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Effect of Diaprepes Root Weevil on Leaf Gas Exchange and Growth of Select Ornamental Tree Species


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EFFECT OF DIAPREPES ROOT WEEV IL ON LEAF GAS EXCHANGE AND GROWTH OF SELECT ORNAMENTAL TREE SPECIES By ALEXANDER P. DIAZ A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2005

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Copyright 2005 by Alexander P. Diaz

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ACKNOWLEDGMENTS I would like to thank Catharine Mannion and Bruce Schaffer for granting me this opportunity to pursue graduate studies and providing funding for this research project. I would also like to thank them along with Susan Webb for their guidance and support throughout this experience. I would like to thank all those who helped me in many different ways while working on my research, especially Holly Glenn, Julio Almanza, Karen Griffin, Mike Gutierrez and Dalia Stubblefield. Finally, I would like to thank my family for their continual encouragement and support. iii

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TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................iii LIST OF TABLES .............................................................................................................vi LIST OF FIGURES ..........................................................................................................vii ABSTRACT .......................................................................................................................ix CHAPTER 1 LITERATURE REVIEW.............................................................................................1 Introduction...................................................................................................................1 Weevils..................................................................................................................2 Root Weevils.........................................................................................................3 Citrus Root Weevils..............................................................................................5 Diaprepes Root Weevil..........................................................................................7 Damage..................................................................................................................9 Arthropod feeding and whole-plant physiology...........................................11 Flooding stress and plant physiology...........................................................16 The ornamental plant industry......................................................................18 Current and potential impact of Diaprepes root weevil on the ornamental plant industry............................................................................................19 Buttonwood, Live Oak and Pygmy Date Palm...................................................20 Research Objectives....................................................................................................22 2 EFFECT OF LARVAL ROOT FEEDING BY DIAPREPES ROOT WEEVIL ON LEAF GAS EXCHANGE AND GROWTH OF SELECT ORNAMENTAL TREE SPECIES.....................................................................................................................23 Introduction.................................................................................................................23 Materials and Methods...............................................................................................25 Plant and Insect Material.....................................................................................26 Experiment 1................................................................................................26 Experiment 2................................................................................................26 Treatments...........................................................................................................27 Experiment 1................................................................................................27 Experiment 2................................................................................................28 iv

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Temperature Measurements................................................................................29 Statistical Analysis..............................................................................................30 Results.........................................................................................................................30 Experiment 1.......................................................................................................30 Experiment 2.......................................................................................................32 Discussion...................................................................................................................35 3 EFFECT OF FLOODING AND DIAPREPES ROOT WEEVIL LARVAL FEEDING ON LEAF GAS EXCHANGE AND GROWTH OF BUTTONWOOD AND LIVE OAK........................................................................................................54 Introduction.................................................................................................................54 Materials and Methods...............................................................................................56 Plant and Insect Material.....................................................................................56 Treatments...........................................................................................................56 Soil Redox Potential............................................................................................57 Leaf Gas Exchange..............................................................................................58 Plant Growth and Larval Recovery.....................................................................58 Statistical Analysis..............................................................................................58 Results.........................................................................................................................59 Soil Redox Potential (Eh)....................................................................................59 Leaf Gas Exchange..............................................................................................59 Plant Growth and Larval Recovery.....................................................................60 Discussion...................................................................................................................61 4 EFFECT OF ADULT DIAPREPES ROOT WEEVIL ON LEAF GAS EXCHANGE AND GROWTH OF BUTTONWOOD AND LIVE OAK.................72 Introduction.................................................................................................................72 Materials and Methods...............................................................................................74 Plant and Insect Material.....................................................................................74 Leaf Gas Exchange..............................................................................................75 Plant biomass.......................................................................................................76 Statistical Analysis..............................................................................................76 Results.........................................................................................................................76 Visible Signs of Herbivory..................................................................................76 Leaf Gas Exchange..............................................................................................77 Plant Biomass......................................................................................................77 Discussion...................................................................................................................78 5 SUMMARY AND CONCLUSIONS.........................................................................86 LIST OF REFERENCES...................................................................................................89 BIOGRAPHICAL SKETCH.............................................................................................97 v

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LIST OF TABLES Table page 2-1 The effect of Diaprepes root weevil larvae on height and trunk diameter of buttonwood and live oak trees..................................................................................51 2-2 The effect of Diaprepes root weevil larvae on buttonwood leaf, stem and root fresh and dry weights at each harvest date...............................................................52 2-3 The effect of Diaprepes root weevil larvae on leaf stem and root fresh and dry weights of live oak and pygmy date palm................................................................53 2-4 Number of Diaprepes root weevil larvae recovered from plants harvested 2, 3, 4 & 5 months after infestation.....................................................................................53 3-1 The effect of flooding (FLD) and insect infestation (INFST) on leaf gas exchange of buttonwood and live oak trees.............................................................70 3-2 The effect of Diaprepes root weevil larvae on leaf, stem, root and total biomass of infested or non-infested buttonwood and live oak trees......................................71 4-1 The effect of adult Diaprepes root weevil on net CO 2 assimilation (A), transpiration (E) and stomatal conductance (g s ) of buttonwood (Expt. 1)...............82 4-2 The effect of adult Diaprepes root weevil on net CO 2 assimilation (A), transpiration (E) and stomatal conductance (g s ) of buttonwood (Expt. 2)...............83 4-3 The effect of adult Diaprepes root weevil on total leaf area of buttonwod..............84 4-4 The effect of adult Diaprepes root weevil leaf feeding on buttonwood fresh and dry weights 2 months after infestation.....................................................................85 vi

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LIST OF FIGURES Figure page 2-1 Mean daily and monthly temperature (A) Air (B) Soil in containers......................40 2-2 Net CO 2 assimilation (A), transpiration (E) and stomatal conductance (g s ) of buttonwood plants infested with medium or large Diaprepes root weevil larvae or non-infested control plants...................................................................................41 2-3 Net CO 2 assimilation (A), transpiration (E) and stomatal conductance (g s ) of live oak plants infested with medium or large Diaprepes root weevil larvae or non-infested control plants (C)........................................................................................42 2-4 The effect of medium or large Diaprepes root weevil larvae on mean root fresh and dry weights of buttonwood trees.......................................................................43 2-5 The effect of medium or large Diaprepes root weevil larvae on mean root fresh and dry weights of live oak trees..............................................................................44 2-6 Net CO 2 assimilation (A), transpiration (E) and stomatal conductance (g s ) measured prior to harvest of buttonwood plants infested with Diaprepes root weevil larvae or not infested....................................................................................45 2-7 Net CO 2 assimilation (A), transpiration (E) and stomatal conductance (g s ) of the buttonwood plants infested monthly with 20 larvae and a total of 80 Diaprepes root weevil larvae by month 4 or not infested..........................................................46 2-8 Net CO 2 assimilation (A), transpiration (E) and stomatal conductance (g s ) measured prior to harvest of live oak plants infested with Diaprepes root weevil larvae or not infested................................................................................................47 2-9 Net CO 2 assimilation (A), transpiration (E) and stomatal conductance (g s ) of live oaks plants infested monthly with 20 larvae and a total of 80 Diaprepes root weevil larvae by month 4 or not infested.................................................................48 2-10 Net CO2 assimilation (A), transpiration (E) and stomatal conductance (g s ) measured prior to harvest of pygmy date palm infested with Diaprepes root weevil larvae or not infested....................................................................................49 vii

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2-11 Net CO 2 assimilation (A), transpiration (E) and stomatal conductance (g s ) of the final harvest set of pygmy date palms infested monthly with 20 larvae and a total of 80 Diaprepes root weevil larvae by month 4 or not infested...............................50 3-1 Soil redox potential (Eh) of flooded buttonwood (A) and live oak (B) in containers.................................................................................................................64 3-2 Net CO 2 assimilation (A), transpiration (E) and stomatal conductance (g s ) of flooded and non-flooded buttonwood trees..............................................................65 3-3 Net CO 2 assimilation (A), transpiration (E) and stomatal conductance (g s ) of flooded and non-flooded live oak trees....................................................................66 3-4 Net CO 2 assimilation (A), transpiration (E) and stomatal conductance (g s ) of buttonwood trees infested with Diaprepes root weevil larvae or non-infested........67 3-5 Net CO 2 assimilation (A), transpiration (E) and stomatal conductance (g s ) of live oak trees infested or non-infested with Diaprepes root weevils...............................68 3-6 Larvae recovered from pre-flooded or non-flooded treatments of buttonwood and live oak..............................................................................................................69 viii

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Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science EFFECTS OF DIAPREPES ROOT WEEVIL ON LEAF GAS EXCHANGE AND GROWTH OF SELECT ORNAMENTAL TREE SPECIES By Alex P. Diaz August 2005 Chair: Catharine Mannion Cochair: Bruce Schaffer Major Department: Entomology and Nematology The Diaprepes root weevil, Diaprepes abbreviatus L. (Coleoptera: Curculionidae), is a serious and economically important pest of citrus and many ornamental plants grown throughout the state of Florida. Studies were conducted to evaluate the effects of root feeding by Diaprepes root weevil larvae on leaf gas exchange and growth of three ornamental tree species commonly grown in south Florida that are known hosts of this weevil. Buttonwood (Conocarpus erectus), live oak (Quercus virginiana) and pygmy date palm (Phoenix roebelenii) in containers were infested with Diaprepes root weevil larvae and leaf gas exchange was measured monthly to determine the effects of larval root feeding on net CO 2 assimilation (A), transpiration (E), stomatal conductance (g s ). Leaf, stem and root fresh and dry weights of each species were also determined. In one of two tests, larval root feeding significantly reduced A, E and g s of infested buttonwood trees. Leaf gas exchange of live oak was not affected by larval infestation. The effects of ix

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multiple infestations of larvae on leaf gas exchange and fresh and dry weights of buttonwood, live oak and pygmy date palm were also tested. Net CO 2 assimilation, E and g s and dry weights of buttonwood were reduced as a result of larval root feeding, whereas there was no effect of multiple larval infestation on leaf gas exchange of live oak or pygmy date palm. There was no effect of multiple larval infestations on dry weights of live oak, but leaf, stem and dry root weight of pygmy date palm was lower for infested plants than non-infested plants. The interaction between pre-flooding (prior to larval infestation) and larval infestation on leaf gas exchange and growth of buttonwood and live oak was also tested. Net CO 2 assimilation, E and g s of buttonwood and live oak were reduced by flooding, but there was no significant interaction between pre-flooding treatment and larval infestation treatment on these variables. Also, after two and three months of infestation there was no significant difference in A, E and g s between infested and non-infested buttonwood or live oak trees. In that study, fresh and dry root weights of buttonwood but not live oak were reduced as a result of larval infestation. The effect of adult Diaprepes root weevil infestation on leaf gas exchange and growth of buttonwood and live oak was tested. Generally, there was no significant effect of adult weevil infestation on A, E or g s Leaf area of buttonwood was less for infested plants than non-infested plants. Weevils fed on both mature and young leaves of buttonwood. However, live oak did not produce any new leaf flushes during the treatment period and adult weevils did not feed on the mature leaves of live oak. It appears from our results that buttonwood is a more suitable host for development of Diaprepes root weevil larvae. x

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CHAPTER 1 LITERATURE REVIEW Introduction The Diaprepes root weevil, Diaprepes abbreviatus L. (Coleoptera: Curculionidae), is a severe pest of citrus, ornamental plants and root crops in Florida. It was first reported in Florida in 1964 from a nursery in Apopka where it was believed to have been introduced in an ornamental plant shipment from Puerto Rico (Woodruff 1968). In Puerto Rico and the Caribbean, where this species is believed to have originated, the weevil has been a major pest of sugarcane since the early 1900s and more recently has become a pest of citrus, sweet potato and ornamentals (Lapointe 2000). Since its introduction into Florida, Diaprepes root weevil has spread throughout most agricultural areas of the state and is causing significant economic loss to Floridas agriculture. Diaprepes root weevil is a polyphagous species associated with about 270 plant species in Florida and is presently infesting approximately 140,000 acres in twenty-two counties (Simpson et al. 2000). The Florida citrus industry is significantly affected by this pest, which costs citrus growers about $72 million annually. Most of the published research with Diaprepes root weevil has focused on citrus and there is almost no information on the effects of this pest on ornamental plants. Ornamental plants are one of the largest agricultural commodity groups in Florida. Nationwide, Florida is ranked second in wholesale value of ornamental plants just after California, which leads in ornamental plant production (USDA Floriculture Crops Summary, 2003). Many of the economically important ornamental plant species grown 1

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2 in Florida are known hosts of Diaprepes root weevil (Mannion et al. 2003). Most of these plant species support all life stages of the weevil including the most damaging and difficult to detect larval stage. Many ornamental trees are able to withstand severe root damage from Diaprepes root weevil before visible symptoms (leaf yellowing, wilting and decline) are apparent (Knapp et al. 2001). There is a general lack of information about Diaprepes root weevil on ornamental crops. It is important to obtain physiological baseline data on the effects of Diaprepes root weevil on these plant species in order to identify the plant species that are most susceptible to predation by this insect and ultimately be able to manage and control this pest on these crops. Weevils The order Coleoptera contains many families of insects. The family Curculionidae (weevils) is one of the most diverse groups containing more than 60,000 described species worldwide (Anderson 2002). This family of beetles, like most others, can be found throughout North America, with most of the diversity in the southern United States. Most weevils feed on living plants; however a few species are saprophagous. Weevils can be found in different plant ecosystems including some species that are associated with freshwater habitats. Weevils are relatively easy to recognize due to their elongate rostrum and characteristic geniculate antennae. The rostrum is highly variable depending on species; long and narrow on some species and short on others. Mouth parts are located at the anterior of the rostrum, body shape and size are widely variable and these characteristics separate this family into subfamilies. The larvae of weevils are legless and have a semi-circular shape. The white, grub-like larvae generally feed on nuts, seeds, fruits, buds, stems or plant roots (Metcalf and Metcalf 1993). Adult and

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3 larval feeding habits can vary and this variation is useful for classification of weevils into separate subfamilies (Anderson 2002). Root Weevils Weevils with polyphagous adults and larvae fall into the subfamily Entiminae (Anderson 2002). Broad-nosed weevils is a common name for this subfamily and according to OBrian and Kovarik (2000) there are about 14,000 known species belonging to this group. They are distinguishable by their relatively short snout compared to other weevils. One recognizable and distinct characteristic of almost all weevils in the subfamily Entiminae is that eclosing adults have a pair of mandibles which is shed after emergence from the soil that leaves a scar at the point of attachment (Anderson 2002). Generally, larvae feed on roots and adults tend to feed on foliage. Most species within this subfamily are generalist feeders, although certain species feed only on specific plant species. Some species in the subfamily Entiminae are parthenogenic and have no known males. This subfamily is the most diverse in North America and contains 124 genera and 23 tribes (Anderson 2002). Root weevil species are relatively similar to each other in their biology and habits. Depending on the species, adults lay their eggs in the soil or on foliage of host plants. The eggs, which are laid on leaves, hatch, the larvae drop to the soil surface and begin moving into the soil to find roots. Most Entiminae larvae begin feeding on small fibrous roots and as they develop damage larger roots. For the most part, adults feed on newly emerging leaf flushes, and characteristically feed on margins of leaves leaving semi-circular notches. Adults of most root weevils emerge from the soil and begin feeding, mating and laying their eggs. Several species in this subfamily are unable to fly (Anderson 2002, Emenegger and Berry 1978).

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4 Many species of root weevils are economically important pests of ornamental, agricultural or forest plants. Root weevils that are economically important include sweet potato weevil Cylas formicarius elegantulus Summers, black vine weevil Otiorhynchus sulcatus F., strawberry root weevil Otiorhynchus ovatus L., and several species of citrus root weevils. The sweet potato weevil is a pest of sweet potato and the larval stage of this weevil causes injury to roots and tubers. These larvae have been known to cause 25 to 75% crop loss (Metcalf and Metcalf 1993). Sweet potato weevil is believed to have been introduced into the United States from Asia and is found in several states including South Carolina, Georgia, Florida, Alabama, Mississippi, Louisiana, Texas and Hawaii. Adult weevils feed on leaves and stems of plants. Females mate and then deposit their eggs in small holes on roots and tubers below ground. Larvae then hatch and feed within the stem or tubers. Larvae generally feed for two to three weeks and cause the sweet potato to develop a foul smell and a bitter taste. The strawberry root weevil is a pest of many crops in North America. Host plants include strawberry, a variety of small fruit crops and ornamental plants. In the Pacific Northwest, Otyrynchus spp. feeds on many agricultural crops such as peppermint, nursery evergreens, strawberries, cane fruits and several ornamental nursery crops (Umble and Fisher 2000). Female strawberry root weevils are parthenogenic and cannot fly (Emenegger and Berry 1978). Larvae move into soil after hatching and begin feeding on roots and underground portions of stems. Adult weevils feed on leaves, but like most root weevil species most damage is caused by root feeding larvae. The black vine weevil is a serious pest of ornamental and small fruit crops in temperate areas around the world (Moorehouse et al. 1992). This pest affects nursery plants and is believed to have originated from temperate areas of Europe. The

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5 spread of this weevil to new areas such as Australia and the United States is thought to have been through infested plant shipments. Infestations can cause serious damage and economic loss to growers. Similar to other root weevil species, most of the damage is caused by larvae feeding on roots. LaLone and Clark (1981) reported that as few as three larvae can kill Rhododendron spp. The life cycle of the black vine weevil is similar to other root weevils. Recently a new root weevil has entered Florida, Myllocerus undecimpustulatus Faust (Thomas 2005). This root weevil from Sri Lanka was first discovered in Broward County and is believed to be established in southeast Florida from the Homestead area north to Boca Raton. The adult weevil has a wide host range that includes citrus and many fruit and ornamental crops. Not much is known about this weevil. However, since it has become established in south Florida and has a wide host range, it has potential to spread and cause damage to many crops. Like most root weevils, larvae remain in the soil and can easily be spread through nursery shipments. Citrus Root Weevils Citrus root weevils comprise several genera that cause serious injury to citrus in several states including Texas and Florida. According to Woodruff (1985) there are about 11 genera of root weevils that are associated with citrus in Florida and the West Indies. Genera of weevils in this subgroup include Artipus, Cleistolophus, Compsus, Diaprepes, Epicaerus, Exophthalmus, Lachnopus, Litostylus, Pachnaeus, Pantomorus, and Tanymecus (Woodruff 1985). Within these genera about five species are pests of citrus in Florida. The Diaprepes root weevil Diaprepes abbreviatus L., Fuller rose beetle Pantomorus cervinus Boheman, citrus root weevil Pachnaeus litus Germar, blue-green

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6 weevil Pachnaeus opalus Oliviar, and the little leaf notcher Artipus floridanus Horn, have been observed on citrus (Knapp 1985). The Fuller rose beetle is a pest of Florida citrus and many ornamental crops. This root weevil is found in many parts of the world including many states in the U.S. In Florida, the Fuller rose beetle is mainly found north of Lake Okeechobee (Woodruff and Bullock 1979). Adult weevils feed on leaves of many host plants, although most damage is caused by the larvae which feed on roots. Two species of root weevils in the genera Pachneus are native to Florida. They are broadly known as citrus root weevils. Both species are blue-green in color but P. opalus, which is called the blue-green weevil, is found in the northern half of Florida, while P. litus is only found in the lower half of southern Florida. Both species are considered serious pests of citrus as larvae and have been responsible for girdling tap roots and killing citrus trees (Woodruff 1981). The little leaf notcher, is another root weevil pest of citrus that is believed to occur on over 150 different host plants (Woodruff 1982). This weevil has a similar life cycle to most root weevils in that the adults feed on leaves and larvae feed underground on roots. This weevil is common in the Bahamas and on the east coast of Florida, from the lower keys up to Daytona Beach. The little leaf notcher is believed to be of West Indian origin (Woodruff 1982). Diaprepes root weevil, is an economically important pest in Florida and in areas of the Caribbean including Puerto Rico were it causes damage to citrus and sugar cane. This is one of the larger root weevils and is known to cause severe damage to a large number of agriculturally important crops, most importantly citrus. Additionally, other plant species, including several tropical and sub-tropical fruit trees in southern

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7 Florida and several economically important ornamental crops in central and southern Florida are affected by this introduced pest. Diaprepes Root Weevil The Diaprepes root weevil was first collected in the West Indies by Carl Linnaeus who described it as Curculio abbreviatus in the tenth edition of Systema Naturae in 1758 (Lapointe 2000). The West Indies, primarily Puerto Rico, is believed to be the center of origin and diversity for this species. In the United States, Diaprepes root weevil was first reported in a nursery in Apoka, Florida in 1964, and was believed to have been introduced in an ornamental plant shipment from a nursery in Puerto Rico (Woodruff 1964). Since the time of its introduction into Florida this weevil has seriously affected citrus orchards and has spread throughout most of central and southern Florida. Diaprepes root weevil is a polyphagous species and is known to feed on a wide variety of plants throughout Florida. Reports show that Diaprepes root weevil has a host range of about 270 plant species (Simpson et al. 1996). In Florida alone it is estimated that this weevil causes about 70 million dollars in damage annually (Weissling et al. 2002). Adult females deposit their eggs in masses protected by leaves that are held together by a secretion released during oviposition. Diaprepes root weevil lay their eggs at night which are deposited in a single layer that usually forms an irregularly shaped cluster. Schroeder (1981) observed in Florida that females deposited eggs mainly during the dark hours between 6 PM and 6 AM. The eggs are oblong to oval in shape, uniformly white and about 1.2 mm long and 0.4 mm wide (Wolcott 1936). Woodruff (1968) reported that the number of eggs can vary between 30 and 264 per cluster, and that a single female may lay up to 5,000 eggs or more during her lifetime. Beavers (1982) reported a slightly larger maximum number of about 6,517 eggs per female. After eggs

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8 hatch, neonates fall to the ground and begin to enter the soil. Soil moisture is believed to affect the ability of neonates to enter the ground. Jones and Schroeder (1983) tested the effects of soil moisture and the ability of neonates to penetrate the soil surface at varying soil moisture contents. Their results showed that neonates did not enter soil that had an extremely low soil moisture content of 0%, and the optimum soil moisture for larvae to enter the soil was between 7.0 and 16.7 %. After entering the soil, neonates begin to feed on fibrous roots, and as they mature, move to larger roots (Knapp et al. 2000). Beavers and Selhime (1975) reported that the developmental period for larvae reared on potted citrus in Florida is highly variable. The number of instars before pupation may vary, but generally after about 11 instars, larvae pupate (Quintela et al. 1998). Adults emerge from the soil year round; however, the peak emergence period is from May through October. As the adults dig their way out of the soil they shed a pair of deciduous mandibles. Adult weevils emerge from the soil and move up the tree canopy to feed and mate. Oviposition generally begins 3-7 days after emergence from the soil (Wolcott 1936). The body length of adult weevils is highly variable (0.95 to 1.90 cm). The color of adult weevils varies between individuals; however black with orange, white or yellowish scales on the elytra is typical of this species. The average longevity reported for adults reared on an artificial diet was about 147 day for females and 135 days for males (Beavers 1982). The average life cycle of Diaprepes root weevil has been reported to last anywhere from less than one year to more than two years (Wolcott 1936). Beavers and Selhime (1976) reported that in Florida overlapping generations of weevils occur throughout the year.

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9 Damage Damage caused by adult weevils is primarily leaf notching. They tend to feed on new leaf flushes and occasionally on fruit. Experiments performed by Syvertsen and McCoy (1985) showed that leaf feeding by adult weevils on citrus caused a reduction in net photosynthesis due to the amount of leaf area consumed. Reduction in transpiration was also related to the percentage of leaf area removed. They reported a 20% reduction in water use efficiency, which was greater than the reduction in photosynthesis. Although leaf feeding may cause damage to citrus and ornamental plants, the most destructive effect of this weevil is caused by the larval stages feeding on roots (Knapp et al. 2001). Larvae tend to feed on all underground portions of the plant including roots and tubers. Neonates enter the soil and begin feeding on fibrous roots. As the larvae mature they feed on larger lateral roots. Diaprepes root weevil has a period of rapid growth starting about 3 to 4 months after eggs hatch, at which time weevils are most active and tend to cause serious injury (Beavers 1982). Rogers et al. (2000) reported that weight gain was greatest for the 6 th to 8 th instars and that larvae at this stage tended to require a large amount of nutrients. For this reason it is believed that weevil larvae at this stage feed on larger roots and cause the greatest amount of damage. The larvae cause damage by feeding their way through the outer bark and cambium layer (Futch and McCoy 1993). Large root weevil larvae have been known to girdle roots and cause tree death by continuous root feeding, which in many cases allows entry for root pathogens that can cause plant disease (McCoy et al. 2003). Open wounds resulting from root feeding allow easy entry for pathogens that would not ordinarily affect plant roots; this feeding has been associated with root diseases by fungal genera such as Fusarium and Phytophthora.

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10 Graham et al. (1997) reported that about 30,000 acres of citrus trees have been devastated by Diaprepes root weevil. Most of the root damage was associated with Phytopthora and the greatest impact was caused in fields of poorly drained, heavy soils in flatwood orchards. They also reported that larval root feeding lessened resistance of relatively resistant rootstocks of Swingle citrumelo. Information on the effects of Diaprepes root weevil on ornamental crops is limited. However, the available information shows that there is a wide variety of host plants. Several plants including dahoon holly (Ilex cassine L.), silver and green buttonwood (Conocarpus erectus L.), pigmy date palm (Phoenix roebelenii OBrien), and many other ornamentals have been observed to be hosts (Knapp et al. 2000, Simpson et al. 1996). In a recent field survey of several ornamental field nurseries in Miami-Dade County, Florida, Mannion et al. (2003) found that several ornamental plant species supported a large number of adult weevils and weevil egg masses. Species observed to have high percentages of egg masses included live oak (Quercus virginiana Mill.), silver buttonwood, and black olive (Bucida buseras L.). Plants that were observed to have the highest presence of adult weevils were dahoon holly, cocoplum (Chrysobalanus icaco L.), black olive, live oak, Bauhinia sp. and Cassia sp. Experiments performed by Schroeder et al. (1979) to test survival of larvae on selected native and ornamental plants in Florida showed that, out of 65 plants, 6 native and 9 non-native ornamental plant species supported larval development. Many plant species support the complete life cycle of Diaprepes root weevil. However, several plants are only hosts for adult weevils, whereas other plant species only support larval feeding. Several tropical fruit crops commonly grown in southern Florida have also been observed to be hosts of Diaprepes

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11 root weevil. Adult weevils have been observed feeding on papaya foliage and fruit (Knapp et al. 2000). Mannion et al. (2003) performed several tests on a variety of different plant species including green bean (Phaseolus vulgaris L.), silver buttonwood, lime (Citrus aurantifolia C.), malanga (Xanthosoma sagittifolium L.), satinleaf (Chrysophillum olivirorme L.), sorghum-sudan (Sorghum Sudanese Piper), cassava (Manihot esculenta L.), and dahoon holly to determine larval survival and the amount of root consumption. Their results showed that silver buttonwood had more larvae with the greatest weights than the other plant species tested. The results suggest that silver buttonwood may be a preferred host for complete development of Diaprepes root weevil from the egg stage to adult. It has been reported that many ornamental trees can support a large amount of larval injury before leaf yellowing and other symptoms occur (Knapp et al. 2000). However there are no studies on how much damage a species can tolerate and which species, if any, can tolerate continuous larval and adult feeding. There are many economically important ornamental species being grown in southern Florida that are affected by Diaprepes root weevil and there are no physiological baseline data that show which species are most susceptible to damage. Pygmy date palm, buttonwood and live oak are all grown in south Florida field nurseries and support all developmental stages of Diaprepes root weevil. Arthropod feeding and whole-plant physiology Herbivory by insects causes economic damage to many agricultural crops. Welter (1989) classified insect herbivores according to the type of feeding damage caused such as, defoliators, gall formers, epidermal feeders, phloem feeders, stem borers and root feeders. There have been many studies on the effects of arthropod herbivory on plant

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12 physiology. Some studies have found beneficial or no effects of insect feeding on plant physiology, while the majority of studies have found negative effects on physiological functions of plants. A basic understanding of the physiological responses of plants to herbivory may provide critical information needed to predict and prevent crop damage (Welter 1989). Measuring plant photosynthesis can provide information about the effects of insect feeding and plant stress prior to the presence of any visible symptoms (Welter 1989, Peterson et al. 1992, Peterson et al. 1998). Results from studies of arthropod herbivory on plant photosynthesis have been variable; some studies of leaf feeding arthropods indicate no significant effects on photosynthetic rates of plants that they feed upon. Peterson et al. (1996) found no significant effects on photosynthesis due to simulated or actual leaf consumption by the larvae of the cecropia moth, Hyalaphora cecropia (L.) on crabapple (Pyrus coronaria L.) and apple (Pyrus malus L.) leaves compared to controls with no feeding damage. Other reports, however, indicate a reduction in net photosynthesis as a result of arthropod herbivory. Li and Proctor (1984) reported simulating defoliation by cutting leaf midribs reduced net photosynthesis more than lateral vein and lamina cuts. Those experiments indicated that the section of the leaf that is fed upon may be critical in determining if net photosynthesis is affected by insect herbivory. Syvertsen and McCoy (1985) determined the effects of leaf feeding by the citrus root weevil, Artipus floridanus, on net CO 2 assimilation, transpiration, and water use efficiency of citrus leaves. Weevil feeding characteristically resulted in leaf notching, which only removed the outer layers of leaf lamina, and no damage to the mid-vein was

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13 observed. The percentage of leaf area removed significantly increased as the weevil population increased, and water loss increased proportionally more than net photosynthesis declined (Syvertsen and McCoy 1985). Results of that study showed that there was a reduction of about 20% in water use efficiency after about 40% of the leaf area was consumed, indicating that the loss of photosynthetic leaf surface area was relatively more important than the changes in net gas exchange of the remaining leaf tissue. Other types of insect herbivores include mesophyll feeders and gall formers. Mesophyll feeding insects damage mesophyll and palisade layers of leaves. Klingeman et al. (2000) studied the effects of azalea lace bug, Stephanitis pyrioides Scott, on whole-plant gas exchange of azalea, Rhododendron sp. L. The azalea lace bug, which damages leaves by feeding within the palisade parenchyma of leaf mesophyll cells caused a reduction in net photosynthesis in plants that had more than 13% of the plant canopy damaged. Gall forming insects have also been shown to alter normal leaf gas exchange (Rilling and Steffan 1978, and Andersen and Mizell 1987). One study showed that gall formation by phylloxera, Dactylosphaera vitifolii Shimer, caused up to a 97.5% reduction in net photosynthesis in gall tissues of grapes (Rilling and Steffan 1978). The net CO 2 assimilation rate, stomatal conductance and transpiration of pecan (Carya illinoensis Wangenh) leaves were all significantly reduced when trees were infested with phylloxera, Phylloxera notabilis Pergande, galls (Andersen and Mizell 1987). Citrus leaf miner, Phyllocnistis citrella Stainton, is another pest that causes damage to leaves and can reduce net photosynthesis. The citrus leaf miner does not actually remove large portions of leaf like other leaf feeding insects. The damage caused by

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14 larvae is mainly due to larval mining through tissues of the leaf surface. Schaffer et al. (1997) reported that the number of days mining and the number of larvae per leaf significantly affected the percentage of leaf area damaged. Net photosynthesis was lower in leaves with leaf miners compared to control plants with no leaf miners. Citrus rust mite, Phyllocoptruta oleivora Ashmead, is another arthropod pest of citrus in Florida that mainly damages fruit by feeding on the epidermal cells and causing discoloration known as sharkskin, russet, or bronzing (Allen 1978). Photosynthetic layers are not damaged on leaves and most of the damage is due to increased transpiration in fruit and increased fruit drop. Allen (1978) reported that water loss was significantly higher in infested fruit than in undamaged fruit of three commonly grown citrus cultivars. Other studies show decreases in net photosynthesis as a result of mite feeding on leaves (Andrews and La Pre 1979, Sances et al. 1981, Hare and Youngman 1987). Sances et al. (1981) found that feeding by large populations of the two spotted spider mite, Tetranychus urticae Koch, reduced transpiration and photosynthesis of strawberry (Fragaria ananassa Duch.). This reduction in photosynthesis was correlated to a reduction in fruit quality and quantity. Feeding damage by the avocado red mite, Oligonichus yothersi McGregor, on avocado (Persea americana Mill), caused a decrease in leaf net photosynthesis, stomatal conductance and transpiration within a few weeks after infestation (Schaffer et al. 1986). Mobley and Marini (1990) tested the effects of feeding of the European red mite, Panonychus ulmi Koch, and the two-spotted spider mite on gas exchange of apple and peach (Prunus persica L.) leaves. Results showed that net photosynthesis, transpiration, and water-use efficiency declined linearly as

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15 European red mite and two-spotted spider mite-days increased, for both apple and peach tree species. Similarly apple (Malus domestica Borkh), infested with European red mite, exhibited a decline in whole canopy net carbon exchange rate with increasing cumulative mite-days (Lakso et al. 1996). Phloem feeding insects such as aphids can be serious pests of many crops. There have been variable results on plant gas exchange due to phloem feeding insects. Feeding injury by three species of pecan aphids caused a 50% reduction in net photosynthesis of pecan seedlings and reduced dark respiration by 25% (Wood et al. 1985). This type of injury appears to be a result of phloem injury that contributed to the problems of irregular bearing and reduced productivity of pecan trees. Stem boring insects have also been reported to affect leaf gas exchange. The dogwood borer, Thamnosphecia scitula Harris, is an example of a pest that enters the stem of plants throught any open wound and begins feeding on phloem and cambial tissue layers. One experiment testing the effects of the dogwood borer on the physiology of dogwood (Cornus florida L.), showed that stomatal resistance increased and net photosynthesis decreased in infested trees compared to healthy, non-infested trees (Heichel and Turner 1973). Published information on the effects of root feeding on whole plant physiology is still limited. A study on the effects of western corn rootworm (Diabrotica virgifera virgifera LeConte) larval injury on net photosynthesis and vegetative growth of several varieties of maize indicated that rootworm infested plants had significantly lower midday leaf photosynthesis compared to controls (Urias-Lopez et al. 2000). Rootworm-infested

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16 plants showed a reduction in net photosynthesis at low and moderate levels of injury. Plant height reduction was also observed in infested plants (Urias-Lopez et al. 2000). Flooding stress and plant physiology Natural environmental occurrences such as flooding can cause plants to become stressed and may predispose them to insect herbivory and pathogens (Syvertsen et al. 1983, Ploetz and Schaffer 1989). The tolerance of plants to flooded conditions varies greatly among species and may even be highly variable within species depending on temperature, duration of flooding and plant age (Kozlowski 1997). Several other factors may affect plant tolerance to waterlogged soil conditions including soil type, soil porosity, soil chemistry, status of pathogens and soil microbes (Schaffer et al. 1992). Flooding results in hypoxia (low oxygen concentration) in soils and plants and if conditions persist, soils may become anoxic (lacking oxygen). Lack of oxygen in flooded soils alters plant growth and function mainly due to reduced O 2 exchange between the root and soil (Li et al. 2004, Dennis et al. 2000). Oxygen in soil pores is displaced by water and the remaining aerobic organisms use up any available oxygen and either become quiescent or die off and are rapidly replaced by anaerobes such as bacteria (Schaffer et al. 1992, Kozlowski 1997). Bacterial by-products, in addition to other chemical changes in flooded conditions, alter the oxidation reduction status of the soil. The soil redox potential is a quantitative measurement of the electrochemical status of the soil, and in well drained soils it is usually 300 mV (Schaffer et al. 1992, Kozlowski, 1997, Pezeshki et al. 1996). Soil redox potentials 200 mV are indicative of anaerobic conditions and may serve as a useful indicator of conditions not conducive to plant survival in flooded soils (Crane and Davies 1989, Pezeshki 1991).

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17 Photosynthesis decreases in many plants after they are flooded for several hours or days depending on species. Flood tolerance of plant species may vary depending on the developmental stage of plants and environmental factors such as temperature, soil or potting media (Crane and Davies 1989). In fruit trees, physiological responses to flooding include reductions in net CO 2 assimilation and stomatal conductance (Larson and Schaffer 1991, Andersen et al. 1984, Syvertsen et al. 1983). Stomatal closure is one of the earliest responses of many plant species to flooding. Leaf conductance of sour orange seedlings was significantly reduced four days after flooding (Syvertsen et al. 1983). In mango, stomatal conductance was reduced three days after trees in containers were flooded (Larson and Schaffer 1991). Reduced stomatal conductance in many species is believed to be correlated with a reduction in net CO 2 assimilation (Kozlowski 1997). Pezeshki et al. (1996) reported a significant reduction in net photosynthesis due to low redox potentials in flooded soils for three tree species, Taxodium distichum L., Quercus falcate Michx and Quercus lyrata Walt. Plants growing in stressful conditions are often more susceptible to insect herbivory than healthy plants. The susceptibility of plants to insect herbivory is often dependant upon the abiotic and biotic conditions in which they grow (Rand 1999). Evidence from agronomic and forest ecosystems suggest a relationship between environmental stress and insect damage to plants (Louda 1986). Louda (1986) tested the response of a native montane crucifer, Cardamine cordifolia Grey, to insect herbivory by three major feeding guilds: chewing, sucking, and mining insects in response to root-cutting and flooding stress. Results showed that insect herbivory was greater on both flood-stressed and root-pruned plants compared to non-stressed control plants. Interactions between flooding and

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18 Diaprepes root weevil feeding were examined on two varieties of citrus rootstocks. Plants that had been previously flooded and then infested with larvae had a higher percentage of larvae recovered from the soil and also a higher percentage of root damage compared to those plants that had not been previously flooded before infestation (Li et al. 2003b). It was also observed that stomatal conductance of non-infested, flooded plants decreased but then increased back to the same level as the non-flooded plants when plants were removed from flooded conditions. However, in plants that had been flooded and infested with larvae, stomatal conductance increased after plants were removed from flooded conditions, and then decreased again following larval infestation. This evidence suggests that flooding predisposes plants to Diaprepes root weevil feeding injury. The ornamental plant industry In the United States nursery and greenhouse crops represent the sixth largest agricultural commodity group and is the fastest growing major segment of U.S. agriculture (Johnson 1999). In 1998, the farm gate value of ornamental plants was estimated to be about $12.12 billion. In 1998, floriculture crops represented 3.93 billion dollars in sales. Woody ornamental trees and shrubs, including sod and unfinished plant products, represented $8.18 billion in sales or just about two-thirds of the industry value (Hodges and Haydu 2000). As of 2002, the wholesale value of floriculture crops increased by 2 percent from the revised 2001 valuation. The total wholesale crop value, summed for all growers with 10,000 dollars or more, was estimated at nearly $4.88 billion (USDA Floriculture Crops Summary 2003). This was an increase from $4.80 billion the previous year. The top five states nationwide in nursery plant sales are California, Florida, Michigan, Texas, and Ohio. These states account for $2.62 billion in

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19 sales, which equals up to 54 percent of the total value (USDA Floriculture Crops Summary, 2003). Nationwide, Florida is ranked second in wholesale value of ornamental plants just behind California, which leads in ornamental plant production with a crop value of nearly $962 million. Floridas wholesale crop value for 2002 was about $877 million. Larson et al. (2000) reported that the wholesale value of sales in 1997 of all floriculture crops in Florida, including cut flowers, house plants, potted flowering plants, bedding/garden plants and cut cultivated greens, accounted for 19.1 % of the total wholesale value of floriculture crops in the nation. The wholesale value of the Florida nursery and greenhouse industry was valued at $1.28 billion in 1998. Florida dominates the United States tropical foliage crops market with more than 85 percent of sales (Hodges and Haydu 2000). Florida has the largest production area of floricultural crops in the United States with about 18,000 acres. This is nearly as large as the combined growing areas of all states in the West including California (Floriculture and Nursery Crops USDA 2003). In 1997, southeastern regions of Florida including Miami-Dade, Broward and Palm Beach Counties, accounted for 53 percent of total foliage sales in Florida. The production value of foliage plants was worth $175.8 million and comprised on nearly 123.9 million square feet (Larson et al. 2000). Current and potential impact of Diaprepes root weevil on the ornamental plant industry Diaprepes root weevil is a serious pest of citrus and ornamental plants in Florida. In Florida, there are about 24 counties known to be infested with Diaprepes root weevil and economic losses in ornamental and citrus crops by this pest amount to more than 75 million dollars annually (Mannion et al. 2003, Lapointe et al. 1999). Diaprepes root

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20 weevil infests over 30,000 acres of citrus in Florida (Muraro 2001). Distribution of infested plant material is the most common way of spread and introduction of this weevil into new areas. Therefore, there is a regulatory process that restricts the movement of plants with the presence of any life stage of this insect (Simpson et al. 1996). In Florida there are nursery quarantines in place which regulate plant pests and require immediate action if pests are found on plant material. Any nursery stock found to be infested with Diaprepes root weevil is quarantined and is not eligible for certification until required treatments are carried out (Simpson et al. 2000). Ornamental field nurseries are also affected by Diaprepes root weevil in south Florida with unknown amounts of crop damage resulting. In 2000, it was reported that 179 nurseries from 11 counties in Florida were quarantined for the presence of Diaprepes root weevil. There are reports of Diaprepes root weevil being established in a citrus grove in the Rio Grande Valley of Texas. Diaprepes root weevils have also been intercepted several times in California from shipments of plants (Grafton-Cardwell et al. 2004). Several Integrated Pest Management options are being used mainly by citrus growers to control this pest. Foliar sprays for adult control and entomopathogenic nematodes and soil barrier treatments for control of larvae along with the use of fungicides are some options farmers have been using (Muraro 2001). Buttonwood, Live Oak and Pygmy Date Palm There are several species of ornamental trees that are known host of Diaprepes root weevil and are commonly grown together in field nurseries. Three species, buttonwood, live oak and pygmy date palm, are known to support both adult and larval feeding. The pigmy date palm is an ornamental palm native to Laos that is commonly grown in southern Florida. This palm grows from 1.83 m to 3.05 m tall and may reach a spread

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21 of up to 3.05 m wide (Gilman and Watson 1993a). Pygmy date palm is a dioecious, single-trunked palm, but is commonly grown together in groups of two or three per container. Optimal growing conditions limit its growing range in the United States to southern Florida or zones 10-11. Leaves of this palm are pinnately compound, up to 1.22 m long and consist of many leaflets. The flowers are unisexual and held on numerous short branches that appear white in color. Flower stalks are found on pygmy date palms year round. The fruit are often about 1.27 cm in diameter and dark red when ripe, they can be found ripening all year round in south Florida (Dehgan 1998). Buttonwood is a native tree of south Florida and is commonly used as a landscape tree mainly in coastal areas. This is a very hardy tree, originally found in coastal swamp areas, and can withstand high salinity. It reaches a height of about 12.19 m (Gilman and Watson 1993b). It is a monoecious evergreen tree that is hardy only in zones 10-11; cold temperature limits its distribution northward. Leaves are simple, alternate, and ovate to elliptic with entire margins and average about 10.16 cm in length. Flowers are found on terminal panicles that are green and cone-like; they are present throughout the year. Fruit appears cone-like, reddish brown, and disintegrates at maturity into small winged seeds. The silver and green buttonwood varieties are commonly grown in south Florida nurseries (Dehgan 1998). Live oak is native to the eastern United States and is a common landscape plant grown throughout Florida. It is probably the most commonly planted landscape plant in the southeastern U.S. (Dehgan 1998). Live oak is mainly used as a shade tree in many places and can grow 18.29 to 24.38 m in height and may reach a spread of up to 36.58 m with a slow to moderate rate. Leaves are simple, spirally arranged, and elliptical to ovate;

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22 they average about 3.81 to 12.70 cm in length and leaf margins are entire but can have 3 lobes. Fruit are nuts that are ovoid in shape and average 2.54 cm in length (Dehgan 1998). Research Objectives The first objective of this research was to determine the effects of Diaprepes root weevil feeding on physiology and growth of pygmy date palm, buttonwood and live oak, three important ornamental plants grown in south Florida. Net CO 2 assimilation, stomatal conductance, and transpiration were measured as physiological indicators of adult and larval insect damage to plants. The second objective was to test the effects of flooding on predisposing live oak and buttonwood to root feeding by Diaprepes root weevil. The third objective was to evaluate the effects of adult Diaprepes root weevil leaf feeding on net CO 2 assimilation and growth of buttonwood and live oak.

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CHAPTER 2 EFFECT OF LARVAL ROOT FEEDING BY DIAPREPES ROOT WEEVIL ON LEAF GAS EXCHANGE AND GROWTH OF SELECT ORNAMENTAL TREE SPECIES Introduction Diaprepes root weevil, Diaprepes abbreviatus L. (Coleoptera: Curculionidae), is a polyphagous weevil species native to the Caribbean. In 1964 it was first reported in the U.S. in a nursery in Apoka, Florida. The weevil is believed to have been introduced into the state in an ornamental plant shipment from a nursery in Puerto Rico (Woodruff 1964). Diaprepes root weevil has become a serious pest and is currently plaguing citrus orchards and many ornamental plant species throughout most of central and southern Florida. This weevil is known to be associated with more than 140,000 acres and is believed to be present in 24 counties throughout the state (Simpson et al. 2000, Mannion et al. 2003). In Florida it is estimated that this weevil causes over $70 million in damage annually (Weissling et al. 2002, Lapointe 2000). Larvae of Diaprepes root weevil cause extensive damage to the root systems of host plants. Larvae tend to feed on all underground portions of the plant including roots and tubers. Neonate larvae enter the soil, feed on fibrous roots, and then begin feeding on larger lateral roots as they mature. Weevil larvae have a period of rapid growth starting about 3 to 4 months after eggs hatch. This is the stage when larvae are most active and cause serious injury (Beavers 1982). Larval growth on select citrus varieties is greatest between the 6 th and 8 th instars when larvae have high nutrient requirements (Rogers et al. 2000). Larvae cause damage by feeding their way through the outer barkand cambium 23

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24 layer (Futch and McCoy 1993). Large root weevil larvae have been known to girdle roots and cause tree death by either continuous root feeding or by allowing entry for root diseases associated with certain root fungi that destroy crops (McCoy et al. 2003). Diaprepes root weevil has a host range of nearly 293 plant species that are known to support either adult or larval stages and in many cases both (Simpson et al. 2000). Several native and ornamental plant species in Florida are known to be hosts of Diaprepes root weevil, and both native and non-native ornamental plant species have been shown to support larval development (Schroeder et al. 1979). This weevil is currently injuring many ornamental nursery plants in southern Florida, but the amount of damage to nursery crops has not been quantified. In 2000 it was reported that 179 nurseries from 11 counties in Florida were quarantined for the presence of Diaprepes root weevil. In addition to ornamental plants, several tropical fruit crops commonly grown in southern Florida have been observed to be hosts of Diaprepes root weevil. It has been reported that many ornamental trees can sustain a large amount of larval feeding before leaf yellowing and other symptoms occur (Knapp et al. 2000). A basic understanding of the physiological responses of plants to herbivory may provide critical information for predicting and preventing crop damage (Welter 1989). Measuring net photosynthesis provides information about the effects of insect feeding on plant physiology and stress often before the appearance of any visual symptoms of stress occur (Welter 1989, Peterson et al. 1992, Peterson et al. 1998). Arthropod herbivory can significantly reduce leaf gas exchange (Schaffer et al. 1997, Godfrey et al. 1993, Schaffer and Mason 1990). The majority of studies on herbivory effects on leaf gas exchange have focused on non-insect arthropods (Schaffer et al. 1986, Mobley and Marini 1990,

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25 Lakso et al. 1996). There are limited reports on the effects of herbivory by insects on leaf gas exchange. Furthermore, the majority of these studies focused on adult insects feeding on leaves (Peterson et al. 1998, Li and Proctor 1984, Johnson et al. 1983) including Diaprepes root weevil (Syvertsen and McCoy 1985). There are a few studies reported on the effect of root damage from insect larvae on leaf gas exchange (Godfrey et al. 1993, Riedell and Reese 1999) and no known reports on the effects of Diaprepes root weevil root herbivory on leaf gas exchange. There are many economically important ornamental plant species grown in southern Florida that are affected by Diaprepes root weevil. Three ornamental plant species, pygmy date palm (Phoenix roebelenii OBrien.), buttonwood (Conocarpus erectus L.), and live oak (Quercus virginiana Mill.) are commonly grown together in south Florida field nurseries. These plant species are known to support larval and adult stages of Diaprepes root weevil and buttonwood has been shown to be a preferred host (Mannion et al. 2003). Serious root injury has been observed on field-grown live oaks and adults and larvae have been found on all three species in several field nurseries in Homestead, FL (Catharine Mannion, University of Florida, Tropical Research and Education Center, pers. comm.). The objective of this study was to evaluate the effects of root feeding by larvae of Diaprepes root weevil on net gas exchange and growth of pygmy date palm, green buttonwood and live oak. Materials and Methods Studies were conducted from October 2003 through July 2004 at the University of Florida, Tropical Research and Education Center, Homestead, FL, 25.5N latitude and 85.5W longitude. Two separate experiments were conducted to determine 1) the effects

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26 of root feeding by larvae of two different ages on leaf gas exchange and plant growth, and 2) the effect of root feeding damage by multiple larval infestations on leaf gas exchange and plant growth. The first experiment was conducted on green buttonwood from October 2003 through March 2004 and on live oak beginning in December of 2003 and ending in May 2004. The second experiment was conducted from January through July 2004 on buttonwood, live oak and pygmy date palm. Plant and Insect Material Experiment 1 Green buttonwood in 3.79-L containers were purchased from a commercial nursery (Princeton Nurseries II, Inc., Homestead, FL) and transplanted into 11.35-L containers with well-drained media consisting of 40% Florida peat, 30% pine bark, 10% sand, 20% cypress sawdust and 6.80 kg dolomite/91.44 cm purchased from a local soil distributor (Lantana Peat & Soil, Boynton Beach, FL). Live oaks in 3.79-L containers were purchased from a commercial nursery (Albert Livingston Tree Farms, Homestead, FL) and transplanted into 11.35-L containers in the same media as described for green buttonwood trees. Experiment 2 Young live oak and pigmy date palms in 3.79-L containers were purchased from a local commercial nursery (Bill Ingram & Grandsons NSY, Homestead, FL). Green buttonwood trees in 3.79-L containers were purchased from another nursery (Princeton Nurseries II, Inc., Homestead, FL). Plants were selected based on relative uniformity of shoot and canopy size within each species. All plants were transplanted into 11.35-L containers in the same media as described for Expt. 1.

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27 All plants in both experiments were fertilized once prior to treatment initiation with PlantacotePlus 14N-9P-15K (Helena Chemical Company, Collierville, TN) controlled release fertilizer. Plants were irrigated twice daily, at 5:00 am and at 5:00 pm for 30 minutes each time. Larvae of Diaprepes root weevil were obtained from the Florida Division of Plant Industry (Division of Agriculture and Consumer Services, Gainesville, FL) and held in containers on artificial diet in the laboratory until treatment initiation. Treatments Experiment 1 Larvae were sorted according to size so that each treatment would have similar age instars. The larvae were first separated into medium (1-1.5 cm) or large (2 cm) size classes, measured from the head to the tip of the abdomen. The head capsules from 20 randomly selected larvae from each size class were measured as described by Quintela et al. (1998) to determine average instars. The medium size class contained 5-6 th instars and the large size class contained 8 th instars. Holes were made in the soil surface around each plant with small spatulas, 4 to 8 cm from the stem, with 2.54 cm spacing between holes. The depth of the holes alternated at 5 and 10 cm from the soil surface. One or two larvae were placed into each hole and then covered with soil. Each container was infested with 20 larvae. All containers were irrigated after infestation to cool the soil and keep larvae from being affected by high soil temperatures. There were three treatments with 16 replications per treatment: 1) non-infested controls, 2) infested with medium larvae, and 3) infested with large larvae. Three months after infestation, 8 replications of each treatment were harvested to determine if larvae

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28 were surviving and feeding on roots. The remaining eight plants in each treatment were re-infested two times at approximately 2-month intervals with 20 larvae per container each time. Larvae were selected and placed into containers as previously described for the first infestation. All remaining plants were harvested 6 months after the initial infestation. Leaf gas exchange. Net CO 2 assimilation (A), stomatal conductance of H 2 O (g s ), and transpiration (E) were measured monthly with CIRAS-2 portable gas analyzer (PP Systems, U.K.) between 1000 and 1200 HR. Leaf gas exchange was measured at a photosynthetic photon flux (PPF) > 900 mol m -2 s -1 with a halogen lamp fitted on the leaf cuvette as the light source. Leaf gas exchange was determined for two fully expanded leaves of each plant and the average of the two leaves was used to represent gas exchange values for each single-plant replication. Root biomass and larval recovery. The roots were carefully separated from the rooting media and rinsed with tap water to remove excess soil. Soil from the roots was placed into bins and carefully inspected for larvae. Recovered larvae were separated and placed into holding containers containing 75% ETOH. Excess water was allowed to drain from roots for 12 hours and root fresh weights were determined. Roots were oven-dried at 70C to a constant weight and root dry weights were determined. Experiment 2 In this experiment, 5 th to 6 th instars (determined by head capsule size as previously described) were used to infest plants. Thirty-two plants of each buttonwood, live oak and pygmy date palm were infested with 20 larvae per container as described for Experiment 1 and 32 plants were left non-infested as controls. Approximately two months after infestation a set of 8 infested plants and 8 non-infested plants of each species were

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29 harvested. Thereafter, 8 plants of each treatment were harvested monthly. All remaining plants in the infested treatment were re-infested with 20 larvae per container as described for Experiment 1. Thus for the infested treatment, the first set of plants harvested were infested with a total of 20 larvae, plants harvested after 3 months were infested with a total of 40 larvae (20 per month), plants harvested after 4 months were infested with a total of 60 larvae, and plants harvested after 5 months were infested with a total of 80 larvae. Leaf gas exchange. Leaf gas exchange was measured monthly prior to each harvest for each set of plants as described for Experiment 1. For the set of plants in the final harvest, leaf gas exchange was measured on the week of infestation and monthly thereafter until the week of harvest. Plant growth and larval recovery. Plant height and trunk diameter was measured just prior to treatment initiation and again prior to the final harvest. Plant height was measured from the base of the stem to the tip of the apical bud. For oak and buttonwood trunk diameter was measured at 10 cm above the soil surface with an electronic digital caliper (Fisher Scientific, Pittsburgh, PA.). Leaves, stems and roots were harvested monthly beginning at the end of the second month and the number of recovered larvae and fresh and dry weights of plant tissues were determined as described for Experiment 1. Temperature Measurements Air and soil temperatures were measured with HOBO StowAway data loggers (Onset Computer Corp., Pocasset, MA). Data loggers used for soil measurements were placed at a depth from the soil surface of about 10 cm in eight 11.35-L containers with no

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30 plants in them and the same media as the containers with plants. Air temperature was measured with a data logger placed under a bench next to the experimental plants. Statistical Analysis Data were analyzed by a standard t-test or Analysis of Variance and means were compared with a Student-Newman-Keuls Range Test or Test for Least Significant Difference using SAS software (SAS Institute, Cary, NC). Results Experiment 1 Leaf gas exchange. Fluctuations in air temperatures were relatively similar to those of soil temperatures throughout the experiment (Fig. 2-1). Leaf gas exchange of buttonwood and live oak fluctuated with temperature, decreasing with dropping temperatures and increasing when temperatures increased for all treatments. In October at the beginning of the experiment, average monthly temperature was about 25C. Average air and soil temperatures decreased by the end of November to 18C. On several days during December and January minimum daily air and soil temperatures were between 4 and 10C (data not shown). Average air and soil temperatures began to increase by February to above 21C (Fig. 2-1). The effect of larval root injury on leaf gas exchange of buttonwood was not observed until average temperatures increased above 21C. At that temperature A, E and g s for each treatment increased from previous levels. However, control trees had significantly higher A, E and g s than plants infested with medium or large larvae four months (A: F=8.70; df=2, 23; P=0.0018; E: F=3.47; df=2, 23; P=0.05; g s : F=5.03; df=2, 23; P=0.016) and five months (A: F=15.74; df=2, 23; P<0.0001; E: F=20.06; df=2, 23; P<0.0001; g s : F=32.97; df=2, P<0.0001) after treatments were initiated (Fig. 2-2).

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31 Leaf gas exchange of live oak fluctuated slightly with temperature. Treatments were initiated during December when temperatures were relatively low and A, E and g s were also relatively low and then increased as average temperature increased. There were no significant differences in A, E and g s among treatments as a result of larval root feeding (Fig. 2-3). One exception was on one measurement date, four months after intial infestation, when g s of both infested treatments were lower than that of the controls four months after treatment initiation (Fig. 2-3). However, only the medium size larvae treatment had significantly lower A than the controls. There was no difference in g s among treatments on the last 2 measurement dates. Root weights and larval recovery. Fresh root weight of both infested treatments of buttonwood were significantly lower than that of the controls (F=3.73; df=2, 23; P=0.04). Dry weights tended to be lower for plants in the infested treatments than control plants but differences were not significant (Fig. 2-4). There were no significant differences in root fresh or dry weights among treatments for live oak (Fig. 2-5). For both plant species, relatively few larvae were recovered from individual plants compared to the amount of total larvae placed in each container. The average number of larvae recovered did not exceed eight per container for either treatment or plant species. For buttonwood, there was no significant difference in the number of larvae recovered between the medium and large larvae treatment (t=-1.79; df=14; P=0.09). The number of larvae recovered was not significantly different among treatments for live oak (t=-0.39; df=14; P=0.70). The number of larvae recovered between plant species was compared and there were no significant differences in the number of larvae recovered between buttonwood and live oak (t=0.69; df=28.6; P=0.49).

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32 Experiment 2 Leaf gas exchange. There was temporal variation in A, E and g s throughout the experimental period within and among plant species. Net CO 2 assimilation, E and g s were more inhibited by larval infestation in buttonwood than in pygmy date palm or live oak. Temperature did not appear to affect leaf A, E or g s For the first set of buttonwood plants there were no significant differences in A, E or g s between treatments prior to harvest. For plants harvested after 3 months, A (t=2.15; df=14; P=0.04), E (t=2.70; df=14; P=0.017) and g s (t=3.23; df=14; P=0.006) were significantly lower in infested plants than the non-infested plants (Fig. 2-6). For plants harvested after 4 5 months A, E and g s were not significantly different between treatments (Fig. 2-6). For the final set of plants where leaf gas exchange was measured monthly over time, A, E and g s of infested plants were lower than those of non-infested controls on every measurement date except the first and last. There was a significant difference in A, E and g s between treatments after each harvest at [1 month: A (t=11.26; df=14; P<0.0001); E (t=7.00; df=14; P<0.0001); g s (t=7.98; df=14; P<0.0001)] [2 months: A (t=5.56; df=9.94; P=0.0002), E (t=2.45; df=13.3; P=0.029); g s (t=2.24; df=13.4; P=0.04)] [3 months: A (t=4.25; df=14; P=0.0008), E (t=5.91; df=13.7; P<0.0001); g s (t=7.22; df=14; P<0.0001)] [4 months: A (t=2.20; df=14; P=0.04); E (t=3.44; df=14; P=0.03); g s (t=3.43; df=14; P=0.004)] (Fig. 2-7). For live oak, there was no effect of treatment on A, E and g s on any measurement date prior to harvest regardless of the number of infestations (Fig. 2-8). For the set of plants harvested last, where leaf gas exchange was measured monthly over time, A was not significantly different between treatments except on the second month when A of the

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33 infested plants was significantly lower than that of the non-infested controls (Fig. 2-9). There were no effects of treatment on g s or E on any measurement date (Fig. 2-9). For pygmy date palms A, E and g s were not significantly different between treatments regardless of the number of infestations (Fig. 2-10). For the set of plants harvested last and measured monthly over time, A and E were not significantly different between treatments (Fig. 2-11). Stomatal conductance was lower in infested plants than in the non-infested plants after 2 months. Although g s remained lower in infested plants than control plants, g s increased in all treatments by the third month and differences between treatments were not significant for the remainder of the experiment (Fig. 2-11). Plant growth and recovery larvae. Plant height and trunk diameter of buttonwood did not differ significantly between treatments on the initial measurement date. On the final measurement date, plant height (t=3.44; df=12.9; P=0.004) and trunk diameter (t=3.52; df=11; P=0.004) were significantly lower for plants in the infested treatments than for the non-infested plants (Table 1). Plant height and trunk diameter of live oak did not differ significantly between treatments on the initial or final measurement dates (Table 1). For buttonwood there was a significant interaction between larval infestation and harvest date for fresh leaf (F=4.17; df=7, 63; P=0.009), stem (F=4.61; df=7, 63; P=0.005) and root (F=4.01; df=7, 63; P=0.011) weights and dry leaf (F=4.52; df=7, 63; P=0.006), stem (F=4.18; df=7, 63; P=0.009) and root (F=6.34; df=7, 63; P=0.0009) weights. Therefore larval effects on leaf, stem and root fresh and dry weights were analyzed separately on each harvest date. Leaf, stem and root fresh weights were lower for infested than non-infested plants at each harvest date except the first when there were

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34 no differences in leaf and stem weights between treatments (Table 2). Leaf dry weight of buttonwood was lower for infested than non-infested plants harvested after 3 and 5 months. Stem dry weights were not significantly different between treatments for plants that were harvested after 2 months. However, stem dry weight of infested plants was significantly lower than that of the control plants harvested after 3, 4 and 5 months (Table 2). Root dry weights on all four harvest dates were significantly lower for infested than control plants (Table 2). There was no significant interactions between treatments and harvest date for leaf, stem and root fresh and dry weights of live oak and pygmy date palm (P > 0.05). Therefore harvest dates were pooled for each species to test the effect of larval root feeding on leaf, stem and root fresh and dry weights. For live oak there was no significant effect of treatment on leaf, stem and root fresh or dry weights (Table 3). Infested pygmy date palms had significantly lower leaf, stem and root fresh and dry weights than non-infested plants (Table 3). For both pygmy palm and live oak the number of larvae recovered, at each harvest, was 10. There was a significant effect of harvest date on the number of larvae recovered for buttonwood (F=27.64; df=3, 31; P<0.0001), live oak (F=10.15; df=3, 31; P=0.0001) and pygmy date palm (F=7.79; df=3, 31; P=0.0006), (Table 4). There tended to be more larvae recovered from buttonwood than from live oak and pygmy date palm, however; the differences were not significant until the last harvest date when more larvae were recovered from buttonwood (F=7.00; df=2, 23; P=0.004) than live oak or pygmy date palm (Table 4).

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35 Discussion In Expt. 1, average monthly temperatures had dropped from about 27C in October to about 18C in December, including several days with minimum temperatures below 10C. In buttonwood, the reduction in A, E and g s of trees in all treatments two months after infestation was presumably the result of low temperature since all treatments including controls were reduced. As the average monthly air and soil temperatures increased to about 21C by the end of February, A, E and g s of all treatments increased. These results are similar to those of (Taylor and Rowley 1971) who observed an immediate reduction in leaf photosynthesis in sorghum (Sorghum Hybrid), maize (Zea mays L.) and pennisetum (Pennisetum typhoides Burm.) as temperatures declined from 25 to 10C and then increased as temperature returned to 25C. Reductions in net CO 2 assimilation have also been observed in tropical woody plant species such as avocado (Persea americana Mill.) and mango (Mangifera indica L.) in response to low temperatures (Whiley et al. 1999). Buttonwood is a tropical plant species native to southern Florida and its distribution into central and northern Florida is limited by low winter temperatures (Tomlinson 1980). The significant difference between treatments in A, E and g s when temperature increased was presumably a result of increased larval activity and root feeding in the infested treatment when root temperature had warmed. Lapointe (2000) found that the growth rate of Diaprepes root weevil larvae increased exponentially with temperature from 15 to 30C and the optimal temperature for larval development was between 22 and 26C. Although A, E and g s were very low during periods of low soil temperature, the average mid day soil temperature presumably warmed enough for some larval root feeding to occur. There are no other known reports on the effects of root feeding by Diaprepes root weevil larvae on A, E and g s However,

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36 for another root-feeding insect, several authors have reported either a significant reduction in A caused by the western corn root worm (Diabrotica virgifera virgifera LeConte) larvae when actively feeding on maize (Zea mays L.) root systems, but did not affect A when the larvae reached the quiescent pupal stage (Godfrey et al. 1993, Riedell and Reese 1999). In Experiment 1, buttonwood plants infested with large (8 th instar) larvae exhibited lower A, E and g s than those infested with medium size (5-6 instars) larvae. It has been reported that weight gain is greatest in larvae between 6 to 9 th instars because these stages require large amounts of nutrients and cause significant injury to plants (Quintela et al. 1998, Rogers et al. 2000). After the first infestation, in Experiment 1, low soil temperatures below 15C may have caused some larval mortality (Lapointe 2000), and some larvae may have become quiescent and pupated during the experimental period thereby reducing the amount of damage caused by larval root feeding. However, re-infestation of larvae may have allowed the larger larvae to continue to cause significantly more damage. Net CO 2 assimilation, E and g s were not as sensitive to temperature or larval infestation in live oak as buttonwood. The oak treatments were initiated later when average temperatures had increased to above 21C. Therefore the temperature was presumably not low enough during the oak treatment period to affect leaf gas exchange. Larval infestation did not appear to affect leaf gas exchange of oak significantly and consistently during the six-month infestation period. In buttonwood, larval infestation resulted in lower root fresh and dry weights in the infested treatments than the controls. However, only fresh root weights were significant. Others have also reported reductions in root weight caused by Diaprepes root weevil

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37 larval feeding (Rogers et al. 2000, Nigg et al. 2001, Mannion et al. 2003). In live oak, root fresh and dry weights tended to be lower for both infested treatments than control trees but differences were not significant. Buttonwood root systems appeared to have more damage than those of the live oak, which may be due in part to the differences in root system anatomy of both species. The root system of buttonwood was more succulent and fibrous than that of live oak. The fast growing succulent roots of buttonwood may be the reason that Diaprepes root weevil larvae prefer buttonwood as a host over many other ornamental plant species (Mannion et al. 2003). Live oak root systems exhibited some root damage in the form of tunneling through the bark and cambium layer on the larger lateral roots and a noticeable reduction in smaller fibrous feeder roots. However most of the root weight was in the large primary tap and lateral roots which were relatively thick and dense and root feeding did not seem to reduce root weight. In Expt 2, buttonwoods were affected by larval root feeding more than pigmy date palm and live oak. Infestation by Diaprepes root weevil larvae caused a reduction in A, E and g s of buttonwood. This is consistent with results from Experiment 1, which showed reductions in A, E and g s of infested plants. Several buttonwood plants were showing signs of leaf yellowing, defoliation and wilting prior to harvest. These symptoms are typical of intense weevil damage (Knapp et al. 2000). Live oak responded as they did in Experiment 1, in that there were no visible signs of damage to the canopy and no significant reductions in A, E and g s caused by larval root feeding. In pygmy date palms there were no visible signs of stress to the canopy and A, E and g s were not significantly affected by treatment. Again, buttonwood may have been more sensitive than live oak

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38 and pygmy date palm because of its fleshier root system, which may have encouraged more continuous larval root feeding. Leaf, stem, and root fresh and dry weights were significantly lower for infested buttonwood than for control plants. Most infested plants, especially those showing signs of damage, when harvested had obvious root feeding damage and very limited root systems. Although only one buttonwood plant showing severe stress died before harvest, all the other buttonwood plants survived. The roots, stems and leaves, however, were noticeably smaller than those of the control plants. Buttonwood plants infested monthly and harvested at the end of the experiment had significantly less trunk diameter and height than the controls. A reduction in root weight and trunk diameter due to Diaprepes root weevil larval root feeding has also been observed by others (Nigg et al. 2001, Mannion et al. 2003). In Experiment 2, buttonwood plants that showed signs of leaf yellowing and wilt had the greatest reduction of growth. As in Experiment 1, infested live oak did not have a significant reduction of root weight. Trunk diameter and plant height of live oak harvested at the end of the Experiment 2 were not significantly affected by treatment. The reasons for this are not clear because larvae were recovered and there were signs of feeding damage. However, it is possible that oaks are more tolerant to root damage than buttonwood and it may take a longer period of feeding to cause significant damage. For pygmy date palm there were significant reductions in root fresh and dry weights between infested and non-infested treatments. Pygmy date palm is a monocot with an adventitious root system (Broschat and Meerow 2000). Although these adventitious root systems are relatively tender and succulent and had obvious signs of

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39 root feeding, there were fewer larvae recovered on each harvest compared to buttonwood and live oak. This may explain why buttonwood had more damage than pygmy date palm despite the succulent adventitious root system. Although both buttonwood and pygmy date palms have succulent roots, there presumably was greater feeding pressure on buttonwood which is a preferred host (Mannion et al. 2003). In contrast, live oak has a much tougher root system which presumably was more resistant to larval feeding than roots of the other species. Although there was obvious root feeding damage found on the root systems of buttonwood, live oak and pigmy date palm, only buttonwood showed signs of severe stress in the form of leaf yellowing, wilt and a significant reduction in leaf gas exchange and plant growth. These tests were performed in containers with young trees and although larval feeding pressure was high due to re-infestation with larvae, the treatment period in these experiments lasted only a few months. The negative effects of larval root feeding most likely are cumulative over time with constant feeding pressure as it occurs in the field with multiple generations feeding year after year. Further studies need to be conducted on plants in the field where Diaprepes root weevils are present year round and there are larvae continually feeding and damaging roots over a long period of time.

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40 Figure 2-1. Mean daily and monthly temperature (A) Air (B) Soil in containers.

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41 Figure 2-2. Net CO 2 assimilation (A), transpiration (E) and stomatal conductance (g s ) of buttonwood plants infested with medium or large Diaprepes root weevil larvae or non-infested control plants. Symbols represent means of 8 replications and bars indicate 1 std. error. Asterisks indicate significant difference among treatments according to ANOVA (P<0.05). 2 4 6 10 12 14 16 18 A (mol CO 2 m 2 s 1 ) Control Medium Large * 0 1 2 3 4 5 6 7 8 9 10 E (mmol H 2 0 m 2 s 1 ) * 350 ** 300 g s (mmol CO 2 m 2 s 1 ) 250 200 150 100 50 0 1 5 2 3 4 Months after infestation

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42 Figure 2-3. Net CO 2 assimilation (A), transpiration (E) and stomatal conductance (g s ) of live oak plants infested with medium or large Diaprepes root weevil larvae or non-infested control plants (C). Symbols represent means of 8 replications and bars indicate 1 std. error. Asterisks indicate significant difference among treatments according to ANOVA (P<0.05). 0 1 2 3 4 5 6 E (mmol H 2 O m 2 s 1 ) 0 2 4 6 8 10 12 14 16 18 A (mol CO 2 m 2 s 1 ) Control Medium Large 350 400 g s (mmol CO 2 m 2 s 1 ) 0 50 100 150 200 250 300 1 6 2 3 4 5 Months after infestation

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43 300 A 250 11Weight (g) 200 50 00 50 0 Medium Large Control 140 B 120 100 Weight (g) 80 60 40 20 0 Medium Large Control Treatment Figure 2-4. The effect of medium or large Diaprepes root weevil larvae on mean root fresh and dry weights of buttonwood trees. Bars represent means of 8 replications and error bars indicate 1 std. error.

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44 350 A 300 Weight (g) 250 200 150 100 50 0 Medium Large Control Figure 2-5. The effect of medium or large Diaprepes root weevil larvae on mean root fresh and dry weights of live oak trees. Bars represent means of 8 replications and error bars indicate 1 std. error. 0 20 40 60 80 100 120 140 160 180 200 B Weight (g) Large Control Medium Treatment

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45 12 Figure 2-6. Net CO 2 assimilation (A), transpiration (E) and stomatal conductance (g s ) measured prior to harvest of buttonwood plants infested with Diaprepes root weevil larvae or not infested. Plants harvested after 2 months were infested once with 20 larvae, plants harvested after 3, 4 and 5 months were infested with a total of 40, 60 and 80 larvae respectively. Bars represent means of 8 replications and error bars indicate 1 std. error. 0 2 46810A (mol CO 2 m2 s 1 ) Infested Non-infested 6 5 E (mmol H 2 O m 2 s 1 ) 4 3 2 1 0 300 250 g s (mmol CO 2 m 2 s 1 ) 200 150 100 50 0 2 5 3 4 Month harvested after initial treatment

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46 14 11A (mol CO 2 * 2 m 2 s 1 ) 0 8 6 4 2 Non-infested Infested 0 -2 6 E (mmol H 2 0 m 2 s 1 ) 5 * 4 3 2 1 0 400 350 g s (mmol CO 2 m 2 s 1 ) 300 250 200 150 100 50 0 0 4 5 1 2 3 Months after treatment initiation Figure 2-7. Net CO 2 assimilation (A), transpiration (E) and stomatal conductance (g s ) of the buttonwood plants infested monthly with 20 larvae and a total of 80 Diaprepes root weevil larvae by month 4 or not infested. Symbols represent means 1 std. error. Asterisks indicate significant difference between treatments according to standard t-test (P< 0.05).

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47 Figure 2-8. Net CO 2 assimilation (A), transpiration (E) and stomatal conductance (g s ) measured prior to harvest of live oak plants infested with Diaprepes root weevil larvae or not infested. Plants harvested after 2 months were infested once with 20 larvae, plants harvested after 3, 4 and 5 months were infested with a total of 40, 60 and 80 larvae respectively. Bars represent means of 8 replications and error bars indicate 1 std. error. 0 2 4 6 8 10 12 14 16 18 Non-infested A (mol CO 2 m 2 s 1 ) Infested 7 6 E (mmol H 2 O m 2 s 1 ) 5 4 3 2 1 0 0 50 1122334g 00 50 00 50 00 50 00 450 s (mmol CO 2 m2 s1 ) 2 5 3 4 Month harvested after initial treatment

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48 Figure 2-9. Net CO 2 assimilation (A), transpiration (E) and stomatal conductance (g s ) of live oaks plants infested monthly with 20 larvae and a total of 80 Diaprepes root weevil larvae by month 4 or not infested. Symbols represent means of 8 replications 1 std. error. Asterisks indicate significant difference between treatments according to standard t-test (P< 0.05). 0 1 2 3 4 5 6 7 E (mmol H 2 O m 2 s1 ) 0 2 4 6 8 111A (mol CO 0 2 4 16 18 20 2 m2 s1 ) Non-infested Infested 0 50 100 150 200 250 300 350 400 450 g s (mmol CO 2 m 2 s 1 ) 0 4 1 2 3 5 Months after treatment initiation

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49 Figure 2-10. Net CO2 assimilation (A), transpiration (E) and stomatal conductance (g s ) measured prior to harvest of pygmy date palm infested with Diaprepes root weevil larvae or not infested. Plants harvested after 2 months were infested once with 20 larvae, plants harvested after 3, 4 and 5 months were infested with a total of 40, 60 and 80 larvae respectively. Bars represent means of 8 replications and error bars indicate 1 std. error. 0 1 2 3 4 5 6 7 8 9 A (mol CO 2 m 2 s 1 ) Non-infested Infested 5 4 E (mmol H 2 O m 2 s 1 ) 3 2 1 0 250 g s (mmol CO 2 m 2 s 1 ) 200 150 100 50 0 2 5 3 4 Month harvested after initial treatment

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50 Figure 2-11. Net CO 2 assimilation (A), transpiration (E) and stomatal conductance (g s ) of the final harvest set of pygmy date palms infested monthly with 20 larvae and a total of 80 Diaprepes root weevil larvae by month 4 or not infested. Symbols represent means of 8 replications 1 std. error. Asterisks indicate significant difference between treatments according to standard t-test (P< 0.05). 0 1 2 3 4 5 6 7 8 9 10 A (mol CO 2 m 2 s 1 ) 5 E (mmol H 2 O m2 s1 ) 4 3 2 1 0 250 200 g s (mmol CO 2 m 2 s 1 ) 150 100 50 0 0 4 5 1 2 3 Months after treatment initiation

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51 Table 2-1. The effect of Diap repes root weevil larvae on he ight and trunk diameter of buttonwood and live oak trees. Species Treatment n Plant hei ght (cm) Trunk diameter (mm) Initial 2/17/04 Final 7/22/04 Initial 2/17/04 Final 7/22/04 Buttonwood Infested Non-infested (P) 8 8 65.54 62.05 (0.14) 71.27 90.47 (0.004) 8.21 8.03 (0.55) 9.40 13.39 (0.004) Live oak Infested Non-infested (P) 8 8 94.61 93.34 (0.65) 121.43 134.28 (0.38) 14.12 13.03 (0.46) 18.23 16.54 (0.35) Significance determined by a sta ndard t-test at the 0.05 level.

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52 Table 2-2. The effect of Diaprepes root weevil larvae on buttonwood leaf, stem and root fresh and dry weights at each harvest date. Significance determined by a standard t-test at the 0.05 level. Plant tissue treatment n Fresh weight (g) Dry weight (g) 4/04 5/04 6/04 7/04 4/04 5/04 6/04 7/04 Leaf Non-infested 8 197.15 219.23 247.59 254.54 73.45 80.28 99.65 97.76 Infested 8 186.27 112.38 188.02 100.28 74.19 43.41 75.94 31.78 (P) (0.47) (0.003) (0.09) (0.001) (0.91) (0.004) (0.11) (0.001) Stem Non-infested 8 86.15 102.48 130.28 243.96 36.04 45.43 66.70 100.43 Infested 8 76.74 58.54 102.99 68.76 32.82 28.51 49.07 31.78 (P) (0.13) (0.006) (0.04) (0.001) (0.28) (0.009) (0.02) (0.001) Root Non-infested 8 143.62 128.01 184.90 243.96 54.50 65.74 84.21 100.43 infested 8 85.03 40.85 109.21 79.42 37.09 25.53 57.49 33.43 (P) (0.0002) (<.0001) (0.007) (0.001) (0.0002) (<.0001) (0.01) (<.0001)

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53 Table 2-3. The effect of Diaprepes root weevil larvae on leaf stem and root fresh and dry weights of live oak and pygmy date palm. Species Treatment n Fresh weight (g) Dry weight (g) leaf stem root leaf stem root Live oak Non-infested 32 89.02 233.40 394.79 45.88 136.75 221.41 Infested 32 84.59 245.43 372.75 41.78 143.77 211.18 (P) (0.55) (0.57) (0.53) (0.31) (0.58) (0.62) Pygmy palm Non-infested 32 171.00 182.92 322.46 67.91 94.46 113.26 Infested 32 148.02 146.24 239.73 58.49 72.89 84.64 (P) (0.06) (0.01) (0.04) (0.05) (0.02) (0.01) Significance determined by standard t-test at the 0.05 level. Table 2-4. Number of Diaprepes root weevil larvae recovered from plants harvested 2, 3, 4 & 5 months after infestation. Species n Buttonwood 8 Live oak 8 Pygmy palm 8 Means within a row followed by different lower case letters and means within a column with different capital letters are significantly different according to ANOVA (P< 0.05). Total number of larvae infested for 1 st harvest=20; 2 nd harvest=40; 3 rd harvest=60; 4 th harvest=80.

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CHAPTER 3 EFFECT OF FLOODING AND DIAPREPES ROOT WEEVIL LARVAL FEEDING ON LEAF GAS EXCHANGE AND GROWTH OF BUTTONWOOD AND LIVE OAK Introduction Diaprepes root weevil, Diaprepes abbreviatus L. (Coleoptera: Curculionidae), is a polyphagous weevil species native to the Caribbean. It was first reported in the United States in 1964 in a nursery in Apopka, Florida. It is believed to have been introduced into Florida in an ornamental plant shipment from a nursery in Puerto Rico (Woodruff 1964). Since its introduction into Florida this weevil has seriously damaged citrus orchards and has spread throughout most of central and southern parts of the state. These weevils cause serious economic damage annually to many plant species including ornamental nursery crops. Recent reports indicate that Diaprepes root weevil affects more than 140,000 acres and is believed to be present in 22 counties in Florida (Simpson et al. 2000, Mannion et al. 2003). Many areas of south Florida where ornamental tree species are grown are prone to flooding. These areas are generally low lying and often have the marl soil types commonly found in Miami-Dade and Monroe Counties, including Everglades National Park. Marl soils have poor to very poor drainage and are formed when the land is cyclically flooded for several months each year. According to a 1996 soil survey, marl soil is classified as Biscayne soil (loamy, carbonatic, hyperthermic, shallow Typic Fluvaquants) (Noble et al. 1996, Li 2001). Ornamental trees in low lying nurseries with marl soil appear to sustain the highest populations of Diaprepes root weevils. 54

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55 Thepreference of these weevils for poorly drained areas has been observed in other heavy soils (Li et al. 2003a). Flooding can cause plants to become stressed and may predispose them to insect herbivory and pathogens (Syvertsen et al. 1983, Ploetz and Schaffer 1989). Tolerance to flooded conditions is highly variable among plant species. Several factors can affect a plants ability to withstand waterlogged soil conditions including soil type, soil porosity, soil chemistry and the status of pathogens and microbes in the soil (Schaffer et al. 1992). In many tree species net CO 2 assimilation and stomatal conductance are negatively impacted by flooding (Larson and Schaffer 1991, Andersen et al. 1984, Syvertsen et al. 1983) or insect predation (Peterson et al. 1998, Schaffer et al. 1997, Schaffer and Mason 1990). These factors may have an additive or synergistic effect on reducing leaf gas exchange. For example, in citrus orchards that were previously flooded and infested with Diaprepes root weevil larvae, a higher percentage of larvae were recovered from the soil and a higher percentage of root damage was observed in orchards that were flooded before insect infestation compared to orchards that had not been previously flooded (Li et al. 2003b). However, there is no published information on the effects of flooding and Diaprepes root weevil feeding on ornamental tree species in south Florida. Live oak (Quercus virginiana Mill.) and green buttonwood (Conocarpus erectus L.) are native to florida and are commonly grown together in south Florida nurseries for sale. These tree species have been observed to support high populations of adult Diaprepes root weevils and their egg masses (Mannion et al. 2003). Diaprepes root weevil larvae and the consequent feeding damage by these insects on buttonwood and live oak root systems have been observed on several occasions at different field nurseries

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56 (Catharine Mannion, University of Florida, Tropical Research and Education Center, pers. comm.). The objective of this study was to evaluate the effects of flooding and herbivory from Diaprepes root weevil larvae on leaf gas exchange and growth of buttonwood and live oak trees. Materials and Methods Studies were conducted from June through November 2004 at the University of Florida, Tropical Research and Education Center, Homestead, FL, 25.5 N Latitude and 85.5 W Longitude. Plant and Insect Material Young live oak and buttonwood trees in 11.35-L containers with well drained media consisting of 40% Florida peat, 30% pine bark and 10% sand were obtained from a commercial nursery (Doug Ingram & Sons NSY, Homestead, FL). Live oaks were pruned slightly at the top of the apical shoots to achieve uniform plant heights. Buttonwoods were already uniform in height so they were not pruned. Plants were irrigated twice daily at 5:00 AM and at 5:00 PM, for 30 minutes each time. Diaprepes larvae were obtained from the Florida Division of Plant Industry (Florida Department of Agriculture and Consumer Services Gainesville, FL) and held in containers with artificial diet in the laboratory until plant infestation treatments were initiated. Treatments Flooding. Live oak and buttonwood trees were subjected to continuous flooding or left non-flooded. Live oaks were flooded from June 1, 2004 to July 7, 2004. Buttonwoods were flooded from July 21, 2004 to August 11, 2004. Sixteen trees of each

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57 species in 11.35-L containers were placed into empty 18.93-L containers and tap water was added to the large containers to about 10 cm above the soil surface. Sixteen trees of each species remained non-flooded. Leaf gas exchange was measured weekly until there was a significant reduction in net CO 2 assimilation (A) on two consecutive measurement dates. After that time, plants were permanently removed from the flooded conditions and infested with larvae. Eight each of the flooded and non-flooded plants of each species were infested with Diaprepes root weevil one day after the flooding treatment was terminated. The other eight plants of each species in each treatment were not infested. Thus there were four treatments: 1) non-flooded, non-infested, 2) non-flooded, infested, 3) flooded, non-infested, and 4) flooded, infested. There were eight single-plant replications per treatment for each species arranged in a completely randomized design. Insect infestation. The head capsules from 20 randomly selected larvae were measured to determine instars as described by Quintela et al. (1998). Larvae used to infest plants were determined to be between the fifth and sixth instars (1-1.5 cm) from the head to the tip of the abdomen. Ten to 15 holes were made in the soil around each plant with small spatulas alternating at 4 to 8 cm from the stem, with 2.54-cm spacing between holes. The depth of the holes alternated at 5 and 10 cm from the soil surface. One or two larvae were placed into each hole and covered with soil for a total of 20 larvae per pot. Plants were re-infested one month after initial infestation and all plants were irrigated immediately after infestation. Soil Redox Potential For plants in the flooded treatments, soil redox potential (Eh), an indirect indicator of oxygen content of the soil (Pezeshki 1991), was measured weekly with a platinum combination electrode (Ag+/AgCl, Accumet, Fisher Scientific, Pittsburgh, Pa.) attached

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58 to a portable pH meter (Accumet AP62, Fisher Scientific, Pittsburgh, Pa.). Measurements were made weekly until Eh readings remained relatively constant. Leaf Gas Exchange Net CO 2 assimilation, stomatal conductance of H 2 O (g s ), and transpiration (E) were measured with a CIRAS-2 portable gas analyzer (PP systems, U.K.) between 1000 and 1200 HR. Leaf gas exchange was measured at a photosynthetic photon flux (PPF) > 900 mol m -2 s -1 with a halogen lamp fitted on the leaf cuvette as the light source. Leaf gas exchange was determined for two fully expanded leaves from each plant and the average of the two leaves was used to represent values for each plant. Leaf gas exchange was measured weekly during flooding and monthly during the larval treatment. Plant Growth and Larval Recovery Four months after treatments began, all plants were harvested and the roots were carefully separated from the potting medium and rinsed with tap water to remove any soil clinging to the root surface. Soil removed from roots was placed into bins and carefully inspected for larvae. Recovered larvae from the soil and roots were placed into containers with 75% ethyl alcohol. Excess water was allowed to drain from the roots overnight and leaf, stem and root fresh weights were determined. Leaves, stems and roots were oven-dried for three days at 70C to a constant weight and leaf, stem and root dry weights were determined. Statistical Analysis Data were analyzed by Analysis of Variance and standard t-test using SAS software (SAS Institute, 2003-2004).

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59 Results Soil Redox Potential (Eh) Within a few hours after buttonwood was flooded, Eh was < 200 mV, indicating that the soil was becoming hypoxic (Li et al. 2004, Kozlowski 1997). After one week of flooding Eh was -250 mV indicating that there was little or no oxygen available to plant roots (Li et al. 2003). At the end of the first week of flooding and continuing through the final two measurements, Eh leveled off at about -280 mV (Fig. 3-1A). Eh values for live oak were negative 3 days after flooding treatments were initiated. Eh values continued to decrease and leveled off at about -250 during the final three weeks of the flooding period (Fig. 3-1B). Leaf Gas Exchange Flooding. Prior to flooding (at day 0), there were no significant differences in A, E or g s between flooded and non-flooded plants (Fig. 3-2). One week after flooding began, flooded plants had significantly lower A (t=-4.03; df=29.5; P=0.0004), E (t=-3.51; df=30; P=0.001), and g s (t=-2.73; df=28.5; P=0.01) than non-flooded plants. Two weeks after flooding began A (t=-6.31; df=29.4; P<0.0001), E (t=-3.70; df=30; P=0.0009) and g s (t=-5.56; df=29.8; P<0.0001) remained lower for flooded plants than non-flooded plants (Fig. 3-2). One, two and three weeks after flooding there were no significant differences in A, E or g s between flooded and non-flooded plants. Four weeks after flooding began A (t=-2.39; df=29.8; P=0.02), E (t=-2.77; df=27.4; P=0.009) and g s (t=-3.02; df=29; P=0.005) were lower for flooded plants than non-flooded plants. At five weeks A (t=-3.94; df=30; P=0.0004), E (t=-2.81; df=27.7; P=0.009) and g s (t=-2.18; df=30; P=0.03) remained

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60 significantly lower in flooded plants than non-flooded plants (Fig. 3-3), and plants were removed from flooding and infested with larvae 24 hours later. Insect infestation. For both buttonwood and live oak, one, two and three months after plants were infested with Diaprepes root weevil larvae, there were no significant interactions between pre-flooding and insect treatments on any measurement date (P>0.05; Table 3-1). Therefore, flooding treatments were pooled to test the effects of Diaprepes root weevil larvae on A, E and g s of each species. One month after buttonwood trees were infested, non-infested trees had higher A (t=-2.77; df=30; P=0.009), E (t=-1.95; df=30; P=0.06) and g s (t=-3.06; df=30; P=0.004) than infested trees (Fig. 3-4). However, two and three months after infestation there was no significant difference in A, E or g s between treatments (Fig. 3-4). In live oak there was no significant difference in A between infested and non-infested plants on all three measurement dates. Although not statistically significant, E tended to be lower for the infested plants than non-infested plants. Two months after larval infestation, g s was lower for infested plants than non-infested plants (t=-2.55; df=29.7; P=0.01; Fig. 3-5). On the final measurement date, there were no significant differences in A, E or g s between the larvae infested and non-infested plants (Fig. 3-5). Plant Growth and Larval Recovery For both buttonwood and live oak, there were no significant effects of flooding on plant growth. There were no significant interactions between pre-flooding and insect treatments on leaf, stem, root and total plant fresh or dry weights (P > 0.05). Therefore, flooding treatments were pooled for testing the effects of larval infestation on plant biomass. Infested buttonwood had significantly lower fresh (t=-4.41; df=29.4; P=0.0001) and dry (t=-3.97; df=29.4; P=0.0004) root weights than non-infested controls (Table 3-2).

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61 Leaf and stem fresh and dry weights were not significantly different between infested and non-infested treatments. Total fresh and dry weights for infested plants tended to be lower than those of the non-infested plants; although, the differences were not statistically significant (Table 3-2). For live oak, leaf, stem, root and total plant fresh and dry weights were not significantly different between infested and non-infested treatments (Table 3-2). There were no differences in the mean number of larvae recovered from infested buttonwood plants that had been pre-flooded compared to that of the non-flooded infested plants. For live oak, the mean number of larvae recovered from plants that were pre-flooded prior to infestation tended to be higher than from non-flooded treatments; however, the difference was not statistically significant (Fig. 3-6). There was no significant difference between oak and buttonwood in the number of larvae recovered from plants that were not pre-flooded. However, there were significantly more larvae recovered from pre-flooded oak than pre-flooded buttonwoods (t=-3.12; df=12.1; P=0.008). Discussion Flooding had a negative effect on leaf gas exchange on both buttonwood and live oak. Live oak is known to tolerate moderately wet conditions (Dehgan 1998). However, the quicker reduction in A, E and g s as a result of flooding in buttonwood compared to live oak was unexpected because buttonwood is a member of the mangrove family and should be able to tolerate flooded conditions (Tomlinson 1980, Gilman and Watson 1993). The difference in the amount of time it took between each plant species to show reductions in A, E and g s may be a result of several factors. Plant responses to flooding may vary depending on genotype, age, properties of flood water and duration of flooding

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62 (Kozlowski 1997). Prior to removal of plants from flooding, flooded buttonwoods exhibited hypertrophied lenticels and adventitious roots growing above the water line. These morphological adaptations to flooding are common in flood tolerant species (Kozlowski, 1984). However, in our tests although buttonwood responded with an initial reduction in A, E and g s under flooded conditions, it is possible that that these morphological adaptations may have aided them in surviving if the flooding period was prolonged. For example, Kozlowski (1984) found that flooding of green ash (Fraxinus pennsylvanica Marsh.) caused immediate stomatal closure of seedlings followed by production of adventitious roots and reopening of stomata after about two weeks. In contrast to our results, Li et al. (2003) found that citrus seedlings flooded prior to infestation were more susceptible to Diaprepes root weevil larval feeding damage than seedlings that were not previously flooded. Pre-flooded and infested seedlings had lower leaf g s than those that were not flooded before infestation. In our study, buttonwood and live oak plants were larger and had a relatively dense root system compared with those of the citrus seedlings used in the previous experiments. Thus, plant species, age and size or a combination of these may affect the time it takes for Diaprepes root weevil larvae to significantly affect its host plant. Unlike Li et al. (2003) who recovered significantly more larvae from seedlings that were pre-flooded prior to infestation, we found no significant difference in the number of larvae recovered between pre-flooded plants or plants that were not pre-flooded treatments. Visible root damage of both buttonwood and live oak was observed and larvae were recovered (although more larvae were recovered from live oak than buttonwood), indicating that larvae were feeding and causing damage to the root systems.

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63 Live oak and buttonwood plants appeared to show initial reductions in leaf gas exchange in response to larval feeding; however, by the final two months of treatment there was no effect of infestation on leaf gas exchange despite the fact that larvae were recovered from buttonwood and live oak roots and there were signs of root feeding damage at the end of the treatment period. There was no significant effect of infestation during the three month treatment period on leaf gas exchange or plant growth with the exception of a reduced root system in buttonwood. However, it is possible that if the tests were continued longer than 3 months, there could have been a negative impact of root feeding on leaf gas exchange and plant growth. Both tree species appeared to recover from flooding and it did not appear as if flooding predisposed the plants to root feeding damage over the 3 month study period. However in the field, continual feeding damage caused by multiple generations of Diaprepes root weevil larvae along with periods of cyclical flooding during the wet summer months presumably can result in reduction in gas exchange and growth. Therefore, further studies are warranted to compare infested plants to non-infested plants in field nurseries.

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64 Figure 3-1. Soil redox potential (Eh) of flooded buttonwood (A) and live oak (B) in containers. Symbols and bars represent means of 16 replications and bars indicate 1 std. error. -300 -250 -200 -150Eh (mV) 100 -50 0 1 2 3 4 5 6 Time (weeks) B -350 -300 -250 -200 -150 -1 Eh (mV) 00 -50 0 50 100 150 A 1 4 2 3 Time (weeks)

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65 Figure 3-2. Net CO 2 assimilation (A), transpiration (E) and stomatal conductance (g s ) of flooded and non-flooded buttonwood trees. Symbols represent means of 16 replications and bars indicate 1 std. error. Asterisks indicate significant differences between treatments according to a standard t-test (P<0.05). 0 2 4 6 8 10 12 14 16 18 20 * A (mol CO 2 m 2 s 1 ) Non-flood Flood 8 7 E (mmol H 2 O m 2 s 1 ) 6 5 4 3 2 1 0 800 700 g s (mmol CO 2 m 2 s 1 ) 600 500 400 300 200 100 0 0 2 1 Weeks after flood treatment commenced

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66 16 18 * A (mol CO 2 m 2 s 1 ) 12 14 10 6 8 Figure 3-3. Net CO 2 assimilation (A), transpiration (E) and stomatal conductance (g s ) of flooded and non-flooded live oak trees. Symbols represent means of 16 replications and bars indicate 1 std. error. Asterisks indicate significant difference between treatments according to a standard t-test (P<0.05). 0 2 4 Non-flood Flood 8 7 E (mmol H 2 0 m2 s 1 ) 6 5 4 3 2 1 0 0 50 100 150 200 250 300 350 400 450 * g s (mmol CO 2 m 2 s 1 ) 0 5 1 2 3 4 Time (weeks)

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67 16 Figure 3-4. Net CO 2 assimilation (A), transpiration (E) and stomatal conductance (g s ) of buttonwood trees infested with Diaprepes root weevil larvae or non-infested. Symbols represent means of 16 replications and bars indicate 1 std. error. Asterisks indicate significant difference between treatments according to a standard t-test (P<0.05). 0 2 4 6 8 101214A (mol CO 2 m2 s 1 ) Non-infested Infested 6 E (mmol H 2 O m 2 s1 ) 5 4 3 2 1 0 350 300 g s (mmol CO 2 m 2 s 1 ) 250 200 150 100 50 0 1 3 2 Months after treatments were infested

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68 16 18 A (mol CO 2 m 2 s 1 ) Figure 3-5. Net CO 2 assimilation (A), transpiration (E) and stomatal conductance (g s ) of live oak trees infested or non-infested with Diaprepes root weevils. Symbols represent means of 16 replications and bars indicate 1 std. error. Asterisks indicate significant differences between treatments according to a standard t-test (P<0.05). 0 2 4 6 8 10 12 14 Non-infested Infested 7 6 E (mmol H 2 O m2 s 1 ) 5 4 3 2 1 0 0 50 100 150 200 250 300 350 400 450 500 g s (mmol CO 2 m 2 s 1 ) 1 3 2 Time (month)

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69 Figure 3-6. Larvae recovered from pre-flooded or non-flooded treatments of buttonwood and live oak. 0 2 4 6 8 10121416Number of larvae recovered 18 oak Non-flood Flood buttonwood

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Table 3-1. The effect of flooding (FLD) and insect infestation (INFST) on leaf gas exchange of buttonwood and live oak trees. Species Measurement treatment One month Two months Three months Buttonwood A (mol CO 2 m -2 s -1 ) SS MS P SS MS P SS MS P FLD 0.31 0.31 0.72 0.09 0.09 0.90 4.61 4.61 0.24 INFST 17.18 17.18 0.01 0.10 0.10 0.89 0.01 0.01 0.94 Fld Infst 0.27 0.27 0.73 0.51 0.51 0.78 0.10 0.10 0.85 E (mmol H 2 O m -2 s -1 ) FLD 0.03 0.03 0.77 0.04 0.04 0.70 0.09 0.09 0.51 INFST 1.45 1.45 0.06 0.44 0.44 0.22 0.04 0.04 0.64 Fld Infst 0.008 0.008 0.88 0.34 0.34 0.29 0.07 0.07 0.55 g s (mmol CO 2 m -2 s -1 ) FLD 166.5 166.5 0.77 882.0 882.0 0.54 582.2 582.2 0.41 INFST 18002.5 18002.5 0.006 488.2 488.2 0.65 8.50 8.50 0.92 Fld Infst 28.12 28.12 0.90 957.0 957.0 0.52 279.0 279.0 0.57 Live Oak A (mol CO 2 m -2 s -1 ) SS MS P SS MS P SS MS P FLD 6.52 6.52 0.29 6.21 6.21 0.36 3.51 3.51 0.58 INFST 0.41 0.41 0.78 16.67 16.67 0.14 1.48 1.48 0.72 Fld Infst 4.92 4.92 0.35 13.78 13.78 0.18 0.07 0.07 0.93 E (mmol H 2 O m -2 s -1 FLD 0.18 0.18 0.58 0.86 0.86 0.29 0.03 0.03 0.76 INFST 0.12 0.12 0.65 2.69 2.69 0.07 0.03 0.03 0.76 Fld Infst 0.61 0.61 0.32 0.09 0.09 0.72 0.05 0.05 0.71 g s (mmol CO 2 m -2 s -1 ) FLD 3.12 3.12 0.98 13304.8 13304.8 0.12 800.0 800.0 0.70 INFST 24.50 24.50 0.96 34485.9 34485.9 0.01 1212.7 1212.7 0.63 Fld Infst 5227.5 5227.5 0.46 446.25 446.25 0.77 406.1 406.1 0.78 70

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71 Table 3-2. The effect of Diaprepes root weevil larvae on leaf, stem, root and total biomass of infested or non-infested buttonwood and live oak trees. Plant species Treatment n Fresh weight (g) Dry weight (g) Buttonwood leaf stem root total leaf stem root total Infested 16 317.00 184.61 162.81 664.42 173.6 99.37 78.96 351.95 Non-infested 16 343.81 216.46 220.77 781.03 200.99 116.86 104.34 422.19 (P) (0.43) (0.14) (0.0001) (0.07) (0.28) (0.12) (0.0004) (0.08) Liveoak Infested 16 54.88 293.57 230.00 578.45 30.62 172.53 135.15 338.31 Non-infested 16 67.35 306.65 272.56 646.57 36.84 175.25 154.19 366.29 (P) (0.12) (0.73) (0.53) (0.52) (0.19) (0.91) (0.62) (0.65) Significance determined by a standard t-test at the 0.05 level.

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CHAPTER 4 EFFECT OF ADULT DIAPREPES ROOT WEEVIL ON LEAF GAS EXCHANGE AND GROWTH OF BUTTONWOOD AND LIVE OAK Introduction Diaprepes root weevil Diaprepes abbreviatus L. (Coleoptera: Curculionidae), is a polyphagous species known to feed on a wide variety of plants throughout Florida. The root weevil has a host range of more than 270 plant species (Simpson et al. 1996). In Florida it is estimated that this weevil causes more than 70 million dollars in damage annually (Weissling et al. 2002). Adult weevils have a voracious appetite and may cause severe defoliation of host plants. Adult Diaprepes root weevils emerge from the soil year round in Florida; however, the peak emergence period is from May through October. As the teneral adults begin to dig their way out of the soil they shed a pair of deciduous mandibles. Adult weevils emerge from the soil and move up the tree canopy to feed and mate. Oviposition generally begins 3 to 7 days after emergence from the soil surface (Wolcott 1936). The average longevity reported for adult weevils reared on an artificial diet is about 147 days for females and 135 days for males (Beavers 1982). Diaprepes root weevil is known to be associated with a wide variety of ornamental plants grown in nurseries throughout Florida and many nurseries in Miami-Dade County, Florida are infested with this pest. In a recent survey of several field nurseries, Mannion et al. (2003) found that characteristic leaf notching and egg masses from adult weevils were widespread on several tree species that are commonly grown together. The plant 72

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73 species with the highest percentage of egg masses were live oak (Quercus virginiana Mill.), silver buttonwood (Conocarpus erectus L.) and black olive (Bucida buseras L.). A basic understanding of plant physiological responses to arthropod herbivory may provide critical information for predicting and preventing crop damage. Leaf gas exchange measurements provide a basis for comparing herbivore effects on different plants and plant parts (Welter 1989, Schaffer and Mason 1990, Peterson et al. 1998). Several studies of arthropod herbivory on plant physiology, in particular leaf feeding effects on leaf gas exchange, have shown reductions in leaf gas exchange as a result of insect feeding (Schaffer et al. 1986, Mobley and Marini 1990, Lakso et al. 1996, Schaffer et al. 1997). However, other studies have shown slight or no effects of leaf feeding on gas exchange (Peterson et al. 1996). Insect herbivory has also been shown to reduce biomass of several plant species (Schaffer and Mason 1990, Welter 1991). Adult Diaprepes root weevils damage plants primarily by leaf notching and generally tend to feed on new leaf flushes and occasionally on fruit. There is not much known about the effects of Diaprepes root weevil feeding on leaf physiology of ornamental plants. Only one previous study examined the effect of adult weevil feeding on citrus leaf photosynthesis (Syvertsen and McCoy 1985). Buttonwood and live oak are known hosts of Diaprepes root weevil and support all stages of this pest from egg to adult (Mannion et al. 2003). Both tree species are commonly grown together in the same field nurseries in south Florida. Larvae alone are known to cause significant root damage to both species and adult weevils have also been observed to cause considerable leaf tissue damage in both young and mature trees. However, there are no published data quantifying the effects of leaf feeding damage on

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74 these ornamental tree species. The objective of this study was to evaluate the effects of adult Diaprepes root weevil leaf feeding on leaf gas exchange and growth of buttonwood and live oak trees. Materials and Methods Three experiments were conducted to assess the effects of adult weevil feeding on live oak and buttonwood trees. All experiments were conducted in spring and summer of 2004 at the University of Florida, Tropical Research and Education Center, Homestead, FL, 25.5 N latitude and 85.5 W longitude. Plant and Insect Material For Experiment 1, green buttonwood trees in 3.79-L containers were purchased from a commercial nursery (Princeton Nurseries II, Inc. Homestead, FL) and repotted into 11.35-L containers with well-drained media purchased from a local soil distributor (Lantana Peat & Soil, Boynton Beach, FL) consisting of 40% Florida peat, 30% pine bark, 10% sand, 20% cypress sawdust (by volume) and 6.80 kg dolomite/91.44 cm. For Experiment 2, green buttonwoods in 3.79-L containers were purchased from a local nursery (Bill Ingram & Grandsons NSY, Homestead, FL) and repotted into 11.35-L containers with the same media described above. For Experiment 3, live oaks in 3.79-L containers were purchased from a commercial nursery (Action Theory Nursery, Homestead, FL) and repotted into 11.35-L containers with the same media described above. All plants where fertilized once prior to treatment initiation with PlantacotePlus 14N-9P-15K (Helena Chemical Company, Collierville, TN) controlled-release fertilizer. Plants were placed in aluminum frame cages constructed to allow suitable light for normal plant growth while preventing insects from escaping. Cages were 1.21 m x 60.96

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75 cm x 60.96 cm with aluminum screening with a mesh size of 1.6 mm. There was one plant per cage. For all three experiments, adult Diaprepes root weevils were collected from a field nursery (Native Tree Nursery, Homestead, FL). Weevils were maintained in acrylic holding cages and fed fresh buttonwood foliage that was replaced every other day with water supplied in small containers with a dental wick. Adult weevils were randomly selected from the holding cages and separated by sex. At the start of each experiment 10 males and 5 females were released into each cage. Each experiment was arranged in a complete block design with two treatments (infested or non-infested) and 7 single-plant replications per treatment. To prevent neonate larvae from entering the soil after eggs hatched, the soil surface of each pot was covered with a 50.8 cm 2 sheet of Weed Block (Easy Gardener, Inc. P.O. Box 21025 Waco, TX), which was secured around the plant stems with duct tape. This material was selected because the pore size was small enough to prevent larvae from passing through, but large enough to allow water to penetrate. Leaf Gas Exchange Net CO 2 assimilation (A), stomatal conductance of H 2 O (g s ), and transpiration (E) were measured prior to infesting plants and then monthly after insect infestation with a CIRAS-2 portable gas analyzer (PP Systems, U.K.) between 1000 and 1200 HR. Leaf gas exchange measurements were made at a photosynthetic photon flux (PPF) > 900 mol m -2 s -1 with a halogen lamp fitted on the leaf cuvette as the light source. Leaf gas exchange was determined for two young flushing leaves and two fully expanded leaves from each plant and the averages of each pair of young and mature leaves were used to represent leaf gas exchange of young and mature leaves for each plant. For experiment 3,

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76 there was an insufficient number of new leaf flushes to measure gas exchange on young leaves. Therefore, gas exchange was measured for only two mature leaves per plant. Plant biomass At the end of each experiment, plants were harvested, all leaves where removed and the total leaf area per plant was determined with a leaf area meter (Li-Cor, Lincoln, NE; model Li-3000). Plants were harvested and roots were washed with tap water to remove media attached to the root hairs. Excess water was allowed to drain from the roots for 24 hrs and leaf, stem and root fresh weights were determined. Plant tissues were then oven-dried at 70C for two days and leaf, stem and root dry weights were determined. Statistical Analysis Data were analyzed by a standard t-test and Analysis of Variance using SAS software (SAS Institute, 2003-2004). Results Visible Signs of Herbivory In both buttonwood experiments the characteristic leaf notching caused by adult Diaprepes root weevil feeding was observed within one day after insects were released into cages. Both young and mature leaves had signs of damage; however, the weevils preferred the younger foliage. Most of the leaf area of young leaves, with the exception of the mid-veins, was completely removed by the first month. Mature leaves also had considerable notching primarily on the edges of the lamina. Live oak plants had very leaf feeding damage. A few live oak leaves had minor damage on the edges of the lamina but no significant damage was caused. In the cages with live oak trees all weevils died within the first month of the experiment.

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77 Leaf Gas Exchange In the first buttonwood experiment there were no significant differences between treatments in A, E or g s of mature leaves. One month after treatments were initiated there were no significant effects of adult Diaprepes root weevil feeding on A, E or g s of young or old leaf tissue. On the final measurement date, two months after infestation, A, E and g s of infested plants tended to be higher than that of the controls but the differences were not significant (Table 1). In the second buttonwood experiment, one month after treatments were initiated, A was significantly lower of young leaves of infested plants than those of non-infested treatments (t=-2.73; df=8.69; P=0.02). However E and g s of young leaves were not significantly different between treatments (Table 2). Net CO 2 assimilation, E and g s of mature leaves were not significantly affected by adult weevil leaf feeding. Two months after treatments were initiated, g s was significantly higher in young leaves of infested than in those of non-infested plants (t=2.14; df=10.7; P=0.05). However, A and E of young leaves were not significantly different between treatments. In mature leaves, A (t=2.66; df=9.95; P=0.02), E (t=2.58; df=11.8; P=0.02) and g s (t=2.79; df=11.4; P=0.01) were higher in infested plants than in non-infested plants (Table 2). For live oak, one month after infestation, there were no significant differences in A, E and g s between treatments. At this time all adult weevils in the cages with oak trees had died and the experiment was terminated. Plant Biomass Mean leaf area for both buttonwood experiments was not significantly different between treatments (Table 3). However, in experiment 1, plants infested with adult

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78 weevils had 18.3% less leaf area than the controls and in experiment 2, plants infested with adult weevils had 27.8% less leaf area than non-infested control plants. Leaf, stem and root fresh and dry weights of buttonwood in experiment 1 were lower in the infested than non-infested plants but the differences were not significant (Table 4). For buttonwood in experiment 2, leaf, stem and root fresh weights for infested treatments were lower than that of non-infested treatments; however, the differences were not statistically significant. Dry weights of leaves and stems were lower for infested plants than non-infested plants but were not significantly different between treatments. Dry root weights were significantly lower (t=-2.12; df=11.1; P=0.05) in the infested than in non-infested treatment (Table 4). Discussion Leaf feeding by adult Diaprepes root weevil had variable effects on leaf gas exchange of buttonwood. Live oak was not affected by adult insect herbivory presumably because mature, hardened-off leaves were too thick for the mandibles of this insect. Adult weevils were observed trying to feed on oak leaves but they were only causing minor scrapes to the leaf margins and all weevils died within a month, most likely because of starvation. Live oak trees were not in a period of leaf flushing during the treatment period and the lack of significant difference in leaf gas exchange and growth between treatments was probably related to the phenological stage of the plants at the time of infestation. In subsequent field observations there appeared to be specific periods of time when relatively large populations of adult Diaprepes root weevils were found in live oak canopies. This feeding period corresponded to periods of leaf flushing when the young leaves were still succulent and the weevils were able to feed on the foliage.

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79 In both buttonwood experiments, one month after treatments were initiated, most young leaves of infested plants had obvious feeding damage and most of the leaf tissue except for the area around the mid-vein, were completely removed. Mature leaves also had signs of feeding damage to the edges of the lamina with no damage to the mid-vein. Similarly, Syvertsen and McCoy (1985) reported considerable leaf feeding damage caused by a citrus root weevil, little leaf notcher (Artipus floridanus Horn.) to tender citrus leaf margins with no injury to the mid-veins. In our study, adult weevils preferred the young tender foliage over mature leaves; however, the majority of leaves of each plant were mature and the outer areas of almost all leaves had feeding damage. Mature leaves appeared to have more leaf area remaining around the mid-vein than younger leaves In the first experiment, A, E and g s of buttonwood were higher for infested than non-infested plants, but the differences were not significantly different after one or two months of feeding. Some authors have reported that leaf mass consumption by insect herbivores does not negatively impact photosynthesis of the remaining leaf tissues (Welter 1989, Peterson et al. 1996). No reduction in leaf gas exchange was reported for actual and simulated herbivory of tomato (Lycopersicon esculentum L.). Actual defoliation by tobacco horn worm (Manduca sexta L.) larvae or simulated defoliation did not change photosynthetic rates per unit area of tomato leaflets (Welter 1991). It has previously been reported that leaf feeding in which there is only removal of leaf tissue without injury to the mid-vein only reduces the amount of photosynthetic leaf area but not photosynthetic rates of the remaining leaf tissue (Li and Proctor 1984, Peterson et al. 1996, Peterson et al. 1992, Peterson et al. 2004). In the first buttonwood experiment,

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80 similar to results of previous studies, we found no significant reduction in leaf gas exchange due to leaf feeding. However, in the second experiment there were variable leaf gas exchange responses. After one month of infestation, there were significant reductions of A, and no reduction of E or g s of younger buttonwood leaves or A, E and g s of mature leaves. After two months of leaf feeding, mature leaves of infested buttonwood trees had greater leaf gas exchange than controls. Although increases in leaf gas exchange have been previously reported for some plant species, other studies have generated contrasting results. Some authors reported no difference in leaf gas exchange as a result of arthropod herbivory, whereas others have reported increases and still others have reported decreases (Welter 1989, Detling et al. 1980, Sances et al. 1981, Mobley and Marini 1990, Peterson et al. 1998). In the majority of studies involving selective tissue feeders such as leafhoppers and mites, showed reduced photosynthesis. Studies involving defoliators, which remove partial or entire leaf tissue, showed a tendency toward increased photosynthesis of remaining leaf tissues (Welter 1989). Both buttonwood experiments were conducted with the same materials and methods, but only the second experiment yielded significant differences in leaf gas exchange between treatments. However, buttonwood experiment 1 leaf gas exchange on the final measurements of both young and mature leaves tended to be higher in the infested plants than in non-infested controls. Our study yielded different results than those of other defoliation studies, which found that leaf gas exchange of infested plants were not significantly affected by simulated or actual insect defoliation (Welter 1991, Peterson et al. 1996, Peterson et al. 2004). These results suggest that plant species may respond differently to defoliation. In our study as with others, gas exchange

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81 measurements were made on single leaves whic h is not a true repres entation of the whole plants response to herbivory. Further studies on the effects of l eaf feeding by Diaprepes root weevils on whole-plant photosynthesis are warranted and should be compared to results with single leaf gas exchange. In both buttonwood experiments, leaf area was less for infested plants than controls, although the differences were not statis tically significant. Leaf, stem and root fresh and dry weights tended to be lower in we evil infested plants than control plants. However, the differences were not signifi cant except for the root dry weights of buttonwoods in the second experiment. Plants were only exposed to adult weevil feeding for a brief (two month) period and although in fested plants weighed less, plant weights were not significantly affected by leaf feed ing. Thus, longer-duration studies may yield statistically significant differences in gas ex change, leaf area a nd dry weights between infested and non-infested buttonwood and oak trees.

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Table 4-1. The effect of adult Diaprepes root weevil on net CO 2 assimilation (A), transpiration (E) and stomatal conductance (g s ) of buttonwood (Expt. 1) Treatment Leaf age Date A (mol CO2 m-2 s-1) E (mmol H2O m-2 s-1) gs (mmol CO2 m-2 s-1) 2/5/04 Pre-Infested young 6.05 2.10 133.36 Non-infested young 8.38 2.31 146.57 (P) (0.06) (0.47) (0.54) Pre-Infested mature 9.10 2.18 145.21 Non-infested mature 8.89 2.22 158.79 (P) (0.90) (0.91) (0.73) 3/9/04 Infested young 3.39 1.46 57.35 Non-infested young 1.41 1.29 46.42 (P) (0.09) (0.70) (0.58) Infested mature 2.77 1.23 49.35 Non-infested mature 1.10 1.13 42.64 (P) (0.21) (0.76) (0.68) 4/19/04 Infested young 5.70 2.55 121.00 Non-infested young 4.85 2.33 115.64 (P) (0.58) (0.66) (0.85) Infested mature 7.40 2.67 129.07 Non-infested mature 6.22 2.08 102.14 (P) (0.46) (0.21) (0.32) Significance determined by a standard t-test at the 0.05 level. 82

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83 Table 4-2. The effect of adu lt Diaprepes root weevil on net CO 2 assimilation (A), transpiration (E) and stomatal conductance (g s ) of buttonwood (Expt. 2) Treatment Leaf age Date A (mol CO2 m-2 s-1) E (mmol H2O m-2 s-1) gs (mmol CO2 m-2 s-1) 5/18/04 Pre-Infested young 4.96 3.92 180.21 Non-infested young 6.13 3.92 192.00 (P) (0.41) (0.99) (0.69) Pre-Infested mature 15.17 5.04 295.57 Non-infested mature 13.63 4.65 260.50 (P) (0.22) (0.29) (0.31) 6/18/04 Infested young 3.08 4.78 254.79 Non-infested young 7.60 5.01 264.71 (P) (0.02) (0.63) (0.73) Infested mature 13.55 5.25 313.71 Non-infested mature 14.40 4.78 285.86 (P) (0.45) (0.49) (0.62) 7/7/04 Infested young 2.47 5.09 200.29 Non-infested young 0.97 4.13 134.71 (P) (0.21) (0.11) (0.05) Infested mature 14.11 6.76 327.93 Non-infested mature 9.18 4.83 187.91 (P) (0.02) (0.02) (0.01) Significance determined by a sta ndard t-test at the 0.05 level.

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84 Table 4-3. The effect of adult Diaprepes root weevil on total leaf area of buttonwod Plant species Treatment leaf area (cm2) Experiment 1 Infested 6419.10 Non-infested 7861.20 (P) (0.10) Experiment 2 Infested 3401.00 Non-infested 4710.20 (P) (0.14) Significance determined by a standard t-test at the 0.05 level.

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85 Table 4-4. The effect of adult Diaprepes root weevil leaf feeding on buttonwood fresh and dry weights 2 months after infestation. Species Treatment Fresh weight (g) Dry weight (g) leaf stem root leaf stem root Exp.1 Infested 293.53 172.89 230.04 109.17 82.51 104.81 Non-infested 339.61 183.1 234.8 120.94 88.48 107.90 (P) (0.19) (0.67) (0.90) (0.34) (0.61) (0.84) Exp. 2 Infested 134.51 36.37 31.94 44.81 11.62 17.32 Non-infested 155.16 39.31 48.05 50.27 13.14 23.60 (P) (0.47) (0.69) (0.07) (0.59) (0.51) (0.05) Significance determined by a standard t-test at the 0.05 level.

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CHAPTER 5 SUMMARY AND CONCLUSIONS Larval root feeding by Diaprepes root weevil caused considerable damage to the root systems of buttonwood, live oak and pygmy date palm. Of the three species tested buttonwood, was affected more by larval root feeding than live oak and pygmy date palm. Buttonwood leaf gas exchange was significantly reduced by larval root feeding in two studies and several plants showed severe symptoms of stress in the form of leaf yellowing and wilt. Although larvae were recovered and larval root damage was observed on all three species, buttonwood appeared to have the most significant root feeding damage and the greatest reduction in root weight. These results, which are similar to those of Mannion et al. (2003), suggest that buttonwood may be a more suitable host for larvae development than live oak or pygmy date palm. However; these studies were conducted for a limited time period. Therefore, although buttonwood was more severely damaged than live oak or pygmy date palm, prolonged larval root feeding damage may cause considerable damage to live oak and pygmy date palm. Flooding is a relatively common occurrence in the low lying ornamental field nurseries, which consist of marl soils, during the summer months in south Florida. These areas also tend to have the highest populations of Diaprepes root weevils. In our study with containerized buttonwood and live oak, leaf gas exchange of both tree species were negatively affected by flooding. However, we found no significant interaction between pre-flood and larval treatments on leaf gas exchange compared with those treatments that were not pre-flooded prior to larval infestation. Larval root feeding did not negatively 86

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87 affect leaf gas exchange of either buttonwood or live oak for the relatively short infestation period. However, in comparison with the first larval root feeding study in which larvae were re-infested and allowed to feed on plant roots over a five month period, this study was conducted over a shorter period of time and larval root feeding was limited to a three month period. Therefore, the length of time in which plants are infested with larvae appears to affect how much damage they may cause to plant root systems. Leaf feeding by adult Diaprepes root weevils causes considerable damage to the leaf tissue of plants. Ornamental plants are grown and sold for the aesthetic appeal and any severely damaged plants are not able to be sold. The effects of leaf feeding damage on physiology and growth of several ornamental tree species, which are hosts of Diaprepes root weevil, have not been previously studied. In this study we measured leaf gas exchange to observe whether leaf feeding damage caused by adult Diaprepes root weevils negatively affects normal physiological functions of plants such as photosynthesis. We found that leaf gas exchange of weevil damaged buttonwood leaves generally had higher net photosynthetic rates on the remaining leaf area compared with controls. However, leaf gas exchange was measured on single leaves and although remaining leaf tissue had higher photosynthesis, there was less leaf area overall so whole plant net photosynthesis may be less. The results from these studies provide us with some basic understanding of the effects of Diaprepes root weevil feeding on physiology and growth of the selected plants. There are many ornamental species affected by this pest and its impact on some of the more economically important tree species is unknown. This information may be useful for future research to expand on the limited information

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88 on the effects of Diaprepes root weevil on ornamental plants and may also improve our knowledge for better management and control this pest.

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LIST OF REFERENCES Allen, J. C. 1978. The effect of citrus rust mite damage on citrus fruit drop. J. Econ. Entomol. 71: 746. Andersen, P., P. B. Lombard, and M. N. Westwood. 1984. Effect of root anaerobiosis on the water relations of several Pyrus species. Physiol. Plant. 62: 245-252. Andersen, P. C., and R. F. Mizell III. 1987. Physiological effects of galls induced by Phylloxera notabilis (Homoptera: Phylloxeridae) on pecan foliage. Environ. Entomol. 16: 264-268. Anderson, R. S. 2002. Curculionidae, pp. 722-806. In R. H. Arnett, M. Thomas, P. Skelley and J. H. Frank (eds.), American beetles. Vol. 2. CRC Press, Boca Raton, FL. Andrews, L. K., and L. F. La Pre. 1979. Effects of pacific spider mite on physiological processes of almond foliage. J. Econ. Entomol. 72: 651-654. Beavers, J. B. 1982. Biology of Diaprepes abbreviatus (Coleoptera: Curculionidae) reared on an artificial diet. Fla. Entomol. 65: 263-269. Beavers, J. B., and A. G. Selhime. 1975. Development of Diaprepes abbreviatus on potted citrus seedlings. Fla. Entomol. 58: 271-272. Beavers, J. B., and A. G. Selhime. 1976. Population dynamics of Diaprepes abbreviatus in an isolated citrus grove in central Florida. J. Econ. Entomol. 69: 9-10. Broschat, T. K., and A. Meerow. 2000. Ornamental Palm Horticulture. University Press of Florida. Gainesville, FL. Crane, J. H., and F. S. Davies. 1989. Flooding responses of Vaccinium species. HortScience 24: 203-209. Dehgan, B. 1998. Landscape Plants for Subtropical Climates. University Press of Florida. Gainesville, FL Dennis, E. S., R. Dolferus, M. Ellis, M. Rahman, Y. Wu, F. U. Hoeren, A. Grover, K. P. Ismond, A. G. Good, and W. J. Peacock. 2000. Molecular strategies for improving waterlogging tolerance in plants. J. Exp. Bot. 51: 89-97. 89

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90 Detling, J. K., D. T. Winn, C. Procter-Gregg and E. L. Painter. 1980. Effects of simulated grazing by below-ground herbivores on growth, CO 2 exchange and carbon allocation patterns of Bouteloua gracilis. J. Appl. Ecol. 17: 771-778. Emenegger, D. B., and R. E. Berry. 1978. Biology of strawberry root weevil on peppermint in western Oregon. Environ. Entomol. 7: 495-498. Futch, S. H., and C. W. McCoy Jr. 1993. Citrus Root Weevils. Circ. 1065. Fla. Coop. Ext. Serv., Inst. Food Agric. Sci., University of Florida, Gainesville, FL. Gilman, E.F., and D.G. Watson. 1993a. Phoenix roebelenii: pygmy date palm. ENH-600. Fla. Coop. Ext. Serv., Inst. Food Agric. Sci., University of Florida, Gainesville, FL. Gilman, E.F., and D.G. Watson. 1993b. Conocarpus erectus: buttonwood. ENH-338. Fla. Coop. Ext. Serv., Inst. Food Agric. Sci., University of Florida, Gainesville, FL. Godfrey, L. D., L. J. Mienke, and R. J. Wright. 1993. Effects of larval injury by western corn rootworm (Coleoptera: Chrysomelidae) on gas exchange parameters of field corn. J. Econ. Entomol. 86: 1546-1556. Grafton-Cardwell, E. E., K. E. Godfrey, J. E. Pea, C. W. McCoy, R. F. Luck. 2004. Diaprepes Root Weevil. University of California, Division of Agriculture and Natural Resources. Publication 8131, 8 pp. Graham, J. H., C. W. McCoy, and J. S. Rogers 1997. The Phytopthora-Diaprepes weevil complex. Citrus Industry 78: 67-70. Hare, J. D., and R. R. Youngman. 1987. Gas exchange of orange (Citrus sinensis) leaves in response to feeding injury by the citrus red mite (Acari: Tetranychidae). J. Econ. Entomol. 80: 1249-1253. Heichel, G. H., and N. C. Turner. 1973. Physiological responses of dogwood (Cornus florida) to infestation by the dogwood borer (Thamnosphecia scitula). Ann. Appl. Biol. 75: 401-408. Hodges, A., and J. Haydu. 2000. Economic Impacts of the Florida Environmental Horticulture Industry, 2000. Report EI 02-3. University of Florida, IFAS, Food and Resource Economics Department. Hou. X., L. J. Mienke and T. J. Arkebaur. 1997. Soil moisture and larval western corn rootworm injury: influences on gas exchange parameters in corn. Agron. J. 89: 709-717. Johnson, M. W., S. C. Welter, N. C. Toscano, I. P. Ting, and J. T. Trumble. 1983. Reduction of tomato leaflet photosynthetic rates by mining activity of Liriomyza sativae (Diptera: Agromyzidae). J. Econ. Entomol. 76: 1061-1063.

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91 Johnson, D. 1999. Floriculture and Environmental Horticulture: Situation and outlook report. USDA/ARS, FLO-1999. Jones, I. F., and W. J. Schroeder. 1983. Study of first-instar Diaprepes abbreviatus (Coleoptera: Curculionidae) activity for control purposes. J. Econ. Entomol. 76: 567-569. Klingeman, W. E., M. W. Van Iersel, G. D. Buntin, S. K. Braman. 2000. Whole-plant CO 2 exchange measurements on azaleas injured by azalea lace bug (Heteroptera: Tingidae) feeding. J. Econ. Entomol. 93: 352-356 Knapp, J. L. 1985. The citrus root weevils in Florida: an Extension Service perspective. Fla. Entomol. 68: 368-370. Knapp, J. L., S. E. Simpson, J. E. Pea, and H. N. Nigg. 2000. Diaprepes root weevil: What Floridians need to know. ENY-640 Fla. Coop. Ext. Serv., Inst. Food Agric. Sci., University of Florida, Gainesville, FL. Knapp, J. L., H. N. Nigg, S. E. Simpson, L. W. Duncan, J. H. Graham, J. E. Pea, C. W. McCoy and C. M. Mannion. 2001. Diaprepes root weevil: a pest of citrus, ornamental and root crops in Florida. ENY-645. Fla. Coop. Ext. Serv., Inst. Food Agric. Sci., University of Florida, Gainesville, FL. Kozlowski, T. T. 1984. Plant response to flooding of soil. Bioscience. 34: 162-167. Kozlowski, T. T. 1997. Response of woody plants to flooding and salinity. Tree Physiol. Monograph No.1. 1-29. Lakso, A. N., G. B. Mattii, J.P. Nyrop, and S. S. Denning. 1996. Influence of European red mite on leaf and whole-canopy carbon dioxide exchange, yield, fruit size, quality, and return cropping in Starkrimson Delicious apple trees. J. Amer. Soc. Hort. Sci. 121: 954-958. LaLone, R. S., and R. G. Clark. 1981. Larval development of Otiorhynchus sulcatus (Coleoptera: Curculionidae) and effects of larval density on larval mortality and injury to rhododendron. Environ. Entomol. 10: 190-191. Lapointe, S. L. 2000. History and importance of Diaprepes to agriculture in the Caribbean region. Diaprepes Short Course, Citrus Research & Education Center, Lake Alfred, FL, March 22, 2000. p. 8-12. Lapointe, S. L., J. P. Shapiro, K. D. Bowman. 1999. Identification of sources of plant resistance to Diaprepes abbreviatus (Coleoptera: Curculionidae) by three bioassays. J. Econ. Entomol. 92: 999-1004. Larson, K. D., and B. Schaffer. 1991. Flooding, leaf gas exchange and growth of mango in containers. J. Amer. Soc. Hort. Sci. 116: 156-160.

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92 Larson Vasquez, B. C., and O. N. Nesheim. 2000. Florida Crop/Pest Management profiles: Ornamentals. Fla. Coop. Ext. Serv., Inst. Food Agric. Sci., University of Florida, Gainesville, FL. Li, H., J.P. Syvertsen, R. J. Stuart, C. W. McCoy, A. W. Schumann and W. S. Castle. 2003a. Correlation of soil characteristics and Diaprepes abbreviatus root weevil populations in a poorly drained citrus grove. Proc. Fla. State Hort. Soc. 116: 242-248. Li, H., J. P. Syvertsen, C. W. McCoy, A. W. Schumann, and J. Dunlop. 2003b. Soil redox potential and leaf stomatal conductance of two citrus rootstocks subjected to flooding and root weevil feeding. Proc. Fla. State Hort. Soc. 116: 252-256. Li, J., and J. T. A. Procter. 1984. Simulated pest injury effects photosynthesis and transpiration of apple leaves. HortScience 19: 815-817. Li, S., S. R. Pezeshki and S. Goodwin. 2004. Effects of soil moisture regimes on photosynthesis and growth in cattail (Typha latifolia). Acta Ecologia 25: 17-22. Li, Y. 2001. Calcareous Soils in Miami-Dade County. SL 183. Fla. Coop. Ext. Serv., Inst. Food Agric. Sci., University of Florida, Gainesville, FL. Louda, S. M. 1986. Insect herbivory in response to root-cutting and flooding stress on a native crucifer under field conditions. Acta Ecologia/ Ecologia Generalis 7: 3753. Mannion, C.M., A. Hunsberger, J.E. Pea, and L.S. Osborne. 2003. Ovipostion and larval survival of Diaprepes abbreviatus (Coleoptera: Curculionidae) on select host plants. Fla. Entomol. 86: 165-173. McCoy, C. W., S. H. Futch, J. H. Graham, L. W. Duncan, and H. N. Nigg. 2003. Florida citrus pest management guide: citrus root weevils. ENY-611. Fla. Coop. Ext. Serv., Inst. Food Agric. Sci., University of Florida, Gainesville, FL. Metcalf. M. R., and R. L. Metcalf. 1993. Destructive and useful insects: Their habits and control, Fifth edition. McGraw-Hill, Inc. New York. Mobley, K. N., and R. P. Marini. 1990. Gas exchange characteristics of apple and peach leaves infested by European red mite and two-spotted spider mite. J. Amer. Soc. Hort. Sci. 115: 757-761. Moorehouse, E. R., A. K. Charnley, and A. T. Gillespie. 1992. A review of the biology and control of the vine weevil, Otiorhynchus sulcatus (Coleoptera: Curculionidae). Ann. Appl. Biol. 121: 431-454. Muraro, R. P. 2001. Cost benefit analysis for controlling Diaprepes. Lake Alfred, FL. University of Florida, IFAS Citrus Research and Education Center: 6 pp.

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93 Nigg, H. N., S. E. Simpson, N. E. El-Gholl, and F. G. Gmitter, Jr. 2001. Response of citrus rootstock seedlings to Diaprepes abbreviatus L. (Coleoptera: Curculionidae) larval feeding. Proc. Fla. State Hort. Soc. 114: 57-64. Noble, C. 1989. Marl Soils in South Florida. Soil Survey Horizons 30: 12-16. OBrian, C.W., and P.W. Kovarik. 2000. The genus Diaprepes: Its origin and geographical distribution in the Caribbean region. Diaprepes Short Course, CREC, Lake Alfred, FL, March 22, 2000. p. 1-6. Peterson, R. K., S. D. Danielson, and L. G. Higley. 1992. Photosynthetic responses of alfalfa to actual and simulated alfalfa weevil (Coleoptera: Curculionidae) injury. Environ. Entomol. 21: 501-507. Peterson, R. K., L. G. Higley, and S. M. Spomer 1996. Injury by Hyalaphora cercopia (Lepidoptera: Saturinidae) and photosynthetic responses of apple and crabapple. Environ. Entomol. 25: 416-422. Peterson, R. K., L. G. Higley, F. J. Haile, and J. A. F. Barrigossi. 1998. Mexican bean beetle (Coleoptera: Coccinellidae) injury affects photosynthesis of Glycine max and Phaseolus vulgaris. Environ. Entomol. 27: 373-381. Peterson, R. K., C. L. Shannon, and A. W. Lenssen. 2004. Photosynthetic responses of legume species to leaf-mass consumption injury. Environ. Entomol. 33: 450-456. Pezeshki, S. R. 1991. Root response of flood-tolerant and flood-sensitive tree species to soil redox conditions. Trees 5: 180-186. Pezeshki, S. R., J. H. Pardue, and R. D. DeLaune. 1996. Leaf gas exchange and growth of flood-tolerant and flood-sensitive tree species under low soil redox conditions. Tree Physiol. 16: 453-458. Ploetz, R. C., and B. Schaffer. 1989. Effects of flooding and Phytopthora root rot on net gas exchange and growth of avocado. Phytopathol. 79: 204-208. Quintela, E. D., J. Fan, and C. W. McCoy. 1998. Development of Diaprepes abbreviatus (Coleoptera: Curculionidae) on artificial and citrus root substrates. J. Econ. Entomol. 91: 1173-1179. Rand, T. A. 1999. Effects of environmental context on the susceptibility of Atriplex patula to attack by herbivorous beetles. Oecologia 121: 39-46. Riedell, W. E. 1990. Rootworm and mechanical damage effects on root morphology and water relations in maize. Crop Sci. 30: 628-631. Riedell, W. E., and R. N. Reese. 1999. Maize morphology and shoot CO 2 assimilation after root damage by western corn rootworm larvae. Crop Sci. 39: 1332-1340.

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94 Rilling, G., and H. Steffan. 1978. Experiments on the carbon dioxide fixation and assimilate import by leaf galls of phyloxera, Dactylosphaera vitifolii, on grapevine, Vitis rupestris 187G. Angew. Bot. 52: 343-354. Rogers, S., C. W. McCoy, and J. H. Graham. 2000. Larval growth of Diaprepes abbreviatus (Coleoptera: Curculionidae) and resulting root injury to three citrus varieties in two soil types. J. Econ. Entomol. 93: 380-387. Sances, F. V., J. A. Wyman, I. P. Ting, R. A. Van Steenwyk, and E. R. Oatman. 1981. Spider mite interactions with photosynthesis, transpiration and productivity of strawberry. Environ. Entomol. 10: 442-448. Schaffer, B., J. Pea, S. P. Lara, and D. Buisson. 1986. Net photosynthesis, transpiration, and stomatal conductance of avocado leaves infested by avocado red mites. Proc. Interamer. Soc. Trop. Hort. 30: 73-77. Schaffer, B., and L. J. Mason. 1990. Effects of scale insect herbivory and shading on net gas exchange and growth of a subtropical tree species (Guaiacum sanctum L.). Oecologia 84: 468-473. Schaffer, B., P. C. Andersen, and R. C. Ploetz. 1992. Response of fruit crops to flooding. Hort. Rev. 13: 257-313. Schaffer, B., J. E. Pea, A. M. Colls, and A. Hunsberger. 1997. Citrus leafminer (Lepidoptera: Gracillaridae) in lime: Assessment of leaf damage and effects on photosynthesis. Crop Protection 16: 337-343. Schroeder, W. J. 1981. Attraction, mating and oviposition behavior in field populations of Diaprepes abbreviatus. Environ. Entomol. 10: 898-900. Schroeder, W. J., R. A. Hamlen, and J. B. Beavers. 1979. Survival of Diaprepes abbreviatus larvae on selected native and ornamental Florida plants. Fla. Entomol. 62: 309-312. Schwarz, P. A., T. J. Fahey and T. E. Dawson. 1997. Seasonal air and soil temperature effects on photosyntheshsis in red spruce (Picea rubens) saplings. Tree Physiol. 17: 187-194. Simpson, S. E., H. N. Nigg, N. C. Coile and R. A. Adair 1996. Diaprepes abbreviatus (Coleoptera: Curculionidae): Host plant associations. Environ. Entomol. 25: 333-349. Simpson, S.E., H. N. Nigg, and J. L. Knapp. 2000. Host plants of Diaprepes root weevil and their implications to the regulatory process. Diaprepes Short Course, Citrus Research & Education Center, Lake Alfred, FL. pp. 19-37. Syvertsen, J. P., R. M. Zablotowicz, and M. L. Smith, jr. 1983. Soil temperature and flooding effects on two species of citrus. Plant and Soil 72: 3-12.

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95 Syvertsen, J. P. and C. W. McCoy. 1985. Leaf feeding injury to citrus by root weevil adults: Leaf area, photosynthesis and water use efficiency. Fla. Entomol. 68: 386-393. Taylor, A. O. and J. A. Rowley. 1971. Plants under climatic stress. Plant. Physiol. 47: 713-718. Tarrnat, C. A., and C. W. McCoy. 1989. Effect of temperature and relative humidity on the egg and larval stages of some citrus root weevils. Fla. Entomol. 72: 117-122. Thomas, M. C. 2005. Pest Alert: Myllocerus undatus Marshall, a weevil new to the Western Hemishpere. http://www.doacs.state.fl.us/pi/enpp/ento/weevil-pestalert.htm Tomlinson, P. B. 1980. The biology of trees native to tropical Florida. pp. 143-148. Harvard Univesity. Alliston, Mass. Umble, J. R., and J. R. Fisher. 2000. Temperature-dependant development of otiorhynchus ovatus (Coleoptera: Curculionidae) pupae. Population Ecol. 29: 758-764. Urias-Lopez, M. A., L. J. Meinke, L. G. Higley, and F. J. Haile. 2000. Influence of western corn rootworm (Coleoptera: Chrysomelidae) larval injury on photosynthetic rate and vegetative growth of different types of maize. Environ. Entomol. 29: 861-867. U.S. Depatment of Agriculture, (USDA). 2003. Floriculture Crops 2002 Summary, USDA. U.S. Department of Agriculture, A.R.S. (USDA). 2003. Floriculture and nursery crops situation outlook yearbook, market and trade economics division, USDA, FLO-2003. Weissling, T. J., J. E. Pea, R. M. Giblin-Davis, and J. L. Knapp Jr. 2002. Sugarcane rootstock borer weevil; Diaprepes abbreviatus (L.). Featured Creatures, University Florida, http://creatures.ifas.ufl.edu/citrus/sugarcane_rootstock_borer_weevil.htm Welter, S. C. 1989. Arthropod impact on plant gas exchange, p. 135-147. In: E. A. Bernays (eds.). Insect-Plant Interactions. Vol 1. CRC Press, Boca Raton, Fla. Welter, S. C. 1991. Responses of tomato to simulated and real herbivory by tobacco hornworm (Lepidoptera: Sphingidae). Environ. Entomol. 20: 1537-1541. Whiley, A. W., C. Searle, B. Schaffer and B. N. Wolstenholme. 1999. Cool orchard temperatures or growing trees in containers can inhibit leaf gas exchange of avocado and mango. J. Amer. Soc. Hort. Sci. 124: 46-51.

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96 Wolcott, G. N. 1936. The life history of Diaprepes abbreviatus L. at Rio Piedras, Puerto Rico. J. Agric. Univ. Puerto Rico 20: 883-914. Wood, B. W., W. L. Tedders, and J. M. Thompson. 1985. Feeding influence of three pecan aphid species on carbon exchange and phloem integrity of seedling pecan foliage. J. Amer. Soc. Hort. Sci. 110: 393-397. Woodruff, R. E. 1964. A Puerto Rican weevil new to the United States (Coleoptera: Curculionidae). Fla. Dept. Agric. Div. Plant Ind. Entomol. Circ. No. 30: 1-2 Woodruff, R. E. 1968. The present status of a West Indian weevil Diaprepes abbreviata L. in Florida (Coleoptera: Curculionidae). Fla. Dept. Agric. Div. Plant Ind. Entomol. Circ. No. 77. Woodruff, R. E. 1981. Citrus root weevils of the genus Pachneaus in Florida (Coleoptera: Curculionidae). Fla. Dept. Agric. Div. Plant Ind. Entomol. Circ. 231 Woodruff, R. E. 1982. Artipus floridanus Horn: another weevil pest of citrus. Fla. Dept. Agric. Div. Plant. Ind. Entomol. Circ. 237: 1-2. Woodruff, R. E. 1985. Citrus weevils in Florida and the West Indies: Preliminary report on systematics, biology and distribution (Coleoptera: Curculionidae). Fla. Entomol. 68: 370-3 Woodruff, R. E., and R. C. Bullock. 1979. Fullers rose weevil Pantomorus cervinus B. In Florida (Coleoptera: Curculionidae). Fla. Dept. Agric. Div. Plant. Ind., Entomol. Circ. 207.

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BIOGRAPHICAL SKETCH Alexander P. Diaz was born on July 8, 1977, in Miami, Florida. In January 1998, he was enrolled in the University of Florida and received a bachelors degree in Environmental Horticulture in May 2001. In August 2002, he was offered a graduate research assistantship to study the effects of Diaprepes root weevil feeding on ornamental plant physiology and growth at the University of Florida, Tropical Research and Education Center. He enrolled in the M.S. degree program in the Entomology and Nematology Department at the University of Florida under the supervision of Dr. Catharine Mannion and Dr. Bruce Schaffer of the Horticultural Sciences Department. After graduation, Alex will pursue a career in the ornamental plant nursery industry in south Florida. 97


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Permanent Link: http://ufdc.ufl.edu/UFE0011363/00001

Material Information

Title: Effect of Diaprepes Root Weevil on Leaf Gas Exchange and Growth of Select Ornamental Tree Species
Physical Description: Mixed Material
Copyright Date: 2008

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
System ID: UFE0011363:00001

Permanent Link: http://ufdc.ufl.edu/UFE0011363/00001

Material Information

Title: Effect of Diaprepes Root Weevil on Leaf Gas Exchange and Growth of Select Ornamental Tree Species
Physical Description: Mixed Material
Copyright Date: 2008

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
System ID: UFE0011363:00001


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Full Text












EFFECT OF DIAPREPES ROOT WEEVIL ON LEAF GAS EXCHANGE AND
GROWTH OF SELECT ORNAMENTAL TREE SPECIES















By

ALEXANDER P. DIAZ


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE

UNIVERSITY OF FLORIDA


2005

































Copyright 2005

by

Alexander P. Diaz















ACKNOWLEDGMENTS

I would like to thank Catharine Mannion and Bruce Schaffer for granting me this

opportunity to pursue graduate studies and providing funding for this research project. I

would also like to thank them along with Susan Webb for their guidance and support

throughout this experience. I would like to thank all those who helped me in many

different ways while working on my research, especially Holly Glenn, Julio Almanza,

Karen Griffin, Mike Gutierrez and Dalia Stubblefield. Finally, I would like to thank my

family for their continual encouragement and support.
















TABLE OF CONTENTS

page

A C K N O W L E D G M E N T S ................................................................................................. iii

LIST OF TABLES ....................................................... ............ ....... ....... vi

L IST O F F IG U R E S .... ...... ................................................ .. .. ..... .............. vii

ABSTRACT ........ .............. ............. ...... .......... .......... ix

CHAPTER

1 LITERATURE REVIEW ............................................................ ................ 1

In tro du ctio n ...................................... ............................. ..... ......... ...... .
W eevils ...................................... ................. ................ ......... 2
R oot W eev ils ............................................... ......................... 3
C itru s R oot W eevils ................................................... .. ........ .......... .. ....
D iaprepes R oot W eevil............................................................... .....................7
D am age .................. ................ ........................................... ...... ..9
Arthropod feeding and whole-plant physiology...........................................11
Flooding stress and plant physiology .........................................................16
The ornam ental plant industry......... ........................................ ............... 18
Current and potential impact of Diaprepes root weevil on the ornamental
plant industry ......................................... ............ ............ 19
Buttonwood, Live Oak and Pygmy Date Palm .................................................20
R research O bjectives.......... .............................................................. ......... ....... 22

2 EFFECT OF LARVAL ROOT FEEDING BY DIAPREPES ROOT WEEVIL ON
LEAF GAS EXCHANGE AND GROWTH OF SELECT ORNAMENTAL TREE
S P E C IE S ........... .... ....... ............................................. .... 2 3

In tro d u ctio n ............. .. .......... .. ................ ................................ 2 3
M materials and M methods ....................................................................... ..................2 5
Plant and Insect M material ............................................................................. 26
E x p erim ent 1 ............................................................2 6
E xperim ent 2 ........................ ....... ............ ..... ..... .... 26
T re a tm e n ts ..................................................................................................... 2 7
E x p erim ent 1 ............................................................2 7
E xperim ent 2 ...................... .................... ................. ..... .. 28










Tem perature M easurem ents ........................................... ........................... 29
Statistical A analysis ...................... .................... .................. ....... 30
R e su lts ...........................................................................................3 0
E x p e rim e n t 1 .................................................................................................. 3 0
E x p e rim e n t 2 .................................................................................................. 3 2
D isc u ssio n .............................................................................................................. 3 5

3 EFFECT OF FLOODING AND DIAPREPES ROOT WEEVIL LARVAL
FEEDING ON LEAF GAS EXCHANGE AND GROWTH OF BUTTONWOOD
A N D L IV E O A K ................................................................................................... 54

In tro d u ctio n ........................................................................................5 4
M materials an d M eth od s .......................................................................................... 56
Plant and Insect Material .................................................56
T re a tm e n ts ...................................................................................................... 5 6
Soil R edox Potential ................................................ ............... 57
L eaf G as E x ch an g e ......................................................................................... 5 8
Plant Growth and Larval Recovery ........................................ ............... 58
Statistical A naly sis ............................................................58
R e su lts .........................................................................................................................5 9
Soil R edox Potential (Eh) ................................................................ ........... 59
L eaf G as E x ch an g e ......................................................................................... 59
Plant Growth and Larval Recovery ........................................ ............... 60
D isc u ssio n .............................................................................................................. 6 1

4 EFFECT OF ADULT DIAPREPES ROOT WEEVIL ON LEAF GAS
EXCHANGE AND GROWTH OF BUTTONWOOD AND LIVE OAK .................72

In tro d u ctio n .......................................................................................7 2
M materials an d M eth od s .......................................................................................... 74
Plant and Insect Material .................................................74
L eaf G as E x ch an g e ......................................................................................... 7 5
P la n t b io m a ss .................................................................................................. 7 6
Statistical A naly sis ...........................................................76
R e su lts ...............................................................................................7 6
V isible Signs of H erbivory ......................................................... ...... .........76
L eaf G as E x ch an g e ......................................................................................... 7 7
P lant B iom ass ...............................................................7 7
D isc u ssio n .............................................................................................................. 7 8

5 SUMMARY AND CONCLUSIONS ............................................................... 86

L IST O F R E F E R E N C E S .............................................................................................. 89

B IO G R A PH IC A L SK E T C H ........................................................................................ 97





v
















LIST OF TABLES


Table p

2-1 The effect of Diaprepes root weevil larvae on height and trunk diameter of
button ood and live oak trees..................................................................... ....... 51

2-2 The effect of Diaprepes root weevil larvae on buttonwood leaf, stem and root
fresh and dry weights at each harvest date. ....................................................... 52

2-3 The effect of Diaprepes root weevil larvae on leaf stem and root fresh and dry
weights of live oak and pygmy date palm............... ............. ............. 53

2-4 Number of Diaprepes root weevil larvae recovered from plants harvested 2, 3, 4
& 5 m months after infestation ........................................................................ ....... 53

3-1 The effect of flooding (FLD) and insect infestation (INFST) on leaf gas
exchange of buttonwood and live oak trees. ................. ............ ............ ...70

3-2 The effect of Diaprepes root weevil larvae on leaf, stem, root and total biomass
of infested or non-infested buttonwood and live oak trees. ................................71

4-1 The effect of adult Diaprepes root weevil on net CO2 assimilation (A),
transpiration (E) and stomatal conductance (gs) of buttonwood (Expt. 1)...............82

4-2 The effect of adult Diaprepes root weevil on net CO2 assimilation (A),
transpiration (E) and stomatal conductance (gs) of buttonwood (Expt. 2)...............83

4-3 The effect of adult Diaprepes root weevil on total leaf area of buttonwod .............84

4-4 The effect of adult Diaprepes root weevil leaf feeding on buttonwood fresh and
dry weights 2 months after infestation. ....................................... ............... 85















LIST OF FIGURES


Figure page

2-1 Mean daily and monthly temperature (A) Air (B) Soil in containers. ...................40

2-2 Net CO2 assimilation (A), transpiration (E) and stomatal conductance (gs) of
buttonwood plants infested with medium or large Diaprepes root weevil larvae
or non-infested control plants........................................................ ............... 41

2-3 Net CO2 assimilation (A), transpiration (E) and stomatal conductance (gs) of live
oak plants infested with medium or large Diaprepes root weevil larvae or non-
infested control plants (C) ............................................ ..... ........................ 42

2-4 The effect of medium or large Diaprepes root weevil larvae on mean root fresh
and dry weights of buttonwood trees ............................................ ...............43

2-5 The effect of medium or large Diaprepes root weevil larvae on mean root fresh
and dry w eights of live oak trees.......................... ......................... ... ............ 44

2-6 Net CO2 assimilation (A), transpiration (E) and stomatal conductance (gs)
measured prior to harvest of buttonwood plants infested with Diaprepes root
w eevil larvae or not infested ............................................................................. 45

2-7 Net CO2 assimilation (A), transpiration (E) and stomatal conductance (gs) of the
buttonwood plants infested monthly with 20 larvae and a total of 80 Diaprepes
root weevil larvae by month 4 or not infested................................................46

2-8 Net CO2 assimilation (A), transpiration (E) and stomatal conductance (gs)
measured prior to harvest of live oak plants infested with Diaprepes root weevil
larvae or not infested. ...................... ................ ............................ 47

2-9 Net CO2 assimilation (A), transpiration (E) and stomatal conductance (gs) of live
oaks plants infested monthly with 20 larvae and a total of 80 Diaprepes root
w eevil larvae by m onth 4 or not infested .................................................................48

2-10 Net CO2 assimilation (A), transpiration (E) and stomatal conductance (gs)
measured prior to harvest of pygmy date palm infested with Diaprepes root
weevil larvae or not infested ...................................................................... 49









2-11 Net CO2 assimilation (A), transpiration (E) and stomatal conductance (gs) of the
final harvest set of pygmy date palms infested monthly with 20 larvae and a total
of 80 Diaprepes root weevil larvae by month 4 or not infested ............................50

3-1 Soil redox potential (Eh) of flooded buttonwood (A) and live oak (B) in
containers. ...........................................................................64

3-2 Net CO2 assimilation (A), transpiration (E) and stomatal conductance (gs) of
flooded and non-flooded buttonwood trees................ ................ ....... ... ........ 65

3-3 Net CO2 assimilation (A), transpiration (E) and stomatal conductance (gs) of
flooded and non-flooded live oak trees.............................................................. 66

3-4 Net CO2 assimilation (A), transpiration (E) and stomatal conductance (gs) of
buttonwood trees infested with Diaprepes root weevil larvae or non-infested ........67

3-5 Net CO2 assimilation (A), transpiration (E) and stomatal conductance (gs) of live
oak trees infested or non-infested with Diaprepes root weevils............................. 68

3-6 Larvae recovered from pre-flooded or non-flooded treatments of buttonwood
an d liv e oak ....................................................... ................. 69















Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

EFFECTS OF DIAPREPES ROOT WEEVIL ON LEAF GAS EXCHANGE AND
GROWTH OF SELECT ORNAMENTAL TREE SPECIES

By

Alex P. Diaz

August 2005

Chair: Catharine Mannion
Cochair: Bruce Schaffer
Major Department: Entomology and Nematology

The Diaprepes root weevil, Diaprepes abbreviatus L. (Coleoptera: Curculionidae),

is a serious and economically important pest of citrus and many ornamental plants grown

throughout the state of Florida. Studies were conducted to evaluate the effects of root

feeding by Diaprepes root weevil larvae on leaf gas exchange and growth of three

ornamental tree species commonly grown in south Florida that are known hosts of this

weevil. Buttonwood (Conocarpus erectus), live oak (Quercus virginiana) and pygmy

date palm (Phoenix roebelenii) in containers were infested with Diaprepes root weevil

larvae and leaf gas exchange was measured monthly to determine the effects of larval

root feeding on net CO2 assimilation (A), transpiration (E), stomatal conductance (gs).

Leaf, stem and root fresh and dry weights of each species were also determined. In one

of two tests, larval root feeding significantly reduced A, E and gs of infested buttonwood

trees. Leaf gas exchange of live oak was not affected by larval infestation. The effects of









multiple infestations of larvae on leaf gas exchange and fresh and dry weights of

buttonwood, live oak and pygmy date palm were also tested. Net CO2 assimilation, E

and gs and dry weights of buttonwood were reduced as a result of larval root feeding,

whereas there was no effect of multiple larval infestation on leaf gas exchange of live oak

or pygmy date palm. There was no effect of multiple larval infestations on dry weights

of live oak, but leaf, stem and dry root weight of pygmy date palm was lower for infested

plants than non-infested plants.

The interaction between pre-flooding (prior to larval infestation) and larval

infestation on leaf gas exchange and growth of buttonwood and live oak was also tested.

Net CO2 assimilation, E and gs of buttonwood and live oak were reduced by flooding, but

there was no significant interaction between pre-flooding treatment and larval infestation

treatment on these variables. Also, after two and three months of infestation there was no

significant difference in A, E and gs between infested and non-infested buttonwood or

live oak trees. In that study, fresh and dry root weights of buttonwood but not live oak

were reduced as a result of larval infestation.

The effect of adult Diaprepes root weevil infestation on leaf gas exchange and

growth of buttonwood and live oak was tested. Generally, there was no significant effect

of adult weevil infestation on A, E or gs. Leaf area of buttonwood was less for infested

plants than non-infested plants. Weevils fed on both mature and young leaves of

buttonwood. However, live oak did not produce any new leaf flushes during the

treatment period and adult weevils did not feed on the mature leaves of live oak. It

appears from our results that buttonwood is a more suitable host for development of

Diaprepes root weevil larvae.














CHAPTER 1
LITERATURE REVIEW

Introduction

The Diaprepes root weevil, Diaprepes abbreviatus L. (Coleoptera: Curculionidae),

is a severe pest of citrus, ornamental plants and root crops in Florida. It was first reported

in Florida in 1964 from a nursery in Apopka where it was believed to have been

introduced in an ornamental plant shipment from Puerto Rico (Woodruff 1968). In

Puerto Rico and the Caribbean, where this species is believed to have originated, the

weevil has been a major pest of sugarcane since the early 1900s and more recently has

become a pest of citrus, sweet potato and ornamentals (Lapointe 2000). Since its

introduction into Florida, Diaprepes root weevil has spread throughout most agricultural

areas of the state and is causing significant economic loss to Florida's agriculture.

Diaprepes root weevil is a polyphagous species associated with about 270 plant

species in Florida and is presently infesting approximately 140,000 acres in twenty-two

counties (Simpson et al. 2000). The Florida citrus industry is significantly affected by

this pest, which costs citrus growers about $72 million annually. Most of the published

research with Diaprepes root weevil has focused on citrus and there is almost no

information on the effects of this pest on ornamental plants.

Ornamental plants are one of the largest agricultural commodity groups in Florida.

Nationwide, Florida is ranked second in wholesale value of ornamental plants just after

California, which leads in ornamental plant production (USDA Floriculture Crops

Summary, 2003). Many of the economically important ornamental plant species grown






2


in Florida are known hosts of Diaprepes root weevil (Mannion et al. 2003). Most of these

plant species support all life stages of the weevil including the most damaging and

difficult to detect larval stage. Many ornamental trees are able to withstand severe root

damage from Diaprepes root weevil before visible symptoms (leaf yellowing, wilting and

decline) are apparent (Knapp et al. 2001).

There is a general lack of information about Diaprepes root weevil on ornamental

crops. It is important to obtain physiological baseline data on the effects of Diaprepes

root weevil on these plant species in order to identify the plant species that are most

susceptible to predation by this insect and ultimately be able to manage and control this

pest on these crops.

Weevils

The order Coleoptera contains many families of insects. The family Curculionidae

(weevils) is one of the most diverse groups containing more than 60,000 described

species worldwide (Anderson 2002). This family of beetles, like most others, can be

found throughout North America, with most of the diversity in the southern United

States. Most weevils feed on living plants; however a few species are saprophagous.

Weevils can be found in different plant ecosystems including some species that are

associated with freshwater habitats. Weevils are relatively easy to recognize due to their

elongate rostrum and characteristic geniculate antennae. The rostrum is highly variable

depending on species; long and narrow on some species and short on others. Mouth parts

are located at the anterior of the rostrum, body shape and size are widely variable and

these characteristics separate this family into subfamilies. The larvae of weevils are

legless and have a semi-circular shape. The white, grub-like larvae generally feed on

nuts, seeds, fruits, buds, stems or plant roots (Metcalf and Metcalf 1993). Adult and









larval feeding habits can vary and this variation is useful for classification of weevils into

separate subfamilies (Anderson 2002).

Root Weevils

Weevils with polyphagous adults and larvae fall into the subfamily Entiminae

(Anderson 2002). Broad-nosed weevils is a common name for this subfamily and

according to O'Brian and Kovarik (2000) there are about 14,000 known species

belonging to this group. They are distinguishable by their relatively short snout

compared to other weevils. One recognizable and distinct characteristic of almost all

weevils in the subfamily Entiminae is that closing adults have a pair of mandibles which

is shed after emergence from the soil that leaves a scar at the point of attachment

(Anderson 2002). Generally, larvae feed on roots and adults tend to feed on foliage.

Most species within this subfamily are generalist feeders, although certain species feed

only on specific plant species. Some species in the subfamily Entiminae are

parthenogenic and have no known males. This subfamily is the most diverse in North

America and contains 124 genera and 23 tribes (Anderson 2002).

Root weevil species are relatively similar to each other in their biology and habits.

Depending on the species, adults lay their eggs in the soil or on foliage of host plants.

The eggs, which are laid on leaves, hatch, the larvae drop to the soil surface and begin

moving into the soil to find roots. Most Entiminae larvae begin feeding on small fibrous

roots and as they develop damage larger roots. For the most part, adults feed on newly

emerging leaf flushes, and characteristically feed on margins of leaves leaving semi-

circular notches. Adults of most root weevils emerge from the soil and begin feeding,

mating and laying their eggs. Several species in this subfamily are unable to fly

(Anderson 2002, Emenegger and Berry 1978).









Many species of root weevils are economically important pests of ornamental,

agricultural or forest plants. Root weevils that are economically important include sweet

potato weevil Cylasformicarius elegantulus Summers, black vine weevil Otiorhynchus

sulcatus F., strawberry root weevil Otiorhynchus ovatus L., and several species of citrus

root weevils. The sweet potato weevil is a pest of sweet potato and the larval stage of

this weevil causes injury to roots and tubers. These larvae have been known to cause 25

to 75% crop loss (Metcalf and Metcalf 1993). Sweet potato weevil is believed to have

been introduced into the United States from Asia and is found in several states including

South Carolina, Georgia, Florida, Alabama, Mississippi, Louisiana, Texas and Hawaii.

Adult weevils feed on leaves and stems of plants. Females mate and then deposit their

eggs in small holes on roots and tubers below ground. Larvae then hatch and feed within

the stem or tubers. Larvae generally feed for two to three weeks and cause the sweet

potato to develop a foul smell and a bitter taste. The strawberry root weevil is a pest of

many crops in North America. Host plants include strawberry, a variety of small fruit

crops and ornamental plants. In the Pacific Northwest, Otyrynchus spp. feeds on many

agricultural crops such as peppermint, nursery evergreens, strawberries, cane fruits and

several ornamental nursery crops (Umble and Fisher 2000). Female strawberry root

weevils are parthenogenic and cannot fly (Emenegger and Berry 1978). Larvae move

into soil after hatching and begin feeding on roots and underground portions of stems.

Adult weevils feed on leaves, but like most root weevil species most damage is caused by

root feeding larvae. The black vine weevil is a serious pest of ornamental and small fruit

crops in temperate areas around the world (Moorehouse et al. 1992). This pest affects

nursery plants and is believed to have originated from temperate areas of Europe. The









spread of this weevil to new areas such as Australia and the United States is thought to

have been through infested plant shipments. Infestations can cause serious damage and

economic loss to growers. Similar to other root weevil species, most of the damage is

caused by larvae feeding on roots. LaLone and Clark (1981) reported that as few as three

larvae can kill Rhododendron spp. The life cycle of the black vine weevil is similar to

other root weevils.

Recently a new root weevil has entered Florida, Myllocerus undecimpustulatus

Faust (Thomas 2005). This root weevil from Sri Lanka was first discovered in Broward

County and is believed to be established in southeast Florida from the Homestead area

north to Boca Raton. The adult weevil has a wide host range that includes citrus and

many fruit and ornamental crops. Not much is known about this weevil. However, since

it has become established in south Florida and has a wide host range, it has potential to

spread and cause damage to many crops. Like most root weevils, larvae remain in the

soil and can easily be spread through nursery shipments.

Citrus Root Weevils

Citrus root weevils comprise several genera that cause serious injury to citrus in

several states including Texas and Florida. According to Woodruff (1985) there are

about 11 genera of root weevils that are associated with citrus in Florida and the West

Indies. Genera of weevils in this subgroup include Artipus, Cleistolophus, Compsus,

Diaprepes, Epicaerus, Exophthalmus, Lachnopus, Litostylus, Pachnaeus, Pantomorus,

and Tanymecus (Woodruff 1985). Within these genera about five species are pests of

citrus in Florida. The Diaprepes root weevil Diaprepes abbreviatus L., Fuller rose beetle

Pantomorus cervinus Boheman, citrus root weevil Pachnaeus litus Germar, blue-green









weevil Pachnaeus opalus Oliviar, and the little leaf notcher Artipusfloridanus Horn,

have been observed on citrus (Knapp 1985).

The Fuller rose beetle is a pest of Florida citrus and many ornamental crops. This

root weevil is found in many parts of the world including many states in the U.S. In

Florida, the Fuller rose beetle is mainly found north of Lake Okeechobee (Woodruff and

Bullock 1979). Adult weevils feed on leaves of many host plants, although most damage

is caused by the larvae which feed on roots. Two species of root weevils in the genera

Pachneus are native to Florida. They are broadly known as citrus root weevils. Both

species are blue-green in color but P. opalus, which is called the blue-green weevil, is

found in the northern half of Florida, while P. litus is only found in the lower half of

southern Florida. Both species are considered serious pests of citrus as larvae and have

been responsible for girdling tap roots and killing citrus trees (Woodruff 1981). The little

leaf notcher, is another root weevil pest of citrus that is believed to occur on over 150

different host plants (Woodruff 1982). This weevil has a similar life cycle to most root

weevils in that the adults feed on leaves and larvae feed underground on roots. This

weevil is common in the Bahamas and on the east coast of Florida, from the lower keys

up to Daytona Beach. The little leaf notcher is believed to be of West Indian origin

(Woodruff 1982). Diaprepes root weevil, is an economically important pest in Florida

and in areas of the Caribbean including Puerto Rico were it causes damage to citrus and

sugar cane. This is one of the larger root weevils and is known to cause severe damage to

a large number of agriculturally important crops, most importantly citrus. Additionally,

other plant species, including several tropical and sub-tropical fruit trees in southern









Florida and several economically important ornamental crops in central and southern

Florida are affected by this introduced pest.

Diaprepes Root Weevil

The Diaprepes root weevil was first collected in the West Indies by Carl Linnaeus

who described it as Curculio abbreviatus in the tenth edition of Systema Naturae in 1758

(Lapointe 2000). The West Indies, primarily Puerto Rico, is believed to be the center of

origin and diversity for this species. In the United States, Diaprepes root weevil was first

reported in a nursery in Apoka, Florida in 1964, and was believed to have been

introduced in an ornamental plant shipment from a nursery in Puerto Rico (Woodruff

1964). Since the time of its introduction into Florida this weevil has seriously affected

citrus orchards and has spread throughout most of central and southern Florida.

Diaprepes root weevil is a polyphagous species and is known to feed on a wide variety of

plants throughout Florida. Reports show that Diaprepes root weevil has a host range of

about 270 plant species (Simpson et al. 1996). In Florida alone it is estimated that this

weevil causes about 70 million dollars in damage annually (Weissling et al. 2002).

Adult females deposit their eggs in masses protected by leaves that are held

together by a secretion released during oviposition. Diaprepes root weevil lay their eggs

at night which are deposited in a single layer that usually forms an irregularly shaped

cluster. Schroeder (1981) observed in Florida that females deposited eggs mainly during

the dark hours between 6 PM and 6 AM. The eggs are oblong to oval in shape, uniformly

white and about 1.2 mm long and 0.4 mm wide (Wolcott 1936). Woodruff (1968)

reported that the number of eggs can vary between 30 and 264 per cluster, and that a

single female may lay up to 5,000 eggs or more during her lifetime. Beavers (1982)

reported a slightly larger maximum number of about 6,517 eggs per female. After eggs









hatch, neonates fall to the ground and begin to enter the soil. Soil moisture is believed to

affect the ability of neonates to enter the ground. Jones and Schroeder (1983) tested the

effects of soil moisture and the ability of neonates to penetrate the soil surface at varying

soil moisture contents. Their results showed that neonates did not enter soil that had an

extremely low soil moisture content of 0%, and the optimum soil moisture for larvae to

enter the soil was between 7.0 and 16.7 %. After entering the soil, neonates begin to feed

on fibrous roots, and as they mature, move to larger roots (Knapp et al. 2000).

Beavers and Selhime (1975) reported that the developmental period for larvae

reared on potted citrus in Florida is highly variable. The number of instars before

pupation may vary, but generally after about 11 instars, larvae pupate (Quintela et al.

1998). Adults emerge from the soil year round; however, the peak emergence period is

from May through October. As the adults dig their way out of the soil they shed a pair of

deciduous mandibles. Adult weevils emerge from the soil and move up the tree canopy

to feed and mate. Oviposition generally begins 3-7 days after emergence from the soil

(Wolcott 1936). The body length of adult weevils is highly variable (0.95 to 1.90 cm).

The color of adult weevils varies between individuals; however black with orange, white

or yellowish scales on the elytra is typical of this species. The average longevity reported

for adults reared on an artificial diet was about 147 day for females and 135 days for

males (Beavers 1982). The average life cycle of Diaprepes root weevil has been reported

to last anywhere from less than one year to more than two years (Wolcott 1936). Beavers

and Selhime (1976) reported that in Florida overlapping generations of weevils occur

throughout the year.









Damage

Damage caused by adult weevils is primarily leaf notching. They tend to feed on

new leaf flushes and occasionally on fruit. Experiments performed by Syvertsen and

McCoy (1985) showed that leaf feeding by adult weevils on citrus caused a reduction in

net photosynthesis due to the amount of leaf area consumed. Reduction in transpiration

was also related to the percentage of leaf area removed. They reported a 20% reduction

in water use efficiency, which was greater than the reduction in photosynthesis.

Although leaf feeding may cause damage to citrus and ornamental plants, the most

destructive effect of this weevil is caused by the larval stages feeding on roots (Knapp et

al. 2001).

Larvae tend to feed on all underground portions of the plant including roots and

tubers. Neonates enter the soil and begin feeding on fibrous roots. As the larvae mature

they feed on larger lateral roots. Diaprepes root weevil has a period of rapid growth

starting about 3 to 4 months after eggs hatch, at which time weevils are most active and

tend to cause serious injury (Beavers 1982). Rogers et al. (2000) reported that weight

gain was greatest for the 6th to 8th instars and that larvae at this stage tended to require a

large amount of nutrients. For this reason it is believed that weevil larvae at this stage

feed on larger roots and cause the greatest amount of damage. The larvae cause damage

by feeding their way through the outer bark and cambium layer (Futch and McCoy 1993).

Large root weevil larvae have been known to girdle roots and cause tree death by

continuous root feeding, which in many cases allows entry for root pathogens that can

cause plant disease (McCoy et al. 2003). Open wounds resulting from root feeding allow

easy entry for pathogens that would not ordinarily affect plant roots; this feeding has been

associated with root diseases by fungal genera such as Fusarium and Phytophthora.









Graham et al. (1997) reported that about 30,000 acres of citrus trees have been devastated

by Diaprepes root weevil. Most of the root damage was associated with Phytopthora and

the greatest impact was caused in fields of poorly drained, heavy soils in flatwood

orchards. They also reported that larval root feeding lessened resistance of relatively

resistant rootstocks of 'Swingle' citrumelo.

Information on the effects of Diaprepes root weevil on ornamental crops is limited.

However, the available information shows that there is a wide variety of host plants.

Several plants including dahoon holly (Ilex cassine L.), silver and green buttonwood

(Conocarpus erectus L.), pigmy date palm (Phoenix roebelenii O'Brien), and many other

ornamentals have been observed to be hosts (Knapp et al. 2000, Simpson et al. 1996). In

a recent field survey of several ornamental field nurseries in Miami-Dade County,

Florida, Mannion et al. (2003) found that several ornamental plant species supported a

large number of adult weevils and weevil egg masses. Species observed to have high

percentages of egg masses included live oak (Quercus virginiana Mill.), silver

buttonwood, and black olive (Bucida buseras L.). Plants that were observed to have the

highest presence of adult weevils were dahoon holly, cocoplum (Chrysobalanus icaco

L.), black olive, live oak, Bauhinia sp. and Cassia sp. Experiments performed by

Schroeder et al. (1979) to test survival of larvae on selected native and ornamental plants

in Florida showed that, out of 65 plants, 6 native and 9 non-native ornamental plant

species supported larval development. Many plant species support the complete life

cycle of Diaprepes root weevil. However, several plants are only hosts for adult weevils,

whereas other plant species only support larval feeding. Several tropical fruit crops

commonly grown in southern Florida have also been observed to be hosts of Diaprepes









root weevil. Adult weevils have been observed feeding on papaya foliage and fruit

(Knapp et al. 2000). Mannion et al. (2003) performed several tests on a variety of

different plant species including green bean (Phaseolus vulgaris L.), silver buttonwood,

lime (Citrus aurantifolia C.), malanga (.X- 1uh,i,/iI, I sagittifolium L.), satinleaf

(Chrysophillum olivirorme L.), sorghum-sudan (Sorghum Sudanese Piper), cassava

(Manihot esculenta L.), and dahoon holly to determine larval survival and the amount of

root consumption. Their results showed that silver buttonwood had more larvae with the

greatest weights than the other plant species tested. The results suggest that silver

buttonwood may be a preferred host for complete development of Diaprepes root weevil

from the egg stage to adult.

It has been reported that many ornamental trees can support a large amount of

larval injury before leaf yellowing and other symptoms occur (Knapp et al. 2000).

However there are no studies on how much damage a species can tolerate and which

species, if any, can tolerate continuous larval and adult feeding. There are many

economically important ornamental species being grown in southern Florida that are

affected by Diaprepes root weevil and there are no physiological baseline data that show

which species are most susceptible to damage. Pygmy date palm, buttonwood and live

oak are all grown in south Florida field nurseries and support all developmental stages of

Diaprepes root weevil.

Arthropod feeding and whole-plant physiology

Herbivory by insects causes economic damage to many agricultural crops. Welter

(1989) classified insect herbivores according to the type of feeding damage caused such

as, defoliators, gall former, epidermal feeders, phloem feeders, stem borers and root

feeders. There have been many studies on the effects of arthropod herbivory on plant









physiology. Some studies have found beneficial or no effects of insect feeding on plant

physiology, while the majority of studies have found negative effects on physiological

functions of plants. A basic understanding of the physiological responses of plants to

herbivory may provide critical information needed to predict and prevent crop damage

(Welter 1989).

Measuring plant photosynthesis can provide information about the effects of insect

feeding and plant stress prior to the presence of any visible symptoms (Welter 1989,

Peterson et al. 1992, Peterson et al. 1998). Results from studies of arthropod herbivory

on plant photosynthesis have been variable; some studies of leaf feeding arthropods

indicate no significant effects on photosynthetic rates of plants that they feed upon.

Peterson et al. (1996) found no significant effects on photosynthesis due to simulated or

actual leaf consumption by the larvae of the cecropia moth, Hyalaphora cecropia (L.) on

crabapple (Pyrus coronaria L.) and apple (Pyrus malus L.) leaves compared to controls

with no feeding damage. Other reports, however, indicate a reduction in net

photosynthesis as a result of arthropod herbivory. Li and Proctor (1984) reported

simulating defoliation by cutting leaf midribs reduced net photosynthesis more than

lateral vein and lamina cuts. Those experiments indicated that the section of the leaf that

is fed upon may be critical in determining if net photosynthesis is affected by insect

herbivory.

Syvertsen and McCoy (1985) determined the effects of leaf feeding by the citrus

root weevil, Artipusfloridanus, on net CO2 assimilation, transpiration, and water use

efficiency of citrus leaves. Weevil feeding characteristically resulted in leaf notching,

which only removed the outer layers of leaf lamina, and no damage to the mid-vein was









observed. The percentage of leaf area removed significantly increased as the weevil

population increased, and water loss increased proportionally more than net

photosynthesis declined (Syvertsen and McCoy 1985). Results of that study showed that

there was a reduction of about 20% in water use efficiency after about 40% of the leaf

area was consumed, indicating that the loss of photosynthetic leaf surface area was

relatively more important than the changes in net gas exchange of the remaining leaf

tissue.

Other types of insect herbivores include mesophyll feeders and gall former.

Mesophyll feeding insects damage mesophyll and palisade layers of leaves. Klingeman

et al. (2000) studied the effects of azalea lace bug, Stephanitispyrioides Scott, on whole-

plant gas exchange of azalea, Rhododendron sp. L. The azalea lace bug, which damages

leaves by feeding within the palisade parenchyma of leaf mesophyll cells caused a

reduction in net photosynthesis in plants that had more than 13% of the plant canopy

damaged. Gall forming insects have also been shown to alter normal leaf gas exchange

(Rilling and Steffan 1978, and Andersen and Mizell 1987). One study showed that gall

formation by phylloxera, Dactylosphaera vitifolii Shimer, caused up to a 97.5% reduction

in net photosynthesis in gall tissues of grapes (Rilling and Steffan 1978). The net CO2

assimilation rate, stomatal conductance and transpiration of pecan (Carya illinoensis

Wangenh) leaves were all significantly reduced when trees were infested with phylloxera,

Phylloxera notabilis Pergande, galls (Andersen and Mizell 1987).

Citrus leaf miner, Phyllocnistis citrella Stainton, is another pest that causes damage

to leaves and can reduce net photosynthesis. The citrus leaf miner does not actually

remove large portions of leaf like other leaf feeding insects. The damage caused by









larvae is mainly due to larval mining through tissues of the leaf surface. Schaffer et al.

(1997) reported that the number of days mining and the number of larvae per leaf

significantly affected the percentage of leaf area damaged. Net photosynthesis was lower

in leaves with leaf miners compared to control plants with no leaf miners.

Citrus rust mite, Phyllocoptruta oleivora Ashmead, is another arthropod pest of

citrus in Florida that mainly damages fruit by feeding on the epidermal cells and causing

discoloration known as sharkskin, russet, or bronzing (Allen 1978). Photosynthetic

layers are not damaged on leaves and most of the damage is due to increased

transpiration in fruit and increased fruit drop. Allen (1978) reported that water loss was

significantly higher in infested fruit than in undamaged fruit of three commonly grown

citrus cultivars.

Other studies show decreases in net photosynthesis as a result of mite feeding on

leaves (Andrews and La Pre 1979, Sances et al. 1981, Hare and Youngman 1987).

Sances et al. (1981) found that feeding by large populations of the two spotted spider

mite, Tetranychus urticae Koch, reduced transpiration and photosynthesis of strawberry

(Fragaria aananssa Duch.). This reduction in photosynthesis was correlated to a

reduction in fruit quality and quantity. Feeding damage by the avocado red mite,

Oligonichus yothersi McGregor, on avocado (Persea americana Mill), caused a decrease

in leaf net photosynthesis, stomatal conductance and transpiration within a few weeks

after infestation (Schaffer et al. 1986). Mobley and Marini (1990) tested the effects of

feeding of the European red mite, Panonychus ulmi Koch, and the two-spotted spider

mite on gas exchange of apple and peach (Prunuspersica L.) leaves. Results showed

that net photosynthesis, transpiration, and water-use efficiency declined linearly as









European red mite and two-spotted spider mite-days increased, for both apple and peach

tree species. Similarly apple (Malus domestic Borkh), infested with European red mite,

exhibited a decline in whole canopy net carbon exchange rate with increasing cumulative

mite-days (Lakso et al. 1996).

Phloem feeding insects such as aphids can be serious pests of many crops. There

have been variable results on plant gas exchange due to phloem feeding insects. Feeding

injury by three species of pecan aphids caused a 50% reduction in net photosynthesis of

pecan seedlings and reduced dark respiration by 25% (Wood et al. 1985). This type of

injury appears to be a result of phloem injury that contributed to the problems of irregular

bearing and reduced productivity of pecan trees.

Stem boring insects have also been reported to affect leaf gas exchange. The

dogwood borer, Thamnosphecia scitula Harris, is an example of a pest that enters the

stem of plants through any open wound and begins feeding on phloem and cambial

tissue layers. One experiment testing the effects of the dogwood borer on the physiology

of dogwood (Cornusflorida L.), showed that stomatal resistance increased and net

photosynthesis decreased in infested trees compared to healthy, non-infested trees

(Heichel and Turner 1973).

Published information on the effects of root feeding on whole plant physiology is

still limited. A study on the effects of western corn rootworm (Diabrotica virgifera

virgifera LeConte) larval injury on net photosynthesis and vegetative growth of several

varieties of maize indicated that rootworm infested plants had significantly lower midday

leaf photosynthesis compared to controls (Urias-Lopez et al. 2000). Rootworm-infested









plants showed a reduction in net photosynthesis at low and moderate levels of injury.

Plant height reduction was also observed in infested plants (Urias-Lopez et al. 2000).

Flooding stress and plant physiology

Natural environmental occurrences such as flooding can cause plants to become

stressed and may predispose them to insect herbivory and pathogens (Syvertsen et al.

1983, Ploetz and Schaffer 1989). The tolerance of plants to flooded conditions varies

greatly among species and may even be highly variable within species depending on

temperature, duration of flooding and plant age (Kozlowski 1997). Several other factors

may affect plant tolerance to waterlogged soil conditions including soil type, soil

porosity, soil chemistry, status of pathogens and soil microbes (Schaffer et al. 1992).

Flooding results in hypoxia (low oxygen concentration) in soils and plants and if

conditions persist, soils may become anoxic (lacking oxygen). Lack of oxygen in

flooded soils alters plant growth and function mainly due to reduced 02 exchange

between the root and soil (Li et al. 2004, Dennis et al. 2000). Oxygen in soil pores is

displaced by water and the remaining aerobic organisms use up any available oxygen and

either become quiescent or die off and are rapidly replaced by anaerobes such as bacteria

(Schaffer et al. 1992, Kozlowski 1997). Bacterial by-products, in addition to other

chemical changes in flooded conditions, alter the oxidation reduction status of the soil.

The soil redox potential is a quantitative measurement of the electrochemical status of the

soil, and in well drained soils it is usually > 300 mV (Schaffer et al. 1992, Kozlowski,

1997, Pezeshki et al. 1996). Soil redox potentials < 200 mV are indicative of anaerobic

conditions and may serve as a useful indicator of conditions not conducive to plant

survival in flooded soils (Crane and Davies 1989, Pezeshki 1991).









Photosynthesis decreases in many plants after they are flooded for several hours or

days depending on species. Flood tolerance of plant species may vary depending on the

developmental stage of plants and environmental factors such as temperature, soil or

potting media (Crane and Davies 1989). In fruit trees, physiological responses to

flooding include reductions in net CO2 assimilation and stomatal conductance (Larson

and Schaffer 1991, Andersen et al. 1984, Syvertsen et al. 1983). Stomatal closure is one

of the earliest responses of many plant species to flooding. Leaf conductance of sour

orange seedlings was significantly reduced four days after flooding (Syvertsen et al.

1983). In mango, stomatal conductance was reduced three days after trees in containers

were flooded (Larson and Schaffer 1991). Reduced stomatal conductance in many

species is believed to be correlated with a reduction in net CO2 assimilation (Kozlowski

1997). Pezeshki et al. (1996) reported a significant reduction in net photosynthesis due to

low redox potentials in flooded soils for three tree species, Taxodium distichum L.,

Quercusfalcate Michx and Quercus lyrata Walt.

Plants growing in stressful conditions are often more susceptible to insect herbivory

than healthy plants. The susceptibility of plants to insect herbivory is often dependant

upon the abiotic and biotic conditions in which they grow (Rand 1999). Evidence from

agronomic and forest ecosystems suggest a relationship between environmental stress and

insect damage to plants (Louda 1986). Louda (1986) tested the response of a native

montane crucifer, Cardamine cordifolia Grey, to insect herbivory by three major feeding

guilds: chewing, sucking, and mining insects in response to root-cutting and flooding

stress. Results showed that insect herbivory was greater on both flood-stressed and root-

pruned plants compared to non-stressed control plants. Interactions between flooding and









Diaprepes root weevil feeding were examined on two varieties of citrus rootstocks.

Plants that had been previously flooded and then infested with larvae had a higher

percentage of larvae recovered from the soil and also a higher percentage of root damage

compared to those plants that had not been previously flooded before infestation (Li et al.

2003b). It was also observed that stomatal conductance of non-infested, flooded plants

decreased but then increased back to the same level as the non-flooded plants when plants

were removed from flooded conditions. However, in plants that had been flooded and

infested with larvae, stomatal conductance increased after plants were removed from

flooded conditions, and then decreased again following larval infestation. This evidence

suggests that flooding predisposes plants to Diaprepes root weevil feeding injury.

The ornamental plant industry

In the United States nursery and greenhouse crops represent the sixth largest

agricultural commodity group and is the fastest growing major segment of U.S.

agriculture (Johnson 1999). In 1998, the farm gate value of ornamental plants was

estimated to be about $12.12 billion. In 1998, floriculture crops represented 3.93 billion

dollars in sales. Woody ornamental trees and shrubs, including sod and unfinished plant

products, represented $8.18 billion in sales or just about two-thirds of the industry value

(Hodges and Haydu 2000). As of 2002, the wholesale value of floriculture crops

increased by 2 percent from the revised 2001 valuation. The total wholesale crop value,

summed for all growers with 10,000 dollars or more, was estimated at nearly $4.88

billion (USDA Floriculture Crops Summary 2003). This was an increase from $4.80

billion the previous year. The top five states nationwide in nursery plant sales are

California, Florida, Michigan, Texas, and Ohio. These states account for $2.62 billion in









sales, which equals up to 54 percent of the total value (USDA Floriculture Crops

Summary, 2003).

Nationwide, Florida is ranked second in wholesale value of ornamental plants just

behind California, which leads in ornamental plant production with a crop value of nearly

$962 million. Florida's wholesale crop value for 2002 was about $877 million. Larson

et al. (2000) reported that the wholesale value of sales in 1997 of all floriculture crops in

Florida, including cut flowers, house plants, potted flowering plants, bedding/garden

plants and cut cultivated greens, accounted for 19.1 % of the total wholesale value of

floriculture crops in the nation.

The wholesale value of the Florida nursery and greenhouse industry was valued at

$1.28 billion in 1998. Florida dominates the United States tropical foliage crops market

with more than 85 percent of sales (Hodges and Haydu 2000). Florida has the largest

production area of floricultural crops in the United States with about 18,000 acres. This

is nearly as large as the combined growing areas of all states in the West including

California (Floriculture and Nursery Crops USDA 2003). In 1997, southeastern regions

of Florida including Miami-Dade, Broward and Palm Beach Counties, accounted for 53

percent of total foliage sales in Florida. The production value of foliage plants was worth

$175.8 million and comprised on nearly 123.9 million square feet (Larson et al. 2000).

Current and potential impact of Diaprepes root weevil on the ornamental plant
industry

Diaprepes root weevil is a serious pest of citrus and ornamental plants in Florida.

In Florida, there are about 24 counties known to be infested with Diaprepes root weevil

and economic losses in ornamental and citrus crops by this pest amount to more than 75

million dollars annually (Mannion et al. 2003, Lapointe et al. 1999). Diaprepes root









weevil infests over 30,000 acres of citrus in Florida (Muraro 2001). Distribution of

infested plant material is the most common way of spread and introduction of this weevil

into new areas. Therefore, there is a regulatory process that restricts the movement of

plants with the presence of any life stage of this insect (Simpson et al. 1996). In Florida

there are nursery quarantines in place which regulate plant pests and require immediate

action if pests are found on plant material. Any nursery stock found to be infested with

Diaprepes root weevil is quarantined and is not eligible for certification until required

treatments are carried out (Simpson et al. 2000). Ornamental field nurseries are also

affected by Diaprepes root weevil in south Florida with unknown amounts of crop

damage resulting. In 2000, it was reported that 179 nurseries from 11 counties in Florida

were quarantined for the presence of Diaprepes root weevil. There are reports of

Diaprepes root weevil being established in a citrus grove in the Rio Grande Valley of

Texas. Diaprepes root weevils have also been intercepted several times in California

from shipments of plants (Grafton-Cardwell et al. 2004).

Several Integrated Pest Management options are being used mainly by citrus

growers to control this pest. Foliar sprays for adult control and entomopathogenic

nematodes and soil barrier treatments for control of larvae along with the use of

fungicides are some options farmers have been using (Muraro 2001).

Buttonwood, Live Oak and Pygmy Date Palm

There are several species of ornamental trees that are known host of Diaprepes root

weevil and are commonly grown together in field nurseries. Three species, buttonwood,

live oak and pygmy date palm, are known to support both adult and larval feeding.

The pigmy date palm is an ornamental palm native to Laos that is commonly grown

in southern Florida. This palm grows from 1.83 m to 3.05 m tall and may reach a spread









of up to 3.05 m wide (Gilman and Watson 1993a). Pygmy date palm is a dioecious,

single-trunked palm, but is commonly grown together in groups of two or three per

container. Optimal growing conditions limit its growing range in the United States to

southern Florida or zones 10-11. Leaves of this palm are pinnately compound, up to 1.22

m long and consist of many leaflets. The flowers are unisexual and held on numerous

short branches that appear white in color. Flower stalks are found on pygmy date palms

year round. The fruit are often about 1.27 cm in diameter and dark red when ripe, they

can be found ripening all year round in south Florida (Dehgan 1998).

Buttonwood is a native tree of south Florida and is commonly used as a landscape

tree mainly in coastal areas. This is a very hardy tree, originally found in coastal swamp

areas, and can withstand high salinity. It reaches a height of about 12.19 m (Gilman and

Watson 1993b). It is a monoecious evergreen tree that is hardy only in zones 10-11; cold

temperature limits its distribution northward. Leaves are simple, alternate, and ovate to

elliptic with entire margins and average about 10.16 cm in length. Flowers are found on

terminal panicles that are green and cone-like; they are present throughout the year. Fruit

appears cone-like, reddish brown, and disintegrates at maturity into small winged seeds.

The silver and green buttonwood varieties are commonly grown in south Florida

nurseries (Dehgan 1998).

Live oak is native to the eastern United States and is a common landscape plant

grown throughout Florida. It is probably the most commonly planted landscape plant in

the southeastern U.S. (Dehgan 1998). Live oak is mainly used as a shade tree in many

places and can grow 18.29 to 24.38 m in height and may reach a spread of up to 36.58 m

with a slow to moderate rate. Leaves are simple, spirally arranged, and elliptical to ovate;









they average about 3.81 to 12.70 cm in length and leaf margins are entire but can have 3

lobes. Fruit are nuts that are ovoid in shape and average 2.54 cm in length (Dehgan

1998).

Research Objectives

The first objective of this research was to determine the effects of Diaprepes root

weevil feeding on physiology and growth of pygmy date palm, buttonwood and live oak,

three important ornamental plants grown in south Florida. Net CO2 assimilation, stomatal

conductance, and transpiration were measured as physiological indicators of adult and

larval insect damage to plants. The second objective was to test the effects of flooding on

predisposing live oak and buttonwood to root feeding by Diaprepes root weevil. The

third objective was to evaluate the effects of adult Diaprepes root weevil leaf feeding on

net CO2 assimilation and growth of buttonwood and live oak.














CHAPTER 2
EFFECT OF LARVAL ROOT FEEDING BY DIAPREPES ROOT WEEVIL ON LEAF
GAS EXCHANGE AND GROWTH OF SELECT ORNAMENTAL TREE SPECIES

Introduction

Diaprepes root weevil, Diaprepes abbreviatus L. (Coleoptera: Curculionidae), is a

polyphagous weevil species native to the Caribbean. In 1964 it was first reported in the

U.S. in a nursery in Apoka, Florida. The weevil is believed to have been introduced into

the state in an ornamental plant shipment from a nursery in Puerto Rico (Woodruff 1964).

Diaprepes root weevil has become a serious pest and is currently plaguing citrus orchards

and many ornamental plant species throughout most of central and southern Florida. This

weevil is known to be associated with more than 140,000 acres and is believed to be

present in 24 counties throughout the state (Simpson et al. 2000, Mannion et al. 2003). In

Florida it is estimated that this weevil causes over $70 million in damage annually

(Weissling et al. 2002, Lapointe 2000).

Larvae of Diaprepes root weevil cause extensive damage to the root systems of host

plants. Larvae tend to feed on all underground portions of the plant including roots and

tubers. Neonate larvae enter the soil, feed on fibrous roots, and then begin feeding on

larger lateral roots as they mature. Weevil larvae have a period of rapid growth starting

about 3 to 4 months after eggs hatch. This is the stage when larvae are most active and

cause serious injury (Beavers 1982). Larval growth on select citrus varieties is greatest

between the 6th and 8th instars when larvae have high nutrient requirements (Rogers et al.

2000). Larvae cause damage by feeding their way through the outer barkand cambium









layer (Futch and McCoy 1993). Large root weevil larvae have been known to girdle

roots and cause tree death by either continuous root feeding or by allowing entry for root

diseases associated with certain root fungi that destroy crops (McCoy et al. 2003).

Diaprepes root weevil has a host range of nearly 293 plant species that are known

to support either adult or larval stages and in many cases both (Simpson et al. 2000).

Several native and ornamental plant species in Florida are known to be hosts of

Diaprepes root weevil, and both native and non-native ornamental plant species have

been shown to support larval development (Schroeder et al. 1979). This weevil is

currently injuring many ornamental nursery plants in southern Florida, but the amount of

damage to nursery crops has not been quantified. In 2000 it was reported that 179

nurseries from 11 counties in Florida were quarantined for the presence of Diaprepes root

weevil. In addition to ornamental plants, several tropical fruit crops commonly grown in

southern Florida have been observed to be hosts of Diaprepes root weevil. It has been

reported that many ornamental trees can sustain a large amount of larval feeding before

leaf yellowing and other symptoms occur (Knapp et al. 2000).

A basic understanding of the physiological responses of plants to herbivory may

provide critical information for predicting and preventing crop damage (Welter 1989).

Measuring net photosynthesis provides information about the effects of insect feeding on

plant physiology and stress often before the appearance of any visual symptoms of stress

occur (Welter 1989, Peterson et al. 1992, Peterson et al. 1998). Arthropod herbivory can

significantly reduce leaf gas exchange (Schaffer et al. 1997, Godfrey et al. 1993, Schaffer

and Mason 1990). The majority of studies on herbivory effects on leaf gas exchange

have focused on non-insect arthropods (Schaffer et al. 1986, Mobley and Marini 1990,









Lakso et al. 1996). There are limited reports on the effects of herbivory by insects on leaf

gas exchange. Furthermore, the majority of these studies focused on adult insects feeding

on leaves (Peterson et al. 1998, Li and Proctor 1984, Johnson et al. 1983) including

Diaprepes root weevil (Syvertsen and McCoy 1985). There are a few studies reported on

the effect of root damage from insect larvae on leaf gas exchange (Godfrey et al. 1993,

Riedell and Reese 1999) and no known reports on the effects of Diaprepes root weevil

root herbivory on leaf gas exchange.

There are many economically important ornamental plant species grown in

southern Florida that are affected by Diaprepes root weevil. Three ornamental plant

species, pygmy date palm (Phoenix roebelenii O'Brien.), buttonwood (Conocarpus

erectus L.), and live oak (Quercus virginiana Mill.) are commonly grown together in

south Florida field nurseries. These plant species are known to support larval and adult

stages of Diaprepes root weevil and buttonwood has been shown to be a preferred host

(Mannion et al. 2003). Serious root injury has been observed on field-grown live oaks

and adults and larvae have been found on all three species in several field nurseries in

Homestead, FL (Catharine Mannion, University of Florida, Tropical Research and

Education Center, pers. comm.).

The objective of this study was to evaluate the effects of root feeding by larvae of

Diaprepes root weevil on net gas exchange and growth of pygmy date palm, green

buttonwood and live oak.

Materials and Methods

Studies were conducted from October 2003 through July 2004 at the University of

Florida, Tropical Research and Education Center, Homestead, FL, 25.50N latitude and

85.50W longitude. Two separate experiments were conducted to determine 1) the effects









of root feeding by larvae of two different ages on leaf gas exchange and plant growth,

and 2) the effect of root feeding damage by multiple larval infestations on leaf gas

exchange and plant growth. The first experiment was conducted on green buttonwood

from October 2003 through March 2004 and on live oak beginning in December of 2003

and ending in May 2004. The second experiment was conducted from January through

July 2004 on buttonwood, live oak and pygmy date palm.

Plant and Insect Material

Experiment 1

Green buttonwood in 3.79-L containers were purchased from a commercial nursery

(Princeton Nurseries II, Inc., Homestead, FL) and transplanted into 11.35-L containers

with well-drained media consisting of 40% Florida peat, 30% pine bark, 10% sand, 20%

cypress sawdust and 6.80 kg dolomite/91.44 cm purchased from a local soil distributor

(Lantana Peat & Soil, Boynton Beach, FL). Live oaks in 3.79-L containers were

purchased from a commercial nursery (Albert Livingston Tree Farms, Homestead, FL)

and transplanted into 11.35-L containers in the same media as described for green

buttonwood trees.

Experiment 2

Young live oak and pigmy date palms in 3.79-L containers were purchased from a

local commercial nursery (Bill Ingram & Grandsons NSY, Homestead, FL). Green

buttonwood trees in 3.79-L containers were purchased from another nursery (Princeton

Nurseries II, Inc., Homestead, FL). Plants were selected based on relative uniformity of

shoot and canopy size within each species. All plants were transplanted into 11.35-L

containers in the same media as described for Expt. 1.









All plants in both experiments were fertilized once prior to treatment initiation with

PlantacotePlus 14N-9P-15K (Helena Chemical Company, Collierville, TN) controlled

release fertilizer. Plants were irrigated twice daily, at 5:00 am and at 5:00 pm for 30

minutes each time.

Larvae of Diaprepes root weevil were obtained from the Florida Division of Plant

Industry (Division of Agriculture and Consumer Services, Gainesville, FL) and held in

containers on artificial diet in the laboratory until treatment initiation.

Treatments

Experiment 1

Larvae were sorted according to size so that each treatment would have similar age

instars. The larvae were first separated into medium (1-1.5 cm) or large (2 cm) size

classes, measured from the head to the tip of the abdomen. The head capsules from 20

randomly selected larvae from each size class were measured as described by Quintela et

al. (1998) to determine average instars. The medium size class contained 5-6th instars

and the large size class contained 8th instars.

Holes were made in the soil surface around each plant with small spatulas, 4 to 8

cm from the stem, with 2.54 cm spacing between holes. The depth of the holes alternated

at 5 and 10 cm from the soil surface. One or two larvae were placed into each hole and

then covered with soil. Each container was infested with 20 larvae. All containers were

irrigated after infestation to cool the soil and keep larvae from being affected by high soil

temperatures.

There were three treatments with 16 replications per treatment: 1) non-infested

controls, 2) infested with medium larvae, and 3) infested with large larvae. Three months

after infestation, 8 replications of each treatment were harvested to determine if larvae









were surviving and feeding on roots. The remaining eight plants in each treatment were

re-infested two times at approximately 2-month intervals with 20 larvae per container

each time. Larvae were selected and placed into containers as previously described for

the first infestation. All remaining plants were harvested 6 months after the initial

infestation.

Leaf gas exchange. Net CO2 assimilation (A), stomatal conductance of H20 (g),

and transpiration (E) were measured monthly with CIRAS-2 portable gas analyzer (PP

Systems, U.K.) between 1000 and 1200 HR. Leaf gas exchange was measured at a

photosynthetic photon flux (PPF) > 900 itmol .m-2 .s-1 with a halogen lamp fitted on the

leaf cuvette as the light source. Leaf gas exchange was determined for two fully

expanded leaves of each plant and the average of the two leaves was used to represent gas

exchange values for each single-plant replication.

Root biomass and larval recovery. The roots were carefully separated from the

rooting media and rinsed with tap water to remove excess soil. Soil from the roots was

placed into bins and carefully inspected for larvae. Recovered larvae were separated and

placed into holding containers containing 75% ETOH. Excess water was allowed to

drain from roots for 12 hours and root fresh weights were determined. Roots were oven-

dried at 700C to a constant weight and root dry weights were determined.

Experiment 2

In this experiment, 5th to 6th instars (determined by head capsule size as previously

described) were used to infest plants. Thirty-two plants of each buttonwood, live oak and

pygmy date palm were infested with 20 larvae per container as described for Experiment

1 and 32 plants were left non-infested as controls. Approximately two months after

infestation a set of 8 infested plants and 8 non-infested plants of each species were









harvested. Thereafter, 8 plants of each treatment were harvested monthly. All remaining

plants in the infested treatment were re-infested with 20 larvae per container as described

for Experiment 1. Thus for the infested treatment, the first set of plants harvested were

infested with a total of 20 larvae, plants harvested after 3 months were infested with a

total of 40 larvae (20 per month), plants harvested after 4 months were infested with a

total of 60 larvae, and plants harvested after 5 months were infested with a total of 80

larvae.

Leaf gas exchange. Leaf gas exchange was measured monthly prior to each

harvest for each set of plants as described for Experiment 1. For the set of plants in the

final harvest, leaf gas exchange was measured on the week of infestation and monthly

thereafter until the week of harvest.

Plant growth and larval recovery. Plant height and trunk diameter was measured

just prior to treatment initiation and again prior to the final harvest. Plant height was

measured from the base of the stem to the tip of the apical bud. For oak and buttonwood

trunk diameter was measured at 10 cm above the soil surface with an electronic digital

caliper (Fisher Scientific, Pittsburgh, PA.). Leaves, stems and roots were harvested

monthly beginning at the end of the second month and the number of recovered larvae

and fresh and dry weights of plant tissues were determined as described for Experiment

1.

Temperature Measurements

Air and soil temperatures were measured with HOBO StowAway data loggers

(Onset Computer Corp., Pocasset, MA). Data loggers used for soil measurements were

placed at a depth from the soil surface of about 10 cm in eight 11.35-L containers with no









plants in them and the same media as the containers with plants. Air temperature was

measured with a data logger placed under a bench next to the experimental plants.

Statistical Analysis

Data were analyzed by a standard t-test or Analysis of Variance and means were

compared with a Student-Newman-Keuls Range Test or Test for Least Significant

Difference using SAS software (SAS Institute, Cary, NC).

Results

Experiment 1

Leaf gas exchange. Fluctuations in air temperatures were relatively similar to

those of soil temperatures throughout the experiment (Fig. 2-1). Leaf gas exchange of

buttonwood and live oak fluctuated with temperature, decreasing with dropping

temperatures and increasing when temperatures increased for all treatments. In October

at the beginning of the experiment, average monthly temperature was about 250C.

Average air and soil temperatures decreased by the end of November to 180C. On

several days during December and January minimum daily air and soil temperatures were

between 40 and 10C (data not shown). Average air and soil temperatures began to

increase by February to above 21C (Fig. 2-1).

The effect of larval root injury on leaf gas exchange of buttonwood was not

observed until average temperatures increased above 210C. At that temperature A, E and

gs for each treatment increased from previous levels. However, control trees had

significantly higher A, E and gs than plants infested with medium or large larvae four

months (A: F=8.70; df=2, 23; P=0.0018; E: F=3.47; df=2, 23; P=0.05; gs: F=5.03; df=2,

23; P=0.016) and five months (A: F=15.74; df=2, 23; P<0.0001; E: F=20.06; df=2, 23;

P<0.0001; gs: F=32.97; df=2, P<0.0001) after treatments were initiated (Fig. 2-2).









Leaf gas exchange of live oak fluctuated slightly with temperature. Treatments

were initiated during December when temperatures were relatively low and A, E and gs

were also relatively low and then increased as average temperature increased. There

were no significant differences in A, E and gs among treatments as a result of larval root

feeding (Fig. 2-3). One exception was on one measurement date, four months after initial

infestation, when gs of both infested treatments were lower than that of the controls four

months after treatment initiation (Fig. 2-3). However, only the medium size larvae

treatment had significantly lower A than the controls. There was no difference in gs

among treatments on the last 2 measurement dates.

Root weights and larval recovery. Fresh root weight of both infested treatments

of buttonwood were significantly lower than that of the controls (F=3.73; df=2, 23;

P=0.04). Dry weights tended to be lower for plants in the infested treatments than

control plants but differences were not significant (Fig. 2-4). There were no significant

differences in root fresh or dry weights among treatments for live oak (Fig. 2-5). For

both plant species, relatively few larvae were recovered from individual plants compared

to the amount of total larvae placed in each container. The average number of larvae

recovered did not exceed eight per container for either treatment or plant species. For

buttonwood, there was no significant difference in the number of larvae recovered

between the medium and large larvae treatment (t=-1.79; df=14; P=0.09). The number of

larvae recovered was not significantly different among treatments for live oak (t=-0.39;

df=14; P=0.70). The number of larvae recovered between plant species was compared

and there were no significant differences in the number of larvae recovered between

buttonwood and live oak (t=0.69; df=28.6; P=0.49).









Experiment 2

Leaf gas exchange. There was temporal variation in A, E and gs throughout the

experimental period within and among plant species. Net CO2 assimilation, E and gs

were more inhibited by larval infestation in buttonwood than in pygmy date palm or live

oak. Temperature did not appear to affect leaf A, E or gs.

For the first set of buttonwood plants there were no significant differences in A, E

or gs between treatments prior to harvest. For plants harvested after 3 months, A (t=2.15;

df=14; P=0.04), E (t=2.70; df=14; P=0.017) and gs (t=3.23; df=14; P=0.006)were

significantly lower in infested plants than the non-infested plants (Fig. 2-6). For plants

harvested after 4 5 months A, E and gs were not significantly different between

treatments (Fig. 2-6). For the final set of plants where leaf gas exchange was measured

monthly over time, A, E and gs of infested plants were lower than those of non-infested

controls on every measurement date except the first and last. There was a significant

difference in A, E and gs between treatments after each harvest at [1 month: A (t- 1.26;

df=14; P<0.0001); E (t=7.00; df=14; P<0.0001); gs (t=7.98; df=14; P<0.0001)] [2

months: A (t=5.56; df=9.94; P=0.0002), E (t=2.45; df=13.3; P=0.029); gs (t=2.24;

df=13.4; P=0.04)] [3 months: A (t=4.25; df=14; P=0.0008), E (t=5.91; df=13.7;

P<0.0001); gs (t=7.22; df=14; P<0.0001)] [4 months: A (t=2.20; df=14; P=0.04); E

(t=3.44; df=14; P=0.03); gs (t=3.43; df=14; P=0.004)] (Fig. 2-7).

For live oak, there was no effect of treatment on A, E and gs on any measurement

date prior to harvest regardless of the number of infestations (Fig. 2-8). For the set of

plants harvested last, where leaf gas exchange was measured monthly over time, A was

not significantly different between treatments except on the second month when A of the









infested plants was significantly lower than that of the non-infested controls (Fig. 2-9).

There were no effects of treatment on gs or E on any measurement date (Fig. 2-9).

For pygmy date palms A, E and gs were not significantly different between

treatments regardless of the number of infestations (Fig. 2-10). For the set of plants

harvested last and measured monthly over time, A and E were not significantly different

between treatments (Fig. 2-11). Stomatal conductance was lower in infested plants than

in the non-infested plants after 2 months. Although gs remained lower in infested plants

than control plants, gs increased in all treatments by the third month and differences

between treatments were not significant for the remainder of the experiment (Fig. 2-11).

Plant growth and recovery larvae. Plant height and trunk diameter of

buttonwood did not differ significantly between treatments on the initial measurement

date. On the final measurement date, plant height (t=3.44; df=12.9; P=0.004) and trunk

diameter (t=3.52; df= 1; P=0.004) were significantly lower for plants in the infested

treatments than for the non-infested plants (Table 1). Plant height and trunk diameter of

live oak did not differ significantly between treatments on the initial or final

measurement dates (Table 1).

For buttonwood there was a significant interaction between larval infestation and

harvest date for fresh leaf (F=4.17; df=7, 63; P=0.009), stem (F=4.61; df=7, 63;

P=0.005) and root (F=4.01; df=7, 63; P=0.011) weights and dry leaf(F=4.52; df=7, 63;

P=0.006), stem (F=4.18; df=7, 63; P=0.009) and root (F=6.34; df=7, 63; P=0.0009)

weights. Therefore larval effects on leaf, stem and root fresh and dry weights were

analyzed separately on each harvest date. Leaf, stem and root fresh weights were lower

for infested than non-infested plants at each harvest date except the first when there were









no differences in leaf and stem weights between treatments (Table 2). Leaf dry weight of

buttonwood was lower for infested than non-infested plants harvested after 3 and 5

months. Stem dry weights were not significantly different between treatments for plants

that were harvested after 2 months. However, stem dry weight of infested plants was

significantly lower than that of the control plants harvested after 3, 4 and 5 months (Table

2). Root dry weights on all four harvest dates were significantly lower for infested than

control plants (Table 2).

There was no significant interactions between treatments and harvest date for leaf,

stem and root fresh and dry weights of live oak and pygmy date palm (P > 0.05).

Therefore harvest dates were pooled for each species to test the effect of larval root

feeding on leaf, stem and root fresh and dry weights. For live oak there was no

significant effect of treatment on leaf, stem and root fresh or dry weights (Table 3).

Infested pygmy date palms had significantly lower leaf, stem and root fresh and dry

weights than non-infested plants (Table 3). For both pygmy palm and live oak the

number of larvae recovered, at each harvest, was < 10.

There was a significant effect of harvest date on the number of larvae recovered for

buttonwood (F=27.64; df=3, 31; P<0.0001), live oak (F=10.15; df=3, 31; P=0.0001) and

pygmy date palm (F=7.79; df=3, 31; P=0.0006), (Table 4). There tended to be more

larvae recovered from buttonwood than from live oak and pygmy date palm, however;

the differences were not significant until the last harvest date when more larvae were

recovered from buttonwood (F=7.00; df=2, 23; P=0.004) than live oak or pygmy date

palm (Table 4).









Discussion

In Expt. 1, average monthly temperatures had dropped from about 270C in October

to about 18C in December, including several days with minimum temperatures below

100C. In buttonwood, the reduction in A, E and gs of trees in all treatments two months

after infestation was presumably the result of low temperature since all treatments

including controls were reduced. As the average monthly air and soil temperatures

increased to about 21C by the end of February, A, E and gs of all treatments increased.

These results are similar to those of (Taylor and Rowley 1971) who observed an

immediate reduction in leaf photosynthesis in sorghum (Sorghum Hybrid), maize (Zea

mays L.) and pennisetum (Pennisetum typhoides Burm.) as temperatures declined from

25 to 100C and then increased as temperature returned to 250C. Reductions in net CO2

assimilation have also been observed in tropical woody plant species such as avocado

(Persea americana Mill.) and mango (Mangifera indica L.) in response to low

temperatures (Whiley et al. 1999). Buttonwood is a tropical plant species native to

southern Florida and its distribution into central and northern Florida is limited by low

winter temperatures (Tomlinson 1980). The significant difference between treatments in

A, E and gs when temperature increased was presumably a result of increased larval

activity and root feeding in the infested treatment when root temperature had warmed.

Lapointe (2000) found that the growth rate of Diaprepes root weevil larvae increased

exponentially with temperature from 15 to 300C and the optimal temperature for larval

development was between 22 and 260C. Although A, E and gs were very low during

periods of low soil temperature, the average mid day soil temperature presumably

warmed enough for some larval root feeding to occur. There are no other known reports

on the effects of root feeding by Diaprepes root weevil larvae on A, E and gs. However,









for another root-feeding insect, several authors have reported either a significant

reduction in A caused by the western corn root worm (Diabrotica virgifera virgifera

LeConte) larvae when actively feeding on maize (Zea mays L.) root systems, but did not

affect A when the larvae reached the quiescent pupal stage (Godfrey et al. 1993, Riedell

and Reese 1999). In Experiment 1, buttonwood plants infested with large (8th instar)

larvae exhibited lower A, E and gs than those infested with medium size (5-6 instars)

larvae. It has been reported that weight gain is greatest in larvae between 6 to 9th instars

because these stages require large amounts of nutrients and cause significant injury to

plants (Quintela et al. 1998, Rogers et al. 2000). After the first infestation, in Experiment

1, low soil temperatures below 150C may have caused some larval mortality (Lapointe

2000), and some larvae may have become quiescent and pupated during the experimental

period thereby reducing the amount of damage caused by larval root feeding. However,

re-infestation of larvae may have allowed the larger larvae to continue to cause

significantly more damage.

Net CO2 assimilation, E and gs were not as sensitive to temperature or larval

infestation in live oak as buttonwood. The oak treatments were initiated later when

average temperatures had increased to above 210C. Therefore the temperature was

presumably not low enough during the oak treatment period to affect leaf gas exchange.

Larval infestation did not appear to affect leaf gas exchange of oak significantly and

consistently during the six-month infestation period.

In buttonwood, larval infestation resulted in lower root fresh and dry weights in the

infested treatments than the controls. However, only fresh root weights were significant.

Others have also reported reductions in root weight caused by Diaprepes root weevil









larval feeding (Rogers et al. 2000, Nigg et al. 2001, Mannion et al. 2003). In live oak,

root fresh and dry weights tended to be lower for both infested treatments than control

trees but differences were not significant. Buttonwood root systems appeared to have

more damage than those of the live oak, which may be due in part to the differences in

root system anatomy of both species. The root system of buttonwood was more

succulent and fibrous than that of live oak. The fast growing succulent roots of

buttonwood may be the reason that Diaprepes root weevil larvae prefer buttonwood as a

host over many other ornamental plant species (Mannion et al. 2003). Live oak root

systems exhibited some root damage in the form of tunneling through the bark and

cambium layer on the larger lateral roots and a noticeable reduction in smaller fibrous

feeder roots. However most of the root weight was in the large primary tap and lateral

roots which were relatively thick and dense and root feeding did not seem to reduce root

weight.

In Expt 2, buttonwoods were affected by larval root feeding more than pigmy date

palm and live oak. Infestation by Diaprepes root weevil larvae caused a reduction in A, E

and gs of buttonwood. This is consistent with results from Experiment 1, which showed

reductions in A, E and gs of infested plants. Several buttonwood plants were showing

signs of leaf yellowing, defoliation and wilting prior to harvest. These symptoms are

typical of intense weevil damage (Knapp et al. 2000). Live oak responded as they did in

Experiment 1, in that there were no visible signs of damage to the canopy and no

significant reductions in A, E and gs caused by larval root feeding. In pygmy date palms

there were no visible signs of stress to the canopy and A, E and gs were not significantly

affected by treatment. Again, buttonwood may have been more sensitive than live oak









and pygmy date palm because of its fleshier root system, which may have encouraged

more continuous larval root feeding.

Leaf, stem, and root fresh and dry weights were significantly lower for infested

buttonwood than for control plants. Most infested plants, especially those showing signs

of damage, when harvested had obvious root feeding damage and very limited root

systems. Although only one buttonwood plant showing severe stress died before harvest,

all the other buttonwood plants survived. The roots, stems and leaves, however, were

noticeably smaller than those of the control plants. Buttonwood plants infested monthly

and harvested at the end of the experiment had significantly less trunk diameter and

height than the controls. A reduction in root weight and trunk diameter due to Diaprepes

root weevil larval root feeding has also been observed by others (Nigg et al. 2001,

Mannion et al. 2003). In Experiment 2, buttonwood plants that showed signs of leaf

yellowing and wilt had the greatest reduction of growth. As in Experiment 1, infested

live oak did not have a significant reduction of root weight. Trunk diameter and plant

height of live oak harvested at the end of the Experiment 2 were not significantly affected

by treatment. The reasons for this are not clear because larvae were recovered and there

were signs of feeding damage. However, it is possible that oaks are more tolerant to root

damage than buttonwood and it may take a longer period of feeding to cause significant

damage.

For pygmy date palm there were significant reductions in root fresh and dry

weights between infested and non-infested treatments. Pygmy date palm is a monocot

with an adventitious root system (Broschat and Meerow 2000). Although these

adventitious root systems are relatively tender and succulent and had obvious signs of









root feeding, there were fewer larvae recovered on each harvest compared to buttonwood

and live oak. This may explain why buttonwood had more damage than pygmy date

palm despite the succulent adventitious root system. Although both buttonwood and

pygmy date palms have succulent roots, there presumably was greater feeding pressure

on buttonwood which is a preferred host (Mannion et al. 2003). In contrast, live oak has

a much tougher root system which presumably was more resistant to larval feeding than

roots of the other species.

Although there was obvious root feeding damage found on the root systems of

buttonwood, live oak and pigmy date palm, only buttonwood showed signs of severe

stress in the form of leaf yellowing, wilt and a significant reduction in leaf gas exchange

and plant growth. These tests were performed in containers with young trees and

although larval feeding pressure was high due to re-infestation with larvae, the treatment

period in these experiments lasted only a few months. The negative effects of larval root

feeding most likely are cumulative over time with constant feeding pressure as it occurs

in the field with multiple generations feeding year after year. Further studies need to be

conducted on plants in the field where Diaprepes root weevils are present year round and

there are larvae continually feeding and damaging roots over a long period of time.



















0
C)
30



S25
E
U,




o
I--

< 20
-c


15
m
t--


10



5


S30
a)


L 25

a
E
I-1

a2
o




15


n'
10
rn


5 I I I I I I I
Oct-03 Nov-03 Dec-03 Jan-04 Feb-04 Mar-04 Apr-04 May-04

Month


Figure 2-1. Mean daily and monthly temperature (A) Air (B) Soil in containers.












- 18
U) 16
N
E 14
0 12
- 10
10
E
6


. . .


3
Months after infestation


Figure 2-2. Net CO2 assimilation (A), transpiration (E) and stomatal conductance (gs) of
buttonwood plants infested with medium or large Diaprepes root weevil larvae
or non-infested control plants. Symbols represent means of 8 replications and
bars indicate + 1 std. error. Asterisks indicate significant difference among
treatments according to ANOVA (P<0.05).


- Control
---- -- Medium
- -- Large

*












18
16
M 14
E 12
O10
U
-66
E 6 --Control
S4 ---m.--- Medium
C Large
2



0
6
















400
S350

0 2

LU


E
E








S 50
400



1 2 3 4 5 6
U 200

E 150
100
0) 50

0
1 2 3 4 5 6
Months after infestation

Figure 2-3. Net CO2 assimilation (A), transpiration (E) and stomatal conductance (gs) of
live oak plants infested with medium or large Diaprepes root weevil larvae or
non-infested control plants (C). Symbols represent means of 8 replications
and bars indicate + 1 std. error. Asterisks indicate significant difference
among treatments according to ANOVA (P<0.05).










300
A
250

200

.150

3100

50

0
Control Medium Large


140
B
120

100

800

60

40

20

0
Control Medium Large
Treatment

Figure 2-4. The effect of medium or large Diaprepes root weevil larvae on mean root
fresh and dry weights of buttonwood trees. Bars represent means of 8
replications and error bars indicate + 1 std. error.






44



350
A
300

S250

5 200

150

100

50

0
Control Medium Large


200
180 B
160
S140
120
.02 100
S80
60
40
20
0
Control Medium Large
Treatment


Figure 2-5. The effect of medium or large Diaprepes root weevil larvae on mean root
fresh and dry weights of live oak trees. Bars represent means of 8 replications
and error bars indicate + 1 std. error.













12
,If- Non-infested 0 Infested
N10

C-4
06








E







2




0





300
E . . . .














o
3100

50
ww















2 3 4 5
Month harvested after initial treatment



Figure 2-6. Net CO2 assimilation (A), transpiration (E) and stomatal conductance (gs)
measured prior to harvest of buttonwood plants infested with Diaprepes root
weevil larvae or not infested. Plants harvested after 2 months were infested
once with 20 larvae, plants harvested after 3, 4 and 5 months were infested
with a total of 40, 60 and 80 larvae respectively. Bars represent means of 8
replications and error bars indicate + 1 std. error.
Month harvste a.eriita.tetmn

Fiue26Ce10 siiato1Atasiain()an tmtlcnutne(s
mesue prior ... ha..es ....nod lnsifstdwt iar sro
....i ....a ... no.netd.Pa t.a.etdatr o tsweeifse
onc .... ... la..ae ....hr ete fe ,4 n o ts eeifse
.... ... total of4,6.nd8.a.erepciey.Br ersntmaso
relcain 2n ro asidct t.err











14
-12
10


06

E4
-I-I
2 -- Non-infested
0 -- Infested
-2



6

5
N, .
E4

I

E
E 2 50



0 200


400
3 50 '
S300 -
E 250

0 200 -
150 -



0

0 1 2 3 4 5
Months after treatment initiation

Figure 2-7. Net CO2 assimilation (A), transpiration (E) and stomatal conductance (gs) of
the buttonwood plants infested monthly with 20 larvae and a total of 80
Diaprepes root weevil larvae by month 4 or not infested. Symbols represent
means + 1 std. error. Asterisks indicate significant difference between
treatments according to standard t-test (P< 0.05).










18
16
E 14
N 12
o 10
0
E 8
6
4
2
0


66
T,
N 5
E
04
3
E2
E
WI
LU 1
0


450
400
N 350
E
2300
0250
5200
E
E150
"100
50
0


O Non-infested
0 Infested


3 4
Month harvested after initial treatment


Figure 2-8. Net CO2 assimilation (A), transpiration (E) and stomatal conductance (gs)
measured prior to harvest of live oak plants infested with Diaprepes root
weevil larvae or not infested. Plants harvested after 2 months were infested
once with 20 larvae, plants harvested after 3, 4 and 5 months were infested
with a total of 40, 60 and 80 larvae respectively. Bars represent means of 8
replications and error bars indicate + 1 std. error.


~I












20
18
' 16
E
N14

0
L12

E 8
46
4
2
0


- Non-infested
- --- Infested


2 3
Months after treatment initiation


Figure 2-9. Net CO2 assimilation (A), transpiration (E) and stomatal conductance (gs) of
live oaks plants infested monthly with 20 larvae and a total of 80 Diaprepes
root weevil larvae by month 4 or not infested. Symbols represent means of 8
replications + 1 std. error. Asterisks indicate significant difference between
treatments according to standard t-test (P< 0.05).







49



9
U)
S8 Non-infested
E 7 Infested
S7














5
43













250
20) 2..0



10
0






= 4-

32 3 4 5





























Figure 2-10. Net CO2 assimilation (A), transpiration (E) and stomatal conductance (g)
S200





0 150
-
















weevil larvae or not infested. Plants harvested after 2 months were infested
0 100 ...





2 3 4 5
Month harvested after initial treatment

Figure 2-10. Net C02 assimilation (A), transpiration (E) and stomatal conductance (gs)
measured prior to harvest of pygmy date palm infested with Diaprepes root
weevil larvae or not infested. Plants harvested after 2 months were infested
once with 20 larvae, plants harvested after 3, 4 and 5 months were infested
with a total of 40, 60 and 80 larvae respectively. Bars represent means of 8
replications and error bars indicate + 1 std. error.











10
f9

E7




3

I
0








E4
E

03

W1









0



250

S 200

E 150

100
E
E 50

0
0 1 2 3 4 5
Months after treatment initiation


Figure 2-11. Net CO2 assimilation (A), transpiration (E) and stomatal conductance (gs) of
the final harvest set of pygmy date palms infested monthly with 20 larvae and
a total of 80 Diaprepes root weevil larvae by month 4 or not infested.
Symbols represent means of 8 replications + 1 std. error. Asterisks indicate
significant difference between treatments according to standard t-test (P<
0.05).






51


Table 2-1. The effect of Diaprepes root weevil larvae on height and trunk diameter of
buttonwood and live oak trees.
Species Treatment n Plant height (cm) Trunk diameter (mm)


Buttonwood



Live oak


Infested
Non-infested
(P)
Infested
Non-infested
(P)


Significance determined by a standard t-test at the 0.05 level.


Initial
2/17/04
65.54
62.05
(0.14)


94.61
93.34
(0.65)


Final
7/22/04
71.27
90.47
(0.004)


121.43
134.28
(0.38)


Initial
2/17/04
8.21
8.03
(0.55)


14.12
13.03
(0.46)


Final
7/22/04
9.40
13.39
(0.004)


18.23
16.54
(0.35)












Table 2-2. The effect of Diaprepes root weevil larvae on buttonwood leaf, stem and root fresh and dry weights at each harvest date.


Plant tissue


treatment


n Fresh weight (g)


Dry weight (g)


Non-infested

Infested

(P)

Non-infested

Infested

(P)

Non-infested

infested

(P)


4/04
8 197.15

8 186.27

(0.47)

8 86.15

8 76.74

(0.13)

8 143.62

8 85.03

(0.0002)


Significance determined by a standard t-test at the 0.05 level.


Leaf


Stem


Root


5/04
219.23

112.38

(0.003)

102.48

58.54

(0.006)

128.01

40.85

(<.0001)


6/04
247.59

188.02

(0.09)

130.28

102.99

(0.04)

184.90

109.21

(0.007)


7/04
254.54

100.28

(0.001)

243.96

68.76

(0.001)

243.96

79.42

(0.001)


4/04
73.45

74.19

(0.91)

36.04

32.82

(0.28)

54.50

37.09

(0.0002)


5/04
80.28

43.41

(0.004)

45.43

28.51

(0.009)

65.74

25.53

(<.0001)


6/04
99.65

75.94

(0.11)

66.70

49.07

(0.02)

84.21

57.49

(0.01)


7/04
97.76

31.78

(0.001)

100.43

31.78

(0.001)

100.43

33.43

(<.0001)










Table 2-3. The effect of Diaprepes root weevil larvae on leaf stem and root fresh and dry
weights of live oak and pygmy date palm.
Species Treatment n Fresh weight (g) Dry weight (g)


Live oak




Pygmy palm


Non-infested
Infested
(P)

Non-infested
Infested
(P)


Significance determined by standard t-test at the 0.05 level.

Table 2-4. Number of Diaprepes root weevil larvae recovered from plants harvested 2, 3,
4 & 5 months after infestation.
Species




Buttonwood


Live oak



Pygmy
palm
Means within a row followed by different lower case letters and means within a column
with different capital letters are significantly different according to ANOVA (P< 0.05).
Total number of larvae infested for 1st harvest=20; 2nd harvest=40; 3rd harvest=60; 4th
harvest=80.


leaf
89.02
84.59
(0.55)

171.00
148.02
(0.06)


stem
233.40
245.43
(0.57)

182.92
146.24
(0.01)


root
394.79
372.75
(0.53)

322.46
239.73
(0.04)


leaf
45.88
41.78
(0.31)

67.91
58.49
(0.05)


stem
136.75
143.77
(0.58)

94.46
72.89
(0.02)


root
221.41
211.18
(0.62)

113.26
84.64
(0.01)














CHAPTER 3
EFFECT OF FLOODING AND DIAPREPES ROOT WEEVIL LARVAL FEEDING
ON LEAF GAS EXCHANGE AND GROWTH OF BUTTONWOOD AND LIVE OAK

Introduction

Diaprepes root weevil, Diaprepes abbreviatus L. (Coleoptera: Curculionidae), is a

polyphagous weevil species native to the Caribbean. It was first reported in the United

States in 1964 in a nursery in Apopka, Florida. It is believed to have been introduced

into Florida in an ornamental plant shipment from a nursery in Puerto Rico (Woodruff

1964). Since its introduction into Florida this weevil has seriously damaged citrus

orchards and has spread throughout most of central and southern parts of the state. These

weevils cause serious economic damage annually to many plant species including

ornamental nursery crops. Recent reports indicate that Diaprepes root weevil affects

more than 140,000 acres and is believed to be present in 22 counties in Florida (Simpson

et al. 2000, Mannion et al. 2003).

Many areas of south Florida where ornamental tree species are grown are prone

to flooding. These areas are generally low lying and often have the marl soil types

commonly found in Miami-Dade and Monroe Counties, including Everglades National

Park. Marl soils have poor to very poor drainage and are formed when the land is

cyclically flooded for several months each year. According to a 1996 soil survey, marl

soil is classified as Biscayne soil (loamy, carbonatic, hyperthermic, shallow Typic

Fluvaquants) (Noble et al. 1996, Li 2001). Ornamental trees in low lying nurseries with

marl soil appear to sustain the highest populations of Diaprepes root weevils.









Thepreference of these weevils for poorly drained areas has been observed in other heavy

soils (Li et al. 2003a). Flooding can cause plants to become stressed and may predispose

them to insect herbivory and pathogens (Syvertsen et al. 1983, Ploetz and Schaffer 1989).

Tolerance to flooded conditions is highly variable among plant species. Several factors

can affect a plant's ability to withstand waterlogged soil conditions including soil type,

soil porosity, soil chemistry and the status of pathogens and microbes in the soil (Schaffer

et al. 1992). In many tree species net CO2 assimilation and stomatal conductance are

negatively impacted by flooding (Larson and Schaffer 1991, Andersen et al. 1984,

Syvertsen et al. 1983) or insect predation (Peterson et al. 1998, Schaffer et al. 1997,

Schaffer and Mason 1990). These factors may have an additive or synergistic effect on

reducing leaf gas exchange. For example, in citrus orchards that were previously flooded

and infested with Diaprepes root weevil larvae, a higher percentage of larvae were

recovered from the soil and a higher percentage of root damage was observed in orchards

that were flooded before insect infestation compared to orchards that had not been

previously flooded (Li et al. 2003b). However, there is no published information on the

effects of flooding and Diaprepes root weevil feeding on ornamental tree species in south

Florida. Live oak (Quercus virginiana Mill.) and green buttonwood (Conocarpus erectus

L.) are native to florida and are commonly grown together in south Florida nurseries for

sale. These tree species have been observed to support high populations of adult

Diaprepes root weevils and their egg masses (Mannion et al. 2003). Diaprepes root

weevil larvae and the consequent feeding damage by these insects on buttonwood and

live oak root systems have been observed on several occasions at different field nurseries









(Catharine Mannion, University of Florida, Tropical Research and Education Center,

pers. comm.).

The objective of this study was to evaluate the effects of flooding and herbivory

from Diaprepes root weevil larvae on leaf gas exchange and growth of buttonwood and

live oak trees.

Materials and Methods

Studies were conducted from June through November 2004 at the University of

Florida, Tropical Research and Education Center, Homestead, FL, 25.50 N Latitude and

85.50 W Longitude.

Plant and Insect Material

Young live oak and buttonwood trees in 11.35-L containers with well drained

media consisting of 40% Florida peat, 30% pine bark and 10% sand were obtained from a

commercial nursery (Doug Ingram & Sons NSY, Homestead, FL). Live oaks were

pruned slightly at the top of the apical shoots to achieve uniform plant heights.

Buttonwoods were already uniform in height so they were not pruned. Plants were

irrigated twice daily at 5:00 AM and at 5:00 PM, for 30 minutes each time.

Diaprepes larvae were obtained from the Florida Division of Plant Industry (Florida

Department of Agriculture and Consumer Services Gainesville, FL) and held in

containers with artificial diet in the laboratory until plant infestation treatments were

initiated.

Treatments

Flooding. Live oak and buttonwood trees were subjected to continuous flooding or

left non-flooded. Live oaks were flooded from June 1, 2004 to July 7, 2004.

Buttonwoods were flooded from July 21, 2004 to August 11, 2004. Sixteen trees of each









species in 11.35-L containers were placed into empty 18.93-L containers and tap water

was added to the large containers to about 10 cm above the soil surface. Sixteen trees of

each species remained non-flooded. Leaf gas exchange was measured weekly until there

was a significant reduction in net CO2 assimilation (A) on two consecutive measurement

dates. After that time, plants were permanently removed from the flooded conditions and

infested with larvae. Eight each of the flooded and non-flooded plants of each species

were infested with Diaprepes root weevil one day after the flooding treatment was

terminated. The other eight plants of each species in each treatment were not infested.

Thus there were four treatments: 1) non-flooded, non-infested, 2) non-flooded, infested,

3) flooded, non-infested, and 4) flooded, infested. There were eight single-plant

replications per treatment for each species arranged in a completely randomized design.

Insect infestation. The head capsules from 20 randomly selected larvae were

measured to determine instars as described by Quintela et al. (1998). Larvae used to

infest plants were determined to be between the fifth and sixth instars (1-1.5 cm) from the

head to the tip of the abdomen. Ten to 15 holes were made in the soil around each plant

with small spatulas alternating at 4 to 8 cm from the stem, with 2.54-cm spacing between

holes. The depth of the holes alternated at 5 and 10 cm from the soil surface. One or two

larvae were placed into each hole and covered with soil for a total of 20 larvae per pot.

Plants were re-infested one month after initial infestation and all plants were irrigated

immediately after infestation.

Soil Redox Potential

For plants in the flooded treatments, soil redox potential (Eh), an indirect indicator

of oxygen content of the soil (Pezeshki 1991), was measured weekly with a platinum

combination electrode (Ag+/AgC1, Accumet, Fisher Scientific, Pittsburgh, Pa.) attached









to a portable pH meter (Accumet AP62, Fisher Scientific, Pittsburgh, Pa.).

Measurements were made weekly until Eh readings remained relatively constant.

Leaf Gas Exchange

Net CO2 assimilation, stomatal conductance of H20 (gs), and transpiration (E)

were measured with a CIRAS-2 portable gas analyzer (PP systems, U.K.) between 1000

and 1200 HR. Leaf gas exchange was measured at a photosynthetic photon flux (PPF) >

900 mrnol .m-2s-1 with a halogen lamp fitted on the leaf cuvette as the light source. Leaf

gas exchange was determined for two fully expanded leaves from each plant and the

average of the two leaves was used to represent values for each plant. Leaf gas exchange

was measured weekly during flooding and monthly during the larval treatment.

Plant Growth and Larval Recovery

Four months after treatments began, all plants were harvested and the roots were

carefully separated from the potting medium and rinsed with tap water to remove any soil

clinging to the root surface. Soil removed from roots was placed into bins and carefully

inspected for larvae. Recovered larvae from the soil and roots were placed into

containers with 75% ethyl alcohol. Excess water was allowed to drain from the roots

overnight and leaf, stem and root fresh weights were determined. Leaves, stems and

roots were oven-dried for three days at 700C to a constant weight and leaf, stem and root

dry weights were determined.

Statistical Analysis

Data were analyzed by Analysis of Variance and standard t-test using SAS

software (SAS Institute, 2003-2004).









Results

Soil Redox Potential (Eh)

Within a few hours after buttonwood was flooded, Eh was < 200 mV, indicating

that the soil was becoming hypoxic (Li et al. 2004, Kozlowski 1997). After one week of

flooding Eh was -250 mV indicating that there was little or no oxygen available to plant

roots (Li et al. 2003). At the end of the first week of flooding and continuing through the

final two measurements, Eh leveled off at about -280 mV (Fig. 3-1A).

Eh values for live oak were negative 3 days after flooding treatments were initiated.

Eh values continued to decrease and leveled off at about < -250 during the final three

weeks of the flooding period (Fig. 3-1B).

Leaf Gas Exchange

Flooding. Prior to flooding (at day 0), there were no significant differences in A, E

or gs between flooded and non-flooded plants (Fig. 3-2). One week after flooding began,

flooded plants had significantly lower A (t=-4.03; df=29.5; P=0.0004), E (t=-3.51; df=30;

P=0.001), and gs (t=-2.73; df=28.5; P=0.01) than non-flooded plants. Two weeks after

flooding began A (t=-6.31; df=29.4; P<0.0001), E (t=-3.70; df=30; P=0.0009) and gs (t=-

5.56; df=29.8; P<0.0001) remained lower for flooded plants than non-flooded plants (Fig.

3-2).

One, two and three weeks after flooding there were no significant differences in A,

E or gs between flooded and non-flooded plants. Four weeks after flooding began A (t=-

2.39; df=29.8; P=0.02), E (t=-2.77; df=27.4; P=0.009) and gs (t=-3.02; df=29; P=0.005)

were lower for flooded plants than non-flooded plants. At five weeks A (t=-3.94; df=30;

P=0.0004), E (t=-2.81; df=27.7; P=0.009) and gs (t=-2.18; df=30; P=0.03) remained









significantly lower in flooded plants than non-flooded plants (Fig. 3-3), and plants were

removed from flooding and infested with larvae 24 hours later.

Insect infestation. For both buttonwood and live oak, one, two and three months

after plants were infested with Diaprepes root weevil larvae, there were no significant

interactions between pre-flooding and insect treatments on any measurement date

(P>0.05; Table 3-1). Therefore, flooding treatments were pooled to test the effects of

Diaprepes root weevil larvae on A, E and gs of each species. One month after

buttonwood trees were infested, non-infested trees had higher A (t=-2.77; df=30;

P=0.009), E (t=-1.95; df=30; P=0.06) and gs (t=-3.06; df=30; P=0.004) than infested

trees (Fig. 3-4). However, two and three months after infestation there was no significant

difference in A, E or gs between treatments (Fig. 3-4).

In live oak there was no significant difference in A between infested and non-

infested plants on all three measurement dates. Although not statistically significant, E

tended to be lower for the infested plants than non-infested plants. Two months after

larval infestation, gs was lower for infested plants than non-infested plants (t=-2.55;

df=29.7; P=0.01; Fig. 3-5). On the final measurement date, there were no significant

differences in A, E or gs between the larvae infested and non-infested plants (Fig. 3-5).

Plant Growth and Larval Recovery

For both buttonwood and live oak, there were no significant effects of flooding on

plant growth. There were no significant interactions between pre-flooding and insect

treatments on leaf, stem, root and total plant fresh or dry weights (P > 0.05). Therefore,

flooding treatments were pooled for testing the effects of larval infestation on plant

biomass. Infested buttonwood had significantly lower fresh (t=-4.41; df=29.4; P=0.0001)

and dry (t=-3.97; df=29.4; P=0.0004) root weights than non-infested controls (Table 3-2).









Leaf and stem fresh and dry weights were not significantly different between infested and

non-infested treatments. Total fresh and dry weights for infested plants tended to be

lower than those of the non-infested plants; although, the differences were not

statistically significant (Table 3-2). For live oak, leaf, stem, root and total plant fresh and

dry weights were not significantly different between infested and non-infested treatments

(Table 3-2).

There were no differences in the mean number of larvae recovered from infested

buttonwood plants that had been pre-flooded compared to that of the non-flooded infested

plants. For live oak, the mean number of larvae recovered from plants that were pre-

flooded prior to infestation tended to be higher than from non-flooded treatments;

however, the difference was not statistically significant (Fig. 3-6). There was no

significant difference between oak and buttonwood in the number of larvae recovered

from plants that were not pre-flooded. However, there were significantly more larvae

recovered from pre-flooded oak than pre-flooded buttonwoods (t=-3.12; df=12.1;

P=0.008).

Discussion

Flooding had a negative effect on leaf gas exchange on both buttonwood and live

oak. Live oak is known to tolerate moderately wet conditions (Dehgan 1998). However,

the quicker reduction in A, E and gs as a result of flooding in buttonwood compared to

live oak was unexpected because buttonwood is a member of the mangrove family and

should be able to tolerate flooded conditions (Tomlinson 1980, Gilman and Watson

1993). The difference in the amount of time it took between each plant species to show

reductions in A, E and gs may be a result of several factors. Plant responses to flooding

may vary depending on genotype, age, properties of flood water and duration of flooding









(Kozlowski 1997). Prior to removal of plants from flooding, flooded buttonwoods

exhibited hypertrophied lenticels and adventitious roots growing above the water line.

These morphological adaptations to flooding are common in flood tolerant species

(Kozlowski, 1984). However, in our tests although buttonwood responded with an initial

reduction in A, E and gs under flooded conditions, it is possible that that these

morphological adaptations may have aided them in surviving if the flooding period was

prolonged. For example, Kozlowski (1984) found that flooding of green ash (Fraxinus

pennsylvanica Marsh.) caused immediate stomatal closure of seedlings followed by

production of adventitious roots and reopening of stomata after about two weeks.

In contrast to our results, Li et al. (2003) found that citrus seedlings flooded prior to

infestation were more susceptible to Diaprepes root weevil larval feeding damage than

seedlings that were not previously flooded. Pre-flooded and infested seedlings had lower

leaf gs than those that were not flooded before infestation. In our study, buttonwood and

live oak plants were larger and had a relatively dense root system compared with those of

the citrus seedlings used in the previous experiments. Thus, plant species, age and size or

a combination of these may affect the time it takes for Diaprepes root weevil larvae to

significantly affect its host plant.

Unlike Li et al. (2003) who recovered significantly more larvae from seedlings

that were pre-flooded prior to infestation, we found no significant difference in the

number of larvae recovered between pre-flooded plants or plants that were not pre-

flooded treatments. Visible root damage of both buttonwood and live oak was observed

and larvae were recovered (although more larvae were recovered from live oak than

buttonwood), indicating that larvae were feeding and causing damage to the root systems.









Live oak and buttonwood plants appeared to show initial reductions in leaf gas

exchange in response to larval feeding; however, by the final two months of treatment

there was no effect of infestation on leaf gas exchange despite the fact that larvae were

recovered from buttonwood and live oak roots and there were signs of root feeding

damage at the end of the treatment period. There was no significant effect of infestation

during the three month treatment period on leaf gas exchange or plant growth with the

exception of a reduced root system in buttonwood. However, it is possible that if the

tests were continued longer than 3 months, there could have been a negative impact of

root feeding on leaf gas exchange and plant growth. Both tree species appeared to

recover from flooding and it did not appear as if flooding predisposed the plants to root

feeding damage over the 3 month study period. However in the field, continual feeding

damage caused by multiple generations of Diaprepes root weevil larvae along with

periods of cyclical flooding during the wet summer months presumably can result in

reduction in gas exchange and growth. Therefore, further studies are warranted to

compare infested plants to non-infested plants in field nurseries.






64



150
100 A
50
> 0
\-50
M -100
-150
-200
-250
-300
-350
1 2 3 4
Time (weeks)



0--
B
-50

> -100

-150

-200

-250

-300
1 2 3 4 5 6
Time (weeks)


Figure 3-1. Soil redox potential (Eh) of flooded buttonwood (A) and live oak (B) in
containers. Symbols and bars represent means of 16 replications and bars
indicate + 1 std. error.







65



20
18 *
16
N 14
12 -. ---
12
O 10
8
E. 6 Non-flood
< 4- -- Flood
2
0






7 -



E 5


I 3
3
E
E 2

1

0



800

700 *

7 600
N
E 500
0 400 --

5 300
E
E 200

O 100

0 -
0 1 2
Weeks after flood treatment commenced


Figure 3-2. Net CO2 assimilation (A), transpiration (E) and stomatal conductance (gs) of
flooded and non-flooded buttonwood trees. Symbols represent means of 16
replications and bars indicate + 1 std. error. Asterisks indicate significant
differences between treatments according to a standard t-test (P<0.05).
















S-----


- Non-flood
- -- Flood


2 3
Time (weeks)


Figure 3-3. Net CO2 assimilation (A), transpiration (E) and stomatal conductance (gs) of
flooded and non-flooded live oak trees. Symbols represent means of 16
replications and bars indicate + 1 std. error. Asterisks indicate significant
difference between treatments according to a standard t-test (P<0.05).











16
14
E12-
o1o
010
E8
Q6
4
4-- Non-infested
2 --- Infested
0


6




-3
o
E
LU

1

0


350
300 *
250
E
6 200
150
E
E 100
c 50

0
1 2 3
Months after treatments were infested

Figure 3-4. Net CO2 assimilation (A), transpiration (E) and stomatal conductance (gs) of
buttonwood trees infested with Diaprepes root weevil larvae or non-infested.
Symbols represent means of 16 replications and bars indicate + 1 std. error.
Asterisks indicate significant difference between treatments according to a
standard t-test (P<0.05).















V)
N
E
0
-5
E













E
0
CM



E
E
I











E


C-
E






E


Time (month)


Figure 3-5. Net CO2 assimilation (A), transpiration (E) and stomatal conductance (gs) of
live oak trees infested or non-infested with Diaprepes root weevils. Symbols
represent means of 16 replications and bars indicate + 1 std. error. Asterisks
indicate significant differences between treatments according to a standard t-
test (P<0.05).


- Non-infested
- -m- Infested


f *
-r










18
16 Flood
14 a Non-flood
> 14


10

12-





oak buttonwood

Figure 3-6. Larvae recovered from pre-flooded or non-flooded treatments of buttonwood
and live oak.
M 1 0 -...... ...... .
J'S 8 . .. .. ', ,"
6_ b ...... .. .. ... .


14- m
z 2 -...... .. ..... ....... .... .


oak buttonwood

Figure 3-6. Larvae recovered from pre-flooded or non-flooded treatments of buttonwood
and live oak.














Table 3-1. The effect of flooding (FLD) and insect infestation (INFST) on leaf gas exchange of buttonwood and live oak trees.


Species
Buttonwood


Measurement
A (tmol C02 m2 s1)


treatment One month


Two months


SS MS P SS


Three months
P SS MS P


E (mmol H2O m2 s'1)


gs (mmol CO2 m2 s1)


Live Oak A ([mol CO2 m2 '1)




E (mmol H2O m2 s'1




gs (mmol CO2 m 2 s1)


0.31
17.18
0.27


0.09
0.10
0.51


FLD
INFST
Fld Infst


FLD
INFST
Fld Infst


FLD
INFST
Fld Infst

FLD
INFST
Fld Infst

FLD
INFST
Fld Infst

FLD
INFST
Fld Infst


0.31
17.18
0.27


0.03
1.45
0.008


166.5
18002.5
28.12
SS
6.52
0.41
4.92

0.18
0.12
0.61

3.12
24.50
5227.5


0.03
1.45
0.008


166.5
18002.5
28.12
MS
6.52
0.41
4.92

0.18
0.12
0.61

3.12
24.50
5227.5


0.77
0.06
0.88


0.77
0.006
0.90
P
0.29
0.78
0.35

0.58
0.65
0.32

0.98
0.96
0.46


0.90
0.89
0.78


0.70
0.22
0.29


0.54
0.65
0.52
P
0.36
0.14
0.18

0.29
0.07
0.72

0.12
0.01
0.77


0.04
0.44
0.34


882.0
488.2
957.0
SS
6.21
16.67
13.78

0.86
2.69
0.09

13304.8
34485.9
446.25


4.61
0.01
0.10


0.09
0.04
0.07


582.2
8.50
279.0
SS
3.51
1.48
0.07

0.03
0.03
0.05

800.0
1212.7
406.1


0.04
0.44
0.34


882.0
488.2
957.0
MS
6.21
16.67
13.78

0.86
2.69
0.09

13304.8
34485.9
446.25


4.61
0.01
0.10


0.09
0.04
0.07


582.2
8.50
279.0
MS
3.51
1.48
0.07

0.03
0.03
0.05

800.0
1212.7
406.1


0.24
0.94
0.85


0.51
0.64
0.55


0.41
0.92
0.57
P
0.58
0.72
0.93

0.76
0.76
0.71

0.70
0.63
0.78












Table 3-2. The effect of Diaprepes root weevil larvae on leaf, stem, root and total biomass of infested or non-infested buttonwood and
live oak trees.
Plant species Treatment n Fresh weight (g) Dry weight (g)
Buttonwood leaf stem root total leaf stem root total
Infested 16 317.00 184.61 162.81 664.42 173.6 99.37 78.96 351.95
Non-infested 16 343.81 216.46 220.77 781.03 200.99 116.86 104.34 422.19
(P) (0.43) (0.14) (0.0001) (0.07) (0.28) (0.12) (0.0004) (0.08)

Live oak
Infested 16 54.88 293.57 230.00 578.45 30.62 172.53 135.15 338.31
Non-infested 16 67.35 306.65 272.56 646.57 36.84 175.25 154.19 366.29
(P) (0.12) (0.73) (0.53) (0.52) (0.19) (0.91) (0.62) (0.65)

Significance determined by a standard t-test at the 0.05 level.















CHAPTER 4
EFFECT OF ADULT DIAPREPES ROOT WEEVIL ON LEAF GAS EXCHANGE
AND GROWTH OF BUTTONWOOD AND LIVE OAK

Introduction

Diaprepes root weevil Diaprepes abbreviatus L. (Coleoptera: Curculionidae), is a

polyphagous species known to feed on a wide variety of plants throughout Florida. The

root weevil has a host range of more than 270 plant species (Simpson et al. 1996). In

Florida it is estimated that this weevil causes more than 70 million dollars in damage

annually (Weissling et al. 2002). Adult weevils have a voracious appetite and may cause

severe defoliation of host plants.

Adult Diaprepes root weevils emerge from the soil year round in Florida; however,

the peak emergence period is from May through October. As the general adults begin to

dig their way out of the soil they shed a pair of deciduous mandibles. Adult weevils

emerge from the soil and move up the tree canopy to feed and mate. Oviposition

generally begins 3 to 7 days after emergence from the soil surface (Wolcott 1936). The

average longevity reported for adult weevils reared on an artificial diet is about 147 days

for females and 135 days for males (Beavers 1982).

Diaprepes root weevil is known to be associated with a wide variety of ornamental

plants grown in nurseries throughout Florida and many nurseries in Miami-Dade County,

Florida are infested with this pest. In a recent survey of several field nurseries, Mannion

et al. (2003) found that characteristic leaf notching and egg masses from adult weevils

were widespread on several tree species that are commonly grown together. The plant









species with the highest percentage of egg masses were live oak (Quercus virginiana

Mill.), silver buttonwood (Conocarpus erectus L.) and black olive (Bucida buseras L.).

A basic understanding of plant physiological responses to arthropod herbivory may

provide critical information for predicting and preventing crop damage. Leaf gas

exchange measurements provide a basis for comparing herbivore effects on different

plants and plant parts (Welter 1989, Schaffer and Mason 1990, Peterson et al. 1998).

Several studies of arthropod herbivory on plant physiology, in particular leaf feeding

effects on leaf gas exchange, have shown reductions in leaf gas exchange as a result of

insect feeding (Schaffer et al. 1986, Mobley and Marini 1990, Lakso et al. 1996, Schaffer

et al. 1997). However, other studies have shown slight or no effects of leaf feeding on

gas exchange (Peterson et al. 1996). Insect herbivory has also been shown to reduce

biomass of several plant species (Schaffer and Mason 1990, Welter 1991).

Adult Diaprepes root weevils damage plants primarily by leaf notching and

generally tend to feed on new leaf flushes and occasionally on fruit. There is not much

known about the effects of Diaprepes root weevil feeding on leaf physiology of

ornamental plants. Only one previous study examined the effect of adult weevil feeding

on citrus leaf photosynthesis (Syvertsen and McCoy 1985).

Buttonwood and live oak are known hosts of Diaprepes root weevil and support all

stages of this pest from egg to adult (Mannion et al. 2003). Both tree species are

commonly grown together in the same field nurseries in south Florida. Larvae alone are

known to cause significant root damage to both species and adult weevils have also been

observed to cause considerable leaf tissue damage in both young and mature trees.

However, there are no published data quantifying the effects of leaf feeding damage on









these ornamental tree species. The objective of this study was to evaluate the effects of

adult Diaprepes root weevil leaf feeding on leaf gas exchange and growth of buttonwood

and live oak trees.

Materials and Methods

Three experiments were conducted to assess the effects of adult weevil feeding on

live oak and buttonwood trees. All experiments were conducted in spring and summer of

2004 at the University of Florida, Tropical Research and Education Center, Homestead,

FL, 25.50 N latitude and 85.50 W longitude.

Plant and Insect Material

For Experiment 1, green buttonwood trees in 3.79-L containers were purchased

from a commercial nursery (Princeton Nurseries II, Inc. Homestead, FL) and repotted

into 11.35-L containers with well-drained media purchased from a local soil distributor

(Lantana Peat & Soil, Boynton Beach, FL) consisting of 40% Florida peat, 30% pine

bark, 10% sand, 20% cypress sawdust (by volume) and 6.80 kg dolomite/91.44 cm. For

Experiment 2, green buttonwoods in 3.79-L containers were purchased from a local

nursery (Bill Ingram & Grandsons NSY, Homestead, FL) and repotted into 11.35-L

containers with the same media described above. For Experiment 3, live oaks in 3.79-L

containers were purchased from a commercial nursery (Action Theory Nursery,

Homestead, FL) and repotted into 11.35-L containers with the same media described

above. All plants where fertilized once prior to treatment initiation with PlantacotePlus

14N-9P-15K (Helena Chemical Company, Collierville, TN) controlled-release fertilizer.

Plants were placed in aluminum frame cages constructed to allow suitable light for

normal plant growth while preventing insects from escaping. Cages were 1.21 m x 60.96









cm x 60.96 cm with aluminum screening with a mesh size of 1.6 mm2. There was one

plant per cage.

For all three experiments, adult Diaprepes root weevils were collected from a field

nursery (Native Tree Nursery, Homestead, FL). Weevils were maintained in acrylic

holding cages and fed fresh buttonwood foliage that was replaced every other day with

water supplied in small containers with a dental wick.

Adult weevils were randomly selected from the holding cages and separated by sex.

At the start of each experiment 10 males and 5 females were released into each cage.

Each experiment was arranged in a complete block design with two treatments (infested

or non-infested) and 7 single-plant replications per treatment.

To prevent neonate larvae from entering the soil after eggs hatched, the soil surface

of each pot was covered with a 50.8 cm2 sheet of Weed Block (Easy Gardener, Inc. P.O.

Box 21025 Waco, TX), which was secured around the plant stems with duct tape. This

material was selected because the pore size was small enough to prevent larvae from

passing through, but large enough to allow water to penetrate.

Leaf Gas Exchange

Net CO2 assimilation (A), stomatal conductance of H20 (gs), and transpiration (E)

were measured prior to infesting plants and then monthly after insect infestation with a

CIRAS-2 portable gas analyzer (PP Systems, U.K.) between 1000 and 1200 HR. Leaf

gas exchange measurements were made at a photosynthetic photon flux (PPF) > 900

mrnol .m-2'-1 with a halogen lamp fitted on the leaf cuvette as the light source. Leaf gas

exchange was determined for two young flushing leaves and two fully expanded leaves

from each plant and the averages of each pair of young and mature leaves were used to

represent leaf gas exchange of young and mature leaves for each plant. For experiment 3,









there was an insufficient number of new leaf flushes to measure gas exchange on young

leaves. Therefore, gas exchange was measured for only two mature leaves per plant.

Plant biomass

At the end of each experiment, plants were harvested, all leaves where removed

and the total leaf area per plant was determined with a leaf area meter (Li-Cor, Lincoln,

NE; model Li-3000). Plants were harvested and roots were washed with tap water to

remove media attached to the root hairs. Excess water was allowed to drain from the

roots for 24 hrs and leaf, stem and root fresh weights were determined. Plant tissues were

then oven-dried at 700C for two days and leaf, stem and root dry weights were

determined.

Statistical Analysis

Data were analyzed by a standard t-test and Analysis of Variance using SAS

software (SAS Institute, 2003-2004).

Results

Visible Signs of Herbivory

In both buttonwood experiments the characteristic leaf notching caused by adult

Diaprepes root weevil feeding was observed within one day after insects were released

into cages. Both young and mature leaves had signs of damage; however, the weevils

preferred the younger foliage. Most of the leaf area of young leaves, with the exception

of the mid-veins, was completely removed by the first month. Mature leaves also had

considerable notching primarily on the edges of the lamina. Live oak plants had very leaf

feeding damage. A few live oak leaves had minor damage on the edges of the lamina but

no significant damage was caused. In the cages with live oak trees all weevils died

within the first month of the experiment.









Leaf Gas Exchange

In the first buttonwood experiment there were no significant differences between

treatments in A, E or gs of mature leaves. One month after treatments were initiated there

were no significant effects of adult Diaprepes root weevil feeding on A, E or gs of young

or old leaf tissue. On the final measurement date, two months after infestation, A, E and

gs of infested plants tended to be higher than that of the controls but the differences were

not significant (Table 1).

In the second buttonwood experiment, one month after treatments were initiated, A

was significantly lower of young leaves of infested plants than those of non-infested

treatments (/=-2.73; df=8.69; P=0.02). However E and gs of young leaves were not

significantly different between treatments (Table 2). Net CO2 assimilation, E and gs of

mature leaves were not significantly affected by adult weevil leaf feeding. Two months

after treatments were initiated, gs was significantly higher in young leaves of infested

than in those of non-infested plants (t=2.14; df=10.7; P=0.05). However, A and E of

young leaves were not significantly different between treatments. In mature leaves, A

(t=2.66; df=9.95; P=0.02), E (t=2.58; df=11.8; P=0.02) and gs (t=2.79; df=11.4; P=0.01)

were higher in infested plants than in non-infested plants (Table 2).

For live oak, one month after infestation, there were no significant differences in A,

E and gs between treatments. At this time all adult weevils in the cages with oak trees

had died and the experiment was terminated.

Plant Biomass

Mean leaf area for both buttonwood experiments was not significantly different

between treatments (Table 3). However, in experiment 1, plants infested with adult









weevils had 18.3% less leaf area than the controls and in experiment 2, plants infested

with adult weevils had 27.8% less leaf area than non-infested control plants.

Leaf, stem and root fresh and dry weights of buttonwood in experiment 1 were

lower in the infested than non-infested plants but the differences were not significant

(Table 4). For buttonwood in experiment 2, leaf, stem and root fresh weights for infested

treatments were lower than that of non-infested treatments; however, the differences were

not statistically significant. Dry weights of leaves and stems were lower for infested

plants than non-infested plants but were not significantly different between treatments.

Dry root weights were significantly lower (t=--2.12; df=l 1.1; P=0.05) in the infested than

in non-infested treatment (Table 4).

Discussion

Leaf feeding by adult Diaprepes root weevil had variable effects on leaf gas

exchange of buttonwood. Live oak was not affected by adult insect herbivory

presumably because mature, hardened-off leaves were too thick for the mandibles of this

insect. Adult weevils were observed trying to feed on oak leaves but they were only

causing minor scrapes to the leaf margins and all weevils died within a month, most

likely because of starvation. Live oak trees were not in a period of leaf flushing during

the treatment period and the lack of significant difference in leaf gas exchange and

growth between treatments was probably related to the phenological stage of the plants at

the time of infestation. In subsequent field observations there appeared to be specific

periods of time when relatively large populations of adult Diaprepes root weevils were

found in live oak canopies. This feeding period corresponded to periods of leaf flushing

when the young leaves were still succulent and the weevils were able to feed on the

foliage.









In both buttonwood experiments, one month after treatments were initiated, most

young leaves of infested plants had obvious feeding damage and most of the leaf tissue

except for the area around the mid-vein, were completely removed. Mature leaves also

had signs of feeding damage to the edges of the lamina with no damage to the mid-vein.

Similarly, Syvertsen and McCoy (1985) reported considerable leaf feeding damage

caused by a citrus root weevil, little leaf notcher (Artipusfloridanus Horn.) to tender

citrus leaf margins with no injury to the mid-veins. In our study, adult weevils preferred

the young tender foliage over mature leaves; however, the majority of leaves of each

plant were mature and the outer areas of almost all leaves had feeding damage. Mature

leaves appeared to have more leaf area remaining around the mid-vein than younger

leaves

In the first experiment, A, E and gs of buttonwood were higher for infested than

non-infested plants, but the differences were not significantly different after one or two

months of feeding. Some authors have reported that leaf mass consumption by insect

herbivores does not negatively impact photosynthesis of the remaining leaf tissues

(Welter 1989, Peterson et al. 1996). No reduction in leaf gas exchange was reported for

actual and simulated herbivory of tomato (Lycopersicon esculentum L.). Actual

defoliation by tobacco horn worm (Manduca sexta L.) larvae or simulated defoliation did

not change photosynthetic rates per unit area of tomato leaflets (Welter 1991). It has

previously been reported that leaf feeding in which there is only removal of leaf tissue

without injury to the mid-vein only reduces the amount of photosynthetic leaf area but

not photosynthetic rates of the remaining leaf tissue (Li and Proctor 1984, Peterson et al.

1996, Peterson et al. 1992, Peterson et al. 2004). In the first buttonwood experiment,









similar to results of previous studies, we found no significant reduction in leaf gas

exchange due to leaf feeding. However, in the second experiment there were variable

leaf gas exchange responses. After one month of infestation, there were significant

reductions of A, and no reduction of E or gs of younger buttonwood leaves or A, E and gs

of mature leaves. After two months of leaf feeding, mature leaves of infested

buttonwood trees had greater leaf gas exchange than controls. Although increases in leaf

gas exchange have been previously reported for some plant species, other studies have

generated contrasting results. Some authors reported no difference in leaf gas exchange

as a result of arthropod herbivory, whereas others have reported increases and still others

have reported decreases (Welter 1989, Detling et al. 1980, Sances et al. 1981, Mobley

and Marini 1990, Peterson et al. 1998). In the majority of studies involving selective

tissue feeders such as leafhoppers and mites, showed reduced photosynthesis. Studies

involving defoliators, which remove partial or entire leaf tissue, showed a tendency

toward increased photosynthesis of remaining leaf tissues (Welter 1989).

Both buttonwood experiments were conducted with the same materials and

methods, but only the second experiment yielded significant differences in leaf gas

exchange between treatments. However, buttonwood experiment 1 leaf gas exchange on

the final measurements of both young and mature leaves tended to be higher in the

infested plants than in non-infested controls. Our study yielded different results than

those of other defoliation studies, which found that leaf gas exchange of infested plants

were not significantly affected by simulated or actual insect defoliation (Welter 1991,

Peterson et al. 1996, Peterson et al. 2004). These results suggest that plant species may

respond differently to defoliation. In our study as with others, gas exchange









measurements were made on single leaves which is not a true representation of the whole

plants response to herbivory. Further studies on the effects of leaf feeding by Diaprepes

root weevils on whole-plant photosynthesis are warranted and should be compared to

results with single leaf gas exchange.

In both buttonwood experiments, leaf area was less for infested plants than

controls, although the differences were not statistically significant. Leaf, stem and root

fresh and dry weights tended to be lower in weevil infested plants than control plants.

However, the differences were not significant except for the root dry weights of

buttonwoods in the second experiment. Plants were only exposed to adult weevil feeding

for a brief (two month) period and although infested plants weighed less, plant weights

were not significantly affected by leaf feeding. Thus, longer-duration studies may yield

statistically significant differences in gas exchange, leaf area and dry weights between

infested and non-infested buttonwood and oak trees.












Table 4-1. The effect of adult Diaprepes root weevil on net CO2 assimilation (A), transpiration (E) and stomatal conductance (gs) of
buttonwood (Expt. 1)
Treatment Leafage Date A (glmol C02 m-2 s-1) E (mmol H20 m-2 s-1) gs (mmol C02 m-2 s-1)

2/5/04


Pre-Infested
Non-infested


young
young
(P)


6.05
8.38
(0.06)


Pre-Infested mature 9.10
Non-infested mature 8.89
(P) (0.90)
3/9/04
Infested young 3.39
Non-infested young 1.41
(P) (0.09)

Infested mature 2.77
Non-infested mature 1.10
(P) (0.21)
4/19/04
Infested young 5.70
Non-infested young 4.85
(P) (0.58)

Infested mature 7.40
Non-infested mature 6.22
(P) (0.46)

Significance determined by a standard t-test at the 0.05 level.


2.10
2.31
(0.47)

2.18
2.22
(0.91)

1.46
1.29
(0.70)

1.23
1.13
(0.76)

2.55
2.33
(0.66)

2.67
2.08
(0.21)


133.36
146.57
(0.54)

145.21
158.79
(0.73)

57.35
46.42
(0.58)

49.35
42.64
(0.68)

121.00
115.64
(0.85)

129.07
102.14
(0.32)












Table 4-2. The effect of adult Diaprepes root weevil on net CO2 assimilation (A), transpiration (E) and stomatal conductance (gs) of
buttonwood (Expt. 2)
Treatment Leaf age Date A (gtmol C02 m-2 s-1) E (mmol H20 m-2 s-1) gs (mmol C02 m-2 s-1)
5/18/04


Pre-Infested
Non-infested


Pre-Infested
Non-infested


Infested
Non-infested


Infested
Non-infested


Infested
Non-infested


Infested
Non-infested


young
young
(P)

mature
mature
(P)

young
young
(P)

mature
mature
(P)

young
young
(P)

mature
mature
(P)


6/18/04


7/7/04


4.96
6.13
(0.41)

15.17
13.63
(0.22)

3.08
7.60
(0.02)

13.55
14.40
(0.45)

2.47
0.97
(0.21)

14.11
9.18
(0.02)


3.92
3.92
(0.99)

5.04
4.65
(0.29)

4.78
5.01
(0.63)

5.25
4.78
(0.49)

5.09
4.13
(0.11)

6.76
4.83
(0.02)


180.21
192.00
(0.69)

295.57
260.50
(0.31)

254.79
264.71
(0.73)

313.71
285.86
(0.62)

200.29
134.71
(0.05)

327.93
187.91
(0.01)


Significance determined by a standard t-test at the 0.05 level.






84


Table 4-3. The effect of adult Diaprepes root weevil on total leaf area of buttonwod
Plant species Treatment leaf area (cm2)


Experiment 1



Experiment 2


Infested
Non-infested
(P)

Infested
Non-infested
(P)


Significance determined by a standard t-test at the 0.05 level.


6419.10
7861.20
(0.10)

3401.00
4710.20
(0.14)









Table 4-4. The effect of adult Diaprepes root weevil leaf feeding on buttonwood fresh
and dry weights 2 months after infestation.
Species Treatment Fresh weight (g) Dry weight (g)


leaf
293.53
339.61


stem
172.89
183.1


root
230.04
234.8


leaf
109.17
120.94


stem
82.51
88.48


root
104.81
107.90


(0.19) (0.67) (0.90) (0.34) (0.61) (0.84)


134.51
155.16


36.37
39.31


31.94
48.05


44.81
50.27


11.62
13.14


17.32
23.60


(0.47) (0.69) (0.07) (0.59) (0.51) (0.05)


Significance determined by a standard t-test at the 0.05 level.


Exp. 1


Exp. 2


Infested
Non-
infested
(P)

Infested
Non-
infested
(P)














CHAPTER 5
SUMMARY AND CONCLUSIONS

Larval root feeding by Diaprepes root weevil caused considerable damage to the

root systems of buttonwood, live oak and pygmy date palm. Of the three species tested

buttonwood, was affected more by larval root feeding than live oak and pygmy date

palm. Buttonwood leaf gas exchange was significantly reduced by larval root feeding in

two studies and several plants showed severe symptoms of stress in the form of leaf

yellowing and wilt. Although larvae were recovered and larval root damage was

observed on all three species, buttonwood appeared to have the most significant root

feeding damage and the greatest reduction in root weight. These results, which are

similar to those of Mannion et al. (2003), suggest that buttonwood may be a more

suitable host for larvae development than live oak or pygmy date palm. However; these

studies were conducted for a limited time period. Therefore, although buttonwood was

more severely damaged than live oak or pygmy date palm, prolonged larval root feeding

damage may cause considerable damage to live oak and pygmy date palm.

Flooding is a relatively common occurrence in the low lying ornamental field

nurseries, which consist of marl soils, during the summer months in south Florida. These

areas also tend to have the highest populations of Diaprepes root weevils. In our study

with containerized buttonwood and live oak, leaf gas exchange of both tree species were

negatively affected by flooding. However, we found no significant interaction between

pre-flood and larval treatments on leaf gas exchange compared with those treatments that

were not pre-flooded prior to larval infestation. Larval root feeding did not negatively









affect leaf gas exchange of either buttonwood or live oak for the relatively short

infestation period. However, in comparison with the first larval root feeding study in

which larvae were re-infested and allowed to feed on plant roots over a five month

period, this study was conducted over a shorter period of time and larval root feeding was

limited to a three month period. Therefore, the length of time in which plants are infested

with larvae appears to affect how much damage they may cause to plant root systems.

Leaf feeding by adult Diaprepes root weevils causes considerable damage to the

leaf tissue of plants. Ornamental plants are grown and sold for the aesthetic appeal and

any severely damaged plants are not able to be sold. The effects of leaf feeding damage

on physiology and growth of several ornamental tree species, which are hosts of

Diaprepes root weevil, have not been previously studied. In this study we measured leaf

gas exchange to observe whether leaf feeding damage caused by adult Diaprepes root

weevils negatively affects normal physiological functions of plants such as

photosynthesis. We found that leaf gas exchange of weevil damaged buttonwood leaves

generally had higher net photosynthetic rates on the remaining leaf area compared with

controls. However, leaf gas exchange was measured on single leaves and although

remaining leaf tissue had higher photosynthesis, there was less leaf area overall so whole

plant net photosynthesis may be less. The results from these studies provide us with

some basic understanding of the effects of Diaprepes root weevil feeding on physiology

and growth of the selected plants. There are many ornamental species affected by this

pest and its impact on some of the more economically important tree species is unknown.

This information may be useful for future research to expand on the limited information






88


on the effects of Diaprepes root weevil on ornamental plants and may also improve our

knowledge for better management and control this pest.
















LIST OF REFERENCES

Allen, J. C. 1978. The effect of citrus rust mite damage on citrus fruit drop. J. Econ.
Entomol. 71: 746.

Andersen, P., P. B. Lombard, and M. N. Westwood. 1984. Effect of root anaerobiosis on
the water relations of several Pyrus species. Physiol. Plant. 62: 245-252.

Andersen, P. C., and R. F. Mizell III. 1987. Physiological effects of galls induced by
Phylloxera notabilis (Homoptera: Phylloxeridae) on pecan foliage. Environ.
Entomol. 16: 264-268.

Anderson, R. S. 2002. Curculionidae, pp. 722-806. In R. H. Arnett, M. Thomas, P.
Skelley and J. H. Frank (eds.), American beetles. Vol. 2. CRC Press, Boca Raton,
FL.

Andrews, L. K., and L. F. La Pre. 1979. Effects of pacific spider mite on physiological
processes of almond foliage. J. Econ. Entomol. 72: 651-654.

Beavers, J. B. 1982. Biology of Diaprepes abbreviatus (Coleoptera: Curculionidae)
reared on an artificial diet. Fla. Entomol. 65: 263-269.

Beavers, J. B., and A. G. Selhime. 1975. Development of Diaprepes abbreviatus on
potted citrus seedlings. Fla. Entomol. 58: 271-272.

Beavers, J. B., and A. G. Selhime. 1976. Population dynamics of Diaprepes abbreviatus
in an isolated citrus grove in central Florida. J. Econ. Entomol. 69: 9-10.

Broschat, T. K., and A. Meerow. 2000. Ornamental Palm Horticulture. University Press
of Florida. Gainesville, FL.

Crane, J. H., and F. S. Davies. 1989. Flooding responses of Vaccinium species.
HortScience 24: 203-209.

Dehgan, B. 1998. Landscape Plants for Subtropical Climates. University Press of
Florida. Gainesville, FL

Dennis, E. S., R. Dolferus, M. Ellis, M. Rahman, Y. Wu, F. U. Hoeren, A. Grover, K. P.
Ismond, A. G. Good, and W. J. Peacock. 2000. Molecular strategies for improving
waterlogging tolerance in plants. J. Exp. Bot. 51: 89-97.









Detling, J. K., D. T. Winn, C. Procter-Gregg and E. L. Painter. 1980. Effects of
simulated grazing by below-ground herbivores on growth, CO2 exchange and
carbon allocation patterns ofBouteloua gracilis. J. Appl. Ecol. 17: 771-778.

Emenegger, D. B., and R. E. Berry. 1978. Biology of strawberry root weevil on
peppermint in western Oregon. Environ. Entomol. 7: 495-498.

Futch, S. H., and C. W. McCoy Jr. 1993. Citrus Root Weevils. Circ. 1065. Fla. Coop.
Ext. Serv., Inst. Food Agric. Sci., University of Florida, Gainesville, FL.

Gilman, E.F., and D.G. Watson. 1993a. Phoenix roebelenii: pygmy date palm. ENH-600.
Fla. Coop. Ext. Serv., Inst. Food Agric. Sci., University of Florida, Gainesville, FL.

Gilman, E.F., and D.G. Watson. 1993b. Conocarpus erectus: buttonwood. ENH-338. Fla.
Coop. Ext. Serv., Inst. Food Agric. Sci., University of Florida, Gainesville, FL.

Godfrey, L. D., L. J. Mienke, and R. J. Wright. 1993. Effects of larval injury by western
corn rootworm (Coleoptera: Chrysomelidae) on gas exchange parameters of field
corn. J. Econ. Entomol. 86: 1546-1556.

Grafton-Cardwell, E. E., K. E. Godfrey, J. E. Pefia, C. W. McCoy, R. F. Luck. 2004.
Diaprepes Root Weevil. University of California, Division of Agriculture and
Natural Resources. Publication 8131, 8 pp.

Graham, J. H., C. W. McCoy, and J. S. Rogers 1997. The Phytopthora-Diaprepes weevil
complex. Citrus Industry 78: 67-70.

Hare, J. D., and R. R. Youngman. 1987. Gas exchange of orange (Citrus sinensis) leaves
in response to feeding injury by the citrus red mite (Acari: Tetranychidae). J. Econ.
Entomol. 80: 1249-1253.

Heichel, G. H., and N. C. Turner. 1973. Physiological responses of dogwood (Cornus
florida) to infestation by the dogwood borer (Thamnosphecia scitula). Ann. Appl.
Biol. 75: 401-408.

Hodges, A., and J. Haydu. 2000. Economic Impacts of the Florida Environmental
Horticulture Industry, 2000. Report El 02-3. University of Florida, IFAS, Food and
Resource Economics Department.

Hou. X., L. J. Mienke and T. J. Arkebaur. 1997. Soil moisture and larval western corn
rootworm injury: influences on gas exchange parameters in corn. Agron. J. 89: 709-
717.

Johnson, M. W., S. C. Welter, N. C. Toscano, I. P. Ting, and J. T. Trumble. 1983.
Reduction of tomato leaflet photosynthetic rates by mining activity of Liriomyza
sativae (Diptera: Agromyzidae). J. Econ. Entomol. 76: 1061-1063.