U.S. patent application number 10/646987 was filed with the patent office on 2004-08-19 for mycopesticides.
This patent application is currently assigned to Myco Pesticides LLC. Invention is credited to Stamets, Paul Edward.
Application Number | 20040161440 10/646987 |
Document ID | / |
Family ID | 24721553 |
Filed Date | 2004-08-19 |
United States Patent
Application |
20040161440 |
Kind Code |
A1 |
Stamets, Paul Edward |
August 19, 2004 |
Mycopesticides
Abstract
The present invention utilizes the non-sporulating mycelial
stage of insect-specific parasitic fungi. The fungus can be present
on grain, attracting the pest, and also infecting it through
digestion. More than one fungus can be used in combination. The
matrix of fungi can be dried or freeze-dried, packaged and
reactivated for use as an effective bioinsecticide.
Inventors: |
Stamets, Paul Edward;
(Shelton, WA) |
Correspondence
Address: |
William R. Hyde
1833 10th Street
Penrose
CO
81240
US
|
Assignee: |
Myco Pesticides LLC
|
Family ID: |
24721553 |
Appl. No.: |
10/646987 |
Filed: |
August 20, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10646987 |
Aug 20, 2003 |
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09678141 |
Oct 3, 2000 |
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6660290 |
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Current U.S.
Class: |
424/405 ;
424/195.15; 424/93.5 |
Current CPC
Class: |
A01N 63/30 20200101;
A01N 63/30 20200101; A01N 63/32 20200101 |
Class at
Publication: |
424/405 ;
424/195.15; 424/093.5 |
International
Class: |
A01N 065/00; A01N
025/00; A01N 063/00 |
Claims
I claim:
1. A method for attracting social insects selected from the group
consisting of carpenter ants, fire ants, Coptotermes Formosan
termites and Reticulitermes termites, consisting essentially of
treating an infested locus with an effective dose of a preconidial
mycelia of an entomopathogenic fungi prior to the formation of
structures leading to the release of air-borne spores, wherein the
preconidial mycelia is grown on a solid culture media selected from
the group consisting of grains, sawdust, sugar cane, corn cobs,
cardboard, paper and cellulose containing substances, and wherein
the preconidial mycelia is provided in an amount sufficient to act
as both an insect attractant and an insect pathogen.
2. The method for attracting social insects of claim 1 wherein
hyphal fragments of the preconidial mycelia act as an initial
vector of parasitization.
3. The method for attracting social insects of claim 1, wherein the
preconidial mycelia is a Beauveria bassiana effective against
carpenter ants.
4. The method according to claim 1 wherein the preconidial mycelia
is metabolically arrested and subsequently metabolically
reactivated.
5. The method according to claim 4 wherein the preconidial mycelia
is metabolically arrested by a method selected from the group
consisting of freeze-drying and drying and is subsequently
metabolically reactivated by rehydration.
6. The method according to claim 4 wherein the preconidial mycelia
is metabolically arrested by a method selected from the group
consisting of refrigeration and cryogenic suspension and
subsequently metabolically reactivated by warming.
7. The method according to claim 1 wherein the preconidial mycelia
additionally comprises Metarhizium anisopliae.
8. A method for attracting carpenter ants, consisting essentially
of treating an infested locus with an effective dose of a
preconidial mycelia of an entomopathogenic fungi prior to the
formation of structures leading to the release of air-borne spores,
wherein the preconidial mycelia is provided in an amount sufficient
to act as both an insect attractant and an insect pathogen, wherein
the preconidial mycelia is a Beauveria bassiana grown on a solid
culture media, wherein the preconidial fungal mycelia is
metabolically arrested by a method selected from the group
consisting of freeze-drying, drying, refrigeration and cryogenic
suspension and subsequently metabolically reactivated by a method
selected from the group consisting of rehydration and warming, and
wherein the solid culture media is selected from the group
consisting of grain, sawdust, sugar cane, corn cobs, cardboard and
paper.
9. A method for attracting carpenter ants consisting essentially of
treating an infested locus with an effective dose of a preconidial
mycelia of an entomopathogenic fungi prior to the formation of
structures leading to the release of air-borne spores, wherein the
preconidial mycelia is a Beauveria bassiana effective against
carpenter ants, wherein the preconidial mycelia is provided in an
amount sufficient to act as both an insect attractant and an insect
pathogen and wherein the preconidial mycelia is grown on a solid
culture media is selected from the group consisting of grain,
sawdust, sugar cane, corn cobs, cardboard and paper.
Description
[0001] This application is a divisional of U.S. application Ser.
No. 09/678,141, filed Oct. 04, 2000, herein incorporated by
reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to the use of fungal mycelium
as a biopesticide. More particularly, the invention relates to the
control and destruction of insects, including carpenter ants, fire
ants, termites, flies, beetles, cockroaches and other pests, using
fungal mycelia as both attractant and infectious agent.
[0004] 2. Description of the Related Art
[0005] The use of chemical pesticides is the cause of many
secondary environmental problems aside from the death of the
targeted pest. Poisoning of soil and underlying aquifers may occur,
along with pollution of surface waters as a result of runoff.
Increases in cancer, allergies, immune disorders, neurological
diseases and even death in agricultural workers and consumers have
been attributable to the use of pesticides. Chemical pesticides are
increasingly regulated and even banned as a health risk to
citizens. Communities are increasingly in need of natural solutions
to pest problems.
[0006] Compounding these problems, many pest type or vermin insects
have developed a broad spectrum of resistance to chemical
pesticides, resulting in few commercially available pesticides that
are effective without thorough and repeated applications. In
addition to being largely ineffective and difficult and costly to
apply, chemical pesticides present the further disadvantage of
detrimental effects on non-target species, resulting in secondary
pest outbreaks. It is believed that widespread use of
broad-spectrum insecticides often destroys or greatly hampers the
natural enemies of pest species, and pest species reinfest the area
faster than non-target species, thereby allowing and encouraging
further pest outbreaks. There is therefore a particular need for
natural alternatives.
[0007] Biological control agents have been tried with varying
results. Bacteria such as Bacillus thuringiensis are used with some
success as a spray on plants susceptible to infestation with
certain insects. Fungal control agents are another promising group
of insect pathogens suitable for use as biopesticides for the
control of insects. However, limited availability, cost and
reliability have hampered the development of such fungal control
agents. Host range and specificity has been a problem as well as an
advantage; a fungal pathogen that is virulent and pathogenic to one
insect species may be ineffective against other species, even those
of the same genus. However, some success has been demonstrated.
[0008] The typical lifecycle of a pathogenic fungi control agent
involves adhesions of the spore(s) to the host insect cuticle,
spore germination and penetration of the cuticle prior to growth in
the hemocoel, death, saprophytic feeding and hyphal reemergence and
sporulation. For example, U.S. Pat. No. 4,925,663 (1990) to Stimac
discloses Beauveria bassiana used to control fire ants
(Solenopsis). Rice, mycelia and spores (conidia) mixture may be
applied to fire ants or used as a bait and carried down into the
nest, thereby introducing spores. U.S. Pat. No. 4,942,030 (1990) to
Osborne discloses control of whiteflies and other pests with
Paecilomyces fumosoroseus Apopka spore conidia formulations. The
Paecilomyces fungus is also useful for control of Diptera,
Hymenoptera, Lepidoptera, Bemisia, Dialeurodes, Thrips, Spodoptera
(beet army worm), Leptinotarsa (Colorado potato beetle), Lymantria
(Gypsy moth), Tetranychus, Frankliniella, Echinothrips, Planococcus
(Citrus mealybug) and Phenaococcus (Solanum mealybug). U.S. Pat.
No. 5,165,929 (1992) to Howell discloses use of Rhizopus nigricans
and other fungus in the order Mucorales as a fungal ant killer.
U.S. Pat. No. 5,413,784 (1995) to Wright et al. discloses
compositions and processes directed to the use of Beauveria
bassiana to control boll weevils, sweet potato whiteflies and
cotton fleahoppers. U.S. Pat. No. 5,683,689 (1997) to Stimac et al.
discloses conidial control of cockroaches, carpenter ants, and
pharaoh ants using strains of Beauveria bassiana grown on rice.
U.S. Pat. No. 5,728,573 (1998) to Sugiura et al. discloses
germinated fungi and rested spore termiticides of entomogenous
fungus such as Beauveria brongniartii, Beauveria bassiana,
Beauveria amorpha, Metarhizium anisopliae and Verticillium lecanii
for use against insects such as termites, cockroaches, ants, pill
wood lice, sow bugs, large centipedes, and shield centipedes. U.S.
Pat. No. 5,989,898 (1999) to Jin et al. is directed to packaged
fungal conidia, particularly Metarhizium and Beauveria. The
scientific journal literature also discusses similar uses of
conidial preparations.
[0009] One disadvantage to such approaches is that the fungal
lifecycle may be particularly sensitive to and dependent upon
conditions of humidity, moisture and free water, particularly
during the stages of germination, penetration of the cuticle prior
to growth, and hyphal reemergence and sporulation after death of
the insect.
[0010] Another continuing problem with existing techniques has been
inconsistent bait acceptance. Baits are often bypassed and left
uneaten. Such may be a particular problem with insects such as
termites, as opposed to house ants and cockroaches, because it is
usually not possible to remove competing food sources for termites.
Attractants and feeding stimulants have sometimes increased the
consistency of bait acceptance, but such increases cost and
complexity, and there remains a continuing need for improved baits
with improved bait acceptance.
[0011] A particular disadvantage with conidial fungal insect
preparations becomes apparent from U.S. Pat. No. 5,595,746 (1997)
to Milner et al. for termite control. Metarhizium anisopliae
conidia are disclosed and claimed as a termite repellant in
uninfested areas and as a termite control method in infested areas.
The difficulties of utilizing conidia or conidia/mycelium as a bait
and/or contact insecticide are readily apparent when considering
that conidia are effective as an insect repellant to termites and
are repellant in varying degrees to most or all targeted insect
pests. A repellant, of course, does not facilitate use as a bait or
contact insecticide. This may be a factor in explaining why fungal
insecticides have all too often proven more effective in the
laboratory, where conidia may be unavoidable in the testing chamber
or even directly applied to insects, than in the field.
[0012] U.S. Pat. No. 4,363,798 (1982) to D'Orazio is for termite
baits utilizing brown rot fungus as an attractant and toxicant
boron compounds in mixtures effectively sufficient to kill termites
without creating bait shyness. Brown-rot inoculated wood is ground
and mixed with cellulosic binder and boron compounds. Such an
approach has the disadvantage of utilizing toxic boron compounds.
In addition, the cultured mycelium must be further processed.
[0013] There is, therefore, a continuing need for enhancing the
effectiveness of entomopathogenic (capable of causing insect
disease) fungal products and methods. There is also a need for
enhancing the attractiveness of such fungal pesticides to insects.
There is also a need for improved packaging, shipping and delivery
methods.
[0014] In view of the foregoing disadvantages inherent in the known
types of fungal biocontrol agents, the present invention provides
improved fungal biocontrol agents and methods of using such
agents.
SUMMARY OF THE INVENTION
[0015] The present invention offers an environmentally benign
approach to insect control by attracting the insects who ingest
latent preconidial mycelium (which may be fresh, dried or
freeze-dried) which then infects the host. The preconidial mycelium
is both the attractant and the pathogenic agent. The infected
insects carrying the fungal hyphae become a vector back to the
central colony, further dispersing the fungal pathogen. Mycelium is
grown in pure culture using standard fermentation techniques for in
vitro propagation. The fermented mycelia is diluted and transferred
into a sterilized grain or a mixture of sterilized grains. Once
inoculated, the fermented mycelia matures to a state prior to
conidia formation. The preconidial mycelium may be utilized as is
or may be arrested in its development through flash chilling (or by
other means such as air-drying or refrigeration) and packaged in
spoilage-proof or sealed packages. The end-user facilitates opening
the package and placing the exposed mycelia-grain contents in the
vicinity of recent pest activity.
[0016] The present invention thus provides improved products and
methods wherein the fungal mycelium acts as bait and attractant and
as an ingested or food insecticide, palatable enough that insects
will readily consume it even in the presence of competing food
sources, with high recruitment of other insects among social
insects that exhibit such behavior. This results in multiple visits
to a highly attractive pathogenic bait, thereby providing effective
individual insect and/or colony inoculation.
[0017] The present invention further provides these and other
advantages with improved control of insect pests using fungal
insecticidal compositions (mycopesticides) having strong attractant
properties and placing these attractant mycopesticides in or around
an object or area to be protected. The present invention also
provides insecticidal baits which use, as a toxicant, relatively
innocuous, naturally occurring materials as the active agent, so as
to control insects without undue effect on the ecology. Finally, by
actively avoiding the use of conidia, the time and expense of
raising conidial stage mycelium and/or separating conidia is
avoided.
[0018] Still further objects and advantages of the present
invention will become more apparent from the following detailed
description and appended claims.
[0019] Before explaining the disclosed embodiments of the present
invention in detail, it is to be understood that the invention is
not limited in its application to the details of the particular
products and methods illustrated, since the invention is capable of
other embodiments. Also, the terminology used herein is for the
purpose of description and not of limitation.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0020] The present invention provides improved mycopesticides
(fungal mycelia utilized as insect biopesticides). The
attractiveness of fungal mycelia to many species is well known.
Black Angus cows have been observed running uphill (a rare event)
to reach spent Oyster mushroom mycelium on straw. Cultured mycelia
such as Morel mycelium is considered a delicacy when added to human
foods; gourmet mushrooms themselves are a form of mycelium
fruitbody. Indeed, the attractiveness of mycelial scents is to a
great degree responsible for the fresh and refreshing scent of a
forest after a rain, a result of the mushroom mycelia responding to
the humid conditions with rapid growth. Mycelium is also known to
be highly attractive to insects. Certain ants, termites and
wood-boring beetles are known to cultivate and raise fungal
mycelium as an exclusive food source ("ambrosia fungi") and
mycelium is a preferred food source of many insect species. As
discussed above, brown rot mycelium (the mycelial stage of a
wood-rotting type of fungus that produces polypore mushrooms) has
been used as an attractant for termites.
[0021] However, for insect control typical use of fungal pathogens
has involved use of either conidia (spores) or a mixture of conidia
and mycelium as a "contact insecticide" control agent. Such
conidial contact insecticides suffer two major disadvantages: 1)
conidia and conidia/mycelium preparations are to some degree
unattractive or even repellant to insects; and 2) such conidia
preparations are highly dependent on free water or humid conditions
for gestation and infestation during the typical life cycle of an
insect fungal control agent. Furthermore, conidia have been found
to be more effective against "stressed" insects and/or insect
populations than against healthy insects and populations. For these
and other reasons, conidia of entomopathogenic fungi have often
been much more effective under laboratory conditions than in the
field.
[0022] Noting that conidia have been utilized as a repellant for
termites, further investigation of the preconidial and conidial
stages were undertaken. The preconidial stage is the vegetative
stage of the fungus, prior to the formation of structures leading
to the release of air-borne spores (which is distinguished from
fragmentation of hyphae which can become airborne if dried). Those
skilled in the art will recognize that mycelia or mycelial hyphal
fragments may form structures such as arthrospores (a preconidial
structure imbedded within the mycelia) and such should be
considered a "preconidial mycelium" as discussed elsewhere. It was
found that the "fragrance signature" of the mycelium is a strong
attractant to insects, but only prior to conidia formation. After
conidia formulation, the conidia/mycelium was found to be repellant
to insects such as carpenter ants. The odor was found to be
similarly pleasing to humans when preconidial and repellant when
post-conidial. It was noted such fragrance signatures are "washed
away" or lost when mycelium is grown via liquid fermentation. It
was also noted liquid fermentation utilizing a typical fermentor
with bubbled air mixing will promote conidia formation, with such
conidia production being even further promoted by the common
commercial practice of utilizing bubbled oxygen.
[0023] It was further found that fungal control agents are much
more effective when preconidial mycopesticidal mycelium is ingested
by the targeted insect as compared to conidia or post-conidial
mycelium/conidia offered to targeted insects for the purpose of
infection by contact. Whereas conidia have little or no effect by
ingestion or vapor, preconidial mycelium has proven to be highly
effective by ingestion, the mycelial hyphae already being in a
state of active growth when ingested. Furthermore, whereas conidial
preparations are more dependent upon humidity in the insect
environments, a preconidial mycopesticidal mycelium which is eaten
by an insect is dependent upon humidity only in the immediate
vicinity of the mycelium, the humidity of the mycelium of course
being much more easily controlled than in the wider general insect
environment.
[0024] It has further been found that the preconidial stage can be
maintained provided carbon dioxide (CO.sub.2) levels are maintained
at an elevated level. The CO.sub.2 levels preferably range from
2,000-200,000 ppm, more preferably in the range of 10,000-50,000+
ppm. Once exposed to fresh air, the mycelium can produce conidia in
just a few days. By preventing conidial formation, the mycelium
continues to accumulate mycelial biomass (sans conidia). Even after
maturation, the mycopesticidal mycelium may be maintained in a
preconidial state via elevated carbon dioxide levels. This
prevention of conidia formation is an active component in this
technology, as conidia formation is actively avoided.
[0025] Mycopesticidal mycelium is grown in pure culture using
standard fermentation techniques well established for in vitro
propagation. The fermented mycelia is diluted and transferred into
a sterilized grain or a mixture of sterilized grains (rice, wheat,
rye, oat, millet, sorghum, corn, barley, etc. The grain is pressure
steam-sterilized at 1 kg/cm.sup.2 (15 psi) for several hours. The
master broth is transferred aseptically manually or by using
peristaltic pumps into the sterilized grain. Growth mediums
containing sawdust, sugar cane, corn cobs, cardboard, paper or
other substances containing cellulose may be utilized for cellulose
loving insects such as termites if desired. A variety of containers
are used for incubation, including high-density polyethylene and
polypropylene bags, glass and polypropylene jars, metal containers,
etc.). Use of such containers provides a convenient method of
maintaining high CO.sub.2 levels, as the growing mycelium gives off
carbon dioxide. CO.sub.2 levels will rise to acceptable levels for
use in the present invention even if filter patches, disks or
materials are utilized to allow some gas exchange. Alternatively,
grow rooms may be maintained at high CO.sub.2 levels. Further
information on such culture techniques may be found in the
applicant's books, Growing Gourmet and Medicinal Mushrooms (1993,
2000) and The Mushroom Cultivator (1983) (with J. Chilton), and in
standard microbiology manuals.
[0026] Once inoculated, the mycelia on grain matures to a state
prior to conidia formation and may be utilized fresh or
metabolically arrested or developmentally arrested through flash
chilling (freeze-drying), drying, refrigeration or by other means.
It will be understood that such metabolic arresting of development
may encompass either a slowing of metabolism and development (such
as refrigeration) or a total suspension or shutdown of metabolism
(freeze-drying, air-drying and cryogenic suspension). When
freeze-drying, drying or other known methods of arresting
development are utilized, it is essential that freeze-drying or
other methods occur at an early stage in the life cycle of these
fungi before the repellant spores are produced. The
mycelium-impregnated grain media may then be fragmented and packed
in appropriate containers for commerce. Fresh mycelium may be
shipped in growing containers such as jars or spawn bags, which
allows easy maintenance of a high carbon dioxide atmosphere and
maintenance of sterile conditions during shipping. It is preferable
that the mycelium be utilized or processed while vigorous, before
it "over-matures" and becomes less viable for lack of new food to
digest and accumulation of waste products.
[0027] When the freeze-dried or dried mycelium is reactivated via
rehydration, the mycelium is preferably allowed to slowly rehydrate
through controlled absorption of atmospheric humidity, with the
result that the mycelium "wakes up" and wicks into the air. This is
a totally different response from immersion, which often results in
bacterial contamination and souring, as the freeze-dried mycelium
suffers when immersed in water. Such rehydration and reactivation
may be carried out on a large scale through high humidity
atmosphere, or may be accomplished by an end user through use of
wet materials such as sponges, wicking materials and/or other
evaporative materials or by atmospheric absorption of humidity from
a remote water reservoir. Such end user rehydration may be carried
out in any suitable container or a bait box if desired. Warming is
suitable for reactivation of refrigerated materials; it is
preferred that the mycelium not be refrigerated for extended
lengths of time.
[0028] Novel features of the invention include the use of a vector
of parasitization that relies on hyphal fragments, not spores or
conidia; the use of a single mycelium as both attractant fungus and
pathogen; the use of high levels of CO.sub.2 to grow and maintain
preconidial mycelium; and the preferred use of various methods to
arrest development at the preconidial stage to facilitate growth,
packaging, shipping and convenient application by an end user. More
than one fungus can be used to create a matrix of characteristics
to increase usefulness as a natural pesticide.
[0029] In general, preferred mycopesticidal species as pathogens
are somewhat slow-acting (that is, not immediately fatal), so as to
avoid bait shyness and to avoid learning effects in social insects
before individuals have distributed mycelium to other members of
the colony. In many applications it may be preferable to utilize a
mixture or matrix of several species of entomopathogenic fungus
with different characteristics, maturation and growth rates,
preferred conditions, virulence and pathogenicity, time to insect
death, etc., while in other applications a single species may be
preferred. Similarly, with reference to a single species, a mixture
of strains or a single strain may be utilized. Those skilled in the
art will recognize that such characteristics can be selected for
utilizing known techniques and bioassays. The mycopesticides
disclosed herein may also be optionally enhanced by the use of
other baits, attractants, arrestants, feeding stimulants, sex
pheromones, aggregating pheromones, trail pheromones, etc.
[0030] There are numerous entomogenous and entomopathogenic fungal
species known. Those skilled in the art will recognize that the
above preconidial fungi methods and products may be favorably
applied to all such insecticidal fungal species, and it is the
intent of the inventor that the invention be understood to cover
such. Suitable entomopathogenic fungi include Metarhizium,
Beauveria, Paecilomyces, Hirsutella, Verticillium and other fungi
imperfecti, the Entomophthoracae and other Phycomycetes, and
sexually reproducing fungi such as Cordyceps and other
Ascomycetes.
[0031] By way of example, but not of limitation, preferred
mycopesticides include Metarhizium anisopliae ("green muscarine"
for pests such as carpenter ants, including Camponotus modoc, C.
vicinus, C. ferrugineus, C. floridanus, C. pennsylvanicus, C.
herculeanus, C. varigatus and C. vicinus, fire ants (Solenopsis
invicta and Solenopsis richteri), termites, including Coptotermes,
Reticulitermes, Cryptotermes, Incisitermes, Macrotermes and
Odontotermes, pasture scarabs such as Adoryphorus couloni, spittle
bug Mahanarva posticata, corn earworm Helicoverpa zea, tobacco
hornworm Manduco sexta, sugar cane froghopper, pill wood lice, sow
bugs, large centipedes, shield centipedes, wheat cockchafer, beetle
grubs, greenhouse pests such as Coleoptera and Lepidoptera, etc.);
Metarhizium flaviride (grasshoppers and locusts); Beauveria
bassiana ("white muscarine" for termites including Formosan
termites, carpenter ants, fire ants, pharaoh ants, cockroaches,
whiteflies, thrips, aphids, mealybugs, boll weevils, sweet potato
whiteflies, cotton fleahoppers, European and Asiatic corn borers
and larvae of other Lepidoptera, codling moth, chinch bug,
soft-bodied insects in the orders Homoptero and Coleoptera,
Heteroptera, etc.); Beauveria brongniartii (white grubs and
cockchafers, Hoplochelis marginalis, Melolontha melontha);
Paecilomyces fumosoroseus (whiteflies, thrips, aphids, spider
mites, mealybugs, beet army worm, Colorado potato beetle, Gypsy
moth, etc.); Verticillium lecanii (greenhouse pests, whiteflies and
aphids); Hirsutella citriformis (rice brown planthopper);
Hirsutella thompsoni (citrus rust mite); and the wide variety of
Cordyceps for baiting and killing pests such as beetles, flies,
cockroaches, earwigs (Forficula auricularia), carpenter ants and
various other insect pests, including Cordyceps variabilis,
including imperfect forms (fly larvae, Xylophagidae family of the
Diptera order), Cordyceps facis and C. subsessilis, (beetle larvae
in the coleopteran family, Scarabaeidae), Cordyceps myrmecophila
(ants); Cordyceps sphecocephala (wasps), Cordyceps entomorrhiza
(beetle larvae), Cordyceps gracilis (larvae of beetles and moths),
Cordyceps militaris, Cordyceps washingtonensis, Cordyceps
melolanthae (beetles and beetle grubs), Cordyceps ravenelii (beetle
grubs), Cordyceps unilateralis (ants, carpenter ants, bees and
wasps) and Cordyceps clavulata (scale insects).
[0032] With regard to the sexually reproducing Cordyceps,
preconidial or pre-sporulation refers to the pre-fruiting state.
The term "preconidial" has a somewhat different meaning than with
most other entomopathogenic fungi, as Cordyceps is a "fungi
perfecti" or mushroom fungi, whereas the other non-mushroom fungi
referred to herein are the more primitive "fungi imperfecti." Some
or all Cordyceps fungi are believed to be anamorphic or dimorphic
and have conidial stages within the imperfect fungal genera
including Beauveria, Metarhizium, Paecilomyces, Hirsutella,
Verticillium, Aspergillus, Akanthomyces, Desmidiospora,
Hymenostilbe, Mariannaea, Nomuraea, Paraisaria, Tolypocladium,
Spicaria (=Isaria) and Botrytis. For example, C. subsessillis is
the perfect form of Tolypocladium inflatum, an anamorph (imperfect)
form which produces cyclosporin. Hodge et al., Tolypocladium
inflatum is the anamorph of Cordyceps subsessilis. Mycologia 88(5):
715-719 (1996). Cordyceps militaris (Fr.) Lk. is also thought to be
dimorphic, the conidial stage of which is believed to be a
Cephalosporium. DNA studies are expected to better elucidate these
relationships. As a further complexity, in addition to possible
anamorphs and dimorphs, Cordyceps species also demonstrate
nonsexual imperfect stages of development. As used herein, unless
otherwise specified, preconidial Cordyceps refers to the
pre-sporulation mycelial stage of the Cordyceps mushrooms,
including any preconidial imperfect stages, but not any conidia
bearing imperfect stages.
[0033] For initial experimentation, a Metarhizium anisopliae from
naturally occurring sources and the carpenter ant were selected. M.
anisopliae was obtained from a public culture collection and used
without further selection for virulence and/or pathogenicity; a
publicly available strain free of proprietary or patent
restrictions on use was selected as offering a preferred source and
a more demanding initial test than strains selected for specific
pathogenicity. It will be understood, of course, that strains
selected for specific characteristics and pathogenicity against
specific insects will in general offer the best mode of practicing
the invention. The carpenter ant offered several advantages: ants
are typically more resistant to spores than termites and other
insects, carpenter ants are a very destructive pest, the effect on
other ant species could also be viewed, and the applicant enjoyed
easy access to an experimental site as his residence was in danger
of collapse due to long term structural infestation by carpenter
ants.
EXAMPLE 1
[0034] Metarhizium anisopliae was grown in pure culture using
standard fermentation techniques and diluted and aseptically
transferred to grain (rice) which had been pressure
steam-sterilized at 1 kg/cm.sup.2 (15 psi). The fermented mycelia
matured to a state prior to conidia formation and the fungus
colonized grain was offered at the site of debris piles caused by
carpenter ants at the 1,100-1,200 sq. ft. house of the applicant's
residence located in Shelton, Wash., U.S.A. Approximately 10-20
grams of preconidial mycelium of Metarhizium anisopliae, grown on
autoclaved rice and having been incubated for two weeks, was
presented at the location of debris piles next to the interior face
of an exterior wall within the house. The non-sporulating mycelium
was presented on a dollhouse dinner dish and left exposed to the
air. Later that night, the applicants' daughter urgently awoke the
applicant when she observed carpenter ants feasting en masse on the
non-sporulating mycelium of the presented Metarhizium. The
applicant and his family observed approximately a dozen carpenter
ants ingesting mycelium and retreating into the wall, carrying the
infectious mycelium with them. In a week's time, the carpenter ant
colony became inactive, killing the nest of ants, and no evidence
of carpenter ant activity was observed henceforth, saving the
structure from further structural damage. Months later, the
ecological niche once occupied by the carpenter ants was taken over
by common household Sugar and Honey ants which were unaffected by
the Metarhizium anisopliae.
EXAMPLE 2
[0035] Cultivate strains of Metarhizium, Beauveria and Cordyceps on
grain as above under high CO.sub.2 conditions to produce
preconidial mycelium. Freeze-dry and rehydrate. Apply as bait and
pathogen at locations infested by insects such as carpenter ants,
termites, beetles, flies, fire ants, cockroaches and other insect
pests and vermin.
EXAMPLE 3
[0036] Drill one or more holes into a termite colony mound or tree
mound. Insert entomopathogenic preconidial mycopesticidal mycelium
into the holes. Cover the holes to prevent entry of marauding
ants.
[0037] No limitations with respect to the specific embodiments
disclosed herein is intended or should be inferred. While preferred
embodiments of the present invention have been shown and described,
it will be apparent to those skilled in the art that many changes
and modifications may be made without departing from the invention
in its broader aspects. The appended claims are therefore intended
to cover all such changes and modifications as fall within the true
spirit and scope of the invention.
* * * * *