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
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 46
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 46
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 47
V
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 47
A. 9 One-way analysis of variance performed on day 13 mortality of Solenopsis invicta maintained in non-sterile soil exposed to polyethylene glycol-treated or untreated alginate pellets 48
A. 10 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 48
A. 11 One-way analysis of variance performed on day 15 mortality of Solenopsis invicta maintained in non-sterile soil exposed to polyethylene glycol-treated or untreated alginate pellets 49
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
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 50
A. 14 One-way analysis of variance performed on day 7 mortality of Solenopsis invicta maintained in non-sterile or sterile soil exposed to polyethylene glycol-treated or untreated alginate pellets 50
A. 15 One-way analysis of variance performed on day 8 mortality of Solenopsis invicta maintained in non-sterile or sterile soil exposed to polyethylene glycol-treated or untreated alginate pellets 51
A. 16 One-way analysis of variance performed on day 9 mortality of Solenopsis invicta maintained in non-sterile or sterile soil exposed to polyethylene glycol-treated or untreated alginate pellets 51
A. 17 One-way analysis of variance performed on day 10 mortality of Solenopsis invicta maintained in non-sterile or sterile soil exposed to polyethylene glycol-treated or untreated alginate pellets 52
A. 18 One-way analysis of variance performed on day 11 mortality of Solenopsis invicta maintained in non-sterile or sterile soil exposed to polyethylene glycol-treated or untreated alginate pellets 52
A. 19 One-way analysis of variance performed on day 12 mortality of Solenopsis invicta maintained in non-sterile or sterile soil exposed to polyethylene glycol-treated or untreated alginate pellets 53
A. 20 One-way analysis of variance performed on day 13 mortality of Solenopsis invicta maintained in non-sterile or sterile soil exposed to polyethylene glycol-treated or untreated alginate pellets 53
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. 24 One-way analysis of variance performed on day 6 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. 30 One-way analysis of variance performed on day 12 mortality of Solenopsis invicta maintained in non-sterile soil exposed to diatomaceous earth, dextrose, and rice powder-amended pellets 58
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 pellets 59
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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20
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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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27
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CO Hi tn C Hi
< ^
CO XJ QJ
QJ O ,
CO QJ H i 4J t n fd a a Hi
- H [l4 Ol <-t O
X o o
CN +1
0 0
X o
CN +1
o r H
u r H CN
CN +1
>43
CO
4J QJ
O SH XJ a O
O U
QJ
o i n II
a in
CO o SH • QJ o 4J 4J V QJ
r H (h\
XJ XJ
a a QJ QJ u u QJ QJ
MH MH MH MH - H - H 13 T3 > , > i
X rH 4-)
QJ fd
O -H
O C MH Cn
- rH
CO CO
fd QJ QJ U S fd
•• CO
Q g CO 6 J Hi n o QJ u 4-)
u c QJ -H 4-1 x ; O 4-> U -H
a. 5
29
CO u O >
-rH
> u
CO
2400 -
2100 -
1800 -
1500 -
UH
O 1200 U QJ X e
fd 01
Control A No Pellets
Control B No Pellets
Fungal
Pellets
7.5g
900 -
600 -
300 -
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
Id QJ
2400 -
2100 -
rn
•ivo
r
u :3
CO
1800
1500
1200 -
900 -
600 -
300 -
Control A No Pellets
Control B No Pellets
Fungal
Pellets
7.5g
Treatment
Figure 3.6 Mean number (± SEM) of surviving Solenopsis mvicta removed from colonies after treatment with diatomaceous earth, dextrose, and rice powder-amended alginate pellets
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
855.4a
375.6b
119.2c
1427.0a
1670.4b
1807.0b
1036.6a
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. (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.
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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.
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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.
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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.
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41
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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
Table A.9. One-way analysis of variance performed on day 13 mortality of Solenopsis invicta maintained in non-sterile soil exposed to polyethylene glycol-treated or untreated alginate pellets.
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
Table A.11. One-way analysis of variance performed on day 15 mortality of Solenopsis invicta maintained in non-sterile soil exposed to polyethylene glycol-treated or untreated alginate pellets.
Source Sum of Deg. of Mean
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.
Source Sum of Deg. of Mean
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.
Source Sum of Deg. of Mean
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
Table A.15 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 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
Table A.18 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 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
Table A.19. 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.
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
Table A.20. One-way analysis of variance performed on day 13 mortality of Solenopsis invicta maintained in non-sterile soil exposed to polyethylene glycol-treated or untreated alginate pellets.
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.
Source Sum of Deg. of Mean
Sauares Freedom Sauares F-Ratio Prob > F
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
Table A.24 One-way analysis of variance performed on day 6 mortality of Solenopsis invicta maintained in non-sterile soil exposed to diatomaceous earth, dextrose, and rice powder-amended alginate pellets.
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
Table 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 alginate pellets.
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
Table A.27 One-way analysis of variance performed on day 9 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
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
Table A.28 One-way analysis of variance performed on day 10 mortality of Solenopsis invicta maintained in non-sterile soil exposed to diatomaceous earth, dextrose, and rice powder-amended alginate pellets.
Source Sum of Deg. of Mean
Sauares Freedom Sauares F-Ratio Prob > F
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
Table A.29 One-way analysis of variance performed on day 11 mortality of Solenopsis invicta maintained in non-sterile soil exposed to diatomaceous earth, dextrose, and rice powder-amended alginate pellets.
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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