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Effects of Herbivory by Diaprepes abbreviatus (L.) (Coleoptera

Permanent Link: http://ufdc.ufl.edu/UFE0024747/00001

Material Information

Title: Effects of Herbivory by Diaprepes abbreviatus (L.) (Coleoptera Curculionidae) and Flooding on the Physiology and Growth of Select Ornamental Plant Species in South Florida
Physical Description: 1 online resource (198 p.)
Language: english
Creator: Martin, Cliff
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: apple, buttonwood, cherry, diaprepes, flooding, mahogany, pond, surinam
Entomology and Nematology -- Dissertations, Academic -- UF
Genre: Entomology and Nematology thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy EFFECTS OF HERBIVORY BY DIAPREPES ABBREVIATUS (L.) (COLEOPTERA: CURCULIONIDAE) AND FLOODING ON THE PHYSIOLOGY AND GROWTH OF SELECT ORNAMENTAL PLANT SPECIES IN SOUTH FLORIDA By Cliff G. Martin August 2009 Chair: Catharine M. Mannion Cochair: Bruce A. Schaffer Major: Entomology and Nematology The objective of this research was to determine effects of herbivory by Diaprepes abbreviatus (L.) (Coleoptera: Curculionidae) and flooding on the physiology and growth of select ornamental plant species in south Florida. Susceptibility to larval feeding was greatest for green buttonwood and Surinam cherry, followed by mahogany, then pond apple. Pond apple supported Diaprepes larvae to pupation and represents a new host plant and family (Annonaceae). Under flooded conditions and/or planted in marl soil, pond apple performed optimally, whereas buttonwood, mahogany, and Surinam cherry had reduced leaf gas exchange and growth. Surinam cherry had the greatest preference of potting medium over marl soil, followed by buttonwood, then mahogany, then pond apple. Pond apple and buttonwood tolerated flooding the best, followed by mahogany, then Surinam cherry. When flooded 38 days, larval survival was lowest in flooded potting medium, followed by flooded marl soil, then non-flooded marl soil and non-flooded potting medium. Larvae from flooded marl soil had smaller head capsule widths and instars than non-flooded larvae in marl soil or potting medium. Flooding more than soil type influenced larval survival, though both factors affected it. Effects on leaf gas exchange and plant growth were cumulative for flooding in green buttonwood and for infestation in Swingle citrumelo. Contrary to similar studies, buttonwood was not flood-adaptated and was the most flood-sensitive species, possibly because of short flood periods, while Swingle citrumelo was the most sensitive species to larval feeding. Flooding with three 2-d cycles seemed more likely to control larvae in Swingle citrumelo than in green buttonwood. In an adult Diaprepes study, green buttonwood adapted to flooding rendering leaf gas exchange, growth, and adult preference the same under flooded and non-flooded conditions. For Surinam cherry and mahogany, leaf gas exchange, growth, and adult feeding damage in infested cages was higher for non-flooded than flooded plants. Pond apple had the lowest adult feeding damage and egg clusters per plant, hence, it seems unlikely to become infested. Surinam cherry was most susceptible to flooding, followed by mahogany, with buttonwood and pond apple least affected, whereas buttonwood and mahogany were most susceptible to adult Diaprepes, followed by Surinam cherry, then pond apple.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Cliff Martin.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: Mannion, Catharine M.
Local: Co-adviser: Schaffer, Bruce A.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024747:00001

Permanent Link: http://ufdc.ufl.edu/UFE0024747/00001

Material Information

Title: Effects of Herbivory by Diaprepes abbreviatus (L.) (Coleoptera Curculionidae) and Flooding on the Physiology and Growth of Select Ornamental Plant Species in South Florida
Physical Description: 1 online resource (198 p.)
Language: english
Creator: Martin, Cliff
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: apple, buttonwood, cherry, diaprepes, flooding, mahogany, pond, surinam
Entomology and Nematology -- Dissertations, Academic -- UF
Genre: Entomology and Nematology thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy EFFECTS OF HERBIVORY BY DIAPREPES ABBREVIATUS (L.) (COLEOPTERA: CURCULIONIDAE) AND FLOODING ON THE PHYSIOLOGY AND GROWTH OF SELECT ORNAMENTAL PLANT SPECIES IN SOUTH FLORIDA By Cliff G. Martin August 2009 Chair: Catharine M. Mannion Cochair: Bruce A. Schaffer Major: Entomology and Nematology The objective of this research was to determine effects of herbivory by Diaprepes abbreviatus (L.) (Coleoptera: Curculionidae) and flooding on the physiology and growth of select ornamental plant species in south Florida. Susceptibility to larval feeding was greatest for green buttonwood and Surinam cherry, followed by mahogany, then pond apple. Pond apple supported Diaprepes larvae to pupation and represents a new host plant and family (Annonaceae). Under flooded conditions and/or planted in marl soil, pond apple performed optimally, whereas buttonwood, mahogany, and Surinam cherry had reduced leaf gas exchange and growth. Surinam cherry had the greatest preference of potting medium over marl soil, followed by buttonwood, then mahogany, then pond apple. Pond apple and buttonwood tolerated flooding the best, followed by mahogany, then Surinam cherry. When flooded 38 days, larval survival was lowest in flooded potting medium, followed by flooded marl soil, then non-flooded marl soil and non-flooded potting medium. Larvae from flooded marl soil had smaller head capsule widths and instars than non-flooded larvae in marl soil or potting medium. Flooding more than soil type influenced larval survival, though both factors affected it. Effects on leaf gas exchange and plant growth were cumulative for flooding in green buttonwood and for infestation in Swingle citrumelo. Contrary to similar studies, buttonwood was not flood-adaptated and was the most flood-sensitive species, possibly because of short flood periods, while Swingle citrumelo was the most sensitive species to larval feeding. Flooding with three 2-d cycles seemed more likely to control larvae in Swingle citrumelo than in green buttonwood. In an adult Diaprepes study, green buttonwood adapted to flooding rendering leaf gas exchange, growth, and adult preference the same under flooded and non-flooded conditions. For Surinam cherry and mahogany, leaf gas exchange, growth, and adult feeding damage in infested cages was higher for non-flooded than flooded plants. Pond apple had the lowest adult feeding damage and egg clusters per plant, hence, it seems unlikely to become infested. Surinam cherry was most susceptible to flooding, followed by mahogany, with buttonwood and pond apple least affected, whereas buttonwood and mahogany were most susceptible to adult Diaprepes, followed by Surinam cherry, then pond apple.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Cliff Martin.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: Mannion, Catharine M.
Local: Co-adviser: Schaffer, Bruce A.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024747:00001


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EFFECTS OF HERBIVORY BY DIAPREPES ABBREVIATUS (L.) (COLEOPTERA: CURCULIONIDAE) AND FLOODING ON TH E PHYSIOLOGY AND GROWTH OF SELECT ORNAMENTAL PLANT SPE CIES IN SOUTH FLORIDA By CLIFF G. MARTIN A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2009 1

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2009 Cliff G. Martin 2

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ACKNOWLEDGMENTS Above all, I would like to thank Cathar ine Mannion for funding me longer than anyone else in my 30 year-work hist ory, Bruce Schaffer for superior mentorship, and both for being flexible to my work habits and accepting me as a student despite my age and previous bad references. I also thank the Tropical Research and Educational Center (TREC) for providing continuous housing at an out standing bargain and for being one of the best places I have lived or worked in my life. I will truly miss living and working at TREC, but I know I must move on. I also thank Eileen Buss and Fred Davies for serving on my committee. Together, the above committee members are really co -authors of this dissertation. I also thank Holly Glenn for excellence in he lp and advice, Julio Almanza for all his help, and Chunfang Li for being probabl y my favorite person to work w ith at TREC. These three not only provided the most technical assistance, but they were my FRIENDS. I also thank Yuqing Fu and Mike Gutierrez for their excellent help an d Luis Bradshaw. I also thank Suzanne Fraiser and the Florida Division of Plant Indust ry, Gainesville, for providing larvae. 3

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TABLE OF CONTENTS page ACKNOWLEDGMENTS ............................................................................................................... 3 LIST OF TABLES ...........................................................................................................................6 LIST OF FIGURES .........................................................................................................................7 ABSTRACT ...................................................................................................................... .............10 CHAPTER 1 INTRODUCTION AND LI TERATURE REVIEW ..............................................................12 Introduction .................................................................................................................. ...........12 Management of Diaprepes Root Weevil .................................................................................24 Flooding Effects on Plants ......................................................................................................30 Plant Species Used in This Dissertation .................................................................................34 Previous Studies with Rutaceae on Soil Moistu re, Nutrients, and Diaprepes Herbivory .......36 Previous Studies with Rutaceae on Effect s of Flooding and Herbivory by Diaprepes ..........37 Previous Studies on Effects of Diaprepes Root Weevil and Fl ooding on Ornamental Plants ...................................................................................................................................41 Objectives .................................................................................................................... ...........42 2 EFFECTS OF HERBIVORY BY DIAPREPES ABBREVIATUS (L.) (COLEOPTERA: CURCULIONIDAE) LARVAE ON FOUR WOODY ORNAMENTAL PLANT SPECIES .................................................................................................................................44 Introduction .................................................................................................................. ...........44 Materials and Methods ...........................................................................................................47 Results .....................................................................................................................................50 Discussion .................................................................................................................... ...........52 3 EFFECTS OF FLOODING AND SOIL TYPE ON THE PHYSIOLOGY AND GROWTH OF FOUR WOODY ORNAMEN TAL PLANT SPECIES IN SOUTH FLORIDA ....................................................................................................................... ........65 Introduction .................................................................................................................. ...........65 Materials and Methods ...........................................................................................................67 Results .....................................................................................................................................70 Discussion .................................................................................................................... ...........77 4 SURVIVAL OF DIAPREPES ABBREVIATUS (COLEOPTERA: CURCULIONIDAE) LARVAE ON GREEN BUTTONWOOD TREE S IN FLOODED MARL SOIL OR POTTING MEDIUM. ...........................................................................................................101 4

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Introduction .................................................................................................................. .........101 Materials and Methods .........................................................................................................102 Results and Discussion ........................................................................................................ .104 5 EFFECTS OF HERBIVORY BY DIAPREPES ABBREVIATUS (L.) (COLEOPTERA: CURCULIONIDAE) LARVAE AND FLO ODING ON LEAF GAS EXCHANGE AND GROWTH OF GREEN BUTTONW OOD AND SWINGLE CITRUMELO PLANTS ........................................................................................................................ .......114 Introduction .................................................................................................................. .........114 Materials and Methods .........................................................................................................116 Results ...................................................................................................................................120 Discussion .................................................................................................................... .........123 6 LEAF DAMAGE AND PHYSIOLOGICAL RESPONSES OF SELECT WOODY ORNAMENTAL PLANTS TO ADULT FEEDING BY DIAPREPES ABBREVIATUS (COLEOPTERA: CURCULIONIDA E) AND SOIL FLOODING ......................................136 Introduction .................................................................................................................. .........136 Materials and Methods .........................................................................................................138 Treatments .................................................................................................................... .140 Data Collection ..............................................................................................................141 Results ...................................................................................................................................145 Discussion .................................................................................................................... .........152 7 CONCLUSIONS ................................................................................................................. .179 REFERENCES .................................................................................................................... ........186 BIOGRAPHICAL SKETCH .......................................................................................................197 5

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LIST OF TABLES Table page 2-1 Effects of Diaprepes root weevil larvae on root, stem, and leaf fresh and dry weights of buttonwood, mahogany, Surinam cherry, and pond apple. ...........................................59 6

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LIST OF FIGURES Figure page 2-1 Average temperature 6 cm below th e soil surface during the experiment. ........................602-2 Effect of Diaprepes larval feeding on net CO2 assimilation ( A ), transpiration ( E ), stomatal conductance (gs), and substomatal partial pressure of CO2 ( Ci ) of green buttonwood plants. .............................................................................................................612-3 Effect of Diaprepes larval feeding on net CO2 assimilation ( A ), transpiration ( E ), stomatal conductance (gs), and substomatal partial pressure of CO2 ( Ci ) of mahogany trees ......................................................................................................................... ...........622-4 Effect of Diaprepes larval feeding on net C O2 assimilation ( A ), transpiration ( E ), stomatal conductance (gs), and substomatal partial pressure of CO2 ( Ci ) of Surinam cherry plants .......................................................................................................................632-5 Average sum of larvae, pupae, and adults of Diaprepes root weev il per plant species .....642-6 Mean head capsule widths and resulti ng instars of Diaprepes root weevil larvae recovered from infested plants ...........................................................................................643-1 Soil redox potential ............................................................................................................833-2 Effects of flooding and soil type on net CO2 assimilation ( A ) of green buttonwood plants ........................................................................................................................ ..........843-3 Effects of flooding and soil type on stomatal conductance ( gs) of green buttonwood plants. ....................................................................................................................... ..........853-4 Effects of flooding and soil type on net CO2 assimilation ( A ) of mahogany plants ..........863-5 Effects of flooding and soil type on stomatal conductance ( gs) of mahogany plants ........873-6 Effects of flooding and soil type on net CO2 assimilation ( A ) of pond apple plants .........883-7 Effects of flooding and soil type on stomatal conductance ( gs) of pond apple plants .......893-8 Effects of flooding and soil type on net CO2 assimilation ( A ) of Surinam cherry plants ........................................................................................................................ ..........903-9 Effects of flooding and soil type on stomatal conductance ( gs) of Surinam cherry plants ........................................................................................................................ ..........913-10 Effects of flooding and soil type on dr y weights of green buttonwood plants ..................923-11 Effects of flooding and soil type on increase of (A, B) stem diameter and (C, D, E, and F) plant height for green buttonwood plants ...............................................................93 7

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3-12 Effects of flooding and soil type on dry weights of mahogany plants ...............................943-13 Effects of flooding and soil type on increases in (A-D) stem diam eter and (E-F) plant height for mahogany plants ................................................................................................953-14 Effects of flooding and soil type on dry weights of pond apple plants ..............................963-15 Effects of flooding and soil type on increas e in stem diameter for pond apple plants ......963-16 Effects of flooding and soil type on dr y weights of Surinam cherry plants ......................973-17 Effects of flooding and soil type on increas e in stem diameter for Surinam cherry plants ........................................................................................................................ ..........983-18 Effects of flooding and soil type on increase in plant height for Surinam cherry plants. ....................................................................................................................... ..........993-19 Effects of flooding and soil type on the number and weight of inflorescences per Surinam cherry plant ........................................................................................................1004-1 Effects of flooding and soil type on percen tages of 15 larvae added to each container that were found live at harvest based on ratios of live/total ............................................1124-2 Mean head capsule widths and inst ars ( SD) of larvae found at harvest .......................1134-3 Soil temperature during the experiment ...........................................................................1135-1 A) temperature and B) soil redox potential during the experiment .................................1325-2 Effects of flooding on A) net CO2 assimilation and B) stomatal conductance for cyclically flooded green buttonwood trees ......................................................................1335-3 Effects of larval infestation or floodi ng on transpiration for A) short-term flooded and B) cyclically flooded green buttonwood plants ........................................................1335-4 Effects of larval infestation on A) tr anspiration and B) stomatal conductance of cyclically flooded Swingle citrumelo trees. .....................................................................1345-5 Effects of flooding and Diaprepes larval infestation on dry weights of cyclically flooded Swingle citrumelo plants ....................................................................................1345-6 Effects of flooding on A) percent survival and B) head capsule width of larvae recovered at harvest .........................................................................................................1355-7 Visual damage ratings for Diaprepes la rvae feeding on Swingle citrumelo roots ...........1356-1 Soil temperatures during the experiment .........................................................................1636-2 Soil redox potential. ..................................................................................................... ....164 8

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6-3 Effect of flooding on dr y weights of infested green buttonwood plants ..........................1656-4 Effects of flooding on increase in A) stem diameter and B) plant height for infested green buttonwood and Surinam cherry plants .................................................................1656-5 Effects of flooding on infe sted green buttonwood trees ..................................................1666-6 A) number of trunks and B) effects of flooding on percen tage of trunks found alive at harvest for infested green buttonwood and Surinam cherry plants ..................................1666-7 Effects of flooding on infest ed green buttonwood plants. ...............................................1676-8 Effects of flooding and infestation on A) number and sex of adult Diaprepes recovered before harvest and B) percentage of adults recovered that were female (a measure of sex ratio) ........................................................................................................1676-9 Effects of flooding on infested Surinam cherry plants ....................................................1686-10 Leaf cholorophyll index for infested Suri nam cherry plants measured 8-Nov-07 (preharvest) ...................................................................................................................... .......1696-11 Effects of flooding on infested Surinam cherry plants ....................................................1696-12 Effect of flooding on infest ed Surinam cherry plants ......................................................1706-13 Effects of flooding on mahogany trees ............................................................................1716-14 Effects of infestation on mahogany trees .........................................................................1726-15 Effects of flooding on mahogany and pond apple plants .................................................1736-16 Effects of flooding and in festation on mahogany trees ...................................................1746-17 Effects of flooding and infestation on adult Diaprepes feeding damage on mahogany trees ......................................................................................................................... .........1756-18 Effects of flooding and infestation on number of Diaprepes egg clusters per mahogany tree ................................................................................................................. .1766-19 Effects of flooding on net CO2 assimilation of infested pond apple trees.. .....................1776-20 Effects of flooding on pond apple plants .........................................................................1776-21 Effect of adult Diaprepes infestation on number of egg clusters per pond apple plant ...178 9

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Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy EFFECTS OF HERBIVORY BY DIAPREPES ABBREVIATUS (L.) (COLEOPTERA: CURCULIONIDAE) AND FLOODING ON TH E PHYSIOLOGY AND GROWTH OF SELECT ORNAMENTAL PLANT SPE CIES IN SOUTH FLORIDA By Cliff G. Martin August 2009 Chair: Catharine M. Mannion Cochair: Bruce A. Schaffer Major: Entomology and Nematology The objective of this research was to determine effects of herbivory by Diaprepes abbreviatus (L.) (Coleoptera: Curc ulionidae) and flooding on th e physiology and growth of select ornamental plant species in south Florida. Susceptibility to larval feeding was greatest for green buttonwood and Surinam cherry, followe d by mahogany, then pond apple. Pond apple supported Diaprepes larvae to pupation and re presents a new host plant and family (Annonaceae). Under flooded conditions and/or pl anted in marl soil, pond apple performed optimally, whereas buttonwood, mahogany, and Surina m cherry had reduced leaf gas exchange and growth. Surinam cherry had the greatest preference of potting medium over marl soil, followed by buttonwood, then mahogany, then pond apple. Pond apple and buttonwood tolerated flooding the best, followed by mahogany, th en Surinam cherry. When flooded 38 days, larval survival was lowest in flooded potting medium, followed by flooded marl soil, then nonflooded marl soil and non-flooded potting medium. Larvae from flooded marl soil had smaller head capsule widths and instars than non-floode d larvae in marl soil or potting medium. Flooding more than soil type influenced larval su rvival, though both factors affected it. Effects on leaf gas exchange and plant growth were cu mulative for flooding in green buttonwood and for 10

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infestation in Swingle citrumelo. Contrary to similar studies, buttonwood was not floodadaptated and was the most flood-sensitive spec ies, possibly because of short flood periods, while Swingle citrumelo was the most sensitive sp ecies to larval feedi ng. Flooding with three 2d cycles seemed more likely to control larvae in Swingle citrumelo than in green buttonwood. In an adult Diaprepes study, green buttonwood adap ted to flooding rendering leaf gas exchange, growth, and adult preference the same under fl ooded and non-flooded conditions. For Surinam cherry and mahogany, leaf gas exch ange, growth, and adult feeding damage in infested cages was higher for non-flooded than flooded plants. Pond apple had the lowest adult feeding damage and egg clusters per plant, hen ce, it seems unlikely to become infested. Surinam cherry was most susceptible to flooding, followed by mahogany, with buttonwood and pond apple least affected, whereas buttonwood and mahogany were most susceptible to adu lt Diaprepes, followed by Surinam cherry, then pond apple. 11

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CHAPTER 1 INTRODUCTION AND LI TERATURE REVIEW Introduction Diaprepes abbreviatus L. (Coleoptera: Curculionidae: Entiminae) commonly called Diaprepes root weevil, was first discovered in Florida in a citrus nursery in Apopka in 1964 (Woodruff 1964). However by 1968, workers had collected hundreds of additional adults and larvae near Apopka (Woodruff 1968). Diaprepes root weevil is believed to have entered Florida from Puerto Rico in a shipment of ornamental plants (Woodruff 1985). Diaprepes root weevil is an abundant and serious pest of citrus and sugarcane in its home range of Puerto Rico (Woodruff 1964). In Florida, it infested approximately 60,000 ac (24,281 ha) of citrus, and control costs and losses have exceeded $1,200 per ac ($2,965 per ha ) (Stanley 1996). The weevil has cost the Florida citrus industry an estimated $72 million annually (Anonymous 1996, Stanley 1996). More recently, the weevil caused about $70 million annually (crops not specified), and about 100,000 ac (40,469 ha) of citrus were infested (Wei ssling et al. 2004). Because of inadequate management strategies and its wide host range, Di aprepes root weevil has th reatened the survival of several agronomic crops (Simpson et al 1996) and nearly put many central Florida ornamental plant nurseries out of business dur ing the 1970s (Schroeder 1994). In Florida, Diaprepes root weevil is currently found in 23 counties in the s outh and central parts of the state, which includes Miami-Dade, Broward, Collier, Palm Beach, Lee, Hendry, Desoto, Glades, Highlands, Martin, St. Lucie, In dian River, Manatee, Hillsborough, Osceola, Polk, Lake, Sumter, Pasco, Orange, Seminole, Volusia, and Mari on (Anonymous 1996, Pea 1997, Weissling et al. 2004). In addition to damage caused by the pest, th ere are regulatory concerns of spreading Diaprepes root weevil into non-infe sted areas, which are particularly important to the ornamental 12

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plant industry because plants are shipped thr oughout the U.S. and abroad (Mannion and Glenn 2003). Diaprepes root weevil has also been found in Texas (Knapp et al. 2001, Skaria and French 2001) and southern California (Klunk 2005), and these weevils likely came from Florida. Hence, there is a need to continually control Diaprepes root weevil to reduce the risk of its introduction into other states and countries. Host range. Diaprepes root weevil is a problematic pest due to its ve ry large host range, which includes at least 317 vari eties, 280 species, 180 genera, and 68 families of plants (Simpson et al. 1996, 2000, Knapp et al. 2000b, Mannion et al. 2003, Godfrey et al. 2006, C.G. Martin personal observation and unpublished data). The plant families with the largest number of varieties, species, and genera atta cked by the pest include Rutaceae (95 varieties, 66 species, and 24 genera) and Fabaceae (43 varieties, 42 species, a nd 31 genera). Both these families are in the order Rutales (Zomlefer 1994). In addition, the four remaining families of Rutales (Meliaceae, Anacardiaceae, Sapindaceae, and Aceraceae) each have representative species on the above list of Diaprepes root weevil host plants. Besides Rutales and other dicots, monocot taxa such as Poaceae and Gymnosperm taxa such as Cupressaceae also have representative species on this list of Diaprepes root weevil host plants (Simpson et al. 1996). Some plants support only one stage of the insect; for example Ardisia crenata Sims (Myrsinaceae), which supports only the larval stage. However, many economically important plan ts support all stages of the weevil from egg to adult, such as sweet potato, Ipomoea batatas (L.) (Convolvulaceae) Lam., and buttonwood, Conocarpus erectus L. (Combretaceae) (Simpson et al. 1996). Mannion et al. (2003) surveyed several ornamental plant nur series in southern Florida and found that egg masses, feeding damage, and adult Diaprepes root weevils were common on field-grown ornamental plants. In the field, highest percentages of plants with egg masses were found on live oak (Quercus virginiana Mill., Fagaceae), silver buttonwood ( C. erectus L. variety 13

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sericeus Fors.Ex DC), and black olive ( Bucida buceras L.) (the latter two species Combretaceae). In addition, adult feeding da mage in the field was commonly found on dahoon holly ( Ilex cassine L., Aquifoliaceae), cocoplum (Chrysobalanus icaco L., Chrysobalanaceae), Bauhinia sp., and Cassia sp. (both Fabaceae). Highest adu lt population densities in the field were found on black olive, dahoon holly, and Bauhinia sp. (Mannion et al. 2003). Mannion et al. (2003) also evaluated Diaprepe s root weevil in choice tests of caged adults. Based on comparisons of mean numbers of egg masses per plant species, silver buttonwood was among the most preferred host. Adair et al. (1998) also compared mean num bers of Diaprepes root weevil egg masses oviposited on two to four different above-ground substrates to determine adult ovipositional preferences. Substrates included you ng and mature grapefruit leaves ( Citrus paradisi Macf., Rutaceae), freezer paper, wax paper, transparency film, and several other materials. Based on mean numbers of egg masses per substrate, Diaprepes root weevil females preferred freezer paper over young or mature grapefru it leaves for oviposition (Adair et al. 1998). Freezer paper is now widely used in laboratories to collect eggs from Diaprepes root weevil adults. Biology, taxonomy, and range. Diaprepes root weevil is part of a complex of problematic root weevils that ar e taxonomically related (Coleopt era: Curculionidae: Entiminae) and share two feeding guilds: r oot feeders (larvae) and direct -leaf-consumers (adults). They have very similar life cycles and behavior wi th the following characteristics (Syvertsen and McCoy 1985, Futch and McCoy 1993): relatively l ong, univoltine life cycles; broad host ranges (70 or more plant species); larvae that feed on roots starting with fibrous roots, channeling the bark, or girdling the plant as they get older; pupation periods of 2 to 4 wk; pre-pupal and postpupal resting periods in the soi l; adult emergence often triggere d by heavy rains; adults that typically notch leaves starting along the edges and progressing to ward midveins; eggs that are 14

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laid in masses between two leaves; and neona tes that drop to the ground upon hatching and burrow into soil. In addition to Diaprepes root weevil this pest complex includes the southern blue-green citrus root weevil, Pachnaeus litus (Germ.), and the northern citrus root weevil, P. opalus (Oliv.) (Coleoptera: Curculionidae: Entiminae) (Woodruff 1981, Anderson 2002). The Entiminae is the broad-nosed or s carred snout beetle s ubfamily (Wolcott 1936, Anderson 2002). Their snouts are shorter and less developed than those in typical long-nosed weevil subfamilies such as Cyclominae (Anderson 2002) Entiminid adults bear scars at the ends of the snouts marking the disappearance of mandibular appendages-prominent, curved, black claws, which are deciduous and larger than the final mandibles of the adult (Wolcott 1936, Anderson 2002). Adults shed the mandibular appendages when burrowing from their pupal chambers to the ground surface (Wolcott 1936, Anderson 2002). Within the genus Diaprepes, Blackwelder (1947) lists 19 sp ecies; 18 from the West Indies and one from Nicaragua. More recently, Pea (1997) noted at l east 20 species (including D. abbreviatus ) in the Caribbean Basin. However, Obri en and Kovarik (2000) recognized 16 species in Diaprepes which is restricted to the Caribbean Basin with one species from Trinidad and Venezuela and the other 15 species from the West Indies. Obrien and Kovarik (2000) examined 1,000 specimens of Diaprepes root weevil and concluded that because of the similarity of Florida and Puerto Rico populations, the weevil was originally introduced from Puerto Rico. This isla nd is probably the center of origin of Diaprepes root weevil since it is the center of diversity, which is suggested by the high degree of phenotypic diversity of the weevil there (Lapointe 2000a, Obrien and Kovarik 2000). In addition to Diaprepes root weevil and two Pachnaeus spp., other problematic root weevils to ornamentals and/or citrus in Florida include two Fuller rose beetles, Pantomorus cervinus (Boheman) and Asynonychus godmani Crotch the little leaf notcher Artipus floridanus Horn and Myllocerus 15

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undatus Marshall (Woodruff and Bullock 1979, Woodruff 1982, Futch and McCoy 1993, DOACS 2002). Although these we evils are frequent pest species, they are less common on citrus based on geographic distribution than Diaprepe s root weevil or the two Pachnaeus spp. suggesting they may be less problematic to citrus and/or ornamentals than the latter two taxa (McCoy et al. 2004). Life cycle. The Diaprepes root weevil life cycle in the field lasts 8 to18 mo from egg hatching to the last egg laid by the female or d eath of the male (Wolcott 1936). On an artificial diet, females required an average of 381 d to develop from oviposition to pupal eclosion (adulthood), and males fully developed in 382 d, he nce, 12.5 mo were needed for both males and females (Beavers 1982). Diaprepes root weevils normally lay eggs in clusters between leaves held together with an adhesive (Wolcott 1936). In the field, the average female produces about 5,000 eggs in her lifetime, and 30 to 264 eggs per cluster (Wol cott 1936, 1948). On an artificial diet, Beavers (1982) found that mated females averaged 6,517 eggs per lifetime, or 69 eggs per cluster. Eggs are oval-oblong, shiny, smooth, 1.2 mm X 0.4 mm, and initially milky-white, but beginning 1 to 2 d after oviposition, they develop cl ear spaces at one end (Wolcott 1936). Eggs are usually laid in single layers, although egg clusters can be up to two layers thick to ward the center (Wolcott 1936). Eggs hatch about 7 d after oviposition (Wolcott 1936). Diaprepes root weevil neonates move across the leaf in a galloping motion and fall off the edge to the ground (Wolcott 1936). On the gr ound, they continue crawli ng until they fall into a crack and work their way down into the ground (Wolcott 1936). Here after searching for up to 2 wk, they find and start feeding on small roots (W olcott 1936). As they grow larger, the larvae prefer larger roots, ideally large enough for them to bu rrow into (Wolcott 1948). Larvae 16

PAGE 17

sometimes burrow into corn kernels, t hough normally only roots and similar underground structures, such as potato tubers, are consumed (Wolcott 1936, Knapp et al. 2001). The rate of larval development for Diaprepe s root weevil varies with the season(s) in which they hatch. A March hatchling reached the sixth instar in 126 d, while a September hatchling took 27 d to reach the same instar. In th e field, 2 to 4 mo is typi cal for the entire larval stage (Wolcott 1936). On an artificial diet, however, Beavers (1982) noted average larval durations of 362 d for females and 363 d for males, or 12 mo for either sex. Diaprepes root weevil larvae have 6-16 molts before pupation, w ith an average of 8 molts (Wolcott 1936). A surplus of food seems to increase the number of instars, wherea s in most other insects, excess larval molting is caused by unfavorable environm ental conditions like cold or lack of food (Wolcott 1936). Diaprepes root weevil larvae normally move horizontally or transversely in the soil except when ready to pupate. They form ver tical pupal chambers by compacting the walls with their caudal ends and then rest in the chambers with heads up (Wolcott 1936). The two periods that vary the most in the Diap repes root weevil life cycle are the prepupal and pre-emergence resting periods (2 to 13 mo combined) (Wolcott 1936). The prepupal resting period lasts 2 to 3 wk after formation of the pupal chamber and is required for Diaprepes root weevil to pupate (Wolcott 1936). The intervening pupal stage is 14 to 26 d (2 to 4 wk) long (Wolcott 1936). Pupation occurs throughout the year, although highest percentage s of annual pupation are in March and October (Wolcott 1936). Upon eclosion from pupa, the adult resting period is 11 to 126 d (0.4 to 4.2 mo), with the average approximately 2 mo, or about half the 4-5 mo mean adult lifespan (Wolcott 1936). In the field, female adu lts usually live longer th an males, often more than twice as long (Wolcott 1936). On an artificial diet, the mean duration of an adult Diaprepes 17

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root weevil lifespan is similar to in the fiel d--147 d (5 mo) for females and 135 d (4.5 mo) for males (Beavers 1982). According to Wolcott (1936), adult emergen ce seems to not be triggered by external factors such as periods of rain. However, according to Grafton-Cardwell et al. (2004), dry, compact soil inhibits adult emergence, whereas irrigation and rainfall tend to promote it. Contrary to the above opinion by Wolcott (1936), large numbers of adult Diaprepes root weevil have been seen emerging after periods of heavy rain in south Florida (C. Mannion pers. comm.). Beavers and Selhime (1976) also observed that heavy rainfall usually preceeded field emergence of adults. Although Diaprepes root weevil is uni voltine, generations overlap throughout its range (Beavers and Selhime 1976, Pea 1997). In central Florida there are two peak adult emergence periods, May to June and August to Septem ber (Beavers and Selhime 1976, Pea 1997). The majority of adult feeding is by female s to meet the requirem ent of egg production. Feeding drops off rapidly when oviposition stops, and males eat much less than females (Wolcott 1936). Diaprepes root weevils typically mate in the foliage, and 3 to 7 d after females emerge from the ground, they begin oviposition (Wolcott 1936, 1948). Duration of oviposition is typically most of an adult females lifespan a nd varies from 2 mo after emergence in May or June to 7 mo at other times of the year (Wolcott 1936, 1948). Diaprepes root weevil adults are gregarious, and some trees may have large numbers congregated in the branches, while nearby trees have none (Wolcott 1936). Pea et al. (2007) sampled tree branches of silver buttonwood a nd dahoon holly trees and found that egg-cluster distribution was aggregate in th e upper and middle branches where most egg clusters were found, whereas lower branches had a random distribut ion. This may reflect that Diaprepes root weevil adults are gregarious and tend to gath er in the upper and middle canopies. In addition, Diaprepes root weevil adults pref er to oviposit on mature, expande d leaves of citrus (Schroeder 18

PAGE 19

and Sutton 1977, Lapointe 2001). On silver butto nwood, mature leaves in the upper and middle canopy tend to be more flattened than the more cu rved, sooty-mold-laiden, mature leaves in the lower canopy. Thus, leaf shapes that favor oviposition in the upper and middle canopies and the gregarious habits of adults may cause greater abundance of egg clusters and the more aggregate distribution in upper and middle branches than in lower branches (Pea et al. 2007). Damage. Neonate Diaprepes root weevils start feeding on small, fibrous roots and work their way up to larger lateral and main roots as they mature (McCoy et al. 2002). Their feeding on the larger roots often forms deep grooves, and they consume outer bark and cambial layers (McCoy et al. 2002). Roots may be girdled causing root death or the crown may be girdled causing tree death (McCoy et al. 2002). Larval girdling often kills small citrus trees by destroying their ability to take up nutrients (Wol cott 1936, 1948; Quintela et al. 1998). Quintela et al. (1998) found that the mean ra te of larval weight gain was highest for fifth through seventh instars, which presumably cause the most dama ge. Effects of root feeding damage from Diaprepes root weevil larvae have been less thoroughly examined fo r ornamental plants than for citrus, although they can be just as severe in either group. For example, root feeding by Diaprepes root weevil larvae resulted in severe damage to many field-grown ornamental plants in south Florida (C. Mannion, pers. comm.). Ad ditionally, Diaprepes root weevil larval feeding and root injury often serve as infection sites for root rot diseases, which exacerbate economic losses compared to those caused by the weevils al one (McCoy et al. 2002). The principle fungi that cause severe root rot are Phytophthora nicotinae Breda de Haan and P. palmivora (Butl.) Butl. (Oomycota) (McCoy et al. 2002). The most obvious feeding damage include s the aforementioned notching from adults feeding along margins of es pecially young, tender leaves (Wolcott 1936, 1948, McCoy et al. 2002). This can result in moderate to severe defoliation of host plants such as young, replanted 19

PAGE 20

citrus trees (Quintela et al. 1998, McCoy et al. 2002, Mannion et al. 2003). Unlike larval root feeding, however, prolonged adult feeding does not seem to economically reduce yields of mature citrus orchards (McCoy et al. 2002). Howe ver, for ornamental plant species, excessive foliar damage makes them less attractive at the time of sale and can reduce sales (C. Mannion pers. comm.). Arthropod effects on photosynthesis. Insect herbivory often affects leaf gas exchange including net CO2 assimilation ( A ), also called net photosynthesis ( Pn ), transpiration ( E ), stomatal conductance (gs), and substomatal partial pressure of CO2, also called internal CO2 concentration ( Ci ) of host plants (Andersen and Mize ll 1987, Schaffer and Mason 1990, Schaffer et al. 1997). Root (1973) and Welter (1989) clas sified arthropods according to their type of feeding damage or guild, such as mesophyll feeder s, phloem feeders, stem borers, root feeders, and direct leaf consumers. The effects of in sect herbivory on leaf gas exchange can vary according to feeding guild (Welter 1989). In a review by Welter (1989) on how arthropod herbivory affects leaf gas excha nge, most studies claimed that he rbivory is detrimental to this process, although insignificant to beneficial effects where herbivor y increased leaf gas exchange have been noted. Diaprepes and other ro ot weevils such as P. litus and the little leaf notcher A. floridanus have two feeding guilds. Larvae are in the root -feeder guild, whereas adu lts are in the directleaf-consumer guild (Syvertsen and McCoy 1985) Syvertsen and McCoy (1985) studied the rates of photosynthesis, transpiration, and water use efficiencies of citrus infested with adult A. floridanus. They found that when weevil population densities exceeded on e weevil per leaf, herbivory reduced water use efficiencies (defin ed as photosynthesis divided by transpiration) up to 20%. Syvertsen and McCoy (1985) also found that when adult A. floridanus increased consumption of citrus leaf ar ea, photosynthesis and water use efficiency declined. Because 20

PAGE 21

water use efficiency decreased more rapidly th an photosynthesis, drought stress from injured leaves may have enhanced the loss of photosynt hesis with increasing leaf area consumed. Hence, drought stress may increase the severity of feeding injury or vice versa (Syvertsen and McCoy 1985). Findings of Syvertsen and McCoy ( 1985) thus exemplify detrimental effects of herbivory on photosynthesis, which are the most common effects caused by root feeders. However according to Welter (1989), insects in th e direct-leaf-consumer feeding guild usually increase photosynthesis measured on a leaf-area basis, but this e ffect is atypical compared to other guilds: for example, stem borers and root feeders tend to decrease photosynthesis (Welter 1989). According to Welter (1989), e ffects of herbivory from the root-feeder guild have been less thoroughly documented than effects of herbivory from above ground guilds, such as phloem feeders and gall formers. In addition, studies of the effects of herbivory by nymphs and larvae on leaf gas exchange, such as those done by Scha ffer et al. (1997) and Di az et al. (2006) have been conducted less frequently than studies of the effects of herbivory by adult insects, such as those conducted by Syvertsen and McCoy (1985) and Boucher and Pfeiffer (1989). Nigg et al. (2001a) tested the responses of seve n varieties of citrus rootstock seedlings to larval feeding by Diaprepes root weevil neonates. They compared fresh and dry root weights, trunk diameters, and larval recovery of infested and non-infested plants of each variety to determine whether it was tolerant to Diaprepes r oot weevil larval feedin g (Nigg et al. 2001a). Hence, none of the seven varie ties were tolerant to larval feeding (Nigg et al. 2001a). Infestation by another root-feeding la rval pest, the western corn rootworm, Diabrotica vergifera vergifera Leconte (Coleoptera: Chrysomelidae) re sulted in varied effects on leaf gas exchange and compensatory growth depending on severity of feeding (Riedell and Reese 1999, Urias-Lopez et al. 2000). During corn vegetativ e growth (48 d after pl anting), photosynthesis 21

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was lower in root-damaged than in n on-damaged plants, and it was lower in severely infested than in moderately infested or non-infested plants (Riedell and Reese 1999) In the tassel stage of corn (54 d after planting), feeding by rootworms significantly lowered stomatal conductance relative to non-infested plants though photosynthesis was unchanged (Riedell and Reese 1999). In addition, adventitious roots gr ew as compensatory growth more often in moderately infested than in severely infested or non-infested plants (Riedell and Reese 1999). Hence, the severity of root damage and amount of compensatory growth each played important roles in mediating shoot growth and the level of photosynt hesis (Riedell and Reese 1999). The stem-borer feeding guild may be the most similar guild to root -feeders because their larvae also consume plant parts such as stems, which are not principle s ites of photosynthesis. Godfrey et al. (1991) found that th e stem-boring European corn borer, Ostrinia nubilalis (Hubner) (Lepidoptera: Pyralidae) significantly reduced corn photosynthesis up to 22% and stomatal conductance up to 28%. As with many fo liage and root feeders, stem borers typically reduce photosynthesis of their host plants such as corn, Zea mays L. (Welter 1989, Godfrey et al. 1991). For the gall-forming guild, all the papers reviewed by Welter (1989) reported that herbivory reduced photosynthesis in the galls and often in surroundi ng tissue. This was the case for galls created by Phylloxera notabilis Pergande (Homoptera: Phylloxeridae) on pecan foliage, Carya illinoinensis (Wangenh.) K. Koch (Juglandaceae ) (Andersen and Mizell 1987). Gall formers reduced photosynthesis by 27% in infested tissue, whereas the galls only occupied 6.4% of the infested area measured (Andersen and Mizell 1987). In addition to reducing photosynthesis, these gall formers significantly re duced transpiration, stomatal conductance, and chlorophyll. They also changed concentrations of some nutrients within galls (e.g., significantly decreasing N, but increasing P and K), and in surrounding tissu e (e.g., significantly decreasing 22

PAGE 23

N, not changing P, and increasing K), and hence a ltered metabolic functioning in areas affected (Andersen and Mizell 1987). Members of another guild, phloem feeders, also tend to reduce photosynthesis according to slightly more than half the papers surv eyed by Welter (1989). The lignum vitae tree Guaiacum sanctum L. (Zygophyllaceae) exemplifies this ma jority because its photosynthesis and other variables were reduced by the phloem-feeding scale insect, Toumeyella sp. (Hemiptera: Coccoidea) (Schaffer and Mason 1990). Toumeyella sp. lowered photosynthesis, transpiration, stomatal conductance, leaf area, and dry weights of the roots, stems, shoots, and leaves of lignum vitae trees (Schaffer and Mason 1990). Leafminers exemplify another feeding guild, the mesophyll feeders. As in most feeding guilds, they usually reduce photosyn thesis of the plants they feed on (Welter 1989). In a study of citrus leafminers, Phyllocnistis citrella Stainton (Lepidoptera: Grac illariidae), which are also herbivorous larvae, a visual estim ation of leaf mining damage pr oved to be rapid, accurate, and practical (Schaffer et al. 1997). Mined leaves were chlorotic or necrotic, which suggested a reduction in photosynthesis that wa s at least partly caused by redu ced chlorophyll content. In plants infested with citrus leafminers, increa sed leafmining duration and increased numbers of the larvae per leaf were each correlated w ith greater leaf area da mage and photosynthesis reduction (Schaffer et al. 1997). Herbivorous m ites are another group of arthropods within the mesophyll feeding guild that usually reduce photosynthesis (Welter 1989). In almond trees, spider mite herbivory caused significantly greater reduction of photosynthesis on water-stressed plants than on properly watered ones (Young man and Barnes 1986, Welter 1989). In pecans infested with the pecan leaf scorch mite, Eotetranychus hicoriae (McGregor) (Acari: Actinedida: Tetranychidae), high levels of nitrogen fertilizer increased photosynthesis of mite-damaged leaves compared to undamaged leaves (Welter 19 89). At moderate (opt imal) nitrogen levels, 23

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however, herbivory by mites significantly decrea sed photosynthesis compared to non-infested plants (Welter 1989). However, at low nitroge n levels, photosynthesis was the same between mite-damaged and undamaged leaves (Welter 19 89). Hence, although mite feeding generally reduces photosynthesis, factors such as levels of water and fertilization can interact with effects of mite herbivory and greatly affect photosynthesis levels. Management of Diaprepes Root Weevil Cultural control. In areas infested with Diaprepes root weevil, the following cultural practices help to optimize growth and maintain health of citrus and other cultivated plants (McCoy et al. 2002): regular fertilization, time ly irrigation, skirt-pr uning of low hanging branches to reduce adult dispersal, and weed cont rol to remove sinks for nutrients and water, and reduce the number of alternate hosts. Borowicz et al. (2005) tested effect s of nutrient supply and below-ground herbivory by Diaprepe s root weevil larvae on citrus growth and mineral nutrient content, which suggests that tolerance to Diap repes root weevil feedi ng is not a function of nutrient status. Borowicz et al. (200 5) also found that application of excess, balanced fertilizer is not likely to offset growth reductions due to root herbivory, and they suggest supplementing specific nutrients to plan ts may be useful. Li et al. (2003b) found that low soil con centrations of Mg, Ca, and H (hydrogen, high acidity), and low cation exchange capacity were correlated with increase d Diaprepes root weevil populations. Li et al. (2003b) also suggested that cultural practic es, such as adding lime to soil with an acidic or neut ral pH, soil erosion by rain or floodwater, and floodi ng could affect field distributions of the weevil. Water applied fo r cultural control may lessen the incidence of Diaprepes root weevil in two ways, either by su pplying water in needed amounts or in extremely large or small quantities (lack or surplus). Adding optimum amounts of water may control the pests indirectly, such as by im proving plant turgidity, which help s in nutrient transport and 24

PAGE 25

photosynthesis. As suggested in the example of spider mites on almonds, careful irrigation may control pests through increased plant development or growth to offset herbivory (Youngman and Barnes 1986, Welter 1989). Sustained flooding can be an important mortality factor for Diaprepes root weevil larvae and ha s been suggested as a possible control tactic in sugarcane fields (Shapiro et al. 1997). Hence, flooding may also be a viable control option for floodtolerant, ornamental plants including buttonwood. Mechanical control. Mechanical control of Diaprepe s root weevil includes restricted movement of materials that spread pests from in fested to non-infested ar eas (Knapp et al. 2000a). This material, commonly called debris, includes so il, plants, leaves, fruit, grass, branches, and stumps that may harbor the pests In addition, shipping and other containers should be cleaned before reuse (Knapp et al. 2000a). After harvesti ng vegetables such as potatoes, the soil should also be disked several times to destroy foci of infestation (Pea 1997). Access to areas with known infestations should be limited; for example, only to crews moving plants within infested zones (Knapp et al. 2000a). Physical control. Gould and Hallman (2004) found that irradiating materials containing adult Diaprepes root weevil at low enough levels not to damage commodities (~50 Gy/min) is a control option because adults are sufficiently sens itive to radiation. Beca use radiation tolerance increases with insect development (Hallman 2001), larvae and eggs should be more sensitive to radiation and easier to c ontrol than adults. Although irradi ation would be detrimental to the growth of nursery stock and is not advisable, this method may be useful for controlling various stages of Diaprepes root weevil in sugar can e pieces, root crops, and similar commodities amenable to this technique (Gould and Hallman 2004). Because ionizing ra diation is a viable disinfestation technique for treatment of exporte d agricultural commodities produced in Florida, this method may help control the spread of Diaprepes root weevil (Gould and Hallman 2004). 25

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Regulatory control. Regulatory control of Diaprepe s root weevil includes quarantine restrictions, which have been im posed to limit the movement of plant materials from infested to non-infested areas. However, these treatments can be expe nsive, labor intensive, and time consuming (Mannion et al. 2003). Sales and customers may be lost because quarantine treatments are in place, hence, plants cannot be shipped to a particular customer or location. Chemical control. Numerous insecticides have been used to control both the larvae and the adults (Pea 1997, McCoy et al. 2004). Trad itionally, broad spectrum insecticides with long residuals have been applied to the soil for larval control (Wolcott 1948). Since the 1980s, imidacloprid (Admire), bifenthrin (Talstar or Capture), and chlo rpyrifos (Suscon Green) have been successfully used to controlled neonates in container-grown citrus (Pea 1997). For small larvae infesting ornamental plants in containers, imidacloprid a nd bifenthrin are widely used (Pea 1997, Pea et al. 2005). McCoy et al. (2004) recommended the followi ng for adult control in citrus: Danitol 2.4EC, Imidan 70WP, diflubenzuron (Micromite 80WGS), acephate (Ort hene 97), carbaryl (Sevin 80S, Sevin 4F, and Sevin XLR). Other adulticides available since the 1980s include azinphos (Guthion), and formetanate hydrochlor ide (Carzol) (Pea 1997). Many of these chemicals are no longer available because their environmental toxi city has resulted in laws which ban their sale. According to Schroeder et al. (1977) and Pea (1997), spray oils such as Florida citrus oil (FC 435-66), are used to separate le aves stuck together by egg masses. Although spray oils did not reduce Diaprepes root weevil fecundity or fert ility according to Schroeder et al. (1977), they significantly reduced its reproductiv e potential due to loss in eggs. Bullock and Pelosi (2002) found that the insect growth regulator, diflubenzuron 25w (Dimilin or Micromite), reduced egg hatching rates an average 80% without oil and 98% with FC 435-66 spray oil added. 26

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Diflubenzuron is highly effectiv e at reducing the reproductive pot ential of not only Diaprepes root weevil, but also other citrus root weevils including P. litus P. opalus, and A. floridanus (Lovestrand and Beavers 1980). As noted, McC oy et al. (2004) also lists diflubenzuron (Micromite) as a pesticide available to combat adult Diaprepes root weevil. Host plant resistance. Host plant resistance has also been suggested to control Diaprepes root weevil. However, according to Lapointe and Bowman (2002), all currently used rootstocks for citrus are suscep tible to the pest. Nevertheless, Bowman et al. (2003) tested resistance of citrus rootstocks to Diap repes root weevil and to infections by Phytophthora palmivora and P. nicotianae under field conditions They found large differences in tolerances of plant varieties to root rot, which was e nhanced by herbivory from Diaprepes root weevil larvae. Based on these citrus studies, host plant resistance may protect ag ainst infections often indirectly caused by Diaprepe s root weevil, such as by Phytophthora spp., but they would not protect against the weevil di rectly (Lapointe and Bowman 2002, Bowman et al. 2003). Biological control. In southern Florida, 75% (3,750) of 5,000 eggs laid by the average Diaprepes root weevil female in her lifetime are k illed by parasitoids (Pea et al. 2005). Of the remaining 1,250 viable eggs, only 1 in 164, or 7 individuals, will survive to adulthood (Nigg et al. 2004). Although parasitoids prob ably do not stop the spread of Diaprepes root weevil, they play a major role in controlling the weevil. Since 1997, 16 egg parasitoids have been introduced, six reared in quarantine, and th ree released in Florida (Pea et al. 2005). The three released species include two endoparasitoids, Quadrastichus [Tetrastichus] haitiensis Gahan (Hymenoptera: Eulophidae) and Ceratogramma etiennei Delvare (Hymenoptera: Trichogrammatidae), and an ectoparasitoid, Aprostocetus vaquitarum (Wolcott) (Hymenoptera: Eulophidae) (Pea et al. 2005). Quadrastichus haitiensis and A. vaquitarum are successfully established in south Florida (Amalin et al. 2004, Pea et al. 2005). 27

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Although Q. haitiensis and A. vaquitarum are successfully establ ished in south Florida (Amalin et al. 2004, Pea et al. 2005), they have not expanded their range into central Florida, where Diaprepes root weevil also abounds (Castill o et al. 2006). Lapointe et al. (2007) examined mean air and soil temperatures at the northern ra nge limit of Diaprepes root weevil in Florida and compared them to corresponding temperatures for development thresholds of Diaprepes root weevil and these parasitoids to predict where they would establish. This method predicted that Diaprepes root weevil could esta blish in many areas in southern California, extreme southern Texas, and extreme southwest Arizona (Lapointe et al. 2007). As noted, the weevils have spread to some of these areas in southern Californi a (Klunk 2005) and Texas (K napp et al. 2001, Skaria and French 2001). However, Lapoi nte et al. (2007) predicted th at the parasitoids would not establish in these new areas, hence, there would be no important biocontrol agents to help control the weevils. Numerous studies have eval uated entomopathogenic nematode s for control of Diaprepes root weevil larvae which include Steinernema [Neoapl ectana] carpocapsae (Weiser) (Mexican strain), Heterorhabditis bacteriophora Poinar (Rhabditida: Steinernematidae and Heterorhabditidae, respectively) (Beavers et al. 1983), and S. riobravis (Cabanillas, Poinar, and Raulston) (Duncan and McCoy 1996, Pea 1997). Heterorhabditis bacteriophora did not affect Diaprepes root weevil populati on densities (Duncan and McC oy 1996). However, Schroeder (1987, 1990) found that S. carpocapsae offers effective, environmen tally safe control. Pea (1997) claimed that S. riobravis adds to the arsenal of biocontrol agents available, and control of Diaprepes root weevil larvae s hould include a fall applica tion of the nematodes. Mannion and Glenn (2003) tested the effects of combining nematodes ( H. bacteriophora or S. riobravis ), a soil drench of bifenthrin, or a comb ination of both to co ntrol large Diaprepes root weevil larvae. They found that combining soil drench and either species of nematode 28

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worked better than nematodes or soil dren ch alone (Mannion and Glenn 2003). This is especially useful considering the need for e fficacious treatments to regulate the spread of Diaprepes root weevil larvae when ornamental plants are shipped. These results also provide a method for controlling older larvae, which are traditionally difficult, and to reduce the cost and use of pesticides (Mannion and Glenn 2003). According to Woodruff (1964), the green muscardine fungus Metarhizium anisopliae (Metschnikoff) Sorokin (Deteromycota), takes a heavy toll on Di aprepes root weevil larvae. However, Wolcott (1936) claims that it sometimes kills larvae that are in jured, mite-infested, or sub-normal, but does not attack healthy larvae in the field. In addition to M. anisopliae, Beavers et al. (1983) found that the following en tomopathogenic fungi were present in soil and infectious to Diaprepes root weevil larvae: Beauveria bassiana (Bals.-Criv.)Vuill. (Ascomycota), Paecilomyces lilacinus (Thom) Samson (Deuteromycota), and Aspergillus ochraceous K.Wilh. (Ascomycota). These fungi were present in nine citrus orchards th roughout the year, though fungi-infested larvae were most preval ent May through July (Beavers et al. 1983). Homestead, Florida, the study site for the research in th is dissertation, has many common predators, which may affect numbers of Diaprepe s root weevil at various stages of their life cycle. These include the Cuban tree frog ( Osteopilus septentrionalis Dumril & Bibron), giant toad ( Bufo marinus L.) (Amphibia: Hylidae and Bufonidae, respectively), brown anole lizard ( Anolis sagrei Dumril & Bibron) (Reptilia: Polychroti dae) (Behler and King 1979), and spiders (many spp., Arachnida: Araneae). Other predators that attack Di aprepes root weevil include several ant species that prey on e ggs and larvae. Examples include Pheidole dentata Mayr and Solenopsis invicta Buren (Hymenoptera: Formicidae) (Wh itcomb et al. 1982, Pea 1997, Pea et al. 2005). The ants consume eggs but may ha ve difficulty finding Diaprepes root weevil neonates on the ground; in addition, neonates produ ce chemicals that repel ants (Jaffe et al. 29

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1990a, b). Also, lady bugs such as Cycloneda sanguinea (L.) (Coleoptera: Coccinellidae) prey upon Diaprepes root weevil eggs and neonates, although the level of control they provide is unclear (Stuart et al. 2002). Other arthropod pred ators of Diaprepes root weevil eggs and larvae include the earwig, Labidura riparia Kirby (Dermaptera: Labiduridae), and the jumping spider Corythalia canosa Walckenaer (Araneae: Sa lticidae) (Whitcomb et al. 1982, Pea et al. 2005). Another predator of Diaprepes root weevil is Euthyrhynchus sp. (Hemiptera: Pentatomidae) (Pea et al. 2005). Although many of the foregoing predators are present in the average citrus orchard or ornamental plant field nursery (Whitcom b et al. 1982), they tend to be most effective at controlling Diaprepes root weevil when more than one species are present simultaneously. Flooding Effects on Plants Tropical agriculture in southern Florida, pa rticularly between Bis cayne and Everglades National Parks, tends to occur in low-lying ar eas with high water tabl es, which are prone to periodic flooding (Schaffer 1998). Changing wate r delivery practices in Everglades National Park have recently mandated the raising of water tables in these areas (Schaffer 1998). This has increased the severity, duration, a nd extent of flooding in regions that produce tropical fruit and ornamental plants. For agriculture to remain vi able in these areas, it is important to understand how flooding affects crop physiology, growth, and yield to help identify flood-adapted crops and production systems (Schaffer 1998). Therefore, considerable agricultural research in extreme southern Florida has focused on testing the respon ses of tropical and subtropical fruit trees, and more recently ornamental plants, to flooding and improving their water use efficiency (Schaffer 1998). This includes an understand ing of how increased water tabl e elevation affects pests such as Diaprepes root weevil and diseases like Phytophthora root rot Elevation of the water ta ble above the root zone t ypically depletes soil O2 levels (Kozlowski 1997). A measure of oxygen abundance in the soil is redox potential. Well-drained, 30

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well oxygenated soils have redox potentials of +3 00 mV or more, whereas flooded soils have redox potentials of +200 mV or less (Ponnamper uma 1972, 1984). Soil redox potential varies with the soil type, pH, and temperature (Patrick et al. 1996). Effects of flooding on the physiology and grow th of a woody, perennial plant species can vary among soil types and is partly based on the rates of soil O2 depletion and other factors like soil pH (Schaffer et al. 1992). For example, when undamaged by Phytophthora root rot, flooding caused more damage to avocado trees in nurse ry potting medium than in calcareous, Krome gravelly loam soil (previously referred to as R ockdale soil), which is native to south Florida (Ploetz and Schaffer 1989). However, when infested with Phytophthora cinnamomi Rands, there was a stronger additive effect between flooding and Phytophthora root rot in Krome soil than in nursery potting medium (Ploetz and Schaffer 1989) Responses of woody, perennial plants to flooding can hence be quite differe nt in calcareous soils with a high pH found in south Florida compared to potting mixes used by ornamental plan t nurseries, or to more acidic natural soils (B. Schaffer, pers. comm.). Gravelly loam and marl soils are collectively high in calcium, have pHs of 7.4 8.4, were derived from Miami limestone, and occur in Monr oe (including the Florid a keys), Miami-Dade, and parts of Broward Counties (N obel et al. 1996, Li 2001). Marl soils cover lowlying terrain and formed in areas with several months of flooding (hydroperiod) combined with several months of non-flooded conditions per year, whereas rocky soils formed in non-flooded, higher terrain (1.5-6 m elevation) (Li 2001). The marl type agricultur al soil in south Florida is classified as Biscayne soil (l oamy, carbonatic, hyperthermic, shallow, typic, fluvaquents) (Nobel et al. 1996, Li 2001). The rocky agri cultural soil in south Florida is classified as Krome very gravelly loam soil (loamy-skeletal, carbonati c, hypothermic, lithic, udorthents) (Nobel et al. 31

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1996, Li 2001). Marl soil is a calcite mud with poor drainage, whereas gravelly soils have good drainage (Li 2001). Responses of woody plants to flooding include senescence, shoot di eback, premature leaf abscission, decreased cambial gr owth, and suppressed formation and expansion of leaves and internodes (Schaffer at al. 1992, Kozlowski 1997). Flooding also inhibits root formation, root branching, and the growth of roots and mycorrhiz ae. Root mortality, lo ss of mycorrhizae, and suppressed root metabolism help to decrease the absorption of macronutrients. In addition, flooding reduces photosynthesis, car bohydrate transport, initiation of flower buds, anthesis, and fruit size, set, quality, and growth (Schaffer et al. 1992, Kozlowski 1997). Flooding often causes a change in the allocation of photosynthates within plants. For example, flooding suppressed height and diameter growth of flooded seedlings of Acer platanoides L. (Aceraceae), whereas bark growth increased, which suggests th at flooding affects carbohydrate partitioning (Yamamoto and Kozlowski 1987). Thus, flooding may reduce photosynthates, which help to produce leaves and root masses that provide food for larvae and adults of Diap repes root weevils. Flooding may indirectly reduce th e initial food available to la rvae and adults as well as subsequent products of photosynt hesis used to repair feedin g damage. Therefore, flooding should reduce the ability of th e plant to repair insect a nd other kinds of damage. Flooding also causes anaerobic decomposition of organic matter and promotes the decay of root systems (Kozlowski 1997). It worsens the severity of diseases by causing a discharge and dispersal of zoospores (Duniway 1983, Wilcox and Mircetich 1985, Schaffer et al. 1992). After zoospores are released from sporangia, their movement thr ough soil depends on high matrix potentials of flooded soils. Flooding thus augments the infection an d mortality of plants by promoting production and dispersal of inoculum (Kenerley et al. 1984). There is often an additive effect or interacti on between flooding and root diseas e, such as between flooding and 32

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Phytophthora root rot with avocado (Ploetz and Schaffer 1989). Flooding does not directly predispose plants to infection from root rot dise ases (Kenerley et al. 1984). As noted in the foregoing discussion of Phytophthora spp., root injury locations, such as from Diaprepes root weevil larval feeding, often serve as infection sites for root rot diseases (McCoy et al. 2002). Many compounds can be produced or their quantity increased in waterlogged soil, such as sulfides, CO2, soluble Fe, and Mn (Wang et al. 1967, Hook et al. 1971, Culbert and Ford 1972) and cyanogenic compounds, acetaldehyde, and ethanol (Fulton and Erickson 1964, Rowe and Catlin 1971, Ponnamperuma 1984). Furthermor e, anaerobic microbes synthesize methane, ethane, propylene, fatty acids, hydroxy-, and decarboxylic acids, unsaturated acids, aldehydes, ketones, diamines, mercaptans, and heterocy clic compounds. These compounds can help to make the flooded environment a toxic one. The foregoing substances in flooded soils can contribute to the injury, reduced growth, and mortality of woody plants (Kozlowski 1997). Injury to flooded plants can be caused by produc ts of anaerobic plant metabolism, such as aldehydes, organic acids, and ethanol (Kozlowski 1997). Considering its effects on plants, flooding can alter or reduce the activity of several metabolic pathways (Kennedy et al. 1991). Flooding affects the pr oduction of proteins, carbohydrates, organic acids, and lipids (Kozlowski 1997). The re sulting stress on metabolic pathways can decrease carbohydr ate production and shoot growth (Kozlowski 1997). A lack of O2, buildup of CO2, added toxins, and other side eff ects of flooding contribute to the phytotoxicity and physiological dysfuncti ons in plants (Kozlowski 1997). To counteract the detrimental effects of flooding, such as reduced photosynthesis, some plants have evolved the follo wing adaptations: development of hypertrophic (swollen) stem lenticels, development of aer enchyma tissue, and production of adventitious (above-ground) roots (Kozlowski 1997). Hypertrophic lenticels benef it flooded plants in two ways: 1) They 33

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exchange gases in flood water through breaks in the closi ng layers (Hook et al. 1970, Hook 1984); and 2) they release potentially toxic com pounds from plants, such as acetaldehyde and ethylene (Chirkova and Gutman 1972) Aerenchyma tissue occurs in root epidermal layers and shoot cortexes of flooded highbush blueberries ( Vaccinium corymbosm L., Ericaceae) (Abbott and Gough 1987, Crane and Davies 1989). Here, aer enchyma tissue facilita tes movement of O2 from shoots to submerged roots. Based on the foregoing discussion, flooding may increase chemicals in the soil and/or plants that tend to be mildly to severely toxic to plants and an imals. In addition, there is a decrease in plant matter such as edible leaves and roots to no urish Diaprepes root weevil. Flooding may therefore reduce herbivory from Diaprepes root weevil because the plants may be more toxic and less nourishing, hence, more repulsi ve and less attractive than non-flooded plants. However, flood-induced production of compounds such as ethanol can also attract insects. For example, Schroeder and Weslien (1994), using traps baited with ethanol and alpha pinene, attracted six phloem-feeding and four predatory insect species in significantly higher numbers than without the baited traps. The attracted in sect species included beet les (Coleoptera) in the families Cerambycidae, Pythidae, Histeridae, and Nitidulidae. Because ethanol attracted beetles in these families (Schroeder a nd Weslien 1994), it may also lure populations of Diaprepes root weevil, which is in the family Curculionidae. Plant Species Used in This Dissertation Green buttonwood ( C. erectus ), mahogany ( Swietenia mahagoni Jacq., Meliaceae), and Surinam cherry ( Eugenia uniflora L., Myrtaceae) are widely grown in south Florida as ornamental plants (Watkins and Sheehan 1975). Surinam cherry is also occasionally grown as a fruit crop. In addition, pond apple ( Annona glabra L., Annonaceae) is a tr ee native to Florida potentially useful as a flood-tolerant rootst ock for Annona species fruit crops (Wunderlin 1998, 34

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Nuez-Elisea et al. 1999). Green buttonwood is a frequent ornamental tr ee or shrub in south Florida and is native to tidal swamps of centr al and south Florida (W atkins and Sheehan 1975, Wunderlin 1998). As suggested from its native range, buttonwood is fair ly tolerant of flooding, though it also thrives in non-floode d, moderately moist soil, wh ich is common for landscape plants. In south Florida, tidal swamps frequently have marl so il, which cover low-lying terrain and originally formed in areas with several months of flooding (hydroperiod) combined with several months of non-flooded conditions per year (Li 2001). However, some tidal swamps may not be covered with marl soil because they are tidally replenished and do not have a chance to dry out for several months per year, and the soil a ppears much darker than marl soil. The native habitat of buttonwood frequently has flooding combin ed with marl soil, but is the environment where buttonwood evolved and should be best adapted. Mahogany is native to coastal hammocks in south Florida (Wunderlin 1998), where it is a widely planted lawn and street tree (Watkins and Sheehan 1975). Surinam cherry is found in disturbed hammocks of south and central Florida, is native to South America (Wunderlin 1998), and is also widely planted as a south Florida shrub, occasionally as a fruit cr op. Although Surinam cherry is named after the country Suriname, its common name is Surina m cherry (Wunderlin 1998). Pond apple is a native tree in swamps of south and central Florida (Wunderlin 1998), parts of the Caribbean, and Central and South America (Popeno 1920, Morton 1987). Although typically not grown as a commercial fruit crop (Popeno 1920, Morton 1987), this species is very tole rant to flooded soil conditions (Schaffer 1998, Nuez-Elisea et al. 1999, Ojeda et al. 2004). When used as a rootstock, pond apple greatly increases the flood tolerance of commercial Annona species (Nuez-Elisea et al. 1999). Hence, commerci al fruit crops in the Annonaceae including cherimoya (Annona cherimoya Mill.), ilama (Annona diversifolia Saff.), sugar apple ( A. 35

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squamosa L.), and atemoya (A. squamosa X A. cherimoya ) (Popeno 1920, Morton 1987) may potentially be grafted onto A. glabra for flood tolerance. Swingle citrumelo rootstock trees were also included for comparison with ornamental plants because most previous research on Diap repes root weevil herbivory was conducted with citrus and its rootstocks (L i et al. 2003a, 2004, 2006, 2007a, b). Other reasons for choosing Swingle included its use by Diaprepe s root weevil as a host plant (Simpson et al. 1996, Li et al. 2003a, 2004, 2006, 2007a, b) and at least half the comme rcial Rutaceous fruit trees in Florida are grown on this rootstock (Auscitrus 2004, F. Davi es pers comm. 2008). According to Hutchison (1974) and Auscitrus (2004), anothe r advantage of Swingle is its tolerance to root rot caused by Phytophthora parasitica Dast. (Oomycota) in Florida (G rimm and Hutchison, unpublished data) and in California (Carpenter and Furr 1962). Hence, Swingle may also tolerate P. nicotinae and P. palmivora associated with feeding injury from Diaprepes root weevil larvae. However, Swingle has moderate to low flood tolerance, th ough it may be more flood-tolerant than another popular rootstock, Carrizo citrange ( Poncirus trifoliata (L.) Raf. x Citrus sinensis (L.) Osb., (Rutaceae) (Auscitrus 2004, Li et al. 2004). A test involving a widely planted and studied citrus rootstock like Swingle may allo w the results of this dissertation to compare with earlier Diaprepes root weevil research. Previous Studies with Rutaceae on Soil Moistu re, Nutrients, and Diaprepes Root Weevil Herbivory Li et al. (2007a, 2007b) conducted studies re lating abundances of Diaprepes root weevils to soil moisture levels and other factors. B ecause of slow dispersal by Diaprepes root weevil adults, areas with high adult popul ations can have high larval populations and high root-feeding damage (Nigg et al. 2001b, McCoy et al. 2003). More adults per tree would presumably increase the egg-laying rate and neonate drop compared to areas with lower adult populations. Li et al. 36

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(2007a) conducted a field study involving two orchards of Hamlin oranges, Citrus sinensis (L.) Osb. (Rutaceae), one in Osceola County on a poorly drained loam soil and the other in Desoto County on a poorly drained sandy soil. At the Osceola site, which was strongly acidic with a pH of 4.9, highest adult weevil populatio n densities occurred in areas lowest in soil Mg and Ca. However at the Desoto site, which had a more ne utral pH of 6.6, adult po pulation densities were highest in areas with the highe st Mg, pH, and lowest sand cont ent (Li et al. 2007a). Weevil density was lowest at a pH of 5.7-6.2, which suggested that adjustment of acidic soil pH by liming may help control Diaprepes root weevil a nd simultaneously render pH more favorable for citrus growth and production (Li et al. 2007a). However, larger, healthier, more densely foliated trees are likely to be good food s ources and attract more weevils per tree than less healthy trees (Lower et al. 2003, Li et al. 2007a ). Management of Diaprepe s root weevil should hence be coordinated on a site-specifi c basis (Li et al. 2007a). Previous Studies with Rutaceae on Effects of Flooding and Herbivory by Diaprepes Root Weevil Studies by Li et al. in 2004 and 2007b were conducted in greenhouses with a sandy loam soil (pH 4.8) from a central Florida citrus orchard infested with Diaprepes root weevil to test combined effects of flood duration and pH on surv ival and/or growth of Diaprepes root weevil larvae. In these studies, pH increased with increasing flood peri od, which was related to reduced oxygen content of flooded soil. Li et al. (2004, 2007b) discovered that flooding increased soil pH 0.3 units above the non-flooded average by the end of the 40-d flood period. In addition, Li et al. (2007b) found that the longe st flood period (40-d) had the lo west survival rate of larvae compared to shorter flood periods, which may be related to higher soil pH at longer flood durations. Here, larval survival and growth were significantly decreased by pre-applied flooding (Li et al. 2007b). Larval surv ival and weight gain observe d by Li et al. (2007b) were 37

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significantly correlated with pH; increasing pH from 4.8 to 5.7 by liming decreased larval survival, and increasing pH from 5.1 to 5.7 significantly decreased larv al weights. In a test when the soil was limed to pH 4.8-5.7, larval survival was highest at pH 5.0 for non-flooded plants (Li et al. 2007b). Similar to results of their 2007b st udy, in 2004, Li et al. found that larval survival and weights were highest when soil pH was near 5.0 for non-flooded plants, and survival tended to decrease as pH dropped below 5.0 or increased a bove 5.1. Larval survival and weight gain in Li et al.s 2004 and 2007b studies were significantly correlated with pH. In 2004, Li et al. noted higher weight gain for larvae in pr eviously flooded than in nonflooded plants, which was different from their 2007b observations where larval survival and growth were significantly decrea sed by pre-applied flooding in both tests. In their 2004 study, soil moisture levels during infestation were hi gher for previously flooded than for non-flooded plants, and higher soil moisture levels of flooded tr eatments were correlated with higher weight gain observed (Li et al. 2004). Other factors such as soil t ype, compaction, bulk density, and water content may also influence larval survival and growth (Riis and Es bjerg 1998, Rogers et al. 2000, Li et al. 2007b). Flooding was recommended as a possible control method for Diaprepes root weevil in citrus by Li et al. (2007b), and it may reduce larval survival while plants are flooded. However, depending on soil pH, water-stressed plants may be more susceptible to Diaprepes root weevil larval feeding when un-flooded th an non-stressed plants that were either never flooded or floodtolerant and previously flooded. Hence, pre-applied flooding may either increase or decrease larval survival based on resulting soil moisture, pH, and plant hea lth while soil is infested. One potentially important conclusion made by Li et al was that increasing the soil pH by at least 1 unit in acid soils is recommended for optimum citrus growth, which occurs at pH 6.0-6.5, and to help control Diaprepes root weevil (Li et al. 2004, 2007b). 38

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Li et al. (2003a, 2006) examined effects of Diaprepes root weevil larval infestation, flooding, or soil type on plant grow th, larval survival, and other characteristics in a greenhouse. Flooding occurred before larval infe station so that both stresses we re not simultaneous (Li et al. 2003a, 2006). They used seedlings of Swingle citrumelo and Smooth Flat Seville citrus rootstock varieties. In both studies, flooding significantly re duced soil redox potential, which dropped from +220 to -100 mV (Li et al. 2006) or from +200 to -100 mV (Li et al. 2003a) within the first 3 d of flooding. Flooding also significantly reduced leaf stomatal conductance in both studies from 260 to 60 mmol m-2 s-1 in the first 30 d of flooding (Li et al. 2003a), and from 260 to 80 mmol m-2 s-1 in the first 20 d of flooding, while signi ficantly reducing shoot growth (Li et al. 2006). Survival of Diaprepes root weevil larvae was significantly higher in previously flooded soil than in non-flooded soil, and flood-da maged seedlings were more susceptible to larval feeding injury than non-flooded seedlings (Li et al. 2003a). Flooding plants for 30 d resulted in more serious root in jury and significantly lower stom atal conductance, and therefore caused greater water stress than fl ooding plants just 10 d (Li et al. 2003a). Similarly, Li et al. (2006) investigated effects of fl ooding and soil type on larval survival. In plants previously flooded for 20 d, larval survival averaged 25% highe r in sandy soil than in loam soil (Li et al. 2006). Soil pH increases with flood duration and coul d adversely affect larval survival (Shapiro et al. 1997, Li et al. 2006). Soil ty pe affects larval growth and surv ival rates, and the effects of soil type on larval survival may be chiefly physical including soil moisture and oxygen levels (Rogers et al. 2000). Flooded and waterlogged soils are also typically denser than non-flooded soils (Saqib et al. 2004), which is a potential problem for larval su rvival in flooded soil (Li et al. 2006). Similar to Li et al. (2003a ), Li et al. (2006) found that pl ants flooded for at least 20 d were more water stressed and more prone to Di aprepes root weevil larval feeding injury after removal of plants from flooding than non-flooded control plants. Their results suggest that 39

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minimizing plant exposure to waterlogging, avoidance of flooding, and early control of Diaprepes root weevil larvae may help pr otect young plants (L i et al. 2006). Lapointe and Shapiro (1999) attempted to determine soil moisture conditions that optimized production of Diaprepes root weevil adults. They initially raised larvae on an artificial diet and transferred them to an artificial so il with varying moisture levels (20-80%) at 25oC. Optimal survival to pupation occurred at 60% soil moisture for the 68-d larvae, and 30-70% for 180-d larvae. About 60-65% of 180-d larvae survived to pupation under these optimal moisture conditions. Both 68-d and 180-d larvae showed th eir poorest survival in low (20%) and in high (80%) soil moisture levels. Li et al. (2003a, 2006), and Diaz (2005) rem oved plants from flooding before infesting the soil with Diaprepes root weev il larvae. In contrast, Shapiro et al. (1997) exposed larvae to flooding to test the effects of va rying temperature (18, 21, 24, and 27oC) and flood periods (0, 1, 2, 3, 4, or 5 wk) on larval mortality. The larvae were large (11.5 mo old), and one larva was placed into each flooded polystyrene box (5.1 cm cube) filled with Immokalee sandy soil and no food (Shapiro et al. 1997). Mean mort ality exceeded 90% by 3 wk at 24 and 27oC and by 5 wk at 21oC, but was only 46% after 5 wk at 18oC (Shapiro et al. 1997). In addition, insect mortality was correlated with pH increase over time, although no such correlation existed between mortality and oxygen level (S hapiro et al. 1997). In other studies, wireworm larvae Melanotus communis (Gyllenhal) (Coleoptera: Elateridae) had 80% mortality after 6 wk of submergence at 27oC (Hall and Cherry 1993). However, sugarcane grubs Tomarus subtropicus (Blatchley) (Coleoptera: Scarabaeidae) had 100% mortality after only 5-10 d (1 wk) of su bmergence (Cherry 1984). Mortality may have been caused by drowning (suffocation) from a l ack of oxygen and surplus carbon dioxide, or by sepsis, from a buildup of microbes in stagnant wa ter and larval cadavers (Shapiro et al. 1997). 40

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Flooding is sometimes used in sugarcane fields of southern Florida to control pests, such as sugarcane grubs (T. subtropicus) (Cherry 1984) and wireworm larvae ( M. communis) (Hall and Cherry 1993). Flooding may hence be useful fo r controlling Diaprepes root weevil larvae in sugarcane fields, although only in the summer and fall when floodwater temp eratures are close to their maximum (27oC) (Hall and Cherry 1993, Shapiro et al. 1997). Previous Studies on Effects of Diaprepes R oot Weevil and Flooding on Ornamental Plants Although the host range of Diap repes root weevil includes ci trus, ornamental plants, and grasses (Simpson et al. 1996), the majority of prev ious research with this insect has focused on citrus. There have been relatively few reports on the effects of Diaprepe s root weevil larval feeding on plant species other than citrus and related genera, su ch as ornamental plants. Diaz (2005) tested the effects of flooding and larval infestation on photosynthesis, transpiration stomatal conductance, and fresh and dr y root weights of buttonwoods and live oaks. Also, Diaz (2005) examined effects of larv al root feeding on photosynthesis, transpiration, and stomatal conductance, leaf, stem, and root fresh and dry weights, plant height and stem diameter, and recovery of larv ae from green buttonwood, live oak ( Q. virginiana), and pygmy date palm (Phoenix roebelenii OBrien, Arecaceae) in 2006 (Diaz et al. 2006). Overall, the measured plant variables seemed to be more af fected by flooding than by larval infestation for both buttonwoods and live oaks (Diaz 2005). There was no significant difference in mean number of larvae recovered from infested pl ants between previously flooded and non-flooded buttonwoods or live oaks (Diaz 2005). In the 2006 study, root feeding by Diaprepes root weevil larvae did not significantly affect photosynthesis, transpiration stomatal conductance, plant wei ghts, or other variables for live oak. In contrast, fresh and dry weights for r oots and stems, and dry leaf weights, were significantly lower for infested th an non-infested plants for pygm y date palm (Diaz et al. 2006). 41

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However, leaf gas exchange and other variables were not signifi cantly different between infested and non-infested treatments of pygmy date palm (Diaz et al. 2006). For green buttonwoods, the weevil significantly reduced photosyn thesis, transpiration, stomatal conductance, plant heights, stem diameters, dry root weight s, and often dry stem and leaf weights (Diaz 2005, Diaz et al. 2006). Larval herbivory hence seemed to a ffect green buttonwoods the most, followed by pygmy date palms, and affected live oaks the least (Diaz 2005, Diaz et al. 2006). Diaz et al. (2005) measured effects of adult Diaprepes root weevil feeding on photosynthesis, transpiration stomatal conductance, root, stem, and leaf fresh and dry weights, and leaf areas of green buttonwood and live oak. Herbivory by adult Diaprepes root weevil caused variable results. A dult herbivory did not affect green buttonwood photosynthesis, transpiration or stomatal conductance in one test, but in another test, all three variables were significantly higher in infe sted than in non-infested mature leav es after 2 mo of infestation (Diaz et al. 2005). This study suggested that as adult Diaprepes root weevil removed more leaf area from infested than non-infested green buttonw oods, though with insignificant differences, root, stem, and leaf weights were also reduced. Although live oaks are a host plant for adult Diaprepes root weevil (Simpson et al. 1996, Ma nnion et al. 2003), adul t herbivory did not significantly affect photos ynthesis, transpiration or stomatal conductance in a test by Diaz et al. (2005), which may have been relate d to lack of young leaves. Objectives Goal. The goal of this project was to dete rmine effects of Diaprepes root weevil herbivory, flooding, and the intera ction of these two stresses on the leaf gas exchange and growth of selected ornamental plant species commonly grown and sold by south Florida nurseries. To help clarify relationships betw een Diaprepes root weev il herbivory, host plant 42

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physiology, and flooding, and to address questions not answered in pr evious research, the following objectives were proposed: 1. To determine effects of larval feeding by Diaprepes root weevil on leaf gas exchange and growth of selected woody ornamental plan ts commonly grown in south Florida. Hypothesis: larval herbivory re duces leaf gas exchange and growth of all plant species tested. 2. To test effects of flooding and of soil type (marl soil and a nursery potting medium) on the growth and physiology of selected woody orname ntal plants, which are potential hosts of Diaprepes root weevil. Hypot hesis: flooding lowers plant bi omass and leaf gas exchange more in marl soil than in nursery potting medium. 3. To determine the survival of Diaprepes root weevil larvae in a flooded marl soil and in a flooded nursery potting medium with green buttonwood as a food source. Hypothesis: flooding reduces larval survival more in marl soil than in potting medium. 4. To investigate effects of intermittent and continuous flooding combined with Diaprepes root weevil larval feeding on plant physiology and growth. H ypothesis: survival rates of Diaprepes root weevil larvae and plant bioma sses are lowered more by continuous than by intermittent flooding. 5. To test effects of flooding and adult herbivor y by Diaprepes root w eevil on leaf damage and physiology of selected woody ornamental plants and effects of flooding on adult host plant preference. Hypothesis: flooding reduces leaf feeding, damage, and attraction to host plants by adult Diaprepes root weevil, and it adversely affects leaf gas exchange of host plants. 43

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CHAPTER 2 EFFECTS OF HERBIVORY BY DIAPREPES ABBREVIATUS (L.) (COLEOPTERA: CURCULIONIDAE) LARVAE ON FOUR WOODY ORNAMENTAL PLANT SPECIES Introduction Diaprepes abbreviatus L. (Coleoptera: Curculionidae: Entiminae) commonly called Diaprepes root weevil, was first found in Flor ida in a citrus nurse ry in Apopka in 1964 (Woodruff 1964). It is believed to have entered Florida from Puerto Rico in a shipment of ornamental plants (Woodruff 1985). Puerto Rico probably is the cen ter of origin of this species (Lapointe 2000a, Obrien and Kovarik 2000), but it also is found in Trinidad, Tobago, Venezuela, and Mexico, though its presence in these other locations is disputed. In th e United States, it is found in Florida, Texas (Knapp et al. 2001, Skar ia and French 2001), and southern California (Klunk 2005). In Florida, Diaprepes root weevil occurs in 23 counties in the south and central parts of the state (Anonymous 1996, Pea 1997, Weissling et al. 2004). Diaprepes root weevil is an abunda nt and a serious pest of citr us and sugarcane in its home range of Puerto Rico (Woodruff 1964). Infestations of this insect nearly put many central Florida ornamental plant nurseries out of busin ess during the 1970s (Schroeder 1994). In 1996 it was reported to infest approximately 24,281 ha (60,000 acres) of citrus, and control costs and losses exceeded $2,965 per ha ($1,200 per acre) (Stanley 1996). Th e weevil has cost the Florida citrus industry an estimated $72 million annua lly (Anonymous 1996, Stanley 1996). Diaprepes root weevil continues to be a long-term threat to several agronomic and horticultural crops because of inadequate management strategies. Diaprepes root weevil is a problematic pest due to its very large host range, which includes at least 317 vari eties, 280 species, 180 genera, and 68 families of plants (Simpson et al. 1996, 2000, Knapp et al. 2000b, Mannion et al. 2003, G odfrey et al. 2006, C.G. Martin personal observation and unpublished data). Some plants support only one stage of the insect; for 44

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example Ardesia crenata Sims, which supports the larval st age. However, many economically important plants support all stag es of the weevil from egg to adult, such as sweet potato, Ipomoea batatas (L.) Lam., and buttonwood, Conocarpus erectus L. (Simpson et al. 1996). Mannion et al. (2003) surveyed se veral ornamental plant nurseries in southern Florida and found that egg masses, feeding damage, and adult Diap repes root weevils were common on field-grown ornamental plants. Plants with the highest percentage of egg masses were live oak ( Quercus virginiana Mill.), silver buttonwood, ( C. erectus L. variety sericeus Fors.Ex DC), and black olive ( Bucida buceras L.). The highest adult population de nsities were found on black olive, dahoon holly ( Ilex cassine L.), and Bauhinia sp. (Mannion et al. 2003). Insect herbivory often affects l eaf gas exchange including net CO2 assimilation, stomatal conductance, transpira tion, and internal CO2 concentration (the partial pressure of CO2 in the substomatal space) of host plants (Ander sen and Mizell 1987, Schaffer and Mason 1990, Schaffer et al. 1997). Measurements of leaf gas exchange can provide quantitative measures of insect damage to plants prior to the appearance of any visual sy mptoms. The effects of insect herbivory on leaf gas exchange can vary accord ing to feeding guild (Welter 1989). Root (1973) and Welter (1989) classified arthropods according to their feeding-damage type or guild, such as mesophyll feeders, phloem feeders, stem borers, root feeder s, or direct leaf consumers. Diaprepes and other ro ot weevils such as Pachneus litus (Germ.) and Artipus floridanus Horn (Coleoptera: Curculionidae) have two feeding guilds. Larvae are in the root-feeder guild, whereas adults are in the direct-leaf-cons umer guild (Syvertsen and McCoy 1985). In a review of arthropod herb ivory effects on leaf gas exchan ge, most studies claimed that herbivory harms this process, although insignificant or beneficial effects of herbivory on leaf gas exchange have been noted (Welter 1989). Syve rtsen and McCoy (1985) found that leaf feeding by A. floridanus adults decreased photosynthesis and transpir ation of citrus tree s. According to 45

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Welter (1989), leaf feeding by adu lts, which are in the direct-lea f-consumer guild, often tend to increase photosynthesis (when measured on a leaf area basis). However, th is effect is atypical compared to the other guilds, such as stem borers and root-feeders, which tend to decrease photosynthesis. As young Diaprepes root weevil larvae work thei r way from small roots to larger lateral and main roots, they often form deep grooves when feeding on the larger roots, and they consume the outer bark and cambial layers (McC oy et al. 2002). Roots may be girdled causing root death, or the crown may be girdled causing tree death (McCoy et al. 2002). Larval girdling often kills small citrus trees by destroying thei r ability to take up nut rients (Wolcott 1936, 1948; Quintela et al. 1998). However once the larvae are controlled, damage especially to smaller roots, which grow more rapidly than larger ro ots, may be outgrown in a few weeks to a few months. Gouges in cambial layers and girdli ng, however, may cause long-term deformity from which plants may never recover. Although the host range of Diaprepes root weevil includes citrus, ornamental plants, and grasses (Simpson et al. 1996), the majority of prev ious research with this insect has focused on citrus and there have been relatively few reports of the effects of larval feeding on plant species other than citrus. In a previ ous study of ornamental plants, root feeding by Diaprepes root weevil larvae did not significantly affect photosynthesis, transpiration stomatal conductance, or root, stem, and leaf dry weights of live oak; however, it did significantly reduce root, stem, and leaf dry weights of pygmy date palm (Phoenix roebelenii OBrien) (Diaz et al. 2006). Diaz et al. (2006) also observed that root feeding by Diaprepes root weevil reduced photosynthesis, transpiration, stomatal conductan ce, root dry weight and often st em and leaf weights of green buttonwood trees. 46

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The purpose of the present study was to furt her examine the effects of Diaprepes root weevil feeding on leaf gas excha nge and growth of woody orname ntal plants. The hypothesis tested was that larval herbivor y reduces leaf gas exch ange and biomass of all the plant species tested. Materials and Methods The experiment was conducted in the spring and summer of 2006 in Homestead, FL, using plants in 11-liter plastic containers placed on ground cl oth in an outdoor site exposed to full sun. There were two treatments (non-infested plants and plants infest ed with Diaprepes root weevil larvae), six replications per treatment, a nd four plant species for a total of 48 plants. Plant material. Mahogany (Swietenia mahagoni Jacq.), Surinam cherry ( Eugenia uniflora L.), pond apple (Annona glabra L.), and green buttonwood trees were purchased in February 2006. Pond apple trees were purchased in 4-liter containers from a commercial nursery (Fort Pierce, FL) and repotted into 11-liter containers 5 d after purchase. Surinam cherry and mahogany were purchased in 11-liter containers from a commercial nursery (Miami, FL). Buttonwood was also purchased in 11-liter containers from a commercial nursery (Homestead, FL). At the time treatments were initiated buttonwood and mahogany plants were each 2 years old, Surinam cherry trees were approximately 1.5 years old and pond apple were approximately 0.5 years. All plants were grown in a nursery mix consisting of 40% Florida peat, 30% pine bark, 20% cypress sawdust, and 10% sand. On th e same day that plants were purchased, about one-third to one-half the foliage of the buttonw ood and Surinam cherry plants was pruned to improve and standardize the size of the plants. On the first infestation date (10 March), plant heights (mean SD) were 43 9, 101 8, 35 5, and 51 14 cm for buttonwood, mahogany, Surinam cherry and pond apple, respectively. The experiment was conducted for 146 d (March July). Plants were irrigated for 30 min twice per day and fertilized 2 d before beginning the 47

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experiment (28 February 2006) with liquid fertilizer (Peters 20-20-20 with micronutrients, United Industries, St Louis, MO) according to the manufacturers recommended rate. Plants were fertilized again on day 69 with the same fertilizer and rate as stated above. Pests other than Diaprepes root weevil were controlled by manual removal of the insects. Larval infestation. Each plant in the infested treatme nt was infested with 20 Diaprepes root weevil larvae on each of two occasions: day 9 (10 March 2006) and day 84 (24 May 2006). The reason for the second infestation was the lack of significant differences in leaf gas exchange (net CO2 assimilation, transpiration stomatal conductance, and internal CO2 concentration) between treatments, and thus the assumption th at the larval populati on may have not been sufficient to detect differences between treatmen ts. To infest the plants, one larva was placed individually into each of 20 holes, 5-10 cm deep made in the potting medium in each container. Holes were 4-8 cm from the stem, and 2.5 cm ap art. Based on mean head capsule widths (Quintela et al. 1998), larvae were second through fifth instars (fourth instar average) for the 10 March infestation and fifth through ninth instar s (seventh instar average) for the 24 May infestation. Control plants were not infested with larvae. Data collection. Data collected included the numb er of larvae, pupae, and adults recovered per plant, larval head caps ule widths, leaf gas exchange (net CO2 assimilation, transpiration stomatal conductance and internal CO2 concentration), plan t height and caliper (trunk diameter), and fresh and dr y weights of roots, stems, and leaves and the number of trunks per container at the end of the study. Trunk caliper 10 cm above so il line and plant height to the top of the highest leaf above th e soil line were measured for all plants prior to Diaprepes root weevil infestation. Trunk diameters were meas ured with a micrometer (0-25 mm Electronic Digital Micrometer, Marathon Co., Richmond Hill, Ontario, Canada). Soil temperature was recorded at 1 h intervals from day 43 (12 April 2006) to day 146 (25 July) with sensors 48

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(StowAway Tidbit temploggers, Onset Co., Pocasset, MA) placed in the soil of four plants that were not included in the experiment but were maintained under the same experimental conditions. Sensors were placed at a soil depth of 6 cm two-thirds the distance from the center to the outer edge of the container. Leaf gas exchange was measured on two fully expanded, recently mature leaves per plant with a CIRAS-2 portable gas analyzer (PP Systems, Amesbury, MA). Recently mature refers to a leaf that has fully expanded and hardened off w ithin the preceding few w eeks of measurement. These leaves usually were between the fourth an d tenth node below the stem tip. Gas exchange values of the two leaves were averaged and the mean value per plant (replication) was used for statistical analyses. All leaf gas exchange measurements were made between 08:30 and 15:45h. However, on each measurement date, all measuremen ts of each plant species (including infested and non-infested treatments) were made within 65 min of each other. During gas exchange measurements, the photosynthetic photon flux was maintained at 1000 mol photons m-2 s-1 with a halogen lamp attached to the leaf cuvette, and the reference CO2 concentration in the cuvette was kept constant at 375 mol mol-1 CO2. Leaf gas exchange wa s measured 8 d (buttonwood and mahogany) and 1d (Surinam cherry and pond appl e) before insect infestation and at 2-wk intervals after infestation. Four weeks after the last leaf gas exchange measurement, trunk diameter and plant height were again recorded, and increases in these vari ables from before the first infestation were calculated. One week later, plants were harvested. Stems were cut off 2-3 cm above the surface of the potting medium. The roots were removed from the potting medium and the medium was placed into bins and carefully inspected for la rvae. Recovered larvae were preserved in 75% ethyl alcohol. Fresh root, stem, and leaf we ights were determined (Mettler PE 3600 Delta Range, Mettler Co., Highstown, NJ) immediately after the plants were harvested. Roots, 49

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stems, and leaves were then dried in an oven (Memmert, Wisconsin Oven Co., East Troy, WI) at 75C for 2 d to a constant weight. Experimental design an d statistical analyses. The experiment was arranged as a completely randomized design with two treatmen ts (infested and non-infested) and six singleplant replications per treatment. There were four plant species te sted for a total of 48. For all plant physiology and growth variables, infested and non-infested treatments were compared within each species with a standa rd T-test. Interactions between species and treatment were not tested because we were only interested in main e ffects. However, larval survival and size were compared among plant species using analysis of variance (ANOVA) a nd Waller-Duncan K-ratio test. Statistical analysis was conducted with SAS statistical software Version 9.1 (PROC TTEST and GLM, SAS Institute, Cary, NC, 2003). Results During the experimental period, mean daily soil temperatures varied between 21 and 32oC with monthly averages between 24 and 28oC (Figure 2-1). For green buttonwood trees, in itially there were no signi ficant differences in net CO2 assimilation, transpiration, stomatal conductance, or internal CO2 concentration between infested and non-infested treatments except at wk 4 when infested plants had significantly higher stomatal conductance (t = 2.4, df = 6, P = 0.048 ) and internal CO2 concentrations ( t = 2.4, df = 10, P = 0.04) than non-infested plants (Figure 2-2c, d). At 14 wk, plants in the non-infested treatment had significantly higher transpiration ( t = -3.3, df = 10, P = 0.009), stomatal conductance ( t = -3.4, df = 10, P = 0.0065), and internal CO2 concentration (t = -2.8, df = 9, P = 0.022) than plants in the infested treatment (Fi gure 2-2b, c, d). By wk 18, infested plants had significantly higher net CO2 assimilation ( t = 3.0, df = 9, P = 0.015) than non-infested plants (Figure 2-2a). Hence, after the second infestat ion (wk 14-18) with larger larvae than the first 50

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infestation, treatment differences in net CO2 assimilation, transpiration, stomatal conductance, and internal CO2 concentration occurred more often than before the second infestation (wk 2-12). Non-infested buttonwood plants had significantly greater root, stem, and leaf fresh and dry weights than infested pl ants (Table 2-1). For mahogany, transpiration (t = -2.7, df = 10, P = 0.023) and stomatal conductance (t = -2.5, df = 9, P = 0.037) were significantly higher for non-infe sted than infested plants at 16 wk (Figure 2-3b, c) and no other leaf gas exchange variables were affected by treatment (Figure 23a, d). Fresh and dry weights of roots, stems, a nd leaves were higher for non-infested plants than infested plants, but these differences were only significant for leav es (Table 2-1). For Surinam cherry, net CO2 assimilation, transpiration, a nd stomatal conductance were generally higher for non-infested than infested pl ants; however, some of these differences were significant only after the second in festation (Figure 2-4). Surinam cherry gas exchange values that were significantly higher for non-infest ed than infested plants included net CO2 assimilation at wk 14 ( t = -5.0, df = 10, P = 0.0005) and at wk 18 ( t = -9.4, df = 10, P = <0.0001), transpiration at wk 14 ( t = -4.1, df = 9, P = 0.0026), and stomatal conductance at wk 14 ( t = -5.0, df = 10, P = 0.0006). Internal CO2 concentration, however, was higher for infested than noninfested plants with significant differences at wk 14 ( t = 2.6, df = 10, P = 0.028) and at wk 18 (t = 2.9, df = 8, P = 0.02) (Figure 2-4d). Fresh and dry wei ghts of roots, stems, and leaves were consistently higher for non-infested than infest ed Surinam cherry plants, but differences were only significant for roots and leaves (Table 2-1). For pond apple, there were no significant differences between infested and non-infested plants in net CO2 assimilation (range 1.8 to 14.9 umol CO2 m-2 s-1), transpiration (range 1.1 to 7.5 mmol H2O m-2 s-1), stomatal conductance (range 28 to 506 mmol CO2 m-2 s-1), or internal CO2 concentration (range 188 to 265 u mol CO2 mol-1). Non-infested pond apple trees tended to 51

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have greater leaf, stem and root fresh and dry weights than in fested plants, though differences were not statistically si gnificant (Table 2-1). Pond appl e did support populations of Diaprepes root weevil larvae (Figure 2-5) and e xhibited damage from root feeding. Mahogany and pond apple each had one trunk, whereas buttonwood and Surinam cherry were multi-trunked. There was no significant effect of treatment on increase in plant height or stem diameter for any of the plant species te sted. The range of plant heights for buttonwood, mahogany, Surinam cherry, and pond apple was 13.7-38.5 cm, 15.6-55 cm, 23.2-37.3 cm, and 0.06-11.6 cm, respectively, and range of stem diameters was 1.5-5.1 mm, 1.2-4.8 mm, 2.9-4.9 mm, and -0.64 to 3.24 mm, respectively. However, the increase in plant height and stem diameter for each species tended to be lower in infested than in non-infested plants. The total number of larvae, pupae, and adults recovered did not differ significantly among plant species ( F = 1.6; df = 3, 23; P = 0.22) and averaged 3.7-7.2 per plant depending on the species (Figure 2-5). No lif e stages were recovered from noninfested plants. There was a significant difference in head capsu le widths of larvae recovered from different plant species ( F = 3.1; df = 3, 23; P = 0.049) with the larges t larvae collected from Surinam cherry (mean 2.59 0.19 mm; 9th instar) and the smallest collect ed from mahogany (2.29 0.06 mm; 8th instar) (Figure 2-6). Discussion Differences in net CO2 assimilation between Diaprepes-infested and non-infested treatments suggest that the order of susceptibility to larval feeding damage of the host plants tested is Surinam cherry followed by buttonw ood, then mahogany with pond apple the least affected. However, differences in biomass (fresh and dry weights) betwee n infested and control treatments indicated a slightly different order of susceptibility to larval feeding damage with reductions in biomass greatest for buttonwood followed by Surinam cherry then mahogany, 52

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again with pond apple the least affected by larval feeding. Diaz et al. (2006) found that buttonwood was more susceptible to larval feedin g than live oak or pygmy date palm. Hence, mahogany, pond apple, and live oak all seem to be less susceptible than buttonwood to Diaprepes root weevil larval feeding. Out of the 40 insects added per pot, the 3.7-7.2 insects recovered is about 9-16% survivorship. The time period was 137 d for th e first 20 insects combined with 62 d for the second 20 insects. Low survivorship similar to levels we found is not uncommon. For example, Diaz et al. (2006) recovered 6-10 Diaprepes root weevil larvae per container 90 d after infesting with 40 fifth-to-sixth-instar larv ae and thus had 15-25% survi vorship. Cannibalism is common among Diaprepes root weevil larvae and may help account for these low survival rates. In addition, predators such as ants, spiders, earwigs, hemipterans, and lady beetles are known to attack Diaprepes root weevil larvae (Whitc omb et al. 1982, Richman et al. 1983a, b, Tryon 1986, Jaffe et al. 1990b, McCoy et al. 2000, Stuart et al. 2002). Predators such as fire ants ( Solenopsis sp.) (Hymenoptera: Formicidae) and spiders (many unknown spp.) (Arachnida: Araneae) have commonly been seen at the study site. Brown anole lizards ( Anolis sagrei Dumril & Bibron) (Reptilia: Polychrotid ae), also abundant at the site, pr ey on soft-bodied, slow-moving insect larvae similar in size to Diaprepes root weev il larvae and hence may also eat Diaprepes root weevil larvae. In addition, younger la rvae seem more inclined than ol der larvae to emigrate from the pots. Hence, emigration of larvae may have been more problematic after the first infestation than after the second infesta tion because younger larvae were used. Cannibalism, predators, pathogens, and emigration may have all contributed to the low larval survi vorship in the present study. We attempted to provide enough larval f eeding pressure to allow significant treatment effects without causing excessive cannibalism and th erefore infested with 20 larvae per container two times. 53

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All recovered adults were found buried in soil presumably in a resting period. Lapointe (2000b) tested the effects of constant 22, 26, and 30oC temperatures on Diaprepes root weevil larval survival and developmenta l rates on an artificial diet an d found the highest survival rates occurred at 22 and 26oC with lowest survival at 30oC. The average monthly soil temperatures for the present study were 24-28oC, or within 2oC of 26oC. Rates of larval development and survival for the present study were thus fairly close to th eir maximum rates. Data for larval recovery were difficult to interpret for plant species vu lnerability to larval feeding injury because differences between treatments were not significa nt for the sum of larvae, pupae, or adults and minimally significant for larval size (head capsule widths). However, they do suggest that buttonwood, Surinam cherry, and mahogany were more vulnerable to larval feeding injury than pond apple. The greater numbers of insects recovered and/or la rger head capsule widths for buttonwood, Surinam cherry and mahogany than pond apple indicates that pond apple is not as strong a host for Diaprepes root weevil larvae as the other three species Overall, buttonwood and Surinam cherry were the most susceptible sp ecies to larval root feeding by Diaprepes root weevil, followed by mahogany and then pond apple. According to Welter (1989), th e effects of herbivory by inse cts in the root-feeding guild has been less thoroughly documented than the effects of herbivory by above-ground guilds, such as phloem feeders and gall formers. In addition, the effects of herbivory by nymphs and larvae on leaf gas exchange (Schaffer et al. 1997, Diaz et al. 2006) has been less frequently studied than the effects of herbivory by adult insects (Syvertsen and McCoy 1985, Boucher and Pfeiffer 1989). Results obtained for green buttonwood in th e present study were generally comparable to those of Diaz et al. (2006) who observed higher net CO2 assimilation, transpiration, and stomatal conductance (on a leaf area basis) for non-infested plants than plants infested with Diaprepes root weevil larvae. However, during 2 to 4 wk following each of two infestations in the present 54

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study, all four gas exchange values for buttonw ood increased in infested compared to noninfested plants, and ended the period mostly hi gher for infested than non-infested plants. Overall, all four gas exchange values for buttonw ood were thus significantly higher for infested than non-infested plants the same number of tim es that values for non-infested plants were significantly higher than those of infested plants. For buttonwoods, a compensatory increase in leaf gas exchange after infestation may have resulted in increased net CO2 assimilation, transpiration, stomatal co nductance, and internal CO2 concentration in infested relative to noninfested plants. Diaz et al. (2006) also found evidence of compensatory response where buttonwood leaf gas exchange values increased in a long-term response to larval feeding injury. Similar to Diaz et al. (2006), we did not observe a compensato ry increase in the other plant species tested. Nigg et al. (2001a) tested the responses of seve n varieties of citrus rootstock seedlings to Diaprepes root weevil larval feeding. They comp ared fresh and dry weights, trunk diameters, and larval recovery of infested and non-infested plants of eac h variety to determine relative tolerance of each variety to la rval feeding on roots. At 90 d, Cleopatra mandarin, Sun Chu Sha mandarin, and sweet lime were tolerant of Diapre pes root weevil larval feeding. However, at 168 d, none of the seven citrus root stock varieties were tolerant or resistant to larval feeding. Hence, instead of using host plan t resistance, Nigg et al. (2001a) recommended controlling adults to reduce larval feeding to tolerable levels. Several studies by Li et al. (2003a, 2006, and 2007b) also used Diaprepes root weevil larvae to test effects of larval feeding (and flooding) on plant gas exchange, biomass, and/or larval growth on citrus varietie s in a laboratory or greenhouse environment. They used much smaller numbers of larvae than Nigg et al. (2001a), Diaz et al (2006), or the present study and much smaller sizes of larvae than Diaz et al. ( 2006) or the present study. However, they also 55

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used much smaller pots (about 5 cm cube) and plants (6 mo old seedlings). Although much fewer and/or smaller larvae were used, they were sufficient to yield the ap propriate results given the smaller plant and pot sizes. Similar results ha ve been reported for an other root-feeding larval pest, the western corn rootworm, Diabrotica vergifera vergifera Leconte (Coleoptera: Chrysomelidae) (Riedell and Reese 1999, Urias-L opez et al. 2000). Riedell and Reese (1999) found that in the tassel stage of corn, larval feeding by r ootworms lowered stomatal conductance significantly relative to non-infested plants, although net CO2 assimilation was unchanged. While larvae were ac tively feeding, net CO2 assimilation was lower in root-damaged than in undamaged corn plants. Net CO2 assimilation was also lower for severely infested than moderately infested or non-infest ed plants. In addition, the devel opment of adventitious roots as a compensatory growth response occurred more in moderately infested than in non-infested or severely infested corn plants. Hence, the severity of root damage and the level of compensatory growth played important roles in mediat ing shoot growth and the level of net CO2 assimilation (Riedell and Reese 1999). Schaffer et al. (1997) also reported sim ilar results for the citrus leafminer, Phyllocnistis citrella Stainton (Lepidoptera: Gracillariidae), whic h are also herbivorous larvae, but in the mesophyll feeding guild (Welter 1989). In plants infested with citr us leafminers, the duration of leafmining and the number of larvae per leaf were each correlated w ith greater leaf area damage and reductions in net CO2 assimilation (Schaffer et al. 1997). Ingham and Detling (1986) artificially defoliated 55% of the fo liage of sideoats gramma grass, Bouteloua curtipendula (Michx.) Torr., and infested roots of a different tr eatment with a root feeding nematode, Tylenchorhynchus claytoni Steiner. Root biomass, shoot bi omass, and tiller number were each reduced by artificial defoliation or by root-feeding nematodes. Net CO2 assimilation and 56

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transpiration, however, significantly increased in the remaining foliage after artificial defoliation, but were unaffected by nematode root feeding. As noted, Welter (1989) stated th at whole-leaf consumption by a dults, which are in the leaf consumer feeding guild, ofte n tends to increase net CO2 assimilation when measured on a leafarea basis, although this e ffect is atypical compared to the other guilds, such as root-feeders, which tend to decrease net CO2 assimilation. With regard to whole-leaf consumption, increased net CO2 assimilation may be explained by the availability of the same quantity of nutrients such as nitrogen delivered to a smaller leaf area after defoliation by the insects. This may render more nitrogen and other nutrients available per remaining leaf area to synthesize chlorophyll and supply other reactions that increase net CO2 assimilation. The results of this study confirmed the fi ndings of Simpson et al (1996) that buttonwood, Surinam cherry, and mahogany are host plants of Diaprepes root weevil. Perhaps most noteworthy, pond apple was able to support Diaprepes root weev il larvae. To our knowledge this is the first report of this species as a host. Furthermore, this is the first member of the entire plant family, Annonaceae, to be reported as a hos t to Diaprepes root weevil larvae. Pond apple has been suggested as a potential floo d-tolerant rootstock for commercial Annona species (Nuez-Elisea et al. 1999). Thus, its ability to host Diaprepes root weevil larvae may be a consideration when selecting rootstocks for commercial Annona species. Furthermore, other commercial fruit crop species in th e Annonaceae such as cherimoya ( Annona cherimoya Mill.), ilama ( Annona diversifolia Saff.), sugar apple ( A. squamosa L.), and atemoya ( A. squamosa X A. cherimoya ) (Popeno 1920, Morton 1987) may be also vul nerable to feeding by Diaprepes root weevil larvae and their susceptibility should be tested in areas where Diaprepes root weevil infestation poses a threat. 57

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The results of this study can help prioriti ze decisions for pest management of buttonwood, Surinam cherry, mahogany, and pond apple plants. For example, pond apple may never require treatment to control Diaprepe s root weevil larvae, while Su rinam cherry and buttonwood may benefit from such a treatment. Future studies sh ould investigate susceptibility to damage from Diaprepes root weevil larval feed ing for other economically valuable plant species often visibly infested with adult weevils, such as Bulnesia arborea (Jacq.) Engl. (Zygophyllaceae) and black olive (B. buceras ). 58

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Table 2-1. Effects of Diaprepes root weevil larv ae on root, stem, and leaf fresh and dry weights of buttonwood, mahogany, Surina m cherry, and pond apple. Fresh Weight (g) (mean SD) Dry Weight (g) (mean SD) Species Treatment Root Stem Leaf Root Stem Leaf Buttonwood N oninfested 78.3 11.4 89.8 9.6 129.0 29.2 68.8 11.7 62.1 7.5 83.7 12.9 Infested 57.3 14.0 48.6 20.3 76.5 21.5 48.7 12.7 35.4 13.7 55.4 12.9 Pa 0.018 0.0027 0.0061 0.018 0.0032 0.0035 Mahogany Noninfested 97.7 47.4 192.1 81.3 61.2 6.1 73.5 34.3 108.6 49.9 47.9 5.4 Infested 53.1 26.9 119.5 65.4 37.1 9.1 44.6 20.4 68.4 36.4 32.0 6.5 P 0.08 0.12 0.0005 0.11 0.14 0.0011 Surinam cherry Noninfested 34.5 10.9 89.0 31.9 135.4 42.1 30.5 9.2 66.8 21.4 75.6 17.0 Infested 15.4 10.0 65.1 26.3 64.0 43.3 12.5 7.4 47.3 19.3 44.0 21.5 P 0.01 0.019 0.016 0.0043 0.13 0.019 Pond apple Noninfested 16.8 16.4 17.7 17.7 3.6 2.5 13.4 12.5 14.3 13.2 16.8 16.4 Infested 11.5 5.0 13.5 7.0 2.9 1.3 9.7 4.1 11.5 5.7 11.5 5.0 P 0.48 0.61 0.56 0.51 0.65 0.55 a Significance level determined with a standard T-test; n=6. 59

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Figure 2-1. Average temperature 6 cm below the soil surface during the experiment. 60

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Figure 2-2. Effect of Diaprepes r oot weevil larval feeding on net CO2 assimilation ( A ), transpiration (E ), stomatal conductance ( gs), and substomatal partial pressure of CO2 ( Ci ) of green buttonwood plants. Symbols repr esent means SD. Asterisks indicate significant differences between treatments at P 0.05, ** P < 0.01, and *** P < 0.001 according to a standard T-test. 61

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Figure 2-3. Effect of Diaprepes r oot weevil larval feeding on net CO2 assimilation ( A ), transpiration (E ), stomatal conductance ( gs), and substomatal partial pressure of CO2 ( Ci ) of mahogany trees. Symbols repres ent means SD. Asterisks indicate significant differences between treatments at P 0.05, ** P < 0.01, and *** P < 0.001 according to a standard T-test. 62

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Figure 2-4. Effect of Diaprepes root weevil larval feeding on net CO2 assimilation ( A ), transpiration (E ), stomatal conductance ( gs), and substomatal partial pressure of CO2 ( Ci ) of Surinam cherry plants. Symbols re present means SD. Asterisks indicate significant differences between treatments at P 0.05, ** P < 0.01, and *** P < 0.001 according to a standard T-test. 63

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Figure 2-5. Average sum of larvae, pupae, and adults of Diaprepes root weev il per plant species. But = buttonwood, Mah = mahogany, SC = Surinam cherry, and PA = pond apple. Bars represent means SD. Differences in total number of insects recovered were not significant among plant species. Figure 2-6. Mean head capsule widths and resulting instars of Diaprepes root weevil larvae recovered from infested plants. But = buttonwood, Mah = mahogany, SC = Surinam cherry, and PA = pond apple. Bars represen t means SD. Different letters indicate significant differences among species according to ANOVA and Waller-Duncan multiple range tests ( P 0.05). 64

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CHAPTER 3 EFFECTS OF FLOODING AND SOIL TYPE ON THE PHYSIOLOGY AND GROWTH OF FOUR WOODY ORNAMENTAL PLANT SPECIES IN SOUTH FLORIDA Introduction A significant portion of agriculture in south Fl orida occurs in low-lying areas with the water table only a few meters below the soil surface (Schaffer 1998). Changes in water delivery practices to Everglades National Park as a resu lt of the Everglades Re storation Project have resulted in elevating the water table in thes e areas (Schaffer 1998). This has increased the severity, duration, and extent of flooding in areas with tropical fruit orchards and ornamental plant nurseries. For agriculture to remain viable in these areas it is important to understand how flooding affects crop physiology, growth, and yield to help identify flood-adapted crops and production systems (Schaffer 1998). Flooding or waterlogging of the root z one typically depletes soil oxygen content (Kozlowski 1997). Oxygen content in soil can be indirectly determined by measuring the redox potential of the soil; redox potentials of +300 mV or more indicate aerobic conditions, whereas redox potentials of less than +200 mV sugge st anaerobic conditions (Ponnamperuma 1972, 1984). The effects of flooding on physiology and gr owth of a woody, perennial plant species can vary among soil types and is partly based on the rates of soil oxygen depletion and other factors such as soil pH (Schaffer et al. 1992, Kozlowsk i 1997). In plant nurseries in south Florida, woody, perennial ornamental plants are grown either in potting medi um in containers or in the field. Field plants are generall y grown in marl soils, which are classified as Biscayne soil (loamy, carbonatic, hyperthermic, shallow Typi c Fluvaquents) (Nobel et al. 1996, Li 2001). These marl soils are derived from limestone in areas that alternate se veral months of flooding during the wet season with several months of non-flooded conditions during the dry season. The 65

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resulting calcite mud soil is high in calci um, has pH of 7.4-8.4, and has poor drainage (Li 2001). Responses of woody, perennial plants to flooding include senescence, shoot dieback, premature leaf abscission, decreased cambial grow th, and the suppression of leaf formation and expansion (Kozlowski 1997). Fl ooding also inhibits root fo rmation and branching, and the growth of roots and mycorrhizae. Root mortality, loss of mycorrhizae, and suppressed root metabolism help to reduce the absorption of macronutrients. In addition, flooding reduces net CO2 assimilation and the transport of carbohydrates within plants, and decreases flower bud initiation, anthesis, and fruit si ze, set, quality, and growth (K ozlowski 1997). Flooding often causes a change in the allocati on of photosynthates within plants. For example, Yamamoto and Kozlowski (1987) found that height and diameter growth of flooded Acer platanoides L. seedlings were suppressed, whereas bark growth was increased, which suggests that flooding affects carbohydrate partitioning. Early meas urable plant responses to flooding include reductions in net CO2 assimilation and stomatal conductance. Therefore, meas uring these leaf gas exchange variables can help quantify damage from flooding prior to the appearance of any visual symptoms. Green buttonwood ( Conocarpus erectus L., Combretaceae), mahogany ( Swietenia mahagoni Jacq., Meliaceae), pond apple ( Annona glabra L., Annonaceae), and Surinam cherry ( Eugenia uniflora L., Myrtaceae) are widely grown in s outhern Florida. Green buttonwood and mahogany trees are native to southern Florida and are grown as lawn and street trees (Watkins and Sheehan 1975). Surinam cherry is also a wide ly planted shrub in southern Florida and is planted on a small scale as a fruit crop. Pond appl e is a tree native to sout h Florida, parts of the Caribbean, and Central and South America. Alt hough typically not grown as a commercial fruit crop (Popeno 1920, Morton 1987), this species is very tolerant of fl ooded soil conditions 66

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(Schaffer 1998, Nez-Elisea et al. 1999, Ojeda et al. 2004) and greatly increases the floodtolerance of commercial Annona species when used as a rootst ock (Nez-Elisea et al. 1999). The purpose of this study was to test effects of flooding on growth and physiology of green buttonwood, mahogany, pond apple, and Surinam cherry trees in a nursery potting medium and in the marl soil typically found in field nurseries in southern Florida. Materials and Methods Two tests were conducted at the Tropical Res earch and Education Ce nter, University of Florida, Homestead, from 2006 to 2007. The firs t test was conducted outside during the spring and summer of 2006 with buttonwood and mahoga ny. The second test was also conducted outside but in the late fall, winter, and spri ng 2006-2007 with pond apple a nd Surinam cherry. Plant material. All plants were obtained from local commercial nurseries. Before initiating treatments, buttonwood, mahogany, and Su rinam cherry trees were approximately 2 years old, and pond apple trees were approximately 1 year old. At the beginning of the experiment, plant heights (mean SD) were 44 11, 96 14, 77 20, and 56 11 cm for buttonwood, mahogany, pond apple, and Surinam cherr y, respectively. Half the plants of each species (12 out of 24 plants) were repotted into 11 -liter plastic containers filled with a marl soil native to south Florida 77 d before initiating the first test. The marl soil was collected from a fallow agricultural field in Homestead, FL and wa s sieved to remove large objects. For each plant species, the remaining half (12 of 24) plants remained in the same 11-L containers in which they were purchased, or if purchased in sma ller containers they were repotted into 11-L containers filled with the same nursery potting medium. The potting media for each species were standard commercial mixes and thus not n ecessarily the same for each species. For green buttonwood, the medium was composed of 60% Florida peat and 40% hardwood chips, by volume. The potting medium for mahogany contai ned 25% Florida peat, 65% pine bark, and 67

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10% coarse sand, by volume. For pond apple and Surinam cherry the medium contained 40% Florida peat, 20% pine bark, 20% cypress sawdust, and 20% sand, by volume. All trees in both tests were fertilized w ith liquid fertilizer (Peters 24-8-16 with micronutrients, United Industries, St Louis, MO) at the manufacturers recommended rate for plants in general. Buttonwood and mahogany trees were each fertilized 9 May 2006 (29 d before beginning the test), while pond apple and Surinam cherry were each fertilized 22 November 2006 (19 d before beginning their test). No pesticides were used; pests were controlled by hand removal. Flooding treatments. For each species, six of the twel ve plants in each soil type were flooded by placing them into 19-liter plastic buckets filled with tap water to 10-cm above the soil surface. The other six plants in each soil type were left unflooded as controls. Flood period durations were 23 d for buttonwood and mahogany and 41 d for pond apple and Surinam cherry. Flood durations were determined by the appearance of physiological indicators of plant stress on at least one of the plant speci es tested, such as reduced phot osynthesis and wilting. All trees were irrigated by overhead spri nkler during time intervals wh en plants were not flooded. Buttonwood and mahogany were irrigated by overh ead sprinklers for 30 min twice a day, whereas pond apple and Surinam cherry were irrigated for 30 min once a day until day 103, when irrigation was changed to 30 min twice a day. During flood periods, however, non-flooded trees were manually irrigated with 0.5 liter of water per plant every 2 d, and flooded trees were not irrigated except by maintaining flood water levels as described above. Soil temperature and redox potential. Soil temperature was measured at 1-h intervals throughout the experiment with sensors lo cated 6 cm below the soil surface (StowAway Tidbit temploggers, Onset Co., Pocasset, MA). Soil temp erature was determined using four sensors for buttonwood and mahogany, and three sensors for pond apple and Surinam cherry. Soil redox potential was measured with a platinum combination electrode attached to a portable volt meter 68

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(Accumet #AP62, Fisher Scientif ic, Pittsburgh, PA). Measurements were made by inserting the electrode into a polyvinyl chlori de (PVC) pipe (20 cm long x 22 mm wide) that protruded 4 cm above the soil surface and was pl aced in the soil 2 cm from th e edge of the pot. Soil redox potential was recorded at a mean depth of 6 cm below soil surface and measured in 3 pots (replications) per flooded treatment (soil type), or six plan ts per plant spec ies. Soil redox potential was measured daily for the first 6 or 7 d of flooding, then at intervals of 3 to 8 d, until plants were unflooded. Leaf gas exchange and chlorophyll index. Net CO2 assimilation and stomatal conductance were measured on two fully expanded, recently matured leaves or leaflets (between the fourth and tenth node below the stem) per plant with a CIRAS-2 portable gas analyzer (PP Systems, Amesbury, MA). For net CO2 assimilation and stomatal conductance measurements, the photosynthetic photon flux was maintained at 1000 mol photons m-2 s-1 with a halogen lamp attached to the leaf cuvette, and the reference CO2 concentration into the cuvette was kept constant at 375 mol mol-1 CO2. Mahogany has pinnately compound leaves with 6-8 similarlysized leaflets per leaf, whereas a ll the other plant species tested ha ve simple leaves (Watkins and Sheehan 1975, Wunderlin 1998). For leaf gas exchange measurements, mahogany leaflets were randomly selected from all positi ons on the leaf. For each plant sp ecies, leaf gas exchange of plants in each treatment was initially measured a few days prior to flooding and periodically until a few days after plants were unflooded. The first measurement of leaf gas exchange for buttonwood, mahogany, pond apple, and Surinam che rry was 6 d, 4 d, 6 d, and 4 d, respectively, before flooding began. Leaf gas excha nge measurements for buttonwood, mahogany, pond apple, and Surinam cherry were spaced at intervals of 2-9 d, 2-9 d, 3-11 d, and 4-11 d, respectively, on a separate day for each plant spec ies. Leaf gas exchange was measured 12 times for buttonwood and mahogany and 19 times for pond apple and Surinam cherry. 69

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Plant growth. For plants in each treatment, stem diameter was measured 10 cm above soil surface, and for plants with multiple stems at th is height, diameter of the largest stem was recorded. Plant height was measured from the so il surface to the plant apex. Stem diameter and plant height were measured prior to flooding and again immediately before harvest. For buttonwood, harvest began 84 d after flooding wa s initiated and 61 d after unflooding. For mahogany, harvest began 88 d after flooding was in itiated and 65 d afte r unflooding. For both pond apple and Surinam cherry, harvest began 128 d after flooding was initi ated and 87 d after unflooding. For all plants, roots, stem s, and leaves were oven-dried at 75oC to a constant weight, and dry weights were determined for each plant organ. For buttonwood, pond apple, and Surinam cherry, leaf dry weights included leaf blades and peti oles, but for mahogany, leaf dry weights included leaflets, petiolules, rachises and petioles. Add itionally, the number of inflorescences per plant was determined for but tonwood and the numbers and weights of flowers and fruits (including pedicels) per plant were determined for pond apple and Surinam cherry. Experimental design an d statistical analysis. Each plant species was analyzed separately. For each plant species, the experi mental design was a 2 (soil type) x 2 (flooding treatment) factorial with six single-plant replicati ons per treatment for a total of 24 plants of each species arranged in a completely randomized design. Data were first analyzed by a two-way analysis of variance (ANOVA) to assess flooding and soil type inter actions. In each soil type, flooded versus non-flooded treatments were compar ed using standard T-te st, and within each flooding treatment, effects of soil type were a ssessed by a standard T-test (PROC T-TEST and GLM, SAS Institute, Cary, NC, 2003). Results Range of mean daily soil temperatures was 24.4-30.5oC for buttonwood and mahogany and 10.3-25.0oC for pond apple and Surinam cherry. So il redox potential for green buttonwood 70

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plants flooded in marl soil ranged from +310 mV on the first day of flooding to mV on day 14 (Figure 3-1a), and in potting medium, soil red ox potential varied from +176 mV on the first day of flooding to -234 mV on day 4 (Figure 3-1a). Soil re dox potential for flooded mahogany in marl soil varied from +296 mV on the fi rst day of flooding to mV on day 21 and in potting medium from +221 mV on the first day of flooding to -239 mV on day 4 (Figure 3-1b). For pond apple, soil redox potentials in flooded ma rl soil ranged from +153 mV on the first day of flooding to mV on day 18 (Figure 3-1c) an d in potting medium ranged from +152 mV on the first day of flooding to -163 mV on day 7. So il redox potentials for Surinam cherry in marl soil ranged from +219 mV on the first day of flooding to mV on day 18 and in potting medium ranged from +152 mV on the first day of flooding to -270 mV on day 7 (Figure 3-1d). Leaf gas exchange and chlorophyll index For green buttonwood, there was a significant interaction between floo ding and soil type ( P 0.05) for net CO2 assimilation and stomatal conductance on one or more measurement dates. Net CO2 assimilation was significantly lower for plants in flooded than in non-flooded marl soil (Figure 3-2a) and in flooded marl soil compared to flooded potting medium (Figure 32c) on several measurement dates. For nonflooded buttonwoods, differences in net CO2 assimilation between pottin g medium and marl soil were not significant in 11 of 12 weeks, but in week 10, net CO2 assimilation was significantly higher for plants in marl soil than in potting medium (Figure 3-2d). Green buttonwood in potting medium performed equally well under floode d and non-flooded conditions: there were no significant differences between flooded and non-flooded plants in net CO2 assimilation (Figure 3-2b) or stomatal conductance (Figure 3-3b). For green butto nwood in marl soil, stomatal conductance was significantly lower for flooded than for non-flooded plants on weeks 3-7 (Figure 3-3a). Also, stomatal conductance of flooded green buttonwood wa s significantly lower in marl soil than potting medium in weeks 6 and 7, but significantl y higher in week 8 (Figure 371

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3c). For non-flooded green buttonwood plants, stomatal conductance was significantly lower for potting medium than marl soil treatment combina tions in weeks 3, 4, 8, and 10 (Figure 3-3d). For mahogany there was a significant intera ction between floodi ng and soil type (P 0.05) for net CO2 assimilation and stomatal conductance on one or more measurement dates. Mahogany net CO2 assimilation was consistently lower for flooded plants in marl soil than for either non-flooded plants in marl soil (Figure 34a) or flooded plants in potting medium (Figure 3-4c). These differences in net CO2 assimilation for mahogany were significant between flooded and non-flooded plants in marl soil in weeks 2-8 and 10 and between plants flooded in marl soil and those flooded in potting medium weeks 3-7 (Figure 3-4 a, c). For mahogany in potting medium, net CO2 assimilation was significantly lower for flooded than non-flooded plants only on week 12, and differences were not significant fo r the other 11 of 12 wk (Figure 3-4b). For non-flooded mahogany, there were no significant differences in net CO2 assimilation between plants in potting medium and t hose in marl soil (Figure 3-4d ). Stomatal conductance of mahogany plants in marl soil was significantly lower for flooded than for non-flooded plants in weeks 2-10, but in potting medium, there was no significant difference between flooded and nonflooded plants (Figure 3-5a and b). St omatal conductance of flooded mahogany was significantly lower in marl soil than in potti ng medium in weeks 3-5 and 7, but for non-flooded plants, there were no significant differences in stomatal conductance between marl soil and potting medium (Figure 3-5c and d). For pond apple, there was a significant in teraction between flooding and soil type ( P 0.05) for net CO2 assimilation and stomatal conductance on one or more measurement dates. For pond apple in marl soil, net CO2 assimilation was significantly higher in flooded than in nonflooded plants on weeks 10, 12, and 14 (Figure 3-6a). Net CO2 assimilation of pond apple plants in potting medium was significantly higher fo r flooded than non-flooded plants on weeks 7, 10, 72

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12, and 14-16 (Figure 3-6b). Net CO2 assimilation of pond apple was significantly higher for plants flooded in marl soil than plants flooded in potting medium on weeks 2 and 3 (Figure 36c). Also, net CO2 assimilation for non-flooded pond apple plants was significantly higher for plants in marl soil than plants in potting me dium on weeks 2-5, 7-8, 10, and 15-16 (Figure 3-6d). There were no significant diffe rences in stomatal conductance between flooded and non-flooded pond apple plants in marl soil or between flooded plants in ma rl soil and flooded plants in potting medium (Figure 3-7a and c). Stomatal conductance of pond apple in potting medium was significantly higher for flooded than non-flooded plants on weeks 7, 12, and 14 (Figure 3-7b). Stomatal conductance of non-flooded pond apple wa s significantly higher for marl soil than potting medium in weeks 2 and 5-9 (Figure 3-7d). For Surinam cherry, there was a significan t interaction be tween flooding and soil type ( P 0.05) for net CO2 assimilation and stomatal conductance on one or more measurement dates. Net CO2 assimilation of Surinam cherry was sign ificantly lower for flooded than non-flooded plants in marl soil on weeks 4-12 (Figure 3-8a). For Surinam cherry plants in potting medium, net CO2 assimilation was significantly lower for flooded th an non-flooded plants in weeks 4-11 (Figure 3-8b). Net CO2 assimilation of flooded Surinam che rry was also significantly lower for plants in marl soil than in potting medium on weeks 4-6 and 8-13 (F igure 3-8c). Net CO2 assimilation for non-flooded Surinam cherry was significantly lower for plants in marl soil than in potting medium, but only in weeks 11 and 18, wh ereas differences were insignificant in the remaining 17 of 19 wk (Figure 3-8d). Stomatal conductance of Surinam cherry in marl soil was significantly lower for flooded than non-flooded plants in weeks 512 (Figure 3-9a). In potting medium, stomatal conductance of Surinam cher ry was significantly lower for flooded than nonflooded plants on weeks 8-9 (Fi gure 3-9b). Stomatal conductan ce for flooded Surinam cherry plants was significantly lower in marl soil than potting medium in weeks 6, 8-13, and 17 (Figure 73

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3-9c). For non-flooded Surinam cherry plants, there was no significant difference in stomatal conductance between plants in potting medium and those in marl soil in 18 of 19 measurements; however, on week 11, stomatal cond uctance for plants in marl so il was significantly lower than those in potting medium (Figure 3-9d). Plant growth. For green buttonwood, there was a significant (P 0.05) interaction between flooding and soil type for leaf dry wei ght and plant height. However, there were no significant interactions between fl ooding and soil type for root, st em, and total plant dry weight, stem diameter, or the number of inflorescences per plant. Therefore, dry weights and plant heights were not pooled, whereas stem diameters, and the number of inflorescences were pooled for statistical analyses. For green buttonwood in marl soil, le af dry weight was significantly lower for flooded than non-flooded plants, but th ere were no significan t differences between flooded and non-flooded plants in root, stem, or total dry weights (Figure 3-10a). In potting medium, there were no significant differences in dry weights due to flooding (Figure 3-10b). Green buttonwood root, stem, leaf, and total dry weights were significa ntly lower for flooded plants in marl soil than for flooded plants in pot ting medium (Figure 3-10c). Root stem, leaf, and total dry weights of non-flooded green buttonwood pl ants were significantly lower in marl soil than in potting medium (Figure 3-10d). Stem diameter of green buttonwood was significantly lower for non-flooded than flooded plants with soil types pooled (Figure 3-11a). Buttonwood stem diameter was also significantly lower for pl ants in marl soil than those in potting medium with flooded and non-flooded plants pooled (Figure 3-11b). Hype rtrophic (swollen) lenticels and small numbers of adventitious roots, fewe r than 10 per plant and up to 15 cm long, were often observed on flooded green buttonwood plants in marl soil and/or potting medium. For green buttonwood in marl soil, plant height wa s lower for flooded than for non-flooded plants, but not significantly (Figure 311c). Also, plant height of flooded green buttonwood was 74

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significantly lower in marl soil than in potting medium (Figure 3-11e). There were no significant differences in plant height between fl ooded and non-flooded green buttonwood in potting medium (Figure 3-11d), nor between marl soil and potting medium for non-flooded plants (Figure 3-11f). There were no significant effects of flooding or soil type on the number of inflorescences per buttonwood plant (range 0-170). For mahogany, there was a significant interaction ( P 0.05) between flooding and soil type for leaf dry weight and stem diameter. However, there was no inte raction for root, stem, and total plant dry weight and plant height. Th erefore, mahogany dry wei ghts and stem diameter were not pooled, whereas plant height was pooled for analyses. For mahogany in marl soil, leaf dry weight was significantly lo wer for flooded than for non-flooded plants (Figure 3-12a). Also, leaf dry weight of flooded plants was significantly lower in ma rl soil than in potting medium (Figure 3-12c). There were no significant differences in root, stem, or total dry weights between flooded and non-flooded mahogany plants in marl so il or between marl soil and potting medium for flooded plants (Figure 3-12a and c). There were no signifi cant differences in dry weights between flooded and non-flooded mahogany plants in potting medium (Figure 3-12b), nor between marl soil and potting medium for non-floode d plants (Figure 3-12d). Stem diameter of mahogany in marl soil was signifi cantly lower for flooded than for non-flooded plants (Figure 313a), and for non-flooded plants in potting medium than non-flooded plants in marl soil (Figure 3-13d). There were no significant differences in stem diameter between flooded and non-flooded mahogany plants in potting medium (Figure 3-13b), nor between marl soil and potting medium for flooded plants (Figure 3-13c). There were no significant differences in plant height between flooded and non-flooded mahogany plants (Figure 3-13e), but plant height was significantly lower in marl soil than in po tting medium (Figure 3-13f). 75

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For pond apple, there was no significant flooding and soil type interaction (P 0.05) for root, stem, leaf, or total plant dry weight, infl orescence number or weight, plant height, or stem diameter. Therefore, all these variables were pooled for analyses. Pond apple root, stem, leaf, and total dry weights were significantly greater in marl soil than in potting medium (Figure 314b), but flooding did not significantly affect thes e variables (Figure 3-14 a). Pond apple stem diameter was significantly greate r in flooded than in non-flooded pl ants (Figure 3-15a), but there were no significant differences between marl soil and potting medium (Figure 3-15b). In addition, flooded pond apple plants exhibited swollen stem lenticels. There was no significant effect of flooding or soil type on height of pond apple plants (ra nge -5 to 5.8 cm), on the number of flowers and fruit per plant (ra nge 0-1), or on weight of flowers and fruit per plant (range 00.55 g). For Surinam cherry, there was a significant interaction ( P 0.05) between flooding and soil type for leaf dry weight, stem diameter, plan t height, and flower and fruit weight. However, there were no significant interactions for root, stem, and total plant dry weights and the number of flowers or fruit per plant. Therefore, data for these variables were not pooled for analyses. Stem, leaf, and total dry weights of Surinam ch erry in marl soil were significantly lower for flooded than non-flooded plants, but there was no significant difference in root dry weight (Figure 3-16a). In potting medium, there were no significant differences between flooded and non-flooded Surinam cherry plants in root, stem, leaf, or total dry wei ghts (Figure 3-16b). Flooded Surinam cherry in marl soil had significan tly lower root, stem, leaf, and total plant dry weights than plants in potting me dium (Figure 3-16c). In addition, Surinam cherry root, stem, and total dry weights were significantly lower for non-flooded plants in marl soil than for nonflooded plants in potting medium, but there was no significant difference in leaf dry weight (Figure 3-16d). Surinam cherry stem diameter in marl soil was significantly lower for flooded 76

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for non-flooded plants (Figure 3-17a). Stem diameter of flooded Surinam cherry was significantly lower for plants in marl soil than for plants in potting me dium (Figure 3-17c). There were no significant di fferences in stem diameter between flooded and non-flooded Surinam cherry plants in potting medium (Fi gure 3-17b), nor between marl soil and potting medium for non-flooded plants (Figur e 3-17d). Height of Surinam ch erry plants in marl soil was significantly lower for flooded than for non-flooded plants (Figure 3-18a). Also, height of Surinam cherry was significantly lower for plants flooded in marl soil than for those flooded in potting medium (Figure 3-18c). There were no si gnificant differences in height between flooded and non-flooded Surinam cherry plants in potting medium (Figure 3-18b), nor between marl soil and potting medium for non-flooded plants (F igure 3-18d). There were no significant differences between any pair of treatment combinations in numb er of Surinam cherry flowers and fruit per plant. Also, for Surinam cherry, th ere were no significant diffe rences in flower and fruit weight per plant between flooded and non-flooded plants in marl soil (Figure 3-19a), nor between marl soil and potting medium for non-floode d plants (Figure 3-19d). However, flower and fruit weight of Surinam cherry in potting medium was significantly higher for flooded than for non-flooded plants (Figure 3-19b). In addition, flower and fruit weight for flooded Surinam cherry plants was significantly lower in marl soil than in potting medi um (Figure 3-19c). Discussion The most apparent trend in gas excha nge data for buttonwood, mahogany, and Surinam cherry was for net CO 2 assimilation and stomatal conductanc e to be lower in flooded marl soil than in non-flooded marl soil or flooded potting medium. For buttonwood, mahogany, and Surinam cherry plants, flooding reduced net CO 2 assimilation and stomatal conductance much more in marl soil than in potting medium. Similarly, growth of green buttonwood, mahogany, and Surinam cherry plants was often significantly less for plants in marl soil than plants in 77

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potting medium, especially when flooded. Flooded marl soil apparently provided a much less favorable environment for net CO2 assimilation and stomatal conductance than did either nonflooded marl soil or flooded potting medium for th ese plant species, and this was manifested in reduced gas exchange and growth. A possible reason is reduced oxygen availability in flooded marl soil than in non-flooded marl soil or flooded potting medium. Green buttonwood was one of two species most compromised by planting in marl soil compared to potting medium, especially when marl soil was flooded (the most compromised species was Surinam cherry). This is surpri sing given the native habitat of green buttonwood, which includes tidal swamps of central and south Florida (Wunderlin 1998), and in south Florida, tidal swamps frequently have marl so il. As suggested from its native range, buttonwood is fairly tolerant of flooding, t hough it also thrives in non-floode d, moderately moist soil, which is common for landscape plants. The native habitat of buttonwood frequently has flooding combined with marl soil, but is the environm ent in which buttonwood evolved and should be best adapted. Flooding green buttonwood initially reduced net CO2 assimilation and stomatal conductance much more in marl soil than in pottin g medium. However, this trend was reversed for stomatal conductance beginning 3 wk after unflooding plants, rendering it higher for plants in flooded marl soil than flooded potting medium. Hence, there was evidence of flood-induced compensatory increase in stomatal conductance in green buttonwood. Compensatory increase in leaf gas exchange of stressed plants has been noted with buttonwood in response to larval feeding by Diaprepes root weevil, Diaprepes abbreviatus (L.) (Coleoptera: Curculionidae) (Diaz et al. 2006, Chapter 2). Buttonwood was the only plan t species that increased leaf gas exchange as a compensatory reaction to a stress because of flooding or insects compared to several other plant species tested in this and othe r studies (Diaz et al. 2006, Chapter 2). 78

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Diaz (2005) tested effects of floodi ng and larval infestation on net CO2 assimilation, stomatal conductance, and fresh and dry root weights of green buttonwood and live oak in potting medium. Diaz (2005) found that flooding significantly reduced net CO2 assimilation and stomatal conductance beginning 1 wk after plants were flooded, although flooding did not significantly affect root, stem, or leaf fresh or dry weights. For green buttonwood in the present study, net CO2 assimilation and stomatal conductance we re not significantly different between flooded and non-flooded plants in potting medium. Minimal differences were also found in the present study between flooded and non-flooded green buttonwood in potting medium in dry weights, plant heights, and number of inflores cences per plant. Results of green buttonwood gas exchange and growth in potting medium in the present study generally ag ree with growth data from Diaz (2005) suggesting the plant grows equally well in flooded and non-flooded potting medium in the time periods of the studies. Ho wever, these results also suggest that green buttonwood in potting medium varied somewhat in response to flooding: Diaz (2005) found that flooding reduced green buttonwood gas exchange, and in the present study and flooding increased plant trunk diameters. Although green buttonwood in potting medium varied in its response to flooding, it generally performed as well in flooded as in non-flooded potting medium in gas exchange and growth. In the present study, pond apple plants developed swollen stems and hypertrophic lenticels in response to flooding. Hypertrophic lenticels have been shown to benefit flooded plants in two ways: 1) they exchange gases in flood water through breaks in the closing layers (Hook et al. 1970, Hook 1984); and 2) they releas e potentially toxic compounds from plants, such as acetaldehyde, ethylene, and ethanol (Chirkova and Gutman 1972). Often, swollen stems of flooded plants indicate increased aerenc hyma, which can facilitate movement of O2 from shoots to submerged roots. Because swollen st em lenticels and aerenchyma tissue presumably 79

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increase the stem girth, a greater stem diameter increase in flooded than in non-flooded soils may indicate adaptation to flooding. Nez-Elisea et al. (1999) tested effects of flooding on net CO2 assimilation and growth of pond apple plants, two other Annona sp. seedlings, and four scion/ rootstock combinations in Krome gravelly soil and potting medium. Marl soil used in the present study and Krome gravelly soil used by Nez-Elisea et al. (1999) ar e similar in pH (7.4-8.4) and both have a very calcium-rich composition, but marl soil is more poorly drained than Kr ome gravelly soil (Li 2001). Nez-Elisea et al. (1999) found that A. squamosa L. and A. reticulata L. did not tolerate flooding as rootstocks or seedlings, but when grafted onto pond apple, A. reticulata and two other scions tolerated flooding. In the present study, pond appl e was very tolerant to soil flooding. Nez-Elisea et al. (1999) found that flooded trees, esp ecially pond apple, developed hypertrophic lenticels and thicker stems that were caused by enlarged xylem cells, but with reduced xylem density. This concurs with obser vations in the present study that flooded pond apple developed hypertrophic lenticels and signifi cantly thicker stems under flooded conditions. Ojeda et al. (2004) investig ated effects of root-zone temperature and flooding on the physiology and growth of pond apple and soursop. Both plant species had roots in temperaturecontrolled chambers with canopies exposed to ambient conditions in a sunny greenhouse with 6 wk flooding for flooded treatments (Oje da et al. 2004). Both studies were similar in their use of a potting medium and their duration of flood peri od. Pond apple was more flood-tolerant than soursop, and only trees with morphological ad aptations such as enlarged trunk bases, hypertrophic stem lenticels, and adventitious roots survived continuous flooding (Ojeda et al. 2004). Overall, the present study, Nez-Elisea et al. (1999), and Ojeda et al. (2004) all agree that pond apple exhibits good fl ood tolerance by developing morphological adaptations in response to flooding, such as thicker trunks and hypertrophic lenticels. 80

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Green buttonwood and pond apple developed si gnificantly larger stem diameters when flooded than when not flooded, and hypertrophic st em lenticels were conspicuous under flooded conditions for both species. For green buttonw ood, more adventitious roots were observed on flooded than non-flooded plants. These data sugg est that green buttonwood and pond apple were flood-adapted by increasing stem diameters and numbers of swollen st em lenticels and/or adventitious roots in response to flooding. Pond apple is well ad apted to flooding and marl soil (Schaffer 1998, Nez-Elisea et al. 1999, Ojeda et al. 2004) and is potentia lly graft-compatible with commercial Annona spp. grown for fruit if compatible interstocks are used (Nez-Elisea et al. 1999). Therefore, pond apple has poten tial as a rootstoc k for commercial Annona species in poorly drained soil, particularly in calcareous soils of south Florida (Schaffer et al., 2006). Mahogany gas exchange and growth were si milar to those of buttonwood and Surinam cherry with lower values in flooded marl soil th an in either non-flooded marl soil or flooded potting medium. Mahogany net CO2 assimilation, stomatal conductance, dry weight, and plant height tended to respond better in potting medium than in marl soil, especially when flooded. However, mahogany stem diameter increase was significantly greater in non-flooded marl soil than in either flooded marl soil or non-fl ooded potting medium, and trees did not develop adventitious roots. Hence, mahogany did not show adaptation to flooding, which may reflect its native habit of coastal hammocks (Wunderlin 199 8), which are typically not flooded and may lack marl soil (Li 2001). The trend for Surinam cherry gas exchange to be significantly great er in non-flooded than flooded potting medium contrasts with bu ttonwood and mahogany which had minimal differences between these treatment combinations. Hence, Surinam cherry seems to be the least flood-tolerant plant species test ed in this study. Similar to mahogany, Surinam cherry did not show adaptation to flooding as shown by the ab sence of adventitious roots when flooded and 81

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significantly larger stem diamet ers in non-flooded marl than in flooded marl soil. This may reflect the habitat in which Surinam cherry is typically found when not cultivated, disturbed hammocks, which are typically non-flooded an d hence do not have marl soil (Wunderlin 1998, Li 2001). Based on leaf gas exchange, growth, a nd signs of morphological adaptation to flooding of the species tested, the order of flood tolerance to marl soil was as follows: pond apple showed the highest, followed by buttonwood, then mahoga ny, and then Surinam cherry; and in the potting media used in this study, the order wa s pond apple and buttonw ood shared the highest flood tolerance followed by mahogany, and then Surinam cherry. 82

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Figure 3-1. Soil redox potential. A) green buttonwood B) mahogany C) pond apple and D) Surinam cherry. Each point represents the mean SD of three measurements per measurement date for each flooded treatme nt combination per plant species. 83

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Figure 3-2. Effects of flooding and soil type on net CO2 assimilation ( A ) of green buttonwood plants. Symbols represent means. Asteri sks indicate significant differences between treatments at P 0.05, ** P < 0.01, *** P < 0.001, and NS non-significant according to a standard T-test. 84

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Figure 3-3. Effects of flooding and soil type on stomatal conductance ( gs) of green buttonwood plants. Symbols represent means. Asteri sks indicate significant differences between treatments at P 0.05, ** P < 0.01, *** P < 0.001, and NS non-significant according to a standard T-test. 85

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Figure 3-4. Effects of flooding and soil type on net CO2 assimilation ( A ) of mahogany plants. Symbols represent means. Asterisks i ndicate significant differences between treatments at P 0.05, ** P < 0.01, *** P < 0.001, and NS non-significant according to a standard T-test. 86

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Figure 3-5. Effects of flooding and soil type on stomatal conductance ( gs) of mahogany plants. Symbols represent means. Asterisks i ndicate significant differences between treatments at P 0.05, ** P < 0.01, *** P < 0.001, and NS non-significant according to a standard T-test. 87

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Figure 3-6. Effects of flooding and soil type on net CO2 assimilation ( A ) of pond apple plants. Symbols represent means. Asterisks i ndicate significant differences between treatments at P 0.05, ** P < 0.01, *** P < 0.001, and NS non-significant according to a standard T-test. 88

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Figure 3-7. Effects of flooding and soil type on stomatal conductance ( gs) of pond apple plants. Symbols represent means. Asterisks i ndicate significant differences between treatments at P 0.05, ** P < 0.01, *** P < 0.001, and NS non-significant according to a standard T-test. 89

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Figure 3-8. Effects of flooding and soil type on net CO2 assimilation ( A ) of Surinam cherry plants. Symbols represent means. Asteri sks indicate significant differences between treatments at P 0.05, ** P < 0.01, *** P < 0.001, and NS non-significant according to a standard T-test. 90

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Figure 3-9. Effects of flooding and soil type on stomatal conductance ( gs) of Surinam cherry plants. Symbols represent means. Asteri sks indicate significant differences between treatments at P 0.05, ** P < 0.01, *** P < 0.001, and NS non-significant according to a standard T-test. 91

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Figure 3-10. Effects of flooding and soil type on dry weights of green buttonwood plants. Bars represent means. Asterisks indicate signi ficant differences between treatments at P 0.05, ** P < 0.01, *** P < 0.001, and NS non-significant a ccording to a standard T-test. 92

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Figure 3-11. Effects of flooding a nd soil type on (A, B) increase in stem diameter and (C, D, E, and F) increase in plant height for green buttonwood plants. Bars represent means. Asterisks indicate significant diffe rences between treatments at P 0.05, ** P < 0.01, *** P < 0.001, and NS non-significant according to a standard T-test. 93

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Figure 3-12. Effects of flooding and soil type on dry weights of mahogany plants. Bars represent means. Asterisks indicate signi ficant differences between treatments at P 0.05, ** P < 0.01, *** P < 0.001, and NS non-significant a ccording to a standard T-test. 94

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Figure 3-13. Effects of flooding and soil type on (A-D) increase in stem diameter and (E-F) increase in plant height for mahogany plan ts. Bars represent means. Asterisks indicate significant differences between treatments at P 0.05, ** P < 0.01, *** P < 0.001, and NS non-significant according to a standard T-test. 95

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Figure 3-14. Effects of flooding and soil type on dry weights of pond apple plants. Bars represent means. Asterisks indicate signi ficant differences between treatments at P 0.05, ** P < 0.01, *** P < 0.001, and NS non-significant a ccording to a standard T-test. Figure 3-15. Effects of flooding and soil type on increase in stem diameter for pond apple plants. Bars represent means. Asterisk s indicate significant differences between treatments at P 0.05, ** P < 0.01, *** P < 0.001, and NS non-significant according to a standard T-test. 96

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Figure 3-16. Effects of flooding and soil type on dry weights of Surinam cherry plants. Bars represent means. Asterisks indicate signi ficant differences between treatments at P 0.05, ** P < 0.01, *** P < 0.001, and NS non-significant a ccording to a standard T-test. 97

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Figure 3-17. Effects of flooding a nd soil type on increase in stem diameter for Surinam cherry plants. Bars represent means. Asterisk s indicate significant differences between treatments at P 0.05, ** P < 0.01, *** P < 0.001, and NS non-significant according to a standard T-test. 98

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Figure 3-18. Effects of flooding and soil type on increase in plant height for Surinam cherry plants. Bars represent means. Asterisk s indicate significant differences between treatments at P 0.05, ** P < 0.01, *** P < 0.001, and NS non-significant according to a standard T-test. 99

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Figure 3-19. Effects of flooding and soil type on the number and weight of inflorescences per Surinam cherry plant. Bars represent means. Asterisks indicate significant differences between treatments at P 0.05, ** P < 0.01, *** P < 0.001, and NS non-significant according to a standard T-test. 100

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CHAPTER 4 SURVIVAL OF DIAPREPES ABBREVIATUS (COLEOPTERA: CURCULIONIDAE) LARVAE ON GREEN BUTTONWOOD TREES IN FLOODED MARL SOIL OR POTTING MEDIUM. Introduction Diaprepes root weevil, Diaprepes abbreviatus (L.) (Coleoptera: Curculionidae), is a serious pest of citrus and sugarcane in its home range of Puerto Rico (Woodruff 1964). Since its introduction to Florida, it has been a continuous problem in citrus (S tanley 1996). Diaprepes root weevil also feeds on a wide host range of ornamental plants and grasses (Simpson et al. 1996) which also commonly provide an avenue of spreading this pest to new locations with the movement of infested plants. Management of th is pest on ornamentals is necessary to reduce the risk of spreading this pest to new areas. Cu rrently, it has been reported in Texas (Knapp et al. 2001, Skaria and French 2001) and sout hern California (Klunk 2005). Flooding is sometimes used in southern Florida sugarcane fields to control various pests (Cherry 1984; Hall and Cherry 1993). It has been suggested that flooding may also be useful for controlling Diaprepes root weevil larvae in sugarcane fields (Ha ll and Cherry 1993, Shapiro et al. 1997). Lapointe and Shapiro (1999) examined the effects of soil moisture on Diaprepes root weevil development and survival and found that th e poorest larval survival would be expected under flooded conditions. Waterlog ged soils are typically denser than non-flooded soils (Saqib et al. 2004), which is a potential problem for surv ival of larvae in flooded soil (Li et al. 2006). On the other hand, flooding of the root zone ma y exacerbate the effects of root feeding by Diaprepes root weevil larvae. Li et al. (2003a) not only found surv ival of Diaprepes root weevil larvae was significantly higher in previously flooded soil than in non-flooded soil, but flooddamaged seedlings were more susceptible to larv al feeding injury than non-flooded seedlings. Similarly, Li et al. (2006) invest igated the effects of flooding and soil type on larval survival and 101

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showed that larval survival was higher in sandy soil than in loam soil. Plants flooded for at least 20 d were more stressed and more prone to Diap repes root weevil larval feeding injury after removal of plants from flooding than non-fl ooded control plants (L i et al. 2006). The objective of this study was to determine th e survival of Diaprepes root weevil larvae in flooded marl soils and in a flooded nursery potting medium w ith green buttonwood ( Conocarpus erectus L ., Combretaceae) serving as a food source. Green buttonwood is widely grown as an ornamental tree or shrub in sout hern Florida and is native to th e tidal swamps of central and south Florida (Watkins and Sheehan 1975, Wund erlin 1998). Green buttonwood has also been reported as a host of both adult and larval Diaprepes root weevil (Mannion et al. 2003). Materials and Methods The experiment was conducted in the winter and spring of 2007 in Homestead, FL, using Diaprepes-infested green buttonwood trees in 4-lite r containers filled with either marl soil or potting medium in an outdoor, open site. Plants were obtained from a commercial nursery in December 2006 and replanted (12 January 2007). At the time treatments were initiated, buttonwood plants were approximately 6-12 mo old. Each plant was repotted into a 4-liter plastic cont ainer with half the plants (16 out of 32) in a nursery potting medium (40% Florida peat, 30 % pine bark, 20% cypress sawdust, and 10% sand) and the other half (16 plants) in marl soil. The marl soil was obtained from a fallow agricultural field (Homestead, FL ). Plants were fertilized (13 February 2007) 10 d before beginning the experiment with liquid fertilizer (Miracle-Gro 15-30-15, Sterns Miracle-Gro Products, Port Washington, NY) at the manufacturer s recommended rate. Insect pests other than Diaprepes root weevil were removed manually. A to tal of 24 plants were us ed in this study with two soil treatments (potting medium and marl soil) and two flood treatments (flooded and nonflooded) arranged in a 2 x 2 factorial design with si x single-plant replicati ons per treatment. An 102

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additional eight monitoring plants (four flooded plants in each so il type) were used for periodic destructive harvest to assess larval survival on one plant in each soil type at each assessment time. The monitoring plants helped determine when to remove plants from flooding and when to harvest and evaluate plants. The monitoring plants were not included in the statistical analysis. Larval infestation. Six weeks after repotting (23 Fe bruary 2007), each container was infested with 15 Diaprepes root weevil larvae ra ised on an artificial diet and supplied by the Florida Division of Plant Industry, Gainesville. Larvae used to in fest plants were fourth through sixth instars (mean fifth instar) based on head capsule width (Qui ntela et al. 1998). Larvae were placed individually into each of 15 holes in the so il, 3-5 cm deep, 4-8 cm from the stem, and 2.5 cm apart, which were then recapped with soil. All containers remained non-flooded for 16 d to allow larvae to become established. Flooding. On day 17 of the experiment (11 March 2007), one-half the number of plants in each soil type (marl soil or potting medium) were flooded by placing the container with each plant into a larger 19-liter plastic container filled with tap water with the level maintained 10 cm above soil surface (24 cm total depth). There were six flooded plants and six non-flooded (control plants) for each soil type One flooded, monitoring plant in each soil type was evaluated after 3, 6, 9, and 23 d to determine when to evalua te the test plants based on the number and size of live larvae found in the soil of the monitoring plants. Test plants in each treatment were harvested when less than 30% of the 15 larvae or iginally added per mon itoring plant were found alive in both soil types (after 38 d of flooding). Non-flooded plants were irrigated by overhead sprinkler 30 min once a day until day 29 (23 Marc h), when irrigation times were changed to 30 min twice a day. Flooded plants were irrigated only when not flooded. Data collection. Data collected included soil temperatur e, numbers of live and dead larvae recovered per plant, and larval head capsule widths. Soil temperature was recorded at 1 h 103

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intervals throughout the experiment with sensors (StowAway Tidbit temploggers, Onset Co., Pocasset, MA). The sensors were placed in th e soil of three non-flooded plants not included in the experiment but held under the same experiment al conditions. Sensors were located at a soil depth of 6 cm two-thirds the distance from the center to the outer edge of the pot. When plants were unflooded and harvested, root s were removed from the soil, and the soil was placed into bins and carefully inspected for la rvae. The number of live and dead larvae were then determined for each plant container and preserved in separate vials of 75% ethanol. Head capsule widths were measured in the laborat ory using a microscope micrometer with 50 micrometer units per mm and 80 x magnification. All data for percentage of larvae found alive were based on live/total ratios, a nd not live/found or found/total, be cause of the presumption that the larvae not found were dead and decomposed. Statistical analyses. A two-way analysis of variance (ANOVA) was used to determine flooding and soil type interaction in a factorial design for percentages of larvae found alive. However, because no larvae were recovered from one treatment combination (flooded potting medium), data for head capsule widths we re analyzed with a one-way ANOVA with three treatments followed by a Duncan -Waller K-ratio test. For pe rcentage of larvae surviving, proportional data based on ratios of live/total were arcsine transformed before analyses by standard T-tests. All statistical analyses were performed with SAS Statistical Software Version 9.1 (PROC T-TEST and GLM, SAS Institute, Cary, NC, 2003). Results and Discussion For plants used for monitoring purposes only, mean percentages of live larvae found in the flooded marl and the flooded potting medium, respectively were 47 and 20 on flood day 4; 87 and 7 on flood day 7; 60 and 7 on flood day 10; and 27 and 0 on flood day 24. The latter sample on day 24 was the first day for which survivorsh ip was less than 30% in both soil types. 104

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Therefore, all flooded test plants were harv ested 2 wk after that date, 38 d after flooding treatments commenced, and no pupae or adults were found in either monitori ng or test plants. For test plants, there was no significant inte raction between effects of flooding and soil type on the numbers of live/total larvae ( F = 3.98; df = 3, 23; P = 0.06). However, because this interaction was nearly significant, data were no t pooled for determination of the percentages of larvae that survived (Figure 4-1). The mean per centage survival out of 15 original larvae per container was significantly lower in flooded pot ting medium than in flooded marl soil ( t = 4.58, df = 5, P = 0.006) (Figure 4-1c). In non-flooded soil, there were no signifi cant effects of soil type on percentages of live larvae recovered (F igure 4-1d). Flooding significantly reduced the mean percentage of larvae surviving comp ared to non-flooded conditions in marl soil ( t = -5.45, df = 9, P = 0.0004) (Figure 4-1a) and in potting medium ( t = -6.36, df = 5, P = 0.0014) (Figure 4-1b). There were significant differences in head capsule widths between treatments ( F = 37.3; df = 2, 17; P < 0.0001). Mean head capsule widths were significantly smaller for larvae in flooded marl soil than for larvae in non-flooded marl so il or non-flooded potting me dium (Figure 4-2). Larval head capsule widths from non-floode d marl and non-flooded potting medium were statistically the same and averag ed eighth instar, whereas flooded marl larvae averaged sixth to seventh instar. Lapointe (2000b) examined the effects of cons tant temperatures on Diaprepes root weevil larval survival and rates of development on an artificial diet. The highest survival rates occurred at 22 and 26oC with lowest survival at 30oC, and the highest deve lopment rate was at 26oC with slower rates at 22 and 30oC (Lapointe 2000b). Mean daily soil temperatures during the treatment period of the present study ranged from 16 to 25oC with monthly averages 17.9 to 21.7oC (Figure 4-3). Thus, average monthly soil temperatures for the present study were 4.3-8.1oC less than 105

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26oC (ideal developmental temperature) and 0.3-4.1oC less than 22oC (ideal survival temperature). Although rates of larval survival may have been close to their maximum rates in the present study, larval development rates we re probably slower than their maximum. The present study focused on larval growth and survival and did not examine flooding and herbivory effects on biomass such as fresh and dry root, stem, and leaf weights, stem diameter, and plant height. However, larval herbivory tends to significantly reduce biomass and gas exchange of buttonwood in potting medium (Diaz 2005, Diaz et al. 2006, Chapter 2). In addition, flooding buttonwoods in potting medi um significantly reduced photosynthesis, transpiration and stomatal conductance beginning 1 wk after flooding (Diaz 2005). In another study with buttonwood, flooding did not cause significant differences in photosynthesis, stomatal conductance, or dry weights of plants grown in potting medium (Chapter 3). However, when grown in marl soil, flooding significantly reduced photosynthesis, stomatal conductance, and leaf dry weight compared to non-fl ooded plants. However, the native tidal-swamp habitat of buttonwood is frequently flooded and on marl soil, but is the environment in which buttonwood evolved and should be best adapted. Comparing Diaprepes root weevil larval surviv al in flooded marl soil with flooded potting medium was difficult because of the high proportion of larvae not recovered. However, this was expected because larvae quickly decompose when they die. Survival of Diaprepes root weevil larvae in flooded marl soil was much higher than its survival in flooded potting medium. In fields of marl soil with mixed nursery stock including flood-sensitive and flood tolerant plants, flooding is probably not a good means to control Diaprepes root weevil larvae because of possible harm to flood-sensitive plants like Surinam cherry ( Eugenia uniflora L., Myrtaceae). For plants grown in a potting medium similar to ours, results of our study suggest root-zone flooding of at least 3 d will help control Diaprepes root weev il larvae in flood-tolerant to 106

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moderately flood-sensitive plant species. This regi me is especially suggested for plants tolerant or moderately tolerant to fl ooding. However, it may not be pr actical to flood plants in containers. When not flooded, soil type such as marl soil or potting medium did not affect Diaprepes root weevil larval surviv al or growth (ie. head capsule widths or instars) during this 54-d experiment. However, when flooded, soil type did signifi cantly affect percent larval survival. In addition, larvae recovered from flooded marl soil had significantly smaller head capsule widths, which indicates they averaged at least one instar smalle r than larvae from nonflooded marl or non-flooded potting medium. Re duced oxygen concentration in flooded soil may have reduced larval respiration and decr eased survival and size of larvae from flooded compared to non-flooded soil of either soil type. Larval survival and growth seem to be more affected by flooding than by soil type, although both treatment e ffects may cause significant differences in larval survival. Flooding is sometimes used in southern Florida sugarcane fields to control pests such as grubs of Tomarus subtropicus (Blatchley) (Coleoptera: S carabaeidae) (Cherry 1984) and wireworm larvae Melanotus communis (Gyllenhal) (Coleoptera: El ateridae) (Hall and Cherry 1993). Shapiro et al. (1997) expos ed Diaprepes root weevil larvae to flooding to test the effects of varying temperature (18, 21, 24, and 27oC) and flood periods (0, 1, 2, 3, 4, or 5 wk) on larval mortality. Mean mortality exceeded 90% by 3 wk at 24 and 27oC and by 5 wk at 21oC, but was only 46% after 5 wk at 18oC (Shapiro et al. 1997). In addi tion, soil pH increas ed significantly with time and mortality (Shapiro et al. 1997). Li et al. (2004 and 2007b) found that pH increased with increasing flood period, which is related to the reduced oxygen content of flooded soil, and not necessarily the Diaprepes root weevil larvae used. In other studies, wireworm larvae ( M. communis) had 80% mortality after 6 wk of submergence at 27oC (Hall and Cherry 1993), whereas scarab grubs ( T. subtropicus) had 100% mortality after ~1 wk (5-10 d) of submergence 107

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(Cherry 1984). Mortality may have been caused by drowning (suffocation) from a lack of oxygen and surplus carbon dioxide, or by sepsis, fr om a buildup of microbe s in stagnant water and larval cadavers (Shapiro et al. 1997). Floodi ng may be useful for controlling Diaprepes root weevil larvae in sugarcane fields, although onl y in the summer and fall when floodwater temperatures are close to their maximum (27oC) (Hall and Cherry 1993, Shapiro et al. 1997). Lapointe and Shapiro (1999) trie d to determine levels of soil moisture that optimized production of Diaprepes root weev il adults in the laboratory. Optimal survival to pupation occurred at 30-70% soil moisture (Lapoint e and Shapiro 1999). About 60-65% of larvae survived to pupation under these ideal moisture conditions (Lapointe an d Shapiro 1999). The poorest survival of larvae occurred in low (20%) and in high (80%) soil moisture levels (Lapointe and Shapiro 1999). T hus, poorest larval survival w ould be expected under flooded conditions, which presumably have over 80% moisture levels, whereas un-flooded plants may have soil moisture levels more fa vorable to larval survival, 30-70%. In the present study and Shapiros et al. (1997) study, larvae of Diaprepes root weevil were exposed directly to flooding. However, Li et al. (2003a, 2006, 2007b) and Diaz (2005) unflooded plants before infestation with larv ae. Although, Diazs (2005) study and the present study were both conducted in Homestead, FL, Diaz (2005) infested soil w ith 40 fifth to sixth instar larvae per container in two infestations of 20 larvae each that were 1 mo apart. Also Diaz unflooded plants 1 d before they were initially infested with la rvae so both stresses were not simultaneous. There were no significant differen ces in larval recovery between previously flooded and non-flooded buttonwoods or live oaks (D iaz 2005). This lack of difference may reflect similar soil moisture contents between previously flooded and non-flooded plants during larval infestation. 108

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Li et al. (2003a, 2006, 2007b) also examined th e effects of Diaprepes root weevil larval infestation and flooding on plant growth, larval survival, and other characteristics in a greenhouse environment. Flooding also occurred before larval infe station so both stresses were not simultaneous (Li et al. 2003a, 2006, 2007b). Th ey used seedlings of Swingle citrumelo ( Citrus paradisi Macf. x Poncirus trifoliata (L.) Raf.) and one other ci trus rootstock. Pot sizes were small (5 cm3) as were plants (seedlings) and sizes a nd numbers of larvae (neonates, 0 or 5 per pot). They also had similar flood periods (0-40 d), larval feeding pe riods (40-56 d), and pots in trays were flooded to 3 cm above soil line. Fo r Li et al. (2003a), surv ival of Diaprepes root weevil larvae was significantly higher in previ ously flooded soil than in non-flooded soil, and flood-damaged seedlings were more susceptibl e to larval feeding injury than non-flooded seedlings. Similarly, Li et al (2006) found that larval survival averaged 25% higher in sandy soil than in loam soil in plants previously flooded for 20 d. Soil type affects larval growth and survival rates, and the effects of soil type on larval survival may be chiefly based on physical characte ristics of the soil which affect soil moisture and oxygen levels (Rogers et al. 2000). Soil pH also increases with flood duration and could adversely affect larval survival (Shapiro et al 1997, Li et al. 2006). Waterlogged soils are also typically denser than non-floode d soils (Saqib et al. 2004), wh ich is a potential problem for survival of larvae in flooded soil (L i et al. 2006). Li et al. (2006) found that plants flooded for at least 20 d were more stressed and more prone to Diaprepes root w eevil larval feeding injury after removal of plants from flooding than non-flooded control plants. Their results suggest that avoidance of flooding and early control of Diap repes root weevil larvae may help protect young plants. Li et al. (2007b) studied the effects of flooding and soil pH on the growth and survival of Diaprepes root weevil larvae. When not lime d, flooding increased the average soil pH up to 0.3 units for the longest flooded (40d) treatment (Li et al. 2007b). In another study by Li et al. 109

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(2007b), citrus seedlings flooded 40 d had the lowest larval survival rates and larval weights compared to seedlings flooded for shorter flood du rations, which may reflect higher soil pH at longer flood durations. Here, la rval survival and growth were significantly decreased by preapplied flooding (Li et al. 2007b). When the so il was limed to pH 4.8-5.7, larval survival was highest at pH 5.0 for no n-flooded plants. Larval survival and weight gain were significantly correlated with pH; increasing pH from 4.8 to 5.7 decreased larval surviv al and increasing pH from 5.1 to 5.7 significantly decreased larval weight s (Li et al. 2007b). Other factors such as soil type, compaction, bulk density, and water content may also influence larval survival and growth (Riis and Esbjerg 1998, Rogers et al. 2000, Li et al. 2007b). Increasing the soil pH by at least 1 unit in acidic soils was recommended for optimum citrus growth, which occurs at pH 6.0-6.5, and to help control Diaprepes root weevil (Li et al. 2007b). Fl ooding was also recommended as a possible control method in citrus (Li et al. 2007b). Flooding may hence reduce larval survival while plants are flooded. Howeve r, depending on soil pH, water-s tressed plants may be more susceptible to Diaprepes root w eevil larval feeding when un-floode d than non-stressed plants that were either never flooded or flood-to lerant and previously flooded. As noted, marl soil native to south Florida has pH of 7.4-8.4 (Li 2001), whereas the potting medium in the present study had a pH of 6.0. As suggested above by Li et al. (2007b), increasing the soil pH from 4.8 to 5.7 decreases larval survival and/or wei ght. Thus, a pH of 6.0 would appear less favorable than 5.0 for Diaprepes r oot weevil survival. Soil pH in the range of marl soil was not investigated in the foregoing studies. Perhaps marl soil offers a pH range more favorable to larval survival and growth than our potting medium. This may help account for higher larval survival rates in flooded marl soil than in flooded potting medium found in the present study. However, this difference in su rvival was not present between non-flooded marl soil and non-flooded potting medium. There is a need to investigate possible survival 110

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advantages to Diaprepes root weevil larvae in th e pH range of marl soil (7.4-8.4) compared to their survival in lower pH (4.8-6.0) of potting medium in the present study and of Florida sandy loam soil used by Li et al. (2007b). 111

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Figure 4-1. Effects of flooding a nd soil type on percentages of 15 Diaprepes root weevil larvae added to each container that were found live at harvest based on rati os of live/total. Bars represent means SD. Asterisks i ndicate significant differences between treatments at P 0.05, ** P < 0.01, and *** P < 0.001 according to a standard Ttest. 112

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Figure 4-2. Mean head capsule widths and inst ars ( SD) of Diaprepe s root weevil larvae found at harvest. Values for live and dead larv ae were pooled. N is the total number of larvae found in each treatment. X-axis sy mbols are Marl-NF (non-flooded marl soil), Marl-FL (flooded marl soil), and PotMed-NF (non-flooded potting medium). Figure 4-3. Soil temperature dur ing the experiment. Each point is the average of three temperature sensors each buried 6 cm below the soil surface in potted plants not used in the experiment but held under the same environmental conditions and with the same media as in the experiment. 113

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CHAPTER 5 EFFECTS OF HERBIVORY BY DIAPREPES ABBREVIATUS (L.) (COLEOPTERA: CURCULIONIDAE) LARVAE AND FLO ODING ON LEAF GAS EXCHANGE AND GROWTH OF GREEN BUTTONWOOD AN D SWINGLE CITRUMELO PLANTS Introduction Diaprepes abbreviatus L. (Coleoptera: Curculionid ae: Entiminae), commonly called Diaprepes root weevil, is an important and economi c pest of citrus and ot her horticultural crops (Simpson et al. 1996). In the United States, it is found in Florida, Texas (Knapp et al. 2001, Skaria and French 2001), and southern Californi a (Klunk 2005). In Florida, Diaprepes root weevil is found in 23 counties in the south and central parts of the state (Anonymous 1996, Pea 1997, Weissling et al. 2004). Diaprepes root weevil is a problematic pest due to its very large host range, which includes at least 317 varieties, 280 species, 180 genera, and 68 families of plants (Simpson et al. 1996, 2000, Knapp et al. 2000b, Mannion et al. 2003, Godfre y et al. 2006). Not all host plants support all stages of Diaprepes root weevil, however many economically important plants support all stages of the weevil from egg to adult, such as sweet potato, Ipomoea batatas (L.) Lam., and (green) buttonwood ( Conocarpus erectus L .) (Simpson et al. 1996). In a survey of ornamental plant nurseries in southern Florida, egg ma sses, feeding damage, and adult weevils were common (Mannion et al. 2003). Di aprepes root weevil larvae feed on the roots of their host plants starting with small roots when they are y oung and move to larger, la teral and main roots as they mature (McCoy et al. 2002). Roots may be girdled causing severe root damage or death which impacts the ability of the plant to take up nutri ents (McCoy et al. 2 002). This type of damage often kills small citrus trees (Wolcott 1936, 1948; Quintela et al. 1998). Tropical agriculture in southern Florida, pa rticularly between Bis cayne and Everglades National Parks, tends to occur in low-lying ar eas with high water tabl es, which are prone to 114

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periodic flooding (Schaffer 1998). Many of the woody ornamental plant nurseries which include green buttonwood are grown in pots in a standard potting medium or in the field in these floodprone areas. Green buttonwood is native to th e tidal swamps of central and south Florida (Watkins and Sheehan 1975, Wunderlin 1998) so it tolerates flooding well, though it also thrives in non-flooded, moderately moist soils, which are common for landscape plants. Considerable research has examined combina tions of Diaprepes root weevil infestation, rootstock variety, soil flooding, so il type, and pH effects on leaf gas exchange and growth of citrus rootstocks and/or Di aprepes root weevil larval su rvival (Li et al. 2003a, 2004, 2006, 2007b). However, there is no published research on interactions between Diaprepes root weevil larval feeding and soil flooding that simultane ously occurs on woody ornamental plants including buttonwood. At least half the Rutaceous fruit trees in commerce in Florida are grown on Swingle citrumelo or Swingle ( Poncirus trifoliata (L.) Raf. x Citrus paradisi Macf.) (Rutaceae) rootstock (Auscitrus 2004, F. Davies pers. comm. 2008). Swingle has moderate to low flood tolerance, though it may be more fl ood-tolerant than another popular rootstock, Carrizo citrange ( Poncirus trifoliata (L.) Raf. x Citrus sinensis (L.) Osb., Rutaceae) (Auscitrus 2004, Li et al. 2004). The objectives of this study were to determine interactions and effects of short-term (continuous) and cyclically flooded soil on damage from Diaprepes root weevil larvae to green buttonwood and Swingle citrumelo plants. An additional objective was to compare effects on green buttonwood with those of Swingle citrumel o, a species known to be sensitive to flooding and larval infestation. The hypothesis was that leaf gas exchange, growth, and Diaprepes root weevil larval survival rates are lowered more by short-term than by cyclical flooding and that flooding exacerbates damage cause d by Diaprepes root weevil. 115

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Materials and Methods The experiment was conducted in Fall 2008 in Homestead, FL with plants in 11-liter plastic containers placed on ground cloth in an out door site exposed to full sun. The two plant species used in these studies were green buttonwood and Swingle citrumelo. Plant material. Green buttonwood and Swingle citrumelo trees (obtained from a commercial nursery) were approximately 2 ye ars old and 1 year old, respectively, when treatments were initiated. Initial plant height s for green buttonwood 6 d before infestation in short-term flooded and cyclically flooded trea tments, respectively, were 126 13 cm and 122 11 cm (mean SD). For Swingle citrumelo, in itial plant heights for short-term flooded and cyclically flooded treatments, respectively, were 141 17 cm and 132 14 cm. The potting medium for green buttonwood and Swingle citr umelo was Fafard mix 2 (70% Canadian peatmoss (porous, fluffy), 20% perlite, and 10% vermiculite). Flooding treatments. For each plant species, plants were exposed to short-term or cyclical flooding by submerging 11-L containers with the plants into 19-L buckets filled with tap water with water levels maintained at 10 cm above the soil surface. Cyclical flooding involved alternating periods of flooding with dry periods. For plants in short-te rm or cyclical flooding treatments, one-half of the total number of plants were left unflooded as controls. Thus, for each test with short-term or cyclical flooding, there were two test plants per replication that were flooded (one infested and one noninfested) and two test plants per replication that were nonflooded (one infested and the other non-infested ). Short-term flooded plants were flooded 2 d consecutively in a single flood cycle followed by 5 d without flooding. Cyc lically flooded plants were flooded for 2 d followed by a 5-d drying period resulting in a 7-d cy cle that was repeated 3 times. Hence tests with short-term and cyclically flooded treatments were performed concurrently during the first 13 d after infestation (31 Oct 13 Nov), but beyond this point, they 116

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were different. Total days flooded for short-term and cyclic flooding was 2 d and 6 d, respectively. All plants (flooded and non-flooded) were irrigated by overhead sprinkler for 30 min twice per day. Larval infestation. For each plant species, one-half the short-term flooded plants and one-half the cyclically flooded plants were infest ed with Diaprepes root weevil larvae on day 4 (31 October 2008). Each of 40 plan t containers was infested with 20 larvae raised on an artificial diet supplied by Florida Division of Plant Industry, Gainesville. La rvae used to infest plants were about 28 d old and averaged 1.15 0.14 mm in head capsule width, hence they were fourth to sixth instar, or late fifth instar on average (Quintela et al 1998). Larvae were placed individually into each of 10-20 holes in the soil, 3-10 cm deep, 4-8 cm from the stem, and 3 cm apart. All containers remained non-flooded for 6 d to allow larvae to become established. Temperature and soil redox potential. Air and soil temperatures were recorded at 1 h intervals throughout the experime nt with two air sensors a nd two soil sensors (StowAway Tidbit temploggers, Onset Co., Pocasset, MA placed in the soil (soil temperature) and canopies (air temperature) of plants not included in the experi ment but in similar potting media, the same container type, and located next to the test plants. The two air sensors were each placed in canopies 66-71 cm above the soil surface. The two soil sensors were placed at a soil depth of 6 cm two-thirds the distance from the cen ter to the outer edge of the pot. Soil redox potential was measured with a me tallic combination electrode (Accumet Model 13-620-115, Fisher Scientific, Pittsburgh, PA) att ached to a portable vol t meter (Accumet model AP62, Fisher Scientific, Pittsburgh, PA). Soil redox potential was measured daily during each flood period for two flooded, infested plants a nd two flooded, non-infested plants. The four measurements were averaged to calculate mean redox potential for short-term flooded or cyclically flooded treatments for each plant sp ecies. Measurements of redox potential were 117

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made by inserting the electrode in to a polyvinyl chloride (PVC) pipe inserted into the soil 2 cm from the edge of the pot. Soil pH was measured for all flooded plants two times per flood period with a platinum combination el ectrode attached to the same portable volt meter used for redox measurements. For each flood cycle, the first pH measurement was on the same day plants were flooded and the second was 2 d later on the da y they were unflooded. The exception was Swingle citrumelo plants during the first flood cy cle, where pH was measured 1 d after plants were flooded, and again 1 d later when plants were unflooded. Plant data collection. Net CO2 assimilation, transpiration and stomatal conductance were measured on two fully expanded, recently mature l eaves or leaflets per plant with a CIRAS-2 portable gas analyzer (PP Systems, Amesbury, MA). Values of the two leaves or leaflets were averaged and the mean value per plant (replication) was used for statistical analyses. Leaf gas exchange was initially measured 2-3 d before infe sting plants with larvae and then periodically throughout the experiment. All leaf gas exchan ge measurements were made between 8:30 and 16:45 h. During gas exchange measurements, th e photosynthetic photon flux was maintained at 1000 mol photons m-2 s-1 with a halogen lamp attached to the leaf cuvette and the reference CO2 concentration in the cuvette was kept constant at 375 mol mol-1. Swingle citrumelo has compound leaves with three leaflets per leaf, and the terminal leaflet is larger than lateral leaflets (Hutchison 1974, Wunderlin 1998), however, buttonw ood has simple leaves. All leaf gas exchange measurements for Swingle citrumelo were taken on the large terminal leaflets. Plant height was measured to the top edge of the highest leaf above th e soil surface prior to infestation and initiation of flooding. Stem di ameter was measured 10 cm above soil surface prior to infestation and initiation of flooding, and for plants with multiple stems at this height, diameter of the largest stem was recorded. Betwee n initial measurements of plant height or stem diameter and harvest, 18-19 d lapsed for short-term flooded plants, which was presumed 118

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insufficient to show a measurable difference in plant height or stem diameter. However, for cyclically flooded plants, 33-34 d lapsed between in itial and final measurement of stem diameter and plant height, which was believe d sufficient to show a difference in these variables. Hence, the second and final measurement of stem diameter and plant height was not made for short-term flooded plants but was performed for cyclically flooded plants 4 d before harvest began, or 28 November 2008. All plants in the short-term flooded test were harvested from days 17 to 18 (13-14 November 2008), or 13-14 d after infesting, 7-8 d after initially flooding pl ants, and 5-6 d after unflooding plants. Plants in the cyclically flooded test were harvested from days 36 to 37 (2-3 December 2008), or 32-33 d after infesting, 26-27 d after initially flooding plants, and 10-11 d after the final unflooding of plan ts. At harvest, stems were cut off 2-3 cm above the surface of the potting medium. The roots were removed from the potting medium and the medium was placed into bins and carefully inspected for larv ae. The number of live and dead larvae were then determined for each plant and preserved in separate vials of 75% ethanol. Head capsule widths were measured in a laboratory with a micr oscope micrometer. Roots, stems, and leaves were then oven dried at 75C for 5 d to a consta nt weight and dry weight s were determined. For buttonwoods, leaf dry weight includ ed leaf blades and petioles, and for Swingle citrumelo, leaf dry weight included leaflets, petio lules, and petioles. Root dama ge was evaluated for infested Swingle citrumelo plants using a visual rati ng system in which 0 = no visible damage, 1 = minimal visible damage, 2 = moderate visibl e damage, and 3 = maximum visible damage. However, root damage was not rated for green buttonwood because larval herbivory generally did not significantly affecting leaf gas exchange or growth in th is species, unlike with Swingle citrumelo. 119

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Experimental design an d statistical analysis. For each plant species, there were four treatments in each short-term and cyclically flooded test: 1) flooded infested, 2) flooded noninfested, 3) non-flooded infested, and 4) non-flooded non-infested in a 2 (flooding treatment) x 2 (infestation treatment) factorial design. For each plant species, there were five single-plant replications per treatment. A two-way analyses of variance (ANOVA) was used to determine flooding and infestation interactions. Standard T-tests were us ed to compare flooded versus nonflooded and infested versus non-infested treatments. For percentages of larvae surviving, proportional data based on ratios of live/total larvae were arcsine transformed prior to statistical analysis. All statistic al analyses were performed with SAS Statistical Software Version 9.1 (PROC T-TEST and GLM, SAS Institute, Cary, NC, 2003). Results Air and soil temperature, soil redox potential, and floodwater pH. During the test period, mean daily temperat ures ranged from 13.0oC to 24.3oC for the air and 16.8oC to 27.7oC in the soil with monthly averages from 15.8oC to 22.8oC for air and 19.8oC to 25.1oC in the soil (Figure 5-1a). Soil redox potential for shortterm flooded green buttonwood plants ranged from +195 mV to +140 mV, and for short-term flooded Swingle citrumelo plants, from +376 mV to +182 mV (Figure 5-1b). For cyclically flooded pl ants, soil redox potenti al during the first flood period ranged from +193 mV to +162 mV for gr een buttonwood and from +378 mV to +165 mV for Swingle citrumelo plants (Figure 5-1b). Du ring the second flood period, soil redox potential ranged from +597 mV to +166 mV for green buttonwood and from +498 mV to +174 mV for Swingle citrumelo (Figure 5-1b). During the third (final) flood period, soil redox potential ranged from +508 mV to +153 mV for green buttonwood and from +523 mV to +193 mV for Swingle citrumelo (Figure 5-1b). For combin ed short-term and cyclically flooded green buttonwood and Swingle citrumelo plants pH of floodwater was 7.21-7.78. 120

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Leaf gas exchange. For Swingle citrumelo or green buttonwood, there were no significant flooding and larval infestation interactions except for net CO2 assimilation for short-term flooded green buttonwood on the first measurement date. To test responses to flooding, treatments for larval infestation were pooled a nd to test responses to larval in festation, flooding treatments were pooled. For cyclically fl ooded green buttonwood, net CO2 assimilation ( t = -2.21, df = 13, P = 0.0451) and stomatal conductance ( t = -2.70, df = 17, P = 0.0154) were significantly higher for non-flooded than flooded plants on the fifth (final ) measurement date (Figure 5-2). However, there were no significant differences between infe sted and non-infested, cy clically flooded green buttonwood plants in net CO2 assimilation (range 1.7-11.9 umol CO2 m-2 s-1) or stomatal conductance (range 24-150 mmol CO2 m-2 s-1). There were also no significant differences between infested and non-infested or fl ooded and non-flooded, short-term flooded green buttonwoods in net CO2 assimilation (range 3.5-12.5 u mol CO2 m-2 s-1) or stomatal conductance (range 39-184 mmol CO2 m-2 s-1). Transpiration was significan tly higher for non-infested than infested, short-term flooded green buttonwood plants on the second measurement date, after infestation but before flooding ( t = -2.50, df = 15, P = 0.0245) (Figure 5-3a). Transpiration for cyclically flooded green buttonwood was signifi cantly higher for non-flooded than flooded plants on the third measurement date, after infest ation and the first flood cycle, but before the second flood cycle (t = -2.24, df = 18, P = 0.0384) (Figure 5-3b). Th ere were no significant differences in transpiration between shor t-term flooded and non-flooded green buttonwood plants (range1.0-4.4 mmol H2O m-2 s-1) nor between infested and non-infested, cyclically flooded plants (range 0.75-4.38 mmol H2O m-2 s-1). There were no significant differences in net CO2 assimilation between flooded and nonflooded or infested and non-infested Swingle citrumelo plants in either short-te rm treatments (range 2.6-11.1 u mol CO2 m-2 s-1) or cyclical treatments (range 1.7-10.5 u mol CO2 m-2 s-1). For 121

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cyclically flooded Swingle citr umelo plants, transpiration ( t = -2.64, df = 17, P = 0.0170) and stomatal conductance (t = -3.10, df = 17, P = 0.0064) were significantly higher for non-infested than infested plants on the fifth (final) measurem ent date (Figure 5-4a and b). However, there were no other significant diffe rences between flooded and non-flooded or infested and noninfested Swingle citrumelo plants in transpir ation or stomatal conduc tance for short-term flooding (ranges in transpiration 1.6-3.9 mmol H2O m-2 s-1 and stomatal conductance 43-143 mmol CO2 m-2 s-1) or cyclical flooding (ranges in transpiration 1.3-3.5 mmol H2O m-2 s-1 and stomatal conductance 32-130 mmol CO2 m-2 s-1). Plant growth. There were no significant flooding and infestation interactions for stem diameter and plant height of e ither plant species. For tissue dry weights, the only significant flooding and larval infestation interaction was for root dry weights of cyclically flooded Swingle citrumelo (F = 4.87; df = 1; P = 0.0422). Therefor e, dry weights of cyclically flooded Swingle citrumelo were not pooled for analysis, whereas a ll other dry weights, stem diameter, and plant height data were pooled for each plant species. There were no significant effect s of cyclical flooding or larv al infestation on increase in stem diameter or plant height for either buttonwood (stem diameter range -0.64 to 2.05 mm, plant height range -2.9 to 7.3 cm ) or Swingle citrumelo (stem diameter range -1.19 to 0.88 mm, plant height range -0.83 to 2.82 cm). There were also no significan t effects of flooding or larval infestation on root, stem, leaf, or total dry we ight for short-term buttonwood (ranges for roots 46100 g, stems 91-178 g, leaves 49-106 g, and total 200-369 g), short-term Swingle (ranges for roots 26-46 g, stems 69-111 g, leaves 10-20 g, a nd total 109-172 g), cyclical buttonwood (ranges for roots 49-103 g, stems 82-204 g, leaves 56-110 g, a nd total 193-407 g), or stem, leaf, or total dry weight for cyclical Swingle (ranges fo r stems 65-112 g, leaves 8.7-20.5 g, and total 102-173 g). However, root dry weight for cyclical Swingle citrumelo was si gnificantly higher for 122

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flooded, infested than for non-flooded, infested plants ( t = 3.16, df = 5, P = 0.0234) (Figure 55a). Root dry weight of Swingle citrumelo was also significantly higher for non-flooded, noninfested than for non-flooded, infested pl ants in the cyclical flooding test ( t = -3.65, df = 8, P = 0.0066) (Figure 5-5b). For green buttonwood, there was no significant eff ect of short-term or cyclical flooding on percent larval survival or head capsule width of recovered larvae (Fi gure 5-6a and b). For Swingle citrumelo, there was also no significant effect of shor t-term flooding on percent larval survival or head capsule width of recovered larvae (Figure 5-6a and b). However, cyclically flooded Swingle citrumelo plants had si gnificantly reduced pe rcent survival ( t = -2.44, df = 6, P = 0.0488) and head capsule width (t = -4.32, df = 5, P = 0.0064) of Diaprepes root weevil larvae than non-flooded plants (Figure 5-6a and b). Root damage rating was significantly higher for non-flooded than cyclically floode d Swingle citrumelo plants ( t = -3.09, df = 5, P = 0.0269) (Figure 5-7), but there was no significant difference between nonflooded and short-term flooded plants. Discussion In the present study, the average monthl y soil temperatur es were 0.9 to 6.2oC below the ideal developmental temperature of 26oC for Diaprepes root weevil and up to 2.2oC below the ideal survival temperatures of 22 to 26oC for this weevil (Lapointe 2000b). Lapointe (2000b) found the highest larval survival rates occurred at 22 and 26oC with lowest survival at 30oC, and the highest development rate was at 26oC with slower rates at 22 and 30oC. In that study, increasing temperatures above 26oC slowed development and decreased survival rates (Lapointe 2000b). Although larval development rates in the present study may have been slower than their maximum, larval survival rates were probably cl ose to or slightly below their maximum levels. 123

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A measure of oxygen abundance in soil is th e redox potential. Effects of flooding on physiology and growth of woody perennial plan t species can vary among soil types and are partly based on rates of O2 depletion in the soil and other factor s such as soil pH (Schaffer et al. 1992). Well-drained, well oxygenated soils have re dox potentials of +300 mV or more, whereas flooded soils have redox potentials of + 200 mV or less (Ponnamperuma 1972, 1984). Redox potentials measured for short-term flooded and cy clically flooded soils in this study indicated that while there was a decline in soil O2 content during flooding periods, soils did not become very depleted of oxygen. This may have been caused by the short durat ion of flooding periods, which included a single 2-d flood period for shor t-term flooded soil and three 2-d flood periods each separated by 5 d without flooding for cyclically flooded soil. All mean soil redox potentials for flooded plants were aerobic (above +300 mV) on the first day of every flood cycle except for short-term flooded and cyclically flooded green buttonwoods on the first flood cycle. On the second day of each flood cycle, all means fo r short-term and cyclically flooded green buttonwood and Swingle citrumelo plants were cumulatively between +193 mV and +281 mV, and they were between +140 and +193 on the third (final) day. All mean soil redox potentials hence ranged from +140 to +597 mV, and were anaerobic to aerobic (Ponnamperuma 1972, 1984). Duration of flooding and larval infestation pe riods in the present study were relatively short compared to previous studies, where gr een buttonwood was exposed to 14-35 d of flooding followed by 90 d infestation (Diaz 2005), and Sw ingle citrumelo was exposed to 20-40 d of flooding followed by 40-56 d of larval infestati on (Li et al. 2004, 2007b) The shorter duration of flooding and larval infestation in the pres ent study may help account for fewer significant differences in leaf gas exchange, growth, and la rval recovery compared to similar tests with flooding and/or Diaprepes root weev il infestation of the same plan t species. However, for short124

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term flooded green buttonwood plants in the presen t study, fourth to sixth instar larvae caused sufficient feeding pressure to significantly re duce transpiration in infested compared to noninfested plants on one measurement 5 d after infe station. This was the onl y significant difference in leaf gas exchange, growth, or larval recovery noted for short-te rm flooded green buttonwood or Swingle citrumelo trees. For cyclically flooded green buttonwood, tran spiration was significantly higher for nonflooded than flooded plants on the third measurement, and net CO2 assimilation and stomatal conductance were significantly higher for non-flooded than flooded plants on the fifth (final) measurement. Transpiration and stomatal conductance for cyclically flooded Swingle citrumelo plants were each significantly higher for non-infest ed than infested plants on the fifth (final) measurement. Most of these significant differences in leaf gas exchange for cyclically flooded green buttonwood and Swingle citrumelo plants occurred on the final measurement, indicating effects of flooding and in sects were cumulative. There were no significant differences between flooded and non-flooded green buttonwood plants in increase of stem diameter and no adventitious roots were observed, but net CO2 assimilation, transpira tion, and stomatal conductance all we re significantly lower for flooded than non-flooded plants on at least one measurement. In another study (Chapter 3), when green buttonwoods in potting medium were flooded for a longer duration (23 d), stem diameter increase was significantly greater in flooded than non-flooded plan ts. Also, adventitious roots and hypertrophic lenticels were seen on flooded plants, but th ere were no significant differences in net CO2 assimilation or stomatal conductance. Increased stem diameter, hypertrophic stem lenticels, and adventitious roots in flooded green buttonwood combined with almost no significant differences in net CO2 assimilation or stomatal conduc tance were also observed in another study (Chapter 6). Effects of flooding may be different when longer flood periods are 125

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applied than those of the present study: for example, over 3 wk of short-term flooding, which is long enough for green buttonwoods to develop thicker stems, adventitious roots, and hypertrophic lenticels (Chapters 3 and 6). In addition, Swingle citrumelo has moderate to low flood tolerance (Auscitrus 2004), whereas green buttonwood is relatively flood tolerant. The short flood durations in the present study may have not allowed sufficient time for green buttonwood to flood-adapt, hence it was less flood tolerant than Swingl e citrumelo; however, flooding reduced infestation more effectiv ely on Swingle than on green buttonwood. Green buttonwood gas exchange and growth was strongly reduced by Diaprepes root weevil larval feeding in other studies with mu ltiple plant species (Diaz 2005, Diaz et al. 2006, Chapter 2). However, Swingle citrumelo was ev en more susceptible than green buttonwood to larval herbivory in the present study. For cyclically flooded Swi ngle citrumelo plants, three 2-d flood periods significantly reduced larval growth and survival. However, these 2-d flood periods did not significantly affect larval growth or surv ival in cyclically flooded green buttonwood or in short-term flooded green buttonwood or Swingle Citrumelo plants. Therefore, three periods of 2-d flooding with 5-d drying peri ods in between, such as may occur in the field from heavy rain, may help control Diaprepes root weevil la rvae without affecting l eaf gas exchange or growth of trees on Swingle citrumelo rootstock. However, these short-term flood periods would probably not benefit green buttonwoods. In the present study, root dry weight of cyc lically flooded Swingle citrumelo plants was significantly greater for flooded infested than fo r non-flooded infested plants and significantly greater for non-flooded non-infested than non-flooded infested plants. This suggests that larval herbivory decreased root dry weight more in non-flooded than in flooded plants. This is supported by significantly higher root damage ra tings for non-flooded th an cyclically flooded Swingle citrumelo root balls. Greater root dr y weight for flooded infested than non-flooded 126

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infested Swingle citrumelo plants suggests that flooding may reduce effects of Diaprepes root weevil larval herbivory. Both h ead capsule width and percent surv ival of recovered larvae were significantly greater for non-flooded than cyclically flooded Swingle citrumelo plants. Reduced larval feeding and growth on cyclically flooded Swingle citrumelo plants may have allowed for increased root dry weight in flooded over non-flooded plants, which, unlike short-term flooded plants, seemed to have sufficient time for these differences to become apparent. In the present study, all data for percentage of larvae found alive we re based on live/total ratios, and not live/found or found/total, because of the presumption th at the larvae not found were dead and decomposed. Previous results suggest that flooding for at least 3 d for plants grown in potting medium, would he lp control Diaprepes root weev il larvae in flood-tolerant to moderately flood-sensitive plan t species (Chapter 4). Shapiro et al. (1997) found that mean mortal ity of flooded unfed Diaprepes root weevil larvae exceeded 90% by 3 wk at 24 and 27oC, and by 5 wk at 21oC, but was only 46% after 5 wk at 18oC (Shapiro et al. 1997). Similarly, larvae of the wireworm Melanotus communis (Gyllenhal) (Coleoptera: Elateridae) had 80% mortality after 6 wk of submergence at 27oC (Hall and Cherry 1993), whereas scarab grubs Tomarus subtropicus (Blatchley) (Coleoptera: Scarabaeidae) had 100% mortality after ~1 wk (5-10 d) of submergence (Cherry 1984). Flooding is sometimes used in southern Florida s ugarcane fields to control the foregoing larvae, T. subtropicus (Cherry 1984) and M. communis (Hall and Cherry 1993). Flooding may be useful for controlling Diaprepes root weevil larvae in sugarcane fields in the summer and fall when floodwater temperatures are close to their maximum (27oC) (Hall and Cherry 1993, Shapiro et al. 1997). Hence, flooding potentially may help c ontrol Diaprepes root weevil larvae in floodtolerant ornamental plants including green buttonwood. 127

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Lapointe and Shapiro (1999) determined that optimal survival to pupation of Diaprepes root weevil occurred at 30-70% soil moisture. About 60-65% of larvae survived to pupation under these ideal moisture conditions (Lapointe and Shapiro 1999). The poorest survival of larvae occurred in low (20%) and in high (80% ) soil moisture levels (Lapointe and Shapiro 1999). Thus, lowest larval survival would be expected under flooded conditions, which presumably have over 80% moisture levels, wher eas un-flooded plants may have soil moisture levels more favorable to larval survival, 30-70%. Larvae of Diaprepes root weevil were exposed directly to flooding in the present study. However, Li et al. (2003a, 2006, 2007b) and Diaz (2005) unflooded plants before infestation with larvae. Diaz (2005) tested effects of flooding and Diaprepes root weevil larval infestation on leaf gas exchange and weight of green but tonwood plants, but unflooded plants 1 d before larval infestation so both stresses were not simultaneous. Overall, leaf gas exchange and plant dry weights observed by Diaz (2005) seemed to decrease more due to flooding than larval infestation in green buttonwood. There were no significant differences in the mean number of larvae recovered between pre-flooded and non-flooded green buttonwood plants, which may reflect similar soil water content between the tr eatments during infestation (Diaz 2005). Hence, whether or not flooding was applied before infest ation with Diaprepes root weevil larvae may be less important than the soil wate r content during larval infest ation, although soil pH and food plant quality may al so affect results. In a previous study (Chapter 4), survival of Diaprepes root weevil larvae was significantly higher in flooded marl soil than in flooded potting medium. This may have been caused by more favorable pH for larval survival in flooded marl soil (pH = 7.4-8.4; Li 2001) than in flooded potting medium (pH = 6.0; Chapter 4). In the present study, the relativ ely high soil pH that increased during the flood period and the well-aer ated potting medium may have allowed similar 128

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larval survival rates and growth in flooded and non-flooded potting media for green buttonwood. A possible reason why pH effects were different in green buttonwood than Swingle citrumelo may be differences in edaphic hab itats in which they are adapted. Li et al. (2003a, 2006, 2007b) examined the e ffects of Diaprepes root weevil larval infestation and flooding on plant gr owth, larval survival, and other characteristics of Swingle citrumelo and one other citrus rootstock in a greenhouse. Flooding occurred before larval infestation so both stresses were not simultaneous. Li et al. ( 2003a) found survival of Diaprepes root weevil larvae was significan tly higher in previously flooded than in non-flooded soil, and flood-damaged seedlings were more susceptibl e to larval feeding injury than non-flooded seedlings. Similarly, Li et al. (2006) investigated the effects of flooding and soil type on larval survival and found that for plants previously fl ooded 20 d, larval surviv al averaged 25% higher in sandy soil than in loam soil. Soil pH increases with flood duration and could adversely affect larval survival (Shapiro et al. 1997, Li et al. 2006) Waterlogged soils are also typically denser than non-flooded soils (Saqib et al. 2004), which is a potential problem for survival of larvae in flooded soil (Li et al. 2006). Li et al. (2006) found that Single citrumelo plants flooded for at least 20 d were more stressed and more prone to Diaprepes root weevil larval feeding injury after removal of plants from flooding than non-flooded control plants. Their results suggest that avoidance of flooding and early control of Diaprepes root weevil larv ae may help protect young plants. Li et al. (2007b) also studied the effects of flooding and soil pH on the grow th and survival of Diaprepes root weevil larvae. Citrus seedlings flooded 40 d had the lowest larval su rvival rates and larval weights compared to seedlings flooded for shor ter flood durations, which may reflect higher soil pH during longer flood durations. Here, larval survival and growth were significantly decreased by pre-applied flooding (Li et al. 2007b). When the soil was limed to pH 4.8-5.7, larval survival 129

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was highest at pH 5.0 for non-flooded plants. Larval survival and weight gain were significantly correlated with pH; increasing pH from 4.8 to 5.7 decreased larval surviv al and increasing pH from 5.1 to 5.7 significantly decreased larval weight s (Li et al. 2007b). Other factors such as soil type, compaction, bulk density, and soil water cont ent may also influence larval survival and growth (Riis and Esbjerg 1998, Rogers et al. 2000, Li et al. 2007b). Increasing the soil pH by at least 1 unit in acidic soils was recommended for optimum citrus growth, which occurs at pH 6.06.5, and to help control Diaprepes root w eevil (Li et al. 2007b). Flooding was also recommended as a possible control method in citrus (Li et al. 2007b). Flooding may hence reduce larval survival while plants are floode d. However, depending on soil pH, flood-stressed plants may be more susceptible to Diaprepes root weevil larval feeding when un-flooded than non-stressed plants that were ei ther never flooded or flood-tolera nt and previously flooded. Hence, pre-applied flooding may either increas e or decrease larval survival based on soil moisture, pH, and plant health while soil is infested. In the present study, flooding slightly increased the soil pH. A pH increase was also noted by Li et al. (2004, 2007b) for previously flooded compared to previously non-flooded soil in a flatwoods Floridana sandy soil (Li et al. 2004) and in Floridana sandy loam (Li et al. 2007b). Here, pH was increased 0.3 units above the m ean pH (5.0) for non-flooded soil after 40 d flooding, resulting in less favorable conditions for growth and survival of Diaprepes root weevil larvae. As suggested above by Li et al. (2007b) increasing the soil pH from 4.8 to 5.7 decreased larval survival and/or weight. Thus, higher pH of floodwater in the present study (7.21-7.78) and in previous studies that used marl soil (Cha pter 4) (7.4-8.4) would a ppear less favorable for Diaprepes root weevil larval surviv al than pH 4.8 to 5.7 in Li et al. (2007b). In Chapter 4, higher Diaprepes root weevil larval su rvival rates were observed in flooded marl soil (pH 7.4-8.4) than in flooded potting medium (pH 6.0). Therefore, the higher pH of marl soil (pH = 7.4-8.4) 130

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compared to potting medium (pH = 6.0) may have been more favorable to larval survival when plants were flooded. In summar y, cyclical flooding for three 2d cycles seems more likely to control larvae in Swingle citrum elo than in green buttonwood. 131

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Figure 5-1. A) temperature and B) soil redox poten tial during the experiment. For temperature, each point is the average of two sensors. For redox potential, each point represents the mean SD of four measurements. Fo r temperature, successi ve flood cycles are denoted by pairs of arrows with the number of the fl ood cycle above the arrows. Each flood cycle in A) corresponds to an individual graph in B). 132

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Figure 5-2. Effects of flooding on A) net CO2 assimilation and B) stomatal conductance for cyclically flooded green buttonwood trees Symbols represent means SD. Successive flood cycles are denoted by pairs of arrows with number of the flood cycle shown above the arrows. Asterisks i ndicate significant differences between treatments at P 0.05 according to a standard T-test. Figure 5-3. Effects of larval infestation or flooding on transpir ation for A) short-term flooded and B) cyclically flooded green buttonwood plants. Symbols represent means SD. For cyclically flooded plants, successive fl ood cycles are denoted by pairs of arrows with the number of the flood cycle show n above the arrows. Asterisks indicate significant differences between treatments at P 0.05 according to a standard T-test. 133

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Figure 5-4. Effects of larval in festation on A) transpiration a nd B) stomatal conductance of cyclically flooded Swingle citrumelo tr ees. Symbols represent means SD. Successive flood cycles are denoted by pairs of arrows with the number of the flood cycle above the arrows. Asterisks indicate significant differences between treatments at P 0.05 and ** P < 0.01 according to a standard T-test. Figure 5-5. Effects of flooding a nd Diaprepes root weevil larval infestation on dry weights of cyclically flooded Swingle citrumelo plants. Bars represent means SD. Asterisks indicate significant differences between treatments at P 0.05 and ** P < 0.01 according to a standard T-test. 134

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Figure 5-6. Effects of flooding on A) percent surv ival and B) head capsule width of Diaprepes root weevil larvae recovered at harvest. X-axis symbols are short-term (continuously) flooded green buttonwood (But-CO), cyclically flooded (intermittent) green buttonwood (But-IN), short-term (continuously) flooded Swingle citrumelo (SwiCO), and cyclically flooded (intermittent) Swingle citrumelo (Swi-IN) plants. Bars represent means SD. Asterisks indicate si gnificant differences between treatments at P 0.05 and ** P < 0.01 according to a standard T-test. Figure 5-7. Visual damage ratings for Diap repes root weevil larvae feeding on Swingle citrumelo roots. Damage rating symbol s are 0 = no visible damage, 1 = minimal visible damage, 2 = moderate visible dama ge, and 3 = maximum visible damage. Xaxis symbols include short-term (continuous ly) flooded Swingle plants (Swi-CO), and cyclically flooded (intermittent) Swingle (S wi-IN). Bars represent means SD. Asterisks indicate significant diffe rences between treatments at P 0.05 and P < 0.01 according to a standard T-test. 135

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CHAPTER 6 LEAF DAMAGE AND PHYSIOLOGICAL RESPONSES OF SELECT WOODY ORNAMENTAL PLANTS TO ADULT FEEDING BY DIAPREPES ABBREVIATUS (COLEOPTERA: CURCULIONI DAE) AND SOIL FLOODING Introduction Diaprepes abbreviatus L. (Coleoptera: Curculionidae: Entiminae) commonly called Diaprepes root weevil, was first found in Flor ida in a citrus nurse ry in Apopka in 1964 (Woodruff 1964). Currently in th e United States, it is found in Florida, Texas (Knapp et al. 2001, Skaria and French 2001), and southern Calif ornia (Klunk 2005). In Florida, Diaprepes root weevil is found in 23 counties in the s outh and central parts of the state (Anonymous 1996, Pea 1997). The host range of Diaprepes root weevil is very large including at le ast 317 varieties, 280 species, 180 genera, and 68 families of plan ts (Simpson et al. 1996, 2000, Knapp et al. 2000b, Mannion et al. 2003, Godfrey et al. 2006). Th e large host range creates many management challenges for this pest. Although not all host plants support all life st ages, many economically important plants support all stages of the weevil from egg to a dult (Simpson et al. 1996). In addition to the management challenges, the wide host range also provides a common avenue of moving this pest to new areas. Many common or namental plant hosts grown in south Florida, such as green buttonwood, are known hosts of Diapre pes root weevil, and if not controlled, may play a role in spreading this pest through the movement of infested plants. Management of Diaprepes root weevil in ornamental plant nurseries is important and necessary to provide plant protection and to reduce the risk of spread. The most obvious feeding damage by adult weevils includes notching along leaf margins of especially young, tend er leaves (McCoy et al. 2002, Wolc ott 1936, 1948). This can result in moderate to severe defoliation of host plants such as young, replanted citrus trees (Quintela et al. 136

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1998, McCoy et al. 2002, Mannion et al. 2003). Un like larval root feed ing, however, prolonged adult feeding does not seem to economically reduce yields of mature citrus trees (McCoy et al. 2002). However, for ornamental plant specie s, excessive foliar damage makes them less attractive at the time of sale and can reduce sales (C. Mannion pers. comm.). Insect herbivory often affects leaf gas exchange of host pl ants (Andersen and Mizell 1987, Schaffer and Mason 1990, Schaffer et al. 1997 ). Syvertsen and McCoy (1985) studied photosynthesis, transpiration, and water use efficiencies of citrus infested with adult A. floridanus, another common weevil in Florida. They found that when weevil population densities were more than one weevil per leaf, herbivory reduced wa ter use efficiency up to 20%. They also found that when adult A. floridanus increased consumption of citrus leaf area, photosynthesis and water use efficiency decrease d. Because water use efficiency decreased more rapidly than photosynthesis, drought stress from injured leaves may have resulted in reduced photosynthesis. Responses of woody plants to flooding include senescence, shoot dieback, premature leaf abscission, decreased cambial growth, and suppression of formation and expansion of leaves and internodes (Schaffer at al. 1992, Kozlowski 1997). In addition, flooding reduces photosynthesis, carbohydrate transport, initiation of flower buds, anthesis, and fru it size, set, quality, and growth (Schaffer at al. 1992, Kozlowski 1997). Flooding often causes a change in the allocation of photosynthates within plants. Fo r example, flooding suppressed he ight and diameter growth of flooded seedlings of Acer platanoides L. (Aceraceae), whereas bark growth increased, which suggests that flooding affects car bohydrate partitioning (Yamamot o and Kozlowski 1987). Thus, flooding may reduce photosynthates, which help to produce leaves and root masses that provide food for insect feeding. Flooding may therefore i ndirectly reduce the initial food available to insects as well as subsequent products of phot osynthesis used to repair feeding damage. 137

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Flooding may therefore reduce herbi vory from insects because the pl ants may be more toxic and less nourishing, hence, more repu lsive and less attrac tive than non-flooded plants. However, flood-induced production of compoun ds such as ethanol can also attract insects (Schroeder and Weslien 1994). The main objectives of our study include investigating effects of flooding and adult Diaprepes root weevil feeding on leaf gas exch ange and to determine if flooding affects host preference by adult weevils or predisposes trees to increased damage or feeding by adult weevils. The host plants included in these studies are green buttonwood Conocarpus erectus L. (Combretaceae), mahogany ( Swietenia mahagoni Jacq., Meliaceae), pond apple ( Annona glabra L., Annonaceae), and Surinam cherry ( Eugenia uniflora L., Myrtaceae) which are widely grown in south Florida as fruit crops or ornament al plants (Watkins and Sheehan 1975, Wunderlin 1998, Nuez-Elisea et al. 1999). Both green buttonwood and pond apple are native to south Florida and can tolerate floodi ng (Watkins and Sheehan 1975, W underlin 1998, Nuez-Elisea et al. 1999). Materials and Methods Three separate tests with four plant species were conducted at the Tropical Research and Education Center, University of Florida, Homest ead, from 2007 to 2008. The first test began in the summer 2007 using green buttonwood and Su rinam cherry; and ended in fall 2007 for Surinam cherry and early winter 2008 for green buttonwood. The second test was conducted in the spring and summer of 2008 using mahogany, a nd the third test wa s conducted during the summer and fall 2008 with pond apple. The objective of these tests was to look at effects on each plant species but not to make comparisons among plant species. Plant material. Green buttonwood trees were purchase d in 4-liter containers from a commercial nursery in February 2007 then tran splanted into 11-liter containers between 19 138

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February and 11 April 2007, about 3-5 mo before initiating the study. Surinam cherry plants were purchased in 11-liter containers from a commercial nursery in February 2006, with multiple plants per container, which is a typical practice in Florida nurserie s. Trunks were divided (each with a separate root system) and transplanted into groups of four trunks per 11-liter container about 4 mo prior to the test. Mahogany trees were obtained by digging s eedlings from the field in Homestead, FL, and repotting them into 11-liter containers before beginning the experiment. Pond apple trees were purchased from a comm ercial nursery in 11-liter containers and transplanted to 15-L plastic containers imme diately after purchasing them. At the time treatments were initiated, approximate trees ag es were 1-year-old for green buttonwood and 3years-old for Surinam cherry, pond apple, and ma hogany. At the beginning of the experiment, plant heights (mean SD) were 91 14, 71 4, 127 11, and 130 17 cm for green buttonwood, Surinam cherry, mahogany, and pond apple, respectively. Green buttonwood, Surinam cherry, and mahogany plants were grown in plastic containers in a standard potting medium (40% Florida peat, 20% pine bark, 20% cypress sawdust, and 20% sand), and pond apple was grown in a similar medium (40% Fl orida peat, 50% pine bark, and 10% sand). Green buttonwood, Surinam cherry, and mahogany were fertilized 8 May 2007, 79 d before initiating treatments, with a ti med release fertilizer (Osmacote Plus 15-9-12, Scotts, Marysville, OH) according to the manufacturers r ecommended rate. On the same date, a foliar iron spray (Sequestrene 138 Fe with 6% chelated iron, Be cker Underwood, Ames, IA) was applied at the manufacturers recommended rate. Surinam cherry was again fertilized 9 July 2007, 16 d before initiating treatments with a liquid ferti lizer (Miracle-gro 15-30-15 with micronutrients, Sterns Mi racle-Gro Products, Port Washington, NY) at the recommended rate. Mahogany trees developed heavy infestations of the woolly whitefly, Aleurothrixus floccosus Maskell (Hemiptera: Al eyrodidae) in fall 2007, about 6 mo before the test began. To 139

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control this pest, plants were sprayed 29 N ovember 2007 with an insect growth regulator (Distance, Valent Co., Walnut Creek, CA) at the ma nufacturers recommended rate. This was mixed with Prescription Treatment Ultra Fine Oil (Whitmire Micro-Gen Research Laboratories, St. Louis, MO) at the manufacturers recommended rate. This combination controlled the insect, and it was a minor pest during the experiment. Pond apple trees were also infested with larvae of the citrus fruit piercer moth, Gonodonta nutrix (Cramer) (Lepidoptera: Noctuidae), which caused most of the foliar damage observed for non-infested plants and similar damage initially for infested plants. These larvae were manually removed as needed. Treatments Infestation treatments. For all four plant species, aluminum screen cages (60 cm x 60 cm x 1.2 m) were used to contain ad ult Diaprepes root weevil. Groups of four screen cages were attached to form cubes, 1.2-m on each side, and placed on 1.2 m x 1.2 m pallets. Two plants (one flooded and one non-flooded) we re placed in each cage. The standard aluminum screen in these cages had an aperture size of 1.5 mm x 1.5 mm and blocked approximately 30% of incoming sunlight (Burger et al. 200 7). All pallets of screen-enclo sed plants were located in an outdoor site exposed to full sun and on la ndscape fabric to control weeds. Twenty Diaprepes root weevil adults (10 males and 10 females) were released into each of six screen cages, one cage per replication. All adults were collected from 3 to 22 d before infestation from canopies of trees growing in fields at commercial plant nurseries in Homestead, FL. Adults for each test were maintained in 30-cm x 30-cm x 30-cm plexiglass cages until their use for infestation. They were provided a diet of green buttonwood leaves and water in 2 vials, and cages were cleaned three times per week. Surinam cherry and mahogany plants were infested with adults after flooded plants exhibite d early signs of physiologi cal stress as indicated by slight reduction in net photosynt hesis. Flooding treatments are described below. Plants were 140

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infested on day 11 of flooding for Surinam cherry and day 31 of flooding for mahogany. Green buttonwoods did not exhi bit reduction in net CO2 assimilation after 25 d of flooding, hence they were found to be relatively flood tolerant and were infested then. Pond apple trees are known to be very flood tolerant (Schaffer 1998, Nez-E lisea et al. 1999, Ojeda et al. 2004, Chapter 3), hence, we did not wait for symptoms of flooding st ress to appear, and trees were infested after 2 d of flooding. Durations of infestation were 81 d, 148 d, 50 d, and 23 d for Surinam cherry, green buttonwood, mahogany, and pond apple, resp ectively. For green buttonwood and Surinam cherry plants, all the cages were infested, whereas for mahogany and pond apple, one-half the total number of cages were infested. Afte r conducting the test for buttonwood and Surinam cherry, it was realized that treat ments without the weevils were necessary for comparison, hence, non-infested treatments were added to the tests with mahogany and pond apple. Flooding treatments. One flooded and one non-flooded plant of the same species were placed together in a screen-enclosed cage. Th e container of each flooded plant was submerged into a 19-liter plastic bucket filled with tap water to 10 cm above soil surface. Plants were flooded July 30 2007 for green buttonwood a nd Surinam cherry, 21 May 2008 for mahogany, and 18 Sep 2008 for pond apple. Flood durations we re 73 d for Surinam cherry, 180 d for green buttonwood, 80 d for mahogany, and 24 d for pond apple. Flood durations were determined by the appearance of physiological indicators of pl ant stress, such as re duced photosynthesis and wilting. All plants were irri gated for 30 min twice per da y by overhead sprinkler. Data Collection Data collected included numbers of egg cl usters, feeding damage to foliage, soil temperature and redox potential, photosynthesis, transpiration, stomatal conductance of water vapor, plant height and stem diam eter (at 10-cm above the soil su rface), and leaf, stem, and root fresh and dry weights. For Suri nam cherry, the leaf chlorophyll index (leaf greenness) was also 141

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measured just before harvest. For green buttonwood, the number of adventitious roots originating above soil level were recorded twice (30 d after treatment initiation and at harvest, day 197). Mahogany has pinnately compound leaves w ith 6-8 leaflets per leaf, whereas all the other plant species tested have simple leaves (Watkins and Sheehan 1975, Wunderlin 1998). For leaf gas exchange measurements, an attempt wa s made to randomly select mahogany leaflets from all positions on the leaf. Soil temperature and redox potential. Soil temperature was recorded at 1 h intervals throughout the experiments with sensors (StowAway Tidbit temploggers, Onset Co., Pocasset, MA) located at a soil depth of 6 cm two-thirds the distance from the center to the outer edge of the pot. Redox potential ( Eh ) of the soil solution was measured with a platinum combination electrode attached to a portable volt meter (Accumet #AP62, Fisher Scientific, Pittsburgh, PA). Measurements were made by inserting the electrode into a polyvinyl chloride (PVC) pipe (20 cm long x 22 mm wide) placed in the soil 2 cm from the edge of the pot. Soil redox potential was recorded at a mean depth of 6 cm below soil su rface. Soil redox potential was measured for four flooded plants per plant species. Redox potential was measured daily during the first 6-7 d of flooding and thereafter at interv als of 3-12 d (mostly 6-8 d) until plants were unflooded. Leaf gas exchange and chlorophyll index. Leaf gas exchange measurements included net CO2 assimilation (photosynthesis), transpira tion, and stomatal conductance. Leaf gas exchange was measured on two recently mature, fu lly expanded, hardened-off leaves or leaflets per plant with a CIRAS-2 portable gas analyzer (PP Systems, Amesbury, MA). For each plant, gas exchange values of two leaves or leaflets we re averaged to provide a single plant value used as a replication. All leaf gas exchange measurements we re made between 10:45 and 17:15 for Surinam cherry, 10:30 and 17:30 for green buttonwood, 14:00 and 19:30 for mahogany, and 14:15 and 18:15 for pond apple. During gas exch ange measurements, the photosynthetic photon 142

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flux in the leaf cuvette was maintained at 1000 mol photons m-2 s-1 with a halogen lamp attached to the cuvette, and the reference CO2 concentration in the cuve tte was kept constant at 375 mol mol-1 CO2. Leaf gas exchange was measured prior to flooding plants and then periodically during the flooding pe riod. A final leaf gas excha nge measurement was made after plants had been unflooded and uninfested. The firs t leaf gas exchange measurements were made 2 d, 5 d, 5 d, and 2 d before flooding green buttonwood, Surinam cherry, mahogany, and pond apple, respectively. Surinam cherry was the only plant species tested in which there was a visible difference in leaf color between fl ooded and non-flooded plants. Hence for Surinam cherry plants, leaf chlorophyll index, a measurem ent of leaf greenness, was determined with a SPAD meter (model 502, Minolta Inc., Japan), on th ree leaves per plant at harvest (day 107, 08 Nov 2007). Egg clusters and leaf damage. Egg clusters were counted and removed from each plant within infested cages and the percentage of leaf damage from feeding was visually estimated for all plants. Relative to release dates of adult Di aprepes root weevil, initial assessments of number of egg clusters and leaf damage during infestatio n were made 5 d, 7 d, 5 d, and 7 d after releasing adults onto green buttonwood, Surinam cherr y, mahogany, and pond apple plants, respectively. Insects remained in cages until no egg clusters were found on at least half th e infested plants, and mean numbers of egg clusters per plant did not differ significantly between flooded and nonflooded treatments for two consecutive weeks. Adults were removed 81 d, 148 d, 50 d, and 23 d after their introduction for Surinam che rry, green buttonwood, mahogany, and pond apple, respectively. Upon removal from cages, all male and female adults were counted from each cage. Final counts of egg clusters and damage were made 15 d after all adults were removed from cages. Egg clusters were removed by det aching two leaves or leaflets en closing them, which resulted in 143

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different numbers of leaves or leaflets removed per treatment (plant) for each replication per measurement date. Neither green buttonwood nor Surinam cherry had significant differences in numbers of egg clusters between flooded a nd non-flooded treatments on any measurement date, and the few leaves removed between treatments were not believed sufficient to significantly affect results, such as leaf fresh weight. For mahogany, however, we compensated for unequal leaf removal among treatments by removing the same number of leaves from each plant as the plant with the highest number of egg clusters in a given replicati on. For pond apple, egg clusters were removed by wiping them off with a cloth in stead of by removing leaves they were attached to because pond apples had much larger leaves than leaves or leaflets of the other plant species. However, pond apple leaves were sensitive to handling and often fell off during evaluation, hence, the same number of leaves were removed from all treatment combinations as in the plant with the most abundant leaf drop to equalize reducti on of leaves. Plant growth. Stem diameter and plant height we re measured for all plants at the beginning and end of each experiment. Stem diameter was measured 10 cm above the soil surface, and for plants with multiple stems at th is height, diameter of the largest stem was recorded. Plant height was measured from the soil surface to the top of the highest leaf or branch. Plants were left floode d until at least one plant exhi bited physical stress and would likely die if not removed from flooding. Suri nam cherry and mahogany were unflooded 73 d and 80d, respectively, after flooding began. Buttonw ood and pond apple, however, did not exhibit physical signs of flooding stress and were unflooded 180 d and 24 d, respectively, after flooding. After adults were removed from screen cages, plants were removed from the cages and placed in the open on weed control cloth for m easurements of leaf damage, number of egg clusters, and leaf gas exchange. At the end of th e treatment period, fresh weights of roots, stems, leaves, and adventitious roots (green buttonw ood), were determined. In addition, for green 144

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buttonwood and Surinam cherry, numbers of fl owers and fruit (including pedicels) were determined per plant. Roots, stems, and leaves were then oven dried at 75C to a constant weight and dry weights were determined. For buttonwood, pond appl e, and Surinam cherry, leaf fresh and dry weights included leaf blades and petioles, but for mahogany, leaf fresh and dry weights included leaflets, petiolules, rachises, and petioles. Experimental design an d statistical analyses. For green buttonwood and Surinam cherry, plants were either flooded or non-flooded and all plants were infested with Diaprepes root weevil adults. For tests with mahogany and pond apple, however, there were two flooding treatments (flooded or non-flooded), and each flooding treatment had two adult infestation treatments (infested or non-infest ed) in a 2 x 2 factoria l design. Data were analyzed separately for each plant species, and there were six single -plant replications per treatment or treatment combination. For each test with mahogany and pond apple, a two-way analysis of variance (ANOVA) was used to determine if there were significant interactions between flooding and insect infestation treatments. Flooding and insect infestati on treatment main effects were compared using standard T-tests. All statistical analyses were done with SAS statistical software Version 9.1 (PROC T-TEST and GLM, SAS Institute, Cary, NC, 2003). Results During the test period for green buttonwood and Surinam cherry, mean daily soil temperatures were 3.5oC to 26.1oC with monthly averages of 14.8oC to 24.6oC (Figure 6-1a). Green buttonwood. Soil redox potential for flooded buttonwood ranged from +123 mV to -288 mV (Figure 6-2a). There were genera lly no significant differe nces between flooded and non-flooded green buttonwoods in net CO2 assimilation (range 0.51-18.8 u mol CO2 m-2 s-1), transpiration (range 0.61-9.10 mmol H2O m-2 s-1), or stomatal conductance (range -117 to 1262 mmol CO2 m-2 s-1). Root dry weight was significan tly lower in flooded than non-flooded 145

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buttonwood plants ( t = -2.79, df = 7, P = 0.0276) (Figure 6-3). There were no significant differences between flooded and non-flooded buttonwood plants in fr esh weight of roots (range 154-358 g), stems (range 304-536 g), leaves (rang e 178-408 g), or total weight (range 673-1247 g), or in stem, leaf, or total dry weight (F igure 6-3). Stem diameter of buttonwood was significantly lower in non-fl ooded than flooded plants ( t = 3.54, df = 6, P = 0.0134), but there was no significant difference in plant height be tween treatments (Figure 6-4). There was no significant effect of flooding on the number of inflorescences per buttonwood plant (range 0109). Significantly more adventitious root s were found on floode d than on non-flooded buttonwoods at 24 d after flooding ( t = 4.19, df = 5, P = 0.0085) and at harvest ( t = 2.92, df = 5, P = 0.0331) (Figure 6-5a). Also, a significantly higher percentage of total fresh weights ( t = 5.45, df = 5, P = 0.0028) and dry weights (t = 5.9, df = 5, P = 0.002) composed of adventitious roots were found on flooded than on non-flooded gr een buttonwoods at harvest (Figure 6-5b). Mean percentage of total weight composed of adventitious roots was 2.1 % for fresh weight and 2.9% for dry weight. Buttonwood plant containers averaged 1.5-1.8 separately rooted plantlets, differences between treatments were not significant and all of them survived to harvest (Figure 6-6). Maximum mean foliar damage (foliage missi ng because of adult feeding) was 32% (nonflooded) and 33% (flooded), and these occurred in the four measurements immediately preceding harvest (Figure 6-7a). There was no significant difference in percent damage from adult Diaprepes root weevil feeding between flooded and non-flooded buttonwoods on any date measured. Mean maximum number of egg cluste rs per plant per evalua tion date for buttonwood was 22 (non-flooded) and 21 (flooded), and these o ccurred during evaluation dates 6-11 (Figure 6-7b). There were no significant differences in number of egg clusters between flooded and nonflooded treatments of buttonwood. At the end of the test, mean ratio of female to total recovered 146

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adults from buttonwood was 0.62, and the differen ce between numbers of males and females was not significant (Figure 6-8). Although not statistica lly compared, buttonwood appeared to have the most egg clusters per plant of four plant sp ecies in the present study, and adult Diaprepes root weevil seemed to live the l ongest on buttonwood compared to the other plant species. Surinam cherry. Soil redox potential for flooded Suri nam cherry ranged from +126 mV to -261 mV (Figure 6-2b). Surinam cherry net CO2 assimilation was significantly lower for flooded than non-flooded plants on 9 of 14 measurement dates (Figure 6-9a). Transpiration and stomatal conductance were each significantly lo wer for flooded than non-flooded plants on 7 of 14 measurement dates (Figure 6-9b and c). Leaf chlorophyll index was significantly lower for flooded than for non-flooded Surinam cherry plants ( t = -2.58, df = 9, P = 0.0296) (Figure 6-10). Surinam cherry leaf fresh weight ( t = -4.19, df = 7, P = 0.0035), total fresh weight ( t = -2.38, df = 9, P = 0.0398), and leaf dry weight ( t = -4.39, df = 8, P = 0.0021) were each significantly lower for flooded than for non-flooded plants (Fig ure 6-11). However, th ere were no significant differences between root or stem fresh weight s or between root, stem, or total dry weights (Figure 6-11). Stem diameter (t = -2.76, df = 10, P = 0.0205) and plant height ( t = -4.55, df = 10, P = 0.0011) were also significantl y lower for flooded than non-flooded Surinam cherry plants (Figure 6-4). There was no signi ficant effect of flooding on the number of flowers and fruit per Surinam cherry plant (range 0-6). Surinam ch erry in non-flooded containers averaged 100% survival of separately rooted plantlets per container, whereas in flooded containers, only 38% survived, and differences between treatments were significant ( t = -7.42, df = 5, P = 0.0007) (Figure 6-6b). Maximum mean percent damage to Surinam cherry foliage from adult Diaprepes root weevil was 33% for non-flooded plants and 20% for flooded plants during the final three measurement dates before harvest (Figure 6-12a). Feeding damage was significantly higher for 147

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non-flooded than flooded Surinam cherry plants in weeks 5 and 7-14 (Fig ure 6-12a). Average maximum number of egg clusters per Surinam cherry plant was 8.0 for non-flooded and 4.7 for flooded plants on measurement dates 2-6, but th ere were no significant differences between treatments (Figure 6-12b). The mean ratio of female to total recovered adults from Surinam cherry at the end of the test was 0.71 with sign ificant differences between numbers of males and females ( t = 2.98, df = 8, P = 0.0169) (Figure 6-8). Among th e four plant species tested, Surinam cherry was the only one with a signifi cant difference between numbers of males and females. In addition to having the highest mean percentage of adults that were female among four plant species tested (71%), Surinam cherry yielded the highest mean total number of adults recovered (8.5 adults), but these differences among plant species were not statistically compared. Mahogany. Mean daily soil temperatures ranged from 24.5oC to 28.9oC with monthly averages of 26.8oC to 27.4oC (Figure 6-1b). Soil redox potenti al for flooded mahogany plants ranged from +67 mV to -297 mV (Figure 6-2c). There were no signifi cant interactions (P 0.05) between flooding and infestation for net CO2 assimilation, transpiration, or stomatal conductance on any measurement date. There were al so no significant interactions between flooding and infestation treatments for stem diameter plant height, root, stem leaf, or total fresh weights or dry weights. However, there were significant flooding and infestation interactions for the percentage of leaf damage from adult feeding and the number of egg clusters per plant on one or more measurement dates. Therefore, percen t damage from adult f eeding and number of egg clusters per plant were not pooled, whereas a ll the other data were pooled for analysis. Net CO2 assimilation was significantly higher for non-flooded than flooded mahogany trees during the final four measuremen ts (Figure 6-13a). Transpiration ( t = -2.59, df = 21, P = 0.0168) and stomatal conductance ( t = -2.53, df = 21, P = 0.0196) were each significantly higher for non-flooded than flooded mahog any during the ninth of 11 m easurements (Figure 6-13b and 148

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c). Net CO2 assimilation, transpiration, and stomat al conductance tended to be higher for infested than for non-infested mahogany during weeks 8-11 with all si gnificant differences occurring in week 9 of 11 meas urements in the test (Figure 6-14). This was 65 d after flooding and 35 d after infesting with adult Diaprepes ro ot weevil (Figure 6-14). At the end of the treatment period, increases in stem diameter ( t = -6.72, df = 22, P = <0.0001) and plant height (t = -2.54, df = 14, P = 0.0233) were each significantly higher for non-flooded than flooded mahogany plants (Figure 6-15). There were no si gnificant differences between infested and noninfested mahogany plants in increase in stem di ameter (range -0.73 to 4.49 mm) or plant height (range 6.5-44.1 cm). Leaf fresh weight (t = -2.99, df = 18, P = 0.0077), total fresh weight ( t = 2.12, df = 21, P = 0.0457), leaf dry weight ( t = -3.09, df = 18, P = 0.0062), and root dry weight ( t = -2.85, df = 18, P = 0.0105) were each significantly higher for non-flooded than flooded mahogany plants (Figure 6-16 a and c). There were no significant differences between nonflooded and flooded mahogany plants in root or stem fresh weight s or in stem or total dry weights (Figure 6-16 a and c). Mahogany leaf fresh weights ( t = -2.44, df = 19, P = 0.0248) and leaf dry weights ( t = -2.31, df = 19, P = 0.0320) were each significantly higher for non-infested than infested plants (Figure 6-16 b and d), but th ere were no significant diffe rences in root, stem, or total fresh or dry weights. Mean maximum percentage of feeding damage from adult Diaprepes root weevil on mahogany foliage was 42% (non-flooded, infested) and 25% (flooded, infested) in weeks 6 and 9, respectively (Figure 6-17). There was significa ntly more leaf damage on non-flooded infested than on flooded infested mahogany plants in week s 1-8 (Figure 6-17a). However, there were no significant differences in leaf damage be tween non-flooded non-infested and flooded noninfested plants (Figure 6-17b). Also, significan tly more damage occurred on flooded, infested than on flooded, non-infested mahogany plants in weeks 3-9 (Figure 6-17c), and on non-flooded, 149

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infested than on non-flooded, non-infested plants in all 9 wk (Figure 6-17d). Mean maximum number of egg clusters per mahogany plant was 14.5 egg clusters per measurement (non-flooded, infested) and 11.2 (flooded, infested), and both maxima occurred on the second measurement date (Figure 6-18). Number of egg clusters per mahogany plant was significantly higher for nonflooded infested than for flooded infested plants in week 1 ( t = -2.32, df = 8, P = 0.0486), with no significant differences between treatments for each of the remaining 8 wk (Figure 6-18a). There were no significant diffe rences in number of egg clusters between non-flooded noninfested and flooded non-infested mahogany plants (F igure 6-18b). Also, numb er of egg clusters per mahogany plant was significantly higher for flooded infested than flooded non-infested plants in weeks 2-4 (Figure 6-18c), and for non-flooded infested than for non-flooded noninfested plants in weeks 1-6 (Figure 6-18d). Mean ratio of female to total recovered adults from mahogany at the end of the test was 0.57 with no significant difference be tween the number of males and females (Figure 6-8). Pond apple. Mean daily soil temperatures ranged from 23.4oC to 27.7oC with monthly averages of 24.9oC to 26.4oC (Figure 6-1c). Soil redox poten tial for flooded pond apple varied from +189 mV to -260 mV (Figure 6-2d). Ther e was a significant inte raction between flooding and infestation treatment ( P 0.05) for net CO2 assimilation on one or more measurement dates. However, there were no significant interactions ( P 0.05) between flooding and infestation treatments for transpiration, stom atal conductance, percent leaf da mage, egg clusters per plant, stem diameter, plant height, root, stem, leaf, or total fresh or dry weight s. Therefore, all pond apple data were pooled for statis tical analysis except for net CO2 assimilation data. Non-flooded, infested pond apple plants had a significantly higher net CO2 assimilation rate than flooded, infested plants, but only on week 5 ( t = -2.90, df = 10, P = 0.0160) (Figure 619). There were no other significant differen ces between flooded and non-flooded or infested 150

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and non-infested pond apple plants in net CO2 assimilation (range 4-22 umol CO2 m-2 s-1). Also, there were no significant diffe rences between flooded and non-flooded or infested and noninfested pond apple plants in transpiration (range 1.5-5.5 mmol H2O m-2 s-1) or stomatal conductance (range 25-196 mmol CO2 m-2 s-1). Fresh root weight ( t = -2.50, df = 16, P = 0.0237) and dry root weight ( t = -4.10, df = 22, P = 0.0005) were each significantly greater for non-flooded than flooded pond apple plants (Figure 6-20). However, there were no significant differences between flooded and non-flooded pond apple plants in stem leaf, or total fresh or dry weight (Figure 6-20). Also, th ere were no significant differe nces between infested and noninfested pond apple plants in root, stem, leaf or total fresh weight (ranges 127-232 g, 306-487 g, 60-176 g, and 543-848 g, respectively) or root, stem leaf, or total dry weight (ranges 41-69 g, 104-162 g, 21-47 g, and 176-273 g, respectively). Th ere were no significant differences in increase in stem diameter or plant height between flooded and non-flooded pond apple plants (Figure 6-15) or between infested and non-infe sted plants (stem diam eter range 0.22-3.96 mm, plant height range -19 to 18 cm ). No adventitious roots we re observed on flooded pond apple, possibly because of the short dur ation of the experiment. Based on diagnosis of a specimen that was co llected from the same nursery on the same date that pond apple trees were purchased, pond apples in the pres ent study were infested with the fungal stem and leaf rot, Phomopsis (sp. or spp.) (Coelomycota) (A. Palmeteer, Plant Disease Clinic, University of Florida Trop ical Research and Education Cent er). This resulted in about 10-30% pond apple foliage loss by the end of th e experiment, whereas average maximum leaf damage from adult Diaprepes root weevil was about 4.1 %. There were no significant differences between flooded and non-flooded or in fested and non-infested pond apple plants in mean feeding damage from adult Diaprepes root weevil (range based on 1 standard deviation was 0.8-6.8%). Mean maximum number of egg clus ters per infested pond apple plant was 0.9 on 151

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the second measurement date, when it was signifi cantly higher than for non-infested plants ( t = 3.53, df = 11, P = 0.0047) (Figure 6-21). There were no ot her significant differences in number of egg clusters between flooded and non-floode d or between infested and non-infested pond apple plants. Mean ratio of female to total r ecovered adults from pond apple at the end of the test was 0.40 with no significant differences between numbers of males and females (Figure 6-8). Pond apple was the only plant species tested with more males than females collected at the end of the treatment period, and it had the lowest survival rate of a dult insects, although there were no statistical comparisons among plant species. Discussion A measure of oxygen abundance in the soil is redox potential. The effects of flooding on the physiology and growth of a woody, perennial plant species can vary among soil types and are partly based on the rates of soil O2 depletion and other factors like soil pH (Schaffer et al. 1992). Well-drained, well oxygenated soils have redox potentials of +300 mV or more, whereas flooded soils have redox potentials of +200 mV or less (Ponnamperuma 1972, 1984). Cumulatively for all plant species in the presen t study, mean soil redox potential of flooded plants ranged from +189 mV to -297 mV, indicating th at flooded soils were hypoxic. To counteract detrimental effects of flooding, such as reduced photosynthesis, many plants have evolved anatomical or morphological adap tations including hypert rophic (swollen) stem lenticels, development of aeren chyma tissue, and adventitious (above-ground) roots (Kozlowski 1997). Hypertrophic lenticels bene fit flooded plants by increasing O2 transport to roots (Hook et al. 1970, Hook 1984) and serving as excretory sites for potentially toxic compounds, such as acetaldehyde, ethylene, and ethanol produced du ring anaerobic root metabolism (Chirkova and Gutman 1972). Aerenchyma tissue occurs in root epidermal layers and shoot cortexes of flooded highbush blueberries ( Vaccinium corymbosm L., Ericaceae) (Abbott and Gough 1987, Crane and 152

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Davies 1989), and it faci litates movement of O2 from shoots to submerged roots. However, according to Nuez-Elisea et al. (1999), floode d pond apple plants did not develop aerenchyma tissue (air pathways or intercellular spaces), but they grew adventitious roots and hypertrophic lenticels on submerged roots. For buttonwoods in the present study, significan tly more adventitious roots were found on flooded than on non-flooded plants. However, root dry weight (i ncluding adventitious roots) was significantly lower for flooded than non-floode d buttonwood plants. This may have resulted from resource partitioning under flooded c onditions (Yamamoto and Kozlowski 1987, Kozlowski 1997). Although root dry weight of buttonwood was significantly lower in flooded than non-flooded plants, stem diameter was si gnificantly lower in nonflooded than flooded plants. Buttonwood developed enlarged stem diameters and adventitious roots when flooded. These adaptations can increase oxygen absorption and transport to flooded roots, and they may indirectly avoid injury from an aerobic compounds and help with nutrient transport (Kozlowski 1997). Thus, enlarged stem diameters and adventitious roots in buttonwood may have contributed to minimal differences observed between flooded and non-fl ooded plants for most gas exchange and growth variables measured. With exception of root dry weight and stem diameter, there were generally no significant differences in net CO2 assimilation transpiration, stomatal conductance, plant height, root fresh weights, or in stem, leaf, or total fresh and dry weights between flooded and non-flooded green buttonwood. Flooded green buttonwood plants were very similar in appearance to non-flooded plants suggesting equal performance and foliage quality. Diaz (2005) found that flooding green buttonwoods in potting medium significantly reduced photosynthesis, transpiration and stomatal conductance be ginning 1 wk after flooding, although flooding did not significantly affect root, stem, or leaf fres h or dry weights. In another study with green buttonwood, flooding did not cause significant differences in photosynthesis, 153

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stomatal conductance, or dry weights of plants grown in potting medium (Chapter 3). The present study found similar results, although floo d periods were longer (180 d for the present study versus 23 d for Chapter 3) and all plants were infested with Diaprepe adults in the present study, unlike in previous chapters. Although we did not compare leaf gas exchange or plant growth between plant species statistically, differences in net CO2 assimilation, transpiration, and stomatal conductance between flooded and non-flooded plants suggest the order of susceptibility to flooding is Surinam cherry, followed by mahogany, with green buttonwood and pond apple the least affected. Differences in plant growth between flooded and non-flooded treatments in infested or pooled cages suggest the order of susceptibility to flooding is Surinam cherry and mahogany most affected and buttonwood and pond apple least affected. Hence, overall differences in gas exchange and growth between flooded and non-fl ooded plants in infested or pooled cages suggest the order of susceptib ility to flooding is Surinam ch erry, then mahogany, with green buttonwood and pond apple least affected. Although pond apple was among the plant species least affected by flooding based on leaf gas exchange and plant growth data, it was stil l more susceptible than in a previous study (Chapter 3), which had 41 d of flooding instead of 24 d in the pr esent study. For example, net CO2 assimilation of pond apple in the previous study was significantly higher in flooded potting medium than non-flooded potting medium on 6 of 19 measurements. However, in the present study, the only significant differenc e was significantly lower net CO2 assimilation for flooded infested than non-flooded infested pond apple plan ts on one measurement. A similar difference was found with pond apple growth, such as stem diameter, which significantly increased with flooding in the previous, but not in the present study. The longer flood duration in the previous 154

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study (41 d) seemed to favor more adaptation and less susceptibility to flooding by pond apple than the shorter duration (24 d) in the present study. Diaz et al. (2005) employed similar methods to the present study in evaluating adult Diaprepes root weevil feeding damage on green buttonwood, but obtained variable results. In one test, feeding by adult Diaprepes root w eevil significantly incr eased photosynthesis, transpiration and stomatal conductance after 2 mo (Diaz et al. 2005). In the second test, feeding by adult Diaprepes root weevil significantly decreas ed root dry weight, but there were otherwise no significant differences in root, stem, or leaf fresh or dr y weights in either test. Mahogany was unique among plant species tested because Diaprepes root weevil adults fed on stems as well as leaves. They left gouge s in the outer stem and bark and chewed up petioles resulting in many more leaves and leaflets killed above the wounds than by feeding alone. Mahogany had the highest maximum mean adult feeding damage and second highest maximum mean number of egg clusters per plant, although adults often av oided older leaves in favor of younger leaves. On buttonwood and Suri nam cherry foliage, however, adults generally ate both young and old leaves, avoided petioles and bark, and tended to not kill uneaten leaves. Diaz et al. (2005) found that Diaprepes root we evil adults fed more on green buttonwood than on live oak leaves, which may have been less suscep tible to adult feeding than buttonwood because the leaves were tougher and more difficult for insects to chew. All adults died within 1 mo on live oaks in the study by Diaz et al. (2005), whereas 96% of adults died after 23 d infestation on pond apple in the present study, probably because of starvation in both studies. In the field, however, adult weevils were obs erved successfully feeding on live oak leaves during flushing, when leaves were less rigid and thus consumable (Diaz et al. 2005). However, on pond apple in the present study, leaves were rege nerated continuously and soft l eaves were always available, which sometimes had a few small notches from Di aprepes root weevil leaf feeding. In addition, 155

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adults tended to congregate on screen tops and sides, also on plant stems, but they avoided pond apple leaves, unlike with the othe r plant species. Live oaks ar e a host plant for adult Diaprepes root weevil (Simpson et al. 1996, Mannion et al 2003, Diaz et al. 2005), but pond apple has not been reported as a host for adults. Although an objective of the pres ent study was not to compare plant species, differences in average maximum feeding damage suggest mahoga ny was most susceptible to damage by adult Diaprepes root weevil, followed by buttonwood a nd Surinam cherry, with pond apple the least affected. However, adults laid the most egg clusters per plant a nd appeared to live the longest on buttonwood compared to the other plant species tested, whereas pond apple had the fewest egg clusters and shortest adult lifes pan. Hence, differences in the maximum number of egg clusters per treatment per plant species indicate a sligh tly different order of susceptibility to adult Diaprepes root weevil: number of egg clusters per plant was greatest for green buttonwood, then mahogany, then Surinam cherry, and was lowest fo r pond apple. Overall differences in these maximum means among treatments and plant spec ies suggest the order of susceptibility to combined feeding damage and number of egg clusters is mahogany and green buttonwood, then Surinam cherry, with pond apple the least affected. Because buttonwood plants are highly attractiv e for adult oviposition (Simpson et al. 1996, Mannion et al. 2003), the resulting larval infestation may be su fficiently high to significantly reduce photosynthesis and growth (Diaz et al 2006, Chapter 2). However, buttonwood is a landscape plant for which aesthetics are quite impor tant. Therefore, adult insects may be more problematic than larvae on buttonwoods because foliar feeding damage makes them less attractive and can reduce sales. Responses of woody plants to flooding include suppressed formation and expansion of leaves, reduced photosynthesis and carbohydrate tr ansport, and changes in the allocation of 156

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photosynthates within plants (Kozlowski 1997). Thus, floodi ng may reduce photosynthates, which help to produce leaves and root masses that provide food for larvae and adults of Diaprepes root weevils. Floodi ng may therefore indir ectly reduce the initial food available to larvae and adults as well as subsequent products of photosynthesis us ed to repair feeding damage. Surinam cherry generally performe d much better in non-flooded than flooded conditions on most gas exchange and growth parameters measured. It was also one of two plants species in the present study in which adults fed significan tly more on non-flooded than on flooded plants in the same cage. According to Bernays and Chapman (1994), most phytophagous insect species are confined to ce rtain plant parts, which typically determine physical and chemical attributes to which ins ects respond. Because adu lt Diaprepes root weevil are foliage feeders, better quality of foliage as food may have caused Diaprepes root weevil adults to prefer non-flooded over flooded Surina m cherry and mahogany fo liage in the present study. There is no indication in the literature that Diapre pes root weevil is attr acted to ethanol or other products of flooding, an aerobic metabolism, or associated microbes. Short plant height and compact foliage of Surinam cherry may have aided within-cage movement of Diaprepes root weev il adults to plants they pref erred for feeding, although they oviposited in statistically equal numbers on non-flooded and flooded plants. Diaprepes root weevil adults significantly pref erred non-flooded over flooded Surinam cherry plants for feeding. Hence, significant differences between non-fl ooded and flooded plants in a given cage for oviposition may have occurred if plants were spa ced farther apart and in larger cages. Possible reasons why non-flooded Surinam cherry plants were preferred over flooded plants include differences in chemicals emitted by the two flood treatments, such as attractants emitted by nonflooded plants or repellents emitted by flooded plan ts, but this was not measured. However, Wee et al. (2008) showed that healthy, non-flooded plants emitted chemicals that attracted another 157

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weevil with similar feeding guild s and phenology to Diaprepes root weevil, the Fullers rose weevil, Pantomorus cervinus (Boheman) (Coleoptera: Curculionidae). Wee et al. (2008) tested behavioral and electrophysiological responses of Fullers rose weev il to host plant volatiles from leaves of sweet lemon ( Citrus limonum Risso., Rutaceae) and white clover ( Trifolium repens L, Fabaceae). Wee et al. (2008) identified eight m onoterpene volatiles and a mixture of them in lemon leaf oil and two green leaf volatiles from white clover leav es, and all these chemicals were detectable by Fullers rose weevil (Wee et al. 2008). However, none of these compounds seemed to be byproducts of flooding, anaerobic me tabolism, or associated microbes (Fulton and Erickson 1964, Wang et al. 1967, Hook et al. 1971, Rowe and Catlin 1971, Culbert and Ford 1972, Ponnamperuma 1984, Kozlowski 1997). Non-flooded mahogany and Surinam cherry plants both tended to have more fresh, whole (mostly uncut) leaf material than flooded plants as shown by their signifi cantly higher leaf fresh and dry weights. Hence, Diaprepes root w eevil adults may have preferred non-flooded over flooded mahogany and Surinam cherry leaves because they produced volatiles in the concentration and mixture needed to attract a dults. In addition, Schroeder and Beavers (1985) found that adult male Diaprepes root weevil produced an aggreg ation pheromone that attracts both males and female adults. Hence, grea ter leaf damage found on non-flooded than flooded Surinam cherry and mahogany plants may have resu lted from initial attr action from both plant volatiles and insect aggregation pheromones causi ng adults to aggregate and feed more on nonflooded than on flooded plants. Ingham and Detling (1986) artifi cially defoliated 55% of the foliage of sideoats gramma grass, Bouteloua curtipendula (Michx.) Torr. Root biomass, s hoot biomass, and tiller number were each reduced by artificial defoliation, though net CO2 assimilation and transpiration significantly increased in the rema ining foliage after artificial defoliation. With mahogany in the 158

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present study, net CO2 assimlation, transpiration, and stomatal conductance were each significantly higher in in fested than non-infested plants on the 9th (of 11) measurement. However, mahogany fresh and dry leaf weights were each signifi cantly higher for non-infested than infested plants, but there we re no significant differences in r oot, stem, or total fresh or dry weights, stem diameter, plant height. As noted, Welter (1989) stated that whole-leaf consumption by adults, which are in the leaf consumer feeding gu ild, often tends to increase net CO2 assimilation when measured on a leaf-area basis, although this effect is atypical compared to the other guilds, such as root-f eeders, which tend to decrease net CO2 assimilation. With regard to whole-leaf co nsumption, increased net CO2 assimilation may be explained by the availability of the same quantity of nutrients su ch as nitrogen delivered to a smaller leaf area after defoliation by the insects. This may render more nitrogen and other nut rients available per remaining leaf area to synthe size chlorophyll and supply other reactions that increase net CO2 assimilation. Mahogany, like Surinam cherry, was not flood-adapted because it had significantly higher net CO2 assimilation, transpiration, stomatal conductan ce, and growth variables such as stem diameter in non-flooded than in flooded treatments Hence, the root environment may have been less aerobic than with buttonw ood or pond apple, which were more flood adapted. Flooded mahogany roots may therefore have created more toxins, such as ethanol and formaldehyde (Kozlowski 1997), which may repel Diaprepes root weevil adults; this is suggested by the feeding preference exhibite d by weevils for non-flooded over flooded mahogany. Mahogany was one of two plant species in which Diaprepe s root weevil adults showed a significant preference of non-flooded over flooded plants base d on foliar damage. Surprisingly, it was the only plant species in which adults also had an ovipositional pref erence of non-flooded over flooded plants based on number of egg clusters, although significan t only for one measurement. 159

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Possible reasons for this preference include a le ss favorable chemical environment in flooded than in non-flooded plants for attracting adults to feed a nd oviposit on leaves. As noted, adult Diaprepes root weevil fed on mahogany differently than on the other plant species by eating the outer stem and bark in addi tion to petioles and leaflet blades. However, adults tended to avoid older leav es in favor of younger leaves. Diaz et al. (2005) showed that adult Diaprepes root weevil pref erred younger over older leaves of live oak suggesting they may have preferred the softer texture of younger leaves because they were easier to consume than the harder, mature leaves. Differences in leaf ch emistry (phagostimulents, etc.) may have also rendered new leaves better tast ing than old leaves. By similar mechanisms, mahogany bark on young stems may be more attractive to adult Diap repes root weevil for feeding than bark on other plant species tested. The results of this study confirmed the fi ndings of Simpson et al. (1996) that green buttonwood, Surinam cherry, and mahogany ar e host plants of adult Diaprepes root weevil. However, pond apple was not found to be a host fo r adult Diaprepes root weevil in the present study. Adults in the present st udy tended to congregate on screen tops instead of on pond apple foliage and were rarely seen feeding on leaves. In addition, very few char acteristic semicircular leaf notches were found along pond a pple leaf margins, and these notches usually appeared much smaller than on the other plant species. Diaprepes root weevil adults also laid the fewest egg clusters and lived the shortest lifespan on pond apple than observed on green buttonwood, Surinam cherry, and mahogany. Hence, the test with pond apple was the shortest of the four tests in the present study. However in anothe r study (Chapter 2), pond apple was found to be a larval host for Diaprepes root weevil, although wit hout significantly affec ting leaf gas exchange or plant growth. Although pond apple can support Di aprepes root weevil larval development, it 160

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does not seem to attract the adults, which are ne eded to oviposit and th ereby infest pond apple with larvae. Nigg et al. (2001a) recommended c ontrolling adult Diaprepes r oot weevil to reduce larval feeding to tolerable levels, and pond apple may al ready have leaf chemistry rendering this step (and accompanying costs) unnecessary. Pond apple is well adapted to flooding and marl soil (Schaffer 1998, Nez-Elisea et al. 1999, Ojeda et al. 2004) and has potentia l as a rootstock for commercial Annona spp., which are not as flood-tolerant as pond apple (Nez-Elisea et al. 1999). The present study provides additional evid ence that pond apple may be a good choice for commercial rootstock of other Annona spp. because adult Diaprepes root weevil rarely oviposited on pond apple, hence, it would seem unlikely to become infested. However, commercial fruit tree species grafted as scions on pond apple rootstock should be tested for adult feeding and oviposition behavior because once used as a root stock, the scion species would determine if adults would oviposit and thus supply roots with la rvae. However, even if this happens, once a scion species is grafted onto pond apple, economic losses may not occur because the larvae may not significantly affect plan t gas exchange or growth (Chapter 2). Flooding has been studied as a means of contro lling Diaprepes root weevil larvae, such as in sugar cane fields, where fl ooding may provide effective contro l, although only in the summer and fall when floodwater temperatures are close to their maximum (27oC) (Hall and Cherry 1993, Shapiro et al. 1997). The results of this study can help prioritize decisions for pest management of green buttonwood, Surinam ch erry, mahogany, and pond apple plants. For example, pond apple may never require treatment to control Diaprepes root weevil larvae, while Surinam cherry and green buttonwood may benefit fr om such a treatment. Future studies should investigate susceptibility to damage from larv ae and adult Diaprepes ro ot weevil feeding for other widely planted landscape plant species of ten visibly infested with weevils, such as Bulnesia 161

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arborea (Jacq.) Engl. (Zygophyllaceae) and black olive ( B. buceras ). Mannion et al. (2003) found adults, feeding damage, and egg masses on B. buceras Because of the tendency of Diaprepes root weevil adults to disperse slowly, and to feed a nd oviposit on the same plants they are found on, the presence of large populations of adult weevils in thes e tree species suggests high production of eggs and larvae. For future Diaprepes root weevil adult choice te sts, particularly in relation to host plant flooding, it would be beneficial to identify Diaprepes root weevil attractants or repellents for flooded plants because flooding sometimes significantly affected adult feeding and/or oviposition preferences. This w ould help determine whether the attractants or repellents are ethanol or something else or could potentially be used to help manage the pest. 162

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Figure 6-1. Soil temperatures during the experiment. A) green buttonwood and Surinam cherry, B) mahogany, and C) pond apple. Each point is the average of one sensor for green buttonwood, Surinam cherry, and mahogany (A-B), and three sensors for pond apple (C). 163

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Figure 6-2. Soil redox potential. A) green buttonwood, B) Surinam cherry, C) mahogany, and D) pond apple. Each point represents the mean SD of four sensors. 164

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Figure 6-3. Effect of flooding on dry weights of infested green buttonwood plants. Bars represent means SD. Asterisks indicate si gnificant differences between treatments at P 0.05, ** P < 0.01, *** P < 0.001 according to a standard T-test. Figure 6-4. Effects of flooding on in creases in A) stem diameter a nd B) plant height for infested green buttonwood and Surinam cherry plants. Bars represent means SD. Asterisks indicate significant differences between treatments within plant species at P 0.05, ** P < 0.01, *** P < 0.001 according to a standard T-test. 165

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Figure 6-5. Effects of flooding on infested green buttonwood trees. A) number of adventitious roots and B) their percentage of total weight. Bars represent means SD. Asterisks indicate significant differences between treatments at P 0.05, ** P < 0.01, *** P < 0.001 according to a standard T-test. Figure 6-6. A) number of trunks and B) effects of flooding on percentage of trunks found alive at harvest for infested green buttonwood and Surinam cherry plants. Mahogany and pond apple each had one trunk. Bars repr esent means SD. Asterisks indicate significant differences between treatments at P 0.05, ** P < 0.01, *** P < 0.001 according to a standard T-test. 166

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Figure 6-7. Effects of flooding on infested green buttonwood plants A) Diaprepes root weevil adult feeding damage and B) number of e gg clusters per plant. Symbols represent means SD. Figure 6-8. Effects of flooding and infestation on A) number and sex of adult Diaprepes root weevil recovered at the end of the test and B) percentage of adults at the end of the test that were female (a measure of sex ra tio). Bars represent means SD. Asterisks indicate significant differences between numbe rs of male and female adults recovered within plant species at P 0.05 according to a standard T-test. But = green buttonwood, Mah = mahogany, SC = Surinam cherry, and PA = pond apple. 167

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Figure 6-9. Effects of flooding on infested Surinam cherry plants. A) net CO2 assimilation, B) transpiration, and C) stomatal conductance. Symbols represent means SD. Asterisks indicate significant diffe rences between treatments at P 0.05, ** P < 0.01, *** P < 0.001 according to a standard T-test. 168

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Figure 6-10. Leaf cholorophyll index for infest ed Surinam cherry plants measured 8-Nov-07 (pre-harvest). Bars represent means SD. Asterisks indicate significant differences between treatments at P 0.05 according to a standard T-test. Figure 6-11. Effects of flooding on infested Surina m cherry plants. A) fresh weights and B) dry weights. Bars represent means SD. Asterisks indicate significant differences between treatments at P 0.05, ** P < 0.01, *** P < 0.001 according to a standard T-test. 169

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Figure 6-12. Effects of flooding on Diaprepes-infested Surinam cherry plants. A) adult feeding damage and B) number of egg clusters pe r plant. Symbols represent means SD. Asterisks indicate significant diffe rences between treatments at P 0.05, ** P < 0.01, *** P < 0.001 according to a standard T-test. 170

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Figure 6-13. Effects of floodi ng on mahogany trees. A) net CO2 assimilation, B) transpiration, and C) stomatal conductance. Symbols re present means SD. Asterisks indicate significant differences between treatments at P 0.05, ** P < 0.01, *** P < 0.001 according to a standard T-test. 171

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Figure 6-14. Effects of infestation on mahogany trees. A) net CO2 assimilation, B) transpiration, and C) stomatal conductance. Symbols represent means SD. Asterisks indicate significant diffe rences between treatments at P 0.05, ** P < 0.01, *** P < 0.001 according to a standard T-test. 172

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Figure 6-15. Effects of flooding on mahogany and pond apple plants. A) increase in stem diameter and B) increase in plant height Bars represent means SD. Asterisks indicate significant differences between treatments at P 0.05, ** P < 0.01, *** P < 0.001 according to a standard T-test. 173

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Figure 6-16. Effects of flooding and infestation on mahogany trees A-B) fresh weights and CD) dry weights. Bars represent means SD. Asterisks indicate significant differences between treatments at P 0.05, ** P < 0.01, *** P < 0.001 according to a standard T-test. 174

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Figure 6-17. Effects of flooding and infestation on adu lt Diaprepes root weevil feeding damage on mahogany trees. Symbols represent means SD. Asterisks indicate significant differences between treatments at P 0.05, ** P < 0.01, *** P < 0.001 according to a standard T-test. 175

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Figure 6-18. Effects of flooding a nd infestation on number of Diap repes root weevil egg clusters per mahogany tree. Symbols represent mean s SD. Asterisks indicate significant differences between treatments at P 0.05, ** P < 0.01, *** P < 0.001 according to a standard T-test. 176

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Figure 6-19. Effects of flooding on net CO2 assimilation of infested pond apple trees. Symbols represent means SD. Asterisks indicate si gnificant differences between treatments at P 0.05, ** P < 0.01, *** P < 0.001 according to a standard T-test. Figure 6-20. Effects of flooding on pond apple plants. A) fresh weights and B) dry weights. Bars represent means SD. Asterisks indicate significant differences between treatments at P 0.05, ** P < 0.01, *** P < 0.001 according to a standard T-test. 177

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Figure 6-21. Effect of adult Diaprepes root w eevil infestation on number of egg clusters per pond apple plant. Symbols represent means SD. Asterisks indicate significant differences between treatments at P 0.05, ** P < 0.01, *** P < 0.001 according to a standard T-test. 178

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CHAPTER 7 CONCLUSIONS The goal of this project was to determine ef fects of Diaprepes root weevil herbivory, flooding, and the interaction of these two stresses on leaf gas exchange and growth of select woody ornamental plant species. Leaf gas exchange included net CO2 assimilation, transpiration, stomatal conductance of water vapor, and internal CO2 concentration. Diaz (2005) and Diaz et al. (2005, 2006) inves tigated effects of larval an d adult Diaprepes root weevil herbivory with and without flooding on leaf gas exchange and growth of woody ornamental plants. However, they left several unanswered questions a bout the growth and physiological responses of ornamental plants to short-term flooding, cyclical flooding, and soil type. These questions included: 1) what are the physiological and growth res ponses of ornamental plants to only flooding; 2) what is the res ponse of Diaprepes root weevil la rvae in marl soil typical of south Florida field nurseries compared to nursery potting soils; 3) would intermittent (cyclical) flooding affect Diaprepes root weevil herbivory differently than short-term (continuous) flooding; 4) what are the effects of long-term feeding and multiple generations of Diaprepes root weevil (at least 2 mo for adults and 8 mo for all stadia combined) ; 5) can a better comparison be made for effects of flooding versus Diaprepes root weevil herbivory on ornamental plants; and 6) does flooding and the resulting anaerobic respirat ion attract adult Diaprepes root weevil. To help clarify relationshi ps between Diaprepes root weevil herbivory, host plant physiology, and flooding, and to answer some of these questions, I proposed the following hypotheses: 1) larval herbivor y reduces leaf gas exchange and growth of green buttonwood ( Conocarpus erectus L.), mahogany ( Swietenia mahagoni Jacq.), Surinam cherry ( Eugenia uniflora L.), and pond apple (Annona glabra L.), which are frequently grown as ornamental or fruit plants in southern Florida; 2) flooding reduces plant growth and leaf gas exchange more in 179

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marl soil than in a standard nursery potting medi um; 3) flooding reduces larval survival more in marl soil than in a standard potting medium; 4) surv ival rates of Diaprepes root weevil larvae and plant biomass are reduced more by short-term th an by cyclical flooding; and 5) flooding reduces leaf gas exchange, growth, leaf feeding damage and attraction to and oviposition on host plants by adult Diaprepes root weevil. Based on differences in leaf gas exchange and plant growth between infested and noninfested plants of the four species tested, green buttonwood and Surinam cherry shared the greatest vulnerability to Diap repes root weevil larval feed ing, followed by mahogany, with pond apple the least affected. For green buttonwood, larval feed ing reduced net CO2 assimilation, transpiration, stomatal co nductance, and internal CO2 concentration by 10-54% just 3 h after infestation with large, seventh instar larvae. However, 4 wk later, net CO2 assimilation, transpiration, and stomatal conduc tance were 11-37% higher for infe sted than non-infested plants on leaf area bases, which may exemplify physiologi cal compensation to ins ect herbivory within leaves. For Surinam cherry plan ts, larval feeding reduced net CO2 assimilation, transpiration, and stomatal conductance by 7-32%. For ma hogany and pond apple, there were few or no significant differences in leaf gas exchange between infested and non-infested plants. For all plant species, mean root and shoot fresh and dry weights were higher for noninfested than infested plants with significant differences most frequent for green buttonwood (3785% higher), followed by Surinam cherry (37143% higher), mahogany (49-84% higher), and pond apple (24-46% higher), which had no signifi cant differences. There were significant differences among plant species in Diaprepes root weevil mean head capsule widths, thus larval instars, of larvae recovered fr om soil with the larg est larvae from Surinam cherry (2.59 0.19 mm) and the smallest from mahogany (2.29 0.06 mm). 180

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Based on combined leaf gas exchange and biomass data, Surinam cherry performed best in potting medium compared to marl soil, followed by buttonwood, mahogany, and pond apple. Based on leaf gas exchange, growth, and signs of morphological adaptation to flooding, order of flood tolerance in marl soil was pond apple with the highest, followed by buttonwood, then mahogany, then Surinam cherry; in potting medium the order was pond apple and buttonwood with the greatest flood tolerance, followed by mahogany, then Surinam cherry. The most apparent trend was for gas exchange and gr owth of green buttonwood, mahogany, and Surinam cherry to be significantly lower for plants growing in flooded marl soil than in either non-flooded marl soil or flooded potting medium. In cont rast to green buttonwood, mahogany, or Surinam cherry, pond apple net CO2 assimilation, stomatal conducta nce, and plant growth (more consistently than the other plant species) tende d to be higher for flooded than non-flooded plants and for plants in marl soil than in pot ting medium. Green buttonwood and pond apple each developed significantly larger stem diameters in flooded than in non-flooded conditions and swollen stem lenticels under flooded conditions. Adventitious roots were also observed on flooded green buttonwood trunks, but were absent from non-flooded plants. Hence, green buttonwood and pond apple were the two most floodadapted plant species possibly because they are native to areas that are often flooded. In marl soil, significantly more Diaprepes r oot weevil larvae surviv ed after 38 d in nonflooded than in flooded conditions. Similarly, more larvae survived in non-flooded than in flooded potting medium, from which no larvae were recovered. Larval survival rates were significantly higher in flooded ma rl soil than flooded potting medium, but there was no difference in survival between non-flooded marl soil and non-flooded potting medium. Larvae recovered from flooded marl soil had significantl y smaller head capsule widths and were therefore at least one instar smaller than larvae recovered from non-flooded marl soil or non181

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flooded potting medium. In summary, flooding marl soil or potting medium reduced survival and often growth of Diaprepes root weevil larvae. Flooding with three 2-d cycles appeared more likely to control Diaprepes root weevil larval infestation in Swingle citrumelo than in green buttonwood plants. Most significant differences noted for each plant species were in response to cyclical fl ooding and after all flood cycles had ended. The effects of flooding ma y be cumulative for green buttonwood and effects of insects may be cumulative for Swingle citrumel o because cyclical flooding caused most of the significant differences noted for green buttonwood and all for Swingle citr umelo. Contrary to similar studies, green buttonwood was the most flood-sensitive plant species, possibly because flood periods were too short for plants to develo p morphological or anatom ical adaptations such as increased stem aerenchyma, hypertrophic stem lenticels, or adventitious roots. Swingle citrumelo was the most sensitive plant species to Diaprepes root weevil larval feeding. Flooding significantly reduced percen t survival and head capsule widths of recovered larvae, but only for cyclically flooded Swingle citrumelo plants, wh ich also had significantly less root damage on flooded than on non-flooded plants. For green buttonwood, there were generally no significant differences between flooded and non-flooded plants in net CO2 assimilation, transpiration, stom atal conductance, plant height, number of inflorescences per plant, root, stem, leaf or total fresh weights, or stem, leaf, or total dry weights. However, root dry weight (including adventitious r oots) was significantly lower for flooded than non-flooded green buttonwood plants. In contrast, the number of adventitious roots and their percentage of fresh and dry weight s were significantly higher in flooded than nonflooded plants. In addition, stem diameter was significantly higher in flooded than in nonflooded green buttonwood plants. These results suggest that green buttonwood adapted to flooding, possibly through increased development of adventitious roots and aerenchyma tissue, 182

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which is suggested by enlarged stem diameter in flooded plants. These adaptations rendered most of the leaf gas exchange, plant growt h, and hence possibly food quality and preference by adult Diaprepes root weevil the same between flooded and non-flooded green buttonwood plants. For Surinam cherry, however, net CO2 assimilation, transpiration, stomatal conductance, leaf chlorophyll index, leaf and total fresh weights, leaf dry weights, plant height, trunk survival, and stem diameter were higher for non-flooded th an flooded plants. Adult feeding damage, but not the number of egg clusters, was significan tly higher for non-flooded than flooded Surinam cherry plants. This may have resulted from non-flooded Surinam cherry plants being healthier and more appealing to the insects. Similar to Surinam cherry, mahogany result s were significantly higher for non-flooded than flooded plants on at least one measurement each for net CO2 assimilation, transpiration, stomatal conductance, plant height, stem diameter leaf and total fresh we ight, and leaf and root dry weight. Net CO2 assimilation, transpiration, and stom atal conductance were significantly greater for infested than non-infested mahoga ny plants, but only on week 9 of 11. Mahogany fresh and dry leaf weights were significantly greate r for non-infested than infested plants. Adult Diaprepes root weevil prefe rred non-flooded over flooded mahogany plants based on feeding damage, which was similar to Surinam cherry. In addition, significantly more egg clusters per plant were laid on non-flooded than flooded mahogany plants on 1 of 9 measurements. Thus, mahogany was the only plant species in which Diaprepes root weevil adults showed an ovipositional preference w ithin infested cages on at least one measurement. Mahogany was also unique because adults fed on stems and petioles, causing more leaf mortality than just from leaf feeding. Although pond apple is a flood-tolerant specie s, it apparently was not flooded long enough to develop enlarged stems or adventitious ro ots as adaptations to flooding. Hence, net CO2 183

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assimilation was significantly hi gher in non-flooded infested th an flooded infested pond apple plants on 1 of 5 measurements, and fresh and dry root weights were significantly higher for nonflooded than flooded plants. Pond apple had the least feeding damage (4.1%) and lowest number of egg clusters (0.9) per plant. Hence, adult Di aprepes root weevil would seem unlikely to infest and oviposit on pond apple plants. Although pond apple can support Diaprepes root weevil larvae through pupation, it is unlikely to become infested with larvae. Based on combined gas exchange and growth data, Surinam cherry showed the highest susceptibility to flooding, followed by mahogany, with buttonwood and pond apple the least affected. Based on combined maximum feeding da mage and number of egg clusters per plant, buttonwood and mahogany shared the highest suscep tibility to adult Diap repes root weevil, followed by Surinam cherry, with pond apple the least affected. Ovipos itional preference of Diaprepes root weevil adults for non-flooded over flooded mahoga ny plants was observed based on egg cluster distribution. Also, a significantly higher percentage of insect damage in nonflooded than flooded plants on most measurem ent dates was unique to mahogany and Surinam cherry, the two plant species most affected by flooding. Significant interaction occurred between flooding and larval in festation during the treatment period, but only for root dry weight of cyclically flooded Swingle citrumelo. For infested plants, root dry wei ght was significantly higher for fl ooded than non-flooded plants, and with non-flooded plants, root dry weight was significantly higher for non-infested than infested plants. These results suggest that flooding for three 2-d cycles decreased loss of root mass from larval feeding, hence, flooding may help contro l Diaprepes root weevil larval infestation in Swingle citrumelo. For adult infestation of pond apple plants, si gnificant flooding and in festation interaction occurred only for net CO2 assimilation on one measurement date, and flooding significantly 184

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decreased net CO2 assimilation on this measurement date for infested plants. Also, pond apple plants had minimal adult feeding damage from Diaprepes root weevil (4.1% mean maximum), and they lacked adaptations to flooding, for exampl e: they had statisticall y equal stem diameters between flooded and non-flooded plants. Hence, th is interaction for pond apple was believed to have resulted mainly from l ack of adaptation to flooding, possibly because of short flood duration (24 d). This flood period would probably not help to control Di aprepes root weevil in pond apples because of low adult feeding dama ge with no significant differences between flooded and non-flooded or infested and non-infested plants. Th is flood duration is also not suggested because of occasional reduction in le af gas exchange and plant growth in flooded compared to non-flooded pond apples. For adult infestation of mahogany plants, significant flooding and infestation interaction occurred for percentage of leaf damage from adult feeding and number of egg clusters per plant on two or more measurement dates. Flooding mahogany plants decreased leaf feeding damage and number of egg clusters per plant. However, flooding is not recommended for Diaprepes root weevil control in mahogany (either) because it reduces leaf gas exchange and plant growth, thus plant health. Overall, plant damage, physiology, and growth of the plants tested were affected by host plant, flooding, and soil type; larval survival and preference were also affected by host plant, flooding, and soil type; and adult host preference was affected by host plant and flooding. 185

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Wang, T. S. C., S. Y. Cheng, and H. Tung. 1967. Dynamics of soil organic acids. Soil Sci. 104: 138-144. Watkins, J. V., and T. J. Sheehan. 1975. Florida landscape plants. University presses of Florida, Gainesville. 420 pp. Wee, S. L., A. M. El-Sayed, A. R. Gibb, V. Mitchell, and D. M. Suckling. 2008. Behavioural and electrophysiological responses of Pant omorus cervinus (Boheman) (Coleoptera: Curculionidae) to host plant volatiles. Australian J. Entomol. 47: 24-31. Weissling, T. J., J. E. Pea, R. M. Giblin-Davis, and J. L. Knapp Jr. 2004. Common name: Diaprepes root weevil. Featured Creatures, Univ. Florida #EENY-24. ( http://creatures.ifas.uf l.edu/citrus/sugarcane_rootstock_borer_weevil.htm ). Welter, S. C. 1989. Arthropod impact on plant gas exch ange, pp. 135-147. In: E. A. Bernays (ed.), Insect-Plant Interactions, Vol. I. CRC Press, Boca Raton, FL. Wilcox, W. F., and S. M. Mircetich. 1985. Influence of soil water matrix potential on the development of Phytophthora root and crown rots of ch erry. Phytopathology 75: 648-653. Whitcomb, W. H., T. D. Gowan, and W. F. Buren. 1982. Predators of Diaprepes abbreviatus (Coleoptera: Curculionidae) larv ae. Fla. Entomol. 65: 150-158. Wolcott, G. N. 1936. The life history of Diaprepes abbreviatus L., at Rio Piedras, Puerto Rico. J. Agr. Univ. Puerto Rico 20: 883-914. Wolcott, G. N. 1948. The insects of Puerto Rico: Coleoptera. J. Agr. Univ. Puerto Rico 32: 225-416. Woodruff, R. E. 1964. A Puerto Rican weevil new to the United States (Coleoptera: Curculionidae). Florida Dept. Agric., Div. Plant Ind. Entomol. Circ. 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. 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. Woodruff, R. E. 1985. Citrus weevils in Florida and th e West Indies: preliminary report on systematics, biology, and distri bution (Coleoptera: Curculioni dae). Fla. Entomol. 68: 370377. Woodruff, R. E., and R. C. Bullock. 1979. Fullers rose weevil, Pantomorus cervinus (Boheman) in Florida (Coleoptera: Curculi onidae). Fla. Dept. Agric. Div. Plant Ind. Entomol. Circ. 207. 195

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Wunderlin, R. P. 1998. Guide to the vascular plants of Florida. University Press of Florida, Gainesville. 806 pp. Yamamoto, F., and T. T. Kozlowski. 1987. Effects of flooding, tilt ing of stems, and ethrel application on growth, stem an atomy, and ethylene production of Acer platanoides seedlings. Scand. J. For. Res. 2: 141-156. Youngman, R. R., and M. M. Barnes. 1986. Interaction of spider mites (Acari: Tetranchidae) and water stress on gas-exchange rates and wa ter potential of almond leaves. Environ. Entomol. 15: 190. Zomlefer, W. B. 1994. Guide to Flowering Plant Families. University of North Carolina Press, Chapel Hill. 430 pp. 196

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BIOGRAPHICAL SKETCH Cliff G. Martin was born in Miami Florida in 195 6. In his early years, he lived in Panama, Canal Zone, which is where his interest in en tomology first blossomed because of the many huge Morpho and beautiful heliconian butterflies, leaf-cu tter and giant Ponerine ants, giant wasps and spiders, and other entomologica l wonders in the jungle. Year s later in Tallahassee, FL, his interest in horticulture ign ited when he took up bonsai as a hobby. He graduated high school in 1975 and earned his B.S. degree from Florid a A&M University in 1979 in ornamental horticulture. His ambition was to work in the nu rsery industry. For 11 years, he worked in the nursery and landscape industry in various horticultural labor positions. In the Fall 1981, however, he obtained an assistan tship and entered an M.S. degree program in horticulture at Iowa State University, but chose to leave afte r one semester. He moved to San Luis Obispo County, CA, where he served as a ranch hand, at a cemetery, and on a fire engine crew for several years, and then to Phoenix, AZ in 1987, where as an employee and a contractor, he improved and diversified his landsca pe skills and experience. In 1991, he got his first entomology job to insp ect cotton boll weevil trap s with the State of Arizona for 10 months. In 1992, he performe d botanical surveys in Saguaro National Monument--one of his favorite jobs. These experiences have shown him the value of independence and creativity as important assets in living a safe, practical, and interesting lifestyle on a limited budget. In 1993, he started a non-thesis M.S. gradua te program. He finished his degree in entomology in 1996 and got a job with the USDA Plant Variety Protection Office in Beltsville, MD (1996-1998). He then obtaine d an assistantship at the Un iversity of Florida in the Department of Entomology in weed biological control. To satisfy a longing for the outdoors and botany, he took as many botany electives as pos sible and identified and prepared hundreds of 197

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198 herbarium specimens to learn plants. Upon gradua ting, he worked on a 3-mo botanical survey at Ozark National Scenic Riverway s in Missouri (2004), and like with Saguaro National Monument (1992), loved the botany. He then moved to Fort Myers, FL, in September 2004 to do clean-up labor after Hurricane Charlie. He subsequently a pplied to the University of Florida to do a Ph.D. and was offered an assistantship to work on Diap repes root weevil and its effects on plants.