N = 30 N = 30
24 0.00a 6.66a 46.66b 28.09 2 * = <.0001 Assail 48 3.33a 16.66a 83.33b 53.07 2 * = <.0001 (Acetamiprid) No. of parasitoids N = 30 N = 30 N = 30
24 0.00a 40.00b 85.70c 54.66 2 * = <.0001 Calypso 48 0.00a 43.33b 92.85c 64.21 2 * = <.0001 (Thiacloprid) No. of parasitoids N = 30 N = 30 N = 30 * - treatments differ significantly at p<0.05 within each date (Pearson Chi-Square)
n.s - there is not significant difference among treatments at p<0.05 (Pearson Chi-Square)
Means followed by different letters within each date differ significantly at p<0.05 (pair wise comparison using Likelihood ratio tests).
Table 4. Population growth parameters measured for Aphelinus mali during sublethal bioassays.
Daily Proportion females Rate of Pop. Growth Treatment Rate N R fecundity F1 0 increase (days) Control water 27 3.32 ± 0.35 0.50 ± 0.05 1.93 0.13 70.84 Rimon (Novaluron) 1x 27 3.12 ± 0.53 0.65 ± 0.05 1.57 0.10 92.10 Control water 29 2.97 ± 0.14 0.70 ± 0.02 2.47 0.17 54.17 Kumulus (Sulfur) 1x 25 2.71 ± 0.18 0.69 ± 0.02 2.96 0.18 51.16 Control water 26 3.02 ± 0.14 0.63 ± 0.03 2.36 0.16 54.18 Kocide + Manzate (Zn+Mn+ Cu OH) 1x 27 2.94 ± 0.19 0.65 ± 0.02 3.18 0.18 51.16 Control water 29 2.91 ± 0.23 0.65 ± 0.02 1.97 0.13 70.84 Cyazapyr (Cyantraniliprole) 1x 30 1.15 ± 0.12 0.60 ± 0.06 1.20 0.05 184.20 Control water 30 2.94 ± 0.23 0.64 ± 0.02 2.13 0.14 65.78
139 Altacor (Chlorantraniliprole) 1x 30 3.00 ± 0.38 0.69 ± 0.02 1.65 0.12 76.75 Control water 29 2.91 ± 0.23 0.65 ± 0.02 1.97 0.13 70.84 Delegate (Spinetoram)* 1x 30 - - 1.03 0.015 614.02 Control water 29 2.91 ± 0.23 0.65 ± 0.02 1.97 0.13 70.84 Warrior II (Lambda-cyhalothrin)* 1x 30 - - 1.11 0.017 541.78 *Matrix model elaborated based on Cyantraniliprole control matrix.
R0 = Net Reproductive Rate.
Figure legends
Fig. 1. Example of experiment set up for the sublethal bioassays: Young potted tree with woolly apple aphid colonies at the top exposed to a female A. mali enclosed by a glass cylinder cage.
Fig. 2. Estimated population growth of A. mali based on stage-structured matrix model from
Poptools. ● = control, ○ = pesticide.
Organdy glued to the top Glass cylinder 45 x 60 mm Parasitoid and aphids were inside
Organdy
Fig. 1. Example of experiment set up for the sublethal bioassays: Young potted tree with woolly apple aphid colonies at the top exposed to a female A. mali enclosed by a glass cylinder cage.
140
7 8
6 Sulfur (Kumulus) Copper hydroxide + zinc + Manganeze (Kocide + Manzate) 6 5
A. mali 4 A. mali 4 3
2
Number of Number of 2
1
0 0
4 Novaluron (Rimon) 4 Cyantraniliprole (Cyazypyr)
3 3
A. mali
A. mali
2 2
Number of
Number of 1 1
0 0
4 4 Spinetoram (Delegate) Chlorantraniliprole (Altacor)
3 3
A. mali
A. mali
2 2
Number of
Number of 1 1
0 0 0 2 4 6 8 10 12 4 Lambda-cyhalothrin (Warrior II) Time
3
A. mali
2
Number of 1
0 0 2 4 6 8 10 12 Time Fig. 2. Estimated population growth of A. mali based on stage-structured matrix model from Poptools. ● = control, ○ = pesticide.
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APPENDICES
Appendix 4.1: Does Aphelinus mali go through the organdy mesh used in the exclusion cage experiments?
One of the main concerns regarding the exclusion cage experiment is the possibility of the mesh size used in treatment 2 to preclude the parasitoids from reaching the aphid colonies inside the cages. Therefore, an independent laboratory study was conducted to assess whether or not A. mali is able to reach aphid colonies enclosed by the different organdy mesh used in treatment 2 in 2010 and 2011. The experiment was conducted at ambient temperature (23 ± 10
C), R.H. of 40 ± 5%, and under fluorescent lights set at a photoperiod of 16:8 L:D. The experimental unit was constituted of a plexi-glass cylinder cage (7 x 20 cm) having both ends covered by fine organdy fabric and containing inside two apple twigs infested with woolly apple aphids. Half bottom of the twigs were individually immersed inside a plastic bottle (2.5 x 6.5 cm) with water and sealed with parafilm to avoid water evaporation and parasitoid falling into it.
One of the twigs inside each cage was enclosed by organdy sleeve cage (2.5 x 10 cm) tied to the bottle with a rubber band. There were five-seven replicates for each treatment (mesh size). The infested twigs that were assigned to be enclosed by the sleeve were first sprayed with a solution of egg whites diluted to 20% in water. Two hours later the twigs were enclosed by the sleeves and six-eight 24-hour old female parasitoids were released inside each cage and allowed to stay for 24 hours. After that, each cage was placed in the freezer for ten minutes and the parasitoids were individually transferred to microvials with the aid of a tooth pick (one for each parasitoid to avoid contamination). Clean parasitoids were used as controls. All parasitoids were subjected to indirect ELISA (see chapter 3) to test for the egg protein, and thus; calculate the percentage that visited the aphid colonies enclosed by the organdy sleeve.
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Females of A. mali were able to reach aphid colonies enclosed by sleeves with both mesh sizes. Approximately 71% of parasitoids visited aphid colonies enclosed by sleeve with the mesh size of 24 x 20/ inch while 81% visited colonies enclosed by sleeves with mesh size of 18 x 13/ inch (Fig. 4.4.1).
100
80
(%)
60
A. mali A. mali
40
Percentage of 20
0 24 x 20/ inch (2010) 18 x 13/ inch (2011)
Fig. 4.1.1. Percentage of female Aphelinus mali that visited the aphid colonies enclosed by organdy sleeves in a 24-hour period.
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Appendix 4.2: Estimation of total tree leaf surface area
Total tree leaf surface area was estimated at the begging and at the end of each exclusion cage experiment by averaging the surface area of 10 leaves/ tree and multiplying it by the total number of leaves/tree. To do this estimation, I measured the midribs of 25 leaves, and then determined the surface area. Leaf surface area was determined by scanning the leaves with a flatbed scanner (HP Photosmart C5508), and calculating the leaf area with imaging software
(Scion Imaging, Scion Corporation, Frederick, Maryland, USA). Midrib length was regressed against surface area, and first order and second order polynomial models were tested. The best
nd 2 fit of the data was by a 2 order polynomial in the form of Y= 0.48 * X – 0.51 * X + 2.29 (Fig.
4.2.1).
50
R2 = 0.96
) P < 0.0001
2 40
30
20
One-side leaf surface area (cm 10
0 0 2 4 6 8 10 12 Leaf mid rib length (cm)
Fig. 4.2.1. Regression between apple leaf surface area and leaf mid rib length. Regression
2 equation: Y= 0.48 * X – 0.51 * X + 2.29.
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Appendix 4.3: Estimation of woolly apple aphid numbers based on colony size
Because of the difficulty and feasibility in counting the precise number of aphids in large colonies, a regression between the surface area covered by the aphid colony and aphid numbers was carried out in order to estimate aphid density on the trees. This approach should work well for woolly apple aphid because the aphids are tightly settled in the colony leaving little or no space between individuals. To obtain the regression equation the surface area of 14 colonies with
200-1500 aphids were measured, and then regressed against the aphid numbers. The best fit of
st the data was by a 1 order equation in the form of Y = 110.27 * X + 276.11 (Fig. 4.3.1). In the cage experiment, colonies were individually measured and the dimensions used to calculate the surface area, which in turn was plugged into the regression formula (Fig. 4.3.1) to estimate the aphid numbers. Surface area measurement was based on two shapes of aphid colonies. The first assumes that the aphid colonies have a more or less quadrilateral shape, either when imaginarily cutting open a fully infested branch (cylinder shape) or when having just an infested lateral side of the branch. Second measurement was based on colonies that have a circumference shape. The colonies were measured with the aid of a ruler and vernier scale.
145
1800
1600 R = 0.89 P< 0.0001
1400
1200
1000
800
No. of WAA 600
400
200
0 0 2 4 6 8 10 12 14
2 Surface area (cm )covered by WAA colony Fig. 4.3.1. Regression between surface area covered by woolly apple aphid colony and aphid numbers. Regression equation: Y = 110.27 * X + 276.11
146
Appendix 6.1: Toxicity of orchard pesticides to woolly apple aphid.
I tested the toxicity of orchard pestices to woolly apple aphid to make sure that aphid mortality woul not confound the sublethal effects of pesticides on Aphelinus mali. Each experimental unit was comprised of a plexi-glass cylinder cage (7 x 20 cm) having both ends covered by organza fabric, and containing inside a 17cm apple twig infested with woolly apple aphid. Twigs infested with woolly apple aphid were collected from either WSU orchards or from our greenhouse colonies. The twigs were inspected for parasitized aphids, and thereafter the numbers of woolly appe aphids were counted. Adult aphids were removed to prevent reproduction, and thus, aphid numbers increasing over time. Each infested twig was individually immersed inside a glass bottle (4 x 8 cm) with water and sealed with parafilm to avoid water evaporation and aphids falling into it. The treatments were: 1) control (distilled water), 2) 1.0x = full field pesticide rate, and 3) 0.1x = 10% of full field pesticide rate. There were 4-5 replicates for each treatment. Each infested twig was individually sprayed with the respective treatment using a180 ml-aerosol spray bottle (Nalgene inc., Rochester, NY). After that the replicates were placed on a lab bench at ambient temperature (24 ± 10 C), R.H. of 40 ± 5%, and under fluorescent lights set at a photoperiod of 16:8 L:D. The aphid mortality was evaluated 48 hours after the spray. Lambda-cyhalothrin (Warrior II) was the most toxic insecticide to woolly apple aphids killing approximately 40% and 60% at the diluted and full rate respectively (Fig. 6.1.1). Thus, this insecticide could negatively affect reproduction of A. mali by killing its host. All the other insecticides tested killed 10% or less at both rates (Fig. 6.1.1)
147
100 100
A B Chlorantraniliprole 80 80 Lambda-cyhalothrin Cyantraniliprole Novaluron Copper hydroxide + (Zinc + Manganese) 60 60 Sulfur
40 40
20 20
Mortality of WAA (%) + SE Mortality of WAA (%) + SE
0 0 Control 0.1x rate 1.0x rate Control 0.1x rate 1.0x rate
Fig. 6.1.1. Percentage mortality of woolly apple aphid at 48 hours after pesticide treatment. A: experiment 1 conducted in 2008, B: experiment 2 conducted in 2011.
148