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Evaluation of a Predator and a Fungus as Biological Control Agents of the Yellowmargined Leaf Beetle, Microtheca ochrolo...

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Material Information

Title: Evaluation of a Predator and a Fungus as Biological Control Agents of the Yellowmargined Leaf Beetle, Microtheca ochroloma Stal (coleoptera Chrysomelidae)
Physical Description: 1 online resource (96 p.)
Language: english
Creator: Montemayor Aizpurua, Cecil
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: biocontrol, infectivity, isaria, microtheca, pfr97, podisus, predation, yellowmargined
Entomology and Nematology -- Dissertations, Academic -- UF
Genre: Entomology and Nematology thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The yellowmargined leaf beetle, Microtheca ochroloma Sta ringl (Coleoptera: Chrysomelidae) is a pest of cruciferous crops in the southern United States since its first detection in the country in 1945. Little information is available in the literature about the natural enemies of this pest. Therefore, the goal of this study was to evaluate the efficacy of the predator Podisus maculiventris (Say) (Hemiptera: Pentatomidae) and the fungus Isaria fumosorosea (Brown and Smith) Wize (Hypocreales: Cordycipitaceae) to control populations of M. ochroloma. Preliminary results demonstrated that P. maculiventris preys on all stages of M. ochroloma. The first experiment of this thesis measured the predation rate, fresh weight, and developmental time of P. maculiventris feeding on M. ochroloma larvae at constant temperatures of 10, 15, 20 and 25degreeC in the laboratory. There was no development of 1st instars or egg hatch at 10degreeC. The nymphal stage of P. maculiventris preyed on a mean total of 65 plus or minus 6.3 4th instars of M. ochroloma during 99 plus or minus 4.6 days at 15degreeC, 53 plus or minus 1.3 4th instars of M. ochroloma during 31 plus or minus 0.3 days at 20degreeC, and 59 4th instars or 741 eggs of M. ochroloma in 23 plus or minus 0.3 and 25 plus or minus 0.3 days, respectively, at 25degreeC. Adults preyed on a mean total of 5.0 plus or minus 2.1 4th instars of M. ochroloma during 10 days at 15degreeC, 45.1 plus or minus 2.3 4th instars at 20degreeC, and 64 plus or minus 2.9 4th instars at 25degreeC. Adult females weighed more than males at 20degreeC. The second experiment of this study was to determine a field release guideline for P. maculiventris by measuring its predation potential on M. ochroloma in field cages. Four, 10, and 16 1st instar P. maculiventris were separately released in cages containing an initial population of 130 1st instars M. ochroloma on six turnip plants. The release rate of 16 P. maculiventris per six large ( > =7 leaves/plant) turnip plants significantly reduced the M. ochroloma population over time compared to the other two release rates. For six small ( < =6 leaves/plant) turnip plants, a release rate of 10 P. maculiventris significantly reduced the M. ochroloma population over time compared to the lowest release rate, but it's pest population suppression capabilities were not significantly different from the higher release rate. The third experiment of this research was to evaluate the infectivity and lethal time (LT) of I. fumosorosea (commercial name: PFR-97TM) on M. ochroloma at the concentration of 1g of PFR-97TM in 100 ml of distilled water in the laboratory. The larval stage is more susceptible to PFR-97TM than eggs, pupae, and adults. Infectivity rates of 17 and 20% were confirmed in the 1st and 3rd instars of M. ochroloma, respectively. The LT10 for 1st and 3rd instars of M. ochroloma were 4 and 3 days, respectively. Concentrations of 1, 2, 3, and 4 g of PFR-97TM in 100 ml of distilled water were applied to 1st instars of M. ochroloma to compare infectivity, LT, and lethal concentrations (LC). Confirmed infectivity rates for 1, 2, 3, and 4 g concentrations were 2, 5, 10, and 27%, respectively. The LT10 and LT25 for the 4 g concentration were 3.4 and 5.7 days, respectively. The LC10 and LC25 were 1.4 g and 5.5 g per 100 ml of distilled water, respectively. The results of my research suggest that P. maculiventris is a promising biological control agent of M. ochroloma. This predator can be used in an augmentative biological control program in cruciferous crops to control M. ochroloma on organic farms. Isaria fumosorosea (PFR-97TM), on the other hand, does not show any clear evidence of being a potentially effective biological control agent of M. ochroloma.
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 Cecil Montemayor Aizpurua.
Thesis: Thesis (M.S.)--University of Florida, 2010.
Local: Adviser: Cave, Ronald D.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0042212:00001

Permanent Link: http://ufdc.ufl.edu/UFE0042212/00001

Material Information

Title: Evaluation of a Predator and a Fungus as Biological Control Agents of the Yellowmargined Leaf Beetle, Microtheca ochroloma Stal (coleoptera Chrysomelidae)
Physical Description: 1 online resource (96 p.)
Language: english
Creator: Montemayor Aizpurua, Cecil
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: biocontrol, infectivity, isaria, microtheca, pfr97, podisus, predation, yellowmargined
Entomology and Nematology -- Dissertations, Academic -- UF
Genre: Entomology and Nematology thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The yellowmargined leaf beetle, Microtheca ochroloma Sta ringl (Coleoptera: Chrysomelidae) is a pest of cruciferous crops in the southern United States since its first detection in the country in 1945. Little information is available in the literature about the natural enemies of this pest. Therefore, the goal of this study was to evaluate the efficacy of the predator Podisus maculiventris (Say) (Hemiptera: Pentatomidae) and the fungus Isaria fumosorosea (Brown and Smith) Wize (Hypocreales: Cordycipitaceae) to control populations of M. ochroloma. Preliminary results demonstrated that P. maculiventris preys on all stages of M. ochroloma. The first experiment of this thesis measured the predation rate, fresh weight, and developmental time of P. maculiventris feeding on M. ochroloma larvae at constant temperatures of 10, 15, 20 and 25degreeC in the laboratory. There was no development of 1st instars or egg hatch at 10degreeC. The nymphal stage of P. maculiventris preyed on a mean total of 65 plus or minus 6.3 4th instars of M. ochroloma during 99 plus or minus 4.6 days at 15degreeC, 53 plus or minus 1.3 4th instars of M. ochroloma during 31 plus or minus 0.3 days at 20degreeC, and 59 4th instars or 741 eggs of M. ochroloma in 23 plus or minus 0.3 and 25 plus or minus 0.3 days, respectively, at 25degreeC. Adults preyed on a mean total of 5.0 plus or minus 2.1 4th instars of M. ochroloma during 10 days at 15degreeC, 45.1 plus or minus 2.3 4th instars at 20degreeC, and 64 plus or minus 2.9 4th instars at 25degreeC. Adult females weighed more than males at 20degreeC. The second experiment of this study was to determine a field release guideline for P. maculiventris by measuring its predation potential on M. ochroloma in field cages. Four, 10, and 16 1st instar P. maculiventris were separately released in cages containing an initial population of 130 1st instars M. ochroloma on six turnip plants. The release rate of 16 P. maculiventris per six large ( > =7 leaves/plant) turnip plants significantly reduced the M. ochroloma population over time compared to the other two release rates. For six small ( < =6 leaves/plant) turnip plants, a release rate of 10 P. maculiventris significantly reduced the M. ochroloma population over time compared to the lowest release rate, but it's pest population suppression capabilities were not significantly different from the higher release rate. The third experiment of this research was to evaluate the infectivity and lethal time (LT) of I. fumosorosea (commercial name: PFR-97TM) on M. ochroloma at the concentration of 1g of PFR-97TM in 100 ml of distilled water in the laboratory. The larval stage is more susceptible to PFR-97TM than eggs, pupae, and adults. Infectivity rates of 17 and 20% were confirmed in the 1st and 3rd instars of M. ochroloma, respectively. The LT10 for 1st and 3rd instars of M. ochroloma were 4 and 3 days, respectively. Concentrations of 1, 2, 3, and 4 g of PFR-97TM in 100 ml of distilled water were applied to 1st instars of M. ochroloma to compare infectivity, LT, and lethal concentrations (LC). Confirmed infectivity rates for 1, 2, 3, and 4 g concentrations were 2, 5, 10, and 27%, respectively. The LT10 and LT25 for the 4 g concentration were 3.4 and 5.7 days, respectively. The LC10 and LC25 were 1.4 g and 5.5 g per 100 ml of distilled water, respectively. The results of my research suggest that P. maculiventris is a promising biological control agent of M. ochroloma. This predator can be used in an augmentative biological control program in cruciferous crops to control M. ochroloma on organic farms. Isaria fumosorosea (PFR-97TM), on the other hand, does not show any clear evidence of being a potentially effective biological control agent of M. ochroloma.
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 Cecil Montemayor Aizpurua.
Thesis: Thesis (M.S.)--University of Florida, 2010.
Local: Adviser: Cave, Ronald D.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0042212:00001


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EVALUATION OF A PREDATOR AND A FUNGUS AS BIOLOGICAL CONTROL
AGENTS OF THE YELLOWMARGINED LEAF BEETLE, Microtheca ochroloma STAL
(COLEOPTERA: CHRYSOMELIDAE)

















By

CECIL 0. MONTEMAYOR AIZPURUA


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

UNIVERSITY OF FLORIDA

2010
































2010 Cecil O. Montemayor Aizpurua





























To my parents, Monty and Lichy; my brothers, Alcides and Joshua; and my family









ACKNOWLEDGMENTS

I would like to acknowledge my advisory committee, my chair Dr. Ronald D. Cave,

Dr. Susan Webb, and Mr. Edward Skvarch. I am thankful to Dr. Cave for his continued

support, encouragement, and guidance during the course of my studies as a graduate

student, and also for providing me financial assistance to participate in state and

national meetings. I am also grateful to Dr. Webb and Mr. Skvarch for their critiques and

suggestions that made substantial improvements to this study. I would like to thank Dr.

Patrick De Clercq at Ghent University in Belgium for providing valuable information to

my thesis. I give thanks to Jose Castillo, Daniel Mancero, Dafne Serrano, and Bradley

Smith for their help conducting my field research.

Thanks go out to the Ministry of Economy and Finances of Panama for sponsoring

my master's education in entomology at the University of Florida, and the Florida

Specialty Crop Foundation to support my field research. Thanks to all my friends and

colleagues at the entomology department in Gainesville, but especially to Veronica

Santillan, Diana Castillo, Daniel Carrillo, Andres Sandoval, and Sebastian Padr6n for

their unconditional friendship. Thanks to Dr. Pasco Avery at IRREC for sharing his

knowledge, and providing equipment. I would also like to thank Valerie Quant for letting

me collect beetles from her organic farm in Vero Beach.

I am grateful to my best friends, Rodrigo Diaz and Ver6nica Manrique, for their

professional advices regarding my research, but also for always being there for me as a

family in Ft. Pierce. I also want to thank Dr. Cave's family, Vilma, Eloise, and Jonathan,

for letting me be part of their special moments during this time. Finally, I would like to

give special thanks to my family for their loving encouragement and unconditional

support, which motivated me to complete my study.









TABLE OF CONTENTS

page

A C K N O W LE D G M E N T S ........................................................................................ .... 4

LIST O F TA BLES ............... ................................................................................... 7

LIST O F FIG U R ES ............................................................................................ 8

LIST O F A BB R EV IA T IO N S .................................................................. ............... 10

A B S T R A C T ........................................................................................... 1 1

CHAPTER

1 G E N ERA L INT R O D U C T IO N ................................... ....................... ............... 14

2 LIT E R A T U R E R EV IEW ........................................ .......................... ............... 16

M icrotheca ochrolom a S tIl ........................................ ........................ ............... 16
O rigin and D distribution ................................................................................... 16
Biology and Host Range ................................................................................ 16
D am age and Sum m er A activity ..................................................... ............... 17
C ontro l M ethods ............................................................................................ 18
P odisus m aculiventris (S ay)..................................... ....................... ............... 20
O rigin and D distribution ................................................................................... 20
Biology and Host Range ................................................................................ 20
S searching Behavior ....................................................................................... 2 1
B biological C control ......................................................................................... 21
Isaria fumosorosea Wize (Brown and Smith) ........................................ ............... 22
O rigin and D distribution ................................................................................... 22
Biology and Host Range ................................................................................ 23
B io lo g ica l C o n tro l ...................................... ............................ .............. 2 4
Objectives of Master of Science Thesis Research............................. ............... 25

3 DEVELOPMENT TIME AND PREDATION RATE OF PODISUS
MACULIVENTRIS (SAY) (HEMIPTERA: PENTATOMIDAE) PREYING ON
MICROTHECA OCHROLOMA STAL (COLEOPTERA: CHRYSOMELIDAE)......... 28

In tro d u c tio n ......................................................................................................... .. 2 8
M materials and M ethods ................................................................. .............. 29
R e s u lts .............................................................................................................. 3 1
D is c u s s io n ......................................................................................................... .. 3 5

4 PREDATION POTENTIAL OF PODISUS MACULIVENTRIS (HEMIPTERA:
PENTATOMIDAE) ON MICROTHECA OCHROLOMA (COLEOPTERA:
C HRYSO M ELIDA E) IN THE FIELD ................................................... ............... 43









Introduction ....................................................................................... ............... 43
M materials and M ethods .......................................................................................... 45
Results ....................................................................... 47
2009 Experim ent ............................................................................................ 47
20 10 Experim ent ............................................................................................ 50
D discussion .............................................................................. ............... ...... 52
2009 Experim ent ............................................................................................ 52
20 10 Experim ent ............................................................................................ 54

5 INFECTIVITY OF MICROTHECA OCHROLOMA STAL (COLEOPTERA:
CHRYSOMELIDAE) BY ISARIA FUMOSOROSEA WIZE (BROWN AND
S M IT H ) ................................................................................................................... 6 3

Introduction ...................................................................................... ............... 63
M a te ria ls a n d M e th o d s ...................................... ...................................................... 6 4
Experiment 1. Susceptibility of Microtheca ochroloma to Infection by PFR-
9 7 TM ......................................... ........ .................. ......... ... ............................. 6 5
Experiment 2. Infectivity of the Most Susceptible Stage of Microtheca
ochroloma by Four Concentrations of PFR-97TM .................................. ....... 66
Results .................................................... .. ............... 68
E x p e rim e n t 1 .................................................................................................... 6 8
E x p e rim e n t 2 .................................................................................................... 6 9
D discussion .............................................................................. ............... ...... 69
E x p e rim e n t 1 .................................................................................................... 6 9
E x p e rim e n t 2 .................................................................................................... 7 1

6 C O NC LUS IO NS ..................................................................................................... 84

LIST O F R EFE R EN C ES ............................................................................................. 88

BIO G RA PH ICA L SKETC H ....................................................................................... 96



















6









LIST OF TABLES


Table page

3-1 Mean ( SE) development time of Podisus maculiventris reared at three
constant temperatures with 4th instar Microtheca ochroloma as prey.............. 39

3-2 Mean ( SE) fresh weight of newly ecdysed Podisus maculiventris reared at
three constant temperatures with 4th instar Microtheca ochroloma as a prey.... 39

3-3 Mean ( SE) daily predation of 4th instar Microtheca ochroloma by Podisus
maculiventris reared at three temperatures.. ................................. ............... 40

3-4 Mean ( SE) total predation of 4th instar Microtheca ochroloma by Podisus
maculiventris reared at three constant temperatures.................... ............... 40

3-5 Mean ( SE) total development time and predation of 4th instar Microtheca
ochroloma per nymph of Podisus maculiventris.. .......................... ............... 41

3-6 Mean ( SE) fresh weight of newly ecdysed adults of Podisus maculiventris
reared with 4th instar Microtheca ochroloma as a prey. ................ ............... 41

3-7 Mean ( SE) predation of eggs of Microtheca ochroloma by Podisus
m aculiventris nym phs reared at 25C ............................................... ............... 42

5-1 Laboratory tests of Isaria fumosorosea in Experiment 2................................ 83









LIST OF FIGURES


Figure page

2-1 Life stages of Microtheca ochroloma............................................................ 26

2-2 Damage by Microtheca ochroloma .............................................................. 27

3-1 Plastic boxes with screened windows used to house experimental insects........ 38

4-1 Cages in the field at the beginning of the 2009 experiment................................ 57

4-2 Plants being gathered for final sampling in the laboratory..................... 57

4-3 Number of Microtheca ochroloma per cage during the 2009 field experiment. .. 58

4-4 Mean number of Podisus maculiventris nymphs per cage during the 2009
fie ld experim ent ......................................................................................... 59

4-5 Mean number of Microtheca ochroloma per cage during the 2010 field
e x p e rim e n t.......................................................................................................... 6 0

4-6 Number of Podisus maculiventris nymphs per cage during the 2010 field
e x p e rim e n t.......................................................................................................... 6 1

4-7 Size of turnip plants in the 2009 experiment................................................. 62

4-8 Size of turnip plants in the 2010 experiment................................................. 62

5-1 Morphological characteristics of Isaria fumosorosea infecting Microtheca
ochrolom a larva......................................................................................... 73

5-2 Laboratory tests of Isaria fumosorosea ........................................................ 74

5-3 Mortality of Microtheca ochroloma by PFR-97TM 20% WDG at 3.0 x 107
blastospores/m l 7 d after application ............................................. ............... 75

5-4 Infectivity of Microtheca ochroloma by PFR-97TM 20 % WDG at 3.0 x 107
blastospores/m l 7 d after application ............................................. ............... 76

5-5 Lethal time of the first and third instars of Microtheca ochroloma treated with
P F R -97TM ............................................................................... 77

5-6 Infectivity of first instar Microtheca ochroloma at four concentrations of PFR-
97TM 7 d after application ............................................... ................ .............. 78

5-7 Lethal time of the first instar Microtheca ochroloma exposed to four
concentrations of P FR -97TM .................................. ..................... ............... 79









5-8 Eggs of Microtheca ochroloma infected by Isaria fumosorosea....................... 80

5-9 Unconfirmed and confirmed infectivity by Isaria fumosorosea in dead larvae
of Microtheca ochrolom a. .................................. .................. ............... 80

5-10 Reduction in the growth of larvae of Microtheca ochroloma infected by Isaria
fu m o s o ro s e a ....................................................................................................... 8 1

5-11 Net-like pupal case of Microtheca ochroloma................................................... 81

5-12 Unsuccessful molting by a larva of Microtheca ochroloma infected with Isaria
fu m o so ro se a ....................................................................................................... 8 2









LIST OF ABBREVIATIONS

LC Lethal concentration

LT Lethal time

PFR Paecilomyces fumosoroseus = Isaria fumosorosea









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

EVALUATION OF A PREDATOR AND A FUNGUS AS BIOLOGICAL CONTROL
AGENTS OF THE YELLOWMARGINED LEAF BEETLE, Microtheca ochroloma STAL
(COLEOPTERA: CHRYSOMELIDAE)

By

Cecil 0. Montemayor Aizpurua

August 2010

Chair: Ronald D. Cave
Major: Entomology and Nematology

The yellowmargined leaf beetle, Microtheca ochroloma Stal (Coleoptera:

Chrysomelidae) is a pest of cruciferous crops in the southern United States since its first

detection in the country in 1945. Little information is available in the literature about the

natural enemies of this pest. Therefore, the goal of this study was to evaluate the

efficacy of the predator Podisus maculiventris (Say) (Hemiptera: Pentatomidae) and the

fungus Isaria fumosorosea (Brown and Smith) Wize (Hypocreales: Cordycipitaceae) to

control populations of M. ochroloma. Preliminary results demonstrated that P.

maculiventris preys on all stages of M. ochroloma. The first experiment of this thesis

measured the predation rate, fresh weight, and developmental time of P. maculiventris

feeding on M. ochroloma larvae at constant temperatures of 10, 15, 20 and 250C in the

laboratory. There was no development of 1st instars or egg hatch at 10C. The

nymphal stage of P. maculiventris preyed on a mean total of 65 6.3 4th instars of M.

ochroloma during 99 4.6 days at 15C, 53 1.3 4th instars of M. ochroloma during 31

0.3 days at 200C, and 59 4th instars or 741 eggs of M. ochroloma in 23 0.3 and 25 +

0.3 days, respectively, at 250C. Adults preyed on a mean total of 5.0 2.1 4th instars of









M. ochroloma during 10 days at 15C, 45.1 2.3 4th instars at 200C, and 64 2.9 4th

instars at 250C. Adult females weighed more than males at 200C.

The second experiment of this study was to determine a field release guideline for

P. maculiventris by measuring its predation potential on M. ochroloma in field cages.

Four, 10, and 16 1st instar P. maculiventris were separately released in cages

containing an initial population of 130 1st instars M. ochroloma on six turnip plants. The

release rate of 16 P. maculiventris per six large (>7 leaves/plant) turnip plants

significantly reduced the M. ochroloma population over time compared to the other two

release rates. For six small (56 leaves/plant) turnip plants, a release rate of 10 P.

maculiventris significantly reduced the M. ochroloma population over time compared to

the lowest release rate, but it's pest population suppression capabilities were not

significantly different from the higher release rate.

The third experiment of this research was to evaluate the infectivity and lethal time

(LT) of I. fumosorosea (commercial name: PFR-97TM) on M. ochroloma at the

concentration of 1g of PFR-97TM in 100 ml of distilled water in the laboratory. The larval

stage is more susceptible to PFR-97TM than eggs, pupae, and adults. Infectivity rates of

17 and 20% were confirmed in the 1st and 3rd instars of M. ochroloma, respectively. The

LT0ofor 1st and 3rd instars of M. ochroloma were 4 and 3 days, respectively.

Concentrations of 1, 2, 3, and 4 g of PFR-97TM in 100 ml of distilled water were applied

to 1st instars of M. ochroloma to compare infectivity, LT, and lethal concentrations (LC).

Confirmed infectivity rates for 1, 2, 3, and 4 g concentrations were 2, 5, 10, and 27%,

respectively. The LTio and LT25 for the 4 g concentration were 3.4 and 5.7 days,









respectively. The LC1o and LC25 were 1.4 g and 5.5 g per 100 ml of distilled water,

respectively.

The results of my research suggest that P. maculiventris is a promising biological

control agent of M. ochroloma. This predator can be used in an augmentative biological

control program in cruciferous crops to control M. ochroloma on organic farms. Isaria

fumosorosea (PFR-97TM), on the other hand, does not show any clear evidence of being

a potentially effective biological control agent of M. ochroloma.









CHAPTER 1
GENERAL INTRODUCTION

Vegetable production is one of the most important sources of income in the

United States. In the Census of Agriculture of 2007, the USDA ranked Florida as the

second highest state with sales in vegetables, melons, potatoes, and sweet potatoes.

From 2002 to 2007, the market value of vegetable production in Florida increased 25%

(USDA-NASS, 2007). The organic vegetable niche has been growing fast since 1997.

According to Florida Organic Grower (2008), 42 farms producing broccoli, turnips,

cabbage, collards, cauliflower, and Chinese cabbage are included on the list of certified

organic growers. The most popular organic vegetables are the "green leaves", in which

the leaves are the main commercial part of the plant. Most of the pests that attack

"green leaves" defoliate their host plants, making them unavailable for the grower to

offer good quality food for the consumers.

Pest management programs on organic farms are based on eco-friendly

strategies. The National Organic Program (NOP) states in the Electronic Code of

federal Regulations (eCFR) (Standards) that "the producer must use management

practices to prevent crop pests, including, but not limited to: 1) augmentation or

introduction of predators or parasites of the pest species; 2) development of habitat for

natural enemies of pests; 3) nonsynthetic controls such as lures, traps, and repellents"

(USDA 2010). My research focused on the first and third of these Federal regulations to

prevent crop pests. This was achieved by evaluating the efficacy of a generalist

predator, Podisus maculiventris (Say), and the infectivity of an entomopathogenic

fungus, Isaria fumosorosea Wize (Brown and Smith), against an invasive pest,









Microtheca ochroloma Stal, commonly called the yellowmargined leaf beetle, in

cruciferous crops.

Podisus maculiventris was chosen because it was observed in cruciferous crops

feeding on larvae and adults of M. ochroloma, and because it can be used for

augmentative biological control (Hough-Goldstein and McPherson 1996; De Clercq et

al. 1998). Isaria fumosorosea is known worldwide as a microbial insecticide due to its

diversity of infective strains and broad host range (Smith 1993). The commercial strain

Apopka 97, registered as PFR-97TM 20% WDG, was chosen for this study because it is

local to Florida (Vidal et al. 1998), and because more information about its host range

needs to be known.









CHAPTER 2
LITERATURE REVIEW

Microtheca ochroloma Stal

Origin and Distribution

The yellowmargined leaf beetle, Microtheca ochroloma Stal (Coleoptera:

Chrysomelidae), was previously known as Microtheca punctigera Achard. Jolivet

(1950) revised the genus Microtheca and recognized genitalic differences between

these two species. Microtheca ochroloma is an invasive insect from South America,

specifically Argentina, Brazil (Rio de Janeiro and Rio Grande do Sul) (Silva et al. 1968;

Racca Filho et al. 1994), Chile, and Uruguay. In the U.S., it was first reported from New

Orleans in 1945, occurring in imported grapes coming from Argentina (Chamberlin and

Tippins 1948). Since then, this pest has spread to Texas (Balsbaugh 1978), Alabama

(Chamberlin and Tippins 1948), Mississippi (Rohwer et al. 1953), North Carolina

(Staines 1999), Louisiana (Oliver 1956), Georgia (Guillebeau 2001), and Florida. In

1972, it was reported on watercress at an aquatic nursery in Tampa, Florida (Woodruff

1974).

Biology and Host Range

Microtheca ochroloma receives the common name "yellowmargined leaf beetle"

because its elytra are brownish to black with yellow margins and four prominent rows of

punctures on each elytron. The eggs are yellow to orange, oval, and often laid in the

soil (Fig. 2- 1A). The larva is yellow to brown, with a sclerotized head capsule

(Woodruff 1974) (Fig. 2-1 B). The immature stage takes 26.6 d at 200C to complete its

development from egg to adult, and the larval stage usually has four instars. However,

laboratory experiments showed that 5% of the population passes through a fifth instar









(Ameen and Story 1997b). When the larvae are ready to pupate, they build a brownish,

net-like pupal case (Fig. 2-1 C) to surround themselves on dry old leaves (Woodruff

1974). The pupal stage lasts from 5 to 6 d at 200C (Ameen and Story 1997b). The adult

of M. ochroloma is about 5 mm long; usually the females are larger than the males (Fig.

2-1 D). Newly emerged adults stay on the dry leaves for 2 d before moving to green

foliage (Capinera 2001). Adults live to as many as 186 d when fed radish plants

(Ameen and Story 1997b).

In Florida, M. ochroloma is present in the field during the cool months of October

to April, which corresponds to the production season of many of its vegetable hosts

(Bowers 2003). The host range of this pest includes all members of the Brassicaceae

family, such as cabbage, collards, turnip, radish, and watercress (Chamberlin and

Tippins 1948). Laboratory studies showed that turnip is the preferred host, since higher

average fecundity (490 eggs per female) was obtained on this host plant compared to

other plant species (198 eggs per female) (Amen and Story 1997a).

Damage and Summer Activity

Microtheca ochroloma is a serious pest of high value crops such as leafy Brassica

greens. Larvae and adults feed on the plant foliage and can completely defoliate their

host plants (Fig. 2-2A, B). When the beetle populations are very high, feeding on the

tubers of turnips can occur (Fig. 2-2C, D). Leaf quality is also affected by the abundant

frass produced by the larvae. Bowers (2003) lists three reasons why M. ochroloma is a

problem in Florida: (1) the host plants thrive in the cool months from October to April,

concurring with the growing season for organic farmers in Florida; (2) hard frosts or

freezes are unlikely to occur; and (3) there are no known predators or parasites in the

U.S.









Although the damage caused by M. ochroloma occurs during in the coolest

months of the year, Bowers (2003) believes that M. ochroloma is present during the

summer in a reproductive quiescence, since adults only were found in the same spots in

which turnips were planted before, also suggesting that the beetle passes the summer

near its feeding sites. To corroborate these observations, some adult beetles were

collected from the field in summer and after 24 hours of exposure to laboratory

conditions females began laying eggs (Bowers 2003). This indicates that M. ochroloma

does not require a lengthy period of time to reactivate its reproductive functions

compared to other insects that do need several days to end the diapause process.

Diapause is a physiological resting period mediated by environmental factors that are

unfavorable to the insect, so that it can survive certain adverse conditions such as

extreme temperatures, and is often triggered by photoperiod (Danks 1987). To resume

activity, the insect first must complete the diapause development mediated by favorable

conditions (Danks 1987). Quiescence is a dormancy that is mediated directly by

extreme environmental factors (e.g., scarcity of food, extreme temperatures). The insect

can respond to favorable environmental factors and resume normal activities

immediately without having to pass through a pre-programmed period as in diapause

(Danks 1987).

Control Methods

Chemical control. Control of M. ochroloma has been reported by growers in

Texas using products such as carbaryl (Sevin) and diazinon or mevinphos (Phosdrin)

(Drees 1990). In Brazil, Bastos-Dequech et al. (2008) studied the effect of plant

extracts on M. ochroloma. One hundred percent larval mortality was achieved after 5 d

in the laboratory at 27 1C by applying p6-de-fumo (Nicotiana tabacum L.,









Solanaceae) and ramo de cinamomo (Melia azedarach L., Meliaceae). Mortality in

adults reached 82 and 74% when DalNeem (a commercial product based on

Azadirachta indica A. Juss, Meliaceae) and p6-de-fumo were applied, respectively.

Basulu and Fadamiro (2008) evaluated the effect of OMRI biorational insecticides

against M. ochroloma in the field. PyGanic (extract of chrysanthemum flowers), Aza-

Direct azadirachtinn from neem plant), and Mycotrol O [entomopathogenic fungus

Beauveria bassiana (Balsmo) Vuillemin] were not effective in reducing populations of M.

ochroloma, but Entrust (spinosad for organic production) did lower pest densities and

mean damage ratings. Similar results were obtained when Overall and Edelson (2007)

evaluated organic insecticides in the field. Results indicated that Entrust and

PyGanic reduced populations of M. ochroloma by 96 and 63%, respectively.

Cultural control. The need to develop ecological methods to control M.

ochroloma is evident to organic growers. Currently, there is little information about the

ecology of the pest. Bowers (2003) studied emergence and host finding behavior and

evaluated intercropping as a tactic for reducing the severity of outbreaks. Results of her

studies showed that there were more adult beetles in the interior of the plots than in the

border (55 versus 8 beetles, respectively), suggesting that a large number of adults

oversummer in the production field. Intercropping failed to be a useful control strategy

for this pest. Bowers (2003) found that intercropping host plants (mizuna: Brassica rapa

L., var. Kyona, Cruciferae) among non-host plants (oak leaf lettuce: Lactuca sativa L.

var. Berenice, Asteraceae) was not effective in preventing M. ochroloma from finding

host plants.









Biological control. Currently, there are no natural enemies of M. ochroloma

reported in the literature (Bowers 2003). However, a generalist predator, the spined

soldier bug, Podisus maculiventris (Say) (Hemiptera: Pentatomidae), was observed in

the field preying on the larvae, adults, and pupae of M. ochroloma. In addition,

laboratory bioassays showed that larvae of the green lacewing, Chrysoperla rufilabris

(Burmeister) (Neuroptera: Chrysopidae) prey on eggs and larvae of M. ochroloma

(personal observation, C. Montemayor).

Podisus maculiventris (Say)

Origin and Distribution

The distribution of P. maculiventris includes North America from Quebec to British

Columbia in Canada and from Florida to Arizona in the U.S.A. (McPherson 1982). In

northern Florida, the occurrence of P. maculiventris is temporal; adults emerge in April-

May and are active in the field throughout the spring and summer until October-

November when the overwintering period begins (Herrick and Reitz 2004).

Biology and Host Range

Various aspects of the biology, behavior, and life history of P. maculiventris are

well documented in the literature (Wiedenmann and O'Neil 1990; O'Neil 1997; Legaspi

2004). Depending on the photoperiod and temperature, the life cycle can vary from 27

to 38 d. Eggs are 1 mm in diameter with long projections around the operculum. The

nymphs pass through five instars of which all except the first are predaceous.

Cannibalism is common from the second instar to adult. Total developmental time from

egg to adult at 27 1 C is 27 d (Richman and Whitcomb 1978). This predator can feed

on more than 75 prey species within 8 orders, mainly Coleoptera and Lepidoptera

(McPherson 1980), on a wide diversity of agricultural crops. Frequently utilized prey









include the Colorado potato beetle [Stilodes decemlineata (Say)], the asparagus beetle

(Crioceris asparagi L.), the three-lined potato beetle (Lema trilinea Olivier), the Mexican

bean beetle (Epilachna varivestis Mulsant), the fall webworm (Hyphantria cunea Drury),

and the diamondback moth (Plutella xylostella Curtis) (Landis 1937).

Searching Behavior

According to O'Neil (1997), the searching strategy of P. maculiventris differs

between laboratory and field conditions. In the laboratory, the attack rate increases as

prey density increases, but the estimated area searched decreases. The decline in the

area searched was associated with accumulated handling time of the prey. In the field,

P. maculiventris maintains a constant low rate of attack even when prey density

increases. In a potato field experiment, he found that the estimated area searched

declined as Colorado potato beetle density increased, but it can not be attributed to the

accumulated handling time of the prey because the predator attacked few prey in the

field (<1 prey per day). This searching behavior in the field may permit P. maculiventris

to survive periods of low prey densities using their stored body lipids and trading off

reproduction for survival (Wiedenmann and O'Neil 1990).

Biological Control

Podisus maculiventris plays an important role in augmentative and conservation

biological control of agricultural pests (Biever and Chauvin 1992; Hough-Goldstein

1996; Tipping et al. 1999). It is commercially available and is commonly used for

augmentative releases in the field to control the Mexican bean beetle (O'Neil, 1988;

Wiedenmann, 1991), the Colorado potato beetle (Stamopoulos and Chloridis, 1994;

Tipping et al. 1999), and the viburnum leaf beetle [Pyrrhalta viburni (Paykull)]

(Desurmont 2008). The efficiency of P. maculiventris as a predator can vary among









host plant species and ecosystems. For example, the presence of allelochemicals in

the host plant or in the prey's diet can directly or indirectly affect the performance of P.

maculiventris due to its facultative plant-feeding behavior (Desurmont 2008). In

addition, the size and shape of the host plant (or leaves) can decrease the efficiency of

the predator by providing more leaf surface or more refugia to the prey (Desurmont

2008). The use of generalist native predators in agroecosystems to suppress pest

populations may reduce risks to non-target organisms by avoiding the introduction of

exotic biological control agents and their non-target impacts in the field. In addition,

growers can easily obtain these generalist predators because they are commercially

available.

Isaria fumosorosea Wize (Brown and Smith)

Origin and Distribution

The entomopathogenic fungus Isaria fumosorosea Wize (Brown and Smith)

(=Paecilomyces fumosoroseus) (Hypocreales:Cordycipitaceae) has a worldwide

distribution in temperate and tropical zones. Different strains of /. fumosorosea are

listed in 27 countries in the catalogue of "USDA-ARS Collection of Entomopathogenic

Fungal Cultures" (ARSEF) (Humber et al. 2009). According to Domsch et al. (1980), /.

fumosorosea is well distributed from Europe to Africa an Asia, and occurs naturally in

soil in the Netherlands, Germany, Canada, and Brazil. It has been isolated from the

surface of dead insects (Greif and Currah 2007). In Florida, it was isolated from

Phenacoccus sp. (Hemiptera: Pseudococcidae), and named the Apopka 97 strain (Vidal

et al. 1998).

Isaria fumosorosea was described in 1832 by Fries and later by Wize in 1904.

After the study of the genus Paecilomyces Samson by Samson (1974), I. fumosorosea









was included in a new section called Paecilomyces section Isariodea, and since then

the fungus was named Paecilomyces fumosorosea (Wize) Brown & Smith for several

years (Zimmerman 2008). However, phylogenetic studies of species belonging to this

new section showed that it is polyphyletic within the Hypocreales (Ascomycota), and the

Isaria clade, which includes I. fumosorosea, was elevated to genus rank (Luangsa-ard

2005).

Biology and Host Range

Abiotic environmental factors are important in the growth, viability, germination,

and insecticidal activity of I. fumosorosea (Vidal and Fargues 2007). Vidal et al. (1997b)

determined that temperature is important in the optimal growth of I. fumosorosea, in

which it can range from 20 to 300C, depending on the isolate. In Europe (French and

Italian isolates), the optimal growth of I. fumosorosea is from 20 to 250C, with higher

tolerance to lower temperatures from 8 to 15C. In tropical and subtropical areas

(Texas, Florida, Cuba, and California isolates), optimal growth is from 25 to 280C, with a

greater tolerance to higher temperatures from 32 to 350C. Indian isolates have optimal

growth at 300C, with higher tolerance to higher temperatures compared to the European

and tropical and subtropical isolates (Vidal et al. 1997b). Solar radiation and low

relative humidity are other abiotic factor than can negatively affect the performance of

this fungus in the field (Fargues et al. 1996). Germination of conidia or blastospore

growth of mycelia are inhibited by allelochemicals (tomatine, solanine, camptothecin,

xanthotoxin, and tannic acid) produced by the host plant (Poprawski and Jones 2001;

Lacey and Mercadier 1998).

Isaria fumosrosea produces the toxin beauvericin, which is also produced by B.

bassiana and Fusarium species to kill insects. In addition, dipicolinic acid was isolated









from /. fumosorosea and confirmed to have an insecticidal effect on third instar Bemisia

tabaci (Gennadius) type B (Bernardini et al. 1975; Asaff et al. 2005). Isaria

fumosorosea has a broad host range in the field, including the beetles S. decemlineata

(Bajan 1973), Hypothenemus hampei Ferrari, and Popilliajaponica Newman (Humber

et al. 2009), the moths Y. maculipennis, Galleria mellonella (L.), and Spodoptera

frugiperda Smith (Smith 1993), and the flies Musca domestic L. (Humber et al. 2009)

and Crossocosmia zebina (Walker) (Smith 1993).

Biological Control

There are several commercial products based on I. fumosorosea available in the

market against arthropod pests. For example, Multiplex Mycomite and Priority are

for control of mites; Micobiol HE is for control of beetles, moths, and nematodes; Pae-

Sin controls whiteflies (Zimmerman 2008); and PFR-97TM 20% WDG (Apopka strain)

controls piercing-sucking insects in greenhouses (Zimmerman 2008).

Isaria fumosorosea strains have been used successfully used against whitefly

populations in enclosed environments such as greenhouses (Osborne et al. 2008). In

field evaluations, the strain CKPF-095 gave effective control of 2nd instar diamondback

moth at 1 x 109 conidia/g (Maketon et al. 2008). In Florida's citrus groves, Meyer et al.

(2008) discovered the strain /frAsCP infesting adults of Asian citrus psyllid (Diaphorina

citri Kuwayama). IfrAsCP was compared to PFR-97TM and results showed that Ifr

AsCP is different from, but related to, PFR-97 TM. Later, Hoy et al. (2010) determined

that /frAsCP is highly pathogenic to the adult Asian citrus psyllids when the insects

were exposed to spores collected from dead psyllids stored at -740C.









PFR-97 TM 20 % WGD is registered in the U.S.A. and manufactured by Certis

USA. It is used in greenhouses and nurseries to control aphids, mites, and whiteflies on

ornamental plants. However, PFR-97 TM has been studied in the laboratory against

various pests in food crops in order to be approved for application in the field. The

overall goal of my thesis research is to provide organic farmers with environmentally

friendly and effective control measures to suppress M. ochroloma populations in the

field by using a native generalist predators and/or an entomopathogenic fungus.

Objectives of Master of Science Thesis Research

1. Measure the development time and predation rate of Podisus maculiventris when
feeding on Microtheca ochroloma in the laboratory

2. Evaluate predation potential of Podisus maculiventris on larvae of Microtheca
ochroloma in the field

3. Assess the infectivity by Isaria fumosorosea on Microtheca ochroloma in the
laboratory








































Figure 2-1. Life stages of Microtheca ochroloma. A) Eggs, B) Larva, C) Pupa, and D)
Adults.


























iAIn .7 fiAfi A L L' iKW iM b, ".^., B




















C D

Figure 2-2. Damage by Microtheca ochroloma. A), Larvae feeding on leaves, B) Total
defoliation of the turnip plant, C) Severe root damage by the larvae, D) Larvae
feeding on the turnip's roots.









CHAPTER 3
DEVELOPMENT TIME AND PREDATION RATE OF PODISUS MACULIVENTRIS
(SAY) (HEMIPTERA: PENTATOMIDAE) PREYING ON MICROTHECA OCHROLOMA
STAL (COLEOPTERA: CHRYSOMELIDAE)

Introduction

The yellowmargined leaf beetle, Microtheca ochroloma Stal, is a pest in crucifer

crops in the southern United States. It is native to Argentina, Uruguay and Brazil

(Chamberlin and Tippins 1948; Dos Anjos et al. 2007). Its preferred host is turnip

(Brassica rapa L.), followed by mustard (Brassicajuncea Cosson) (Ameen and Story

1997a). The main damage caused by M. ochroloma is defoliation, but when infestations

are severe and leaves are entirely consumed, the beetle will feed on the roots (C.

Montemayor, personal observation). There are no specialist natural enemies reported

in the literature that can contribute to the control of this pest on organic farms (Fasulo

2005). Nevertheless, there are generalist predators present in agricultural ecosystems

that may contribute to the management of M. ochroloma.

The spined soldier bug, Podisus maculiventris (Say) (Hemiptera: Pentatomidae:

Asopinae), is a generalist predator present in many agroecosystems, where it preys

primarily on Coleoptera and Lepidoptera larvae (McPherson 1980). In laboratory

studies, nymphs and adults of P. maculiventris showed high potential predation rates

against different life stages of the beet armyworm, Spodoptera exigua (Hcbner) (De

Clercq and Degheele 1994). On organic farms, P. maculiventris has been observed on

crucifer crops, preying on all stages of M. ochroloma. The use of P. maculiventris in

augmentative biological control may serve as a tool to reduce populations of M.

ochroloma. However, no studies have been conducted to evaluate the efficacy of this

predator as a biological control agent of this pest. Therefore, the objective of this study









was to know if M. ochroloma would be a suitable prey by quantifying the rate of

predation of M. ochroloma larvae by P. maculiventris and the developmental time of the

predator at four constant temperatures under laboratory conditions.

Materials and Methods

Stock Colonies. Adults and larvae of M. ochroloma were brought from White

Rabbit Acres certified organic farm located in Vero Beach, FL to the laboratory at the

Biological Control Research and Containment Laboratory (BCRCL) at the Indian River

Research and Educational Center (IRREC) in Ft. Pierce. The colony was established

and maintained in plastic boxes (27 x 15 x 8 cm, Ziploc) with screen mesh openings in

the walls for ventilation. Boxes were kept in an environmentally controlled chamber at

25C, 50% RH, and 10L:14D photoperiod.

A bottle of 250 eggs of P. maculiventris was purchased online from Rincon-Vitova

Insectaries, Inc. (Ventura, CA [www.rinconvitova.com]) and shipped with overnight

delivery. After arrival, eggs were divided into groups of 30 and each group was placed

in a Petri dish (60 x 75 mm, Fisherbrand) with moistened filter paper (55 mm 0

[diameter], Whatman). The Petri dishes were sealed with Parafilm and stored in an

environmentally controlled chamber at 250C, 50% RH, and 10L:14D photoperiod.

Plant Material. Turnip Seven Top (Greens) (Brassica rapa L. var. rapifera) seeds

were seeded in 72-hole trays containing sterilized soil mix (Fafard germination mix,

Agawam, MA) inside a greenhouse. Seedlings were transplanted 2 weeks later into 3.8

L plastic pots containing soil mixture (Fafard #3B mix). The plants were fertilized

weekly with 400 ml per pot of liquid fertilizer (Miracle Grow 24N-8P-16K).

Experimental Design. An individual neonate P. maculiventris was housed in a 7-

cm3 plastic box (Fig. 1) with a hole in the top sealed with screen mesh. To maintain









proper humidity in the box, a piece of white filter paper (90 mm 0, Whatman)

moistened with water was placed on the bottom of the cage. Each box held a 60 mm 0

piece of turnip leaf that was replaced daily. The number of 4th instar M. ochroloma

provided daily to P. maculiventris varied among instars: five prey were provided to 2nd

and 3rd instar P. maculiventris and 10 prey were provided to 4th and 5th instar P.

maculiventris. The boxes with insects were held in environmentally controlled

chambers at each of four constant temperatures, 10, 15, 20, and 250C, with 50% RH

and 10L:14D photoperiod. HOBO data loggers were placed inside each chamber to

monitor the temperature and humidity. Each treatment had at least 12 replicates.

Number of M. ochroloma larvae killed daily by each predator nymph was recorded.

Total predation per instar was thus determined and the total predation per nymph was

measured as the total predation through all instars. Total development time for each

predator instar and for the total nymphal stage was measured in days. Predation by the

adult stage of P. maculiventris was evaluated for the first 10 d of adult life. Fresh

weights of newly ecdysed nymphs and adults were measured using an Ohaus

Adventurer AR2140 analytical scale. A control treatment with three replicates consisted

of five to ten 4th instar M. ochroloma (number varied gradually with P. maculiventris

development in other treatments) in the absence of P. maculiventris to record natural

mortality. Dead larvae in the control were replaced daily. Mortality in the control

treatment was used as a correction factor for the mortality in the predator treatment.

Development time and daily and total predation of eggs of M. ochroloma by 2nd to

5th instar P. maculiventris were measured in Petri dishes at 250C. Each Petri dish (60 x

75 mm, Fisherbrand) contained one predator, eggs of M. ochroloma, and a white









moistened filter paper. The number of eggs of M. ochroloma provided daily to P.

maculiventris varied among instars: 50 eggs were provided to 2nd, 100 eggs to 3rd instar,

200 eggs to 4th instar, and 500 eggs to 5th instar P. maculiventris. Turnip leaves were

not provided.

Statistical Analysis. Data were analyzed using analysis of variance (ANOVA).

Treatment means were separated using the Student-Newman-Keuls (SNK) test (SAS

Institute, 1999) at a significance level of 5%. Means are reported with their standard

error.

Results

Two-way ANOVA indicated a significant interaction between temperature and

instar of P. maculiventris for all variables evaluated (developmental time, fresh weight,

daily and total predation) (F = 25.03; df = 17,181; P < 0.0001 for developmental time; F

= 12.49; df = 14,150; P< 0.0001 for fresh weight; F= 39.72; df = 14,128 P< 0.0001 for

daily predation; F = 53.95; df = 14,128; P < 0.0001 for total predation). At 10C, eggs

did not hatch. First instar P. maculiventris reared at this same temperature died about

26 2.9 d after hatching at 25C, therefore no data was obtained to measure

developmental time at 10C. The 1st instar of P. maculiventris is a non-feeding stage,

thus no daily and total predation rates were determined for this stadium.

Development Time. Mean development times of P. maculiventris eggs and

instars varied with temperature (Table 3-1). Mean developmental times of eggs, 1st, 2nd,

3rd, 4th, and 5th instars reared at 15C were significantly higher than those at 20 and

25C (F = 6170.37; df = 2,31; P < 0.0001 for egg; F = 503.16; df = 2,42; P < 0.0001 for

1st instars; F = 131.88; df = 2,30; P <0.0001 for 2nd instars; F = 315.93; df = 2,27; P

<0.0001 for 3rd instars; F = 57.10; df = 2,26; P = < 0.0001 for 4th instars; F = 926.75; df =









2,25; P < 0.0001 for 5th instars). Mean development times of eggs, 1st instars, and 5th

instars at 20C were significantly higher than those at 25C; there were no differences

between these two temperatures for the 2nd, 3rd, and 4th instars. Mean total

development times of nymphs varied significantly with temperature (F = 814.5; df= 2,25;

P < 0.0001) (Table 3-5); the development time at 25C was significantly less than the

development time at 20C, which was significantly lower than the development time at

150C.

There were significant differences in development time among instars within each

temperature (Table 3-1). At all experimental temperatures the mean development time

of the 5th instar was significantly higher than the mean development times of the other

four instars. At 15C, mean development times of eggs, 2nd instars, 3rd instars, and 4th

instars were not significantly different from each other, but they were significantly higher

than that of the 1st instar (F = 30.12; df = 5,49; P < 0.0001). At 20 and 25C, mean

development time of eggs was significantly higher than those of 2nd and 4th instars,

which were significantly higher than mean development times of 1st and 3rd instars (F =

99.02; df = 5,66; P = < 0.0001 for 20C; F = 57.34; df = 5,66; P < 0.0001 for 25C).

Mean development times of 2nd to 5th instar P. maculiventris feeding on M.

ochroloma eggs were 3.9 0.1 d, 3.8 + 0.1 d, 4.0 0.0 d, and 5.7 0.2 d, respectively.

These means were not significantly different from those of nymphs feeding on M.

ochroloma larvae at 25C (Table 3-1) (P = 0.1325). However, the total development

time of the nymphal stage of the predator feeding on 4th instars of M. ochroloma was

significantly shorter (23 0.3 d) than when feeding on eggs (25 0.3 d) (F= 36.51; df =

1,20; P< 0.001).









Fresh Weight. Mean fresh weights of some instars varied significantly with

temperature (Table 3-2). Mean fresh weights of 3rd and 5th instars reared at 25C were

significantly higher than those at 20C, which were significantly higher compared to

those at 15C (F = 14.23; df =2,29; P < 0.0001 for 3rd instars; F = 19.29; df = 2,26; P <

0.0001 for 5th instars). There were no significant differences in mean fresh weight of 4th

instars and adults between 20 and 25C, but their mean fresh weights at these two

temperatures were significantly higher compared to those at 15C (F = 11.66; df = 2,27;

P = 0.0002). There were no significant differences in mean fresh weight of 2nd instars

among the three temperatures. As with development time, there were significant

differences in mean fresh weights among stages within each temperature (Table 3-2).

Mean fresh weight at all experimental temperatures increased significantly with each

successive stage.

There was no significant interaction between temperature and sex for fresh weight

of P. maculiventris adults. Mean fresh weights of adult females and males were not

significantly different at 25C. However, at 20C, the mean fresh weight of females was

significantly higher than the mean fresh weight of males (Table 3-6) (F = 16.76, df =

1,10; P = 0.0022). Mean fresh weights of females and males reared at 15C were not

statistically compared due to low sample sizes, yet the female adult weight tended to be

higher than male adult weight.

Daily Predation. There were no significant differences in mean daily predation by

the 2nd and 3rd instars between 20 and 25C (Table 3-3). However, mean daily

predation by these instars at those two temperatures were significantly higher than

mean daily predation at 15C. Mean daily predation by 4th instars, 5th instars, and adults









was significantly higher at 25C than that at 20C, and mean daily predation was

significantly higher at 20C than at 15C (F = 36.82; df = 2,26; P < 0.0001 for 4th instars;

F = 78.92; df = 2,25; P < 0.0001 for 5th instars; F = 79.03; df = 2,19; P < 0.0001 for

adults).

Mean daily predation also varied significantly among stages at each temperature

(Table 3-3). At 15C, the adult stage had significantly lower mean daily predation

compared to that of the 5th instar, however, it was not significantly different from the

mean daily predation by the 2nd, 3rd, and 4th instars (F = 3.00; df = 4,24; P = 0.0387). At

20C, mean daily predation increased significantly with each successive stage (F =

123.00; df = 5,66; P < 0.0001). At 25C, there was no significant difference in mean

daily predation between the 5th instar and adult (F = 186.99; df = 5,60; P < 0.0001 ).

However, their rates were significantly higher than predation by the 4th instar, which was

higher than that by the 3rd instar. Second instar mean daily predation was significantly

the lowest among all stages at 25C. Mean daily predation of eggs of M. ochroloma by

P. maculiventris nymphs increased significantly with each successive instar (F = 159.14;

df = 3,36; P < 0.0001) (Table 3-7). Embryonic fluids within the eggs were totally

consumed by the predator.

Total predation. Mean total number of M. ochroloma larvae consumed by each

P. maculiventris stage also varied significantly among temperatures (Table 3-4). There

was significantly higher mean total predation by the 2nd instar at 15C compared to 20

and 25C (F = 50.80; df = 3,31; P < 0.0001 ). Mean total predation by the 3rd instar was

significantly higher at 25C than at 15 and 20C (F = 6.64; df = 2,27; P = 0.0045 ). Mean

total predation by the adults was significantly higher at 25C than at 20C which was









higher compared to predation at 15C (F = 79.03; df = 2,19; P < 0.001 ). On the other

hand, there were no significant differences in mean total predation by 4th and 5th instars

at 15, 20, and 25C. Mean total predation of prey by the nymphal stage varied

significantly with temperature (F = 4.72; df = 2,25; P = 0.0182) (Table 3-5); predation at

20C was significantly less than predation at 15C, but predation at 25C was not

significantly different from predation at the lower two temperatures.

Similar to mean daily predation, mean total predation by P. maculiventris nymphs

varied significantly among stages at each temperature (Table 3-4). Mean total

predation by 5th instars at 15C was significantly higher than predation by 4th instars,

which was significantly higher than that by the adult stage; predation by the latter did not

vary significantly from predation by the 2nd and 3rd instars (F = 10.88; df = 4,24; P <

0.0001). Mean total predation at 20C was significantly higher by the adult stage

compared to the 5th instar, which was higher than the 4th and 3rd instars, and these

consumed significantly more prey than the 2nd instar (F = 210.30; df = 5,66; P < 0.0001).

At 25C, mean total predation was significantly higher by the adult stage than by the 5th

instar, which was higher than predation by the 4th instar; the latter was significantly

higher compared to the 2nd and 3rd instars (F = 349.78; df = 5,60, P < 0.0001 ). Mean

total predation of eggs of M. ochroloma by P. maculiventris nymphs increased

significantly with each successive instar (F = 250.29; df = 3,36; P < 0.0001) (Table 3-7).

Total consumption of eggs of M. ochroloma during the nymphal stage of P.

maculiventris was 741.0 24.8.

Discussion

The 7-cm3 plastic boxes used as cages provided more space for P. maculiventris

nymphs and adults to seek and attack the larvae of M. ochroloma in a more natural way









compared to Petri dishes. According to Wiedenmann and O'Neil (1991), attacks rates

in Petri dishes will be limited by handling time, but in the field or more complex arenas

they will be limited by search behavior. The M. ochroloma larvae that were attacked in

my study were most often killed, then either abandoned unconsumed or partially

consumed. De Clercq and Degheele (1994) reported similar observations when nymphs

preyed on 5th instars of the beet armyworm.

Neither development of 1st instars nor egg hatching of P. maculiventris was

successfully completed at 10C. These findings match with the estimated lower

developmental threshold for eggs 10.7C and nymphs 11.7C of P. maculiventris, as

determined by De Clercq and Degheele (1992). There was a well defined inverse

interaction between developmental time and temperature. Developmental time of all

stages of P. maculiventris is longer at 15C compared to higher temperatures (Table 3-

1). The fifth instar of P. maculiventris requires more time to develop than earlier instars,

regardless temperature (Table 3-1). More time in the 5th instar allows the daily and total

predation rates of the predator to be greater compared to those of the earlier instars

(Tables 3-3 and 3-4). Development times of nymphs reared at 20 and 25C and fed 4th

instar M. ochroloma are comparable to development times of nymphs reared on beet

armyworm larvae at 23C (De Clercq and Degheele 1994). Mahdian et al. (2006)

reported similar trends of consumption rate and developmental time of P. maculiventris

feeding on 4th instar Spodoptera littoralis (Boisduval) at 18, 23, and 27C. Desurmont

and Weston (2008) found that nymphs of P. maculiventris consumed an average of 101

larvae and 17 adults of Pyrrhalta viburni (Paykull) (Coleoptera: Chrysomelidae) and the









total developmental time from 2nd instar to adult was 22 d at 22C, which is very similar

to the total developmental time (21 d) at 20C in my study.

Podisus maculiventris is able to complete its life cycle by feeding on eggs only of

M. ochroloma. This suggests that the egg of M. ochroloma is an adequate food source

as is the 4th instar, and probably, too, the adult and pupa of the beetle since P.

maculiventris also feeds on them (C. Montemayor, personal observation). It is yet

unclear whether or not nymphs will search for eggs on dry leaves and in the soil;

however, if they do, the population of M. ochroloma could be significantly reduced due

to the predator's high predation of eggs (Table 3-6). Two other studies have reported

lower mean total consumption of other prey eggs by the nymphal stage of P.

maculiventris: 293 eggs of Stilodes decemlineata Say (Coleoptera: Chrysomelidae)

(Gusev et al. 1983) and 160 eggs of Epilachna varivestis (Mulsant) (Coleoptera:

Coccinellidae) (Waddill and Shepard 1975). However, De Clercq and Degheele (1994)

reported total nymphal consumption of 1,200 eggs of S. exigua.

Regarding to the weight measured of P. maculiventris in this study. The mean

fresh weight of newly ecdysed 5th instar P. maculiventris fed on 4th instar M. ochroloma

at 25C is 30.98 mg. This weight was comparable to the 27.3 mg in the study of De

Clercq and Degheele (1994), who fed the predator 4th instar S. exigua at 230C.

The numbers of M. ochroloma larvae killed by nymphs and adults suggest that P.

maculiventris has great potential to reduce populations of M. ochroloma under field

conditions in Florida, in which the natural temporal occurrence of the predator (spring-

summer) matches well with outbreaks of the pest in late spring (Herrick and Reitz

2004). In order to enhance the control of M. ochroloma in the field, augmentative









releases of the predator can be made in the late fall and early spring. Therefore, it is

essential to evaluate its predation potential under field conditions (see Chapter 4).


Figure 3-1. Plastic boxes with screened windows used to house experimental insects.









Table 3-1. Mean ( SE) development time of Podisus maculiventris reared at three constant temperatures with 4th instar
Microtheca ochroloma as prey. Number within parentheses equals sample size.
Mean development time (days)
Temp*
(C) Egg 1st instar 2nd instar 3rd instar 4th instar 5th instar
15 17.0 0.0 (10) Ba 10.0 0.2 (21) Ca 18.7 1.4 (9) Ba 15.0 0.9 (6) Ba 18.6 3.1 (5) Ba 25.5 1.0 (4)Aa
20 5.7 0.1 (12) Bb 4.0 0.0 (12) Db 4.5 0.3 (12) Cb 3.8 0.1 (12) Db 4.8 0.2 (12) Cb 8.1 0.1 (12) Ab
25 4.0 0.0 (12) Bc 3.0 0.0 (12) Dc 3.7 0.1 (12) Cb 3.4 0.1 (12) Db 3.9 0.1 (12) Cb 5.4 0.1 (12) Ac
Means followed by the same uppercase letter within a row are not significantly different (P > 0.05). Means followed by the
same lowercase letter within a column are not significantly different (P > 0.05). (*) Temperature.


Table 3-2. Mean ( SE) fresh weight of newly ecdysed Podisus maculiventris reared at three constant temperatures with
4th instar Microtheca ochroloma as a prey. Number within parentheses equals sample size.
Mean fresh weight (mg)
Temperature
(C) 2nd instar 3rd instar 4th instar 5th instar Adult (10 days)
15 0.77 0.0 (21) Da 2.01 0.2 (9) Dc 6.25 0.5 (6) Cb 14.20 2.7 (5) Bc 41.28 0.2 (5) Ab
20 0.84 0.0 (12) Da 2.69 0.2 (12) Db 8.96 0.6 (12) Ca 25.41 1.2 (12) Bb 59.15 2.3 (12) Aa
25 0.82 0.0 (12) Da 3.63 0.2 (12) Da 10.70 0.5 (12) Ca 30.98 1.6 (12) Ba 58.00 2.1 (12) Aa
Means followed by the same uppercase letter within a row are not significantly different (P > 0.05). Means followed by the
same lowercase letter within a column are not significantly different (P > 0.05).









Table 3-3. Mean ( SE) daily predation of 4th instar Microtheca ochroloma by Podisus maculiventris reared at three
temperatures. Number within parentheses equals sample size.
Mean daily predation of prey
Temperature
(C) 2nd instar 3rd instar 4th instar 5th instar Adult (10 days)
15 0.7 0.1 (10) ABb 0.7 0.1 (6) ABb 0.8 0.1 (5) ABc 1.2 0.2 (4) Ac 0.5 0.2 (4) Bc
20 1.1 0.1 (12) Da 2.3 0.2 (12) Ca 2.5 0.2 (12) Cb 3.4 0.1 (12) Bb 4.5 0.2 (12)Ab
25 1.0 0.1 (12) Da 1.9 0.1 (12) Ca 3.4 0.2 (12) Ba 6.5 0.3 (12) Aa 6.4 0.3 (6) Aa
Means followed by the same uppercase letter within a row are not significantly different (P > 0.05). Means followed by the
same lowercase letter within a column are not significantly different (P > 0.05).


Table 3-4. Mean ( SE) total predation of 4th instar Microtheca ochroloma by Podisus maculiventris reared at three
constant temperatures. Number within parentheses equals sample size.
Mean total predation of prey
S Temperature
S(C) 2nd instar 3rd instar 4th instar 5th instar Adult (10 days)
15 12.9 1.1 (10) CBa 11.0 1.8 (6) CBa 14.8 2.4 (5) Ba 30.0 5.8 (4) Aa 5.0 2.1 Cc
20 4.9 0.5 (12) Db 9.0 0.7 (12) Ca 12.0 0.9 (12) Ca 27.2 1.0 (12) Ba 45.1 2.3 (12) Ab
25 3.8 0.2 (12) Db 6.5 0.4 (12) Db 13.2 0.7 (12) Ca 35.6 2.0 (12) Ba 64.1 2.9 (6) Aa
Means followed by the same uppercase letter within a row are not significantly different (P > 0.05). Means followed by the
same lowercase letter within a column are not significantly different (P > 0.05).









Table 3-5. Mean ( SE) total development time and predation of 4th instar Microtheca
ochroloma per nymph of Podisus maculiventris. Number within parentheses
equals sample size.
Temperature (oC) Total development (days) Total predation (prey)
15 99 4.6 (4) a 65 6.3 (4) a
20 31 0.3 (12) b 53 1.3 (12) b
25 23 0.3 (12) c 59 2.1 (12) ab
Means followed by the same lowercase letter within a column are not significantly
different (P > 0.05).


Table 3-6. Mean ( SE) fresh weight of newly ecdysed adults of Podisus maculiventris
reared with 4th instar Microtheca ochroloma as a prey. Number within
parentheses equals sample size.
Mean fresh weight (mg)
Temperature (oC) Female Male
15 43.00 2.4 (2) 39.55 2.0 (2)
20 63.50 1.2 (8) a 50.45 4.0 (4) b
25 61.98 2.9 (6) a 54.00 2.3 (6) a
Means followed by the same lowercase letter within a row are not significantly different
(P> 0.05).









Table 3-7. Mean ( SE) predation of eggs of Microtheca ochroloma by Podisus maculiventris nymphs reared at 250C.
Number within parentheses equals sample size.


Mean predation of eggs
Predation 2nd instar 3rd instar 4th instar
Daily 6.3 0.6 (10) d 18.2 1.0 (10) c 50.2 3.1 (10) b
Total 24.2 2.1 (10) d 68.3 3.6 (10) c 187.7 12.6 (10) b
Means followed by the same lowercase letter within a row are not significantly different (P > 0.05).


5'" instar
83.5 4.4 (10) a
460.7 13.8 (10) a









CHAPTER 4
PREDATION POTENTIAL OF PODISUS MACULIVENTRIS (HEMIPTERA:
PENTATOMIDAE) ON MICROTHECA OCHROLOMA (COLEOPTERA:
CHRYSOMELIDAE) IN THE FIELD

Introduction

The yellowmargined leaf beetle, Microtheca ochroloma Stal, is a cool seasonal

pest in crucifer (Brassicaceae) crops, with turnips (Brassica rapa L.) and mustard

(Brassicajuncea Cosson) its preferred host (Amen and Story 1997a). In 1945, the first

specimen to be found in the United States was reported in New Orleans on grapes

coming from Argentina where it may also be a serious problem (Chamberlin and Tippins

1948). Since then, M. ochroloma has spread to and established in several southern

states in the United States (Staines 1999), including Florida where the beetle was found

on watercress in 1972 (Woodruff 1974). In Florida, M. ochroloma is present in the field

during the coolest months (October through April), which corresponds with the primary

production season for leafy greens (Bowers 2003).

For organic crucifer growers, it has been a challenge to maintain populations of M.

ochroloma under tolerable levels, given that growers are restricted to using insecticides

on the Organic Materials Review Institute (OMRI) list, and no known native specialist

natural enemies have been reported (Bowers 2003). The manipulation of generalist

predator populations to enhance control of M. ochroloma should be considered in

developing an integrated pest management program for this pest.

Predatory stink bugs (Hemiptera: Pentatomidae: Asopinae) are commonly used in

augmentative releases to control pests in agricultural ecosystems (Biever and Chauvin

1992; Hough-Goldstein 1996; Tipping et al. 1999). The spined soldier bug, Podisus

maculiventris (Say), is a generalist predator native to North America (McPherson 1982).









Coleoptera and Lepidoptera larvae are its main prey (McPherson 1980). This generalist

predator has been released in the field and in greenhouses to control pests in tomato

and cotton with successful results (Lopez et al. 1976; Ables and McCommas 1982; De

Clercq et al. 1998). Hough-Goldstein and McPherson (1996) reported that

augmentative releases of P. maculiventris in small field plots reduced the larval

population of the Colorado potato beetle, Stilodes decemlineata (Say). The study of

predation rates in field-cage experiments demonstrated that the 5th instar of P.

maculiventris could kill an average number of 15 3rd instar S. decemlineata

(Stamopoulos and Chloridis 1994).

In central Florida, P. maculiventris has been observed preying on M. ochroloma

(C. Montemayor, personal observation). However, little is known about its predation

potential on field populations of M. ochroloma. In northern Florida, P. maculiventris is

present beginning in March and starts overwintering in October (Herrick and Reitz

2004). This period of time permits natural populations of P. maculiventris to interact

with the pest for the last couple of months of late spring in the crucifer growing season,

since M. ochroloma is a problem during the cooler months of the year in Florida. Early

augmentative releases of P. maculiventris in cruciferous crops in the field may

contribute to control of M. ochroloma.

The present study evaluates the predation capacity of P. maculiventris at different

densities in field cages containing M. ochroloma. The goal of this study is to provide

growers a guideline for releasing P. maculiventris. This way the predator can be used

as a new integrated pest management tool to control M. ochroloma.









Materials and Methods

Stock Colonies. Adults and larvae of M. ochroloma were obtained from the field

and a laboratory colony was established and maintained as described in Chapter 2.

The releases of P. maculiventris were designed to simulate as much as possible the

way that growers would release the predator based on the recommendations of the

vendor. Therefore, eggs of P. maculiventris were purchased and held in the laboratory

as described in Chapter 2. When the first 100 1st instars emerged, they were held for 5

d at 15C and then released into the field cages.

Plant Material. Small plants of Seven Top (Greens) variety turnips (B. rapa var.

rapifera) were grown as described in Chapter 2. Three weeks after transplanting in

pots, seedlings were transplanted into field beds at the IRREC. Four beds, each 100 m

long and 0.70 m wide, were covered by white plastic mulch. Granulate fertilizer (8N-

12P-20K with minor elements; Howard Fertilizer Co., Inc., Orlando, FL) was placed in

the center of the beds at a rate of 85 g/m. Distance between plants was 30 cm,

distance between rows was 30 cm, and distance between beds was 90 cm. Irrigation

was provided by flooding between beds.

Experimental Design. Cage frames 1.35 m long, 0.90 m wide, and 0.70 m high

were constructed of 1.904 cm (3/4 inch) PVC (polyvinyl chloride) tubing. Fine mesh

cloth was sewn in a manner to fit tightly over each frame; around the base of the cloth

cage was a 15-cm skirt. Each cage was placed over a field bed with six turnip plants.

Soil was heaped on the skirt to avoid the entrance or exit of animals and anchor the

cage in place. One hundred thirty-two 1st instars of M. ochroloma were introduced into

each cage (22 larvae per plant). In 2009, the insects were introduced into the cage 20 d

after the turnip plants were transplanted into the field; in 2010, the insects were









introduced into the cage 10 d after transplanting. On the same day as the introduction

of M. ochroloma into the cages, one of three densities (4 = low, 10 = medium, or 16 =

high) of 1st instar P. maculiventris was released evenly among the six plants in a cage.

Each treatment (P. maculiventris density) was replicated four times. Rincon-Vitova

Insectaries recommends releasing 25 eggs per "hot-spot" or 25 eggs per 10 m2 for

caterpillar control. In other words, Rincon-Vitova's release recommendation for my

study would be three 1st instars per 1.21 m2 (area of the cage). However, by

comparison, the actual release rates in my study were 33 1st instars in the low predator

density treatment, 83 1st instars in the medium predator density treatment, and 132 1st

instars in the high predator density treatment per 10 m2

Treatment arrangement was randomized within each replicate block (Fig. 1). The

experiment was conducted twice, once in February-March 2009 and once in February-

March 2010. All plants in each cage were monitored every four days for four weeks in

2009 and for five weeks in 2010, and the numbers of M. ochroloma larvae, pupae, and

adults and P. maculiventris nymphs and adults per plant were recorded. At the end of

both experiments, all plants were pulled out of the cages and brought into the laboratory

for the last sampling (Fig. 2). A HOBO data logger (Onset Computer Corporation,

Bourne, Massachusetts) was placed inside a cage to monitor temperature and relative

humidity during the experiments.

Statistical Analysis. The effect of predator density on the caged M. ochroloma

population was analyzed by comparing the number of live M. ochroloma per cage with

analysis of variance with repeated measures over time. Treatment means were









separated by time using the Student-Newman-Keuls (SNK) test (SAS Institute, 1999)

at a significance level of 5%. Means are reported with their standard error.

Results

2009 Experiment

The mean temperature during the experiment was 16.70C 0.1. The mean

maximum temperature was 23.20C 0.6. Fifteen days had a maximum above this

temperature, with 27.9C as the highest recorded temperature for three days between

1:00 and 1:30 p.m. The mean minimum temperature was 10.80C 0.9. Fourteen days

had a minimum below this temperature, with -0.2C as the lowest temperature on one

day between 6:00 and 6:15 a.m.

Although 132 1st instars were introduced into each cage at the beginning of the

experiment, the highest mean number of M. ochroloma larvae per cage on the first

sampling date was 33 3.1 in the medium predator density treatment. On 18 February,

the mean number of larvae per cage in the high predator density treatment was

significantly lower than in the medium predator density, low predatory density, and

control treatments (Fig. 3-Larvae) (F= 5.69; df = 3,12; P = 0.0116). On 22 February, the

mean number of M. ochroloma pupae per cage in the high and medium predator density

treatments was significantly lower than in the low predatory density and control

treatments (F= 8.58; df = 3,12; P = 0.0026) (Fig. 3-Pupae). On 3 March, the mean

numbers of pupae per cage in all treatments with predators were significantly lower than

in the control treatment (F= 4.37; df = 3,12; P = 0.0268) (Fig. 3-Pupae). Although SNK

test could not separate the mean number of pupae per cage among treatments on 18

and 26 February, ANOVA did show significance differences among them. A real effect

by the predator was apparent in the medium and high predator density treatments









because these treatments consistently had lower mean number of pupae per cage

compared to the low predator density and control treatments (F= 4.08; df = 3,12; P =

0.0326; F = 4.34; df = 3,12; P = 0.0273) (Fig. 3-Pupae). On 3 March, the mean number

of adult M. ochroloma in the two higher predator density treatments was significantly

lower than in the low predatory density and control treatments (F= 8.87; df = 3,12; P =

0.0023) (Fig. 3-Adults).

Overall, there were differences in the number of M. ochroloma larvae + pupae +

adults per cage among treatments over time. The mean number per cage was

significantly lower in the high predator density treatment than in the medium predator

density, which was significantly lower compared to the low predator density treatment.

There was no significant difference in the mean number per cage between the low

predator density and the control. However, there was a significant interaction between

treatment and time (treatment x time: F = 5.10; df = 3,72; P = 0.0167; treatment: F=

18.37; df = 3,72; time: F= 61.07; df = 3,72; P < 0.0001) (Fig. 3-Total). The medium and

high predator treatments had the lowest mean number of M. ochroloma per cage from

18 February until the end of the experiment. On 26 February, there was a significant

difference, according to the ANOVA, in the mean number of M. ochroloma per cage

among the treatments, however, no differences were detected when the means were

separated with SNK test (F= 3.54; df = 3,12; P = 0.0482).

Excluding eggs, the population of M. ochroloma per cage in the high predator

density treatment at the end of the experiment was 91.7% 8.3 adults and 8.3% 8.3

pupae. In the medium predator density treatment, the proportions per cage at the end

of the experiment were 88.1% 7.9 adults and 11.9% 7.9 pupae. In the low predator









density treatment, the M. ochroloma population (excluding eggs) per cage was 89%

3.8 adults and 9.9% 3.7 pupae, whereas in the control treatment the proportions were

75.3% 8.4 adults, 24.2% 8.2 pupae, and 0.5% 0.5 larvae.

Overall, there were differences in the number of P. maculiventris nymphs per cage

among treatments (F = 3.87; df = 3,72; P < 0.0001) (Fig. 4). The number of nymphs per

cage was significantly higher in the medium predator density treatment than in the high

predator density treatment which was significantly higher than in the low predator

density treatment; the control treatment, with no predators released, was significantly

lower than all other treatments. However, there was a significant interaction between

treatment and time (treatment x time: F = 3.87; df = 3,72; P < 0.0001; treatment: F=

79.12; df = 3,72; P< 0.0001; time: F= 7.10; df = 3,72; P< 0.0001) (Fig. 4).

On only one date, 6 and 22 February (4 d after introducing predators into the

cages), the mean number of predator nymphs per cage was significantly higher in the

medium predator density treatment compared to the high predator density treatment; on

all the other sampling dates there was no significant difference between these two

treatments. Beginning 3 March, the two higher predator density treatments maintained

significantly higher numbers of P. maculiventris nymphs per cage compared to the low

predator density treatment during the remainder of the study (F =18.63; df = 3,12; P <

0.0001) (Fig. 4).

At the end of the experiment, the overall survivorship ([number of nymphs

recovered/number of nymphs released]*100) of P. maculiventris per cage among the

three predator release treatments was 51.3% 5.4. In the low predator density

treatment, predator survivorship was 62.5% 12.5; 75.0% 14.4 of the nymphs were in









the 3rd instar and 25% 14.4 were in the 4th instar. In the medium predator density

treatment, predator survivorship was 52.5% 4.8; 65.4% 16.7 of the nymphs were in

the 3rd instar and 34.6% 16.7 were in the 4th instar. Predator survivorship in the high

predator density treatment was 39.1% 6.9; 83.7% 10.3 of the nymphs were in the 3rd

instar and 16.3% 10.3 were in the 4th instar.

2010 Experiment

The mean temperature during the experiment was 14.80C 0.6. The mean

maximum temperature was 21.60C 0.6. Twenty days had a maximum above this

temperature, with 26.9C as the highest recorded temperature for one day between

2:30 and 2:45 p.m. The mean minimum temperature was 8.30C 0.8. Seventeen days

had a minimum below this temperature, with 0.3C as the lowest temperature on one

day between 5:30 and 5:45 a.m.

The highest mean number of M. ochroloma larvae per cage on the first sampling

date was 27 7.6 in the control treatment. On 1 March, the mean number of larvae per

cage in the high, medium, and low predator density treatments was significantly lower

than in the control treatment (F= 12.54; df = 3,12; P = 0.0005) (Fig. 5-Larvae). On 9

March, pupae were observed in the low predator density and control treatments, but not

in the high and medium predator density treatments. However, no significant

differences were detected among the four treatments (Fig. 5-Pupae). On 13 March

pupae were seen only the in the control treatment, but again no significant differences

were detected among all treatments (Fig. 5-Pupae). On 17, 22, and 25 March, the

mean number of adults per cage in the three predator density treatments was









significantly lower than in the control treatment (F>21.75; df= 3,12; P < 0.0001) (Fig. 5-

Adults). All M. ochroloma became reproductive adults in all treatments after 36 d.

Overall, there were differences in the number of M. ochroloma larvae + pupae +

adults per cage among treatments over time. The mean number of M. ochroloma per

cage was significantly lower in the medium and high predator density treatments than in

the low predatory density treatment, which was significant lower compared to the control

treatment (Fig. 5-Total). However, there was a significant interaction between

treatments and time (treatment x time: F = 4.64; df = 3,96; P < 0.0001; treatment: F =

58.97; df = 3,96; P < 0.0001; time: F = 29.56; df = 3,96; P < 0.0001) (Fig. 5-Total). On

1, 17, 22, and 25 March, there were no significant differences in the mean number of M.

ochroloma per cage among the low, medium, and high predator density treatments, but

all of them were significantly different from the control treatment (F=12.54; df = 3,12; P

= 0.0005; F=21.75; df = 3,12; P< 0.0001; F=28.47; df= 3,12; P< 0.0001; F= 38.35; df

= 3,12; P < 0.0001) (Fig. 5-Total).

Overall, there were differences in the number of P. maculiventris nymphs per

cage among treatments (F = 1.79; df = 3,96; P < 0.0001) (Fig. 6). The number of

nymphs per cage was significantly higher in the high and medium predator density

treatments than in the low predator density treatment, which was significantly higher

than the control treatment with no predators released (F = 1.79; df = 3,96; P < 0.0001).

However, there was a significant interaction between treatment and time (treatment x

time: F = 1.79; df = 3,96; P < 0.0001; treatment: F = 34.70; df = 3,96; P < 0.0001; time:

F= 5.11; df = 3,96; P = 0.01) (Fig. 6). Significant differences were found amount

treatments on all sampling dates except for 9 March (F= 2.63; df = 3,96; P = 0.09). The









mean number of nymphs per cage in the high predator density treatment decreased

over time. On 25 March, the low predator density treatment was the only treatment

significantly different from the control (F= 4.24; df = 3,96; P = 0.0293).

At the end of the experiment, the overall survivorship per cage of P. maculiventris

was 26% 8.1. In the low predator density treatment, survivorship per cage was 56.3

% 11.9; 25.0% 28.9 were in the 3rd instar, 50.0% 28.9 were in the 4th instar, 12.5%

12.5 were in the 5th instar, and 12.5% 12.5 were adults (molting to adult on 25

March). In the medium predator density treatment, survivorship per cage was 17.5% +

8.5; 66.7% 16.7 were in the 4th instar, and 33.3% 16.7 were in the 5th instar. In the

high predator density treatment, survivorship per cage was 3.1% 1.8; 50.0% 50.0

were in the 4th instar and 50.0 % 50.0 were in the 5th instar.

Discussion

2009 Experiment

Only seven sampling dates (five weeks) were evaluated due to the very large

turnip plants in the cages at the end of the experiment (Fig. 7), which also made

sampling more difficult. The notable reduction in the number of larvae of M. ochroloma

in the control treatment on 6 February, four days after the release of the insects into the

cages, was likely due to the difficulty in detecting the small first instars by the visual

sampling method. Temperature probably did not have a major influence on the

mortality of M. ochroloma, since temperature ranged from 10.8 to 23.3C during the

experiment. Cold tolerance studies indicate that 1st instar M. ochroloma can survive at

least 2 d exposed to 0C (unpublished data). The decrease in the number of M.

ochroloma per cage in all treatments from 6 to 18 February (Fig. 3-Total) can be

attributed, in part, to the transition of larvae to pupae (Fig. 3-Larvae), but in the predator









density treatments it also can be attributed to predation by P. maculiventris, as

evidenced by the significantly greater decrease in number of M. ochroloma in the

medium and high predator density treatment versus the low predator density and control

treatments. On 18 February, the difference between the mean number of larvae and

pupae is probably due to the larvae behavior when it is ready to pupate (Fig. 3-Larvae

and Pupae). When the larvae are ready to pupate, they move to dry leaves, tight

places, and soil (Woodruff 1974; Bower 2003). In this study, the larvae moved under

the plastic, making it difficult to find them. This confirms the disappearance of larvae

after 22 February in the control treatment was due to pupation. In the treatments with

predators, the reduced number of M. ochroloma larvae can be attributed to predation

and pupation. On the last sampling date, the predation effect of P. maculiventris on the

reduction in the number of M. ochroloma was more noticeable in the medium and high

predator density treatments compared to the low predator density treatment (Fig.3

Total). However, the overall number of M. ochroloma was kept lowest in the high

predator density treatment during the 30-day experiment.

There was a drastic reduction of 1st instar P. maculiventris on 6 February, probably

due to the visual sampling method or unexplained causes not measured. Temperature

likely did not influence the number of 1st instars observed because De Clercq and

Degheele (1992) estimated the lower threshold for development of P. maculiventris

nymphs is 11.7C, well below temperatures experienced during my field study.

Although the survival of 1st instar P. maculiventris feeding on 1st instar M. ochroloma in

the field has not been studied, a reduction in the survivorship could be expected. The

observed survival of the predator per cage on 6 February was 40% 8.6, whereas the









actual survival determine on 3 March was 51% 5.4, suggesting that the survivorship of

the predator at the beginning of the experiment was underestimated by the sampling

method. Cannibalism may have been minimal or may not have occurred at all in this

experiment since the search area was high due to the large plant size and because the

populations of M. ochroloma were maintained at low levels, i.e. never driven to

extinction. If populations of M. ochroloma were to become limited, P. maculiventris

would have no problem surviving on other prey since it is a generalist predator, or even

on plants since it is a facultative herbivore (McPherson 1982; Valicente and O'Neil

1995). According to Wiedenmann and O'Neil (1990), the presence of plant material can

enhance survival of P. maculiventris at very low prey inputs.

2010 Experiment

Plant size (about four leaves per plant) in the 2010 experiment was considerably

smaller than it was in 2009 experiment (about seven leaves per plant), both at the

moment of the insect releases and at the end of the experiment (Fig. 8), because the

releases were made 10 d earlier after transplanting in 2010 than in 2009. This

difference extended the experiment to nine sampling dates in 2010. Plants in the

control treatment had to be replaced on 25 February due to complete defoliation by the

M. ochroloma larvae. Plants in the other treatments, on the other hand, did not have to

be replaced. This is evidence that predation by the P. maculiventris nymphs on M.

ochroloma significantly reduced the number of larvae in those treatments and,

therefore, the feeding damage on the plants.

On 21 February, four days after releasing the insects in the cages, there was a

drastic reduction in the number of M. ochroloma larvae as there was in 2009, again

probably due to the lack of detection by the sampling method. From 21 February to 5









March, the number of M. ochroloma decreased in all treatments (Fig. 5-Total), just as it

did in 2009 and for the same possible reasons. It is important to observe that after 9

March, the number of M. ochroloma was virtually zero in the medium and high predator

treatments for all stages (Fig. 5-Total), probably due to the effect of predation by P.

maculiventris. This phenomenon did not happen in the 2009 experiment because the

leaf surface area (larger plants) was higher than in 2010, therefore reducing the

probability of the predator encountering and feeding upon the prey. Desurmont and

Weston (2008) studied the effect of two host plant species, arrowwood (Viburnum

dentatum L., Caprifoliaceae) and American cranberrybush (Viburnum opulus L. var.

americanum Aiton, Caprifoliaceae), on consumption of the viburnum leaf beetle,

Pyrrhalta viburni (Paykull), by P. maculiventris. Their results showed that prey

consumption was inversely related to leaf surface area on one of the host plants. They

hypothesized that the searching efficiency of the predator decreases as leaf surface

increases because of the ability of the prey to hide or move around.

The 44% survivorship of P. maculiventris on 21 February is similar to the observed

survival in 2009; however, survivorship rates at the end of the two experiments are quite

different. The final survivorship in 2010 was 26% compared to 51% in 2009. Lower

survival in 2010 could have been due to cannibalism since the leaf surface area was

smaller and the number of M. ochroloma larvae was driven to zero after 9 March in the

high and medium predator density treatments. Dispersal would be an option for the

predators in an open field scenario, but not to a great extent because the nymphs do

not have wings. According to the data gathered in this study, 1st instar P. maculiventris

would still be in the nymphal stage five weeks after being released in the field.









On the last sampling date (25 March), the effect of P. maculiventris nymphal

predation on the number of M. ochroloma per cage is remarkable in all the predator

density treatments. However, the number of M. ochroloma was kept lower for 36 d with

at least 10 nymphs released per six plants. The release rate of three P. maculiventris

1st instars per 1.21 m2 or 25 1st instars per 10 m2 as recommended by Rincon-Vitova

Insectaries is not adequate to control M. ochroloma. Therefore, two recommendations

emerge from the overall results of my field-cage study:

* Release 16 1st instars of P. maculiventris per six plants if the plants are expected
to be large (27 leaves/plant) with at least 130 1st instars of M. ochroloma.

* Release 10 1st instars of P. maculiventris per six plants if the plants are expected
to be small (56 leaves/plant) with at least 130 1st instars of M. ochroloma.

Although there was no significant overall difference between the medium and high

predator density treatments, the second recommendation is made from an economic

point of view and the probability of cannibalism. In addition, increasing the number of

biological control agents in the field does not always translate into greater pest control,

but does increase the cost of using biological control (Van Driesche et al. 2002; Collier

and Van Steenwyk 2004). Consequently, releasing an optimal number of biological

control agents should result in a more efficient and economic augmentative biological

control program (Crower 2007).































Figure 4-1. Cages in the field at the beginning of the 2009 experiment.


Figure 4-2. Plants being gathered for final sampling in the laboratory.










M Control
H Low
D Mediul
E High


ebbL


a

Lb


bb


10-Feb 14-Feb 18-Feb 22-Feb 26-Feb 3-Mlar
Sampling date


Figure 4-3. Number of Microtheca ochroloma per cage during the 2009 field
experiment. Means followed by the same letter within each sampling date are
not significantly different (P>0.05). An asterisk (*) over the bars on the same
date indicates that differences among treatments were detected by ANOVA,
but the means could not be separated by the SNK test.


Total


Larvae




a


Pupae


Adults


6-Feb










Control a
0 Low
O MNedium
a
a 0 High

T. a .


6-Feb 10-Feb 14-Feb 18-Feb 22-Feb 26-Feb 3-Mar
Sampling date

Figure 4-4. Mean number of Podisus maculiventris nymphs per cage during the 2009
field experiment. Means followed by the same letter within each sampling
date are not significantly different (P>0.05).










5 Total
30 o a
25 m Cortrol a
20 fT 1 ledin .L
15 0 T High

bLs bb
0. b b b
10 1|) L 1



35 Larvae
30
S25
-H
E 20 -






0.4 Pupae
1.2







0


30 a
25
ES I I












10


0 Ibb lb b
o 1










21-Feb 25-Feb 1-Mar 5-1inri 9-1ir 13-Mar 17-Mar 22-Mar 25-Mar
Sampinpg date


Figure 4-5. Mean number of Microtheca ochroloma per cage during the 2010 field
experiment. Means followed by the same letter within each sampling date are
not significantly different (P>0.05).
exeiet ensfloe yth aelte wti ahsmpigdt r
no iniiatl ifeet(P00)











* Control
E Low
O Medium
M High


a
ab


aab


21-Feb 25-Feb 1-Mar 5-Mar 9-Mar 13-Mar 17-Mar 22-Mar 25-Mar
Sampling date

Figure 4-6. Number of Podisus maculiventris nymphs per cage during the 2010 field
experiment. Means followed by the same letter within each sampling date are
not significantly different (P>0.05).






























Figure 4-7. Size of turnip plants in the 2009 experiment.


Figure 4-8. Size of turnip plants in the 2010 experiment.









CHAPTER 5
INFECTIVITY OF MICROTHECA OCHROLOMA STAL (COLEOPTERA:
CHRYSOMELIDAE) BY ISARIA FUMOSOROSEA WIZE (BROWN AND SMITH)

Introduction

Microtheca ochroloma Stal, the yellowmargined leaf beetle, is a serious pest in

crucifer crops during the late fall and winter months in Florida. Since 1947, this

adventive species has been established in most of the southern US states. Ameen and

Story (1997a) studied the feeding preferences of the larvae and adults and concluded

that turnip and mustard are their preferred host plants. Its main damage is caused by

defoliation; however, roots can also be damaged when infestations are severe. On

large-scale commercial farms, the most common and easy way to control this beetle is

by applying insecticides, but the overuse of insecticides can lead to the development of

resistance over time, as has occurred with of the Colorado potato beetle Stilodes

decemlineata (Say) (Alyokhin et al. 2008). On organic farms, it is more difficult to

control M. ochroloma due to the restricted use of insecticides, in addition to the lack of

specific natural enemies of M. ochroloma in the United States (Fasulo 2005). Currently,

there is no pest management program available for growers to control this pest in the

United States. Bowers (2003) evaluated whether intercropping between host (mizuna)

and non-host (oak leaf lettuce) plants can reduce the severity of M. ochroloma

outbreaks, but still the beetles were able to find and colonize host plants among the

non-host plants.

Biological control by entomopathogenic fungi may potentially be used to control M.

ochroloma on organic farms. Anjos et al. (2007) reported infection of M. ochroloma in

the field by Beauveria bassiana (Bals.) Vuilleman in Rio Grande do Sul, one of more

southern states in Brazil. Isaria fumosorosea (=Paecilomyces fumosoroseus) Wize









(Brown and Smith) has a worldwide distribution and its effectiveness against many pest

insects, especially whiteflies, is well documented (Osborne and Landa 1992; Vidal et al.

1997a; Lacey et al. 1999). This fungus has a broad host range, including chrysomelid

beetles such as S. decemlineata (Bajan 1973), Pyrrhalta luteola (Mueller), and

Spaethiella sp. (Humber et al. 2009).

In 1986, a strain of /. fumosorosea named Apopka 97 was isolated in Apopka

(Orange County), FL from Phenacoccus sp. (Hemiptera: Pseudococcidae) (Vidal et al.

1998). The strain is registered under the commercial name PFR-97 TM 20% WDG

[chemical family: microbial insecticide, chemical name: Paecilomyces fumosoroseus

Apopka Strain 97 (ATCC 20874)] by the manufacturer Certis USA, Columbia, MD. It is

recommended for use in greenhouses against aphids, citrus psyllid, spider mites, thrips,

and whitefly [www.certisusa.com], but it is still being evaluated against field pests in

food crops.

The use of microbial insecticides as a tool to control pests in agricultural

ecosystems is becoming more popular among growers. Although there is no

information available on commercial products to specifically control M. ochroloma,

products that control other pest beetles should be evaluated on M. ochroloma. In this

study, the infectivity of Apopka 97 was evaluated as a potential biological control agent

of M. ochroloma.

Materials and Methods

Stock Colony. Eggs, larvae, and adults of M. ochroloma were obtained from the

field and a laboratory colony was established and maintained as described in Chapter 2.

Fungus. PFR-97TM 20% WDG (a.i. Paecilomyces fumosoroseus Apopka strain 97

20%, inert ingredients 80%) was provided for research by Certis USA in a 0.45 kg bag









(Lot: 0833004401) in the form of desiccated granules of I. fumosorosea blastospores.

The bag contains 1 x 109 colony-forming units (CFU)/g.

Plant Material. Plants of Seven Top (Greens) turnips (Brassica rapa L. var.

rapifera, Brassicaceae) were grown as described in Chapter 2.

Experiment 1. Susceptibility of Microtheca ochroloma to Infection by PFR-97TM

The goal of this experiment was to determine the susceptibility of various stages of

M. ochroloma to infection by PFR-97TM. Five stages of M. ochroloma, egg, 1st and 3rd

instars, pupa, and adult, were removed from the laboratory colony for exposure to a

concentration of 1 g of PFR-97TM in 100 ml of sterile distilled water at 250 C, 10L:14D

photoperiod, and 60% RH. The fungal suspension was prepared in a beaker and

allowed to settle for 20 min until the supernatant containing blastospores and the inert

sediment of the product separated. The suspension was applied (see below for

method) to groups of 10 insects per stage housed in separate and sealed plastic Petri

dishes (60 x 75 mm, Fisherbrand) with moistened filter paper (55 mm 0 [diameter],

Whatman) on the bottom dish. A 2.5 cm2 0 piece of turnip leaf was placed on top of

the filter paper. The Petri dishes were sealed with Parafilm.

All fungal treatments consisted of 10 replicates with 10 pseudo-replicates per

replicate. A pseudo-replicate was a single insect. A control treatment consisted of five

replicates in which the test insect stages were sprayed with sterile distilled water only.

Mortality was checked daily during the 7 d following the fungal application. Infectivity

rate was determined by using the control mortality as a correction factor for the mortality

in the fungus treatment. Morphological traits unique to I. fumosorosea in dead insects

(see below for method) were used to confirm infection. The experiment was repeated

one time.









Experiment 2. Infectivity of the Most Susceptible Stage of Microtheca ochroloma
by Four Concentrations of PFR-97TM

The goal of this experiment was to compare the infectivity of four concentrations of

PFR-97TM in the most susceptible stage of M. ochroloma, which was determined in

Experiment 1. The concentrations were 1, 2, 3, and 4 g of PFR-97TM per 100 ml of

sterile distilled water. Each concentration was applied (see below for method) to groups

of 10 1st instars housed in separate and sealed plastic Petri dishes with moistened filter

paper on the bottom. A 2.5 cm2 piece of turnip leaf was placed on top of the filter paper.

The Petri dishes were then housed in an environmentally controlled chamber set to 250

C, 10L: 14D photoperiod, and 60% RH. Each fungal treatment consisted of 10

replicates with 10 pseudo-replicates per replicate. A control treatment consisted of five

replicates in which the test insects were sprayed with sterile distilled water only.

Mortality was checked daily during the 7 d following the fungal application. Infectivity

rate was determined by using the control mortality as a correction factor for the mortality

in the fungus treatment. Confirmed infectivity rate was determined by morphological

traits unique to I. fumosorosea in dead insects (see below for method). The experiment

was repeated one time.

For both experiments, the initial blastospore concentration was determined by

counting the number of blastospores per ml using a disposable plastic Neubauer

hemocytometer, C-Chip DHC-N01, manufactured by Incyto (Korea). Each suspension

was poured separately into 180-ml NalgeneTM (Rochester, NY) spray bottles for

application to test insects. Each group of insects with their respective piece of leaf

received 3 sec of application (~2.5 ml) on each side of the leaf. The sprayed leaf was

not removed from the Petri dish and starting 3 d after treatment new non-sprayed









leaves were added daily to each Petri dish but not removed. Blastospore deposition

density was determined by placing a plastic cover slip among the test insects during the

application of the fungus, after which the number of blastospores per mm2 was counted.

Viability of blastospores was determined by taking 100L from the 10-3 serial dilution,

spreading it on potato dextrose agar (PDA) in Petri dishes, maintaining the dishes under

the same environmental conditions as the insects, and counting the number of CFU

after 7 d.

To confirm infectivity in Experiment 1, dead insects were removed daily and

transferred directly to a Petri dish containing a mixture of PDA, odine, streptomycin, and

chlrophenacol; dishes were then sealed with Parafilm and stored at 25C. The

presence of hyphae morphologically unique to I. fumosorosea (Fig. 5-1) was recorded.

In Experiment 2, dead insects were surface sterilized in 70% ethanol for a few seconds

before being placed on the PDA-mixture plates. Unconfirmed infectivity was assigned

to dead insects in which I. fumosorosea could not be identified because of its absence

or contamination by other fungi.

Statistical Analysis. Data were arcsine-transformed prior to analysis. Mortality

and infectivity rates were analyzed using analysis of variance, and treatment means

were separated using Student-Newman-Keuls test. All tests were performed with PROC

GLM in SAS v. 9.2 (SAS Institute Inc. 2002), with a significance level of 5%. Lethal

concentration (LC25) and lethal times (LTlo and LT25) were analyzed using PROC

PROBIT (SAS Institute Inc. 2002), and significant differences between treatments were

identified using 95% confidence intervals (Tabashnick and Gushing 1987).









Results


Experiment 1

For 1 g of PFR-97TM in 100 ml of water, the concentration of the suspension was

3.0 0.1 x 107 blastospores/ml (Fig. 5-2A). Mean blastospore deposition density was

1,043 181.5 blastospores/mm2 (Fig 5-2B). Viability was 49 1.0 CFU/ml-1 in 10-4

serial dilution (Fig.5- 2C).

The most susceptible stage of M. ochroloma to the Apopka 97 strain of /.

fumosorosea was the larval stage. The mean mortality rates of eggs, pupae and adults

were not significantly different from their respective controls (Fig. 5-2). In contrast, mean

mortality rates of the 1st and 3rd instars were significantly higher (4.4 and 0.8 times,

respectively) than their respective controls (F = 14.39; df = 1,13; P = 0.0022; F = 8.12;

df = 1,13; P = 0.0137) (Fig. 5-3). Mean infectivity rate was at least 6.3 times higher in

the 1st and 3rd instars than in the egg, pupal and adult stages (F=12.19; df = 4,35; P <

0.0001) (Fig. 5-3). However, only 17 and 20% of the infectivity in the 1st and 3rd instar,

respectively, was confirmed (Figs. 5-4 and 5-9). Mortality of the 1st instar was observed

beginning 3 d after treatment. By that time thelst instars had already molted to 2nd

instar. Mortality in the 3rd instar was observed beginning 1 day after treatment.

The LTio for the 1st instar (4 d) was significantly higher than the 3rd instar (2 d) (P <

0.05). No significant difference was apparent for the LT25 between the two instars (P >

0.05) (Fig. 5-5). The LT50 could not be determined due to the low mortality rate of

larvae exposed to the 1 g treatment concentration of PFR-97TM; however the LT50

predicted by the model for the 1st and 3rd instar were 8.6 fiduciall limits: 7.6-10.5) and

21.2 d fiduciall limits: 12.9-60.7), respectively. The LT models were not significant for

eggs, pupae, and adults (P > 0.05).









Experiment 2

For this experiment, the concentration of blastospores, blastospore deposition,

and viability for the four experimental concentrations of PFR-97TM are reported in Table

5-1. The 1st instar of M. ochroloma was selected for this experiment based on the

results of Experiment 1. There was no mortality in the control treatment. Therefore, all

mortality in the fungus treatments was considered to be caused by infection with /.

fumosorosea. Mean infectivity rate was significantly higher by 2.6 times in the 4 g

concentration treatment than in the 1, 2, and 3 g concentration treatments (F = 3.76, df

= 3,36; P=0.0191) (Fig. 5-6). Confirmed infectivity rates were 2, 5, 10, and 27% in the

1, 2, 3, and 4 g concentration treatments, respectively (Fig. 5-6).

Based on fiducial limits (95%), the LTlo and LT25 in the 4 g concentration

treatment were significantly lower compared to the other treatments (P < 0.05). The

LT50 predicted by the model was 10 d fiduciall limits: 8.4-13.7). There was no significant

difference among the 1, 2, and 3 g concentration treatments (P > 0.05) (Fig. 5-7).

The LClo and LC25 of PFR-97TM applied to 1st instar M. ochroloma were 1.4

fiduciall limits: 0.3-2.0) and 5.5 g fiduciall limits: 3.6-39.0) per 100 ml of distilled water,

respectively, on day 7. The LC5o predicted by the model was 25.6 g per 100 ml of

distilled water, on day 7 (intercept = -1.42 + 0.16; slope = 1.0 0.36; X2 = 1.0 0.36; P

= < 0.0001).

Discussion

Experiment 1

The fungus had a low, insignificant ovicidal effect with an egg mortality rate of

3% (Figs. 5-4 and 5-8). Although the ovicidal effect was low in this experiment, the

fungal residues on the eggs and on the leaf surface may have a significant impact on









the emerging neonates. There is a great deal of variability and discussion concerning

the ovicidal effect of I. fumosorosea. Rodrigues-Rueda and Fargues (1980) showed that

P. fumosoroseus has high ovicidal activity on eggs of the moths Mamestra brassicae

(Linneaus) and Spodoptera littoralis (Boisduval). In contrast, Lacey et al. (1999)

reported a low, but significant mortality (10-20%) of eggs of Bemisia tabaci (Gennadius)

treated with of PFR-97TM, but no significant ovicidal effect was reported on eggs of

Yponomeuta xylostella (Linneaus) (Maketon et al. 2008).

Larvae of M. ochroloma in the 1st and 3rd instars experienced the highest

infection rates among all the insect's life stages. The unconfirmed infections may be

attributed to the procedure of transferring dead insects to Petri dishes without first

surface sterilizing the insects. This may have resulted in the rapid growth of

saprophagous fungi, thus slowing the growth of I. fumosorosea (if there was any) and

not allowing its appearance of diagnostic morphological features (Fig. 5-9). Larvae

infected by I. fumosorosea exhibited noticeable reduced growth (Fig. 5-10) and

unsuccessful molting in which the exuvium remained attached to the new integument

(Fig. 5-12). Similar studies by Hussain et al. (2009) have also shown a reduction in the

consumption and growth of all instars of Ocinara varians Walker (Lepidoptera:

Bombycidae) when I. fumosorosea strain 03011-C3.19A was applied. A reduction in

feeding was also reported by Fargues et al. (1994) in S. decemlineata attacked by B.

bassiana. Mortality and growth rate reduction may be attributed to the production of

toxins by the fungus, mechanical disruption of the structural integrity of membranes by

the growth of hyphae, and dehydration of cells from the loss of fluids (Ferron 1981;

Tefera and Pringle 2003; Assaf et al. 2005).









Microtheca ochroloma pupates within a net-like case which only has direct

contact with the cuticle of the pupa at the apex of the body (Fig. 5-11). For this reason,

low mortality and infectivity rates of pupae were observed, since the net-like case

apparently serves as a physical barrier to the deposition of blastospores on the cuticle

of the pupa, which is necessary to initiate infection.

Adults of M. ochroloma were not affected by I. fumosorosea, probably because

the hard cuticle is composed primarily of a higher degree of cross-linked proteins and

chitin than that of the immature stages, which provides greater strength and hardness to

the exoskeleton and functions as a formidable barrier to blastospore germination

(Klowden 2007). Only 4% mortality was recorded in the fungus treatment, compared to

none in the control treatment (Fig. 5-3), but the mortality in the treatment cannot be

confidently attributed to the fungus since there was no confirmed infectivity (Fig. 5-4).

Michalaki et al. (2007) reported low mortality of adults of Tribolium confusum Jacquelin

du Val (Coleoptera: Tenebrionidae) exposed to I. fumosorosea.

Experiment 2

There was a well defined positive correlation between fungus concentration and

mortality rates of 1st instars of M. ochroloma. The highest infectivity and confirmed

infectivity rates were achieved with the 4 g concentration treatment, which

corresponded to the highest concentration of blastospores/ml, deposition of

blastospores/mm2, and viability among all treatments (Table 5-1). However, the 4 g

concentration treatment achieved only 29% infectivity in the laboratory; infectivity rates

in the field may be expected to be lower. The model predicts the LC25 is 5.5 g per 100

ml of water, which is equivalent to approximately 1.6 x 108 blastospores/ml, and the

predicted LCso is 25.6 g per 100 ml of water, which is equivalent to approximately 7.3 x









108 blastospores/ml. Higher concentrations of PFR-97TM should be tested in the

laboratory, since it seems that infectivity rates in the 1st instar increase as the

concentration of blastospores/ml increases (Fig. 5-6), and the LTlo and LT25 are

achieved faster with increasing concentration (Fig. 5-7). Once the product is registered

for use in the field for fruiting crops, the concentration of CFU per bag probably will be

higher; therefore, the amount of grams per 100 ml required for high infection rates

would be lower.

The unconfirmed infectivity rate in Experiment 2 was lower compared to the

unconfirmed infectivity rate in Experiment 1 because when the insects died in the former

they were sterilized with alcohol for few seconds before placing them on the PDA. The

unconfirmed infectivity rate in Experiment 2 may be reduced even more by using the

polymerase chain reaction technique to identify the presence of PFR-97TM strain in dead

insects, as has been done in other studies (Meyer 2007; Meyer et al. 2008; Hoy et al.

2010).

Once blastospores are deposited and germinate on the integument of the insect,

death of the host will most likely occur within 3 d at any concentration. In both

experiments, the appearance of fungal infection in the 1st instar of M. ochroloma began

3 d following application. Similar results were reported by Tounou et al. (2003) in

nymphs of the green leafhopper, Empoasca decipiens Paoli (Hemiptera: Cicadellidae),

which began dying 3 d after treatment with I. fumosorosea strain Pfr12. However, there

will be a higher probability of deposition and germination of blastospores at higher

concentrations of the fungus, thereby killing a greater number of insects compared to

lower concentrations.









In summary, the larval stage of M. ochroloma is the most susceptible stage to /.

fumosorosea. However, a concentration of PFR-97TM greater than those tested here is

required to reach the LT50 and LC50 in 1st instars under laboratory conditions. Higher

concentrations than those tested in the laboratory will need to be applied in the field as

well.


Figure 5-1. Morphological characteristics of Isaria fumosorosea infecting Microtheca
ochroloma larva.







w i U,..le
RiUWDW BIWWd-

- mmm r4 men
a m Ima Cms

mmei amns
Hi'a M~'.IBIeU


Figure 5-2. Laboratory tests of Isaria fumosorosea. A) Blastospore concentration
(blastospores/ml), B) Blastospore deposition density(blastospore/mm2, and
C) Viability (CFU/g).













* PFR-97 T

E Control


TJ


Pupae


Adults


.licrotheca ochroloma stage

Figure 5-3. Mortality of Microtheca ochroloma by PFR-97TM 20% WDG at 3.0 x 107
blastospores/ml 7 d after application. Bars with different letter within each
stage are significantly different (SNK test, P < 0.05).


80 -


.40

S30


10

0


Eggs


1st instars


3rd instars










45 -


40 -

35

S30



20-
C
15



51
0-


* I ncontli rmed

0 Confirmed


b

Rim


F


1st instars


3rd instars


Pupae


Adults


Microtheca ochroloma stage

Figure 5-4. Infectivity of Microtheca ochroloma by PFR-97TM 20 % WDG at 3.0 x 107
blastospores/ml 7 d after application. Bars with the same letter are not
significantly different (SNK test, P > 0.05).


I












instar


X Observed

- Predicted


LT --------

X


10 H LTlo


2 4 6 8 10 12
Days


Figure 5-5. Lethal time of the first and third instars of Microtheca ochroloma treated
with PFR-97TM. LT10 and LT25 values within the same box are not
significantly different in their 95% confidence intervals.


4011

35 -

30


Zo'



20


ist instar










40

35


30


25
2 0

S20 -

S15-

10-


* Unconfirmed

I Confirmed


I I
2 3
g of PFR-97 100 nil of distilled water


Figure 5-6. Infectivity of first instar Microtheca ochroloma at four concentrations of
PFR-97TM 7 d after application. Bars followed by the same lowercase letter
are not significantly different (P>0.05).












25 LT ----------- ------------
S 4g
20 g


15 -


10- LT1o --- 2g Observed

-- Predicted
5 -



0 1 2 3 4 5 6 7 8 9 10 11 12 13
Days

Figure 5-7. Lethal time of the first instar Microtheca ochroloma exposed to four
concentrations of PFR-97TM. LTio and LT25 values within the same box are
not significantly different in their 95% confidence intervals.




























Figure 5-8. Eggs of Microtheca ochroloma infected by Isaria fumosorosea.


Figure 5-9. Unconfirmed and confirmed infectivity by Isaria fumosorosea in dead larvae
of Microtheca ochroloma.






















Figure 5-10. Reduction in the growth
Isaria fumosorosea.


of larvae of Microtheca ochroloma infected by


Figure 5-11. Net-like pupal case of Microtheca ochroloma.


I UNNFECED























...... ii.l.... A B
















C

Figure 5-12. Unsuccessful molting by a larva of Microtheca ochroloma infected with
Isaria fumosorosea. A) Head partially out, B) Exuvia attached to the dorsal
part of the body, and C) Larva starting to pull out from the tip of the abdomen.










Table 5-1. Laboratory tests of Isaria fumosorosea in Experiment 2.
PFR-97TM Concentration SEa Deposition SE CFUb + SE
(g) (blastospores/ml) (blastospores/mm2) (CFU/ml at 10-4)
1 2.2 0.1 x 107 779 150.5 24.5 2.5
2 3.8 0.2 x 107 1088 174.1 55.5 2.5
3 8.4 0.7 x 107 4157 962.6 84.5 1.5
4 1.1 0.0 x 108 6658 881.6 146.5 1.5
a standard error. bColony-forming units.









CHAPTER 6
CONCLUSIONS

My thesis study focused on the evaluation of two potential biological control

agents of the yellowmargined leaf beetle, M. ochroloma. The predator, P. maculiventris,

was chosen because it 1) occurs naturally in the field, 2) can be acquired commercially

by growers, and 3) preys on many agricultural pests. The fungus, I. fumosorosea (PFR-

97TM), was chosen because it 1) is a local strain and 2) has been under previous study

for application to food crops in the field.

There was no development of 1st instars or egg hatching of P. maculiventris at

10C. Development of the predator from egg to adult is shorter at 20 and 250C (31 and

23 d, respectively) than at 150 C (99 d). The daily predation by P. maculiventris starting

from the 3rd instar until 10 d of adulthood was higher at 25C compared to 20C, which

was higher than at 15C. However, the total predation by P. maculiventris on 4th instar

M. ochroloma was higher at 250C compared to 200C, which was higher than at 15C

only during the 10 d of adulthood. Podisus maculiventris nymphs killed 59, 53, and 65

4th instars of M. ochroloma at 15, 20 and 250C, respectively. Fresh adult females weigh

more than males at 200C, but not at 250C. Predator nymphs also preyed on an average

of 741 eggs of M. ochroloma, completing their development in 25 d at 250C. Therefore,

P. maculiventris can develop successfully on a diet of M. ochroloma eggs or larvae,

despite the presence of secondary compounds (glucosinolates) in crucifers consumed

by M. ochroloma. The nymphal stage of P. maculiventris develops faster and preys on

more 4th instars of M. ochroloma at higher temperatures.

A two-year cage experiment addressed the field predation potential of P.

maculiventris when 1st instars were released at three densities (4=low, 10=medium, and









16=high) on six turnip plants with a known initial population of M. ochroloma larvae. In

2009, the plants grew larger (27 leaves/plant). The high predator density treatment

reduced the M. ochroloma population significantly more than the medium predator

density treatment, which reduced the pest population significantly more than the low

predator density treatment. The high predator density treatment reduced the M.

ochroloma population by 96% and the overall survivorship of P. maculiventris was 51%.

In 2010, the plants were smaller (56 leaves/plant). The medium and high predator

density treatments reduced equally the M. ochroloma population, but significantly more

than the low predator density treatment. The medium and high predator density

treatments reduced the M. ochroloma population by 99% and the overall survivorship of

P. maculiventris was 26%. Therefore, depending on the plant size, 10 or 16 nymphs

per 1.21 m2 (the approximate area covered by six plants) are recommended to release

in the field to control M. ochroloma population

Two experiments addressed the evaluation of the fungus on M. ochroloma. In

Experiment 1 the larval stage was shown to be the most susceptible stage to PFR-97TM

In Experiment 2 the most susceptible stage from Experiment 1 (i.e. 1st instar) was

exposed to four concentrations of PFR-97TM (1, 2, 3, and 4 g of PFR-97TM each in 100

ml of distilled water). The 4 g concentration caused the highest infection rate (27%

confirmed infectivity) on the 1st instars of M. ochroloma compared to the 1, 2, and 3 g

concentration (2, 5, and 10% infectivity, respectively). Therefore, PFR-97TM is not

recommended for use to control M. ochroloma in the field, due to the low rate of

infection observed in the laboratory.









For the Growers

Podisus maculiventris is recommended for use in the field to control populations

of M. ochroloma. The initial density of M. ochroloma used in this study is relatively low

compared to densities that may be observed in organic farms. Therefore, releases of P.

maculiventris should be made when infestations of M. ochroloma are low so that the

predator may provide preventive control of increasing pest populations. For big plants

(27 leaves/plant), a release of 16 1st instar P. maculiventris per six plants or 1.21 m2 is

recommended. For small plants (56 leaves/plant), a release rate of 10 1st instar P.

maculiventris per six plants or 1.21 m2 is recommended. A bottle of 250 eggs of P.

maculiventris can be purchased online at www.riconvitova.com ($112.74), www.arbico-

organics.com ($133.50), or www.planetnatural.com ($ 118.95). The costs of releasing

10 and 16 nymphs per 1.21 m2 are $4.50 and $7.21, respectively. A bottle of 250 eggs

of P. maculiventris will cover 30 m2 for the release density of 10 nymphs and 19 m2 for

the release density of 16 nymphs. A cheaper alternative for obtaining eggs is by

collecting adults from the field and holding them indoors with prey. The prey can be

mainly caterpillars or beetle larvae. However, the availability of prey and the time to

feed the predators can be a disadvantage to this alternate method of obtaining eggs.

The product PFR-97TM containing blastospores of I. fumosorosea is not

recommended for use in the field to control M. ochroloma, since higher concentrations

than those tested in the laboratory are required to kill more than 50% of the population.

The suspension preparation of PFR-97TM at concentrations higher than 4 g per 100 ml

will increase pest management costs significantly, and the higher concentrated material

could clog the nozzle during application of the product. The manufacturer, Certis









(www.certisusa.com), sells a 9.5-kg bag of PFR-97TM for $35.00. Even though PFR-

97TM did not have a high rate of infection in M. ochroloma, OMRI listed products such as

Entrust and PyGaniccan be another option to control populations of M. ochroloma.









LIST OF REFERENCES


Ables, J. R., and D. W. McCommas. 1982. Efficacy of Podisus maculiventris as a
predator of variegated cutworm on greenhouse cotton. J. Ga. Entomol. Soc. 17:
204-206.

Alyokhin, A., M. Baker, D. Mota-Sanchez, G. Dively, and E. Grafius. 2008. Colorado
potato beetle resistant to insecticides. Am. J. Pot. Res. 85:395-413.

Ameen, A. 0., and R. N. Story. 1997a. Feeding preferences of larval and adult
Microtheca ochroloma (Coleoptera: Chrysomelidae) for crucifer foliage. J. Agric.
Entomol. 14: 363-368.

Ameen, A. 0., and R. N. Story. 1997b. Biology of the yellowmargined leaf beetle
(Coleoptera: Chrysomelidae) on crucifers. J. Agric. Entomol. 32: 478-486.

Asaff, A., C. Cerda-Garcia-Rojas, and M. de la Torre 2005. Isolation of dipicolinic acid
as an insecticidal toxin from Paecilomyces fumosoroseus. Applied Microbiology
and Biotechnology. 68: 542-547.

Bajan, C. 1973. Paecilomyces fumoso-roseus (Wize) pathogenic agent of the Colorado
beetle (Leptinotarsa decemlineata Say), Ekologia Polska. 21: 705-713.

Bastos-Dequech, S. T., C. D. Sausen, C. G Lima, and R. Egewarth. 2008. Efeito de
extratos de plants com atividade inseticida no control de Microtheca
ochroloma Stal (Col: Chrysomelidae), em laborat6rio. Biotemas. 21: 41-46.

Balsbaugh, E. U. 1978. A second species of Microtheca Stal (Coleoptera:
Chrysomelidae) found in North America. Coleopts. Bull. 32: 219-222.

Bernardini, M., A. Carilli, G. Pacioni, and B. Santurbano 1975. Isolation of
Beauvericin from Paecilomyces fumosoroseus, Phytochemistry. 14: 1865.

Biever, K. D., and R. L. Chauvin. 1992. Suppression of the Colorado potato beetle
(Coleoptera : Chrysomelidae) with augmentative releases of predaceous
stinkbugs (Hemiptera : Pentatomidae). J. Econ. Entomol. 85: 720-726.

Bowers, K. 2003. Effects of within-field location of host plants and intercropping on the
distribution of Microtheca ochroloma (Stal) in Mizuna. M. S. thesis, University of
Florida, Gainesville.

Basulu, R. R., and H. Fadamiro. 2008. Field evaluation of select OMRI biorational
insecticides against yellowmargined leaf beetle, Microtheca ochroloma
(Coleoptera: Chrysomelidae) in organic crucifer vegetables. Entomological
Society of America. Annual meeting. Tuesday, November 18, 2008. D0227.
Plant-Insect Ecosystems Section.
(http://esa.confex.com/esa/2008/techprogram/paper_37405. htm)









Capinera, J. L. 2001. Handbook of Vegetables Pests. Academic Press, San Diego, CA.

Chamberlin, F. S., and H. H. Tippins. 1948. Microtheca ochroloma, an introduced pest
of crucifers, found in Alabama. J. Econ. Entomol. 41: 979-980.

Collier T., and R. Van Steenwyk. 2004. A critical evaluation of augmentative biological
control. Biol. Control 31: 245-256.

Crower, D. 2007. Impact of release rates on the effectiveness of augmentative
biological control agents. J. Insect Sci. 15:1-11.

Danks, H. V. 1987. Insect dormancy: An ecological Perspective. Biological Survey of
Canada (Terrestrial Artropods), Ottawa, Canada.

De Clercq, P., and D. Degheele. 1992. Development and survival of Podisus
maculiventris (Say) and Podisus sagitta (Fab.) (Heteroptera: Pentatomidae) at
various constant temperatures. Can. Entomol. 124: 125-133.

De Clercq, P., and D. Degheele. 1994. Laboratory measurement of predation by
Podisus maculiventris and Podisus sagitta (Hemiptera: Pentatomidae) on beet
armyworm (Lepidoptera: Noctuidae). J. Econ. Entomol. 87: 76-83.

De Clercq, P., F. Merlevede, I. Mestdagh, K. Vandendurpel, J. Mohaghegh, and D.
Degheele. 1998. Predation on the tomato looper Chrysodeixis chalcites (Esper)
(Lep., Noctuidae) by Podisus maculiventris (Say) and Podisus nigripinus (Dallas)
(Het., Pentatomidae). J. Appl. Entomol. 122: 93-98.

Desurmont, G., and P. A. Weston. 2008. Influence of prey size and environmental
factors on predation by Podisus maculiventris (Hemiptera: Pentatomidae) on
viburnum leaf beetle (Coleoptera: Chrysomelidae). Can. Entomol. 140: 192-202.

Dos Anjos, J., P. Rosalino, C. D. Sausen, L. Do Prado, R. Egewarth, V. Soares, and
S. T. Bastos. 2007. Fungos entomolatogenicos em Diabrotica speciosa e
Microtheca ochroloma (Col., Chrysomelidae) em hortalizas. Informe Tecnico.
Universidade Federal de Santa Maria, Santa Maria, Brasil.

Drees, B. M. 1990. Yellowmargined leaf beetle on leafy greens in Texas. Texas A&M
University. Texas agricultural extension services. UC-006.
(http://insects.tamu.edu/extension/bulletins/uc/uc-006.html)

Fargues, J., J. C. Delmas, R. A. Lebrun.1994. Leaf consumption by larvae of the
Colorado potato beetle (Coleoptera: Chrysomelidae) infected with the
entomopathogen, Beauveria bassiana. J. Econ. Entomol. 87: 67-71.









Fargues, J., M. S. Goettel, N. Smits, A. Ouedraogo, C. Vidal, L. A. Lacey, C. J.
Lomer, and M. Rougier. 1996. Variability in susceptibility to simulated sunlight
of conidia among isolates of entomopathogenic hyphomycetes. Mycopathologia
135: 171-181.

Fasulo, T. R. 2005. Yellowmargined leaf beetle, Microtheca ochroloma Stal (Insecta:
Coleoptera: Chrysomelidae). University of Florida. Florida Cooperative Extension
service. IFAS. EDIS. EENY 348. (http://edis.ifas.ufl.edu/pdffiles/IN/IN62500.pdf)

Ferron, P. 1981. Pest Control by The Fungi Beauveria bassiana and Metarhizium: In
Microbial Control Pests and Plant diseases. Ed by H. D. Burges. Academic
Press, 1970-1980. New York and London.

(FOG) Florida Organic Grower. 2008. List of Certified Growers & Handlers. Florida.
(http://www.foginfo.org/)

Guillebeau, P. 2001. Crop profile for leafy greens in Georgia. USDA crop profiles.
(http://www.ipmcenters.org/cropprofiles/docs/GAleafgreen. pdf)

Greif, M. D., and R. S. Currah. 2007. Patterns in the occurrence of saprophytic fungi
carried by arthropods caught in traps baited with rotted wood and dung.
Mycologia 99: 7-19.

Gusev, G. V., Yu. V. Zayats, E. M. Topashchenko, and G. K. Rzhavina. 1983.
Control of the Colorado potato beetle (Coleoptera: Chrysomelidae). Zash. Rast.
9: 38-39.

Herrick, N. J., and S. R. Reitz. 2004. Temporal occurrence of Podisus maculiventris
(Hemiptera: Heteroptera: Pentatomidae) in North Florida. Fla. Entomol. 87: 587-
590.

Hough-Goldstein, J. 1996. Use of predaceous pentatomids in integrated management
of the Colorado potato beetle (Coleoptera: Chrysomelidae), in M. Coil and J.
Ruberson (eds.), Predatory Heteroptera in agroecosystems: their ecology and
use in biological control. Thomas Say Publ., Entomol. Soc. America, Lanham,
MD.

Hough-Goldstein, J., and D. McPherson. 1996. Comparasion of Perillus bioculatus
and Podisus maculiventris (Hemiptera: Pentatomidae) as potential control agents
of the Colorado potato beetle (Coleoptera: Chrysomelidae). J. Econ. Entomol.
89: 1116-1123.

Hoy, M. A., R. Singh, M. E. Rogers. 2010. Evaluations of a novel isolate of Isaria
fumosorosea for control of the Asian citrus psyllid, Diaphorina citri (Hemiptera:
Psyllidae). Fla. Entomol. 93: 24-32.









Humber, R. A., K. S. Hansen, and M. M. Wheeler. 2009. USDA-ARS Collection of
entomopathogenic fungal cultures (ARSEF), ARSEF-Catalog of Species.
(http://arsef.fpsnl.cornell.edu/mycology/catalogs/Catalog.pdf )

Hussain, A., M. Tian, Y. He, and S. Ahmed. 2009. Entomopathogenic fungi disturbed
the larval growth and feeding performance of Ocinara varians (Lepidoptera:
Bombycidae) larvae. Insect Sci. 16: 511-517

Jolivet, P. 1950. Contribution a I'etude des Microtheca StWl (Coleoptera,
Chrysomelidae). Bulletin de I'lnstitut royal des sciences naturelles de Belgique
26: 1-27.

Klowden, M. J. 2007. Physiological Systems in Insects. Integumentary Systems.
Academic Press Elsevier, Moscow, Idaho.

Lacey, L. A., and G. Mercadier. 1998. The effect of selected allelochemicals on
germination of conidia and blastospores and mycelial growth of the
entomopathogenic fungus, Paecilomyces fumosoroseus (Deuteromycotina:
Hyphomycetes). Mycopathologia, 142: 17-25.

Lacey, L. A., A. A. Kirk, L. Millar, G. Mercadier, and C. Vidal. 1999. Ovicidal and
larvicidal activity of conidia and blastospores of Paecilomyces fumosoroseus
(Deuteromycotina: Hyphomycetes) against Bemisia argentifolii (Homoptera:
Aleyrodidae) with a description of a bioassay system allowing prolonged survival
of control insects. Biocontrol Sci. Technol. 9: 9-18.

Landis, B. J. 1937. Insect hosts and nymphal development of Podisus maculiventris
Say and Perillus bioculatus F. Ohio J Sci. 37:252-259

Legaspi, J. C. 2004. Life history of Podisus maculiventris (Heteroptera: Pentatomidae)
adult females under different constant temperatures. Environ. Entomol. 33:1200-
1206.

Luangsa-ard, J. J., N. L. Hywel-Jones, L. Manoch, and R. A. Samson. 2005. On the
relationships of Paecilomyces sect. Isarioidea species. Mycological Research.
109: 581-589.

Maketon, M., P. Orosz-Coghlan, J. Jaengarun. 2008. Field evaluation of Isaria
fumosorosea on controlling the diamondback moth (Plutella xylostella) in
Chinese kale. Phytoparasitica 36: 260-263.

Michalaki, M. P., C. G. Athanassiou, T. Steenberg, and C. T. Buchelos. 2007. Effect
of Paecilomyces fumosoroseus (Wize) Brown and Smith (Ascomycota:
Hypocreales) alone or in combination with diatomaceous earth against Tribolium
confusum Jacquelin du Val (Coleoptera: Tenebrionidae) and Ephestia kuehniella
Zeller (Lepidoptera: Pyralidae), Biol. Control. 40: 280-286.









McPherson, J. E. 1980. A list of the prey species of Podisus maculiventris (Hemiptera:
Pentatomidae). Great Lakes Entomol. 13: 17-24.

McPherson, J. E. 1982. The Pentatomoidea (Hemiptera) of northeastern North
America. Southern Illinions University Press, Carbondale and Edwardsville. IL.

Meyer, J. M. 2007. Microbial associates of the Asian citrus psyllid and its two
parasitoids: symbionts and pathogens. Ph.D. Dissertation, University of Florida,
Gainesville, 145 pp.

Meyer, J. M., M. A. Hoy, and D. G. Boucias. 2008. Isolation and characterization of an
Isaria fumosorosea isolate infecting the Asian citrus psyllid in Florida. J. Invertr.
Pathol. 99: 96-102.

Oliver, A. D. 1956. Yellow-margined leaf beetle (Microtheca ochroloma). Coop. Econ.
Insect Report. 6:351-353.

Osborne, L. S., and Z. Landa. 1992. Biological control of whiteflies with
entomopathogenic fungi. Fla. Entomol. 75: 456-471

Osborne, L., Z. Landa, A. Bohata, and C. McKenzie. 2008. Potential of
entomopathogenic fungus Isaria fumosorosea to protect potted ornamental
plants against Bemisia tabaci during shipping. International Organization for
Biological Control/WPRS Bulletin 32: 159-165.

Overall, L., and J. Edelson. 2007. Field evaluation of organic insecticides to control the
harlequin bug, Murgantia histrionica, and the yellowmargined leaf beetle,
Microtheca ochroloma, on leafy greens in southern Oklahoma. Entomological
Society of America Annual meeting. Monday, December 10, 2007 10:29 AM
0517 (http://esa.confex.com/esa/2007/techprogram/paper_30387.htm)

Poprawski, T. J., and W. J. Jones. 2001. Host plant effects on activity of the
mitosporic fungi Beauveria bassiana and Paecilomyces fumosoroseus against
two populations of Bemisia whiteflies (Homoptera: Aleyrodidae). Mycopathologia.
151: 11-20.

Racca Filho, F., I. L. Rodrigues-Filho, C. A. C. Santos, and C. N. Rodrigues. 1994.
Microtheca ochroloma (Coleoptera: Chrysomelidae): aspects taxonomicos e
biol6gicos. Rev. Univ. Rural. 16: 29-35.

Richman, D. B., and W. H. Whitcomb. 1978. Comparative life cycles of four species of
predatory stink bugs (Hemiptera: Pentatomidae). Fla. Entomol. 61: 113-119.

Rohwer, K. S., F. E. Guyton, and F. S. Chamberlin. 1953. Status of the yellow-
margined leaf beetle. Coop. Econ. Insect Report 3: 194-195.









Rodrigues-Rueda, D., and J. Fargues. 1980. Pathogenicity of entomopathogenic
hyphomycetes, Paecilomyces fumosoroseus and Nomuraea rilei, to eggs of
noctuids, Mamestra brassicae and S podoptera littoralis. J. Invert. Pathol. 36:
399-408.

Samson, R. A. 1974. Paecilomyces and some allied Hyphomycetes. Studies in Mycol.
6: 32-41.

Silva, A. G. D'A., C. R. Gongalves, D. M. Galvao, A. J. L. Gongalve, J. Gomes, M. N.
Silva, and L. Simoni. 1968. Quarto catalogo dos insetos que vivem nas plants
do Brasil, seus parasitas e predadores. Ministerio da Agricultura, Rio de Janeiro,
Brasil.

Smith, P. 1993. Control of Bemisia tabaci and the potential of Paecilomyces
fumosoroseus as a biopesticide. Biocontrol News & Inform. 14: 71-78.

Staines, C. L. 1999. Chrysomelidae (Coleoptera) new to North Carolina. Coleopts. Bull.
53: 27-29.

Stamopoulos, D. C., and A. Chlroridis. 1994. Predation rates, survivorship and
development of Podisus maculiventris (Het.: Pentatomidae) on larvae of
Leptinotarsa decemlineata (Col.: Chrysomelidae) and Pieris brassicae
(Lep.:Pieridae), under field conditions. Entomophaga. 39: 3-9.

Tefera, T., and K. L. Pringle. 2003. Food consumption by Chilo partellus (Lepidoptera:
Pyralidae) larvae infected with Beauveria bassiana and Metarhizium anisopliae
and effects of feeding natural versus artificial diets on mortality and mycosis. J.
Invertebr. Pathol. 84: 220-225.

Tabashnick, B. E., and N. L. Cushing.1987.Quantitative genetic analysis of insecticide
resistance: Variation in fenvalerate tolerance in a diamondback moth
(Lepidoptera: Plutellidae) population. J. Econ. Entomol. 82: 5-10.

Tipping, P. W., C. A. Holko, A. A. Abdul-Baki, and J. R. Aldrich. 1999. Evaluating
Edovum puttleri Grissell and Podisus maculiventris (Say) for augmentative
biological control of Colorado potato beetle in tomatoes. Biol. Control. 16: 35-42.

Tounou, A. K., K. Agboka, H. M. Poehling, J. K. Raupach, J. Langewald, G.
Zimmermann, and C. Borgemeister. 2003. Evaluation of the entomopathogenic
fungi Metarhizium anisopliae and Paecilomyces fumosoroseus
(Deuteromycotina: Hyphomycetes) for control of the green leafhopper empoasca
decipiens (Homoptera: Cicadellidae) and potential side effects on the egg
parasitoid Anagrus atomus (Hymenoptera: Mymaridae). Biocontrol Sci. and
Technol. 13: 715-728.









(USDA) U. S. Department of Agriculture. 2010. National Organic Program. Resource
Center: Regulations.
(http://www.ams.usda.gov/AMSv1.0/ams.fetchTemplateData.do?template=Templ
ateF&navlD=NationalOrganicProgram&leftNav=NationalOrganicProgram&page=
NOPResourceCenterRegulations&description=NOP%20Regulations&acct=nopru
lemaking)

(USDA-NASS) U. S. Department of Agriculture National Agricultural Statistics
Service. 2009. 2007 Census of Agriculture. United Sates Department of
Agriculture, National Agriculture Statistics, Washington, D.C.
(http://www.agcensus.usda.gov/Publications/2007/Online_Highlights/County_Pro
files/Florida/cp99012.pdf.).

Van Driesche, R. D., S. Lyon, K. Jacques, T. Smith, and P. Lopes. 2002.
Comparative cost of chemical and biological whitefly control In poinsettia: Is there
a gap?. Fla. Entomol. 85: 488-493.

Valicente, F. H., and R. J. O'Neil. 1995. Effects of host plants and feeding regimes on
selected life history characteristics of Podisus maculiventris (Say) (Heteroptera:
Pentatomidae). Biol. Control 5:449-461

Vidal, C., and J. Fargues. 2007. Climatic Constraints for Fungal Biopesticides, pp. 39-
55. In S. Ekesi, and N. K. Maniania (eds.), Use of Entomopathogenic Fungi in
Biological Pest Management. Kerala, India. Research Signpost.

Vidal, C., L. A. Lacey, and J. Fargues. 1997a. Pathogenicity of Paecilomyces
fumosoroseus (Deuteromycotina: Hyphomycetes) against Bemisia argentifolii
(Homoptera: Aleyrodidae) with a Description of a Bioassay Method. J. Econ.
Entomol. 90: 765-772.

Vidal, C., J. Margues, and L. A. Lacey. 1997b. Intraspecific variability of Paecilomyces
fumosoroseus: effect of temperature on vegetative growth. J. Invert. Pathol. 70:
18-26.

Vidal, C., L.S. Osborne, L.A. Lacey, and J. Fargues, 1998. Effect of host plant on the
potential of Paecilomyces fumosoroseus (Deuteromycotina: Hyphomycetes) for
controlling the silverleaf whitefly, Bemisia argentifolii (Homoptera: Aleyrodidae) in
greenhouses. Biol. Control. 12: 191-199.

Waddill, V., and M. Shepard. 1975. A comparison of predation by the pentatomids,
Podisus maculiventris (Say) and Stiretrus anchorage (F.), on the Mexican bean
beetle, Epilachna varivestis Mulsant. Ann. Entomol. Soc. Am. 68: 1023-1027.

Wiedenmann, R. N., and R. J. O'Neil. 1990. Effects of low rates of predation on
selected life-history characterisitcs of Podisus maculiventris (Say) (Heteroptera:
Pentatomidae). Can. Entomol. 122: 271-283.









Wiedenmann, R. N., and R. J. O'Neil. 1991. Laboratory measurement of the functional
response of Podisus maculiventris (Say) (Heteroptera: Pentatomidae). Environ.
Entomol. 20: 610-614.

Woodruff, R. E. 1974. A South American leaf beetle pest of crucifers in Florida
(Coleoptera: Chrysomelidae). FDACS-DPI Entomol. Cir. 148.









BIOGRAPHICAL SKETCH

Cecil O. Montemayor Aizpurua was born in David, Panama. She received her

bachelor's degree at the Panamerican School of Agriculture, Zamorano, in Honduras in

2005. She conducted her degree internship at Chiquita Brands International Co. in La

Ceiba, Honduras, working with entomopathogenic fungi to control pests in bananas. In

2006, she was an intern at the University of Minnesota, where she worked in wetlands

restoration and in the biological control of soybean aphids. Since 2007, she has worked

at the University of Florida's Biological Control Research and Containment Laboratory

(BCRCL) at the Indian River Research and Education Center in Ft. Pierce. At the

BCRCL, she first worked as a short-term scholar, conducting research on biological

control of insects, specifically the yellowmargined leaf beetle, and processing

specimens collected for an inventory of the arthropods on tree islands in the South

Florida Water Management District Conservation Area. She then began her Master of

Science degree program in the Entomology and Nematology Department in August

2008. She received a scholarship grant from the Ministry of Economy and Finances of

Panama to support her during her study program. She is a member of the

Entomological Society of America, Florida Entomological Society, and Florida State

Horticultural Society. She was the president of the Statewide Student Association at

University of Florida from 2009 to 2010. She has presented talks about her research at

the annual meetings of the Florida State Horticultural Society (third place in the student

competition), the Florida Entomological Society (first place in the student competition),

and the Entomological Society of America.





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1 EVAL UATION OF A PREDATOR AND A FUNGUS AS BIOLOGICAL CONTROL AGENTS OF THE YELLOWMARGINED LEAF BEETLE, Microtheca ochroloma STL (COLEOPTERA: CHRYSOMELIDAE) By CECIL O. MONTEMAYOR AIZPURA A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2010

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2 2010 Cecil O. Montemayor Aizpura

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3 T o my parents, Monty and Lichy ; my brothers, Alcides and Joshua; and my family

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4 ACKNOWLEDGMENTS I would like to acknowledge my advisory committee, my chair Dr. Ronald D. Cave, Dr. Susan Webb, and Mr. Edward Skvarch. I am thankful to Dr. Cave for his continued support encourageme nt, and guidance during the course of my studies as a graduate stud ent and also for providing me financial assistance to participate in state and national meetings. I am also grateful to Dr. Webb and Mr. Skvarch for their critiques and suggestions that ma de substantial improvements to this study. I would like to thank Dr. Patrick De Clercq at Ghent University in Belgium for providing valuable information to my thesis. I give thanks to Jos Castillo, Daniel Mancero, Dafne Serrano, and Bradley Sm ith for thei r help conducting my field research. T hank s go out to the Minist r y of Economy and Finances of Panama for sponsor ing my masters education in entomology at the University of Florida and the Florida Specialty Crop Foundation to support my field research. Thanks to all my friends and colleagues at the entomology department in Gainesville, but especially to Veronica Santillan, Diana Castillo, Daniel Carrillo, Andres Sandoval, and Sebastian Padrn for their unconditional friendship T hank s to Dr. Pasco Avery at IRREC for sharing his knowledge, and providing equipment I would also like to thank Valerie Quant for letting me collect beetles from her organic farm in Vero Beach. I am grateful to my best friends Rodrigo Daz and Vernica Manrique, for their profes sional advic es regarding my research, but also for always being there for me as a family in Ft. P ierce I also want to thank Dr. Caves family Vilma, Eloise, and Jonathan, for letting me be part of their special moments during this time. Finally, I would like to give special thank s to my family for their loving encouragement and unconditional support which motivated me to complete my study.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 7 LIST OF FIGURES .......................................................................................................... 8 LIST OF ABBREVIATIONS ........................................................................................... 10 ABSTRACT ................................................................................................................... 11 CHAPTER 1 GENERAL INTRODUCTION .................................................................................. 14 2 LITERATURE REVIEW .......................................................................................... 16 Microtheca ochroloma Stl ..................................................................................... 16 Origin and Distribution ...................................................................................... 16 Biology and Host Range ................................................................................... 16 Damage and Summer Activity .......................................................................... 17 Control Methods ............................................................................................... 18 Podisus maculiventris (Say) .................................................................................... 20 Origin and Distribution ...................................................................................... 20 Biology and Host Range ................................................................................... 20 Searching Behavior .......................................................................................... 21 Biological Control ............................................................................................. 21 Isaria fumosorosea Wize (Brown and Smith) .......................................................... 22 Origin and Distribution ...................................................................................... 22 Biology and Host Range ................................................................................... 23 Biological Control ............................................................................................. 24 Objectives of Master of Science Thesis Research .................................................. 25 3 DEVELOPMENT TIME AND PREDATION RATE OF PODISUS MACULIVENTRIS (SAY) (HEMIPTERA: PENTATOMIDAE) PREYING ON MICROTHECA OCHROLOMA STL (COLEOPTERA: CHRYSOMELIDAE) ......... 28 Introduction ............................................................................................................. 28 Materials and Methods ............................................................................................ 29 Results .................................................................................................................... 31 Discussion .............................................................................................................. 35 4 PREDATION POTENTIAL OF PODISUS MACULIVENTRIS (HEMIPTERA: PENTATOMIDAE) ON MICROT HECA OCHROLOMA (COLEOPTERA: CHRYSOMELIDAE) IN THE FIELD ........................................................................ 43

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6 Introduction ............................................................................................................. 43 Materials and Methods ............................................................................................ 45 Results .................................................................................................................... 47 2009 Experiment .............................................................................................. 47 2010 Experiment .............................................................................................. 50 Discussion .............................................................................................................. 52 2009 Experiment .............................................................................................. 52 2010 Experiment .............................................................................................. 54 5 INFECTIVITY OF MICROTHECA OCHROLOMA STL (COLEOPTERA: CHRYSOMELIDAE) BY ISARIA FUMOSOROSEA WIZE (BROWN AND SMITH) ................................................................................................................... 63 Introduction ............................................................................................................. 63 Materials and Methods ............................................................................................ 64 Experiment 1. Susceptibility of Microtheca ochroloma to Infection by PFR 97TM ............................................................................................................... 65 Experi ment 2. Infectivity of the Most Susceptible Stage of Microtheca ochroloma by Four Concentrations of PFR 97TM........................................... 66 Results .................................................................................................................... 68 Expe riment 1 .................................................................................................... 68 Experiment 2 .................................................................................................... 69 Discussion .............................................................................................................. 69 Experiment 1 .................................................................................................... 69 Experiment 2 .................................................................................................... 71 6 CONCLUSIONS ..................................................................................................... 84 LIST OF REFERENCES ............................................................................................... 88 BIOGRAPHICAL SKETCH ............................................................................................ 96

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7 LIST OF TABLES Table page 3 1 Mean ( SE) development time of Podisus macul iventris reared at three constant temperatures with 4th instar Microtheca ochroloma as prey. ................ 39 3 2 Mean ( SE) fresh weight of newly ecdysed Podisus maculiventris reared at three constant te mperatures with 4th instar Microtheca ochroloma as a prey. ... 39 3 3 Mean ( SE) daily predation of 4th instar Microtheca ochroloma by Podisus maculiventris reared at three temperatures.. ...................................................... 40 3 4 Mean ( SE) total predation of 4th instar Microtheca ochroloma by Podisus maculiventris reared at three constant temperatures. ........................................ 40 3 5 Mean ( SE) total development time and predation of 4th instar Microtheca ochroloma per nymph of Podisus maculiventris .. ............................................... 41 3 6 Mean ( SE) fresh weight of newly ecdysed adults of P odisus maculiventris reared with 4th instar Microtheca ochroloma as a prey. ..................................... 41 3 7 Mean ( SE) predation of eggs of Microtheca ochroloma by Podisus maculiventris nymphs reared at 25C. ................................................................ 42 5 1 Laboratory tests of Isaria fumosorosea in Experiment 2. .................................... 83

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8 LIST OF FIGURES Figure page 2 1 Life stages of Microtheca ochroloma .................................................................. 26 2 2 Damage by Microtheca ochroloma. .................................................................... 27 3 1 Plastic boxes with screened windows used to house experimental insects. ....... 38 4 1 Cages in the field at the beginning of the 2009 experiment. ............................... 57 4 2 Plants being gathered for final sampling in the laboratory. ................................. 57 4 3 Number of Microtheca ochroloma per cage during the 2009 field experiment. .. 58 4 4 Mean number of Podisus maculiventris nymphs per cage during the 2009 field experiment .................................................................................................. 59 4 5 Mean number of Microtheca ochroloma per cage during the 2010 field experiment .......................................................................................................... 60 4 6 Number of Podisus maculiventris nymphs per cage during the 2010 field experiment. ......................................................................................................... 61 4 7 Size of turnip plants in the 2009 exper iment. ...................................................... 6 2 4 8 Size of turnip plants in the 2010 experiment. ...................................................... 62 5 1 Morphological characteristics of Isaria fumosorosea infecting Microtheca ochroloma larva. ................................................................................................. 73 5 2 Laboratory tests of Isaria fumosorosea. ............................................................. 74 5 3 Mortality of Microtheca ochroloma by PFR 97TM 20% WDG at 3.0 107 blastospores/ml 7 d after application .................................................................. 75 5 4 Infectivity of Microtheca ochroloma by PFR 97TM 20 % WDG at 3.0 107 blastospores/ml 7 d a fter application. ................................................................. 76 5 5 Lethal time of the first and third instars of Microtheca ochroloma tr eated with PFR 97TM ............................................................................................................ 77 5 6 Infectivity of first instar Microt heca ochroloma at four concentrations of PFR 97TM 7 d after application. ................................................................................... 78 5 7 Lethal time of the first instar Microtheca ochroloma exposed to four concentrations of PFR 97TM ............................................................................... 79

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9 5 8 Eggs of Microtheca ochroloma infected by Isaria fumosorosea. ......................... 80 5 9 Unconfirmed and confirmed infectivity by Isaria fumosorosea in dea d larvae of Microtheca ochroloma. ................................................................................... 80 5 10 Reduction in the growth of larvae of Microtheca ochroloma infected by Isaria fumosorosea. ...................................................................................................... 81 5 11 Net like pupal case of Microtheca ochroloma. .................................................... 81 5 12 Unsuccessful molting by a larva of Microtheca ochroloma infected with Isaria fumosorosea ....................................................................................................... 82

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1 0 LIST OF ABBREVIATION S LC Lethal concentrat ion LT Lethal time PFR P aeci lomyces fumosoroseus = Isaria fumosorosea

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11 Abstrac t of Thesis Presented to the Graduate School of the University of Florida i n Partial Fulfillment of the Requi rements for the Degree of Master of Science EVAL UATION OF A PREDATOR AND A FUNGUS AS BIOLOGICAL CONTROL AGENTS OF THE YELLOWMARGINED LEAF BEETLE, Microtheca ochroloma STL (COLEOPTERA: CHRYSOMELIDAE) By Cecil O. Montemayor Aizpur a August 2010 Chair: Ronald D Cave Major: Entomology and Nematology The yellowmargined leaf beetle, Microtheca ochroloma Stl (Coleoptera: Chrysomelidae) is a pest of cruciferous crops in the southern United States since its first detection in the country in 1945. Little informat ion is available in the literature about the natural enemies of this pest. Therefore, the goal of this study was to evaluate the efficacy of the predator Podisus maculiventris (Say) (Hemiptera: Pentatomidae) and the fungus Isaria fumosorosea (Brown and Sm ith) Wize ( Hypocreales : Cordycipitaceae) to control populations of M. ochroloma. Preliminary results demonstrated that P. maculiventris preys on all stages of M. ochroloma. The first experiment of this thesis measured the predation rate fresh weight and developmental time of P. maculiventris feeding on M. ochroloma larvae at constant temperatures of 10, 15, 20 and 25C in the laboratory. T here was no development of 1st instar s or egg hatch at 10 C. The nymphal stage of P. maculiventris preyed on a mean total of 65 6.3 4th instars of M. ochroloma during 99 4.6 days at 15 C, 53 1.3 4th instars of M. ochroloma during 31 0.3 days at 20C, and 59 4th instars or 741 eggs of M. ochroloma in 23 0.3 and 25 0.3 days, respectively, at 25 C A dults preyed on a mean total of 5.0 2.1 4th instars of

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12 M. ochroloma during 10 days at 15C, 45.1 2.3 4th instars at 20C, and 64 2.9 4th instars at 25C. Adult females weighed more than males at 20C. The second experiment of this study was to determine a fi eld release guideline for P. maculiventris by measuring its predation potential on M. ochroloma in field cages. Four, 10, and 16 1st instar P. maculiventris were separately released in cages containing an initial population of 130 1st instar s M. ochroloma on six turnip plants The release rate of 16 P. maculiventris per six large ( 7 leaves/plant) turnip plants significantly reduced the M. ochroloma population over time compared to the other two release rates. For six small ( 6 leaves/plant) turnip plants a release rate of 10 P. maculiventris significantly reduced the M. ochroloma population over time compared to the lowest release rate, but its pest population suppression capabilities were not significantly different from the higher release rate. The th ird experiment of this research was to evaluate the infectivity and lethal time (LT) of I. fumosorosea (commercial name: PFR 97TM) on M. ochroloma at the concentration of 1g of PFR 97TM in 100 ml of distilled water in the laboratory. The larval stage is mo re susceptible to PFR 97TM than eggs, pupae, and adults. Infectivity rates of 17 and 20% were confirmed in the 1st and 3rd instars of M. ochroloma, respectively. The LT10 for 1st and 3rd instars of M. ochroloma w ere 4 and 3 days, respectively. Concentrations of 1, 2, 3, and 4 g of PFR 97TM in 100 ml of distilled water were applied to 1st instars of M. ochroloma to compare i nfectivity LT, and lethal concentrations (LC). Confirmed infectivity rates for 1, 2, 3, and 4 g concentrations were 2, 5, 10, and 27%, respectively. The LT10 and LT25 for the 4 g concentration were 3.4 and 5.7 days,

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13 respectively. The LC10 and LC25 were 1.4 g and 5.5 g per 100 ml of distilled water, respectively. The results of my research suggest that P. maculiventris is a promising bio logical control agent of M. ochroloma. This predator can be use d in an augmentative biological control program in cruciferous crops to control M. ochroloma on organic farms I saria fumosorosea (PFR 97TM), on the other hand, does not show any clear evidenc e of being a potentially effective biological control agent of M. ochroloma.

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14 CHAPTER 1 GENERAL INTRODUCTION Vegetable production is one of the most important sources of income in the United States. In the Census of Agriculture of 2007, the USDA ranked Fl orida as the second highest state with sales in vegetables, melons, potatoes and sweet potatoes. From 2002 to 2007, the market value of vegetable production in Florida increased 25% (USDA NASS, 2007). The organic vegetable niche has been growing fast since 1997. According to Florida Organic Grower (2008), 42 farms producing broccoli, turnips, cabbage, collards, cauliflower, and Chinese cabbage are included on the list of certified organic growers. The most popular organic vegetables are the green leaves in which the leaves are the main commercial part of the plant. Most of the pests that attack green leaves defoliate their host plants, making them unavailable for the grower to offer good quality food for the consumers. Pest management programs on or ganic farms are based on ecofriendly strategies. The National Organic Program (NOP) states in the Electronic Code of f ederal Regulations (eCFR) (Standards) that the producer must use management practices to prevent crop pests including but not limited to: 1) a ugmentation or introduction of predators or parasites of the pest species; 2) d evelopment of habitat for natural enemies of pests; 3) n onsynthetic controls such as lures, traps, and repellents (USDA 2010). My research focused on the first and thir d of these Federal regulations to prevent crop pests. This was achieved by evaluating the efficacy of a generalist predator, Podisus maculiventris (Say), and the infectivity of an entomopathogenic fungus, Isaria fumosorosea Wize (Brown and Smith) against an invasive pest,

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15 Microtheca ochroloma Stl, commonly called the yellowmargined leaf beetle, in cruciferous crops. Podisus maculiventris was chosen because it was observed in cruciferous crops feeding on larvae and adults of M. ochroloma, and because it c an be used for augmentative biological control (HoughGol d stein and McPherson 1996; De Clercq et al. 1998). Isaria fumosorosea is known worldwide as a microbial insecticide due to its diversity of infective strains and broad host range (Smith 1993). The commercial strain Apopka 97, registered as PFR 97TM 20% WDG, was chosen for this study because it is local to Florida (Vidal et al. 1998), and because more information about its host range needs to be known.

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16 CHAPTER 2 LITERATURE REVIEW Microtheca ochrolo ma Stl Origin and D istribution The yellowmargined leaf beetle, Microtheca ochroloma Stl (Coleoptera: Chrysomelidae), was previously known as Microtheca punctigera Achard. Jol i v et (1950) revised the genus Microtheca and recognized genitalic differences between these two species. Microtheca ochroloma is an invasive insect from South America, specifically Argentina, Brazil (Rio de Janeiro and Rio Grande do Sul) (Silva et al. 1968; Racca Filho et al. 1994), Chile, and Uruguay. In the U.S., it was first reported from New Orleans in 1945, occurring in imported grapes coming from Argentina (Chamberlin and Tippins 1948). Since then, this pest has spread to Texas (Balsbaugh 1978), Alabama (Chamberlin and Tippins 1948), Mississippi (Rohwer et al. 1953), North Carolina (Staines 1999), Louisiana (Oliver 1956), Georgia (Guillebeau 2001), and Florida. In 1972, it was reported on watercress at an aquatic nursery in Tampa, Florida (Woodruff 1974). Biology and Host R ange Microtheca ochroloma receives the common name yellowmargined leaf beetle because its elytra are brownish to black with yellow margins and four prominent rows of punctures on each elytron. The eggs are yellow to orange, oval, and often laid in the soil (Fig. 2 1A). The larva is yellow to brown, wi th a sclerotized head capsule (Woodruff 1974) (Fig. 2 1 B). The immature stage takes 26.6 d at 20C to complete its development from egg to adult, and the larval stage usually has four instars. However, laboratory experiments showed that 5% of the populati on passes through a fifth instar

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17 (Ameen and Story 1997b). When the larvae are ready to pupate, they build a brownish, net like pupal case (Fig. 2 1 C) to surround themselves on dry old leaves (Woodruff 1974). The pupal stage last s from 5 to 6 d at 20 C (A meen and Story 1997b). The adult of M. ochroloma is about 5 mm long; usually the females are larger than the males (Fig. 2 1D ). New ly emerged adults stay o n the dry leaves for 2 d before moving to green foliage (Capinera 2001). Adults live to as many as 186 d when fed radish plants (Ameen and Story 1997b) In Florida, M. ochroloma is present in the field during the cool months o f October to April, which corresponds to the production season of many of its vegetable hosts (Bowers 2003) The host range of t his pest includes all members of the Brassicaceae family such as cabbage, collards, turnip, radish, and wat ercress (Chamberlin and Tippins 1948). Laboratory studies showed that turnip is the preferred host, since higher average fecundity (490 eggs per fe male ) was obtained on this host plant compared to other plant species (198 eggs per female) (Amen and Story 1997a). Damage and S umme r A ctivity M icrotheca ochroloma is a serious pest of high value crops such as leafy Brassica greens. Larvae and adults feed on the plant foliage and can completely defoliate their host plants (Fig. 2 2 A, B). When the beetle populations are very high, feeding on the tubers of turnips can occur (Fig. 2 2 C, D). Leaf quality is also affected by the abundant frass produced by t he larvae. Bowers (2003) list s three reasons why M. ochroloma is a problem in Florida: (1) the host plants thrive in the cool months from October to April concurring with the growing season for organic farmers in Florida; (2) h ard frosts or freezes are u nl ikely to occur; and (3) there are no known predators or parasites in the U.S.

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18 Although the damage caused by M. ochroloma occurs during in the coolest months of the year, Bowers (2003) believes that M. ochroloma is present during the summer in a reproduc tive quiescence, since adults only were found in the same spots in which turnips were planted before, also suggesting that the beetle passes the summer near its feeding sites. To corroborate these observations, some adult beetles were collected from the f ield in summer and after 24 hours of exposure to laboratory conditions females began laying eggs (Bowers 2003). This indicates that M. ochroloma does not require a lengthy period of time to reactivate its reproductive functions compared to other insects t hat do need several d ays to end the diapause process. Diapause is a physiological resting period mediated by environmental factors that are unfavorable to the insect, so that it can survive certain adverse conditions such as extreme temperatures, and is o ften triggered by photoperiod (Danks 1987). To resume activity, the insect first must complete the diapause development mediated by favorable conditions (Danks 1987). Quiescence is a dormancy that is mediated directly by extreme environmental factors ( e. g ., scarcity of food, extreme temperatures). The insect can respond to favorable environmental factors and resume normal activities immediately without having to pass through a preprogrammed period as in diapause (Danks 1987). Control M ethods Chemical co ntrol. Control of M. ochroloma has been reported by growers in Texas using products such as carbaryl (Sevin) and diazinon or mevinphos (Phosdrin) (Drees 1990). In Brazil, Bastos Dequech et al. (2008) studied the effect of plant extracts on M. ochroloma. One hundred percent larval mortality was achiev ed after 5 d in the laboratory at 27 1 C by applying pdefumo ( Nicotiana tabacum L.,

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19 Solanaceae) and ramo de cinamomo ( Melia azedarach L., Meliaceae). Mortality in adults reached 82 and 74% when DalNe em (a commercial product based on Azadirachta indica A. Juss, Meliaceae) and p defumo were applied, respectively. Basulu and Fadamiro (2008) evaluated the effect of OMRI biorational insecticides against M. ochroloma in the field. PyGanic ( extract of ch rysanthemum flowers ), Aza Direct (azadirachtin from neem plant), and Mycotrol O [entomopathogenic fungus Beauveria bassiana (Balsmo) Vuillemin] were not effective in reducing populations of M. ochroloma, but Entrust (spinosad for organic production) did lower pest densities and mean damage ratings. Similar results were obtained when Overall and Edelson (2007) evaluated organic insecticides in the field. Results indicated that Entrust and PyGanic reduced populations of M. ochroloma by 96 and 63%, respectively. Cultural control. The need to develop ecological methods to control M. ochroloma is evident to organic growers. Currently, there i s little information about the ecology of the pest. Bowers (2003) studied emergence and host finding behavior and evaluated intercropping as a tactic for reducing the severity of outbreaks. Results of her studies showed that there were more adult beetles in the interior of the plots than in the border (55 versus 8 beetles, respectively), suggesting that a large numb er of adults oversummer in the production field. Intercropping failed to be a useful control strategy for this pest. Bowers (2003) found that intercropping host plants (mizuna: Brassica rapa L., var. Kyona, Cruciferae) among nonhost plants (oak leaf lettuce: Lactuca sativa L. var. Berenice, Asteraceae) was not effective in preventing M. ochroloma from finding host plants.

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20 Biological control. Currently, there are no natural enemies of M. ochroloma re ported in the literature (Bowers 2003). However, a generalist predator, the spined soldier bug, Podisus maculiventris (Say) (Hemiptera: Pentatomidae), was observed in the field preying on the larvae, adults, and pupae of M. ochroloma. In addition, laboratory bioassays showed that larvae of the green lacewing Chrysoperla r ufilabris (Burmeiste r) (Neuroptera: Chrysopidae) prey on eggs and larvae of M. ochroloma (personal observation, C. Montemayor). Podisus maculiventris (Say) Origin and D istribution The distribution of P. maculiventris includes North America f rom Quebec to British Columbia in Canada and from Florida to Arizona in the U S .A. (McPherson 19 8 2). In northern Florida, the occurrence of P. maculiventris is temporal ; a dults emerge in April May and are active in the field throughout the spring and summ er until October November when the overwintering period begins (Herrick and Reitz 2004). Biology and Host R ange Various aspects of the biology, behavior and life history of P. maculiventris are well d ocumented in the literature (Wiedenmann and ONeil 1990 ; ONeil 1997 ; Legaspi 2004). Depending on the photoperiod and temperature, the life cycle can vary from 27 to 38 d Eggs are 1 mm in diameter with long projections around the operculum. The nymphs pass through five instars of which all except the fir st are predaceous. Cannibalism is common from the second instar to adult. Total developmental ti me from egg to adult at 27 1 C is 27 d ( Richman and Whitcomb 1978). This predator can feed on more than 75 prey species within 8 orders mainly Coleoptera and Lepidoptera (McPherson 1980), o n a wide diversity of agricultural crops. Frequently utilized prey

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21 include the Colorado potato beetle [ Stilodes decemlineata ( Say)], the asparagus beetle ( Crioceris asparagi L.), the three lined potato beetle ( Lema trilinea Olivier), the Mexican bean beetle ( Epilachna varivestis Mulsant), the fall webworm ( Hyphantria cunea Drury), and the diamondback moth ( Plutella xylostella Curtis) (Landis 1937). Searching B ehavior According to ONei l (1997), the searching strategy of P. maculiventris differs between laboratory and field conditions. In the la boratory, the attack rate increases as prey dens ity increases, but the estimated area searched decreases. The decline in the area searched was associated with accumulated handling t ime of the prey. In the field, P. maculiventris maintains a constant low rate of attack even when prey densit y increases In a potato field experiment, he found that the estimated area searched declined as Colorado potato beetle density increased, but it can not be attributed to the accumulated handling time of the prey because the predator attacked few prey in the field (<1 prey per day). This searching behavior in the field may permit P. maculiventris to s urvive periods of low prey densities using their stored body lipids and trading off reproduction for survival (Wie denmann and ONeil 1990). Biological C ontrol Podisus maculiventris plays an important role in augmentative and conservation biological control of agricultural pests (Biever and Chauvin 1992; Hough Goldstein 1996; Tipping et al. 1999) It is commercially available and is commonly used for augmentative releases in the field to control the Mexican bea n beetle (ONeil, 1988; Wie denmann 1991), the C olorado potato beetle (Stamopoulos and Chloridi s, 1994; Tipping et al. 1999), and the viburnum leaf beetle [ Pyrrhalta viburni (Paykull)] ( Desurmont 2008). The effici ency of P. maculiventris as a predator can vary among

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22 host plant species and ecosystems. For example, the presence of allelochemicals in the host plant or in the preys diet can directly or indirectly affect the performance of P. maculiventris due to its facultative plant feeding behavior (Desurmont 2008). In addition, the size and shape of the host plant (or leaves) can decrease the effi ciency of the predator by providing more leaf surface or more refugia to the prey (Desurmont 2008). The use of generalist native predators in agroecosystems to suppress pest populations may reduce risks to nontarget organisms by avoiding the introduction of exotic biological control agents and their nontarget impacts in the field. In addition, growers can easily obtain these generalist predators because they are commercially available. Isaria fumosorosea Wize (Brown and Smith) Origin and D istribution T he entomopathogenic fungus Isaria fumosorosea Wize (Brown and Smith) (= Paecilomyces fumosoroseus ) (Hypocreales : Cordycipitaceae) has a worldwide distribution in temperate and tropical zones. Different strains of I. fumosorosea are listed in 27 countries in the catalogue of USDA ARS Collection of Entomopathogenic Fungal Cultures (ARSEF) (Humber et al. 2009). According to Domsch et al. (1980), I. fumosorosea is well distributed from Europe to Africa an Asia, and occurs naturally in soil in the Netherlands, Germany, Canada, and Brazil. It has been isolated from the surface of dead insects (Greif and Currah 2007). In Florida, it was isolated from Phenacoccus sp. (Hemiptera: Pseudococcidae), and named the Apopka 97 strain (Vidal et al. 1998). Isaria fumosor osea was described in 1832 by Fries and later by Wize in 1904. After the study of the genus Paecilomyces Samson by Samson (1974), I. fumosorosea

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23 was included in a new section called Paecilomyces section Isariodea and since then the fungus was named Paecil omyces fumosorosea (Wize) Brown & Smith for several years (Zimmerman 2008). However, phylogenetic studies of species belonging to this new section showed that it is polyphyletic within the Hypocreales (Ascomycota), and the Isaria clade, which includes I. fumosorosea, was elevated to genus rank (Luangsaard 2005). Biology and Host R ange Abiotic environmental factors are important in the growth, viability, germination, and insecticidal activity of I. fumosorosea (Vidal and Fargues 2007). Vidal et al. (1997b ) determined that temperature is important in the optimal growth of I. fumosorosea, in which it can range from 20 to 30C, depending on the isolate. In Europe (French and Italian isolates), the optimal growth of I. fumosorosea is from 20 to 25C with hi gher tolerance to lower temperatures from 8 to 15 C. In tropical and subtropical areas (Texas, Florida, Cuba, and California isola tes), optimal growth is from 25 to 28C, with a greater tolerance to higher temperatures from 32 to 35 C. Indian isolates have optimal growth at 30 C, with higher tolerance to higher temperatures compared to the European and tropical and subtropical isolates (Vidal et al. 1997b ). Solar radiation and low relative humidity are other abiotic factor than can negatively affect the performance of this fungus in the field (Fargues et al. 1996). G ermin ation of conidia or blastospore growth of mycelia are inhibited by allelochemicals (tomatine, solanine, camptothecin, xanthotoxin, and tannic acid) produced by the host plant (Poprawski and Jones 2001 ; Lacey and Mercadier 1998). Isa r ia fumosrosea produces the toxin beauvericin, which is also produced by B bass iana and Fusariu m species to kill insects. In addition, dipicolinic acid was isolated

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24 from I. fumosorosea and confirmed to have a n insecticidal effect on third instar Bemi sia tabaci ( Gennadius) type B ( Bernardini et al. 1975; Asaff et al. 2005). Isaria fumosorosea has a broad host range in the field, including the beetles S. decemlineata (Bajan 1973), Hypothenemus hampei Ferrari a nd Popillia japonica Newman (Humber et al. 2009) the moths Y. maculipennis Galleria mellonella (L.), and Spodoptera frugiperda Smith (Smith 1993) and the flies Musca domestica L. (Humber et al. 2009) and Crossocosmia zebina (Walker) (Smith 1993) Biol o gical C ontrol There are several commercial products based on I. fumosorosea available in the market against arthropod pests. For example, Multiplex Mycomite and Priority are for control of mites; Micobiol HE is for control of beetles, moths, and nemato des; Pae Sin controls whiteflies ( Zimmerman 2008) ; and PFR 97TM 20% WDG (Apopka strain) controls piercing sucking insects in greenhouses ( Zimmerman 2008 ) Isaria fumosorosea strains have been used successfully used against whitefly populations in enclos ed environments such as greenhouses (Osborne et al 2008). In field evaluations, the strain CKPF 095 gave effective control of 2nd instar diamondback moth at 1 109 conidia/g (Maketon et al. 2008). In Floridas citrus groves, Meyer et al. ( 2008) discove red the strain Ifr AsCP infesting adults of Asian citrus psyllid ( Diaphorina citri Kuwayama). Ifr AsCP was compared to PFR 97TM and results showed that Ifr AsCP is different from, but related to, PFR 97 TM. Later, Hoy et al. (2010) determined that Ifr As CP is highly pathogenic to the adult Asian citrus psyllids when the insects were exposed to spores collected fr om dead psyllids stored at 74 C.

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25 PFR 97 TM 20 % WGD is registered in the U.S. A. and manufactured by Certis USA It is use d in greenhouses and nurseries to control aphids, mites, and whiteflies on o rnamental plants. However, PFR 97 TM has been studied in the laboratory against various pests in food crops in order to be approved for application in the field. T he overall goal of my thesis research is to provide organic farmers with environmentally friendly and effective control measures to suppress M. ochroloma populations in the field by using a native generalist predators and/or an entomopathogenic fungus. Objectives of Master of Science Thesis Research 1. Measure the development time and predation rate of Podisus maculiventris when feeding on Microtheca ochroloma in the laboratory 2. Evaluate predation potential of Podisus maculiventris on larvae of Microtheca ochroloma in the field 3. Assess the infect ivity by Isaria fumosorosea on Microtheca ochroloma in the laboratory

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26 A B C D Figure 21 Life stages of Microtheca ochroloma. A) Eggs, B) Larva, C) Pupa and D) Adults.

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27 A B C D Figure 22 Damage by Microtheca ochr oloma. A), Larvae feeding on leaves, B) Total defoliation of the turnip plant, C) Se vere root damage by the larvae, D) Larv ae feeding on the turnips roots

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28 CHAPTER 3 DEVELOPMENT TIME AND PREDATION RATE OF PODISUS MACULIVENTRIS (SAY) (HEMIPTERA: PENTATOMI DAE) PREYING ON MICROTHECA OCHROLOMA STL (COLEOPTERA: CHRYSOMELIDAE) Introduction The yellowmargined leaf beetle, Microtheca ochroloma Stl, is a pest in crucifer crops in the southern United States. It is native to Argentina, Uruguay and Brazil (Chamberl in and Tippins 1948; Dos Anjos et al. 2007). Its preferred host is turnip ( Brassica rapa L.), followed by mustard ( Brassica juncea Cosson) (Ameen and Story 1997a). The main damage caused by M. ochroloma is defoliation, but when infestations are severe and leaves are entirely consumed, the beetle will feed on the roots (C. Montemayor, personal observation). There are no specialist natural enemies reported in the literature that can contribute to the control of this pest on organic farms (Fasulo 2005). Nev ertheless, there are generalist predators present in agricultural ecosystems that may contribute to the management of M. ochroloma. The spined soldier bug, Podisus maculiventris (Say) (Hemiptera: Pentatomidae: Asopinae), is a generalist predator present i n many agroecosystems, where it preys primarily on Coleoptera and Lepidoptera larvae (McPherson 1980). In laboratory studies, nymphs and adults of P. maculiventris showed high potential predation rates against different life stages of the beet armyworm, S podoptera exigua (Hbner) (De Clercq and Degheele 1994). On organic farms, P. maculiventris has been observed on crucifer crops, preying on all stages of M. ochroloma. The use of P. maculiventris in augmentative biological control may serve as a tool to reduce populations of M. ochroloma. However, no studies have been conducted to evaluate the efficacy of this predator as a biological control agent of this pest. Therefore, the objective of this study

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29 was to know if M. ochroloma would be a suitable prey by quantifying the rate of predation of M. ochroloma larvae by P. maculiventris and the developmental time of the predator at four constant temperatures under laboratory conditions. Materials and Methods Stock Colonies Adults and larvae of M. ochroloma were brought from White Rabbit Acres certified organic farm located in Vero Beach, FL to the laboratory at the Biological Control Research and Containment Laboratory (BCRCL) at the Indian River Research and Educational Center (IRREC) in Ft. Pierce. The colony was established and maintained in plastic boxes (27 15 8 cm, Ziploc) with screen mesh openings in the walls for ventilation. Boxes were kept in an environmentally controlled chamber at 25C, 50% RH, and 10L:14D photoperiod. A bottle of 250 eggs of P. maculiventris was purchased online from RinconVitova Insectaries, Inc. (Ventura, CA [ www.rinconvitova.com ]) and shipped with overnight delivery. After arrival, eggs were divided into groups of 30 and each group was placed in a Petri dish (60 75 mm, Fisherbrand) with moistened filter paper (55 mm [diameter], Whatman). The Petri dishes were sealed with Parafilm and stored in an environmentally controlled chamber at 25C, 50% RH, and 10L:14D photoperiod. Plant Material Turnip Seven Top (Greens) (Brassica rapa L. var. rapifera) seeds were seeded in 72hole trays containing sterilized soil mix (Fafard germination mix, Agawam, MA) inside a greenhouse. Seedlings were transplanted 2 weeks later into 3.8 L plastic pots containing soil mixture (Fafard #3B mix). The plants were fertilized weekly with 400 ml per pot of liquid fertilizer (Miracle Grow 24N 8P 16K). Experimental Design An i ndividual neonate P. maculiventris was housed in a 7cm3 plasti c box ( Fig. 1) with a hole in the top sealed with screen mesh. To maintain

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30 proper humidity in the box, a piece of white filter paper (90 mm Whatman) moistened with water was placed on the bottom of the cage. Each box held a 60 mm piece of turnip le af that was replaced daily. The number of 4th instar M. ochroloma provided daily to P. maculiventris varied among instars: five prey were provided to 2nd and 3rd instar P. maculiventris and 10 prey were provided to 4th and 5th instar P. maculiventris Th e boxes with insects were held in environmentally controlled chambers at each of four constant temperatures, 10, 15, 20, and 25oC, with 50% RH and 10L:14D photoperiod. HOBO data loggers were placed inside each chamber to monitor the temperature and humidity. Each treatment had at least 12 replicates. Number of M. ochroloma larvae killed daily by each predator nymph was recorded. Total predation per instar was thus determined and the total predation per nymph was measured as the total predation through all instars. Total development time for each predator instar and for the total nymphal stage was measured in d ays Predation by the adult stage of P. maculiventris was evaluated for the first 10 d of adult life. Fresh weights of newly ecdysed nymphs and adults were measured using an Ohaus Adventurer AR2140 analytical scale. A control treatment with three replicates consisted of five to ten 4th instar M. ochroloma (number varied gradually with P. maculiventris development in other treatments) in the absenc e of P. maculiventris to record natural mortality. Dead larvae in the control were replaced daily. Mortality in the control treatment was used as a correction factor for the mortality in the predator treatment. Development time and daily and total predat ion of eggs of M. ochroloma by 2nd to 5th instar P. maculiventris were measured in Petri dishes at 25oC. Each Petri dish (60 75 mm, Fisherbrand) contained one predator, eggs of M. ochroloma, and a white

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31 moistened filter paper. The number of eggs of M. ochroloma provided daily to P. maculiventris varied among instars: 50 eggs were provided to 2nd, 100 eggs to 3rd instar, 200 eggs to 4th instar, and 500 eggs to 5th instar P. maculiventris Turnip leaves were not provided. Statistical Analysis. Data wer e analyzed using anal ysis of variance (ANOVA). T reatment means were separated using the Student Newman Keuls (SNK) test (SAS Institute, 1999) at a significance level of 5%. Means are reported with their standard error. Results Two way ANOVA indicated a s ignificant interaction between temperature and instar of P. maculiventris for all variables evaluated (developmental time, fresh weight, daily and total predation) ( F = 25.03; df = 17,181; P < 0.0001 for developmental time; F = 12.49; df = 14,150; P < 0.00 01 for fresh weight; F = 39.72; df = 14,128 P < 0.0001 for daily predation; F = 53.95; df = 14,128; P < 0.0001 for total predation). At 10 C, eggs did not hatch. First instar P. maculiventris reared at this same temperature died about 2 6 2.9 d after hat ching at 25C, therefore no data was obtained to measure developmental time at 10C The 1st instar of P. maculiventris is a nonfeeding stage, thus no daily and total predation rates were determined for this stadium. Development Time Mean development t imes of P. maculiventris eggs and instars varied with temperature (Table 31). Mean developmental times of eggs, 1st, 2nd, 3rd, 4th, and 5th instars reared at 15C were significantly h igher than those at 20 and 25C ( F = 6170.37; df = 2,31; P < 0.0001 for egg; F = 503.16; df = 2,42; P < 0.0001 for 1st instar s; F = 131.88; df = 2,30; P <0.0001 for 2nd instar s; F = 315.93; df = 2,27; P <0.0001 for 3rd instar s; F = 57.10; df = 2,26; P = < 0.0001 for 4th instar s; F = 926.75; df =

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32 2,25; P < 0.0001 for 5th insta rs). Mean development times of eggs, 1st instars, and 5th instars at 20C were significantly higher than those at 25C; there were no differences between these two temperatures for the 2nd, 3rd, and 4th instars. Mean total development times of nymphs var ied significantly with temperature ( F = 814.5; df= 2,25 ; P < 0.0001) (Table 35 ); the development time at 25C was significantly less t han the development time at 20C, which was significantly lower t han the development time at 15C. There were significan t differences in development time among instars within each temperature (Table 31). At all experimental temperatures the mean development time of the 5th instar was significantly higher than the mean development times of the other four instars. At 15 C, mean development times of eggs, 2nd instars, 3rd instars, and 4th instars were not significantly different from each other, but they were significantly higher than that of the 1st instar ( F = 30.12; df = 5,49 ; P < 0.0001). At 20 and 25 C, mean development time of eggs was significantly higher than those of 2nd and 4th instars, which were significantly higher than mean development times of 1st and 3rd instars ( F = 99.02; df = 5,66; P = < 0.0001 for 20C; F = 57.34; df = 5,66; P < 0.0001 for 25C ). Mean dev elopment times of 2nd to 5th instar P. maculiventris feeding on M. ochroloma eggs were 3.9 0.1 d, 3.8 0.1 d, 4.0 0.0 d, and 5.7 0.2 d, respectively. These means were not significantly different from those of nymphs feeding on M. ochroloma larvae at 25C (Table 31) (P = 0.1325). However, the total development time of the nymphal stage of the predator feeding on 4th instars of M. ochroloma was significantly shorter (23 0.3 d) than when feeding on eggs (25 0.3 d) ( F = 36.51; df = 1,20 ; P < 0.001)

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33 Fresh W eight Mean fresh weights of some instars varied significantly with temperature (Table 32). Mean fresh weights of 3rd and 5th instars reared at 25C were significantly higher than those at 20 C, which were significantly higher compared to thos e at 15C ( F = 14.23; df =2,29; P < 0.0001 for 3rd instars; F = 19.29; df = 2,26; P < 0.0001 for 5th instars ). There were no significant differences in mean fresh weight of 4th instars and adults between 20 and 25C, but their mean fresh weights at these two temperatures were significantly higher compared to those at 15C ( F = 11.66; df = 2,27; P = 0.0002). There were no significant differences in mean fresh weight of 2nd instars among the three temperatures. As with development time, there were signifi cant differences in mean fresh weights among stages within each temperature (Table 32). M ean fresh weight at all experimental temperatures increased significantly with each successive stage There was no significant interaction between temperature and s ex for fresh weight of P. maculiventris adults. Mean fresh weights of adult females and males were not significantly di fferent at 25C. However, at 20 C, the mean fresh weight of females was significantly higher than the mean fresh weight of males (Table 3 6 ) ( F = 16.76, df = 1,10 ; P = 0.0022). Mean fresh weights of females and males reared at 15C were not statistically compared due to low sample sizes, yet the female adult weight tended to be higher than male adult weight. Daily P redation. There were no significant differences in mean daily predation by the 2nd and 3rd instars between 20 and 25C (Table 33). However, mean daily predation by these instars at those two temperatures were significantly higher than mean daily predation at 15 C. Mean daily predation by 4th instars, 5th instars, and adults

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34 was significantly higher at 25C than that at 20C, and mean daily predation was significantly higher at 20C than at 15 C ( F = 36.82; df = 2,26; P < 0.0001 for 4th instars; F = 78.92; df = 2,25; P < 0.0001 for 5th instars; F = 79.03; df = 2,19; P < 0.0001 for adults). Mean daily predation also varied significantly among stages at each t emperature (Table 33). At 15 C, the adult stage had significantly lower mean daily predation compared to that of the 5th instar, however, it was not significantly different from the mean daily predation by the 2nd, 3rd, and 4th instars ( F = 3.00 ; df = 4,24; P = 0.0387 ). At 20C, mean daily predation increased significantly with each successive stage ( F = 123.00; df = 5,66 ; P < 0.0001). At 25 C, there was no significant difference in mean daily predation between the 5th instar and adult ( F = 186.99; df = 5,60 ; P < 0.0001 ). However, their rates were significantly higher than predation by the 4th instar, which was higher than that by the 3rd instar. Second instar mean daily predation was significantly the lowest among all stages at 25C. Mean daily predation of eggs of M. ochroloma by P. maculiventris nymphs increased significantly with each successive instar ( F = 159.14; df = 3,36; P < 0.0001) (Table 37 ). Embryonic fluids within the eggs were totally consumed by the predator. Total predation. Mean total number of M. ochroloma larvae consumed by each P. maculiventris stage also varied significantly among temperatures (Ta ble 34). There was significantly higher mean total predation by the 2nd instar at 15 C compared to 20 and 25C ( F = 50.80 ; df = 3,31 ; P < 0.0001 ). Mean total predation by the 3rd instar was significantly higher at 25C than at 15 and 20 C ( F = 6.64; df = 2,27; P = 0.0045 ). Mean total predation by the adults was significantly higher at 25C tha n at 20C which was

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35 higher compared to predation at 15 C ( F = 79.03 ; df = 2,19 ; P < 0.001 ). On the other hand, there were no significant differences in mean tot al predation by 4th and 5th instars at 15, 20, and 25C. Mean total predation of prey by the nymphal stage varied significantly with temperature ( F = 4.72; df = 2,25 ; P = 0.0182) (Table 35); predation at 20C was significant ly less than predation at 15C but predation at 25C was not significantly different from predation at the lower two temperatures. Similar to mean daily predation, mean total predation by P. maculiventris nymphs varied significantly among stages at each temperature (Table 34). Mean total predation by 5th instars at 15C was significantly higher than predation by 4th instars, which was significantly higher than that by the adult stage; predation by the latter did not vary significantly from predation by the 2nd and 3rd instars ( F = 1 0.88 ; df = 4,24; P < 0.0001). Mean total predation at 20 C was significantly higher by the adult stage compared to the 5th instar, which was higher than the 4th and 3rd instars, and these consumed significantly more prey than the 2nd instar ( F = 210.30 ; d f = 5,66; P < 0.0001 ). At 25 C, mean total predation was significantly higher by the adult stage than by the 5th instar, which was higher than predation by the 4th instar; the latter was significantly higher compared to the 2nd and 3rd instars ( F = 349.78; df = 5,60, P < 0.0001 ). Mean total predation of eggs of M. ochroloma by P. maculiventris nymphs increased significantly with each successive instar ( F = 250.29; df = 3,36 ; P < 0.0001) (Table 37 ). Total consumption of eggs of M. ochroloma during the ny mphal stage of P. maculiventris was 741.0 24.8. Discussion The 7 cm3 plastic boxes used as cages provided more space for P. maculiventris nymphs and adults to seek and attack the larvae of M. ochroloma in a more natural way

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36 compared to Petri dishes. Acc ording to Wiedenmann and ONeil (1991), attacks rates in Petri dishes will be limited by handling time, but in the field or more complex arenas they will be limited by search behavior. The M. ochroloma larvae that were attacked in my study were most often killed, then either abandoned unconsumed or partially consumed. De Cler cq and Degheele (1994) reported similar observations when nymphs preyed on 5th instars of the beet armyworm Neither development of 1st instars nor egg hatching of P. maculiventris wa s successfully completed at 10C. These findings match with the estimated lower developmental threshold f or eggs 10.7C and nymphs 11.7C of P. maculiventris as determined by De Cler cq and Degheele (1992). There was a well defined inverse interaction bet ween developmental time and temperature. Developmental time of all stages of P. maculiventris is longer at 15 C compared to higher temperatures (Table 31). The fifth instar of P. maculiventris requires more time to develop than earlier instars, regardles s temperature (Table 31). More time in the 5th instar allows the daily and total predation rates of the predator to be greater compared to those of the earlier instars (Tables 33 and 34). Developme nt times of nymphs reared at 20 and 25C and fed 4th in star M. ochroloma are comparable to development times of nymphs reare d on beet armyworm larvae at 23 C (De Cler cq and Degheele 1994). Mahdian et al. (2006) reported similar trends of consumption rate and developmental time of P. maculiventris feeding on 4t h instar S podoptera littoralis (Boisduval ) at 18, 23, and 27C. Desurmont and Weston (2008) found that nymphs of P. maculiventris consumed an average of 101 larvae and 17 adults of Pyrrhalta viburni (Paykull) (Coleoptera: Chrysomelidae) and the

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37 total devel opmental time from 2nd instar to adult was 22 d at 22C which is very similar to the total developm ental time (21 d) at 20 C in my study. Podisus maculiventris is able to complete its life cycle by feeding on eggs only of M. ochroloma. This suggests th at the egg of M. ochroloma is an adequate food source as is the 4th instar, and probably, too, the adult and pupa of the beetle since P. maculiventris also feeds on them (C. Montemayor, personal observation). It is yet unclear whether or not nymphs will s earch for eggs on dry leaves and in the soil; however, if they do, the population of M. ochroloma could be significantly reduced due to the predators high predation of eggs (Table 36 ). Two other studies have reported low er mean total consumption of othe r prey eggs by the nymphal stage of P. maculiventris : 293 eggs of Stilodes decemlineata Say (Coleoptera: Chrysomelidae) (Gusev et al. 1983) and 160 eggs of Epilachna varivestis (Mulsant) (Coleoptera: Coccinellidae) (Waddill and Shepard 1975). However, De Cler cq and Degheele (1994) reported total nymphal consumption of 1,200 eggs of S. exigua. Regarding to the weight measured of P. maculiventris in this study. The mean fresh weight of newly ecdysed 5th instar P. maculiventris fed on 4th instar M. ochroloma at 25 C is 30.98 mg. This weight was comparable to the 27.3 mg in the study of De Cler cq and Degheele (1994), who fed the predator 4th instar S. exigua at 23 C. The numbers of M. ochroloma larvae killed by nymphs and adults suggest that P. maculiventri s has great potential to reduce populations of M. ochroloma under field conditions in Florida, in which the natural temporal occurrence of the predator (spring summer) matches well with outbreaks of the pest in late spring (Herrick and Reitz 2004). In order to enhance the control of M. ochroloma in the field, augmentative

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38 releases of the predator can be made in the late fall and early spring. Therefore, it is essential to evaluate its predation potential under field conditions (see Chapter 4). Figure 3 1. Plastic boxes with screened windows used to house experimental insects.

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39 Table 3 1. Mean ( SE ) development time of Podisus maculiventris reared at three constant temperatures with 4th instar Microtheca ochroloma as prey. Number within parentheses equals sample size. Mean development time (days) Temp* ( C) Egg 1 st instar 2 nd instar 3 rd instar 4 th instar 5 th instar 15 17.0 0.0 (10) Ba 10.0 0.2 (21) Ca 18.7 1.4 (9) Ba 15.0 0.9 (6) Ba 18.6 3.1 (5) Ba 25.5 1.0 (4)Aa 20 5.7 0.1 ( 12) Bb 4.0 0.0 (12) Db 4.5 0.3 (12) Cb 3.8 0.1 (12) Db 4.8 0.2 (12) Cb 8.1 0.1 (12) Ab 25 4.0 0.0 (12) Bc 3.0 0.0 (12) Dc 3.7 0.1 (12) Cb 3.4 0.1 (12) Db 3.9 0.1 (12) Cb 5.4 0.1 (12) Ac Means followed by the same uppercas e letter within a row are not significantly different ( P > 0.05). Means followed by the same lowercase letter within a column are not significantly different ( P > 0.05). ( ) Temperature. Table 3 2. Mean ( SE) fresh weight of newly ecdysed Podisus maculiventris reared at three constant temperatures with 4th instar Microtheca ochroloma as a prey. Number within parentheses equals sample size. Mean fresh weight (mg) Temperature ( C) 2 nd instar 3 rd instar 4 th instar 5 th instar Adult (10 days) 15 0.77 0.0 (21) Da 2.01 0.2 (9) Dc 6.25 0.5 (6) Cb 14.20 2.7 (5) Bc 41.28 0.2 (5 ) Ab 20 0.84 0.0 (12) Da 2.69 0.2 (12) Db 8.96 0.6 (12) Ca 25.41 1.2 (12) Bb 59.15 2.3 (12) A a 25 0.82 0.0 (12) Da 3.63 0.2 (12) Da 10.70 0.5 (12) C a 30.98 1.6 (12) Ba 58.00 2.1 (12) A a Means followed by the same uppercase letter within a row are not significantly different ( P > 0.05). Means followed by the same lowercase letter within a column are not significantly different ( P > 0.05).

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40 Tab le 3 3. Mean ( SE) daily predation of 4th instar Microtheca ochroloma by Podisus maculiventris reared at three temperatures. Number within parentheses equals sample size. Mean daily predation of prey Temperature ( C) 2 nd instar 3 rd instar 4 th inst ar 5 th instar Adult (10 days) 15 0.7 0.1 (10) ABb 0.7 0.1 (6) ABb 0.8 0.1 (5) ABc 1.2 0.2 (4) Ac 0.5 0.2 (4) Bc 20 1.1 0.1 (12) Da 2.3 0.2 (12) Ca 2.5 0.2 (12) Cb 3.4 0.1 (12) Bb 4.5 0.2 (12) A b 25 1.0 0.1 (12) Da 1.9 0.1 (12) Ca 3.4 0.2 (12) Ba 6.5 0.3 (12) Aa 6.4 0.3 (6) A a Means followed by the same uppercase letter within a row are not significantly different ( P > 0.05). Means followed by the same lowercase letter within a column are not si gnificantly different ( P > 0.05). Table 3 4. Mean ( SE) total predation of 4th instar Microtheca ochroloma by Podisus maculiventris reared at three constant temperatures. Number within parentheses equals sample size. Mean total predation of prey Te mperature ( C) 2 nd instar 3 rd instar 4 th instar 5 th instar Adult (10 days) 15 12.9 1.1 (10) CBa 11.0 1.8 (6) CBa 14.8 2.4 (5) Ba 30.0 5.8 (4) Aa 5.0 2.1 Cc 20 4.9 0.5 (12) Db 9.0 0.7 (12) Ca 12.0 0.9 (12) Ca 27.2 1.0 (12) Ba 45.1 2.3 (12) A b 25 3.8 0.2 (12) Db 6.5 0.4 (12) Db 13.2 0.7 (12) Ca 35.6 2.0 (12) Ba 64.1 2.9 (6) A a Means followed by the same uppercase letter within a row are not significantly different ( P > 0.05). Means followed by the same lowercase letter within a column are not significantly different ( P > 0.05).

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41 Table 35. Mean ( SE) total development time and predation of 4th instar Microtheca ochroloma per nymph of Podisus maculiventris Number within parentheses equals sample size. Temperature ( C) Total development (days) Total predation (prey) 15 99 4.6 (4) a 65 6.3 (4) a 20 31 0.3 (12) b 53 1.3 (12) b 25 23 0.3 (12) c 59 2.1 (12) ab Means followed by the same lowercase letter within a column are not significantly different ( P > 0.05). Table 3 6 Mean ( SE) fresh weight of newly ecdysed adults of Podisus maculiventris reared with 4th instar Microtheca ochroloma as a prey. Number within parentheses equals sample size. Mean fresh weight (mg) Temperature ( C) Female Male 15 43.00 2.4 (2) 39.55 2.0 (2) 20 63.50 1.2 (8) a 50.45 4.0 (4) b 25 61.98 2.9 (6) a 54.00 2.3 (6) a Means followed by the same lowercase letter within a row are not significantly different ( P > 0.05).

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42 Table 37 Mean ( SE) predation of eggs of Microtheca ochroloma by Podisus maculiventris nymphs reared at 25C Number within parentheses equals sample size. Mean predation of eggs Predation 2 nd instar 3 rd instar 4 th instar 5 th instar Daily 6.3 0.6 (10) d 18.2 1.0 (10) c 50.2 3.1 (10) b 83.5 4.4 (10) a Total 24.2 2.1 (10) d 68.3 3.6 (10) c 187.7 12.6 (10) b 460.7 13.8 (10) a Means followed by the same lowercase letter within a row are not significantl y different ( P > 0.05).

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43 CHAPTER 4 PREDATION POTENTIAL OF PODISUS MACULIVENTRIS (HEMIPTERA: PENTATOMIDAE) ON MICROTHECA OCHROLOMA (COLEOPTERA: CHRYSOMELIDAE) IN THE FIELD Introduction The yellowmargined leaf beetle, Microtheca ochroloma Stl, is a cool seasonal pest in crucifer (Brassicaceae) crops, with turnips ( Brassica rapa L.) and mustard ( Brassica juncea Cosson) its preferred host (Amen and Story 1997a). In 1945, the first specimen to be found in the United States was reported in New Orleans on grapes coming from Argentina where it may also be a serious problem (Chamberlin and Tippins 1948). Since then, M. ochroloma has spread to and established in several southern states in the United States (Staines 1999), including Florida where the beetle was found on watercress in 1972 (Woodruff 1974). In Florida, M. ochroloma is present in the field during the coolest months (October through April), which correspond s with the primary production season for leafy greens (Bowers 2003). For organic crucifer growers, it has been a challenge to maintain populations of M. ochroloma under tolerable levels, given that growers are restricted to using insecticides on the Organic Materials Review Institute (OMRI) list, and no known native specialist natural en emies have been reported (Bowers 2003). T he manipulation of generalist predator populations to enhance control of M. ochroloma should be considered in developing an i ntegrated pest management program for this pest. Predatory stink bugs (Hemiptera: Pentatomidae: Asopinae) are commonly used in augmentative releases to control pests in agricultural ecosystems (Biever and Chauvin 1992; HoughGoldstein 1996; Tipping et al. 1999). The spined soldier bug, Podisus maculiventris (Say), is a generalist predator native t o North America (McPherson 1982).

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44 Coleoptera and Lepidoptera larvae are its main prey (McPherson 1980). This generalist predator has been released in the field and in greenhouses to control pests in tomato and cotton with successful results (Lopez et al. 1976; Ables and McCommas 1982; De Clercq et al. 1998). HoughGol d stein and McPherson (1996) reported that augmentative releases of P. maculiventris in small field plots reduced the larval population of the Colorado potato beetle, Stilodes decemlineata (Sa y). The study of p redation rates in fieldcage experiments demonstrated that the 5th instar of P. maculiventris could kill an average number of 15 3rd instar S. decemlineata (Stamopoulos and Chloridis 1994). In central Florida, P. maculiventris has been observed preying on M. ochroloma (C. Montemayor, personal observation). However, little is known about its predation potential on field populations of M. ochroloma. In northern Florida, P. maculiventris is present beginning in March and starts overwinter ing in October (Herrick and Reitz 2004). This period of time permits natural populations of P. maculiventris to interact with the pest for the last couple of months of late spring in the crucifer growing season, since M. ochroloma is a problem during the cooler months of the year in Florida. Early augmentative releases of P. maculiventris in cruciferous crops in the field may contribute to control of M. ochroloma. The present study evaluates the predation capacity of P. maculiventris at different densit ies in field cage s containing M. ochroloma. The goal of this study is to provide growers a guideline for releasing P. maculiventris This way the predator can be used as a new integrated pest management tool to control M. ochroloma.

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45 Materials and Methods Stock Colonies Adults and larvae of M. ochroloma were obtained from the field and a laboratory colony was established and maintained as described in Chapter 2. The releases of P. maculiventris were designed to simulate as much as possible the way that growers would release the predator based on the recommendations of the vendor. Therefore, eggs of P. maculiventris were purchased and held in the laboratory as described in Chapter 2. When the first 100 1st instars emerged, they were held for 5 d at 15C and then released into the field cages. Plant Material Small plants of Seven Top (Greens) variety turnips ( B. rapa var. rapifera) were grown as described in Chapter 2. Three weeks after transplanting in pots, seedlings were transplanted into field beds at the IRREC Four beds, each 100 m long and 0.70 m wide, were covered by white plastic mulch. Granulate fertilizer (8N 12P 20K with minor elements; Howard Fertilizer Co., Inc., Orlando, FL) was placed in the center of the beds at a rate of 85 g/m. Dis tance between plants was 30 cm, distance between rows was 30 cm, and distance between beds was 90 cm. Irrigation was provided by flooding between beds. Experimental Design Cage frames 1.35 m long, 0.90 m wide, and 0.70 m high were constructed of 1.904 cm (3/4 inch) PVC (polyvinyl chloride) tubing. Fine mesh cloth was sewn in a manner to fit tightly over each frame; around the base of the cloth cage was a 15cm skirt Each cage was placed over a field bed with six turnip plants. Soil was heaped on th e skirt to avoid the entrance or exit of animals and anchor the cage in place. One hundred thirty two 1st instars of M. ochroloma were introduced into each cage (22 larvae per plant). In 2009, the insects were introduced into the cage 20 d after the turnip plants were transplanted into the field; in 2010, the insects were

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46 introduced into the cage 10 d after t ransplanting. On the same day as the introduction of M. ochroloma into the cages, one of three densities (4 = low, 10 = medium, or 16 = high) of 1st instar P. maculiventris was released evenly among the six plants in a cage. Each treatment ( P. maculiventris density) was replicated four times. RinconVitova Insectaries recommends releasing 25 eggs per hot spot or 25 eggs per 10 m2 for caterpillar c ontrol. In other words, RinconVitovas release recommendation for my study would be three 1st instars per 1.21 m2 (area of the cage). However, by comparison, the actual release rates in my study were 33 1st instars in the low predator density treatment, 83 1st instars in the medium predator density treatment, and 132 1st instars in the high predator density treatment per 10 m2. Treatment arrangement was randomized within each replicate block (Fig. 1). The experiment was conducted twice, once in February March 2009 and once in February March 2010. All plants in each cage were monitored every four days for four weeks in 2009 and for five weeks in 2010, and the numbers of M. ochroloma larvae, pupae, and adults and P. maculiventris nymphs and adults per pla nt were recorded. At the end of both experiments, all plants were pulled out of the cages and brought into the laboratory for the last sampling (Fig. 2). A HOBO data logger (Onset Computer Corporation, Bourne, Massachusetts) was placed inside a cage to monitor temperature and relative humidity during the experiments. Statistical Analysis. The effect of predator density on the caged M. ochroloma population was analyzed by comparing the number of live M. ochroloma per cage with analysis of variance with repeated measures over time. Treatment m eans were

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47 separated by time using the Student Newman Keuls (SNK) test (SAS Institute, 1999) at a significance level of 5%. Means are reported with their standard error. Results 2009 Experiment The mean temperature during the experiment was 16.7 C 0.1. The mea n maximum temperature was 23.2 C 0.6. Fifteen days had a maximum above this temperature, with 27.9 C as the highest recorded temperature for three days between 1:00 and 1:30 p.m. The mean minimum temperat ure was 10.8 C 0.9. Fourteen days had a minimum bel ow this temperature, with 0.2 C as the lowest temperature on one day between 6:00 and 6:15 a.m. Although 132 1st instars were introduced into each cage at the beginning of the experiment, the highest m ean number of M. ochrolo m a larvae per cage on the first sampling date was 33 3.1 in the medium predator density treatment. On 18 February, the mean number of larvae per cage in the high predator density treatment was significantly lower than in the mediu m predator density, low predatory density, and control treatments (Fig. 3Larvae) ( F = 5.69; df = 3,12; P = 0.0116). On 22 February, the mean number of M. ochroloma pupae per cage in the high and medium predator density treatments was significantly lower t han in the low predatory density and control treatments ( F = 8.58; df = 3,12; P = 0.0026) (Fig. 3Pupae). On 3 March, the mean number s of pupae per cage in all treatments with predators were significantly lower than in the control treatment ( F = 4.37; df = 3,12; P = 0.0268) (Fig. 3Pupae). Although SNK test could not separate the mean number of pupae per cage among treatments on 18 and 26 February, ANOVA did show significance differences among them. A real effect by the predator was apparent in the mediu m and high predator density treatments

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48 because these treatments consistently had lower mean number of pupae per cage compared to the low predator density and control treatments ( F = 4.08; df = 3,12; P = 0.0326; F = 4.34; df = 3,12; P = 0.0273) (Fig. 3 Pupa e). On 3 March, the mean number of adult M. ochroloma in the two higher predator density treatments was significantly lower than in the low predatory density and control treatments ( F = 8.87; df = 3,12; P = 0.0023) (Fig. 3Adults). Overall, there were dif ferences in the number of M. ochroloma larvae + pupae + adults per cage among treatments over time. The mean number per cage was significantly lower in the high predator density treatment than in the medium predator density, which was significantly lower compared to the low predator density treatment. There was no significant difference in the mean number per cage between the low predator density and the control. However, there was a significant interaction between treatment and time (treatment time: F = 5 .10; df = 3,72; P = 0.0167; treatment: F = 18.37; df = 3,72; time: F = 61.07; df = 3,72; P < 0.0001) (Fig. 3 Total). The medium and high predator treatments had the lowest mean number of M. ochroloma per cage from 18 February until the end of the experiment. On 26 February, there was a significant difference, according to the ANOVA, in the mean number of M. ochroloma per cage among the treatments, however, no differences were detected when the means were separated with SNK test ( F = 3.54; df = 3,12; P = 0.0482). Excluding eggs, t he population of M. ochroloma per cage in the high predator density treatment at the end of the experiment was 91.7% 8.3 adult s and 8.3% 8.3 pupae. I n the medium predator density treatment the proportions per cage at the end of the experiment w ere 88.1% 7.9 adult s and 11.9% 7.9 pupae. In the low predator

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49 density treatment the M. ochroloma population (excluding eggs) per cage was 89% 3.8 adult s and 9.9% 3.7 pupae, whereas in the control treatment the proportions w ere 75.3% 8.4 adult s 24.2% 8.2 pupae, and 0.5% 0.5 larvae. Overall, there were differences in the number of P. maculiventris nymphs per cage among treatments ( F = 3.87; df = 3,72; P < 0.0001) (Fig. 4). The number of nymphs per cage was significant ly higher in the medium predator density treatment than in the high predator density treatment which was significantly higher than in the low predator density treatment; the control treatment, with no predators released, was significantly lower than all ot her treatments. However, there was a significant interaction between treatment and time (treatment time: F = 3.87; df = 3,72; P < 0.0001; treatment: F = 79.12; df = 3,72; P < 0.0001; time: F = 7.10; df = 3,72; P < 0.0001) (Fig. 4). On only one date, 6 and 22 February (4 d after introducing predators into the cages), the mean number of predator nymphs per cage was significantly higher in the medium predator density treatment compared to the high predator density treatment; on all the other sampling dat es there was no significant difference between these two treatments Beginning 3 March, the two higher predator density treatments maintained significantly higher numbers of P. maculiventris nymphs per cage compared to the low predator density treatment d uring the remainder of the study ( F =18.63; df = 3,12; P < 0.0001) (Fig. 4). At the end of the experiment, the overall survivorship ([number of nymphs recovered/number of nymphs released]*100) of P. maculiventris per cage among the three predator release treatments was 51.3% 5.4. In the low predator density treatment, predator survivorship was 62.5% 12.5; 75.0% 14.4 of the nymphs were in

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50 the 3rd instar and 25% 14.4 were in the 4th instar. In the medium predator density treatment, predator survivorship was 52.5% 4.8; 65.4% 16.7 of the nymphs were in the 3rd instar and 34.6% 16.7 were in the 4th instar. Predator survivorship in the high predator density treatment was 39.1% 6.9; 83.7% 10.3 of the nymphs were in the 3rd instar and 16.3% 10.3 were in the 4th instar. 2010 Experiment The mean temperature during the experiment was 14.8 C 0.6. The mea n maximum temperature was 21.6 C 0.6. Twenty days had a maximum above this temperature, with 26.9 C as the highest recorded temperature for on e day between 2:30 and 2:45 p.m. The mean minimum temperature was 8. 3 C 0.8. Seventeen days had a minimum below this temperature, with 0.3 C as the lowest temperature on one day between 5:30 and 5:45 a.m. The highest mean number of M. ochroloma larvae per cage on the first sampling date was 27 7.6 in the control treatment. On 1 March, the mean number of larvae per cage in the high, medium, and low predator density treatments was significantly lower than in the control treatment ( F = 12.54; df = 3,12; P = 0.0005) (Fig. 5Larvae). On 9 March, pupae were observed in the low predator density and control treatments, but not in the high and medium predator density treatments. However, no significant differences were detected among the four treatments (Fig. 5 Pupae). On 13 March pupae were seen only the in the control treatment, but again no significant differences were detected among all treatments (Fig. 5Pupae). On 17, 22, and 25 March, the mean number of adults per cage in the three predator density treatments was

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51 significantly lower than in the control treatment ( F >21. 75; df= 3,12; P < 0.0001) (Fig. 5Adults). All M. ochroloma became reproductive adults in all treatments after 36 d Overall, there were differences in the number of M. ochroloma larvae + pupae + adults per cage among treatments over time. The mean number of M. ochroloma per cage was significantly lower in the medium and high predator density treatments than in the low predatory density treatment, which was significant lower compared to the control treatment (Fig. 5 Total). However, there was a significant interaction between treatments and time (treatment time: F = 4.64; df = 3,96; P < 0.0001; treatment: F = 58.97; df = 3,96; P < 0.0001; time: F = 29.56; df = 3,96; P < 0.0001) (Fig. 5 Total). On 1, 17, 22, and 25 March, there were no significant differences in the mean number of M. ochroloma per cage among the low, medium, and high predator density treatments, but all of them were significantly different from the control treatment ( F =12.54; df = 3,12; P = 0.0005; F =21.75; df = 3,12; P < 0.0001; F =28.47; df = 3,12; P < 0.0001; F = 38.35; df = 3,12; P < 0.0001) (Fig. 5 Total). Overall, there were differences in the number of P. maculiventris nymphs per cage among treatments ( F = 1. 79; df = 3,96; P < 0.0001) (Fig. 6). The number of nymphs per cage was significantly higher in the high and medium predator density treatments than in the low predator density treatment which was significantly higher than the control treatment with no pr edators released ( F = 1.79; df = 3,96; P < 0.0001). However, there was a significant interaction between treatment and time (treatment time: F = 1.79; df = 3,96; P < 0.0001; treatment: F = 34.70; df = 3,96; P < 0.0001; time: F = 5.11; df = 3,96; P = 0.01 ) (Fig. 6 ). Significant differences were found amount treatments on all sampling dates except for 9 March ( F = 2.63; df = 3,96; P = 0.09). The

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52 mean number of nymphs per cage in the high predator density treatment decreased over time. On 25 March, the low predator density treatment was the only treatment significantly different from the control ( F = 4.24; df = 3,96; P = 0.0293). At the end of the experiment, the overall survivorship per cage of P. maculiventris was 26% 8.1. In the low predator density treatment, survivorship per cage was 56.3 % 11.9; 25.0% 28.9 were in the 3rd instar, 50.0% 28.9 were in the 4th instar, 12.5% 12.5 were in the 5th instar, and 12.5% 12.5 were adults (molting to adult on 25 March). In the medium predator density treatment, survivorship per cage was 17.5% 8.5; 66.7% 16.7 were in the 4th instar and 33.3% 16.7 were in the 5th instar. In the high predator density treatment, survivorship per cage was 3.1% 1.8; 50.0% 50.0 were in the 4th instar and 50.0 % 50.0 were in the 5th instar. Discussion 2009 Experiment Only seven sampling dates (five weeks) were evaluated due to the very large turnip plants in the cages at the end of the experiment ( Fig. 7), which also made sampling more difficult. The notable reduction in the number of larvae of M. ochroloma in the control treatment on 6 February, four days after the release of the insects into the cages, was likely due to the difficulty in detecting the small first instars by the visual sampling method. Temperature probably did not have a major influence on the mortality of M. ochroloma, since temperature ranged from 10.8 to 23.3 C during the experiment. Cold tolerance studies indicate that 1st instar M. ochroloma can survive at least 2 d exposed to 0 C (unpublis hed data). The decrease in the number of M. ochroloma per cage in all treatments from 6 to 18 February (Fig. 3Total) can be attributed, in part, to the transi tion of larvae to pupae (Fig. 3Larvae), but in the predator

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53 density treatments it also can be at tributed to predation by P. maculiventris as evidenced by the significantly greater decrease in number of M. ochroloma in the medium and high predator density treatment versus the low predator density and control treatments. On 18 February, the differenc e between the mean number of larvae and pupae is probably due to the larvae behavior when it is ready to pupate (Fig. 3Larvae and Pupae). When the larvae are ready to pupate, they move to dry leaves, tight places, and soil (Woodruff 1974; Bower 2003). I n this study, the larvae moved under the plastic, making it difficult to find them. This confirms the disappearance of larvae after 22 February in the control treatment was due to pupation. In the treatments with predators, the reduced number of M. ochrol oma larvae can be attri buted to predation and pupation. On the last sampling date, the predation effect of P. maculiventris on the reduction in the number of M. o chrol oma was more noticeable in the medium and high predator density treatments compared to t he low predator density treatment (Fig.3 Total). However, the overall number of M. ochroloma was kept lowest in the high predator density treatment during the 30day experiment. There was a drastic reduction of 1st instar P. maculiventris on 6 February, probably due to the visual sampling method or unexplained causes not measured. Temperature likely did not influence the number of 1st instars observed because De Cler cq and Degheele (1992) estimated the lower threshold for development of P. maculiventris nymphs is 11.7 C, well below temperatures experienced during my field study. Although the survival of 1st instar P. maculiventris feeding on 1st instar M. ochroloma in the field has not been studied, a reduction in the survivorship could be expected. The observed survival of the predator per cage on 6 February was 40% 8.6, whereas the

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54 actual survival determine on 3 March was 51% 5.4, suggesting that the survivorship of the predator at the beginning of the experiment was underestimated by the sampling method. Cannibalism may have been minimal or may not have occurred at all in this experiment since the search area was high due to the large plant size and because the populations of M. ochroloma were maintained at low levels, i.e never driven to extinct ion. If populations of M. ochroloma were to become limited, P. maculiventris would have no problem surviving on other prey since it is a generalist predator, or even on plants since it is a facultative herbivore (McPherson 1982; Valicente and ONeil 1995) According to Wiedenmann and ONeil (1990), the presence of plant material can enhance survival of P. maculiventris at very low prey inputs. 2010 Experiment Plant size (about four leaves per plant) in the 2010 experiment was considerably smaller than it was in 2009 experiment (about seven leaves per plant), both at the moment of the insect releases and at the end of the experiment (Fig. 8), because the releases were made 10 d earlier after transplanting in 2010 than in 2009. This difference extended the experiment to nine sampling dates in 2010. Plants in the control treatment had to be replaced on 25 February due to complete defoliation by the M. ochroloma larvae. Plants in the other treatments, on the other hand, did not have to be replaced. This is evidence that predation by the P. maculiventris nymphs on M. ochroloma significantly reduced the number of larvae in those treatments and, therefore, the feeding damage on the plants. On 21 February, four days after releasing the insects in the cages, there was a drastic reduction in the number of M. ochroloma larvae as there was in 2009, again probably due to the lack of detection by the sampling method. From 21 February to 5

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55 March, the number of M. ochroloma decreased in all treatments (Fig. 5Total), j ust as it did in 2009 and for the same possible reasons. It is important to observe that after 9 March, the number of M. ochroloma was virtually zero in the medium and high predator treatments for all stages (Fig. 5Total), probably due to the effect of p redation by P. maculiventris This phenomenon did not happen in the 2009 experiment because the leaf surface area (larger plants) was higher than in 2010, therefore reducing the probability of the predator encountering and feeding upon the prey Desurmont and Weston (2008) studied the effect of two host plant species, arrowwood ( Viburnum dentatum L., Caprifoliaceae) and American cranberrybush ( Viburnum opulus L. var. americanum Aiton, Caprifoliaceae), on consumption of the viburnum leaf beetle, Pyrrhalta viburni (Paykull), by P. maculiventris Their results showed that prey consumption was inversely related to leaf surface area on one of the host plants. They hypothesized that the searching efficiency of the predator decreases as leaf surface increases because of the ability of the prey to hide or move around. The 44% survivorship of P. maculiventris on 21 February is similar to the observed survival in 2009; however, survivorship rates at the end of the two experiments are quite different. The final sur vivorship in 2010 was 26% compared to 51% in 2009. Lower survival in 2010 could have been due to cannibalism since the leaf surface area was smaller and the number of M. ochroloma larvae was driven to zero after 9 March in the high and medium predator density treatments. Dispersal would be an option for the predators in an open field scenario, but not to a great extent because the nymphs do not have wings. According to the data gathered in this study, 1st instar P. maculiventris would still be in the nym phal stage five weeks after being released in the field.

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56 On the last sampling date (25 March), the effect of P. maculiventris nymphal predation on the number of M. o chrol oma per cage is remarkable in all the predator density treatments. However, the number of M. ochroloma was kept lower for 36 d with at least 10 nymphs released per six plants. The release rate of three P. maculiventris 1st instars per 1.21 m2 or 25 1st instars per 10 m2 as recommended by RinconVitova Insectaries is not adequate to control M. ochroloma. Therefore, two recommendations emerge from the overall results of my fieldcage study: Release 16 1st instars of P. maculiventris per six plants if the plants are expected to be large ( 7 leaves/plant) with at least 130 1st instars of M. ochroloma. Release 10 1st instars of P. maculiventris per six plants if the plants are expected to be small ( 6 leaves/plant) with at least 130 1st instars of M. ochroloma. Although there was no signific ant overall difference between the medium and high predator density treatments, the second recommendation is made from an economic point of view and the probability of cannibalism. In addition, increasing the number of biological control agents in the field does not always translate into greater pest control, but does increase the cos t of using biological control (V an Dri esche et al. 2002; Collier and V an Steenwyk 2004). Consequently, releasing an optimal number of biological control agents should result in a more efficient and economic augmentative biological control program (Crower 2007).

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57 Figure 41. Cages in the field at the beginning of the 2009 experiment. Figure 42. Plants being gathered for final sampling in the laboratory.

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58 Figure 43. Number of Microtheca ochroloma per cage during the 2009 field experiment. Means followed by the same letter within each sampling date are not significantly different (P>0.05). An asterisk (*) over the bars on the same date indicates that differences am ong treatments were detected by ANOVA, but the means could not be separated by the SNK test.

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59 Figure 44. Mean number of Podisus maculiventris nymphs per cage during the 2009 field experiment. Means followed by the same letter within each sampling date are not significantly different (P>0.05).

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60 Figure 45. Mean number of Microtheca ochroloma per cage during the 2010 field experiment. Means followed by the same letter within each sampling date are not significantly different (P>0.05).

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61 Figure 4 6. Number of Podisus maculiventris nymphs per cage during the 2010 field experiment. Means followed by the same letter within each sampling date are not significantly different (P>0.05).

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62 Figure 47. Size of turnip plants in the 2009 experiment. Figure 48. Size of turnip plants in the 2010 experiment.

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63 CHAPTER 5 INFECTIVITY OF MICROTHECA OCHROLOMA STL (COLEOPTERA: CHRYSOMELIDAE) BY ISARIA FUMOSOROSEA WIZE (BROWN AND SMITH) Introduction Microtheca ochroloma Stl, the yellowmargined leaf beetle, is a serious pest in crucifer crops during the late fall and winter months in Florida. Since 1947, this adventive species has been established in most of the southern US states. Ameen and Story (1997a) studied the feeding preferences of the larvae and adults and concluded that turnip and mustard are their preferred host plants. Its main damage is caused by defoliat ion; however, roots can also be damaged when infestations are severe. On large scale commercial farms, the most common and easy way to contr ol this beetle is by applying insecticides, but the overuse of insecticides can lead to the development of resistance over time, as has occurred with of the Colorado potato beetle Stilodes decemlineata (Say) (Alyokhin et al. 2008). On organic farms, it is more difficult to control M. ochroloma due to the restricted use of insecticides, in addition to the lack of specific natural enemies of M. ochroloma in the United States (Fasulo 2005). Currently, there is no pest management program available for growers to control this pest in the United States. Bowers (2003) evaluated whether intercropping between host (mizuna) and nonhost (oak leaf lettuce) plants can reduce the severity of M. ochroloma outbreaks, but still the beetles were able to find and colonize host plants among the nonhost plants. Biological control by entomopathogenic fungi may potentially be used to control M. ochroloma on organic farms. Anjos et al. (2007) reported infection of M. ochroloma in the field by Beauveria bassiana (Bals.) Vuillem an in Rio Grande do Sul, one of more southern states in Brazil. Isaria fumosorosea (= Paecilomyces fumosoroseus ) Wize

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64 (Brown and Smith) has a worldwide distribution and its effectiveness against many pest insects, especially whiteflies, is well documented ( Osborne and Landa 1992; Vidal et al. 1997a ; Lacey et al. 1999). This fungus has a broad host range, including chrysomelid beetles such as S. decemlineata (Bajan 1973), Pyrrhalta luteola (Mueller), and Spaethiella sp. (Humber et al. 2009). In 1986, a strain of I. fumosorosea named Apopka 97 was isolated in Apopka (Orange County), FL from Phenacoccus sp. (Hemiptera: Pseudococcidae) (Vidal et al. 1998). The strain is registered under the commercial name PFR 97 TM 20% WDG [chemical family: microbial insecticide, chemical name: Paecilomyces fumosoroseus Apopka Strain 97 (ATCC 20874)] by the manufacturer Certis USA, Columbia, MD. It is recommended for use in greenhouses against aphids, citrus psyllid, spider mites, thrips, and whitefly [www.certisusa.com], bu t it is still being evaluated against field pests in food crops. The use of microbial insecticides as a tool to control pests in agricultural ecosystems is becoming more popular among growers. Although there is no information available on commercial products to specifically control M. ochroloma, products that control other pest beetles should be evaluated on M. ochroloma. In this study, the infectivity of Apopka 97 was evaluated as a potential biological control agent of M. ochroloma. Materials and Meth ods Stock Colony Eggs, larvae, and adults of M. ochroloma were obtained from the field and a laboratory colony was established and maintained as described in Chapter 2. Fungus PFR 97TM 20% WDG (a.i. Paecilomyces fumosoroseus Apopka strain 97 20%, inert ingredients 80%) was provided for research by Certis USA in a 0.45 kg bag

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65 (Lot: 0833004401) in the form of desiccated granules of I fumosorosea bla stospores. The bag contains 1 109 colony forming units (CFU)/g. Plant Material Plants of Seven Top (Gre ens) turnips ( Brassica rapa L. var rapifera, Brassicaceae) were grown as described in Chapter 2. Experiment 1. Susceptibility of Microtheca ochroloma to I nfection by PFR 97TM The goal of this experiment was to determine the susceptibility of various stages of M. ochroloma to infection by PFR 97TM. Five stages of M. ochroloma, egg, 1st and 3rd instars, pupa, and adult, were removed from the laboratory colony for exposure to a concentration of 1 g of PFR 97TM in 100 ml of sterile distilled water at 25o C 10L:14D photoperiod, and 60% RH. The fungal suspension was prepared in a beaker and allowed to settle for 20 min until the supernatant containing blastospores and the inert sediment of the product separated. The suspension was applied (see below for met hod) to groups of 10 insects per stage housed in separate and sealed plastic Petri dishes (60 75 mm, Fisherbrand) with moistened filter paper (55 mm [diameter], Whatman) on the bottom dish A 2.5 cm2 piece of turnip leaf was placed on top of the f ilter paper. The Petri dishes were sealed with Parafilm. All fungal treatments consisted of 10 replicates with 10 pseudoreplicates per replicate. A pseudoreplicate was a single insect. A control treatment consisted of five replicates in which the test insect stages were sprayed with sterile distilled water only. Mortality was checked daily during the 7 d following the fungal application. Infectivity rate was determined by using the control mortality as a correction factor for the mortality in the fungu s treatment. Morphological traits unique to I. fumosorosea in dead insects (see below for method) were used to confirm infection. The experiment was repeated one time.

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66 Experiment 2. Infectivity o f the Most Susceptible S tage of Microtheca ochroloma by F our C oncentrations of PFR 97TM The goal of this experiment was to com pare the infectivity of four concentrations of PFR 97TM in the most susceptible stage of M. ochroloma, which was determined in Experiment 1. The concentrations were 1, 2, 3, and 4 g of P FR 97TM per 100 ml of sterile distilled water. Each concentration was applied (see below for method) to groups of 10 1st instars housed in separate and sealed plastic Petri dishes with moistened filter paper on the bottom. A 2.5 cm2 piece of turnip leaf w as placed on top of the filter paper. The Petri dishes were then housed in an environmentally controlled chamber set to 25o C 10L:14D photoperiod, and 60% RH. Each fungal treatment consisted of 10 replicates with 10 pseudoreplicates per replicate. A c ontrol treatment consisted of five replicates in which the test insects were sprayed with sterile distilled water only. Mortality was checked daily during the 7 d following the fungal application. Infectivity rate was determined by using the control mortality as a correction factor for the mortality in the fungus treatment. Confirmed infectivity rate was determined by morphological traits unique to I. fumosorosea in dead insects (see below for method). The experiment was repeated one time. For both experiments, the initial blastospore concentration was determined by counting the number of blastospores per ml using a disposable plastic Neubauer hemocytometer, C Chip D HCN01, manufactured by Incyto ( Korea). Each suspension was poured separately into 180ml Nalgene ( Rochester, NY) spray bottles for application to test insects. Each group of insects with their respective piece of leaf received 3 sec of application ( ~ 2.5 ml) on each side of the leaf. The sprayed leaf was not removed from the Petri dish and starting 3 d after treatment new nonsprayed

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67 leaves were added daily to each Petri dish but not removed. B lastospore deposition density was determined by placing a plastic c over slip among the test insects during the application of the fungus, after which the number of blastospores per mm2 was counted. Viability of blastospores was determined by taking 100L from the 103 serial dilution, spreading it on potato dextrose agar (PDA) in Petri dishes, maintaining the dishes under the same environmental conditi ons as the insects, and counting the number of CFU after 7 d To confirm infectivity in Experiment 1, dead insects were removed daily and transferred directly to a Petri dis h containing a mixture of PDA, odine, streptomycin, and chlrophenacol; dishes were then sealed with Parafilm and stored at 25C The presence of hyphae morphologically unique to I. fumosorosea (Fig. 5 1) was recorded. In Experiment 2, dead insects were surface sterilized in 70% ethanol for a few seconds before being placed on the PDA mi xture plates Unconfirmed infectivity was assigned to dead insects in which I. fumosorosea could not be identified because of its absence or contamination by other fungi Statistical Analysis. Data were arcsinetransformed prior to analysis. M ortality and infectivity rates were analyzed using analysis of variance, and treatment means were separated using Student NewmanKeuls test. All tests were performed with PROC GLM in SAS v. 9.2 ( SAS Institute Inc. 2002) with a significance level of 5%. Lethal con centration (LC25) and lethal times (LT10 and LT25) were analyzed using PROC PROBIT (SAS Ins t itute Inc. 2002), and significant differences between treatments were identified using 95% confidence intervals (Tabashnick and Cushing 1987).

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68 Results Experiment 1 For 1 g of PFR 97TM in 100 ml of water, the concentration o f the suspension was 3.0 0.1 107 blastospores/ml (Fig. 5 2 A) M ean blastos pore deposition density was 1 043 181.5 blastospores/mm2 (Fig 5 2 B) Viability was 49 1.0 CFU/ml1 in 104 seri al dilution (Fig. 5 2 C). The most susceptible stage of M. ochroloma to the Apopka 97 strain of I fumosorosea was the larval stage. The mean mortality rates of eggs, pupae and adults were not significantly different from their respective controls (Fi g. 5 2). In contrast, mean mortali ty rates of the 1st and 3rd instars were significantly higher (4.4 and 0.8 times, respectively) than their respective controls ( F = 14.39; df = 1,13; P = 0.0022; F = 8.12; df = 1,13; P = 0.0137) (Fig. 5 3). Mean infectivity r ate was at least 6.3 times higher in the 1st and 3rd instars than in the egg, pupal and adult stages ( F =12.19; df = 4,35; P < 0.0001) (Fig. 5 3). However, only 17 and 20% of the infectivity in the 1st and 3rd instar, respectively, was confirmed (Figs. 5 4 and 59). Mortality of the 1st instar was observed beginning 3 d after treatment. By that time the1st instars had already molted to 2nd instar. Mortality in the 3rd instar was observed beginning 1 day after treatment. The LT10 for the 1st instar ( 4 d) w as significantly higher than the 3rd instar (2 d) ( P < 0.05). N o significant difference was apparent for the LT25 between the two instars ( P > 0.05) (Fig. 5 5). The LT50 could not be determined due to the low mortality rate of larvae exposed to the 1 g treatment concentration of PFR 97TM; however the LT50 predicted by the model for the 1st and 3rd instar we re 8.6 (fiducial limitis: 7.610.5) and 21.2 d (fiducial limits: 12.9 60.7), respectively. The LT models were not significant for eggs, pupae, and adults ( P > 0.05).

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69 Experiment 2 For this experiment, the concentration of blastospores blastospore deposition, and viability for the four experimental concentrations of PFR 97TM are reported in Table 5 1. The 1st instar of M. ochroloma was selected for this experiment based on the results of Experiment 1. There was no mortality in the control treatment. Therefore, all mortality in the fungus treatments was considered to be caused by infection with I. fumosorosea. Mean infectivity rate was significant ly higher by 2.6 times in the 4 g concentration treatment than in the 1, 2, and 3 g concentration treatments ( F = 3.76, df = 3,36; P =0.0191) (Fig. 5 6). Confirmed infectivity rates were 2, 5, 10, and 27% in the 1, 2, 3, and 4 g concentration treatments, r espectively (Fig. 5 6). Based on fiducial limits (95%), the LT10 and LT25 in the 4 g concentration treatment were significantly lower compared to the other treatments ( P < 0.05). The LT50 predicted by the model was 10 d (fiducial limits: 8.413.7). There was no significant difference among the 1, 2, and 3 g concentration treatments ( P > 0.05) (Fig. 5 7). The LC10 and LC25 of PFR 97TM applied to 1st instar M. ochroloma were 1.4 (fiducial limit s: 0.3 2.0) and 5.5 g (fiducial limitis: 3.6 39.0) per 100 ml of distilled water, re spectively, on day 7. The LC50 predicted by the model was 25.6 g per 100 ml of distilled water, on day 7 (i ntercept = 1.42 0. 16; s lope = 1.0 0.36; X2 = 1.0 0.36; P = < 0.0001). Discussion Experiment 1 The fungus had a low, insignificant ovicidal effect with an egg mortality rate of 3% (Figs. 5 4 and 5 8). Although the ovicidal effect was low in this experiment, the fungal residues on the eggs and on the leaf surface may have a significant impact on

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70 the emerging neonates. T here is a great deal of variability and discussion concerning the ovicidal effect of I. fumosorosea. Rodrigues Rueda and Fargues (1980) showed that P. fumosorose us has high ovicidal activity on eggs of the moths Mamestra brassicae (Linneaus) and Spodoptera littoralis (Boisduval) In contrast, Lacey et al. (1999) reported a low, but significant mortalit y (10 20%) of eggs of Bemisia tabaci (Gennadius) treated with of PFR 97TM, but no significant ovicidal effect was reported on eggs of Yponomeuta xylostella ( Linneaus) (Maketon et al. 2008). Larvae of M. ochroloma in the 1st and 3rd instars experienced the h ighest infection rates among all the insects life stages. The unconfirmed infectioins may be attributed to the procedure of transferring dead insects to Petri dishes without first surface sterilizing the insects This may have resulted in the rapid growth of saprophagous fungi, thus slowing the growth of I. fumosorosea (if there was any) and not allowing its appearance of diagnostic morphological features (Fig. 5 9). Larvae infected by I. fumosorosea exhibited noticeable reduced growth (Fig. 5 10) and unsuccessful molting in which the exuvium remained attached to the new integument (Fig. 5 12). Similar studies by Hussain et al. (2009) have also shown a reduction in the consumption and growth of all instars of Ocinara varians Walker (Lepidoptera: Bombycidae) when I. fumosorosea strain 03011 C3.19A was applied. A reduction in feeding was also reported by Fargues et al. (1994) in S. decem lineata attacked by B. bassiana. Mortality and growth rate r eduction may be attributed to the production of toxins by the fungus, mechanical disruption of the structural integrity of membranes by the growth of hyphae, and dehydration of cells from the loss of fluids ( Ferro n 1981; Tefera and Pringle 2003 ; Assaf et al. 2005 ).

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71 M icrotheca ochroloma pupates within a net like case which only has direct contact with the c uticle of the pupa at the apex of the body (Fig. 511). For this reason, low mortality and infectivity rates of pupae were observed, since the net like case apparently serves as a physical barrier to the deposition of blastospores on the cuticle of the pu pa, which is necessary to initiate infection. Adults of M. ochroloma were not affected by I. fumosorosea, probably because the hard cuticle is composed primarily of a higher degree of crosslinked proteins and chitin than that of the immature stages, whic h provides greater strength and hardness to the exoskeleton and functions as a formidable barrier to blastospore germination (Klowden 2007). Only 4% mortality was recorded in the fungus treatment, compared to none in the control treatment (Fig. 5 3), but the mortality in the treatment cannot be confidently attributed to the fungus since there was no confirmed infectivity (Fig. 5 4). Michalaki et al. (2007) reported low mortality of adults of Tribolium confusum Jacquelin du Val (Coleoptera: Tenebrionidae) exposed to I. fumosorosea. Experiment 2 There was a well defined positive correlation between fungus concentration and mortality rates of 1st instars of M. ochroloma. The highest infectivity and confirmed infectivity rates were achieved with the 4 g c oncentration treatment, which corresponded to the highest concentration of blastospores/ml, deposition of blastospores/mm2, and viability among all treatments (Table 51). However, the 4 g concentration treatment achieved only 29% infectivity in the labor atory; infectivity rates in the field may be expected to be lower. The model predicts the LC25 is 5.5 g per 100 ml of water, which is equivalent to approximately 1.6 108 blastospores/ml, and the predicted LC50 is 25.6 g per 100 ml of water, which is equivalent to approximately 7.3

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72 108 blastospores/ml. Higher concentrations of PFR 97TM should be tested in the laboratory, since it seems that infectivity rates in the 1st instar increase as the concentration of blastospores/ml increases (Fig. 5 6), and th e LT10 and LT25 are achieved faster with increasing concentration (Fig. 5 7). Once the product is registered for use in the field for fruiting crops the concentration of CFU per bag probably will be higher; therefore, the amount of grams per 100 ml requi red for high infection rates would be lower. The unconfirmed infectivity rate in Experiment 2 was lower compared to the unconfirmed infectivity rate in E xperiment 1 because when the insects died in the former they were sterilized with alcohol for few sec onds before plac ing them o n the PDA. The unconfirmed infectivity rate in E xperiment 2 may be reduced even more by using the polymerase chain reaction technique to identify the presence of PFR 97TM strain in dead insects, as has been done in other studies (Meyer 2007; Meyer et al. 2008; Hoy et al. 2010). Once blastospores are deposited and germinate on the integument of the insect, death of the host will most likely occur within 3 d at any concentration. In both experiments, the appearance of fungal infection in the 1st instar of M. ochroloma began 3 d following application. Similar results were reported by Tounou et al. (2003) in nymphs of the green leafhopper, Empoasca decipiens Paoli (Hemiptera: Cicadellidae), which began dying 3 d after treatment wit h I fumosorosea strain Pfr12. However, there will be a higher probability of deposition and germination of blastospores at higher concentrations of the fungus, thereby killing a greater number of insects compared to lower concentrations.

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73 In summary, t he larval stage of M. ochroloma is the most susceptible stage to I. fumosorosea. However, a concentration of PFR 97TM greater than those tested here is required to reach the LT50 and LC50 in 1st inst ars under laboratory conditions. Higher concentrations than those tested in the laboratory will n eed to be applied in the field as well. Figure 51. Morphological characteristics of Isaria fumosorosea infecting Microtheca ochroloma larva. Larva Fungus

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74 A B C Figure 52. Laboratory tests of Isaria fumosorosea A) Blastospore concentration (blastospores/ml) B) Blastospore deposition density (blastospore/mm2, and C) Viability (CFU/g)

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75 Figure 53. Mortality of Microtheca ochroloma by PFR 97TM 20% WDG at 3.0 107 blastospores/ml 7 d after application. Bars with different letter within each stage are significantly different ( SNK test, P < 0.05).

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76 Figure 54. Infectivity of Microtheca ochroloma by PFR 97TM 20 % WDG at 3.0 107 blastospores/ml 7 d after application. Bars with the same letter are not sig nificantly different ( SNK test, P > 0.05).

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77 Figure 55. Lethal time of the first and third instars of Microtheca ochroloma treated with PFR 97TM. LT10 and LT25 values within the same box are not significantly different in their 95% confidence interval s.

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78 Figure 5 6. Infectivity of first instar Microtheca ochroloma at four concentrations of PFR 97TM 7 d after application. Bars followed by the same lowercase letter are not significantly different (P>0.05).

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79 Figure 57. Lethal time of the f irst instar Microtheca ochroloma exposed to f our concentrations of PFR 97TM. LT10 and LT25 values within the same box are not significantly different in their 95% confidence intervals.

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80 Figure 58. Eggs of Microtheca ochroloma infected by Isar ia fumosorosea. Figure 59. Unconfirmed and confirmed infectivity by Isaria fumosorosea in dead larvae of Microtheca ochroloma.

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81 Figure 510. Reduction in the growth of larvae of Microtheca ochroloma infected by Isaria fumosorosea. Figure 511. Net like pupal case of Microtheca ochroloma.

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82 A B C Figure 512. Unsuccessful molting by a larva of Microtheca ochroloma infected with Isaria fumosorosea. A) Head partially out, B) Exuvia attached to the dorsal part of the body, and C) Larva st arting to pull out from the tip of the abdomen.

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83 Table 51. Laboratory tests of Isaria fumosor osea in Experiment 2. PFR 97 TM Concentration SE a Deposition SE CFU b SE (g) (blastospores/ml) (blastospores/mm 2 ) (CFU/ml at 10 4 ) 1 2.2 0.1 10 7 779 150.5 24.5 2.5 2 3.8 0.2 10 7 1088 174.1 55.5 2.5 3 8.4 0.7 10 7 4157 962.6 84.5 1.5 4 1.1 0.0 10 8 6658 881.6 146.5 1.5 a standard error. bColony forming units

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84 CHAPTER 6 C ONCLUSIONS My thesis study focused on the evaluation of two potential biological control agents of the yellowmargined leaf beetle, M. ochroloma. The predator, P maculiventris was chosen because it 1) occurs naturally in the field, 2) can be acquired commercially by growers, and 3) preys on many agricultural pests. The fungus, I fumosorosea (PFR 97TM), was chosen because it 1) is a local strain and 2) has been under previous study for application to food crops in the field. There was no development of 1st instars or egg hatching of P. maculiventris at 10C. Development of the predator from egg to adult is shorter at 20 and 25C (31 and 23 d, respectively) than at 15 C (99 d). The daily predation by P. maculiventris starting from the 3rd instar until 10 d of adulthood was higher at 25C compared to 20C, which was higher than at 15 C. However, the total predation by P. maculiventris on 4th instar M. ochroloma was higher at 25C compared to 20C, which was higher than at 15C only during the 10 d of adulthood. Podisus maculiventris nymphs killed 59, 53, and 65 4th instars of M. ochroloma at 15, 20 and 25 C, respectively. Fresh adult females weigh more than males at 20 C, but not at 25C. Predator nymphs also preyed on an average of 741 eggs of M. ochroloma, completing their development in 25 d at 25C. Therefore, P maculiventris can develop successfully on a diet of M. ochroloma eggs or larvae, despite the presence of secondary compounds (glucosinolates) in crucifers consumed by M. ochroloma. The nymphal stage of P maculive ntris develops faster and preys on more 4th instars of M. ochroloma at higher temperatures A two year cage experiment addressed the field predation potential of P. maculiventris when 1st instars were released at three densities (4=low, 10=medium, and

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85 16=high) on six turnip plants with a known initial population of M. ochroloma larvae. In 2009, the plants grew larger ( 7 leaves /plant). The high predator density treatment reduced the M. ochroloma population significantly more than the medium predator density treatment, which reduced the pest population significantly more than the low predator density treatment. The hig h predator density treatment reduced the M. ochroloma population by 96% and the overall survivorship of P. maculiventris was 51%. In 2010, the plants were smaller ( 6 leaves/plant). The medium and high predator density treatments reduced equally the M. oc hroloma population, but significantly more than the low predator density treatment. The medium and high predator density treatments reduced the M. ochroloma population by 99% and the overall survivorship of P. maculiventris was 26%. Therefore, depending on the plant size, 10 or 16 nymphs per 1.21 m2 (the approximate area covered by six plants) are recommended to release in the field to control M. ochroloma population Two experiments addressed the evaluation of the fungus on M. ochroloma. In Experiment 1 t he larval stage was shown to be the most susceptible stage to PFR 97TM In Experiment 2 the most susceptible stage from Experiment 1 ( i.e 1st instar) was exposed to four concentrations of PFR 97TM (1, 2, 3, and 4 g of PFR 97TM each in 100 ml of distilled w ater). The 4 g concentration caused the highest infection rate (27% confirmed infectivity) on the 1st instars of M. ochroloma compared to the 1, 2, and 3 g concentration (2, 5, and 10% infectivity, respectively). Therefore, PFR 97TM is not recommended for use to control M. ochroloma in the field, due to the low rate of infection observed in the laboratory.

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86 For the Growers Podisus maculiventris is recommended for use in the field to control populations of M. ochroloma. The initial density of M. ochrolom a used in this study is relatively low compared to densities that may be observed in organic farms. Therefore, releases of P. maculiventris should be made when infestations of M. ochroloma are low so that the predator may provide preventive control of inc reasing pest populations For big plants ( 7 leaves/plant), a release of 16 1st instar P. maculiventris per six plants or 1.21 m2 is recommended. For small plants ( 6 leaves/plant), a release rate of 10 1st instar P. maculiventris per six plants or 1.21 m2 is recommended. A bottle of 250 eggs of P maculiventris can be purchased online at www.riconvitova.com ($112.74), www.arbico organics.com ($133.50), or www.planetnatural.com ($ 118.95). The costs of releasing 10 and 16 nymphs per 1.21 m2 are $4.50 and $7.21, respectively. A bottle of 250 eggs of P. maculiventris will cover 30 m2 for the release density of 10 nymphs and 19 m2 for the release density of 16 nymphs. A cheaper alternative for obtaining eggs is by collecting adults from the field and holding them indoors with prey. The prey can be mainly caterpillars or beetle larvae. However, the availability of prey and the ti me to feed the predators can be a disadvantage to this alternate method of obtaining eggs. The product PFR 97TM containing blastospores of I. fumosorosea is not recommended for use in the field to control M. ochroloma, since higher concentrations than tho se tested in the laboratory are required to kill more than 50% of the population. The suspension preparation of PFR 97TM at concentrations higher than 4 g per 100 ml will increase pest management costs significantly, and the higher concentrated material could clog the nozzle during application of the product. The manufacturer, Certis

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87 ( www.certisusa.com ), sells a 9.5kg bag of PFR 97TM for $35.00. Even though PFR 97TM did not have a high rate of infection in M. ochro loma OMRI listed products such as Entrust and PyGanic can be another option to control populations of M. ochroloma.

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88 LIST OF REFERENCES Ables, J. R., and D. W. McCommas. 1982. Efficacy of Podisus maculiventris as a predator of variegated cutworm on gree nhouse cotton. J. Ga. Entomol. Soc. 17: 204206. Alyokhin, A., M. Baker, D. Mota Sanchez, G. Dively, and E. Grafius. 2008. Colorado potato beetle resistant to insec ticides. Am. J. Pot. Res. 85:395413. Ameen, A. O., and R. N. Story. 1997a. Feeding pref erences of larval and adult Microtheca ochroloma (Coleoptera: Chrysomelidae) for crucifer foli age. J. Agric. Entomol. 14: 363368. Ameen, A. O., and R. N. Story. 1997b Biology of the yellow margined leaf beetle (Coleoptera: Chrysomelidae) on crucifers. J. Agric. Entomol. 32: 478486. Asaff, A., C. Cerda Garcia Rojas, and M. de la Torre 2005. Isolation of dipicolinic acid as an insecticidal toxin from Paecilomyces fumosoroseus Applied Microbiology and Bi otechnology. 68: 542547. Bajan, C. 1973. Paecilomy ces fumoso roseus (Wize) pathogenic agent of the Colorado beetle ( Leptinotarsa decemlineata Say), Ekologia Polska. 21: 705713. BastosDequech, S. T., C. D. Sausen, C. G Lima, and R. Egewarth. 2008. Efeito de extratos de plantas com atividade inseticida no controle de Microtheca ochroloma Stal (Col: Chrysomelidae), em laboratrio. Biotemas. 21: 4146. Balsbaugh, E. U. 1978. A second species of Microtheca Stl (Coleoptera: Chrysomelidae) found in North America. Coleopts. Bull. 32: 219222. Bernardini, M., A. Carilli, G. Pacioni, and B. Santurbano 1975 Isolation of Beauvericin from Paecilomyces fumosoroseus Phytochemistry. 14: 1865. Biever, K. D., and R. L. Chauvin. 1992 Suppression of the Colorado potato beetle (Coleoptera : Chrysomelidae) with augment ative releases of predaceous stinkbugs (Hemiptera : Pentatomi dae). J. Econ. Entomol. 85: 720726. Bowers, K. 2003. Effects of within field location of host plants and intercropping on the distribution of Microtheca ochroloma (Stl) in Mizuna. M. S. thesis University of Florida, Gainesville. Basulu, R. R., and H. Fadamiro. 2008. Field evaluation of select OMRI biorational insecticides against yellowmargined leaf beetle, Microtheca ochroloma (Coleoptera: Chrysomelidae) in organic crucifer vegetables. Entomological Society of America. Annual meeting. Tuesday, November 18, 2008. D0227. Plant Insect Ecosystems Section. ( http://esa.confex.com/esa/2008/techprogram/paper_37405.htm )

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89 Capin era, J. L. 2001. Handbook of Vegetables Pests. Academic Press, San Diego, CA. Chamberlin, F. S., and H. H. Tippins. 1948. Microtheca ochroloma, an introduced pest of crucifers, found in Alabama. J. Econ. Entomol. 41: 979980. Collier T., and R. Van Stee nwyk. 2004. A critical evaluation of augmentative biological control. Biol. Control 31: 245256 Crower, D. 2007. Impact of release rates on the effectiveness of augmentative biological control agents. J. I nsect Sci. 15:111. Danks, H. V. 1987. Insect dormancy: An ecological Perspective. Biological Survey of Canada (Terrestrial Artropods), Ottawa, Canada. De Clercq, P., and D. Degheele. 1992. Development and survival of Podisus maculiventris (Say) and Podisus sagitta (Fab.) (Heteroptera: Pentatomidae) at various constant temperatures. Can. Entomol. 124: 125133. De Clercq, P., and D. Degheele. 1994. Laboratory measurement of predation by Podisus maculiventris and Podisus sagitta (Hemiptera: Pentatomidae) on beet armyworm (Lepidoptera: Noctuidae). J. Econ. Entomol. 87: 7683. De Clercq, P., F. Merlevede, I. Mestdagh, K. Vandendurpel, J. Mohaghegh, and D. Degheele. 1998. Predation on the tomato looper Chrysodeixis chalcites (Esper) (Lep., Noctuidae) by Podisus maculiventris (Say) and Podisus nigripinus ( Dallas) (Het., Pentatomi dae). J. Appl. Entomol. 122: 9398. Desurmont, G., and P. A. Weston. 2008 Influence of prey size and environmental factors on predation by Podisus maculiventris (Hemiptera: Pentatomidae) on viburnum leaf beetle (Coleoptera: Chrysomelidae). Can. Entomol. 140: 192202. Dos Anjos, J., P. Rosalino, C. D. Sausen, L. Do Prado, R. Egewarth, V. Soares, and S. T. Bastos. 2007. Fungos entomolatognicos em Diabrotica speciosa e Microtheca ochroloma (Col., Chrysomelidae) em hortalizas. Inform e Tcnico. Univ ersidade Federal de Santa Maria, Santa Maria, Bras il. Drees, B. M. 1990. Yellowmargined leaf beetle on leafy greens in Texas. Texas A&M University. Texas agricultural extension services. UC 006. ( http://insects.tamu.edu/extension/bulletins/uc/uc 006.html ) Fargues J. J. C. Delmas, R. A. Lebrun. 1994. Leaf consumption by larvae of the Colorado potato beetle (Coleoptera: Chrysomelidae) infected with the entomopathogen Beauveria bassiana. J. Econ. Entomol. 87 : 67 71

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90 Fargues, J., M. S. Goettel, N. Smits, A. Ouedraogo, C. Vidal, L. A. Lacey, C. J. Lomer, and M. Rougier. 1996. Variability in susceptibility to simulated sunlight of conidia among isolates of entomopathog enic hyphomy cetes. Mycopathologia 135: 171181. Fasulo, T. R. 2005. Yellowmargined leaf beetle, Microtheca ochroloma Stl (Insecta: Coleoptera: Chrysomelidae). University of Florida. Florida Cooperative Extension service. IFAS. EDIS. EENY 348. ( http://edis.ifas.ufl.edu/pdffiles/IN/IN62500.pdf ) Ferron, P. 1981. Pest Control by The Fungi Beauveria bassiana and Metarhizium : In Microbial Control Pests and Plant diseases. Ed by H. D. Burges. Ac ademic Press, 19701980. New York and London. (FOG) Florida Organic Grower. 2008. List of Certified Growers & Handlers. Florida. ( http://www.foginfo.org/) Guillebeau, P. 2001. Crop profile for leafy greens in Georgia. USDA crop profiles. ( http://www.ipmcenters.org/cropprofiles/docs/GAleafgreen.pdf ) Greif, M. D., and R. S. Currah. 2007 Patterns in the occurrence of saprophytic fungi carried by arthropods caught in traps baited with rotted wood and dung. Mycologia 99: 719. Gusev, G. V., Yu. V. Zayats, E. M. Topashchenko, and G. K. Rzhavina. 1983. Control of the Colorado potato beetle (Coleoptera: Chry somelidae). Zash. Rast. 9: 3839. He rrick, N. J., and S. R. Reitz. 2004. Temporal occurrence of Podisus maculiventris (Hemiptera: Heteroptera: Pentatomidae) in North Florida. Fla. Entomol. 87: 587590. HoughGoldstein, J. 1996. Use of predaceous pentatomids in integrated management of the C olorado potato beetle (Coleoptera: Chrysomelidae), in M. Coll and J. Ruberson (eds.), Predatory Heteroptera in agroecosystems: their ecology and use in biological control. Thomas Say Publ., Entomol. Soc. America, Lanham, MD. HoughGoldstein, J., and D. Mc Pherson. 1996. Comparasion of Perillus bioculatus and Podisus maculiventris (Hemiptera: Pentatomidae) as potential control agents of the Colorado potato beetle (Coleoptera: Chrysomelidae). J. Econ. Entomol. 89: 11161123. Hoy, M. A., R. Singh, M. E. Roger s. 2010. Evaluations of a novel isolate of Isaria fumosorosea for control of the Asian citrus psyllid, Diaphorina citri (Hemiptera: Psyllidae). Fla. Entomol. 93: 2432.

PAGE 91

91 Humber, R. A., K. S. Hansen, and M. M. Wheeler. 2009. USDA ARS Collection of entomopathogenic fungal cultures (ARSEF), ARSEF Catalog of Species. ( http://arsef.fpsnl.cornell.edu/mycology/catalogs/Catalog.pdf ) Hussain, A., M. Tian, Y. He, and S. Ahmed. 2009. Ento mopathogenic fungi disturbed the larval growth and feeding performance of Ocinara varians (Lepidoptera: Bombycidae) larvae. Insect Sci. 16: 511517 Jol i v et, P. 1950. Contribution a l'tude des Microtheca Stl (Coleoptera, Chrysomelidae). Bulletin de l'Ins titut royal des scienc es naturelles de Belgique 26: 127. Klowden, M. J. 2007. Physiological Systems i n Insects. Integumentary Systems. Academic Press Elsevier, Moscow, Idaho. Lacey, L. A., and G. Mercadier. 1998. The effect of selected allelochemicals on germination of conidia and blastospores and mycelial growth of the entomopathogenic fungus, Paecilomyces fumosoroseus (Deuteromycotina: Hyphomy cetes). Mycopathologia, 142: 1725. Lacey, L. A., A. A. Kirk, L. Millar, G. Mercadier, and C. Vidal. 1999. Ov icidal and larvicidal activity of conidia and blastospores of Paecilomyces fumosoroseus (Deuteromycotina: Hyphomycetes) against Bemisia argentifolii (Homoptera: Aleyrodidae) with a description of a bioassay system allowing prolonged survival of control ins ects. Biocontrol Sci. Technol 9 : 9 18. Landis, B. J. 1937. Insect hosts and nymphal development of Podisus maculiventris Say and Perillus bioculatus F. Ohio J Sci. 37:252259 Legaspi, J. C. 2004. Life history of Podisus maculiventris (Heteroptera: Pentatomidae) adult females under different constant temperat ures. Environ. Entomol. 33:12001206. Luangsa ard, J. J., N. L. HywelJones, L. Manoch, and R. A. Samson. 2005. On the relationships of Paecilomyces sect. Isarioidea species. Mycological Research. 109: 581589. Maketon, M., P. Orosz Coghlan, J. Jaengarun. 2008. Field evaluation of Isaria fumosorosea on controlling the diamondback moth ( Plutella xylostella ) in Chines e kale. Phytoparasitica 36: 260263. Michalaki, M. P., C. G. Athanassiou, T. Steenber g, and C. T. Buchelos. 2007 Effect of Paecilomyces fumosoroseus (Wize) Brown and Smith (Ascomycota: Hypocreales) alone or in combination with diatomaceous earth against Tribolium confusum Jacquelin du Val (Coleoptera: Tenebrionidae) and Ephestia kuehniell a Zeller (Lepidoptera: Pyr alidae), Biol. Control. 40: 280286.

PAGE 92

92 McPherson, J. E. 1980. A list of the prey species of Podisus maculiventris (Hemiptera: Pentatomidae). Great Lakes Entomol. 13: 1724. McPherson, J. E. 1982. The Pentatomoidea (Hemiptera) of northeastern North America. Southern Illinions University Press, Carbondale and Edwardsville. IL. Meyer, J. M. 2007. Microbial associates of the Asian citrus psyllid and its two parasitoids: symbionts and pathogens. Ph.D. Dissertation, University of Flori da, Gainesville, 145 pp. Meyer, J. M., M. A. Hoy, and D. G. Boucias. 2008. Isolation and characterization of an Isaria fumosorosea isolate infecting the Asian citrus psyllid in Florida. J. Invertr. Pathol 99: 96 102 Oliver, A. D. 1956. Yellow margi ned leaf beetle ( Microtheca ochroloma). Coop. Econ. Insect Report. 6:351353. Osborne, L. S., and Z. Landa. 1992. Biological control of whiteflies with entomopathogenic fungi. Fl a Entomol 75 : 456 471 Osborne, L., Z. Landa, A. Bohata, and C. McKenzie. 2 008. Potential of entomopathogenic fungus Isaria fumosorosea to protect potted ornamental plants against Bemisia tabaci during shipping. International Organization for Biologica l Control/WPRS Bulletin 32: 159165. Overall, L., and J. Edelson. 2007. Field evaluation of organic insecticides to control the harlequin bug, Murgantia histrionica and the yellowmargined leaf beetle, Microtheca ochroloma, on leafy greens in southern Oklahoma. Entomological Society of America Annual meeting. Monday, December 10, 2007 10:29 AM 0517 ( http://esa.confex.com/esa/2007/techprogram/paper_30387.htm) Poprawski, T. J., and W. J. Jones. 2001. Host plant effects on activity of the mitosporic fungi Be auveria bassiana and Paecilomyces fumosoroseus against two populations of Bemisia whiteflies (Homoptera: Aleyrodidae). Mycopathologia. 151: 1120. Racca Filho, F., I. L. Rodrigues Filho, C. A. C. Santos, and C. N. Rodrigues. 1994 Microtheca ochroloma (Co leoptera: Chrysomelidae): aspectos taxonmicos e biolgicos. Rev. Univ. Rural. 16: 2935. Richman, D. B., and W. H. Whitcomb. 1978. Comparative life cycles of four species of predatory stink bugs (Hemiptera: Pentatomidae). Fla. Entomol. 61: 113119. Rohw er, K. S., F. E. Guyton, and F. S. Chamberlin. 1953. Status of the yellow m argined leaf beetle. Coop. Econ. Insect Report 3: 194195.

PAGE 93

93 Rodrigues Rueda, D., and J. Fargues. 1980. Pathogenicity of entomopathogenic hyphomycetes, Paecilomyces fumosoroseus and Nomuraea rilei to eggs of noctuids, Mamestra brassicae and S podoptera littoralis J. Invert. Pathol. 36: 399408. Samson, R. A. 1974. Paecilomyces and some allied Hyphomycetes. Studies in M yco l 6: 3241. Silva, A. G. DA., C. R. Gonalves, D. M. Galv o, A. J. L. Gonalve, J. Gomes, M. N. Silva, and L. Simoni. 1968. Quarto catlogo dos insetos que vivem nas plantas do Brasil, seus parasitas e predadores. Ministrio da A gricultura, Rio de Janeiro, Bras il. Smith, P. 1993. Control of Bemisia tabaci and t he potential of Paecilomyces fumosoroseus as a biopesticide. Biocontrol News & Inform. 14: 7178. Staines, C. L. 1999. Chrysomelidae (Coleoptera) new to North Carolina. Coleopts. Bull. 53: 2729. Stamopoulos, D. C., and A. Chlroridis. 1994. Predation rat es, survivorship and development of Podisus maculiventris (Het.: Pentatomidae) on larvae of Leptinotarsa decemlineata (Col.: Chrysomelidae) and Pieris brassicae (Lep.:Pieridae), under field con ditions. Entomophaga. 39: 39. Tefera, T., and K. L. Pringle. 2003 Food consumption by Chilo partellus (Lepidoptera: Pyralidae) larvae infected with Beauveria bassiana and Metarhizium anisopliae and effects of feeding natural versus artificial diets on mortality and mycosis. J Invertebr. Pathol. 84: 220225. Tabashnick, B. E., and N. L. Cushing.1987. Quantitative genetic analysis of insecticide resistance: Variation in fenvalerate tolerance in a diamondback moth (Lepidoptera: Plutellidae) populat ion. J. Econ. Entomol. 82: 510. Tipping, P. W., C. A. Holko A. A. Abdul Baki, and J. R. Aldrich. 1999. Evaluating Edovum puttleri Grissell and Podisus maculiventris (Say) for augmentative biological control of C olorado potato beetle in tomatoes. Biol. Control. 16: 3542. Tounou, A. K. K. Agboka, H. M. Poehling, J. K. Raupach, J. Langewald, G. Zimmermann, and C. Borgemeister 2003. Evaluation of the entomopathogenic fungi Metarhizium anisopliae and Paecilomyces fumosoroseus (Deuteromycotina: Hyphomycetes) for control of the green leafhopper empoasca decipiens (Homopter a: Cicadellidae) and potential s ide effects on the egg parasitoid Anagrus atomus (Hymenoptera: Mymaridae). Biocontr ol Sci. and Technol 13: 715728

PAGE 94

94 (USDA) U. S. Department of Agriculture. 2010. National Organic Program. Resource Center: Regulations. ( http://www.ams.usda.gov/AMSv1.0/ams.fetchTemplateData.do?template=Templ ateF&navID=NationalOrganicProgram&leftNav=NationalOrganicProgram&page= NOPResourceCenterRegulations&description=NOP%20Regulations&acct=nopru lemaking ) (USDA NASS) U. S. Department of Agriculture National Agricultural Statistics Service. 2009. 2007 Census of Agriculture. United Sates Department of Agriculture, National Agriculture Statistics, Washington, D.C. ( http://www.agcensus.usda.gov/Publications/2007/Online_Highlights/County_Pro files/Florida/cp99012.pdf. ). Van Driesche, R. D., S. L yon, K. Jacques, T. Smith, and P. Lopes. 2002 Comparative cost of chemical and biological whitefly control In poinsettia: Is there a gap?. Fla. Entomol. 85: 488 493 Valicente, F. H. and R. J. ONeil. 1995 Effects of host plants and feeding regimes on selected life history characteristics of Podisus maculiventris (Say) (Heteroptera: Pentatomidae). Biol Control 5:449 461 Vidal, C., and J. Fargues. 2007. Climatic Constraints for Fungal Biopesticides, pp. 3955. In S. Ekesi and N. K. Maniania (eds.), U se of Entomopathogenic Fungi in Biological Pest Management. Kerala, India. Research Signpost. Vidal, C., L. A. Lacey, and J. Fargues. 1997a. Pathogenicity of Paecilomyces fumosoroseus (Deuteromycotina: Hyphomycetes) against Bemisia argentifolii (Homopter a: Aleyrodidae) with a Description of a Bioassay Method. J. Econ. Entomol. 90: 765772. Vidal, C., J. Margues, and L. A. Lacey. 1997b. Intraspecific variability of Paecilomyces fumosoroseus : effect of temperature on vegetative gr owth. J. Invert. Pathol. 7 0: 1826. Vidal, C., L.S. Osborne, L.A. Lacey, and J. Fargues, 1998. Effect of host plant on the potential of Paecilomyces fumosoroseus ( Deuteromycotina: Hyphomycetes) for controlling the silverleaf whitefly, Bemisia argentifolii (Homoptera: Aleyrodidae) in gree nhouses. Biol. Control. 12: 191199. Waddill, V., and M. Shepard. 1975. A comparison of predation by the pentatomids, Podisus maculiventris (Say) and Stiretrus anchorago (F.), on the Mexican bean beetle, Epilachna varivestis Mulsant. Ann. Entomol. Soc. Am. 68: 10231027. Wiedenmann, R. N., and R. J. O'Neil. 1990. Effects of low rates of predation on selected lifehistory characterisitcs of Podisus maculiventris (Say) (Heteroptera: Pentatomidae). Can. Entomol. 122: 271283.

PAGE 95

95 Wiedenmann, R. N., and R J. O'Neil. 1991. Laboratory measurement of the functional response of Podisus maculiventris (Say) (Heteroptera: Pentatomi dae). Environ. Entomol. 20: 610614. Woodruff, R. E. 1974. A South American leaf beetle pest of crucifers in Florida (Coleoptera: Chrysomelidae). FDACS DPI Entomol. Cir. 148.

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96 BIOGRAPHICAL SKETCH Cecil O. Montemayor Aizpura was born in David, Panam. She received her bachelor s degree at the Panamerican School of Agriculture, Zamorano, in Honduras in 2005. She conducted her degree internship at Chiquita Brands International Co. in La Ceiba, Honduras, work ing with entomopathogenic fungi to control pests in bananas. In 2006, she was an intern at the University of Minnesota, where she work ed in wetlands restoration and in the biologi cal control of soybean aphids Since 2007, she has work ed at the University of Florida s Biological Control Research and Containment Laboratory (BCRCL) at the Indian River Research and Education Center in Ft. Pierce. At the BCRCL, she first worked as a s hort term scholar, conducting research on biological control of insects, specifically the yellowmargined leaf beetle, and processing specimens collected for an inventory of the arthropods on tree islands in the South Florida Water Management District Conservation Area S he then began her Master of Science degree program in the Entomology and Nematology Department in August 2008. She received a scholarship grant from the Ministry of Economy and Finances of Panama to support her during her study program. S he is a member of the Entomological Society of America, Florida Entomological Society, and Florida State Horticultural Society She was the president of the Statewide Student Association at University of Florida from 2009 to 2010 She has presented talks about her research at the annual meetings of the Florida State Horticultural Society (third place in the student competition), the Florida Entomological Society (first place in the student competition), and the Entomological Society of America.