alginate pellet formulation of beauverja imported fire …

y

ALGINATE PELLET FORMULATION OF Beauverja

bassiana PATHOGENIC TO THE RED

IMPORTED FIRE ANT

by

HERSHEL E. WHITE, JR., B.A.

A THESIS

IN

ENTOMOLOGY

Submitted to the Graduate Faculty of Texas Tech University in

Partial Fulfillment of the Requirements for

the Degree of

MASTER OF SCIENCE

Aooroved

December, 1995

^^-

ACKNOWLEDGMENTS

I would first and foremost like to extend my deepest love and

appreciation to my family. Although I have often been inattentive and

distracted, they have continued to support me. Without them, I would

never have made it.

I am also indebted to Dr. Harlan Thorvilson and Dr. Sherman

Phillips, Jr. Dr. Thorvilson has been a stable and responsible

supervisor; his support and advice have been invaluable. I thank Dr.

Phillips for encouraging me to enter the field of entomology and for

allowing me to be a part of his many adventures. Dr. John Zak has been

instrumental in providing insights on the subject of mycology; his

laboratory skills and enthusiasm made the intimidating subject of

microbiology fun and informative.

I would also like to extend a special thanks to Dr. Amadou Ba.

When I was still a lost and overwhelmed newcomer, he provided me with

assistance and guidance that proves him a very special person.

Camille Landry has also been a source of great help to me. She

has shared with me all the pains and gains of graduate school life.

Without her, this journey would have been much lonelier and far less

rich.

11

11

V

1 >:

TABLE OF CONTENTS

ACKNOWLEDGMENTS

LIST OF TABLE

LIST OF FIGURES

CHAPTER

I - LITERATURE REVIEW 1

Introduction and Range Expansion of the Red Imported

Fire Ant the United States 1

Microbial Pathogens of Solenopsis invicta 2

Beauveria bassiana as a Control Agent of Imported Fire Ants 3

Encapsulation of Biocontrol Agents in Alginate Pellets 6

Alginate Formulations and the Nursery Industry 9

Research Objectives 10

II. MATERIALS AND METHODS 11

Growth and Culture of Beauveria bassiana 11

Production of Alginate Pellets 11

Preparation of Alginate Pellets for Electron Microscopy 12

Treatment of Alginate Pellets with Polyethylene Glycol 12

Collection and Maintenance of Red Imported Fire Ants 13

Mortality of Solenopsis invicta Maintained in Non-sterile Soil 13 Mortality of Solenopsis invicta Maintained in Sterile or Non-sterile Potting Soil 14

Production of Alginate Pellets with Diatomaceous Earth, Dextrose, and Rice Powder 14

Mortality of Solenopsis invicta Treated with Diatomaceous Earth, Dextrose and Rice Powder-Amended , Alginate Pellets with B. bassiana 15

Pellet-Induced Motality of Small RIFA Colonies in

Large Containers 15

III. RESULTS 18

Electron Microscopy of Alginate Pellets 18

Mortality of Solenopsis invicta Maintained in Non-sterile Soil 18

111

Mortality of Solenopsis invicta Maintained in Sterile or Non-sterile Potting Soil 18

Mortality of Solenopsis invicta Treated with Diatomaceous Earth, Dextrose and Rice Powder-Amended Alginate Pellets with B. bassiana 26

Pellet Induced Mortality of Small RIFA Colonies

in Large Containers 26

Bioassay of Plated Ants 33

IV. SUMMARY AND CONCLUSIONS 37

REFERENCES CITED 39

APPENDIX 43

IV

LIST OF TABLES

3.1 Mean cumulative percent mortality of Solenopsis invicta in response to soil conditions and polyethylene glycol 21

3.2 Mean cumulative percent mortality of Solenopsis invicta in response to soil conditions and polyethylene glycol 24

3.3 Mean cumulative percent mortality of Solenopsis invicta in response to non-sterile soil and diatomaceous earth, dextrose, and rice powder-amended alginate pellets 28

3.4 Mean number of Solenopsis invicta removed from refuse piles and mean number of survivors after exposure to diatomaceous earth, dextrose, and rice powder-amended alginate pellets 32

3.5 Comparison among treatment means number from refuse piles and mean number of survivors after exposure to diatomaceous earth, dextrose, and rice powder-amended alginate pellets 32

3.6 Mean percent of live Solenopsis invicta removed weekly that tested positive for Beauveria bassiana infection 34

3.7 Mean percent of surviving Solenopsis invicta floated out of tubs that tested positive for Beauveria bassiana infection 3 5

3.8 Mean percent of live Solenopsis invicta removed weekly that were positive for Beauveria bassiana infection

36 A.l One-way analysis of variance performed on day 5

mortality of Solenopsis invicta maintained in non-sterile soil exposed to polyethylene glycol-treated or untreated alginate pellets 44

A.2 One-way analysis of variance performed on day 6 mortality of Solenopsis invicta maintained in non-sterile soil exposed to polyethylene glycol-treated or untreated alginate pellets 44

A. 3 One-way analysis of variance performed on day 7 mortality of Solenopsis invicta maintained in non-sterile soil exposed to polyethylene glycol-treated or untreated alginate pellets 45

A. 4 One-way analysis of variance performed on day 8 mortality of SolenopsT ^ invicta maintained in non-sterile soil exposed to polyethylene glycol-treated or untreated alginate pellets 45




V





A. 12 One-way analysis of variance performed on day 5 mortality of Solenopsis invicta maintained in non-sterile or sterile soil exposed to polyethylene glycol-treated or untreated alginate pellets 49









VI

A

A.21 One-way analysis of variance performed on day 14 mortality of Soleriopsis invicta maintained in non-sterile or sterile soil exposed to polyethylene glycol-treatrd or untreated alginate pellets 54

,22 One-way analysis of variance performed on day 4 mortality of Solenopsis invicta maintained in non-sterile exposed to diatomaceous earth, dextrose, and rice powder-amended pellets 54

A. 23 One-way analysis of variance performed on day 5 mortality of Solenopsis invicta maintained in non-sterile soil exposed to diatomaceous earth, dextrose, and rice powder-amended pellets 55


A. 25 One-way analysis of variance performed on day 7 mortality of Solenopsis invicta maintained in non-sterile soil exposed to diatomaceou earth, dextrose, and rice powder-amended pellets 56

A. 26 One-way analysis of variance performed on day 8 mortality of Solenopsis invicta maintained in non-sterile soil exposed to diatomaceous earth, dextrose, and rice powder-amended pellet 56

A. 27 One-way analysis of variance performed on day 9 mortality of Solenopsis invicta maintained in non-sterile exposed to diatomaceous earth, dextrose, and rice powder-amended-pellets 57

A. 28 One-way analysis of variance performed on day 10 mortality of Solenopsis invicta maintained in non-sterile exposed to diatomaceous earth, dextrose, and rice powder-amended pellets 57

A. 29 One-way analysis of variance performed on day 11 mortality of Solenopsis invicta maintained in non-sterile exposed to diatomaceous earth, dextrose, and rice powder-amended pellets 58



A. 32 One-way analysis of variance performed on refuse piles counts taken from 7.5g alginate pellet treatments 59

A. 33 One-way analysis of variance performed on surviving Solenopsis invicta floated out of 7.5g alginate pellets treatments 60

A. 34 One-way analysis of variance performed on refuse piles counts taken from 7.5g and 70.Og alginate pellets treatments 60

A. 3 5 One-way analysis of variance performed on surviving Solenopsis invicta floated out of 70.Og alginate pellets treatments 61

A. 36 One-way analysis of variance performed on refuse piles counts taken from 7.5g and 70.Og alginate pellet treatments 61

Vll

A. 37 One-way analysis of variance performed on surviving Solenopsis invicta taken from 7.5g and 70.Og alginate pellet treatments 62

A. 38 One-way analysis of variance performed on live Solenopsis invicta removed weekly from large containers after treatment with diatomaceous earth, dextrose, and rice powder-amended alginate pellets (week two) b2

A. 39 One-way analysis of variance performed on live Solenopsis invicta removed weekly from large containers after treatment with diatomaceous earth, dextrose, and rice powder-amended alginate pellets (week three) 63

A. 40 One-way analysis of variance performed on percent of Solenopsis invict are removed from refuse piles that tested positive for B. Bassiana infection 63

.41 One-way analysis of variance performed on percent of surviving Solenopsis invicta that tested positive foj B. Bassiana infection 64

A. 42 Daily cumulative percent mortality of Solenopsis invicta maintained in non-sterile soil and exposed to Polyethylene glycol-treated or untreated alginate pellets 65

A. 43 Daily cumulative percent mortality of Solenopsis invicta in response to non-sterile soil conditions and polyethylene glycol 66

A. 44 Daily cumulative percent mortality of Solenopsis invicta in response to soil conditions and polyethylene glycol 67

A. 45 Daily cumulative percent mortality of Solenopsis invicta in response to non-sterile soil and diatomaceous earth, dextrose, and rice powder-amended pellets 69

VI11

LIST OF FIGURES

3.1 Scanning electron micrograph of an alginate pellet of B.bassiana showing it to be a matrix of alginate and mycelia

19 3.2 Mean cumulative mortality of Solenopsis invicta after

treatment with PEG-treated or untreated alginated B.bassiana in non-sterile soil 20

3.3 Mean cumulative mortality of Solenopsis invicta maintained in sterile or non-sterile soil after treatment with PEG-treated or non-treated alginated B. bassiana 23

3.4 Mean cumulative mortality of Solenopsis invicta maintained in non-sterile soil after treatment with diatomaceous earth, dextrose, and rice powder-amended B. bassiana 27

3.5 Mean number of Solenopsis invicta removed from refuse piles after treatment with diatomaceous earth, dextrose, and rice powder-amended alginate B. bassiana 3 0

3.6 Mean number of survivors removed from tubs after treatment with diatomaceuos earth, dextrose, and rice powder-amended alginated B. bassiana 31

IX

CHAPTER I

LITERATURE REVIEW

Introduction and Range Expansion of The Red Imported Fire Ant in the United States

Solenopsis invicta Buren (Hymenoptera: Formicidae), an ant

species native to the Matto Grasso area of Brazil, was accidentally

introduced into the United States between 1928 and 1945 (Buren, 1972).

Unimpeded by natural enemies (Quattlebaum, 1980) and unwittingly spread

by shipment of infested nursery stock, the red imported fire ant (RIFA)

rapidly expanded its range (Lofgren, 1986). Within approximately 60

years, Solenopsis invicta has attained major pest status and has

negatively impacted both humans and the environment. The sting of

Solenopsis invicta is not only painful and irritating, but in some

cases, it can be fatal (Rhoades, 1977). Solenopsis invicta is also a

maj.or agricultural pest in the U.S.A. Fire ants are responsible for

damage to a variety of crops such as okra, corn, soybeans, potatoes, and

peanuts (Adams, 1986). Fire ants also damage electrical devices. Air

conditioners, electric meters, signs, lights, and circuit boxes are just

some of the electrical equipment that may be shut down by fire ant

intrusions (Coplin, 1989). Environmentally, fire ants are responsible

for destruction of wildlife and displacement of native ant species

(Lutz, 1992; Camilo & Phillips, 1990; Porter & Savignano, 1990) .

Although invasion by Solenopsis invicta has been challenged with a

succession of insecticides, their numbers and range continue to

increase. This has led many researchers to focus their attention on the

search for biological control agents (Jouvenaz, 1990).

Solenopsis invicta was first detected in Texas in 1953. Although

this infestation was promptly eradicated, a survey in 1957 revealed

infestation of five more counties (Summerlin, 1977). From this modest

beginning, Solenopsis invicta has spread across much of the state,

bringing with it the same problems that were brought to much of the rest

1

of the southern USA. Solenopsis invicta in Texas has been blamed for

death of wildlife (Lutz, 1992; Allen, 1993), for the decimation of

native ant populations (Porter & Savignano, 1990; Camilo & Phillips,

1990), and for the shorting-out of electrical equipment (Vinson &

Mackay, 1990). Cokendolpher and Phillips (1989) documented the rate of

spread of RIFA in Texas and concluded that by the year 2000, 63% of the

total area of the state would be colonized by S. invicta. Ultimately,

the spread of this species into western and southern Texas could enable

Solenopsis invicta to substantially increase its range by infesting New

Mexico, Arizona, California, and Mexico (Allen et al., 1993). The rapid

expansion of Solenopsis invicta along with the problems associated with

their presence makes formulation of new control mechanisms a crucial

need.

Microbial Pathogens of Solenopsis invicta

Microbes account for the majority of reported natural enemies of

Solenopsis invicta (Pereira, 1991); however, their occurrence in the

United States is apparently very low (Beckham et al., 1982). Beckham

(1980) surveyed 3 0 counties in central and eastern Texas and found no

apparent occurrences of RIFA pathogens. Jouvenaz et al. (1977) examined

fire ant populations in South Carolina, Georgia, Florida, Mississippi,

and Louisiana discovered what was thought to be a mildly pathogenic

yeast (later identified as a endozoic mold) and a microsporidian

infecting less than 10% of the colonies. Broome (1974) extensive""y

surveyed fire ant mounds in Mississippi and was unable to find any major

infestations of pathogens. A survey of nearly 1000 RIFA founding queens

collected in College Station, Texas detected the occurrence of two

fungi, Conidiobolus (probably macrosporus) and Metarhizium anisopliae

(Sanchez-Pena, 1992). Both fungi were in very low incidence, and only

one, M. anosopliae, was proven in subsequent laboratory tests to be

pathogenic to Solenopsis invicta.

The absence of any appreciable natural enemies in the United

States led many researchers to begin searching the South American

homeland of Solenopsis invicta for pathogens (Quattlebaum, 1980) . In

contrast to RIFA populations in the United States, fire ants in South

America are beset by a number of naturally occurring pathogens

(Jouvenaz, 1986). In fact, Beckham (1980) stated that it was not

unusual to find some species of ants in South America with up to 25% of

the colonies infected with pathogens. Endemic pathogens including

protozoa, viruses, and fungi have regularly been found infecting RIFA

removed from mounds in Brazil (Allen & Buren, 1974; Jouvenaz, 1986) .

Allen and Buren (1974) discovered masses of microsporidia in the gasters

of S. invicta worker ants. This microsporidium was later identified as

belonging to the genus Thelohanis Henneguy. Knell et al. (1977)

discovered a previously unknown microsporidian, Thelohanis solenopsae,

in samples of S. invicta collected in Brazil. In 1974, Allen and Buren

reviewed the literature concerning fire ant diseases and concluded that

the fungi Metarhizium anisopliae and Beauveria bassiana were important

natural controls in ant populations in South America.

Beauveria bassiana as a Control Agent of Imported Fire Ants

Beauveria bassiana (Fungi: Deuteromycotina) has been demonstrated,

both in the laboratory and under field conditions, to cause considerable

mortality when applied to imported fire ants (IFA).

Laboratory Trials

Broome (1974) studied the susceptibility of adult and larval fire

ants {Solenopsis richteri Forel, the black imported fire ant) to

Beauveria bassiana administered both orally and topically. By feeding

the fungus to ants in a sucrose solution, he attained 90% mortality of

larvae and 67% mortality of adults. Cuticular applications of B.

bassiana conidia to adults and early stage larvae caused much lower

rates of mortality than did oral applications (35% and 72%,

respectively). Stimac et al. (1990) evaluated transmission of B.

bassiana within populations of Solenopsis invicta both in nest soil and

without soil. Treatments consisted of populations of B. bassiana-

infected ants mixed with uninfected ants in laboratory colonies. The

researchers demonstrated that ants infected with B. bassiana were able

to transmit the fungus to uninfected ants in the absence of soil.

However, in the presence of soil this transmission was absent. Stimac

et al. (1993) evaluated three methods of applying B. bassiana to

laboratory colonies of S. invicta. He achieved 70% to 92% mortality by

injection of a powder formulation containing both conidia and

diatomaceous earth into the soil.

Pereira et al.(1993) evaluated the ability of B. bassiana to

infect and kill RIFA workers in both sterile and non-sterile nest soils.

Results of this research showed large differences in effects of sterile

and non-sterile soil on fungal infectivity. A concentration of 10^

conidial/g soil resulted in 90% mortality in sterile soil as compared to

no significant mortality in non-sterile soil.

An isolate of B. bassiana obtained from Atta mexicana (the Mexican

leaf-cutting ant) caused mortality in the laboratory when applied to

RIFAs as both conidia and dried mycelia (Sanchez-Pena, 1992). Fire ant

workers were exposed to conidia by immersion in a conidial suspension

for 10 seconds (concentration between 1x10^ to Ix 10^ conidia/ml).

Using tl.is method, Sanchez-Pena attained a LT g in 3.51 days and a LT,

in 4.5 days. Dried mycelia were added to commercial potting soil and

tested against fire ant founding queens. As little as 0.005% of B.

bassiana mycelia added to soil (w:w) caused 63.3% mortality after 19

days.

4

Field Trials

The first research to demonstrate the ability of B. bassiana to

cause mortality of imported fire ants in the field was that of Broome

(1974). Broome infected S. richteri colonies in Mississippi by feeding

B. bassiana grown on sterile, moist, crushed corn. Mortality of treated

fire ant colonies 22 weeks post-treatment was 35%. In addition to

mortality, population growth in the treated colonies was less than 1%

compared to a population growth of 31% in the untreated colonies.

Quattlebaum (1980) successfully infected fire ant colonies in South

Carolina by feeding B. bassiana-infected Heliothis sp. (Lepidoptera:

Noctuidae) larval cadavers. Mortality of treated colonies ranged from

21.7 to 47.1% as compared to the control mounds where mortality ranged

from 4.3 to 12.3%.

Oi et al. (1994) applied B. bassiana to fire ant mounds in Florida

pastures in one of three ways: (a) rice inoculations, a culture of B.

bassiana grown on 200 grams of cooked rice and applied to the tops of

mounds, (b) injections of conidia powder formulations, and (c)

injections of B. bassiana conidia mixed with diatomaceous earth.

Controls consisted of rice without B. bassiana and diatomaceous earth

without B. bassiana. Rice inoculations resulted in a maximum infection

rate of 55% of live ants sampled. In areas within which mounds were

injected with B. bassiana conidia or B. bassiana and diatomaceous earth

formulations, fire ant foraging was reduced, and foraging by other ant

species increased. These studies clearly demonstrated that B. bassiana

may be a significant biological control agent of S. invicta. However,

the efficacy of B. bassiana as a biological control has traditionally

been limited by the lack of formulation and application technologies

(McCoy, 1990).

Encapsulation of Biocontrol Agents in Alginate Pellets

Churchill (1982) and Lisansky (1985) defined several desirable

characteristics of biocontrol formulations: (1) ease of preparation and

application, (2) stability, (3) adequate shelf life, (4) abundant and

viable propagules, and (5) low cost of production. Alginate

formulations may meet many of these requirements.

Alginate is an easily gelled polysaccharide gum extracted from

seaweed and are widely used in food products, cosmetics, agriculture,

and industry (McNeeley & Pettitt, 1973). Living biological control

organisms such as fungi, bacteria, and nematodes can be safely entrapped

in a nontoxic alginate matrix by a process known as iontrophic gelation

(Connick, 1988). Sodium alginate used as an encapsulating device for

living biological control agents may offer important advantages over

more traditional methods of formulation (Lewis & Papavizas, 1985; Fravel

et al., 1985; Lewis & Papavizas, 1987).

1. Enhancement of shelf life,

2. Uniform pellet size,

3. Biodegradability,

4. Easy preparation using common laboratory equipment,

5. Easy application using conventional agricultural equipment,

6. Environmental stability after application (including resistance to solar radiation),

7. Enhanced conidial production as compared to pure dry mycelia,

8. Combinations and concentrations of ingredients can be adjusted to favor the biological agent in whatever environment it is applied.

The first reported use of sodium alginate to encapsulate a living

biological control agent was to create a mycoherbicide by encapsulating

five different fungi, including a Phyllosticta sp. (Walker & Connick,

1983). All fungi encapsulated in this study sporulated under field

conditions when rehydrated. Since that time, other researchers have

successfully used the alginate process to pelletize and deliver living

biological control agents. Bashan (1986) used alginate to encapsulate

Azospirillum brasilense, a bacterium that aids plant growth, and then

used it to successfully inoculate roots of common wheat plants (Triticum

aestivum cv. Deganit) in the laboratory. Kaya and Nelson (1985)

encapsulated parasitic nematodes (5teinerne/natid and Heterorhabditid)

and fed them to beet armyworm larvae {Spodoptera exigua). These

nematode formulations produced 99% mortality in beet armyworm larvae.

The nematode formulations maintained their population density and

infectivity even after storage for eight months.

Entomopathogenic fungi have also been encapsulated and applied to

target insect pest populations. An isolate of B. bassiana pathogenic to

cereal aphids {Scizaphis grawinum) was alginate-encapsulated (Knudsen et

al., 1990) and caused substantial aphid mortality under laboratory

conditions. After 9-15 days, 3-44% of the aphid population was killed

by B. bassiana versus 0% aphid mortality on wheat where no pellets had

been placed. In addition, pellets had excellent shelf life (Knudsen et

al., 1990). Alginate pellets have been used successfully to combat the

pine wood nematode in Japan (Shimazu, 1992). Pellets containing B.

bassiana were implanted in the trunks of trees infected with the

nematode Monochamus alternatus and the average mortality of nematodes

was 43-45% in treated, standing trees.

Addition of a nutrient source to pellets may increase

proliferation of fungi in soil. Lewis et al. (1987) reported that the

biocontrol fungi Trichodenna viride and T. harzianum proliferated

abundantly in soil when added as pellets composed of mycelia on wheat

bran. This represented the first reported instance of such fungal

proliferation in soil that was not first sterilized (Lewis et al.,

1987). Knudsen et al. (1990) found that the addition of wheat bran to

an alginate formulation of Trichoderma harzianum significantly increased

fungal biomass in soil as compared to formulations without bran. Also,

hyphae from alginate pellets containing bran sporulated more quickly

than those pellets without bran (Knudsen et al., 1990).

Pereira and Roberts (1990) demonstrated that Beauveria bassiana

mycelia treated with a dextrose solution before being dried and stored,

survived storage better and produced significantly more conidia after

storage than did mycelia not treated with dextrose or treated with a

sucrose solution or a sterile water solution. The Beauveria bassiana

mycelial mats were obtained from liquid cultures and treated with one of

three 10% sugar solutions (dextrose, sucrose, or maltose). Control

treatments consisted of either pure deionized water or no treatment.

Mycelia were then stored for 135 days in tightly sealed plastic bags

under refrigeration (22° C) . After this storage period, mycelia v;ere

removed and allowed to sporulate. The results demonstrated that mycelia

treated with either the dextrose or maltose solutions had greater

conidia production than did preparations treated with pure water or not

treated at all.

Although this study did not involve the pelletization of the

fungus, it demonstrated that addition of dextrose to a mycelial

preparation improved sporulation after long-term storage.

Beauveria bassiana alginate pellets treated with a 40% aqueous

polyethylene glycol (PEG) solution produced conidia 48 hours more

quickly than did untreated pellets (Knudsen et al., 1991). Although the

precise manner in which PEG improves speed of sporulation is unknown, it

may operate as an osmo-regulant, controling the rate and amount of water

absorbed by pellets.

Although most alginate pellets are gelled in a calcium chloride

solution, Fravel et al. (1985) compared the survivability of various

biocontrol organisms, including a bacterium and four species of fungi,

by gelling in either a 0.1 M calcium gluconate solution or in a 0.25 M

calcium chloride solution. Encapsulated bacteria and fungi had greater

survivability and viability in alginate pellets gelled in calcium

gluconate than they did when gelled in calcium chloride. Survival of

all the organisms gelled with calcium gluconate after 12 weeks was

significantly greater than those gelled with calcium chloride.

Alginate Formulations and the Nurserv Industry

Early spread of RIFA in the southeastern United States was mainly

caused by shipment of infested nursery stock (Lofgren, 1986). That fact

has led to attempts to quarantine infested shipments.

At the present time, nurseries that wish to ship nursery stock

across quarantine areas must treat the plants in one of five ways

(Imported Fire Ant Quarantine Treatments for Nursery Stock and Other

Regulated Articles. U.S. Dept. of Agric, Supplement. 25th Avenue,

Gulfport, MS 39501. January 1994.

1. Immersion--Soil around the plant must be totally immersed in

an emulsifiable chlorpyrifos solution.

2. Drenching--Plants in containers must be treated to the point

of saturation by a solution of either chlorpyrifos, diazinon,

or bifenthrin. Plants in burlap must be treated with a

chlorpyrifos solution on a twice daily schedule three

consecutive days.

3. Topical Application--This method is approved for the

treatment of nursery stock in 3-4 liter containers only, and

the only approved insecticide for this purpose is bifenthrin

(Talstar, FMC, Philadelphia, PA).

4. Incorporation of granular insecticides--Granular bifenthrin is

incorporated into potting soil media in which containerized

plants are grown.

5. In-field treatments--Based on a sequential application of

fenoxycarb or hydramethylnon bait followed by an application

if granular chlorpyrifos. This combination of treatments is

•J.^^Jm-ltf' «* i^'.^"*'

necessary because the chlorpyrifos may not eliminate large,

mature fire ant colonies.

Although chlorpyrifos is an important treatment of nursery stock

and is, in fact, the only chemical insecticide currently approved for

the immersion method and drenching of balled-and-burlaped plants,

Jouvenaz and Martin (1992) reported that chlorpyrifos may pose worker

safety problems and is expensive.

A treatment that poses little or no environmental hazard, which

can be safely and easily applied, and is cost-effective would be very

helpful to the nursery industry. Entomopathogenic fungi in an alginate

formulation may offer promise in meeting this concern.

Research Objectives

Alginate formulations of Beauveria bassiana have caused mortality

to a wide range of pests. Although finding effective biological control

formulations to combat Solenopsis invicta is important, alginate

formulations have not been tested against them. Therefore,- this

research will attempt to accomplish the following objectives.

1. To evaluate an alginate formulation of Beauveria bassiana to

cause mortality of Solenopsis invicta in the laboratory.

2. To select ingredients that provide the most efficient and

practical alginate formulation possible. Formulations will be

judged in their ability to control fire ant populations in

laboratory trials.

3. To evaluate B. bassiana formulations to control fire ant

populations in nursery-size containers of soil.

10

CHAPTER II

MATERIALS AND METHODS

Growth and Culture of Beauveria bassiana

All experiments were conducted using Beauveria bassiana

(ARSEF#2484), hereafter, BB 2484 originally isolated from workers of the

leaf-cutting ant, Atta mexicana. The strain was collected on the

Pacific Plains of the Mexican state of Sinaloa near El Fuerte in 1986

(Sanchez-Pena, 1990). Prior to our first experiments, the fungus was

reisolated several times from infected Solenopsis invicta. BB 2484 was

maintained on Sabouraud's dextrose agar medium + 1% yeast extract

(SDAY). This fungus was used to inoculate 100 ml of Sabouraud dextrose

broth plus 1% yeast extract (SDBY) in 300 ml flasks. Flasks were then

plugged with cotton and incubated at room temperature (24°C) on a rotary

platform shaker (100 RPM) for 10-14 days. Mycelia were harvested by

straining the liquid culture through white, cotton muslim cloth which

retained the hyphal biomass.

Production of Alginate Pellets

After harvesting the fungal biomass, alginate pellets were

produced utilizing the methods of Knudsen et al. (1990) and modified as

follows. Mycelia were added to a 1% aqueous sodium alginate solution

(2.5 grams of sodium alginate [Bio-Serv, Frenchtown, New Jersey]

dissolved in 10 ml of 95% ethanol in 500 ml of spent SDBY media) at the

rate of 37 g of wet mycelia per 100 ml of sodium alginate solution. To

this suspension, 2 g of wheat bran were added and the resulting mixture

was gently stirred with a magnetic stirrer until the solution was evenly

dispersed. The solution was subsequently blended in an electric blender

(about 25 seconds) in order to break the alginate mycelial particles.

Pellets were produced by adding the mycelium-alginate mixture drop-wise

with a pipette into one liter of a 0.25 M aqueous solution of calcium

chloride (36.8 grams per liter of sterile, reverse osmosis [RO] water).

11

The resulting pellets were removed within five minutes from the solution

with a kitchen sieve and allowed to air-dry on double thickness sheets

of waxed paper for approximately 24 hours. After drying, the pellets

were stored in air-tight plastic vials.

Preparation of Alginate Pellets For Electron Microscopy

Approximately ten alginate pellets were prepared for scanning

electron microscope (SEM) viewing utilizing the following protocol: (a)

primary fixation in a 2% glutaraldehyde solution in a phosphate buffer;

(b) wash in a phosphate buffer for three changes; (c) dehydrate in

successive ethanol series; (d) critical point drying; (e) mount on a

metal stub; and (f) sputter metal coating. After preparation, the

pellets were viewed and photographed.

Treatment of Alginate Pellets with Polyethylene Glycol

Alginate pellets were prepared as previously described. However,

after partial air-drying for approximately 16 hours on double sheets of

waxed paper, units of approximately 100 pellets were placed in 100 ml of

a 40% aqueous solution of polyethylene glycol (40 grams PEG [Spectrum

Chemical MFG. Corp., Gardena, CA] per liter of RO water) in 500-ml

Erlenmeyer flasks and were incubated at 24° C on a rotary, platform

shaker (100 RPM) for approximately 24 hours. PEG treatment of alginate

pellets increases the rate and amount of conidia production in fungi

(Knudsen et al., 1991). Subsequently, pellets were removed from the PEG

solution and allowed to air-dry on a double layer of waxed paper for

approximately 24 hours, then after drying, the pellets were stored in

air-tight vials.

12

Collection and Maintenance of Red Imported Fire Ants

RIFA were collected in June, 1993 from field populations in

Abilene, Taylor County, Texas. Colonies were collected and handled

according to Banks et al. (1981). Ants were normally fed three times

each week, but one day prior to experimentation, colonies were fed extra

quantities. After removal from soil, ants were maintained in the

laboratory in plastic trays, were fed laboratory-reared cockroaches and

canned dog food, and were provided a constant source of water. Colonies

were maintained for approximately two months before use.

Mortality of Solenopsis invicta Maintained in Non-sterile Soil

Ten Solenopsis invicta workers were transferred to separate moist

chambers composed of Petri dishes (85 mm dia) with a single disk of

filter paper (Whatman no. 1), two g of non-sterile potting soil (Perma

Grow organic potting soil, Houston, TX), and approximately 0.25 grams of

either PEG-treated or non-treated alginate pellets containing B.

bassiana. Three ml of sterilized RO water were added to each dish, and

Petri dishes were sealed with laboratory film (Parafilm ®, American

National Can, Greenwich, CT). Ants were maintained in Petri dishes at

25°C. Control treatments consisted of ants in Petri dishes with soil,

but without any alginate pellets. Ten replicates (dishes) of each

treatment were prepared, and the mortality of ants was checked daily.

At the termination of the experiment, daily percent mortality was

calculated. Analysis of Variance (ANOVA) and Fisher's Protected LSD

(PLSD) were used to detect differences in mortality produced in each

treatment for each day

Mortality of Solenopsis invicta Maintained in Sterile or Non-sterile Potting Soil

Moist chambers were assembled as previously described. However, in

order to compare the effects of sterile versus non-sterile soil on the

13

ability of alginate pellets to cause mortality of RIFA, two grams of

either sterile or non-sterile potting soil (Perma Grow organic potting

soil, Houston, TX) and 0.25 grams of PEG-treated or untreated alginate

pellets were added to each of the experimental units. Control

treatments consisted of ants in Petri dishes with soil, but without any

alginate pellets. Ten replicates (dishes) of each treatment were

prepared. Mortality of ants was recorded and analyzed as in the

previous experiments.

Production of Alginate Pellets With Diatomaceous Earth, Dextrose, and Rice Powder

Alginate pellets with B. bassiana were produced as before (pp. 13-

14); however, in an effort to increase the efficacy of the alginate

pellets the following modifications were made: Two grams of rice

powder were added in place of wheat bran. Rice has been included as an

ingredient in Beauveria bassiana formulations applied to field

populations of RIFA by Oi et al.(1994). In addition, 2 g of

diatomaceous earth were also added. Diatomaceous earth is a well known

biological control agent that causes RIFA mortality when included in B.

bassiana formulations (Oi et al. 1994; Stimac et al., 1993). One gram

of dextrose was added to the formulation to provide an easily accessible

nutrient source for B. bassiana. Pereira and Roberts (1990)

demonstrated that mycelial preparations of B. bassiana treated v;ith a

dextrose solution had enhanced conidial production after long-term

storage.

Fravel et al. (1985) demonstrated that fungi in alginate pellets

gelled in calcium gluconate had greater survivability and viability than

did fungi gelled in calcium chloride. Therefore, pellets were gelled in

a 0.1 M calcium gluconate solution (43 grams per liter of sterile, RO

water). Pellets used as control treatments were prepared exactly as

14

described previously; however, shredded filter paper was substituted for

B. bassiana mycelia.

Mortality of Solenopsis invicta Treated With Diatomaceous Earth. Dextrose, and

Rice Powder-Amended Alginate Pellets With B. bassiana

Moist chambers were constructed exactly as in the previous

experiments. Treatments were replicated ten times and consisted of one

B. bassiana treatment and two controls: (a) pellets containing shredded

filter paper in place of the B. bassiana, and (b) ants in Petri dishes

with soil, but without any alginate pellets. Mortality of ants was

recorded and analyzed as in the previous experiments.

Pellet-Induced Mortality of Small RIFA Colonies in Large Containers

For this experiment, RIFAs were collected in March 1994, from

Abilene, Taylor County, Texas. The RIFA were separated from soil

utilizing the methods of Markin (1968) modified as follows. Field

colonies were collected in the field in 19 liter plastic buckets and

returned to the laboratory. Mound soil containing ants from each bucket

was spread in a layer over the bottom of an escape-proof, table top and

allowed to dry. As the soil dried, ants entered a moist chamber,

comprised of a plastic box v/ith a water-moistened, plaster-of-paris

(Humco Laboratory, Texarkana, TX) bottom. To facilitate ant exodus from

soil, colony queens were located and placed into the plastic box. In

addition, a supply of sugar water was provided in the chamber, and after

the majority of the ants had entered the box, ants were removed and

placed in plastic trays.

After ants had been forced from soil, dried soil was gathered from

the drying table and sieved to remove ant cadavers. To retain the mound

soil to as nearly as original field conditions as possible, all organic

15

material (grass, sticks, leaves, etc.) and inorganic material (such as,

rocks) were re-introduced to the soil after removal of ant cadavers.

Approximately 2000 worker ants and two queens were placed in each

37.9 liter plastic tub containing 17.0 kilograms of mound soil. Prior

to addition of the ants, mound soil was rehydrated with 3 1 of RO water.

The upper edges of tubs were lined with Fluon (Northern Products, Inc.,

Woonsocket, RI) to provide a slippery barrier to prevent ants from

escaping. Ants were allowed ten days to construct galleries in the soil

before the mortality experiments were begun.

Experimental treatments consisted of two control treatments and

two B. bassiana treatments. Fungal treatments consisted of the alginate

pellets containing B. bassiana. The surfaces of each experimental unit

were sprinkled with either 7.5 or 70.0 grams of one of the pellet

treatments. Treatments were randomly assigned to each experimental unit

(tub), and each treatment was replicated five times. Controls were: (a)

alginate pellets containing shredded filter paper in the place of the

fungus, and (b) no pellets. During the course of the experiment, RIFAs

were fed laboratory-reared cockroaches three times each week, and a

sugar-water solution (10% glucose) was available at all times. RO water

was added to container soil when it appeared almost dry to maintain a

slightly moist condition. Air humidity in the laboratory was maintained

between 50 and 60% during the experiment, and laboratory temperature

ranged between 24 and 26° C. Once each week for the duration of the

experiment, ten worker ants were removed from each experimental unit,

surface-sterilized with 30% hydrogen peroxide for several minutes and

were placed on potato dextrose agar in Petri dishes to test for B.

bassiana infection.

Ten days after introduction of the pellets to the soil, ant

refuse-pile cadavers were removed and counted. Twenty percent of these

cadavers were plated on potato dextrose agar to test for B. bassiana

infection. Twenty-one days after the experiment was begun, ants were

16

floated from soil in each tub, and surviving ants were counted. Ten

percent of these survivors were surface-sterilized, with 30% hydrogen

peroxide and plated on potato dextrose agar to test for B. bassiana

infection.

17

CHAPTER III

RESULTS

Electron Microscopy of Alginate Pellets

Viewed under the SEM, alginate pellets containing B. bassaiana

appeared as a mixture of mycelia and alginate forming a sodium alginate-

B. bassiana matrix (Figure 3.1).

Mortality of Solenopsis invicta Maintained in Non-Sterile Soil

Daily cumulative mortality of ants exposed to alginate pellets is

shown in Figure 3.2. PEG-treated alginate pellets produced conidia

faster than did pellets not treated with PEG (personal observation). In

non-sterile soil, PEG-treated pellets caused significantly greater

mortality than non-PEG-treated pellets beginning on day 5 which

continued throughout the remainder of the experiment (Tables 3.1,and

A.l-A.ll). Subsequently, L,T^Q was reached in 12 days in the PEG-treated

pellet dishes versus 14 days in the un-treated pellets dishes. In the

control treatments RIFA mortality reached 26% at the termination of the

experiment. Control mortality was assumed to be the result of natural

causes including injury and age.

Mortality of Solenopsis invicta Maintained in Sterile or Non-Sterile Potting Soil

The progression of mortality of fire ants exposed to alginated B.

bassiana is shown in Figure 3.3. In sterile soil, LT50 of ants was

reached more quickly for both PEG-treated and untreated pellets than in

non-sterile soil. In both soils PEG-treated pellets produced 50%

mortality of fire ants faster than did pellets not treated with PEG. On

day 5 when the first day mortality occurred, there were significant

differences in percent mortality between ants in non-sterile and sterile

soil environments (Table 3.2 and A.12-A.21), with greater

18

Figure 3.1 Scanning electron micrograph of an alginate pellet of B. bassiana showing it to be a matrix of alginate and mycelia measuring bar = .30 mm

19

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15 20

Figure 3.2 Mean cumulative percent mortality of Solenopsis invicta after treatment with polyethylene glycol-treated or untreated alginate pellets in non-sterile or sterile soil

20

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14 16

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23

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mortality occurring in the sterile soil. This significantly greater

mortality persisted throughout the remainder of the experiment. In both

sterile and non-sterile soil, sodium alginate pellets produced greater

mortality than occurred in the controls (Table 3.2).

Mortality of Solenopsis invicta Treated With Diatomaceous Earth, Dextrose, and Rice

Powder-Amended Alginate Pellets With B. bassiana

The progression of mortality of fire ants exposed to diatomaceous

earth, dextrose, and rice powder-amended alginate pellets is shown in

Figure 3.4. These pellets produced a LT50 of fire ants in 10 days

(Figure 3.4). By day 5 the alginate pellets containing B. bassiana

caused significantly greater mortality of ants than did either of the

control treatments. This difference in mortality persisted throughout

the remainder of the experiment (Table 3.3 and tables A.22-A.31).

Pellet Induced Mortality of Small RIFA Colonies in Large Containers

Results of these experiments are shown in Figure 3.5 and 3.6.

There was significant differences in both refuse pile counts and ending

survivor counts for the fungal pellet treatments and control treatments

for both experiments (Tables 3.4 and A.32-A.35).

Significant differences were also detected between the 7.5 g

pellet treatment and 70.0 g pellet treatment, both for the control

pellets and the fungal pellets (Table 3.4). The 70.0 g control pellet

treatment containing diatomaceous earth, dextrose, and rice powder with

shredded filter paper in place of the B. bassiana caused mortality that

was not significantly greater than that caused by the 7.5 g fungal

treatments (Tables 3.5 and A.36-A.37).

26

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Days Post-Treatment

Figure 3.4 Mean cumulative mortality of Solenopsis invicta maintained in non-sterile soil after treatment with diatomaceous earth, dextrose, and rice powder-amended alginated Beauveria bassiana

27

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Fungal

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Treatment

Figure 3.5 Mean number (± SEM)of surviving Solenopsis invicta removed from colonies after treatment with diatomaceous earth, dextrose, and rice powder-amended alginate pellets

QJ Xi

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31

Table 3.4. Mean number of Solenopsis invicta removed from refuse piles and mean number of survivors after exposure to diatomaceous earth, dextrose, and rice powder-amended alginate pellets

Treatment

B. bassiana

Pellets(paper)

No Pellets A&B

Mean Bone-pile Counts^ 7.5 grams 70.0 grams

Mean Survivor Counts'" 7 . 5 grams 7 0.0 grams

357.0a

121.4b

105.6b

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375.6b

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1807.0b

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1440.2b

1860.0c

Mean = Total number of Solenopsis invicta removed divided by number of replications (r=5). Mean = Total number of survivors divided by number of replications (r=5). ANOVA Protected LSD: Means followed by different letters within columns are significantly different (P < 0.05).

Table 3.5. Comparison among treatments means number from refuse piles and mean niimber of survivors after exposure to diatomaceous earth, dextrose, and rice powder-amended alginate pellets

Treatment Mean refuse piles Count^

B. bassiana (7.5 grams) 357.0 a

B. bassiana (70.0 g) 855.4 b

Control Pellets (7.5 g) 121.4 c

Control Pellets (70.0 g) 375.6 a

No Pellets A 105.6 c

No Pellets B 119.2 c

Mean Survivor Count"

1427.0 c

1036.6 d

1670.4 b

1439.0 c

1807.0 ab

1860.0 a

Mean = Total number of Solenopsis invicta removed divided by number of replications 15) . Mean = Total number of survivors divided by number of replications ( 5 > • • . .

ANOVA Protected LSD: Means followed by different letters withm columns are significantly different (£ < 0.05).

32

Bioassay of Plated Ants

The percentage of live ants, removed weekly, testing positive for

B. bassiana infection are shown in Table 3.6. For week one, no positive

signs of infection were found for any of the ants removed. By week two,

a small percentage of live ants removed from the alginated B. bassiana-

treated tubs tested positive for fungal infection. The percentage of

ants testing positive for infection was directly correlated with the

amount of alginated B. bassiana applied, with a greater percentage of

positives found in the 70.Og treatments than in the 7.5g treatments

(Tables 3.6 and A.38, A.39). At no time were ants in either of the

control treatments found to be positive for B. bassiana infection.

The percentage of dead ants removed from refuse-piles testing

positive for fungal infection is shown in Tables 3.7. and A.40 The only

positive B. bassiana infections were detected in ants removed from B.

bassiana pellets treatments. There was a positive correlation between

the amounts of alginated B. bassiana added and number of infected ants

(Tables 3.7 and A.4), with a greater percentage of ants testing positive

for infection taken from the 70.0 g fungal treatments.

The percentage of surviving ants that were floated out at the

termination of the experiment are shown in Tables 3.8. and A.42. The

only positive B. bassiana infections were detected in the B. bassiana

pellet treatments. There was a positive correlation between amount of

B. bassiana added and number of ants testing positive for fungal

infection (Table 3.8), with a greater percentage of ants testing

positive for infection taken from the 70.0 g fungal treatments.

33

Table 3.6 Percent of live Solenopsis invicta removed weekly that tested positive for Beauveria bassiana infection

Treatment^

Week Number

B. bassiana Pellets 7.5grams 70.Ograms

0 .0 0 .0

5 . 0 a 1 2 . 5 b

1 5 . 0 a 3 0 . 0 b

Non B. bassiana Pellets 7. 5grams 70.Ograms

0.0 0.0

0.0c 0.0c

0.0c 0.0c

Control A B

0.0 0.0

0.0c 0.0c

0.0c 0.0c

n = 50 per replication per week Protected LSD: Means followed by different letters within columns are significantly different (P < 0.05).

34

Table 3.7. Percent of ants removed from refuse-piles that tested positive for B. bassiana infection

Treatment No. of Ants^ Percent Positive

Fungal Pellets (7.5) 356 52.0b

Control Pellets (7.5) 100 0.0c

Control (A) 104 0.0c

Fungal Pellets (70. 852 59.46a

Control Pellets (70.0) 372 O.c

Control (B) 96 0.0c

\ 20% of bone-pile cadavers Protected LSD: Means fol. are significantly different (P < 0.05) ^ Protected LSD: Means followed by different letters within columns

35

Table 3.8. Mean percent of surviving Solenopsis invicta that tested positive for Beauveria bassiana infection

Treatment No. of Ants^ Percent Positive"

Fungal Pellets (7.5) 712 13.0a

Control Pellets (7.5) 833 O.Ob

Control A 900 O.Ob

Fungal Pellets (70.0) 517 21.8c

Control Pellets (70.0) 717 O.Ob

Control B 928 O.Ob

^ 10% of surviving Solenopsis invicta were plated ^ Protected LSD: Means followed by different letters within columns are significantly different (£ < 0.05).

36

CHAPTER IV

SUMMARY AND CONCLUSIONS

This research provides the first evidence that alginated B.

bassiana can cause substantial mortality of the RIFA in the laboratory.

This research also provides proof that ingredients in an alginate

formulation can be manipulated to increase the rate of mortality among

colonies of the RIFA and strongly suggests that future research should

focus on combinations of ingredients that could improve the efficacy of

the alginated B. bassiana even further.

The use of PEG-treatment to increase the rate of Solenopsis

invicta mortality was also demonstrated in this study. When tested in

both sterile and non-sterile soil, PEG-treated pellets were observed to

sporulate sooner (personal observation) , and subsequently to cause

mortality more quickly, than alginate pellets not treated with PEG.

This supports the observations of increased rate of sporulation reported

by Knudsen et al. (1991). Because PEG treatment increases the amount

and speed of sporulation from the pelletized matrix, it should be part

of any future formulations.

The soil antagonism documented in other studies (Stimac et al.,

1990; Pereira et al., 1993) was also observed in the second experiment

described in this thesis. The major limiting factor in the use of B.

bassiana for controlling RIFA populations seems to be inhibition from

soil microbes or through fungistatic compounds.

Because past studies have demonstrated that other living

biological control mechanisms such as nematodes and bacteria can be

successfully encapsulated in a sodium alginate matrix (Shimazu, 1992;

Bashan, 1986; Kaya & Nelson, 1985), it might be interesting to combine

several different biological control agents in one alginate pellet. In

this way combinations could be manipulated in such a way that one

37

organism might make up for the weaknesses of other organisms and vice

versa.

In an effort to stop or at least slow the spread of the RIFA, a

quarantine of nursery stock has been initiated. Because most

researchers now think that eradication of the RIFA is impossible, this

quarantine remciins the single best weapon we have to combat the spread

of the RIFA. Although a major part of the quarantine calls for

treatment of infested nursery stock with the chemical pesticide

chlorpyrifos, some researchers have called its safety into cjuestion

(Jouvenaz & Martin, 1992). Given the questions raised about the safety

of chlorpyrifos and other chemical pesticides and, the importance of

enforcing the quarantine, it seems safe to assume that more attention

and interest will be focused on safer, more effective, and less

expensive biological control agents. Benefits of sodium alginate

formulation include the ability to combine numerous ingredients in one

application, safety, increased storage life, and ease of preparation and

application. These benefits combined with concerns about the future of

chemical insecticides should lead to greater interest in the use of B.

bassiana in an alginated pellets form.

38

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39

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Jouvenaz, D. P., Allen, G. E., Banks, W. A., & Wojcik, D. P. (1977). A survey for pathogens of fire ants, Solenopsis invicta spp., in the southeastern United States. Florida Entomologist. 60: 275-279.

Jouvenaz, D. P., Lofgren, C. S., & Banks, W. A. (1981). Biological control of imported fire ants: A review of current knowledge- The Bulletin of the Entomological Society of America. 27: 203-208.

Jouvenaz, D. P. (1986). Disease of fire ants: Problems and opportunities, pp. 327 338. in C. S. Lofgren & R. K. Vander Meer [eds.] Fire ants and leaf-cutting ants: Biology and management. Boulder, Colorado: Westview Press.

Jouvenaz, D. P. (1990). Approaches to biological control in the United States (pp. 620-627). In R. K. Vander Meer, K. Jaffe, & A. Cedeno [eds.]. Applied mvrmecology a world perspective. Boulder, Colorado: Westview Press.

Jouvenaz, D. P, & Martin, W. R. (1992). Evaluation of the nematode Steinernema carpocapsae to control fire ants in nursery stock. Florida Entomolocrv. 75: 148-151.

Jouvenaz, D. P. (1992). Natural enemies of fire ants. Fla. Entomol.. 66: 111-121. Kaya, H. K., & Nelson, C. E. (1985). Encapsulation of Steinerematid and Heterorhabditid nematodes with calcium alginate: A new approach for insect control and other applications. Environmental Entomology. lA: 512-514.

Knell, J. D., Allen, G. E., & Hazard, E. I. (1977). Liynt and electron microscope study of Thelohania solenopsae n. sp. (MicrcDsporidia: Protozoa) in the red imported fire ant, Solenopsis invicta. Journal Invertebrate Pathology. 29: 192-200.

Knudsen, G. R. , Johnson, J. B., & Eschen, D. J. (1990). Alginate pellet formulation of a Beauveria bassiana (Fungi: Hyphomycetes) isolate pathogenic to cereal aphids. J. Econ. Entomol.. £3.: 2225-2228.

Knudsen, G. R., & Bin, L. (1990). Effects of temperature, soil moisture, and wheat bran on growth of Trichoderma harzianum from alginate pellets. Phytopathology. M : 724-727.

Knudsen, G. R., Eschen, D. J., Dandurand, L. M., & Wang, Z. G. (1991). Method to enhance growth and sporulation of pelletized biocontrol fungi. Applied and Environmental Microbiology. 51: 2864-2867.

40

Lewis, J. A., & Papavizas, G. C. (1985). Characteristics of alginate pellets formulated with Trichoderma and Gliocladium and their effect on the proliferation of the fungi in soil. Plant Pathology. 34: 571-577.

Lewis, J. A., & Papavizas, G. C. (1987). Application of Trichoderma and Giliocladium in alginate pellets for control of Rhizoctonia damping-off. Plant Pathology. 36: 438-446.

Lisansky, S. G. (1985). Production and commercialization of pathogens, pp. 210-218. In N. W. Hussey & N. Scopes [eds.] Biological Pest Control. Poole, England : Blandford Press.

Lofgren. C. S., Banks, W. A., & Glancey, B. M. (1975). Biology and control of imported fire ants. Annu. Rev. Entmol.. 20: 1-30.

Lofgren, C. S. (1986). History of imported fire ants in the United States (pp. 36-47), in C. S. Lofgren and R. K. Vander Meer, [eds.] Fire ants and leaf-cutting ants biolocry and management, Boulder, Colorado: Westview Press.

Lutz, Markin, G. P. (1968). Handling techniques for large quantities of ants. Journal of Economic Entomology. 61: 1744-1746.

McCoy, C. W. (1990). Entomogenous fungi as microbial pesticides (pp. 139-159) in New directions in biological control: Alternatives for suppressing agricultural pests and diseases. Alan R. Liss, Inc.

McNeeley, W. H., & Pettitt, D. J. (1973). In R. L. Whistler [ed.] Industrial Gums.. New York, New York: Academic Press.

Oi, D. H., Pereira, R. M., Stimac, J. L., & Wood, L. A. (1994). Field applications of Beauveria bassiana for control of the red imported fire ant (Hymenoptera: Formicidae) . J. Econ. Entomol. . Sl_: 623-630.

Pereira, R. M., & Roberts, D. W. (1990). Dry mycelium preparations of entomopathogenic fungi, Metarhizium anisopliae and Beauveria bassiana. Journal of Invertabrate Pathology.. M : 39-46.

Pereira, R. M. (1991). Evaluation of the entomopathogenic fungus Beauveria bassiana on the red imported fire ant, Solenopsis invicta. Ph.D. dissertation. University of Florida, Gainesville, Florida.

Pereira, R. M., & Roberts, D. W. (1991). Alginate and cornstarch mycelial formulations of entomological fungi, Beauveria bassiana and Metarhizium anisopliae. J. Econ. Entomol.. M : 1657-1661.

Pereira, R. M., & Stimac, J. L. (1992). Transmission of Beauveria bassiana within artificial nests of Solenopsis invicta (Hymenoptera: Formicidae) in the laboratory. Environmental Entomology, 21: 1427-1432.

Pereira, R. M. , Stimac, J. L., & Alves, S. B. (1993). Soil antagonism affecting the dose-response of workers of the red imported fire ant, Solenopsi invicta to Beauveria bassiana conidia. Journal of Invertebrate Pathology. 61: 156-161.

41

Porter, S. D., & Savignano, D. A. (1990). Invasion of polygyne fire ants decimates native ants and disrupts arthropod community. Ecolocjv. 71: 2095-2106.

Quattlebaum, E. C. (1980). Evaluation of fungal and nematode pathogens to control the red imported fire ant, Solenopsis invicta Buren. Ph.D. dissertation, Clemson University, Clemson, SC.

Rhodes, R. B. (1977) . Medical aspects of the imported fire ants. Gainesville, Florida: University Presses of Florida.

Sanchez-Pena, S. (1992). Entomopathogenic fungi against red imported fire ant, Solenopsis invicta Buren (Hymenoptera: Formicidae). M. S. thesis, Texas Tech, Lubbock, Texas.

Shimazu, M. , Kushida, T., Tsuchiya, D. and Mitsuhashi, W. (1992). Microbial control of Monochamus alternatus Hope (Coleoptera: Cerambycidae) by implanting wheat-bran pellets with Beauveria bassiana in infested tree trunks. J. Jon. For. Soc.. 74: (4) 325-330.

Stimac, J. L. , Pereira, R. M., Alves, S. B., & Wood, L. A. (1990). Field evaluation of a Brazilian strain of Beauveria bassiana for control of the red imported fire ant. Solenopsis invicta, in Florida. Proc. 5th Int. Collog, Invertebr. Pathol,, p. 337 (abstr.)

Stimac, J. L. , Pereira, R. M., Alves, S. B., & Wood, L. A. (1993). ' Beauveria bassiana (Balsamo) Vuillemin (Deuteromycetes) applied to laboratory colonies of Solenopsis invicta Buren (Hymenoptera: Formicidae) in soil. Journal of Economic Entomology. 86: (2) 348-352.

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42

APPENDIX

43

Table A.1 One-way analysis of variance performed on day 5 mortality of Solenopsis invicta maintained in non-sterile soil exposed to polyethylene-treated or untreated alginate pellets.

Source

Between Treatment

Error

Total

LSD Value = t, </2

Sum of Sauares

106.67

240.00

346.67

n/ ' = 2 .742

Deg. of Freedom

2

27

Mean Sauares

53.33

8.99

F-Ratio

6.00

Prob > F

0.007

Table A.2. One-way analysis of variance performed on day 6 mortality of Solenopsis invicta maintained in non-sterile soil exposed to polyethylene glycol-treated or untreated alginate pellets.

Source Sum of

Squares Deg. of Freedom

Mean Squares F-Ratio Prob > F

Between Treatment

Error

346.67

320.00

2

27

173.33

11.85

14.63 <0.001

Total 666.67

LSD Value =3 .148

29

44

Table A.3 One-way analysis of variance performed on day 7 mortality of Solenopsis invicta maintained in non-sterile soil exposed to polyethylene glycol-treated or untreated alginate pellets.

Source Between Treatment

Error

Sum of Squares

Deg. of Freedom

Mean Squares F-Ratio Prob > F

560.00

760.00

2

27

280.00

28.15

9.95 <0.001

Total 1320.00 29

LSD Value = 4.852

Table A 4. One-way analysis of variance performed on day 8 mortality of Solenopsis invicta maintained in non-sterile soil exposed to polyethylene glycol-treated or untreated alginate pellets.

Source

Between Treatment

Error

Total

Sum of Squares

986.67

960.00

1946.67

Deg. of Freedom

2

27

29

Mean Squares

493.33

35.56

F-Ratio

13.88

Prob > F

<0.001

LSD Value = 5.457

45

Table A.5. One-way analysis of variance performed on day 9 mortality of Solenopsis invicta maintained in non-sterile soil exposed to polyethylene glycol-treated or untreated alginate pellets

Source Sum of

Squares

Between Treatment

Error

1680.00

1040-00

Total 2720.00

LSD Value = 5.676

Deg. of Mean Freedom Squares F-Ratio Prob > F

2

27

29

840.00

38.52

21.81 <0.001

Table A. 6. One-way analysis of variance performed on day 10 mortality of Solenopsis invicta maintained in non-sterile soil exposed to polyethylene glycol-treated or untreated alginate pellets.

Source Sum of

Squares Deg., of Mean Freedom Squares F -Ratio Prob > F

Between Treatment

Error

2000.00

2000.00

2

27

1000.00

74.07

13.50 <0.001

Total

LSD Value = 3.360

4000.00 29

46

Table A.7. One-way analysis of variance performed on day 11 mortality of Solenopsis invicta maintained in non-sterile soil exposed to polyethylene glycol-treated or untreated alginate pellets

Source

Between Treatment

Error

Total

Sum of Squares

2906.67

19600.00

24866.67

Deg. of Freedom

2

27

29

Mean Squares

1453.33

72.59

F -Ratio

20.02

Prob > F

<0.001

LSD Value = 4.092

Table A 8. One-way analysis of variance performed on day 12 mortality Of Solenopsis invicta maintained in non-sterile soil exposed to polyethylene glycol-treated or untreated alginate pellets.

• ni] rce

Between Treatment

Error

T o t P> 1

Sum of Squares

5488.67

2930.00

8418.67

Deg. of Freedom

2

27

29

Mean Squares

2743.33

108.52

F -Ratio

25.28

Prob > F

<0-001

LSD Value = 4 . 598

47


Source Sum of Deg. of Mean

Squares Freedom Squares F -Ratio Prob > F

Between Treatment

Error

6206.67

2890.00

2

27

3103.33

107.04

28.99 <0.001

Total 9096.67

LSD Value = 4.924

29

Table A.IO.

Source

One-way analysis of variance performed on day 14 mortality of Solenopsis invicta maintained in non-sterile soil exposed to polyethylene glycol-treated or untreated alginate pellets.

Sum of Squares

Deg. of Mean Freedom Squares F -Ratio Prob > F

Between Treatment

Error

Total

6206.67

2130.00

8336.67

LSD Value = 5.736

2

27

3103.33

78.89

39.34 <0.001

29

48



Squares Freedom Sauares F -Ratio Prob > F

Between Treatment

Error

8886.67

1660.00

2

27

4443.33

61.48

Total 10546.67

72.27 >0.001

29

LSD Value = 7.170

Table A.12 One-way analysis of variance performed on day 5 mortality of Solenopsis invicta maintained in non-sterile or sterile soil exposed to polyethylene glycol-treated or untreated alginate pellets.


Squares Freedom Sauares F-Ratio Prob > F

Between Treatment 3333.33 666.67 11.61 >0.001

Error 3100.00 54 57 .41

Total 6433.33 59

LSD Value = 6.777

49

Table A.13. One-way analysis of variance performed on day 6 mortality of Solenopsis invicta maintained in non-sterile or sterile soil exposed to polyethylene glycol-treated or untreated alginate pellets.

Source

Between Treatment

Error

Total

LSD Value = -- 5.

Sum of Sauares

22555.00

1930.00

24485.00

.470

Deg. of Freedom

5

54

59

Mean Sauares

4511.00

35.74

F-Ratio

126.21

Prob > F

<0.001

Table A. 14. One-way analysis of variance performed on day 7 mortality of Solenopsis invicta maintained in non-sterile soil exposed to polyethylene glycol-treated or untreated alginate pellets.


Sauares Freedom Sauares F-Ratio Prob > F

Between Treatment

Error

19733.33

3540.00

5

54

3946.67

65.56

60.20 <0.001

Total 23273.33 59

LSD Value = 6.939

50


Source Sum of

Sauares Deg. of Mean Freedom Sauares F-Ratio Prob > F

Between Treatment

Error

Total

.

28520.00

3820.00

32340.00

5

54

59

5704.00

70.74

80.63 <0.001

LSD Value = 7.523

Table A. 16 One-way analysis of variance performed on day 9 mortality of Solenopsis invicta maintained in non-sterile soil exposed to polyethlene glycol-treated or untreated alginate pellets.

Source

Between Treatment

Error

Total

Sum of Sauares

27235.00

2890.00

30125.00

Deg. of Freedom

5

54

59

Mean Sauares

5447.00

53.52

F-Ratio

101.78

Prob > F

<0.001

LSD Value = 6.543

51

Table A.17. One-way analysis of variance performed on day 10 mortality of Solenopsis invicta maintained in non-sterile soil

exposed to polyethylene glycol-treated or untreated alginate pellets.

Source

Between Treatment

Error

Total

Sum of Sauares

53792.33

3218.40

57010.73

Deg. of Freedom

5

54

59

Mean Sauares

10758.47

59.60

F-Ratio

180.51

Prob > F

<0.001

LSD Value = 6.905


Source

Between Treatment

Error

Total

Sum of Sauares

36873.33

1900.00

38773.33

Deg. of Freedom

5

54

59

Mean Squares

7374.67

35.19

F-Ratio

209.60

Prob > F

<0.001

LSD Value = 5.470

52


Source

Between Treatment

Error

Total

Sum of Sauares

48593.33

4300.00

52893.33

Deg. of Freedom

5

54

59

Mean Sauares

9718.67

79.63

F-Ratio

122.05

Prob > F

<0.001

LSD Value = 5.121


Source

Betweem Treatment

Error

Total

Sum of Sauares

49608.33

2010.00

51618.33

Deg. of Freedom

5

54

59

Mean Sauares

9921.67

37.22

F-Ratio

266.55

Prob > F

<0.001

LSD Value = 5.457

53

Table A.21. One-way analysis of variance performed on day 14 mortality of Solenopsis invicta maintained in non-sterile soil exposed to polyethylene glycol-treated or untreated

alginate pellets.

Source

Between Treatment

Error

Total

Sum of Sauares

52620.00

1920.00

54540.00

LSD Value = 5.361

Deg. of Mean Freedom Sauarf^s

5 10524.00

54 35.56

59

F-Ratio Prob > F

295.99 <0.001

Table A.22 One-way analysis of variance performed on day 4 mortality of Solenopsis invicta maintained in non-sterile soil exposed to diatomaceous earth, dextrose,and rice powder-amended alginate pellets.

Deg. of Source

Mean Squares Freedom Sauares F-Ratio Prob > F

Between Treatment

Error

20.00

250.00

2

27

10.00

9.26

1.08 0.354

Total 270.00

LSD Value = 20783

29

54

Table A.23 One-way analysis of variance performed on day 5 mortality of Solenopsis invicta maintained in non-sterile soil exposed to diatomaceous earth, dextrose, and rice powder-amended alginate pellets.



Between Treatment

Error

Total

486.67

580.00

1066.67

LSD Value = 4.239

2

27

243.33

21.48

11.33 <0.001

29


Source Sum of

Sauares Deg. of Freedom

Mean Sauares F-Ratio Prob > F

Between Treatment

Error

Total

8 2 6 .

5 2 0 .

1 3 4 6 .

.67

.00

,67

2

27

29

4 1 3 ,

19,

. 33

. 26

21.46 <0.001

LSD Value = 4.014

55

Table A.25. One-way analysis of variance performed on day 7 mortality of Solenopsis invicta maintained in non-sterile soil exposed to diatomaceous earth, dextrose, and rice powder-amended alginate pellets.

Source

Between Treacment

Error

Total

Sum of Sauares

2580.00

500.00

3080.00

LSD Value = 3.936

Deg. of Freedom

2

27

29

Mean Sauares

1290.00

18.52

F-Ratio Prob > F

69.66 >0.001


Source Sum of Deg. of Mean Sauares Freedom Squares F-Ratio Prob > F

Between Treatment

Error

Total

5286.

1300.

6586.

,67

.00

.67

2

27

29

2643.

48,

.33

.15

54.90 <0.001

LSD Value = 6.346

56


Source

Between Treatment

Error

Total

Sum of Sauares

11060.00

1420.00

12480.00

LSD Value = 6.632

Deg. of Mean Freedom Sauares

2

27

29

F-Ratio Prob > F

5530.00

52.59

105.15 :0.001




Between Treatment

Error

Total

11540.00

1210.00

12750.00

2

27

5770.00

44.81

128.75 <0.00

29

LSD Value - 10.377

57


Source Sum of Deg. of Mean Sauares Freedom Sauares F-Ratio Prob > F

Between Treatment

Error

16086.67

3300.00

2

27

8043.33

122.22

65.81 <0.001

Total 19386.67 29

LSD Value = 10.196

Table A.30. One-way analysis of variance performed on day 12 mortality of Solenopsis invicza maintained in non-sterile soil exposed to diatomaceous earth, dextrose, and rice powder-amended alginate pellets.

Source

Between Treatment

Error

Total

Sum of Sauares

21740.00

2730.00

24470-00

Deg. of Freedom

2

27

2

Mean Sauares

1087.00

101.11

F-Ratio

107.51

Prob > F

<0.001

LSD Value = 9.191

58

Table A.31. One-way analysis of variance performed on day 13 mortality of Solenopsis invicta maintained in non-sterile soil exposed to diatomaceous earth, dextrose, and rice powder-amended alginate pellets.

Source

Between Treatment

Error

Total

Sum of Sauares

31820.00

1410.00

33230.00

Deg. of Freedom

2

27

29

Mean Sauares

15910.00

52.22

F-Ratio

304.66

Prob > F

<0.001

LSD Value = 6.609

Table A.32. One-way analysis of variance performed on refuse pile counts taken from 7. 5g alginate pellet treatments.

Source

Between Treatment

Error

Total

Sum of Sauares

198264.93

109596.40

307861.33

Deg. of Freedom

2

12

14

Mean Sauares

99132.46

9133.03

F-Ratio

10.85

P>F

0.002

LSD= 129.646

59

Table A.33. One-way analysis of variance performed on surviving Solenopsis invicta from 7.5g alginate pellets treatments

Sum of Deg. of Mean ^^^^^^ Squares Freedom Squares F- Ratio Prob>F

Between Treatment 370505.20 2 185252.60 11.16 0.002

Error 199113.20 12 16592.77

Total 569618.40 14

LSD= 174.750

Table A.34. One-way analysis of variance performed on refuse pile counts taken from 70.0 g alginate pellets treatments.

Sum of Deg. of Mean Source Sauares Freedom Sauares F-Ratio Prob>F

Between Treatment 1396565.73 2 698282.87 11.16 <0.001

Error 194575.20 12 16214-60

Total 1591140.93 14

LSD= 172.747

60

Table A.35. One-way analysis of variance performed on surviving Solenopsis invicta from 70.Og alginate pellet treatments

Source

Between Treatment

Error

Total

LSD= 123.841

Sum of Sauares

1698504.93

99998.80

1798503.73

Deg. of Freedom

Mean Sauares F-Ratio Prob >F

2

12

14

849252.47 101.91

8333.23

<0.001

Table A.36. One-way analysis of variance performed on refuse pile counts taken from 7.5g and 70.Og alginate pellet treatments.

Source Sum of Deg. of Mean Sauares Freedom Sauares F-Ratio P>F

Between Treatment

Error

2084049.37

304171.60

5 416809.87

24 12673.82

32.89 <0.001

Total

LSD= 152.725

2388220-97 29

61

Table A.37. One-way analysis of variance performed on surviving Solenopsis invicta taken from 7.5g and 70.Og alginate pellet treatments.

Source

Between Treatment

Error

Total

LSD= 152.725

Sum of Deg. of Mean Squares Freedom Squares F-Ratio

2636863.47

P>F

2337936.27 5

298927.20 24

29

416809.87 32.89

12673.82

<0.001

Table. A.38. One-way analysis of variance performed on live Solenopsis invicta removed weekly from large containers after treatment with diatomaceous earth, dextrose, and rice powder-amended alginate pellets (week two)

Source Sum of

Sauares Deg. of Freedom

Mean Sauares F-Ratio Prob >F

Between Treatment

Error

7.072

3.60

5

24

1.41

0 15

9.42 <0.001

Total 10.67 29

LSD=.501

62

Table. A.39 One-way analysis of variance performed on live Solenopsis mvicta removed weekly from large containers after treatment with diatomaceous earth, dextrose, and rice powder-amended alginate pellets (week three)

Source Sum of Sauares

Deg. of Mean Freedom Squares F-Ratio Prob >F

Between Treatment

Error

40.1667

3.20

24

5

8.033

0.133

60.25 <0.001

Total 42.76 29

LSD=.484

Table, A.40. One-way analysis of variance performed on percent of Solenopsis invicta removed from refuse piles testing positive for Beauveria bassiana infection

Source

Between Treatment

Error

Total

Sum of Sauares

21460.275

146.725

21607.000

Deg. of Freedom

5

24

29

Mean Squares

4292.0553

6.0885

F-Ratio

704.94

Prob >F

<0.001

LSD= 3.191

63

Table. A.41. One-way analysis of variance performed on percent of surviving ants that tested positive for Beauveria bassiana infection

Source Sum of Degree of Mean

Sauares Freedom Sauares F-Ratio 'roo

Between Treatment

Error

2233.45

70.52

5 446.6903 152.02

24 2.9383

<0.001

Total 2303.97 29

LSD = 2.217

64

Table A. 42 Daily cumulative percent mortality of Solenopsis invicta in response to non-sterile soil conditions and polyethylene glycol-treatment _^_^_

Percent Mortality at Day

Treatments 8

1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3

0 0 , 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 - 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0

0 0 . 0 0 0 . 0 1 0 . 0 0 0 . 0 1 0 . 0 1 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 1 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0

1 0 . 0 0 0 . 0 1 0 . 0 1 0 . 0 1 0 . 0 10 0 1 0 . 0 1 0 . 0 0 0 . 0 1 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 1 0 . 0 0 0 . 0 1 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0

1 0 . 0 1 0 . 0 1 0 . 0 2 0 . 0 2 0 . 0 1 0 . 0 2 0 . 0 1 0 . 0 0 0 . 0 1 0 . 0 1 0 . 0 0 0 . 0 0 0 - 0 0 0 . 0 0 0 . 0 1 0 . 0 0 0 . 0 0 0 - 0 0 0 . 0 0 0 . 0 1 0 . 0 0 0 . 0 1 0 . 0 1 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 1 0 . 0 0 0 . 0 0 0 . 0

2 0 . 0 2 0 - 0 2 0 - 0 2 0 . 0 2 0 . 0 1 0 . 0 2 0 . 0 1 0 . 0 1 0 . 0 1 0 . 0 1 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 1 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 1 0 . 0 0 0 . 0 2 0 . 0 2 0 . 0 0 0 . 0 1 0 . 0 1 0 . 0 1 0 . 0 0 0 . 0 0 0 . 0

3 0 . 0 3 0 - 0 2 0 . 0 2 0 . 0 2 0 . 0 3 0 . 0 3 0 . 0 3 0 . 0 1 0 . 0 2 0 - 0 1 0 . 0 1 0 . 0 1 0 . 0 1 0 - 0 0 0 . 0 1 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 1 0 . 0 1 0 . 0 1 0 . 0 2 0 . 0 2 0 . 0 0 0 . 0 1 0 . 0 2 0 . 0 1 0 . 0 1 0 . 0 1 0 . 0

65

Table A. 42. (Continued)

Treatments

1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3

10

30.0 30.0 30.0 20.0 20.0 40.0 30.0 50.0 30.0 20.0 20.0 10.0 10.0 10.0 00.0 10.0 00.0 20.0 20.0 00.0 20.0 20.0 30.0 30.0 20.0 30.0 20.0 10.0 10.0 10.0

11

50.0 40.0 40.0 30.0 30.0 40.0 30.0 50.0 50.0 20.0 30.0 10.0 10.0 10.0 00.0 10.0 00.0 20.0 20.0 10.0 20.0 20.0 30.0 30.0 20.0 30.0 20.0 20.0 20.0 10.0

Percent

12

50.0 60.0 40.0 60.0 40.0 50.0 40.0 50.0 60.0 40.0 30.0 10.0 10.0 10.0 00.0 10.0 10.0 20.0 20.0 40.0 30.0 20.0 30.0 40.0 20.0 50.0 40.0 30.0 20.0 20.0

. Mortal

13

50.0 60.0 40.0 60.0 40.0 50.0 60.0 70.0 70.0 40.0 30.0 10.0 10.0 20.0 10.0 20.0 20.0 20.0 20.0 40.0 50.0 30.0 40.0 50.0 50.0 60.0 50.0 40.0 50.0 30.0

ity at

14

70.0 50.0 60.0 50.0 70.0 50.0 50.0 70.0 70.0 60.0 30.0 20.0 20.0 30.0 20.0 20.0 30.0 30.0 20.0 40.0 50.0 60.0 60.0 50.0 60.0 60.0 50.0 40.0 50.0 30.0

Day

15

80.0 60.0 60.0 60.0 70.0 70.0 60.0 80.0 80.0 70.0 40.0 30.0 20.0 30.0 20.0 20.0 30.0 30.0 20.0 40.0 50.0 70.0 60.0 50.0 60.0 60.0 50.0 60.0 50.0 60.0

1 = PEG-treated 2 = No pellets 3 = No PEG treatment

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alginate pellet formulation of beauverja imported fire …

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