U.S. patent application number 11/901547 was filed with the patent office on 2009-03-19 for composition of entomopathogenic fungus and method of production and application for insect control.
Invention is credited to Mark A. Jackson, Stefan T. Jaronski.
Application Number | 20090074809 11/901547 |
Document ID | / |
Family ID | 40452785 |
Filed Date | 2009-03-19 |
United States Patent
Application |
20090074809 |
Kind Code |
A1 |
Jackson; Mark A. ; et
al. |
March 19, 2009 |
Composition of entomopathogenic fungus and method of production and
application for insect control
Abstract
Microsclerotia of entomopathogenic fungi, including Metarhizium
species, Beauveria species, and Lecanicillium species, may be
produced. These microsclerotia are effective for the control of a
wide variety of insect pests, particularly soil-dwelling insect
pests.
Inventors: |
Jackson; Mark A.; (Peoria,
IL) ; Jaronski; Stefan T.; (Sydney, MT) |
Correspondence
Address: |
USDA-ARS-OFFICE OF TECHNOLOGY TRANSFER;NATIONAL CTR FOR AGRICULTURAL
UTILIZATION RESEARCH
1815 N. UNIVERSITY STREET
PEORIA
IL
61604
US
|
Family ID: |
40452785 |
Appl. No.: |
11/901547 |
Filed: |
September 13, 2007 |
Current U.S.
Class: |
424/195.15 ;
435/256.8 |
Current CPC
Class: |
A01N 65/00 20130101;
A01N 63/30 20200101; A01G 18/00 20180201 |
Class at
Publication: |
424/195.15 ;
435/256.8 |
International
Class: |
A01N 63/00 20060101
A01N063/00; A01P 7/04 20060101 A01P007/04; C12N 1/14 20060101
C12N001/14 |
Claims
1. Isolated microsclerotia of an entomopathogenic fungus selected
from the group consisting of Metarhizium species, Beauveria
species, and Lecanicillium species.
2. The isolated microsclerotia of claim 1 wherein said
entomopathogenic fungus comprises a Metarhizium species.
3. The isolated microsclerotia of claim 2 wherein said
entomopathogenic fungus comprises Metarhizium anisopliae.
4. The isolated microsclerotia of claim 1 wherein said
entomopathogenic fungus is substantially biologically pure.
5. A composition comprising microsclerotia of an entomopathogenic
fungus selected from the group consisting of Metarhizium species,
Beauveria species, and Lecanicillium species with an agronomically
acceptable carrier which said microsclerotia, upon rehydration,
germinate hyphally or sporogenically to produce infective, aerial
conidia.
6. The composition of claim 5 wherein said entomopathogenic fungus
comprises a Metarhizium species.
7. The composition of claim 6 wherein said entomopathogenic fungus
comprises Metarhizium anisopliae.
8. The composition of claim 5 wherein said entomopathogenic fungus
is substantially pure.
9. The composition of claim 5 wherein said microsclerotia are
present in an insecticidally effective amount.
10. The composition of claim 5 wherein said microsclerotia are
produced by liquid culture and are present in the recovered biomass
in a concentration at least about 1.times.10.sup.6 microsclerotia
per gram of said biomass.
11. A method for insect control comprising applying to the locus of
said insects an insecticidally effective amount of microsclerotia
of an entomopathogenic fungus selected from the group consisting of
Metarhizium species, Beauveria species, and Lecanicillium
species.
12. The method of claim 11 wherein said entomopathogenic fungus
comprises a Metarhizium species.
13. The method of claim 12 wherein said entomopathogenic fungus
comprises Metarhizium anisopliae.
14. The method of claim 11 wherein said insects are selected from
the group consisting of root weevils, soil grubs, rootworms,
wireworms, fruit flies, and root maggots.
15. The method of claim 11 wherein said insects are selected from
the group consisting of subterranean termites (Reticulitermes and
Coptotermes spp.), corn rootworms (Diabrotica spp), black vine
weevils (Otiorhynchus sulcatus), wireworms (larvae of family
Elateridae), citrus root weevils (Diaprepes abbreviates), sugarbeet
root maggots (Tetanops myopaeformis), cabbage/turnip/onion/seed
corn maggots (Delia spp.), carrot rust fly (Psila rosae), sweet
potato weevils (Cylas formicarius), Japanese beetles (Popillia
japonica), and European chafers (Rhizotrogus majalis).
16. The method of claim 14 wherein said applying comprises applying
said microsclerotia to soil or greenhouse soilless potting mix.
17. The method of claim 11 wherein said insects are plant foliage
or tree bark inhabiting insects selected from the group consisting
of emerald ash borer (Agrilus planipennis), and gypsy moth
(Lymantria dispar), and pecan weevil (Curculio caryae).
18. The method of claim 17 wherein said microsclerotia are applied
to the bark and canopy of plants and trees.
19. A method of producing from an en entomopathogenic fungus a high
concentration of desiccation tolerant fungal microsclerotia,
comprising the steps of: a) inoculating a liquid culture medium
comprising a carbon source and a nitrogen source with fungal
propagules of an entomopathogenic fungus selected from the group
consisting of Metarhizium species, Beauveria species, and
Lecanicillium species, said nitrogen source having a concentration
between 8.1 grams/liter and 50 grams/liter and said carbon source
having a concentration greater than 20 grams/liter; b) incubating
the propagules for a sufficient time to allow for production of
microsclerotia; and c) collecting the resulting microsclerotia.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The invention relates to the formation of microsclerotial
propagules by entomopathogenic fungi and the use of those
microsclerotia for the control of insects.
[0003] 2. Description of the Prior Art
[0004] Chemical pesticides have been used for control of insect and
weed control for over 60 years. Interest in the use of
biologically-based pest control measures has been brought about by
the development of pest resistance to many chemical pesticides
coupled with public concerns about the adverse impact of widespread
chemical use on human health, food safety and the environment
(Gillespie and Moorhouse, 1989, Biotechnology of Fungi for
Improving Plant Growth, pp 85-125; Hajek, 1993, New options for
insect control using fungi, In, Pest Management: Biologically Based
Technologies, (R. D. Lumsden and J. Vaugn, eds.) Amer. Chem. Soc.,
Washington, D.C.; Leathers et al., 1994, J. Industrial Microbiology
12:69-75). In the late 19.sup.th century, Metchnikoff was the first
to describe Metarhizium anisopliae "green muscardine" infections on
the cereal cockchafer and suggested the use of the microorganism as
a biological control agent for insects (Zimmermann et al., 1995,
Biocontrol Science and Technology, 5:527-30). Subsequent studies
showed that an application of M. anisopliae spores could kill the
cereal cockchafer and the sugarbeet weevil via direct infection.
Initial production methods for this fungus focused on the use of
the host insect or artificial media as a growth vehicle for
producing conidia of the pathogen.
[0005] The choice of pursuing soil-dwelling insects as targets for
biological control, versus insects on the phylloplane, is tempting.
Among these targets are root weevils, soil grubs, rootworms,
wireworms, fruit flies, and root maggots (Bruck, 2005, Biological
Control, 32:155-163; Krueger and Roberts, 1997, Biological Control,
9:67-74; Chandler and Davidson, 2005, Journal of Economic
Entomology, 98:1856-1862; Vanninen et al., 1999, Journal of Applied
Entomology, 123:107-113; Kabaluk et al., 2005, IOBC/wprs Bulletin,
28:109-115). UV radiation, which can result in a very short
persistence on plant surfaces, is avoided. Rainfall, washing the
infectious conidia off foliage shortly after an application, is not
a concern. Soil temperatures are moderated by its insulative value,
and soil moistures above the permanent wilting point of plants are
well within the optimal range for microorganisms.
[0006] The entomopathogenic fungus Metarhizium anisopliae has been
registered as a biological insecticide for the control of
soil-dwelling and cryptic insect pests in the United States and
many other countries. Metarhizium anisopliae has been reported to
infect more than 100 insects including the soil-dwelling insects
listed: subterranean termites (Reticulitermes and Coptotermes
spp.), corn rootworms (Diabrotica spp), black vine weevils
(Otiorhynchus sulcatus), citrus root weevils (Diaprepes
abbreviatus), Japanese beetles (Popillia japonica), and European
chafers (Rhizotrogus majalis) (Krueger et al., 1992, Journal of
Invertebrate Pathology, 59: 54-60; Schwarz, 1995. Metarhizium
anisopliae for soil pest control. In Biorational Pest Control
Agents; Formulation and Delivery, F. R. Hale and J. W. Barry, eds.,
ACS Symposium Series 595, American Chemical Society, Washington,
D.C. p. 183-196; Krueger and Roberts, 1997, ibid; Bruck, 2005,
ibid). Commercial interest in using M. anisopliae to control
soil-dwelling insects has resulted in the development of granular
pest control formulations based on liquid culture-produced mycelial
pellets or solid substrate-produced conidia on a nutritive or
non-nutritive carrier, or fungus on the spent solid substrate
itself (Schwarz, 1995, ibid; Storey et al., 1990, Conidiation
kinetics of the microsclerotial granules of Metarhizium anisopliae
(Bio 1020) and its biological activity against different soil
insects. Proceedings of the Vth International Colloquium on
Invertebrate Pathology and Microbial Control, Adelaide, Australia.
p. 320-325; Andersch et al., 1995, U.S. Pat. No. 5,418,164), the
most practical formulation being the mycelial pellet. The fungus on
these granular formulations must necessarily grow out from the
carrier and resporulate to produce the infectious conidia. Since
the infective propagules (conidia) of M. anisopliae must contact
and infect the insect host, the number, distribution and
persistence of conidia, as produced by fungus on a granular
carrier, in the soil is of utmost importance (Bruck, 2005, ibid; Hu
and St. Ledger, 2002). Practical application of these formulations
has been limited because of product physical characteristics
precluding use in conventional farm equipment, high production
costs, and/or poor practical shelf life. Mycelial pellets, such as
disclosed in US Pat. No. 5,418,164, have generally poor,
room-temperature shelf life or must be lyophilized, an expensive
process. Conidia, blastospores or mycelium in sodium alginate (U.S.
Pat. No. 5,360,607; Knudsen et al., 1990, J. Econ. Entomol.,
83(6):2225-2228; Meyer 1994, Fund. Applied Nematology,
17(6):563-567) have been commercialized, but this formulation is
too expensive for general use in field crops, and suffers from poor
room temperature shelf-life. Conidia (produced in solid substrate
fermentation) bound to a granular carrier generally have poor shelf
life. Spent solid substrate fermentation granules (typically rice,
barley, wheat grains) containing residual fungus after harvest of
conidia, cannot be applied using conventional farm equipment nor
can they be readily ground to the proper size without killing the
fungus, even though this formulation is readily available as a
by-product of conidia production.
[0007] For persistence in soil and decaying plant material, many
plant pathogenic fungi produce sclerotia; i.e., melanized, compact
hyphal aggregates that are highly resistant to desiccation. These
propagules often serve as the overwintering structure for the
fungus (Cooke, 1983, Morphogenesis of sclerotia. In "Fungal
Differentiation: A Contemporary Synthesis" Smith, J. E., ed. pp
397-418. Marcel Dekker, Inc., New York, N.Y., U.S.A.; Coley-Smith
and Cooke, 1971, Survival and germination of fungal sclerotia. In
"Annual Review of Phytopathology", Horsfall, J. G., Baker, K. F.,
Zentmyer, G. A., eds. pp 65-92. Annual Reviews Inc., Palo Alto,
Calif., U.S.A.). Microsclerotia (small sclerotial particles,
200-600 um) of fungal plant pathogens such as Colletotrichum
truncatum and Mycoleptodiscus terrestris have been produced in high
concentration in submerged liquid culture fermentation (Jackson and
Schisler, 1995, Mycological Research,99:879-884; Shearer and
Jackson, 2003, U.S. Pat. No. 6,569,807). Microsclerotia of these
pathogens of weedy plants have shown value as persistent, infective
propagules in soil and aquatic environments (Shearer and Jackson,
2006, Biological Control. 38:298-306; Boyette et al., 2007,
BioControl 52:413-426). However, to date, microsclerotia have not
been reported for any fungal pathogens of insects.
SUMMARY OF THE INVENTION
[0008] We have now discovered the novel, hitherto undescribed,
formation of microsclerotia by entomopathogenic fungi, which are
effective for the control of insect pests, as well as techniques
for the production of these microsclerotial propagules. In
accordance with this discovery, microsclerotia may be produced from
entomopathogenic fungi including Metarhizium species, Beauveria
species, and Lecanicillium species. These microsclerotia are
desiccation tolerant, survive low-cost, air-drying processes to low
moisture levels, exhibit excellent shelf-life at room as well as
refrigerated temperatures, and can be processed to formulation
sizes which are compatible with conventional granular pesticide
applicators. In use, the microsclerotia sporulate profusely (thus
producing large number of insect-infectious conidia) upon
rehydration such as in normal soil, and may exhibit comparable or
even higher levels and rates of infectivity against insect pests in
comparison to conventional conidia-based granular formulations.
[0009] In accordance with this discovery, it is an object of this
invention to provide entomopathogenic fungi in the form of
microsclerotial propagules.
[0010] Another object of this invention is to provide
microsclerotia propagules of entomopathogenic fungi which are
effective as biological control agents against economically
important insect pests.
[0011] A further object of this invention is to provide
microsclerotia propagules of entomopathogenic fungi which are
desiccation tolerant and storage stable, while retaining efficacy
as biological control agents against insect pests.
[0012] Yet another object of this invention is to provide a method
for producing these microsclerotia propagules of entomopathogenic
fungi in high yields in submerged liquid culture. Other objects and
advantages of the invention will become readily apparent from the
ensuing description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 shows the relative efficacy of two types of
Metarhizium anisopliae F52 granules against third instar Tetanops
myopaeformis (Sugarbeet Root Maggot) larvae in a soil incorporation
assay as described in Example 4. The granules consisted of either
20/30 mesh microsclerotia-containing granules prepared from liquid
fermentation, or a more conventional, 16/30 mesh corn-grit carrier
coated with conidia, using 10% polyoxyethylene sorbitan monooleate
(TWEEN 80) binder. Granules were incorporated into a clay soil at
the rate of 1.8 mg/g soil, and the soils subsequently wetted to the
desired moisture endpoints with water. Soil water activities were
determined after 48 hours using a water activity meter following
the manufacturer's instructions. Each treatment had three
replicates of 10 larvae and the entire test was replicated
twice.
DETAILED DESCRIPTION OF THE INVENTION
[0014] As used herein, the term "microsclerotia" refers to small
sclerotial bodies which are some resting state of the fungi.
Microsclerotia are stable, viable, sometimes melanized, compact
hyphal aggregates of the fungus. The microsclerotia per se are not
infective, but when rehydrated such as by exposure to moisture in
the soil or within the crevices in the bark of trees, the
microsclerotia will germinate hyphally or sporogenically to produce
conidia which are infective to the target insects. The
microsclerotia are extremely desiccation tolerant, are capable of
germinating both sporogenically and vegetatively, and also retain
the insecticidal capabilities of their native or normal forms
(i.e., hyphae, blastospores, and/or conidia of the same
entomopathogenic fungus). Morphologically, the microsclerotia may
be present as an agglomerated group of cells. The term
"insecticide" refers to a material or mixture of materials which
induce mortality, disrupt or impede growth, interfere with
metamorphosis or other morphogenic functions, effect sterilization,
or interfere with reproduction of the targeted insects. The terms
"controlling" or "control of the target insect" is used herein to
mean that the population of the insect is reduced, principally
through mortality, at a level that is significantly greater than an
untreated population. "Significant mortality" is defined herein to
mean that the percentage of insects that die within a given period
of time after coming into contact with the insecticide is
significantly greater than the number of insects not contacted with
the insecticide that die during the same period of time, based on
standard statistical analyses.
[0015] The invention described herein is effective for producing
microsclerotia from any species, strain or variety of
entomopathogenic fungi from the genus Metarhizium, although it is
also envisioned that the invention may be used to produce
microsclerotia from species from the genera Beauveria or
Lecanicillium. Preferred species for use herein include Beauveria
bassiana, Metarhizium flavoviride, and particularly Metarhizium
anisopliae sensuo lato.
[0016] Production of the microsclerotia of this invention is
preferably effected in liquid-culture, and large scale production
is preferably conducted by deep-tank liquid-culture fermentation.
It is also envisioned that solid culture media may be utilized. The
liquid medium used in the preparation of the melanized
microsclerotia is critical, as their formation and yield are medium
dependent. Generally, liquid media having high carbon and nitrogen
concentrations are necessary for high yields of microsclerotia of
M. anisopliae. For use herein, the medium preferably contains a
nitrogen source at a concentration between 8.1 grams nitrogen
source/liter and less than 50 grams nitrogen source/liter, and a
carbon source at a concentration greater than 20 grams of
carbohydrate/liter, preferably greater than 30 grams
carbohydrate/liter. Suitable nitrogen sources include, but are not
limited to hydrolyzed casein, yeast extract, hydrolyzed soy
protein, hydrolyzed cottonseed protein, and hydrolyzed corn gluten
protein. Suitable carbon sources include, but are not limited to
carbohydrates, including glucose, fructose, and sucrose, and
glycerol. The preferred liquid-culture media for use herein is
described by Jackson (U.S. Pat. No. 5,968,808, the contents of
which are incorporated by reference herein). We have surprisingly
discovered that the above-mentioned entomopathogenic fungi produce
microsclerotia when grown in submerged culture on the Jackson
medium. These microsclerotia have not been hitherto described from
these fungi. In contrast, species of the entomopathogenic fungus
Paecilomyces produce blastospores rather than microsclerotia when
grown in the same medium under the same conditions. The
fermentation may be conducted using conventional aerobic
liquid-culture techniques with agitation and aeration. Agitation is
preferred to inhibit mycelial growth on the vessel wall. Suitable
temperatures may range from about 20.degree. C. to about 32.degree.
C., and the pH may range from about 4 to about 8. Once a
sufficiently heavy growth of the fungus has been obtained, usually
in about 2-4 days, microsclerotia begin to form and the
fermentation is then continued until a sufficiently high
concentration of the microsclerotia is obtained. Without being
limited thereto, in a preferred embodiment, the fermentation is
continued until a major proportion of the viable fungi in the
culture (i.e., greater than 30% by weight), and more preferably
until a predominant proportion of the viable fungi in the culture
(i.e., greater than 50% by weight) are in the form of
microsclerotia. Following completion of the fermentation, the
microsclerotia may be recovered using conventional techniques, such
as by filtration or centrifugation. The microsclerotia may be
dried, such as by air-drying, to a low moisture level, and stored
at room temperature or lower. In a preferred embodiment, the
biomass recovered from the fermentation, following drying, will
contain approximately 1.times.10.sup.6 or higher microsclerotia per
gram of biomass (based on dry weight of the biomass), particularly
at least 9.times.10.sup.6 microsclerotia per gram of biomass.
[0017] Commercial formulations for use as a biological insect
control agent may be prepared from microsclerotia that have been
harvested from the culture medium such as described hereinabove. As
a practical matter, it is envisioned that commercial formulations
may be prepared directly from the culture, thereby obviating the
need for any purification steps. While liquid cultures may be used
directly, in the preferred embodiment the water is removed from the
cultures to partial or substantial dryness as described above, and
the dried culture broken or ground into small particles suitable
for application through conventional granule applicators, using
techniques conventional in the art. To facilitate application and
subsequent fungal outgrowth and conidiation, the harvested
microsclerotia may alternatively be formulated in a suitable,
agronomically acceptable, nutritional or inert carrier or vehicle
for application as wettable powders, dusts, granules, baits,
solutions, emulsifiable concentrates, emulsions, suspension
concentrates and sprays (aerosols). For example, for liquid
applications, the microsclerotia may be formulated as a suspension
or emulsion. In this embodiment, preferred carriers include but are
not limited to water, buffers, or vegetable or plant oils. In an
alternative, preferred embodiment particularly suited for solid
granular applications, the microsclerotia may be formulated with
solid inert carriers or diluents such as diatomaceous earth, talc,
clay, vermiculite, CaCO.sub.3, corn cob grits, alginate gels,
starch matrices or synthetic polymers, or they may be incorporated
into conventional controlled release microparticles or
microcapsules. The skilled practitioner will recognize that the
fungi may also be formulated in combination with conventional
additives such as sticking agents or adherents, emulsifying agents,
surfactants, foams, humectants, or wetting agents, antioxidants, UV
protectants, nutritive additives, fertilizers, insecticides, or
even with fungicides which exhibit low toxicity to the subject
fungi. For application onto the bark or canopy of trees and plants,
the microsclerotia are also preferably formulated with a
hygroscopic or hydrophilic adjuvant.
[0018] The absolute amount of the microsclerotia and their
concentration in the final composition are selected to provide an
effective reduction in the population of the target insect as
compared to an untreated control. The actual amount is not critical
and is a function of practical considerations such as the
properties of the vehicle or carrier, the density of the target
insect population, and the method and site of application, and may
be readily determined by routine testing. As the microsclerotia of
this invention serve to produce and deliver a high concentration of
the infective conidia to control the target insects by infection
and death, for purposes of formulation and application, an
"effective amount" is defined to mean any quantity of
microsclerotia sufficient to subsequently produce enough conidia in
the target habitat to infect and kill the target insect relative to
an untreated control. By way of example and without being limited
thereto, it is envisioned that suitable formulations will typically
contain about 1.times.10.sup.6 or higher microsclerotia per gram of
biomass recovered from the liquid culture (based on the dried
weight of the biomass), preferably at least 1.5.times.10.sup.7
microsclerotia per gram of biomass, For application to typical row
crops, without being limited thereto, it is envisioned that
suitable application rates are 1.times.10.sup.9 microsclerotia per
acre, applied in furrow.
[0019] In use, the microsclerotia of this invention may be applied
to the locus or vicinity of the target insects or on the surface of
the plants to be protected, e.g., onto tree bark, or as a seed
coating, using conventional techniques. In a preferred embodiment,
the microsclerotia are applied to the soil, or to soil-less potting
mixes such as are used in greenhouses, in a granular form.
Depending upon the target insect pest, the microsclerotia may be
applied in agricultural fields, orchards, greenhouses, gardens or
lawns, or on or in the vicinity of ornamental plants, trees, or
commercial or residential structures.
[0020] The microsclerotia of the entomopathogenic fungi of this
invention produce the infective propagules (aerial conidia)
effective for infecting and killing a wide variety of economically
important insects, particularly soil-born insects, but also
including some ground- and canopy-dwelling insects. Without being
limited thereto, insects which may be controlled by the
microsclerotia of this invention include root weevils, rootworms,
wireworms, fruit flies, soil grubs, root maggots, termites, and
ants, particularly corn rootworm (Diabrotica spp), black vine
weevil (Otiorhynchus sulcatus), citrus root weevil (Diaprepes
abbreviatus), sweet potato weevil (Cylas formicarius), sugarbeet
root maggot (Tetanops myopaeformis), cabbage maggot (Delia
radicum), onion maggot (Delia antigua), turnip maggot (Delia
floralis), seedcorn maggot (Delia platura), carrot rust fly (Psila
rosae), Japanese beetle (Popillia japonica), European chafer
(Rhizotrogus majalis), subterranean termite (Reticulitermes and
Coptotermes spp.). In addition, certain canopy dwelling, especially
bark dwelling, insects may be controlled by microsclerotia of this
invention. These insects include emerald ash borer (Agrilus
planipennis), gypsy moth (Lymantria dispar), and the pecan weevil
(Curculio caryae).
[0021] The following examples are intended only to further
illustrate the invention and are not intended to limit the scope of
the invention that is defined by the claims.
EXAMPLE 1
Production of Microsclerotia
[0022] In this example we have evaluated different liquid culture
nutritional environments and measured biomass accumulation and
blastospore and microsclerotia yields. The desiccation tolerance of
microsclerotia was measured by evaluating their ability to
germinate vegetatively and/or sporogenically upon rehydration.
[0023] Three strains of Metarhizium anisopliae var. anisopliae
(Metchnikoff) Sorokin were used in this study: a commercial strain,
F52 (ATCC 90448, (Earth Biosciences, now Novozyme Biologicals,
Salem, Va., reisolated from Tetanops myopaeformis larvae), MA1200
(ATCC 62176, passaged through T. myopaeformis larvae), and TM109
(ARSEF5520 reisolated from T. myopaeformis larvae). All isolates
were stored at -80.degree. C. at USDA ARS NPARL and at USDA ARS
NCAUR. Stock cultures of each strain of M. anisopliae were grown as
single spore isolates on potato dextrose agar (PDA) for three weeks
at room temperatures. The sporulated plate was cut into 1 mm.sup.2
agar plugs and stock cultures of these agar plugs stored in 10%
glycerol at -80.degree. C. Conidial inocula for liquid culture
experiments were produced by inoculating PDA plates with a conidial
suspension from the frozen stock cultures and growing these
cultures at room temperature (.about.22.degree. C.) for 2-3 wks.
All liquid cultures were inoculated at an initial concentration of
5.times.10.sup.6 conidia ml.sup.-1 culture broth.
[0024] The six liquid media tested were composed of a basal salts
medium supplemented with trace metals, vitamins (Jackson et al.,
1997, Mycological Research, 101:35-41) and various combinations of
glucose and acid hydrolyzed casein, and casamino acids. The defined
basal salts solution used in all liquid cultures contained per
liter of deionized water: KH.sub.2PO.sub.4, 4.0 g;
CaCl.sub.2.2H.sub.2O, 0.8 g; MgSO.sub.4.7H.sub.2O, 0.6 g;
FeSO.sub.4.7H.sub.2O, 0.1 g; CoCl.sub.2.6H.sub.2O, 37 mg;
MnSO.sub.4.H.sub.2O, 16 mg; ZnSO.sub.4.7H.sub.2O, 14 mg; thiamin,
riboflavin, pantothenate, niacin, pyridoxamine, thiotic acid, 500
microgram each; and folic acid, biotin, vitamin B.sub.12, 50
microgram each. In Table 1, the amounts of glucose and
acid-hydrolyzed casein, and the corresponding carbon concentration
and carbon-to-nitrogen are given for each medium tested. Carbon
concentrations and carbon to nitrogen ratios calculations were
based on 40% carbon in glucose and 53% carbon, 8% nitrogen in acid
hydrolyzed casein.
[0025] All cultures were grown as 100 ml cultures in 250 ml
baffled, Erlenmeyer flasks at 28.degree. C. and 300 rpm in a rotary
shaker incubator. Flasks were hand-shaken frequently to inhibit
mycelial growth on the flask wall. At two, four, and eight days
post inoculation, samples were taken to measure biomass
accumulation, blastospore concentrations, and microsclerotia
concentrations. For each experiment, duplicate samples were made
from each flask on each sampling date, and three replicate flasks
for each media were used. All experiments were repeated at least
twice.
[0026] For biomass accumulation measurements, one ml of whole
culture broth was collected from culture flasks and the biomass was
separated from the spent medium by vacuum filtration onto
pre-weighed filter disks (Whatman GF/A, Maidstone, England). Dry
weight accumulation was determined by drying the biomass and filter
disk at 60.degree. C. to a constant weight prior to measurement. To
determine microsclerotia concentrations, culture broth was diluted
appropriately and a drop placed on a glass microscope slide,
overlaid with a coverslip and the number of microsclerotia counted
in 100 microliters. Microsclerotia were counted when compact,
sometimes melanized, hyphal aggregates were larger than 200
microns. Only well formed microsclerotia were counted. Culture
broth was diluted as appropriate for ease of counting. During
culture broth sampling, microsclerotial suspensions were constantly
vortexed to ensure homogeneity
[0027] After growing the M. anisopliae cultures for eight days,
diatomaceous earth (HYFLO, Celite Corp., Lompoc, Calif.) was added
to the combined fungal biomass of the three flasks in each
treatment at a concentration of 5 g diatomaceous earth/100 ml
culture broth. The microsclerotia-diatomaceous earth mixture was
vacuum-filtered in a Buchner funnel using Whatman No. 54 filter
paper. The filter cake was broken up by pulsing in a blender (MINI
PREP Plus, Cuisinart) and layered in shallow aluminum trays and
air-dried overnight in an operating biological containment hood.
The moisture content of the microsclerotia-diatomaceous earth
preparation was determined with a moisture analyzer. When M.
anisopliae formulations dried to a moisture content approx. 5%,
they were vacuum packed in synthetic polyethylene bags with a
vacuum packer and stored at 4.degree. C. Upon rehydration,
microscierotia of M. anisopliae germinated hyphally (germ tube
formation) and conidiated (produced conidial masses on the surface
of the microsclerotium). Microsclerotia viability (hyphal
germination) and spore production (sporogenic germination) were
determined on dried microsclerotia preparations by sprinkling 25 mg
of the dried microsclerotia formulation onto the surface of water
agar plates. Two water agar plates were used for each treatment.
Following a 24 hr incubation period at 28.degree. C., 100
microsclerotia were microscopically examined on each plate for
hyphal germination as a measure of viability. To enumerate spore
production, the incubation of the water agar plates was continued
for eight days at 28.degree. C. Each water agar plate was flooded
with 5 ml of sterile water and the conidia were dislodged from the
microsclerotia with a sterile loop. After the conidia were
dislodged, the available liquid was pipetted from each plate and
the liquid volume measured. Conidia were counted using a
hemacytometer. To determine the number of conidia of M. anisopliae
produced per g of dried microsclerotia formulation, the number of
conidia harvested per plate was divided by the weight of the dried
microsclerotia preparation added to each plate (0.025 g).
Results
[0028] Biomass accumulation by the three strains of M. anisopliae
followed the predicted pattern where those grown in media that
contained 8 g/l carbon produced lower biomass concentrations when
compared to those grown in media with 36 g/l carbon (Table 2). When
comparing cultures grown in media with differing carbon-to-nitrogen
ratios, biomass accumulation was not affected by nitrogen content
for those grown in media with a carbon concentration of 8 g/l
suggesting that the medium was carbon-limited. For all strains of
M. anisopliae grown in media containing 36 g/l carbon, biomass
accumulation was significantly higher after 4 and 8 days growth for
cultures grown in lower carbon-to-nitrogen (higher nitrogen
content) media suggesting that nitrogen was growth limiting (Table
2).
[0029] The formation, yield, and melanization of microsclerotia by
M. anisopliae were strain and medium dependent (Table 3). While
microsclerotia formation could be seen in all media and with all
strains of M. anisopliae, highest microsclerotia concentrations
were measured on days 4 and 8 post inoculation in rich media (36
g/l carbon) by M. anisopliae strain F52. On day 8, rich media with
carbon-to-nitrogen ratios of 30:1 and 50:1 yielded 2.7 and
2.9.times.10.sup.5 microsclerotia/ml, respectively (Table 3).
Microsclerotia formed by M. anisopliae strain F52 in media with a
carbon-to-nitrogen ratio of 50:1 were more highly melanized
compared to microsclerotia formed in media with higher
carbon-to-nitrogen ratios.
[0030] The desiccation tolerance of air-dried, microsclerotia from
8-day-old cultures showed that all cultures and strains of M.
anisopliae produced microsclerotia that survived the drying process
with no significant loss in viability except those cultures grown
in weak media with low nitrogen content [8 g/l carbon, 50:1
carbon-to-nitrogen ratio (Table 4)]. Conidia production by
air-dried microsclerotia for all strains of M. anisopliae,
regardless of media, was greater than 1.times.10.sup.8 conidia/g
dried formulate (Table 3). In general, dried microsclerotia
formulations from the rich media (media 4, 5, 6) produced higher
numbers of conidia when compared to microsclerotia formulates
derived from the media with lower carbon concentrations. In
addition, cultures of M. anisopliae grown in rich media produced
more biomass (Table 2) which resulted in higher yields of dried
microsclerotia formulations.
EXAMPLE 2
Assessment of Fungal Outgrowth and Sporulation From Microsclerotia
on Different Soils
[0031] The fungal outgrowth and sporulation of M. anisopliae strain
MA1200 from microsclerotial granules produced from Medium 4, 5, and
6 were evaluated on moist soil plates. The granules were prepared
as described earlier, sieved to a 0.6-1.7 mm particle size, and
stored in sealed plastic bags at 5-7> C. for 9 months prior to
use. A clay soil from a sugarbeet field in Sidney Mo., a clay loam
soil collected from a sugarbeet field near St. Thomas, N.D., and a
sandy-loam soil from Torrington, Wyo. were separately air dried to
a moisture content less than 2%, pulverized and sieved through as
20-mesh (U.S.) sieve to a uniform particle size range. Soil texture
was determined by standard methods (Sheldrick and Wang, 1993,
Particle size distribution. In, Soil Sampling and Methods of
Analysis, M. R. Carter, Ed. Can. Soc. Soil Science, Lewis
Publishers, Boca Raton, Fla., pp. 499-512). All soils tested were
non-sterile. The three soils were placed in Petri dishes and wetted
with reverse osmosis water to 20% field capacity (as previously
determined for each soil). Microsclerotia-containing granules of M.
anisopliae from each production medium were sprinkled onto the
surface of three replicate plates of each soil. The plates were
placed in resealable plastic bags and incubated at 2520 C. Granules
were visually examined for fungal outgrowth and conidiation 3 and 7
days later.
[0032] Fungal outgrowth and sporulation of M. anisopliae strains
MA1200, F52 and TM109 produced from Medium 5 and corn-grit based
granules of M. anisopliae strain F52 (see bioassays, below) were
also evaluated on moist, clay soil plates. The non sterile clay
soil was the same as used earlier. The evaluation protocol was as
described previously, with visual observations daily, beginning on
Day 3.
Results
[0033] Medium 4 (10:1 C:N Ratio, 36g Carbon/L) granules: By Day 3 a
compact fungal outgrowth typical of M. anisopliae was present on
all microsclerotial granules incubated in the three soils tested.
Less than 10% had a greenish tinge indicative of initial
conidiation. On Day 7, the microsclerotial granules were largely
covered with a fungal hymenium but conidiation was not
prominent.
[0034] Medium 5 (30:1 C:N Ratio, 36g Carbon/L) granules: On Day 3,
compact fungal outgrowth and conidiation was present on all
granules on all three soils. There was also a small amount of more
erect, filamentous growth. By Day 7, intense conidiation of M.
anisopliae was present on all granules, on all three soils.
[0035] Medium 6 (50:1 C:N Ratio, 36g Carbon/L) granules: On Day 3,
fungal outgrowth was weak and spotty on the clay loam and sandy
loam soils. On the clay soil, fungal outgrowth was very sparse. By
Day 7, fungal outgrowth and the typical green conidiation had
occurred on essentially all granules on all three soils. While the
extent of conidiation was not quantified, levels of conidiation for
the various soils tested followed the pattern; sandy loam soil
>clay loam soil> clay soil.
[0036] By Day 3, microsclerotial granules of MA1200, F52 and TM109
had fungal outgrowth, unlike corn grit granules of M. anisopliae
strain F52. Strain TM109 granules had compact hymenium on their
surfaces with areas of profuse conidiation. Outgrowth on the strain
F52 and MA1200 granules was less robust; strain F52 granules had
more visible conidiation than strain MA1200. There was very little
outgrowth on the strain F52 corn grit granules with most growth
consisting of simple scattered mycelial strands. On Day 4,
conidiation was visibly underway with strain Ma1200 and TM109 on
corn grit granules, but absent from the corn grit granules
inoculated with strain F52. By Day 5, all microsclerotial granules
of M. anisopliae had profuse compact green conidiation. Compared to
microsclerotial granules, the corn grit granules of all the strains
of M. anisopliae tested continued to have sparse fungal outgrowth
and little conidiation until Day 7-8 (FIG. 2). Subsequent to Day 8,
sporulation became more robust but never achieved the same visual
extent as the microsclerotial granules.
EXAMPLE 3
Relative Efficacy of Microsclerotial Granules Produced by the Six
Media in Example 1
[0037] The relative biological efficacy of the microsclerotial
granules produced by strain F52 in all six media was evaluated
using soil-based bioassays with larval sugarbeet root maggots
(SBRM). Granules (20/30 mesh) of F52 from all six media were
incorporated into a dry, sieved, non-sterile clay soil used earlier
at the rate of 14 mg granules/60 g soil. Two separate production
batches of granules were evaluated. The granules had been stored in
sealed plastic bags at 5-9.degree. C. for 7 months prior to use.
The soils were moistened with reverse osmosis water to an end point
of 15% Field Capacity (previously determined) and the water
potentials determined with an AQUALAB moisture meter (Decagon
Products, Pullman, Wash.). Resulting soil moistures were
0.982-0.983 A.sub.w (-2.32 to -2.47 MPa matric potential), which
moistures were sufficient for fungal outgrowth and sporulation.
Permanent Wilting Point for most plants is 0.989 A.sub.w. An
untreated control soil was prepared simply by wetting an additional
aliquot of soil, without any granules, with the same amount of
water. Each treated and control soil was then dispensed equally
into three 60 cc, lidded, plastic, condiment cups. The cups were
sealed and placed on a layer of water-moistened paper towel (to
maintain humidity) in a large, lidded plastic container, and
incubated at 24.degree. C. After 1 week, the soils were infested
with 10 third-instar SBRM larvae per cup. These larvae were
field-collected, in diapause yet motile and non-feeding, and had
been stored in moist sterile sand at 3-4> C. for several months
prior to use. Each treatment was replicated three times. Larval
mortality was determined weekly for three weeks. Each week, all
cadavers were removed and placed at 95-100% high humidity for three
days to elicit the presence of mycosis. Two separate production
lots were evaluated in this manner.
[0038] For statistical analyses of bioassay data, all mortality
data were adjusted for control mortality, when necessary, by
application of Abbott's correction (Abbott, 1925) and then
subjected to angular transformation before further analysis. Data
were then subjected to ANOVA and Tukey's HSD mean separation test
when significant treatment effects were identified.
Results
[0039] There were no significant differences in efficacy between
the two production batches of microsclerotial granules for any of
the media tested at 1 and 3 weeks (F=0.06, p=0.83 for week 1;
F=1.51 p=0.34 for week 3), and a barely significant difference at
Week 2 (F=23.94, p=0.04), due to mortalities from Medium 1
microsclerotial granules being significantly different between the
two batches. Control mortality for SBRM was 0% even after three
weeks. Data are presented in Table 5. Significant differences
existed among the early mortalities from granules produced on the
six media, in both batches, one week after treated soils were
infested with larvae (F=6.41, 5 df, p=0.004 for Batch E050509, and
F=4.94, 5 df, p=0.011 for Batch E050516). When the data for both
batches were pooled, granules from Medium 4 and 5 were
significantly better than the rest (Tukey's HSD, p=0.05). By three
weeks after infestation, mortalities of larvae had reached 100% in
most of the treatments with significant differences among Media 2-6
disappearing; Medium 1 granules performed more poorly than the
rest.
EXAMPLE 4
Comparison of Efficacy of Microsclerotial Granules With
Conventional Nutritive Carrier Granules in Two Different Soils
[0040] A bioassay was also conducted to compare the strain F52
microsclerotial granules from Medium 5 with more conventional corn
grit-based granules that have been used in laboratory work and
field trials against the SBRM (Jaronski et al., 2006, Challenges in
using Metarhizium anisopliae for control of Sugarbeet Root Maggot,
Tetanops myopaeformis. Bulletin IOBC/wprs 30(7):119-124; Campbell
et al., 2006, Environmental Entomology. 35(4):986-991; Jaronski
& Campbell, 2006, 2005 Sugarbeet Research and Extension
Reports. 36:185-189; Majumdar et al., 2006, 2005 Sugarbeet Research
and Extension Reports. 36: 222-227). The evaluation was conducted
in two soils, a clay-loam soil collected from a sugarbeet field
near St. Thomas, N.D., and the clay soil used earlier. The corn
grit granules consisted of a 16/30 mesh corn grit carrier (Bunger
Milling, St. Louis, Mo.) coated with conidia using a 10% aqueous
polyoxyethylene sorbitan monooleate (TWEEN 80) binder. Target
concentration of conidia on these granules was 1-2.times.10.sup.5
conidia/granule. These corn grit-based granules were freshly
prepared using dry conidia produced with solid substrate
fermentation and refrigerated until use. The dry microsclerotial
granules from Medium 5 were sieved to 20/32 mesh size before use.
When placed in a sufficiently moist (A.sub.w>0.95) environment
such as water agar, moist soil, or moist filter paper, the corn
grit granules become covered with a second generation of conidia
within 7-10 days, while the microsclerotial granules sporulated
profusely within 3-4 days. The bioassay was conducted as described
earlier but with a rate of 112 mg granules/60 gram dry soil. Soils
were hydrated to 15% Field Capacity for each soil. This level of
moisture resulted in measured A.sub.w of 0.983 and 0.984 for the
two soils as determined by the Aqualab meter. Three replicate cups
of 10 larvae each were used for each treatment. SBRM larvae were
added after the soils had been incubated for 1 week at 24> C.
Larval mortalities were determined after 1, 2, and 3 weeks, as
described earlier.
Results
[0041] In both clay-loam and clay soils, the microsclerotial
granules from Medium 5 had significantly greater efficacy than the
more traditional conidia-covered corn grit granule. Mortality from
the microsclerotial granule was 100% within 1 week of infesting
treated soils with larvae (Table 6). In the clay soil, the corn
grit-based granules caused only a low larval mortality.
EXAMPLE 4
Comparison of Efficacy of Microsclerotial Granules With
Conventional Nutritive Carrier Granules At Different Soil
Moistures
[0042] Additional bioassays were conducted to compare the granules
from Medium 5 with the corn grit granules at several soil moisture
levels. Granules were incorporated into a clay soil at the rate of
1.8 mg/g soil, and the soils subsequently wetted to the desired
moisture endpoint with water. These assays were conducted as
described previously, with 3 replicate cups of 10 larvae each, per
treatment, except that clay soil was moistened to either 7.5%
(A.sub.w=0.836), 10% (A.sub.w=0.919), 15% (A.sub.w=0.983), or 20%
(A.sub.w=0.991) Field Capacity. Moisture levels were verified two
days after inoculation with conidia and hydration using an AQUALAB
water activity meter (Decagon Devices, Inc.) The entire assay was
replicated twice. Larval mortality was determined by destructive
sampling 3 weeks later. Any cadavers without sporulating fungus on
their exteriors were removed and incubated at high humidity for
three day to evince presence of mycosis.
Results
[0043] When the efficacy of microsclerotial granules from Medium 5
were compared with the corn grit granules at several soil moisture
levels, the former caused a significantly higher SBRM mortality at
moisture levels of 0.919 A.sub.w and above (FIG. 1). Larval
mortality was 100% vs. 20 and 30% for the corn grit based granules
at A.sub.w levels of 0.983 and 0.991. At a moisture of 0.919 Water
Activity units, larval mortality from exposure to the
microsclerotial granules was 20% vs. 6% for the corn grit granules.
Control mortality was less than 10% at all moisture levels.
Overall, the microsclerotial granules caused a much higher
mortality in these undersaturated soils because the microsclerotia
produced more infectious conidia faster than the conventional
granule formulation. These data underscore the superiority of
microsclerotial granules over conidia-containing nutritive
substrates.
[0044] It is understood that the foregoing detailed description is
given merely by way of illustration and that modifications and
variations may be made therein without departing from the spirit
and scope of the invention.
TABLE-US-00001 TABLE 1 Carbon concentration (g L.sup.-1) and
carbon-to-nitrogen ratio in liquid cultures used to assess the
growth and yields of different strains of M. anisopliae. Casamino C
Glucose Acids (g L.sup.-1) C:N (g L.sup.-1) (g L.sup.-1) 8 10:1
10.0 10.0 8 30:1 16.6 3.4 8 50:1 18.0 2.0 36 10:1 45.0 45.0 36 30:1
75.0 15.0 36 50:1 81.0 9.0
TABLE-US-00002 TABLE 2 Comparison of various media on the
production of biomass by Metarhizium anisopliae in liquid culture
after 2, 4, and 8-days growth. Metarhizium Carbon Carbon-to-
Biomass anisopliae Conc Nitrogen (mg/ml) Isolate Medium (g/l) Ratio
Day 2 Day 4 Day 8 Ma 1200 1 8 10:1 2.5a 5.5d 3.8d 2 8 30:1 2.5a
4.5d 5.0d 3 8 50:1 1.5a 4.5d 4.4d 4 36 10:1 2.7a 22.2a 26.6a 5 36
30:1 3.5a 14.8b 18.9b 6 36 50:1 2.1a 10.0c 13.3c F52 1 8 10:1 1.1c
8.2d 3.2c 2 8 30:1 3.4a 5.4e 5.5c 3 8 50:1 1.7c 4.0e 3.5c 4 36 10:1
2.0b,c 22.6a 33.0a 5 36 30:1 3.8a 19.7b 21.6b 6 36 50:1 3.2a,b
11.8c 18.3b TM109 1 8 10:1 0.8c 5.2c 4.5d 2 8 30:1 0.7c 3.1c,d 3.8d
3 8 50:1 0.6c 2.2d 4.0d 4 36 10:1 1.0b,c 12.7a,b 30.5a 5 36 30:1
1.7a 13.7a 24.0b 6 36 50:1 1.5a,b 10.6b 14.0c For each isolate,
mean values followed by different letters are significantly
different using Tukey-Kramer HSD. Mean values are derived from 6
values (3 separate experiments run in duplicate for each
treatment).
TABLE-US-00003 TABLE 3 Comparison of various media on the
production of microsclerotia by Metarhizium anisopliae in liquid
culture after 2, 4, and 8-days growth. Metarhizium Carbon
Carbon-to- Microsclerotia anisopliae Conc Nitrogen
(microsclerotia/ml .times. 10.sup.4) Isolate Medium (g/l) Ratio Day
2 Day 4 Day 8 Ma 1200 1 8 10:1 3.1a 10.6a,b 15.3a 2 8 30:1 0.5b
2.7c 6.4b,c 3 8 50:1 0.5b 2.4c 4.9c 4 36 10:1 1.7a,b 5.7b,c 12.0a,b
5 36 30:1 2.3a,b 7.7b,c 9.3a,b,c 6 36 50:1 2.1a,b 15.3a 14.7a F52 1
8 10:1 0.8b 10.5a 5.3b 2 8 30:1 1.7b 6.4a,b 11.7b 3 8 50:1 2.3b
5.0b 8.5b 4 36 10:1 8.0a 10.3a 9.3b 5 36 30:1 7.9a 11.0a 27.0a 6 36
50:1 9.5a 6.8a,b 29.0a TM109 1 8 10:1 0.0a 2.0a,b 1.2a 2 8 30:1
0.7a 1.2b 1.8a 3 8 50:1 0.6a 0.2b 1.0a 4 36 10:1 0.2a 1.9a,b 5.3a 5
36 30:1 0.5a 2.0a,b 3.7a 6 36 50:1 0.3a 4.9a 3.9a *For each
isolate, mean values followed by different letters are
significantly different using Tukey-Kramer HSD. Mean values are
derived from 6 values (3 separate experiments run in duplicate for
each treatment).
TABLE-US-00004 TABLE 4 Evaluation of the desiccation tolerance and
conidia production capability of air-dried microsclerotia of
Metarhizium anisopliae. Sporogenic Germination Hyphal (conidia/g
Metarhizium Carbon Carbon-to- Germination dried anisopliae Conc
Nitrogen (% formulate .times. Isolate Medium) (g/l) Ratio
microsclerotia) 10.sup.7) Ma 1 8 10:1 100a 24.0b 1200 2 8 30:1 100a
29.5b 3 8 50:1 87b 28.0b 4 36 10:1 100a 42.3b 5 36 30:1 100a 97.5a
6 36 50:1 99a 21.5b F52 1 8 10:1 99a 53.5b 2 8 30:1 96a 64.5b 3 8
50:1 99a 46.5b 4 36 10:1 100a 114.5a 5 36 30:1 100a 82.5a,b 6 36
50:1 97a 82.3a,b TM109 1 8 10:1 85a,b 18.1b 2 8 30:1 93a,b 15.4b 3
8 50:1 46b 16.0b 4 36 10:1 100a 81.3a,b 5 36 30:1 100a 94.3a 6 36
50:1 100a 63.8a,b For each isolate, mean values followed by
different letters are significantly different using Tukey-Kramer
HSD. Mean values are derived from 4 values (2 separate experiments
run in duplicate for each treatment).
TABLE-US-00005 TABLE 5 Mortality of third-instar Tetanops
myopaeformis larvae exposed to microsclerotial granule-treated
soils, 1, 2, and 3 weeks post-infestation. Microsclerotial granules
of Metarhizium anisopliae Strain F52 prepared from liquid cultures
produced in media 1-6. 1 Week 2 Weeks 3 Weeks Treatment Mortality*
Mycosis Mortality Mycosis Mortality Mycosis Test 1 (Batch 050509)
Medium 1 0% b -- 23.3% b 71.4% 46.7% b 71.4% Medium 2 6.7% ab 50.0%
50% ab 100% 96.7% a 100% Medium 3 10.0% ab 66.7% 63.3% a 100% 100%
a 100% Medium 4 36.7% a 100% 76.7% a 100% 100% a 100% Medium 5
26.7% a 75% 56.7% a 100% 96.7% a 100% Medium 6 6.7% ab 100% 70.0% a
100% 100% a 100% ANOVA F = 6.41, 5 df, F = 7.55, 5 df, F = 19.9, 5
df, statistics p = 0.004 p = 0.002 p < 0.001 Test 2 (Batch
050516) Medium 1 0.0% b 63.3% a 94.7% 86.7% a 85.7% Medium 2 6.7% b
100% 76.7% a 81% 96.7% a 100% Medium 3 0.0% b 53.3% a 81.3% 96.7% a
100% Medium 4 26.7% a 100% 70% a 100% 100% a 100% Medium 5 16.7% ab
80% 70% a 100% 90% a 100% Medium 6 13.3% ab 75% 73% a 100% 96.7% a
100% ANOVA F = 4.94, 5 df, F = 0.66, 5 df, F = 0.93, 5 df,
statistics p = 0.011 p = 0.66 p = 0.49 *Mortalities are the mean of
three replicates and if followed by different letter in a column,
are significantly different (Tukey's HSD, P < .05).
TABLE-US-00006 TABLE 6 Mortality of third instar larvae of Tetanops
myopaeformis exposed to microsclerotial granule-treated soils, 1 2,
and 3 weeks post-infestation. Mortalities are the mean of three
replicates (S.D.) and if followed by different letter in a column,
are significantly different (Tukey's HSD, P < .01). Mycosis was
100% among all cadavers. Mortality Soil Treatment 1 Week 2 Weeks 3
Weeks Clay Untreated 0% a 0% a 0% a Microsclerotial 100% b 100% c
100% c Granule Corn Grit 0% a 23.3% (15.3%)b 26.7% (15.3%)b Granule
Clay- Untreated 0% a 0% a 0% a loam Microsclerotial 100% b 100% c
100% c Granule Corn Grit 0% a 0% a 0% a Granule
* * * * *