U.S. patent application number 14/247207 was filed with the patent office on 2014-08-07 for integrative fungal solutions for protecting bees and overcoming colony collapse disorder (ccd): methods and compositions.
The applicant listed for this patent is Paul Edward Stamets. Invention is credited to Paul Edward Stamets.
Application Number | 20140220150 14/247207 |
Document ID | / |
Family ID | 49477505 |
Filed Date | 2014-08-07 |
United States Patent
Application |
20140220150 |
Kind Code |
A1 |
Stamets; Paul Edward |
August 7, 2014 |
Integrative fungal solutions for protecting bees and overcoming
colony collapse disorder (CCD): methods and compositions
Abstract
The present invention is based on a plurality of benefits from
the extracts of mycelia of individual fungal species, and their
mixtures, to provide an armamentarium of defenses from multiple
stressors in order to help bees survive and more particularly
overcome a complex of symptoms collectively called colony collapse
disorder (CCD). The methods and compositions of this designed
fungal bioshield helps tilt the balance in favor of bee colony
survival, improving the health conditions, disease resistance,
pesticide tolerance, pollution tolerance, drought tolerance,
pollination abilities, and quality of the honey produced by
bees.
Inventors: |
Stamets; Paul Edward;
(Shelton, WA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Stamets; Paul Edward |
Shelton |
WA |
US |
|
|
Family ID: |
49477505 |
Appl. No.: |
14/247207 |
Filed: |
April 7, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13986978 |
Jun 20, 2013 |
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14247207 |
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13066566 |
Apr 18, 2011 |
8501207 |
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13986978 |
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12288535 |
Oct 20, 2008 |
7951389 |
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13066566 |
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10853059 |
May 24, 2004 |
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12288535 |
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09969456 |
Oct 1, 2001 |
7122176 |
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10853059 |
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09678141 |
Oct 4, 2000 |
6660290 |
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09969456 |
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13998914 |
Dec 20, 2013 |
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09678141 |
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12284646 |
Sep 24, 2008 |
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13998914 |
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11728613 |
Mar 27, 2007 |
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12284646 |
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11386402 |
Mar 22, 2006 |
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11728613 |
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11145679 |
Jun 6, 2005 |
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11386402 |
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11029861 |
Jan 4, 2005 |
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11145679 |
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13373719 |
Nov 28, 2011 |
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11029861 |
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13317613 |
Oct 24, 2011 |
8753656 |
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13373719 |
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13066566 |
Apr 18, 2011 |
8501207 |
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13317613 |
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12288535 |
Oct 20, 2008 |
7951389 |
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13066566 |
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10853059 |
May 24, 2004 |
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12288535 |
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09969456 |
Oct 1, 2001 |
7122176 |
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10853059 |
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09678141 |
Oct 4, 2000 |
6660290 |
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09969456 |
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12284646 |
Sep 24, 2008 |
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13317613 |
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11728613 |
Mar 27, 2007 |
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12284646 |
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11386402 |
Mar 22, 2006 |
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11728613 |
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11145679 |
Jun 6, 2005 |
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11386402 |
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11029861 |
Jan 4, 2005 |
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11145679 |
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61967117 |
Mar 10, 2014 |
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60994972 |
Sep 24, 2007 |
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60534776 |
Jan 6, 2004 |
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60994972 |
Sep 24, 2007 |
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60534776 |
Jan 6, 2004 |
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Current U.S.
Class: |
424/537 ;
424/195.15 |
Current CPC
Class: |
A61K 45/06 20130101;
A01N 63/30 20200101; A61K 35/644 20130101; A01N 63/30 20200101;
A01N 63/30 20200101; A01N 2300/00 20130101; A01N 63/30 20200101;
A01N 2300/00 20130101; A01N 63/30 20200101; A61K 36/07 20130101;
A01N 65/00 20130101 |
Class at
Publication: |
424/537 ;
424/195.15 |
International
Class: |
A61K 36/07 20060101
A61K036/07; A61K 45/06 20060101 A61K045/06; A61K 35/64 20060101
A61K035/64 |
Claims
1. A composition for improving bee health and helping to prevent
colony collapse disorder among bees comprising a nutraceutical
product selected from the group consisting of an extract of a
mushroom mycelium, a derivative of an extract of a mushroom
mycelium and combinations thereof, combined with a bee product
selected from the group consisting of sugar syrup, high fructose
corn syrup water, bee candy, pollen patties, grease patties,
propolis, bees wax, bee sprays, bee feed, sticky strips and
combinations thereof.
2. The composition of claim 1 wherein the extract of a mushroom
mycelium and the derivative of an extract of a mushroom mycelium is
selected from the group of mycelium derivatives consisting of an
aqueous extract of mushroom mycelium, an aqueous ethanolic extract
of mushroom mycelium, precipitate from an aqueous extract of
mushroom mycelium precipitated by addition of ethanol, supernatant
remaining after the precipitate from an aqueous extract of mushroom
mycelium precipitated by addition of ethanol is removed,
precipitate from an aqueous ethanolic extract of mushroom mycelium
allowed to sit, concentrate from aqueous ethanolic extract of
mushroom mycelium which has had a portion of the solvent removed,
supernatant from aqueous ethanolic extract which has had a portion
of solvent removed, an extract of mushroom mycelium from which
solvent has been removed and combinations thereof.
3. The composition of claim 1 wherein the extract of a mushroom
mycelium is an extract of Stropharia rugoso-annulata mycelium.
4. The composition of claim 1 wherein the mushroom mycelium is
selected from the group consisting of Polyporales mushroom
mycelium, Hymenochaetales mushroom mycelium, Agaricales mushroom
mycelium and combinations thereof.
5. The composition of claim 4 wherein Polyporales and
Hymenochaetales are selected from the group consisting of Antrodia,
Coriolus, Daedalea, Fomes, Fomitopsis, Fomitoporia, Ganoderma,
Grifola, Inonotus, Heterobasidion, Irpex, Ischnoderma, Laetiporus,
Lenzites, Merilipus, Phaeolus, Phanerochaete, Pycnoporus, Phaeolus,
Phellinus, Polyporus, Schizophyllum, Sparassis, Stereum, Trametes,
Trichaptum, Tyromyces, Wolfiporia and combinations thereof, and
Agaricales are selected from the group consisting of Agaricus,
Agrocybe, Amanita, Armillaria, Clitocybe, Conocybe, Coprinus,
Coprinopsis, Galerina, Gymnopus, Hypholoma, Hypsizygus, Lentinus,
Lepiota, Lepista, Mycena, Panaeolus, Pleurotus, Panellus, Pluteus,
Psathyrella, Psilocybe, Stropharia, Termitomyces, Volvaria,
Volvariella and combinations thereof.
6. The composition of claim 4 wherein Polyporales and
Hymenochaetales are selected from the group consisting of Antrodia
cinnomonea, Ganoderma applanatum, Ganoderma atrum, Ganoderma
brownii, Ganoderma curtisii, Ganoderma lucidum, Ganoderma lingzhi,
Ganoderma oregonense, Ganoderma resinaceum, Ganoderma tsugae, Fomes
fomentarius, Fomitopsis officinalis, Fomitopsis pinicola,
Fomitiporia robusta, Heterobasidion annosum, Inonotus hispidus,
Inonotus johnsonii, Inonotus obliquus, Laetiporus cincinnatus,
Laetiporus sulphureus, Laetiporus conifericola, Lenzites betulina,
Phanerochaete chrysosporium, Phaeolus schweinitzii, Phellinus
igniarius, Phellinus linteus, Phellinus pini, Polyporus elegans,
Phanerochaetes chrysosporium, Phaeolus schweitnitzii, Schizophyllum
commune, Stereum complicatum, Stereum hirsutum, Stereum ostrea,
Trametes elegans, Trametes versicolor, Trametes gibbosa, Trametes
hirsuta, Trametes villosa, Tramets cingulata, Wolfiporia cocos and
combinations thereof, and Agaricales are selected from the group
consisting of Agaricus augustus, Agaricus blazei, Agaricus
bonardii, Agaricus brasiliensis, Agaricus campestris, Agaricus
lilaceps, Agaricus placomyces, Agaricus subrufescens, Agaricus
sylvicola, Agrocybe pediades, Agrocybe aegerita, Agrocybe arvalis,
praecox, Amanita muscaria, Amanita gemmata, Amanita pantherina,
Amanita phalloides, Amanita virosa, Amanita pachycolea, Amanita
vaginata, Clitocybe odora, Clitocybe dealbata, Clitocybe dilitata,
Conocybe cyanopus, Conocybe lacteus, Conocybe rickenii, Conocybe
smithii, Conocybe tenera, Coprinopsis atrementaria, Coprinopsis
nivea, Coprinopsis lagopus, Coprinus comatus, Coprinus micaceus,
Galerina autumnalis, Galerina marginata, Galerina venenata,
Gymnopus hydrophilus, Gymnopilus peronatus, Hypholoma aurantica
(Leratiomyces ceres), Hypholoma capnoides, Hypholoma fasciculare,
Hypholoma sublateritium, Hypsizygus marmoreus, Hypsizygus
tessulatus, Hypsizygus ulmarius, Lentinus ponderosus, Lepiota
procera (Macrolepiota procera), Lepiota rachodes (Chlorophyllum
rachodes), Lepista nuda, Mycena alcalina, Mycena pura, Mycena
aurantiadisca, Panellus serotinus, Panaeolus foenisecii, Panaeolus
subbalteatus, Pleurotus columbinus, Pleurotus ostreatus, Pleurotus
cystidiosus, Pleurotus pulmonarius, Pleurotus sapidus, Pleurotus
tuberregium, Panellus stipticus, Panellus serotinus, Pluteus
cervinus, Psathyrella aquatica, Psathyrella condolleana,
Psathyrella hydrophila, Psilocybe allenii, Psilocybe coprophila,
Psilocybe cubensis, Psilocybe cyanescens, Psilocybe
ovoideocystidiata, Stropharia aeruginosa, Stropharia coronilla,
Stropharia rugoso-annulata, Stropharia semiglobata, Stropharia
semigloboides, Stropharia squamosa, Stropharia thrausta, Stropharia
umbonotescens, Termitomyces robusta, Volvaria bombycina,
Volvariella volvacea and combinations thereof.
7. The composition of claim 1 wherein the extract of a mushroom
mycelium is selected from the group consisting of extract of
Ganoderma resinaceum mycelium, extract of Ganoderma lucidum
mycelium and combinations thereof, wherein the extract is
concentrated into a form with variable viscosities resembling a
form of honey and is used as a honey substitute.
8. The composition of claim 1 where the extract of a mushroom
mycelium is concentrated to the consistency of honey and is used as
a honey substitute.
9. The composition of claim 1 wherein the mushroom mycelium is
mycelium of a mushroom species that will not form primordia unless
the mushroom mycelium is exposed to light selected from the group
consisting of ultraviolet light and 360 nanometer or lower
wavelength light.
10. The composition of claim 1 wherein the mushroom mycelium is a
brightly colored mushroom mycelium.
11. The composition of claim 10 wherein the brightly colored
mushroom mycelium is yellow.
12. The composition of claim 1 wherein the composition additionally
comprises a compound selected from the group consisting of extracts
from Neem trees, derivatives from Neem trees and combinations
thereof.
13. The composition of claim 1 where in the composition
additionally comprises tree resins.
14. The composition of claim 1 wherein the composition additionally
comprises a miticide, wherein the miticide is selected from the
group consisting of synthetic miticides, natural miticides and
combinations thereof, and wherein the natural miticides are
selected from the group consisting of oxalic acid, formic acid,
lactic acid, spores of entomopathogenic fungi pathogenic to mites,
hyphae of entomopathogenic fungi pathogenic to mites, preconidial
mycelium of entomopathogenic fungi pathogenic to mites, extracts of
preconidial mycelium of entomopathogenic fungi pathogenic to mites
and combinations thereof, and wherein the spores of
entomopathogenic fungi pathogenic to mites are selected from the
group consisting of Metarhizium anisopliae spores, Beauveria
bassiana spores, Entomophthorales spores and combinations
thereof.
15. A composition for improving and up-regulating the metabolic,
immune and detoxification systems of bees, including antiviral,
antibacterial, antifungal and antiprotozoal properties comprising a
derivative of a mycelium of a mushroom fungus combined with a
compound that benefits bees selected from the group consisting of
sugar syrup, high fructose corn syrup water, bee candy, pollen
patties, grease patties, bee sprays, plant sprays for plants of
benefit to bees, neem extracts, neem derivatives, tree resins,
miticides, bees wax, spores and preconidial mycelium of
entomopathogenic fungi, sticky strips and combinations thereof.
16. The composition of claim 15 wherein the derivative of a
mycelium of a mushroom fungus is selected from the group of
mycelium derivatives consisting of an aqueous extract of mushroom
mycelium, an aqueous ethanolic extract of mushroom mycelium,
precipitate from an aqueous extract of mushroom mycelium
precipitated by addition of ethanol, supernatant remaining after
the precipitate from an aqueous extract of mushroom mycelium
precipitated by addition of ethanol is removed, precipitate from an
aqueous ethanolic extract of mushroom mycelium allowed to sit,
concentrate from aqueous ethanolic extract of mushroom mycelium
which has had a portion of solvent removed, supernatant from
aqueous ethanolic extract which has had a portion of solvent
removed, an extract of mushroom mycelium from which solvent has
been removed and combinations thereof.
17. The composition of claim 15 wherein the derivative of a
mycelium of a mushroom fungus is an extract of Stropharia
rugoso-annulata mycelium.
18. The composition of claim 15 wherein the derivative of a
mycelium of a mushroom fungus is selected from the group consisting
of derivatives of Polyporales mushroom mycelium, Hymenochaetales
mushroom mycelium, Agaricales mushroom mycelium and combinations
thereof, wherein Polyporales and Hymenochaetales are selected from
the group consisting of Antrodia, Coriolus, Daedalea, Fomes,
Fomitopsis, Fomitoporia, Ganoderma, Grifola, Inonotus,
Heterobasidion, Irpex, Ischnoderma, Laeitoporus, Lenzites,
Merilipus, Phaeolus, Phanerochaete, Pycnoporus, Phaeolus,
Phellinus, Polyporus, Schizophyllum, Sparassis, Stereum, Trametes,
Trichaptum, Tyromyces, Wolfiporia and combinations thereof, and
wherein Agaricales are selected from the group consisting of
Agaricus, Agrocybe, Amanita, Armillaria, Clitocybe, Conocybe,
Coprinus, Coprinopsis, Galerina, Gymnopus, Hypholoma, Hypsizygus,
Lentinus, Lepiota, Lepista, Mycena, Panaeolus, Pleurotus, Panellus,
Pluteus, Psathyrella, Psilocybe, Stropharia, Termitomyces,
Volvaria, Volvariella and combinations thereof.
19. The composition of claim 15 wherein the derivative of a
mycelium of a mushroom fungus is selected from the group consisting
of Polyporales mushroom mycelium, Hymenochaetales mushroom
mycelium, Agaricales mushroom mycelium and combinations thereof,
and wherein Polyporales and Hymenochaetales are selected from the
group consisting of Antrodia cinnomonea, Ganoderma applanatum,
Ganoderma brownii, Ganoderma curtisii, Ganoderma lucidum, Ganoderma
lingzhi, Ganoderma oregonense, Ganoderma resinaceum, Ganoderma
tsugae, Fomes fomentarius, Fomitopsis officinalis, Fomitopsis
pinicola, Fomitiporia robusta, Heterobasidion annosum, Inonotus
hispidus, Inonotus johnsonii, Inonotus obliquus, Laetiporus
cincinnatus, Laetiporus sulphureus, Laetiporus conifericola,
Lenzites betulina, Phanerochaete chrysosporium, Phaeolus
schweinitzii, Phellinus igniarius, Phellinus linteus, Polyporus
elegans, Phanerochaetes chrysosporium, Phaeolus schweitnitzii,
Stereum complicatum, Stereum hirsutum, Stereum ostrea, Trametes
elegans, Trametes versicolor, Wolfiporia cocos and combinations
thereof, and wherein Agaricales are selected from the group
consisting of Agaricus augustus, Agaricus blazei, Agaricus
bonardii, Agaricus brasiliensis, Agaricus campestris, Agaricus
lilaceps, Agaricus placomyces, Agaricus subrufescens, Agaricus
sylvicola, Agrocybe pediades, Agrocybe aegerita, Agrocybe arvalis,
praecox, Amanita muscaria, Amanita gemmata, Amanita pantherina,
Amanita phalloides, Amanita virosa, Amanita pachycolea, Amanita
vaginata, Clitocybe odora, Clitocybe dealbata, Clitocybe dilitata,
Conocybe cyanopous, Conocybe lacteus, Conocybe rickenii, Conocybe
smithii, Conocybe tenera, Coprinopsis atrementaria, Coprinopsis
nivea, Coprinopsis lagopus, Coprinus comatus, Coprinus micaceus,
Galerina autumnalis, Galerina marginata, Galerina venenata,
Gymnopus hydrophilus, Gymnopilus peronatus, Hypholoma aurantica
(Leratiomyces ceres), Hypholoma capnoides, Hypholoma fasciculare,
Hypholoma sublateritium, Hypsizygus marmoreus, Hypsizygus
tessulatus, Hypsizygus ulmarius, Lentinus ponderosus, Lepiota
procera (Macrolepiota procera), Lepiota rachodes (Chlorophyllum
rachodes), Lepista nuda, Mycena alcalina, Mycena pura, Mycena
aurantiadisca, Panellus serotinus, Panaeolus foenisecii, Panaeolus
subbalteatus, Pleurotus columbinus, Pleurotus ostreatus, Pleurotus
cystidiosus, Pleurotus pulmonarius, Pleurotus sapidus, Pleurotus
tuberregium, Panellus stipticus, Panellus serotinus, Pluteus
cervinus, Psathyrella aquatica, Psathyrella condolleana,
Psathyrella hydrophila, Psilocybe allenii, Psilocybe coprophila,
Psilocybe cubensis, Psilocybe cyanescens, Psilocybe
ovoideocystidiata, Stropharia aeruginosa, Stropharia coronilla,
Stropharia rugoso-annulata, Stropharia semiglobata, Stropharia
semigloboides, Stropharia squamosa, Stropharia thrausta, Stropharia
umbonotescens, Termitomyces robusta, Volvaria bombycina,
Volvariella volvacea and combinations thereof.
20. The composition of claim 15 wherein the derivative of a
mycelium of a mushroom fungus is selected from the group consisting
of extract of Ganoderma resinaceum mycelium, extract of Ganoderma
lucidum mycelium and combinations thereof, wherein the extract is
concentrated into a form with variable viscosities resembling a
form of honey and is used as a honey substitute.
21. The composition of claim 15 where the derivative of a mycelium
of a mushroom fungus is an extract concentrated to the consistency
of honey and used as a honey substitute.
22. The composition of claim 15 wherein the mushroom fungus is a
mushroom species that will not form primordia unless the mushroom
mycelium is exposed to light selected from the group consisting of
ultraviolet light and light of 360 nanometer or lower
wavelength.
23. The composition of claim 15 wherein the mycelium is a brightly
colored mushroom mycelium
24. The composition of claim 23 wherein the brightly colored
mushroom mycelium is yellow.
25. The composition of claim 15 wherein the composition
additionally comprises a compound selected from the group
consisting of extracts from Neem trees, derivatives from Neem
trees, tree resins and combinations thereof.
26. The composition of claim 15 wherein the composition
additionally comprises a miticide, wherein the miticide is selected
from the group consisting of synthetic miticides, natural miticides
and combinations thereof, and wherein the natural miticides are
selected from the group consisting of oxalic acid, formic acid,
lactic acid, spores of entomopathogenic fungi pathogenic to mites,
hyphae of entomopathogenic fungi pathogenic to mites, preconidial
mycelium of entomopathogenic fungi pathogenic to mites, extracts of
preconidial mycelium of entomopathogenic fungi pathogenic to mites
and combinations thereof, and wherein the spores of
entomopathogenic fungi pathogenic to mites are selected from the
group consisting of Metarhizium anisopliae spores, Beauveria
bassiana spores, Entomophthorales spores and combinations
thereof.
27. A composition to benefit bees comprising a mushroom product
selected from the group consisting of an extract of fungal
mycelium, a derivative of an extract of fungal mycelium and
combinations thereof, wherein the fungal mycelium is selected from
the group consisting of entomopathogenic fungi mycelium and
mushroom fungi mycelium, combined with a spray selected from the
group consisting of sprays applied to bees and sprays applied to
plants that bees visit.
28. A composition of benefit to bees comprising an extract of
mycelium of a mushroom, honey, and a composition selected from the
group consisting of oxalic acid, neem extracts and derivatives and
spores of entomopathogenic fungi.
29. The composition of claim 28 wherein the neem extracts and
derivatives are selected from the group consisting of neem
insecticides and edible human health products.
30. The composition of claim 29 wherein the honey is obtained from
bees that have been fed mushroom mycelium products.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. provisional
patent application No. 61/967,117, filed Mar. 10, 2014, which is
hereby incorporated by reference in its entirety. This application
is also a continuation-in-part of U.S. patent application Ser. No.
13/986,978, filed Jun. 20, 2013, currently co-pending, which is a
continuation-in-part of U.S. patent application Ser. No.
13/066,566, filed Apr. 18, 2011 (now issued as U.S. Pat. No.
8,501,207), which is a divisional of U.S. patent application Ser.
No. 12/288,535, filed Oct. 20, 2008 (now issued as U.S. Pat. No.
7,951,389), which is a divisional of U.S. patent application Ser.
No. 10/853,059, filed May 24, 2004, which is a divisional of U.S.
patent application Ser. No. 09/969,456, filed Oct. 1, 2001 (now
issued as U.S. Pat. No. 7,122,176), which is a continuation-in-part
of U.S. patent application Ser. No. 09/678,141, filed Oct. 4, 2000
(now issued as U.S. Pat. No. 6,660,290), all of which are hereby
incorporated by reference in their entirety. This application is
also a continuation-in-part of U.S. patent application Ser. No.
13/998,914, filed Dec. 20, 2013, currently-copending, which is a
continuation-in-part of U.S. patent application Ser. No.
12/284,646, filed Sep. 24, 2008, which claims the benefit of U.S.
provisional patent application 60/944,972, filed Sep. 24, 2007 and
is a continuation-in-part of U.S. patent application Ser. No.
11/728,613, filed Mar. 27, 2007, which is a continuation in part of
U.S. patent application Ser. No. 11/386,402, filed Mar. 22, 2006,
which is a continuation in part of U.S. patent application Ser. No.
11/145,679, filed Jun. 6, 2005, which is a continuation-in part of
U.S. patent application Ser. No. 11/029,861, filed Jan. 4, 2005,
which is a application claiming the benefit under 35 USC 119(e) of
U.S. provisional patent application Ser. No. 60/534,776, filed Jan.
6, 2004, all of which are hereby incorporated by reference in their
entirety. This application is also a continuation-in-part of U.S.
patent application Ser. No. 13/373,719, filed Nov. 28, 2011,
currently co-pending, which is a continuation-in-part of U.S.
patent application Ser. No. 13/317,613, filed Oct. 24, 2011, which
is a continuation-in-part of U.S. patent application Ser. No.
13/066,566 (see above) and also a continuation in part of U.S.
patent application Ser. No. 12/284,646 (see above), all of which
are hereby incorporated by reference in their entirety.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] Not Applicable.
THE NAMES OF PARTIES TO A JOINT RESEARCH AGREEMENT
[0003] Not Applicable.
INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ON A COMPACT
DISC
[0004] Not Applicable.
BACKGROUND OF THE INVENTION
[0005] 1. Field of the Invention
[0006] The present invention relates to compositions and methods
for utilizing fungal mycelium products to provide an armamentarium
of defenses from multiple stressors in order to help bees survive,
and more particularly to overcome a complex of symptoms
collectively called colony collapse disorder ("CCD").
[0007] 2. Description of Related Art Including Information
Disclosed Under 37 CFR 1.97 and 1.98
[0008] Approximately 100,000 species of insects, birds and mammals
are involved in the pollination of flowering plants. This includes
almost 20,000 known species of bees. The Food and Agriculture
Organization of the United Nations estimates that of the slightly
more than 100 crop species that provide 90 percent of food supplies
for 146 countries, 71 are bee-pollinated (mainly by wild bees), and
several others are pollinated by thrips, wasps, flies, beetles,
moths and other insects. The annual monetary value of pollination
services in global agriculture could be as high as $200 billion.
Protecting the Pollinators, Food and Agriculture Organization of
the United Nations, December 2005,
http://www.fao.org/ag/magazine/0512sp1.htm. The co-evolution of
plants and bees (Apis species) is fundamental to their mutual
survival. The bees spread pollen and many plants produce rich
nectar in return.
[0009] Approximately 4,000 bee species are native to North America.
With the introduction of European (or "western") honey bees (Apis
mellifera) to North America by colonists, commercial orchards and
farms that would not normally be able to survive have thrived,
although many New World crops and native flowering plants are
primarily dependent upon native bee species for pollination. Asian
agriculture is similarly dependent upon the Asian (or "eastern")
honey bee (Apis cerana), although typically on a smaller and more
regionalized scale (A. mellifera has also been introduced).
Throughout agriculture the number of fruit, nut and vegetable crops
benefitting from bee pollination is staggering, as are the number
of flowering trees, shrubs and wildflowers. Indeed it is difficult
to overstate the role of bees in the commercial production of food.
The loss of bees we are experiencing now is unprecedented and a
huge threat to food security worldwide. In some regions of China,
for instance, the loss of bees has necessitated hand pollination to
save crops, a dauntingly difficult task.
[0010] A honey bee hive is a warm, moist, densely populated
environment inhabited by closely related individuals--the perfect
setting for viruses, bacteria, fungi, protozoa and mites. Bees have
successfully protected themselves for millions of years from such
threats with unique colony-level and individual-level host defense
systems and immune responses, but these defenses may be breaking
down as the result of intense domestication of the European honey
bee and multiple threats, including new anthropogenic stressors,
resulting in a precipitous decline in the number of feral honey
bees and native bees in areas including North America, Europe and
China from 1972 to 2006, and the emergence of Colony Collapse
Disorder ("CCD") in honey bees in 2006.
[0011] Colony losses and bee disappearances have occurred
throughout the history of beekeeping ("apiculture"), including
various honey bee syndromes in the 1880s, the 1900s through the
1920s, the 1960s and the 1990s, such as "disappearing disease,"
"spring dwindle," "fall dwindle," "autumn collapse" and "mystery
disease." In 2006, some beekeepers began reporting unusually high
losses of 30-90 percent of their hives. This disappearing bee
affliction was renamed "colony collapse disorder" (CCD, sometimes
referred to as spontaneous hive collapse or Mary Celeste syndrome
in the UK). CCD may or may not be related to the prior colony loss
syndromes; it may be a genuinely new disorder or a known disorder
that previously only had a minor impact.
[0012] CCD is now approaching 40% with many beekeepers; with the
`factory farms,` where up to 84,000 beehives are kept in one
location, CCD can claim more than 60%. This has raised the costs
for almond tree pollination, for example, from $25-30 per bee
colony per 1/2 to 1 acre of almond orchard for 3 weeks to more than
$250. More than 1/3 of all the non-animal food Americans consume is
dependent upon pollination from bees. Should this upward trend in
bee colony losses continue, the economic and societal expenses
could run into the hundreds of billions of dollars.
[0013] The loss of the services provided by bees has other
far-reaching implications. For example, neem trees, the source of
thousands of popular health, beauty and insecticide products, are
dependent upon pollination from bees, who are not adversely
affected. Interestingly, neem products that contain the active
ingredient, azadirachtin, are useful for limiting or killing mites,
including Varroa mites that transmit diseases to bees, and
including mites that transmit diseases to other animals and plants.
Should bees be lost, so too will this vast resource of health
products and a natural insecticide.
[0014] The main symptoms of CCD are the disappearance of the worker
class (resulting in very few or no adult "worker" bees in the
hive), a live queen and few to no dead bees on the ground around
the colony. Often there is still honey in the hive, immature capped
brood bees are present (bees will not normally abandon a hive until
the capped brood have all hatched) and the hive contains honey and
bee pollen that was not immediately robbed by neighboring bees. The
hive is also slow to be robbed by colony pests such as wax moths or
small hive beetles. Varroa mites, a virus-transmitting parasite of
honey bees, have frequently been found in hives hit by CCD.
Collapsing colonies typically do not have enough bees to maintain
colony brood and have workers that consist of younger adult bees;
the progression of symptoms may be rapid or slow (up to two years).
The colony may have ample food stores and be reluctant to eat food
provided by the beekeeper. See, for example, Honey Bees and Colony
Collapse Disorder, United States Department of Agriculture
Agricultural Research Service
http://www.ars.usda.gov/news/docs.htm?docid=15572 (2013).
[0015] The reasons for increasing colony collapse are complex and
appear to be the result of multiple factors. Suggested causes
include increasing urbanization and loss of biodiversity,
particularly wildflower meadows and "weeds" that provided high
quality bee forage, poor nutrition and malnutrition,
immunodeficiencies, microbial pathogens including viruses,
bacteria, fungi and protozoa, both lethal and sub-lethal exposure
to pesticides including insecticides, fungicides and herbicides,
beekeeper applied miticides and antibiotics, parasitic mites
(Varroa destructor and V. jacobsoni mites and Acarapis woodi
tracheal mites), the fungi Nosema ceranae and N. apis, heavy
metals, toxic pollutants, natural plant toxins, biting insects,
selective breeding in apiculture and loss of genetic diversity,
climate change and increased environmental stresses from drought
and cold snaps, and combinations of these factors. Another factor
is the new nature of the bee business and changing beekeeping
practices. In the USA, there are now virtually no feral bees and
domesticated bee colonies are often trucked hundreds of miles from
factory bee `livestock` apiaries, conferring additional stress
factors to colony health and facilitating wider spread of
infections and parasites amongst bee populations.
[0016] Although the exact cause(s) and mechanisms of CCD remain to
be elucidated, it appears the combination of stressors is of
importance, particularly 1) microbial viral and fungal pathogens
such as Israeli Acute Paralysis Virus ("IAPV") and Deformed Wing
Virus ("DWV") and Nosema (a pathogenic fungi); 2) parasitic mites
(particularly Varroa mites); 3) pesticides at lethal or sub-lethal
doses, including neonicitinoid insecticides (such as clothianidin,
thiamethoxam, and imidacloprid) and beekeeper-applied miticides
("BAM") and other environmental stressors; 4) the management
stressors of beekeeping including increasing viral exchange from
trucked bees (particularly in the midwinter almond pollination
migration to California), and 5) honey bee diets including use of
honey substitutes and exposure to pollen of low nutritional value
as opposed to native diverse pollen and nectar of high nutritional
value. Research suggests that honey bee diets, parasites, diseases
and multiple pesticides interact to have stronger negative effects
on managed honey bee colonies, while nutritional limitation and
exposure to sublethal doses of pesticides, in particular, may alter
susceptibility to or the severity of bee parasites and pathogens.
Pettis et al., Crop Pollination Exposes Honey Bees to Pesticides
Which Alters Their Susceptibility to the Gut Pathogen Nosema
ceranae, PLOS ONE, Published: Jul. 24, 2013, DOI:
10.1371/journal.pone.0070182,
http://www.plosone.org/article/fetchObject.action?uri=info%3Adoi%2F1
0.1371%2Fjournal.pone.0070182&representation=PDF.
Honey Bee Host Defense and Immune System:
[0017] Colonies of bees may be infected by several species of
parasites or diseases at any time, but the colony-level and
individual-level immune systems generally deal with the infections
(with the possible exception of parasitic Varroa destructor mites)
provided that environmental conditions are favorable. In the case
of colony collapse, that normally effective immune function is
clearly faltering.
[0018] Honey bees have numerous physical, chemical and behavioral
defenses at the local population, colony hive, cell and individual
bee levels. The first line of colony and individual defense is to
avoid allowing parasites to gain a foothold--bees spend large
amounts of energy on cooperative "social immunity" behaviors
including grooming their body surfaces (both self auto-grooming and
allo-grooming of a nestmate), cooperative hygienic behavior to
detect and remove diseased brood and corpses of adult bees from the
hive, cleaning the inner surfaces of the nest cavity and
sterilizing all surfaces with antimicrobial secretions in their
saliva (such as glucose oxidase), and utilizing (sometimes called
"stealing") components of the plant immune system by gathering the
highly antimicrobial resins found at leaf buds and wounds,
incorporating them into propolis and using the propolis to form an
antimicrobial barrier around for the colony, including heavy use at
the entrance, coating inner surfaces of the cavity and face of the
comb and sealing cracks and crevices.
Individual Systemic Immune Response:
[0019] Insects possess innate immunity, which is characterized by
non-specific immune reactions against invading pathogens, while
lacking the complex "adaptive" or "acquired" immunity such as
formation of antibodies specific to new pathogens. The defense
mechanism in insects consists of cellular and humoral immunity. In
the cellular defense mechanism, plasmocytes and granulocytes are
the major haemocytes that react to foreign invaders either by
phagocytosis and/or encapsulation. A hallmark of the humoral
reactions is the synthesis and secretion of anti-microbial peptides
(AMPs) that accumulate in the hemolymph against invading pathogenic
bacteria. Yoshiyama, Innate immune system in the honey bee,
Honeybee Research Group, National Institute of Livestock and
Grassland Science,
http://www.scj.go.jp/en/sca/activities/conferences/conf.sub.--8_projects/-
pdf/b4.pdf. This "induced" response of antimicrobial peptides can
last for weeks, and it appears these peptides can be passed to
nestmates to confer resistance prior to infection. Oliver,
http://scientificbeekeeping.com/sick-bees-part-3-the-bee-immune-system/
[0020] The bee antiviral response is based upon RNA interference
(RNAi). RNAi "silences" the expression of genes between the
transcription of the genetic code and its translation into
functional proteins. MicroRNA (miRNA, small non-coding RNAs that
function in networks of protein-coding genes and cell physiological
processes via transcriptional and post-transcriptional regulation
of gene expression) and small interfering RNA (siRNA, short
double-stranded fragments) bind to specific messenger RNA (mRNA)
molecules and increase or decrease their activity, for example
protein production or defending cells against viral nucleotide
sequences. The miRNAs are a well-conserved, evolutionarily ancient
component of genetic regulation found in many eukaryotic
organisms.
[0021] RNAi is initiated by the enzyme Dicer, which cleaves long
double-stranded (dsRNA) molecules into short double stranded
fragments of siRNAs. Each siRNA is unwound into two single-stranded
ssRNAs, the passenger strand and the guide strand. The guide strand
is incorporated into the RNA-induced silencing complex (RISC).
After integration into the RISC, siRNAs base-pair to their target
mRNA and cleave it, thereby preventing it from being used as a
translation template. When the dsRNA is exogenous (for example,
coming from infection by a virus), the RNA is imported directly
into the cytoplasm and cleaved to short fragments by Dicer.
[0022] Bees possess more RNAi pathway components relative to flies
and appear to more readily mount a systemic RNAi response than do
flies. It follows that bees should be quite capable of battling
viruses and arguably other pathogens through knockdowns based on
double-stranded RNAs of pathogen-expressed genes (Evans/Spivak
2009). Notably, this form of response to viral attack provides a
long-term memory similar to that resulting from the antibodies
produced in mammals. Oliver,
http://scientificbeekeeping.com/sick-bees-part-4-immune-response-to-virus-
es/.
Viruses, Nosema and Microbial Pathogens:
[0023] Bees are host to at least 18 viruses, nearly all being
single-stranded RNA viruses. Some are "emerging" pathogens, such as
Deformed Wing Virus and Acute Bee Paralysis Virus, which were once
considered to be "economically irrelevant" (Genersch 2010) and
then, with the arrival of Varroa as a vector, began to devastate
colonies. Oliver, Sick Bees--Part 4: Immune Response to Viruses,
http://scientificbeekeeping.com/sick-bees-part-4-immune-response-to-virus-
es/
[0024] Viral diseases include chronic paralysis virus (CPV), acute
bee paralysis virus (ABPV), Israeli acute paralysis virus (IAPV),
Kashmir bee virus (KBV), Black queen cell virus (BQCV), Cloudy wing
virus (CWV), Sacbrood virus (SBV), deformed wing virus (DWV),
Kakugo virus, invertebrate iridescent virus type 6 (IIV-6), Lake
Sinai viruses (LSV1 and LSV2) and tobacco ringspot virus (TRSV).
Within these viruses are many subtypes whose virulence towards bees
is currently being investigated. More pathogenic viruses will
likely be discovered. The co-occurrence of more than one
internalized virus further challenges the immunological health of
bees. Hence, there is a need for advantageous remedies which are
non-toxic yet active against more than one virus. This invention
creates the scientific pathways towards such discoveries: wood
rotting fungi confer a broad spectrum of antiviral benefits to
bees.
[0025] Bees are also vulnerable to pathogen host shifts. The
tobacco ringspot virus can replicate and produce virions in Apis
mellifera honeybees, resulting in infections throughout the entire
body, including extensive infection of the nervous system and
likely impacts on colony survival. TRSV was also found in the
gastric cecum of Varroa mites, suggesting that Varroa mites may
facilitate the spread of TRSV in bees while avoiding systemic
invasion. Li et al., Systemic Spread and Propagation of a
Plant-Pathogenic Virus in European Honeybees, Apis mellifera, mBio
5(1):e00898-13. doi:10.1128/mBio.00898-13. The virus, first
observed in infected tobacco, is spread through infected pollen of
numerous plant species including soy and numerous crops, weeds and
ornamentals.
[0026] Nosema apis is a microsporidium, recently reclassified as a
fungus, which invades the intestinal tracts of adult bees and
causes Nosema disease, also known as nosemosis. Nosema infection is
also associated with black queen cell virus and Kashmir bee virus.
Nosema ceranae is becoming an increasing problem on both the Asian
honey bee Apis cerana and the western honey bee.
[0027] Some honey bee viruses (DWV and KBV) and the fungi Nosema
ceranae are able to infect other species of bees and wasps, and
possibly Varroa gut cells; honeybees are likely the source of the
bumblebee pathogens. Furst et al., Disease associations between
honeybees and bumblebees as a threat to wild pollinators, Nature,
Volume:506, 364-366, (2014). This new bee-to-bee vector could be a
tipping point, causing wide scale collapse of many native bee
species, with consequences well beyond our control, or imagination.
From a historical and biological perspective, this is an `all hands
on deck` moment. What evolution has provided us over millions of
years can be lost in decades due to the human interventions whose
incentives are short term in view at the expense of the long
term.
[0028] Bacterial diseases of bees include American foulbrood (AFB),
caused by Paenibacillus larvae, and European foulbrood (EFB),
caused by the bacterium Melissococcus plutonius. Fungal diseases
include Chalkbrood, caused by Ascosphaera apis, and Stonebrood, a
fungal disease caused by Aspergillus fumigatus, Aspergillus flavus,
and Aspergillus niger. New, as yet unidentified, fungal pathogens
are expected to co-occur or become a primary cause of bee diseases
in the future as humans further alter the natural environment and
cause unintended consequences from the use of transgenic crops,
more broadly known as GMOs--genetically modified organisms. Such
potential fungal pathogens include Candida, Cryptococcus,
Coccidiodes and other yeast-like organisms. And yet, many of these
so-called pathogens, especially, for instance, the pre-sporulating
forms of entomopathogenic fungi, have properties that can confer
benefits to insects, including bees, provided that their endogenous
toxins are eliminated, reduced or altered so to not harm bees,
thereby reducing the threat to bees by disease-causing,
disease-bearing or disease-spreading organisms.
[0029] All honey bees are infected by more than one species of
bacteria, including beneficial endosymbionts that offer protection
against yeasts, chalkbrood and foulbrood. Apparently healthy bees
may also be infected by more than one species of virus. The
dynamics of bee-bacteria, bee-virus and virus-virus interactions
are complex and poorly understood. Certain bee viruses may enhance
the virulence of other viruses while some bee viruses may
competitively suppress the replication of others. So too there are
likely bacteria-to-bacteria, bacteriophage-to-bacteria,
fungi-to-bacteria and fungi-to-virus interrelationships scientists
have yet to discover. Many virulent bee viruses can exist in an
"unapparent" infection--one can detect the presence of the virus in
bees, but there are no noticeable negative effects due to the
infection. An infection by a second virus or other stressor may
cause a dormant virus to start replicating. A number of researchers
have found that the mere action of a Varroa mite feeding upon a bee
(which includes injection of immune suppressants by the mite) may
induce or activate the replication of unapparent and normally
non-pathological virus infections. It is common for collapsing
colonies to be simultaneously infected with three or four viruses,
Varroa mites, Nosema (ceranae and especially apis), and
trypanosomes. See Oliver,
http://scientificbeekeeping.com/sick-bees-part-3-the-bee-immune-system/
[0030] Crithidia bombi is a trypanosomatid protozoan bee parasite
known to have serious effects on bumblebees, particularly under
starvation conditions. The related Crithidia mellificae may be
contributing to mortality in the honey bee. Ravoet et al.,
Comprehensive Bee Pathogen Screening in Belgium Reveals Crithidia
mellificae as a New Contributory Factor to Winter Mortality (2013),
PLoS ONE 8(8): e72443,
http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pon
e.0072443.
Varroa Mites and Other Parasites:
[0031] Varroa destructor and Varroa jacobsoni are parasitic mites
that feed on the bodily fluids of bee adults, pupae and larvae.
Acarapis woodi is a tracheal mite that infests the airways of the
honey bee. The Asian parasitic brood mites Tropilaelaps clareae and
T. mercedesae are considered serious potential threats to
honeybees, although they have not been found in the United States
or Canada to date.
[0032] The Asian honey bee Apis cerana is the natural host to the
Varroa jacobsoni mite and the parasite Nosema ceranae. Having
co-evolved with these parasites, A. cerana exhibits more careful
grooming than A. mellifera, and thus has a more effective defense
mechanism against Varroa and Nosema, which are becoming
increasingly serious pests of the western honey bee.
[0033] Varroa mites breaching bees' hygienic, mechanical and
physiological barriers to invasion have increasingly acted as a
vector for viruses as well as causing major stress to bees.
Widespread colony losses have only been reported from countries is
which Varroa is a problem (Neumann 2010). Colonies without mites
may be virus free (Highfield 2009), but up to 100% of colonies with
Varroa may be infected by one or more viruses, even if there are no
apparent symptoms (Tentcheva 2004). Oliver,
http://scientificbeekeeping.com/sick-bees-part-1/
[0034] Varroa mites have been found to be far more susceptible to
acids than are honey bees. Organic acids such as oxalic acid,
formic acid and lactic acid can be used as "natural miticides" or
mite treatments in the hive, as they are all naturally found in
honey. Oxalic acid is typically mixed with distilled water to
prevent the formation of salts, resulting in an acidic solution
with pH often times <1. That the bees can tolerate such a low pH
while mites cannot is significant. The oxalic acid will capture
calcium and other minerals from the exoskeleton of the mites to
form oxalates. When direct contact of oxalic or formic acid with
the chitonous like exoskeleton of the mites pulls out calcium, the
exoskeleton is weakened, thus making the mites susceptible to other
stressors, including but not limited to infection or toxin exposure
from entomopathogenic fungi.
[0035] Besides known colony insect pests, such as the greater and
lesser wax moths and the small hive beetle, the phorid fly,
previously known to parasitize bumblebees, may be emerging as a
threat to honey bees. Core et al., A New Threat to Honey Bees, the
Parasitic Phorid Fly Apocephalus borealis (2012), PLoS ONE 7(1):
e29639. doi:10.1371/journal.pone.0029639,
http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pon
e.0029639; Ravoet, supra.
Pesticides:
[0036] Pesticides cause multiple forms of stress to bees.
Agricultural spraying may affect honey bees and large-scale
spraying programs for mosquitoes, gypsy moths, spruce worms and
other insect pests may cause direct or indirect bee kills including
native bumblebees and solitary bees. There is also a shift in the
types of pesticides applied--many, such as neonicitinoids, are less
toxic to vertebrates and the necessity of repeated application is
reduced, but they act systemically and are absorbed and distributed
throughout the plant upon seed or soil treatment, including
distribution to the pollen and nectar.
[0037] Sub-lethal pesticide exposure, including exposure to
cholinergic neonicitinoid insecticides (nicotinic receptor
agonists) and/or cholinergic organophosphate miticides
(acetylcholinesterase inhibitors), has been found to alter bee
activity, development, oviposition, behavior, offspring sex ratios,
flight and mobility, navigation and orientation ability, feeding
behavior, learning, memory and immune function, population dynamics
and increase susceptibility to and mortality from diseases,
including Nosema. See, for example, Pettis, Crop Pollination
Exposes Honey Bees to Pesticides Which Alters Their Susceptibility
to the Gut Pathogen Nosema ceranae, supra at 1. Fungicides and
miticides used by beekeepers can have a pronounced ability on bees'
ability to withstand parasite infection. Pettis, supra at 4. Often
bees are exposed to a variety of pesticides, which may have
interactive effects. See, for example, Di Prisco et al.,
Neonicitinoid clothianidin adversely affects insect immunity and
promotes replication of a viral pathogen in honey bees, PNAS Early
Edition,
http://www.pnas.org/content/early/2013/10/18/1314923110.full.pdf;
Pettis et al., Crop Pollination Exposes Honey Bees to Pesticides
Which Alters Their Susceptibility to the Gut Pathogen Nosema
ceranae, PLoS ONE (2013),
http://www.plosone.org/article/fetchObject.action?uri=info%3Adoi%/2F1
0.1371%2Fjournal.pone.0070182&representation=PDF; Palmer et
al., Cholinergic pesticides cause mushroom body neuronal
inactivation in honeybees, Nature Communications, 4:1634, (2013),
http://www.nature.com/ncomms/journal/v4/n3/pdf/ncomms2648.pdf;
Williamson et al., Exposure to multiple cholinergic pesticides
impairs olfactory learning and memory in honeybees, The Journal of
Experimental Biology 216, 1799-1807 (2013),
http://jeb.biologists.org/content/216/10/1799.full.pdf+html;
Derecka et al., Transient Exposure to Low Levels of Insecticide
Affects Metabolic Networks of Honeybee Larvae, PLoS ONE 8(7),
e68191 (2013),
http://www.plosone.org/article/fetchObject.action?uri=info%3Adoi%2F1
0.1371%2Fjournal.pone.0068191&representation=PDF.
[0038] Exposure to fungicides also kills or reduces the beneficial
fungi found on pollen--the result likely being a higher incidence
of disease in honeybees, including Nosema infections and chalkbrood
(ironically, fungal diseases).
[0039] The bee genome has relatively few genes that are related to
detoxification compared to solitary insects such as flies and
mosquitoes. Some of the most marked differences between bees and
other insects occur in three superfamilies encoding xenobiotic
detoxifying enzymes. Whereas most other insect genomes contain 80
or more cytochrome P450 (CYP) genes, A. mellifera has only 46
cytochrome P450 genes, whilst humans host about 60 CYP genes. Honey
bees have only about half as many glutathione-S-transferases (GSTs)
and carboxyl/cholinesterases (CCEs), compared to most insect
genomes. This includes 10-fold or greater shortfalls in the Delta
and Epsilon GSTs and CYP4 P450s, members of which clades have been
linked to insecticide resistance in other species. Claudianos et
al., A deficit of detoxification enzymes: pesticide sensitivity and
environmental response in the honeybee, Insect Molecular Biology,
15(5), 615-636 (2006),
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1761136/.
[0040] Whereas bees evolved to deal with plant phytochemicals and
natural toxins, they now must additionally metabolize and detoxify
anthropogenic insecticides, miticides, herbicides, fungicides and
environmental pollutants, an unprecedented evolutionary
challenge.
Management Stressors of Beekeeping:
[0041] Use of honey or pollen substitutes (such as sugar syrup;
high fructose corn syrup; bee candy; "grease patties" containing
grease, sugar and optionally salt or essential oils; or "pollen
patties" containing soy, yeast and nonfat dry milk, which may have
added pollen, possibly from areas contaminated with pesticides) may
be a contributing factor to declining bee populations and CCD for
several reasons. Malnutrition is likely a major factor in declining
bee populations. Synthesized bee diets simply do not provide the
nutritional value obtained by bees from a mixture of quality
pollens. Although quality proteins, carbohydrates and vitamins can
be provided to honey bees in the lab, we still cannot keep them
alive more than two months in confinement on our best diets.
Mussen, Posts Tagged: from the UC Apiaries newsletter--The
California Backyard Orchard,
http://homeorchard.ucanr.edu/?blogtag=from%20the%20UC%20Apiarie
s%20newsletter&blogasset=455388&sharing=yes.
[0042] Honey contains several substances that activate nutrient
sensing, metabolic, detoxification and immune processes in the
European honey bee Apis mellifera, plus other chemicals useful to
honey bee health. The enzymes are found on the pollen walls of
flowers and enter the honey by sticking to the bees' legs.
Ingestion of tree resins, balsams and tree saps via incorporation
into propolis or bee glue is also known to reduce bee
susceptibility to both insecticides and microbial pathogens and
up-regulate the transcription of the detoxification genes. Honey
substitutes or pollen patties, which don't contain these chemicals,
may therefore contribute to colony collapse disorder. See Mao,
Wenfru, Schuler, Mary A. and Berenbaum, May R., Honey constituents
up-regulate detoxification and immunity genes in the western honey
bee Apis mellifera, Proceedings of the National Academy of
Sciences, www.pnas.org/cgi/doi/10.1073/pnas.1303884110,
http://www.pnas.org/content/110/22/8842.full. Mao et al. found that
constituents in honey derived from pollen and tree exudates,
including p-coumaric acid (=4-hydroxycinnamic acid), pinocembrin,
pinobanksin and pinobanksin 5-methyl ether, are strong inducers of
cytochrome P450 genes detoxification genes via a number of CYP6 and
CYP9 family members. Massively parallel RNA sequencing and RNA-seq
analysis revealed that p-coumaric acid specifically up-regulates
all classes of detoxification genes as well as select genes for
antimicrobial peptides required for defense against pesticides and
pathogens.
[0043] Those species of honey bees that nest in tree cavities use
propolis to seal cracks in the hive, as do bees in domestic hives,
although feral honey bees coat the entire inner surface of their
nesting cavity, whereas domesticated honey bees lay down
comparatively little resin in beekeeping hives. The coating of
propolis has been demonstrated to inhibit AFB (Ant nez 2008),
fungi, and wax moth; Spivak has demonstrated that propolis from
some regions is effective against Varroa, and is investigating its
effect on viruses. Of great interest is the finding (Simone 2009)
that the abundance of propolis appears to decrease the necessary
investment in immune function of bees--thus, the bee colony, by
self-medicating with antimicrobial chemicals from plants, incurs
less of a metabolic cost in fighting pathogens. Oliver,
http://scientificbeekeeping.com/sick-bees-part-3-the-bee-immune-system/.
Bears, Mushrooms and Bees:
[0044] The inventor noticed, on one of his many forays in the old
growth forests of the Olympic Peninsula, a conifer tree scratched
by a bear (a photograph appears in the book he authored, Mycelium
Running: How Mushrooms Can Help Save the World, 2005, pg. 70, FIG.
75. Ten Speed Press, Berkeley). The research literature on the
inter-relationships between bears and mushrooms stated that
Fomitopsis species, brown rotting polypore wood conks, including
the frequently seen Fomitopsis pinicola and the rarely seen
Fomitopsis officinalis, were the most common fungal species to grow
after bear scratchings in conifer forests of the Pacific Northwest
and elsewhere. Forest scientists showed that when bears scratch a
living tree, they leave an open wood, and the Fomitopsis species
opportunistically gain an entry site for infection. After a
scratching, sugar-rich resin often beads out as droplets,
attractive to bears and bees. Indeed, when the author returned a
few years later to the same tree deep in the old growth forests
along the south fork of the Hoh River, Olympic Peninsula of
Washington State, Fomitopsis pinicola mushrooms were fruiting from
the now-fallen tree.
[0045] "On young conifers, particularly Douglas-fir trees, bears
will rip strips of bark off with their teeth to reach insects or
the sweet-tasting sap found inside. The bear's teeth leave long
vertical grooves in the sapwood and large strips of bark are found
around the bases of trees they peel. These marks are typically made
from April to July, but the results may be seen all year. This
foraging activity is common in tree plantations where large stands
of trees are similarly aged and of a single species." Living with
Wildlife: Black Bears, Washington State Dept. of Fish and Wildlife,
http://wdfw.wa.gov/living/bears.html.
[0046] For this reason, a bounty was placed upon bears by forest
stakeholders since the bears were thought to reduce the
profitability of forests for timber. Tens of thousands of bears
were killed by hunters hired by the timber companies. In the 1990s,
it was discovered that bears actually benefit the forests by bring
sea minerals, particularly phosphorus and nitrogen, due to their
foraging for salmon and trout in the rivers adjacent to the
forests. One reason the lowland old growth forests are so much
larger than old growth forests several thousand feet up in
elevation, above the limit of the migrating fish, is that bears
brought the carcasses of fish onto shore, benefitting the adjacent
trees. Humans are particularly adept at making decisions contrary
to their long-term best interests due to a fundamental
misunderstanding about the interconnectedness of nature.
[0047] In Stamets, Growing Gourmet and Medicinal Mushrooms, 1993,
p. 42-43, the current inventor stated "For 6 weeks one summer our
bees attacked a King Stropharia bed, exposing the mycelium to the
air, and suckled the sugar-rich cytoplasm from the wounds. A
continuous convoy of bees could be traced, from morning to evening,
from our beehives to the mushroom patch, until the bed of King
Stropharia literally collapsed. When a report of this phenomenon
was published in Harrowsmith Magazine (Ingle, 1988), bee keepers
across North America wrote me to explain that they had been long
mystified by bees' attraction to sawdust piles." Although it may
not have been clear to one of ordinary skill in the art if the bees
were attracted to the mycelium, the lignin within the sawdust or
wood resins within the sawdust, the inventor concluded "Now it is
clear the bees were seeking the underlying sweet mushroom
mycelium."
[0048] An urgent solution is needed.
BRIEF SUMMARY OF THE INVENTION
[0049] The present inventor sees the intersection and interplay of
several mycological methods and compositions as a possible
integrated solution to CCD. Each one of these elements may be
sufficient to cause an effect leading to preventing or reducing
CCD. As an integrated platform of partial solutions, the totality
of these methods will achieve a synergistic benefit.
[0050] The basis of these compositions and methods include the
extracellular exudates and extracts made therefrom, of the
mycelium, prior to fruitbody formation, in the mushroom species of
the Agaricales, Polyporales and Hymenochaetales in combination or
independently. Extracts of the pre-conidial mycelium of
entomopathogenic fungi may optionally be used to control mites and
other bee and hive parasites. Mixtures of these extracts and bee
products such as bee food or bee treatment sprays offer multiple
solutions to help prevent CCD or help bees overcome CCD.
Sustainable solutions to problems plaguing bees will be derived
from promoting their natural defenses through habitat enhancement
via beneficial fungi.
[0051] The inventor has isolated various strains of mushroom fungi,
including Pleurotus ostreatus, Trametes versicolor, and Psilocybe
azurescens that have demonstrated superior abilities to
"bioremediate" or "mycoremediate" various toxins including oil,
pesticides and nerve gases such as Sarin, Soman and VX
(dimethylmethylphosphonate), working with Battelle Laboratories, a
public report of which was published in Jane's Defence Weekly.
(Fungi could combat chemical weapons, Jane's Defence Weekly, 1999.
32(7):37.)
[0052] The inventor has also isolated various strains of fungi,
including Fomitopsis officinalis, Fomitopsis pinicola, Ganoderma
applanatum, Ganoderma annularis, Ganoderma lucidum, Ganoderma
resinaceum, Inonotus obliquus, Irpex lacteus, Phellinus linteus,
Piptoporus betulinus, Pleurotus ostreatus, Polyporus umbellatus,
and Trametes versicolor that have demonstrated superior antiviral,
antibacterial, antifungal and antiprotozoal properties. Without
being bound to any theory, the inventor would hypothesize that
these mushroom species are rich in compounds that up-regulate genes
for detoxification and defense against pollutants, pesticides and
pathogens in animals, including humans and bees. By repeatedly
culturing and expanding non-sporulating sectors of entomopathogenic
fungi, the inventor also discovered that such "pre-sporulating" or
"preconidial" mycelium and extracts of preconidial mycelium odors
(ranging from Metarhizium anisopliae and Aspergillus flavus
"butterscotch" to Beauveria bassiana "vanilla cola" and "hard
Christmas candy") and tastes are attractive to animals including
humans and both non-social and social insects, which offer
advantages in control of pests such as Varroa mites.
[0053] The inventor now hypothesizes that the Fomitopsis
colonization of the wood from bear foraging and the entry wound
site (see above) would lead to the production of enzymes (laccases,
lignin peroxidases, cellulases), ergosterols and other sterols,
mycoflavonoids and especially arrays of nutritious complex
polysaccharides that would not only soften the wood, provide water,
nutrition, and emit fragrances, all of which would attract bees,
while the extracellular exudates being secreted by the mycelium
would be rich in p-coumaric acids and coumarins and the glycosides
of unsubstituted and substituted benzoic, cinnamic and coumaric
acids, all stimulating the up-regulation of innate cytochrome p450
genes and enzymes and also providing antiviral and antibacterial
agents, all expressed during the decomposition of the infected
tree. A complex fungal tree nectar is exuded, one that provides
physiological benefits and boosts the innate immunity of bees via
numerous pathways as the trees decompose. In some instances, bees
nest within these logs or in the ground beneath them, benefitting
from long-term contact. The bees can then incorporate these
beneficial agents into their honey, propolis and combs so to as to
protect the brood, the queen and ultimately the colony.
[0054] The inventor also hypothesizes that combinations of the
fungal species including but not limited to their resident phenols
above and below will have additive or even synergistic
consequences, including regulation and up-regulation of
nutrient-sensing, metabolic, detoxification, immunity and
antimicrobial peptide genes and systems. This invention speaks
directly to the link between the contact bees have with fungi that
are beneficial, not only nutritionally, antivirally, antifungally,
antibacterially, but especially in activating the cytochrome P450
pathways for deactivating and metabolizing xenobiotic and
anthropogenic toxins.
[0055] The current invention provides a plurality of partial
solutions to provide scientists, farmers, biotechnologists, policy
makers and thought leaders with biological tools of practical and
scalable remedies before ecological collapse forces us to
ever-limiting options as biodiversity plummets. The combination of
these partial solutions cumulatively and synergistically provide
that which is necessary for bees to overcome CCD.
[0056] As Albert Einstein noted, "We cannot solve our problems with
the same thinking we used when we created them." This patent
follows this philosophy by offering a complex platform of
synergistic solutions addressing a multiplicity of problems, which
ultimately help bees overcome colony collapse disorder.
DETAILED DESCRIPTION OF THE INVENTION
[0057] Bees are increasingly dealing with new anthropogenic
stressors. Over hundreds of millions of years fungi have evolved to
fight viruses, bacteria and other fungi; have evolved to infect
parasites, including insects; have evolved enzymes to break down
toxins; and have evolved substances to up-regulate such processes.
This means they offer a potential nutraceutical treasure trove of
compounds useful for protecting bees and other pollinators from
such threats, including a plurality of antiviral, antibacterial,
antifungal and antiprotozoal compounds and compounds useful for
up-regulating the digestive, detoxification and immune systems of
bees.
[0058] Without being held to any one theory, the inventor
hypothesizes that the fungal mycelium extracts specifically
modulate, induce and increase the expression of detoxification and
xenobiotic metabolizing genes, specifically to up-regulate all
classes of detoxification genes, increase midgut metabolism of
pesticides, function as a nutraceutical regulating immune and
detoxification processes, up-regulate immune, metabolic and
nutrient pathways (lipid and glucose-metabolizing pathways) and
up-regulate genes encoding antimicrobial peptides. Moreover, select
fungal species support the microbiome of beneficial microorganisms
in the digestion systems of bees, and their compatibility is an
important species-to-species bridge, matching beneficial wood
rotting fungi to the beneficial microbes resident in the hindgut of
bees.
[0059] Since bees are under assault from multiple pathogens--mites,
viruses, microsporidia, protozoa, phorid flies and exposure to
airborne pollutants--finding a robust broad-based platform of
protection to help bolster the host immune defense of bees is of
paramount importance. Using the extracellular exudates of the
mycelium of select species of fungi, including but not restricted
to Stropharia rugoso-annulata and other members of the
Strophariaceae, Fomitopsis officinalis and other members of the
Fomitopsidaceae and Metarhizium anisopliae and other members of the
Clavicipitaceae, helps prevent colony collapse disorder. Many other
species of basidiomycetes and ascomycetes are also expected to
confer similar benefits in the course of research into the benefits
of bee-beneficial exudates secreted by the mycelium.
[0060] With regard to fungal extracts, mycelial extracts are
preferred to "mushroom extracts" because the hyphae produce
extracellular exudates that are not only rich in accessible water,
oils, polysaccharides, amino acids, B vitamins, coumarins,
p-coumaric acids, phenols and polyphenols, as well as ergosterols,
enzymes, acids, including fatty acids, antibacterials and
antivirals. The individual hyphal threads of the mycelium emits
complex scents that volatilize into the air whereas the mushrooms
tend to be nutritionally dense but do not have the extensive,
exposed cellular surface area as the same mass of mycelium. The
mushroom fruitbody is composed of cellularly compacted hyphae,
laminated together, so only a small fraction of the mycelial mass
in the fruitbody is exposed to the atmosphere. Hence the mushroom
fruitbodies lack the fragrance attributes of the mycelium from
which they form. Since these extracellular exudates can readily
dissolve into solution, these exudates can be more usefully
incorporated into amendments, such as pollen patties, sugar
solutions or water, bee sprays or foliar plant sprays, and are
better attractants to bees and other insects than the mushroom
fruitbodies. This is not currently obvious to those skilled in the
arts of mycology or entomology.
[0061] Only recently, research has discovered that the mycelium has
more genes turned on than the mushrooms that ultimately are formed
from same. As was noted by Li et al., 2013, "The protein-coding
genes were expressed higher in mycelia or primordial stages
compared with those in the fruiting bodies." Li, J., Zhang, J.,
Chen, H., Chen, X., Lan, J., Liu, C. 2013. "Complete mitochondrial
genome of the medicinal mushroom Ganoderma lucidum." PLoS ONE
8(8):e72038. doi:10.1371/journal.pone.0072038.
[0062] Moreover, the network-like structure of the mycelium allows
for epigenetic evolution of strains that can be evolved to emit
substances targeted specifically for the benefit of bees. Such
improvements are anticipated by the inventor as a method for making
strains and compositions more attractive to bees and more
appropriate for helping bees overcome CCD.
[0063] In essence, the inventor has devised a novel nutraceutical
which is rich is a wide array of coumarins, phenols and
polyphenols; and anti-viral, anti-fungal, anti-bacterial and
anti-protozoal agents, and a wide diversity of specialized
metabolites such as antioxidants and antimutagens, which are
generated as a result of mycelium digesting grains or wood and are
attractive to bees and supportive of their host defense against
stressors and diseases. The extracts of mushrooms used medicinally
for human health have an unexpected benefit for bee health too.
Indeed, the fungal contribution to propolis and honey, as well as
to pollen, augments the immune systems of bees, and by extension to
people, on specific, fundamental, complex levels. The inventor
notes extracts of mycelium grown on grain inoculated wood are
expected to contain more polyphenols, coumarins and compounds that
up-regulate detoxification and immunity genes in the bees, as
opposed to extracts of mycelium grown via liquid fermentation.
[0064] Since nature may require decades, even centuries, before new
beneficial associations can be established, with bees unable to
react quickly enough to the recent advent of new herbicides,
pesticides, fungicides and miticides, we can jump start this
process by giving these beneficial fungal species a primary role in
the pathways of bee biology and biochemistry to bolster their host
defenses and prevent CCD. The chemical composition of fungal
mycelium is complex and variable within and among the various
mushroom phyla, families and genera, traits that makes fungal
extracts a good defense against rapidly evolving pests and
pathogens.
Attractants:
[0065] One component of the invention is the use of fungal
extracts, whereby the extracts are generated from the mycelium of
polyporoid, basidiomycetous and ascomycetous species, to attract
bees. The bees are attracted to the polysaccharide-rich
extracellular and intracellular metabolites secreted by the
mycelium. Within these exudates are compounds that attract bees,
feed them with sugar rich and other nutrients, provide antiviral,
antifungal and antibacterial protection, while bolstering their
resistance to pesticides and improving colony health and honey
production. In fact, honeys holding these fungal components could
proffer medicinal benefits to bees and other species, including
humans.
[0066] These extracellular exudates from, for instance, the King
Stropharia or the Garden Giant mushroom (Stropharia
rugoso-annulata), have an attractive effect on bees, especially
during the time when flowering plants of their preference are
limited. Bees are attracted both to the extracellular extracts as
well as living mycelium. Other mushroom species, including gourmet
and medicinal mushrooms, are expected to attract bees to varying
degrees in a similar fashion.
[0067] The pleasant fragrance of Stropharia rugoso-annulata
out-gassing from the mycelium is one component of its attractancy
to bees. The present inventor has discovered Stropharia
rugoso-annulata mycelium emits a rich, attracting flower-like
essence, while providing an immune-enhancing `mycelium nectar,` the
combination of which makes it an exceptional species for aiding
bees when challenged by multiple stressors that can lead to CCD.
Oyster mushrooms in the genus Pleurotus, especially Pleurotus
ostreatus, P. pulmonarius, P. lignatilis, P. sapidus, P. eryngii,
P. populinus and other related species emit a pleasing anise-like
fragrance, as does Clitocybe odora. Another candidate is the
split-gill polypore, Schizophyllum commune, one of the most common
of all woodland Basidiomycetes, which produces a potent, sweet
fragrance in culture, at times overwhelming the olfactory senses of
lab personnel, and is a source of coumarins and coumaric acids.
Interestingly, only those growing Schizophyllum commune in mass, in
vitro, on cereal grains or wood would ever know about this potent
outgassing fragrance. The inventor knows of no one else in his 40
years of experience who has mentioned or reported on this fragrance
phenomenon. Schizophyllum commune is one of the most prominent
white rot, woodland species across the temperate and tropical
regions of the world, and creates softened, sweet wood from which
bees can benefit. Many other species probably emit attractive
fragrances to bees, which are undetectable to humans or not
noticeably enticing.
[0068] The inventor hypothesizes that Basidiomycetes fungi,
particularly those that produce large, forking rhizomorphs,
classically known as "cord producers," may be better suited for
bees harvesting the fungal nectars. One family of fungi cord
producers is the Strophariaceae or the Stropharioideae.
Interestingly, the mycelium of Stropharia rugoso-annulata does not
produce these rhizomorphic cords until making contact with the
bacterial genome resident in woody soils. This dynamic is likely to
confer bees with more robust nutritional and immune support.
[0069] The extracts of the Agaricomycetes are the source of new bee
attractants. The Agaricomycetes are the only fungi that decompose
lignin, and includes the gilled mushrooms, such as Stropharia
rugoso-annulata, and the polypores, such as those related to
Fomitopsis species. The Agaricomycetes encompasses .about.16,000
described species. Many of the Agaricomycetes dually decompose
cellulose and lignin. Native bees use rotten logs for nesting, as
discussed above in connection with bears, fungi and bees, which the
inventor hypothesizes provides bees with the sugar rich, antiviral,
antifungal, antibacterial, antiprotozoal and cytochrome P450 coding
and up-regulating compounds via water droplets and nectar secreted
by the mycelium of Agaricomycetes as extracellular exudates which
helps the bee's host defenses to survive assaults from multiple
pathogens and toxins. Unfortunately for bees, these assaults can be
co-occurring, depressing the bee's natural host defenses, and
increasing their susceptibility to CCD. Currently, our regenerated
forests have about 10-15% of the wood debris compared to native
woodlands! This relatively recent loss of decomposable wood debris
limits the availability of these beneficial fungi to native and
imported bees, introducing a heretofore unreported, additional
stress factor. The continued constriction of debris fields further
erodes the foodwebs essential not only to bees, but to most
organisms that are dependent upon healthy and sustainable
ecosystems.
[0070] For instance, fungal extracts of non-Agaricomycetes fungi
have been shown by the inventor to attract Phorid flies (and other
insects) to the extracts of the preconidial (pre-sporulation)
mycelium of Metarhizium anisopliae and Aspergillus flavus (See U.S.
Pat. Nos. 6,660,290, 7,122,176, 7,951,388, 7,951,389 and
8,501,207), arresting their migration, and thus prevent these flies
from vectoring diseases. Moreover, pathogen hosting mites are also
attracted and stopped from moving into the bee colonies using these
mycelium-based extracts, thus reducing not only the pathogen
payloads mites carry, but also reducing the numbers of mites which
might otherwise infect the bees. Similar approaches may be used to
control beehive pests, such as the greater and lesser wax moths and
the small hive beetle, if needed. Moreover, strains of these
pre-sporulation entomopathogenic fungi can be selected for their
high thermal tolerance and their abilities for attracting and
killing mites and flies which harm bees or vector pathogens.
Research into post-sporulation and spore-based Metarhizium
anisopliae technologies (which may have the disadvantage of
repelling mites and/or insects as compared to the attractancy of
preconidial mycelium) have demonstrated the relative ease with
which strains may be selected for thermal tolerance to high hive
temperatures and high pathogenicity and/or mortality to Varroa
mites. Rodriguez et al., Selection of entomopathogenic fungi to
control Varroa destructor (Acari: Varroidae), Chilean J. Agric.
Res., 69(4): 534-540 (2009); Rodriguez et al., Evaluation of
Metarhizium anisopliae var. anisopliae Qu-M845 isolate to control
Varroa destructor (Acari: Varroidae) in laboratory and field
trials, Chilean J. Agric. Res., 69(4): 541-547 (2009); Boyle,
Integrated Pest Management--Compatible Biological Control of Varroa
Mite of Honey Bee, www.gnb.ca/0389/2007/03892007001-e.asp; Fungi
help combat honeybee killer, BBC News Science/Nature,
http://news.bbc.co.uk/2/hi/science/nature/2182948.stm.
[0071] Humans are limited to perceiving color wavelengths of light
from approximately 390 to 750 nanometers (nm). Bees, like many
insects, see colors from approximately 300 to 650 nm. Many mushroom
species like Oyster mushrooms (Pleurotus ostreatus) are triggered
into fruiting around 360 nanometers, beyond the far end of our
ability to detect. (See ACTION SPECTRA FOR HYPHAL AGGREGATION, THE
FIRST STAGE OF FRUITING, IN THE BASIDIOMYCETE Pleurotus ostreatus,
Richartz and Maclellan in Photochemistry and Photobiology Volume
45, Issue Supplement 51, pages 815-820, May 1987.
(http://onlinelibrary.wiley.com/doi/10.1111/j.1751-1097.tb.07888.x/abstra-
ct).
[0072] When mycelium growing deep within wood or the ground reaches
the surface of ground or wood, and is exposed to light, a phase
change occurs in the mushroom's life cycle, going from mycelium to
the first stages of mushroom formation, hyphal aggregation and
primordia (`baby mushroom`) formation. The mycelium in many species
will not form primordia unless there is light exposure near to the
ultraviolet or 360 nanometer or lower wavelengths. This is well
within the range bees can detect but beyond the limits of what
humans can.
[0073] Attractiveness to mycelium stimulated by blue light
invisible to humans but visible to bees is highly significant
discovery as bees are most easily trained to associate food in the
ultraviolet wavelengths of color. As Menzel and Backhaus determined
in 1989, bees could learn faster when the food was associated with
violet light was used compared to all other colors. Menzel, R. and
Backhaus, W. 1989. "Color vision in honey bees: Phenomena and
physiological mechanisms". In D. Stavenga and R. Hardie (eds):
Facets of vision. Berlin-Heidelberg-New York: 281-297.
[0074] Hence, bees finding surfacing mycelium, at the time when
nutrients are being up-channeled into the pre-primordia or
primordia forming mycelium in response to violet light wavelengths,
and when this light is critical for stimulating mycelium to switch
into mushroom formation, such detection by bees would be an
opportune time to find surfacing mycelium and capture dense
nutrition when mycelium is so metabolically active. Since bees can
be trained to discover food based on light spectra associations,
this invention can accelerate the learning process of bees for
finding new food sources using the attributes of mycelium. As a
result, the embodiments of this invention also provide the benefit
of enhancing the usefulness and attractiveness of other forms of
foods for helping the health of bees using these aforementioned
mycelial properties, particularly helping bees discover mycelium at
the primordia formation stages.
[0075] Surfacing mycelium outgasses carbon dioxide and exudates
fragrances, and this inventor hypothesizes that bees can detect
mycelium not only from its scent, but are also attracted to the
mycelium's response to this blue spectrum light, whereupon mushroom
mycelium begins to pack protein, vitamins, and sugar-rich nutrients
at the interface between the high carbon dioxide environment within
substrates and the highly oxygenated environments just above, and
in doing so builds nutritionally dense but accessible
primordia--the first stage of mushroom formation or basidiospores
formation (as in the case of resupinate polypores like Inonotus
species, forming exposed hymenial surfaces, or crusts, that are
brightly colored such as Inonotus andersonii). Many of the brightly
colored fungal pigments, especially but not limited to, yellowish
ones, exhibited by mycelium can be composed of fungal
bioflavonoids, Exploring this rich interface environment--the
surface of yellowish fungal mycelial membranes exposed to the
atmosphere--is anticipated by the inventor to be a rich reservoir
for bees to harvest extracellular and intracellular metabolites
endowed with nutrients and immune-supporting compounds, including
"mycoflavonoids" and "mycosterols" including phenols and
polyphenols not limited to coumarins and benzoic and cinnamic acid
derivatives including coumaric acids and their glycosides.
[0076] By way of example, but not of limitation, mycelia of some
species, especially in the genus Phellinus and Inonotus, produce
brightly colored, yellowish pigments in their mycelium including
polyphenols, for example hispolons such as
6-(3,4-Dihydroxyphenyl)-4-hydroxyhexa-3,5-dien-2-one, (C12H12O4,),
a bright yellow bioactive group of compounds with antioxidant and
immune enhancing properties derived from polypore species such as
Inonotus hisipidus and Phellinus linteus. The inventor hypothesizes
these bright yellowish-colored mycelia would additionally attract
bees foraging for sugars, polyphenols, moisture, natural nutrients
and other secretions that have immune-building antiviral,
antibacterial, antifungal and antiprotozoal properties. Since bees
are especially attracted to yellow colors, those species of fungi,
such as Phellinus and Inonotus, which produce bright yellowish
colors, could preferentially attract bees and also are directly
associated with the yellowish polyphenols containing coumarins to
help bees activate their cytochrome P450 enzyme pathways. This
inventor sees the growing of these wood-decomposing species that
produce brightly pigmented mycelia as preferred candidates for
designing mycelial platforms and extracts for helping bees.
Consequently, extracts of mycelium forming primordia and extracts
of colored mycelium are preferred bee attractants.
[0077] The mycelium in many fungal species will not form
sporulating structures, including but limited to mushroom
formation; such fungi are also preferred for studying their
mycelial extracts for bee attractancy and health.
Viruses, Fungi, Bacteria and Protozoa:
[0078] Bees infected by viruses can lose immune function, as well
as the ability to perform other metabolic functions, as a result of
the viruses "hijacking" the ribosomal machinery to their benefit,
chemically interfering with the crucial phenoloxidase cascade,
suppressing immune responses before they are initiated,
manipulating the host's immune signaling network, disabling the
host's antimicrobial peptides, interfering with the RNAi response
and/or creating "superantigens" that can overwhelm the host immune
system and hijacking.
[0079] The exclusive dependence of viruses on the host cellular
machinery for their propagation and survival make them highly
susceptible to the characteristics of the cellular environment like
short RNA mediated interference. It also gives the virus an
opportunity to fight and/or modulate the host to suit its needs.
Thus the range of interactions possible through miRNA-mRNA cross
talk at the host-pathogen interface is large. These interactions
can be further fine-tuned in the host by changes in gene
expression, mutations and polymorphisms. In the pathogen, the high
rate of mutations adds to the complexity of the interaction
network. Viruses either produce micro-RNAs or target host
micro-RNAs essential to the host immune system. Scaria et al.
(2006) Host-virus interaction: a new role for microRNAs.
www.retrovirology.com/content/3/1/68; Oliver,
http://scientificbeekeeping.com/sick-bees-part-4-immune-response-to-virus-
es/.
[0080] Mushroom mycelium produces a wide array of compounds that
can be anti-bacterial or anti-viral. U.S. patent application Ser.
No. 12/284,646 has been allowed for the antiviral activity of
Fomitopsis officinalis, which includes activity against avian flu
viruses and herpes simplex I & II. Other viruses are
anticipated to be sensitive to the antivirals being coded and
expressed by the mycelium of Fomitopsis officinalis, and indeed
many species in the polyporaceae and Basidiomycetes fungi. The
mycelial extracts are anticipated to be active against viruses that
harm bees, particularly but not limited to IAPV (Israeli Apiary
Virus), DWV (Deformed Wing Virus), the Tobacco Ringspot Virus, and
their relatives. The active ingredients limiting viruses within
extracts are varied, but two groups are polyphenols including
coumarins and sterols including dehydrosulpherinic acids, eburicoic
acids and related compounds. Synergistic benefits between these
polyphenols and sterols can further boost the host defense of bees.
These compounds are resident within the complexes that include
fatty acids, lipids and sterols. As such, many other active
ingredients related to fatty acids, lipids and sterols having
antiviral properties are expected to be of bee benefit. Many of
these aforementioned compounds known as bioflavonoids, and the
species that produce them, are of interest because some of these
species produce mycelium with bright yellowish colors, which may
also serve to attract bees. Very little work, if any, has been done
by mycologists to detect the "colors" of myceliated wood visible to
bees but invisible, or nearly so, to the human eye, especially
light reflected in the ultraviolet bands.
[0081] The inventor has also discovered the antibacterial
properties of Fomitopsis officinalis mycelial extracts against
staph, tuberculosis and E. coli bacteria. This antibacterial
activity is likely to confer an additional layer of protection from
diseases carried by other organisms. These extracts will similarly
have a positive influence in limiting the deleterious effects from
known and yet undiscovered bacteria that are harmful to bees,
animals and plants. See U.S. patent application Ser. No. 13/998,914
and related applications above.
[0082] It is expected that medicinal mushroom species substances
useful in humans will similarly prove useful in up-regulating of
immune genes and benefitting the bee's immune system. Since many
such genes are evolutionarily conserved or similar, it is expected
that the extracts of the mycelium of such mushrooms will similarly
be useful in up-regulating genes and systems in bees to degrade and
deal with infections.
[0083] Useful and preferred fungal genera include, by way of
example but not of limitation: the gilled mushrooms (Agaricales)
Agaricus, Agrocybe, Armillaria, Clitocybe, Collybia, Conocybe,
Coprinus, Coprinopsis, Flammulina, Giganopanus, Gymnopilus,
Hypholoma, Inocybe, Hypsizygus, Lentinula, Lentinus, Lenzites,
Lepiota, Lepista, Lyophyllum, Macrocybe, Marasmius, Mycena,
Omphalotus, Panellus, Panaeolus, Sarcomyxa, Pholiota, Pleurotus,
Pluteus, Psathyrella, Psilocybe, Schizophyllum, Stropharia,
Termitomyces, Tricholoma, Volvariella, etc.; the polypore mushrooms
(Polyporaceae) Albatrellus, Antrodia, Bjerkandera, Bondarzewia,
Bridgeoporus, Ceriporia, Coltricia, Coriolus, Daedalea,
Dentocorticium, Echinodontium, Fistulina, Flavodon, Fomes,
Fomitopsis, Ganoderma, Gloeophyllum, Grifola, Heterobasidion,
Inonotus, Irpex, Laetiporus, Meripilus, Oligoporus, Oxyporus,
Phaeolus, Phellinus, Piptoporus, Polyporus, Poria, Schizophyllum,
Schizopora, Trametes, Wolfiporia; the toothed mushrooms Hericium,
Sarcodon, Hydnum, Hydnellum etc.; Basidiomycetes such as
Auricularia, Calvatia, Ceriporiopsis, Coniophora, Cyathus,
Lycoperdon, Merulius, Phlebia, Serpula, Sparassis and Stereum;
Ascomycetes such as Cordyceps, Ophiocordyceps, Morchella, Tuber,
Peziza, etc.; `jelly fungi` such as Tremella; the mycorrhizal
mushrooms (including both gilled and polypore mushrooms); fungi
such as Phanerochaete (including those such as P. chrysosporium
with an imperfect state and P. sordida).
[0084] Suitable fungal species and genera include by way of example
only, but not of limitation: Agaricus augustus, A. blazei, A.
brasiliensis, A. brunnescens, A. campestris, A. lilaceps, A.
placomyces, A. subrufescens and A. sylvicola, Acaulospora delicata;
Agrocybe aegerita, A. praecox and A. arvalis; Albatrellus hirtus
and A. syringae; Alpova pachyploeus; Amanita muscaria; Antrodia
carbonica, A. cinnamomea and A. radiculosa; Armillaria bulbosa, A.
gallica, A. matsutake, A. mellea and A. ponderosa; Astraeus
hygrometricus; Athelia neuhoffii; Auricularia auricula and A.
polytricha; Bjerkandera adusta and B. adusta; Boletinellus
merulioides; Boletus punctipes; Bondarzewia berkeleyi; Bridgeoporus
nobilissimus; Calvatia gigantea; Cenococcum geophilum; Ceriporia
purpurea; Ceriporiopsis subvermispora; Clitocybe odora, Collybia
albuminosa and C. tuberosa; Coltricia perennis; Coniophora puteana;
Coprinus comatus, C. niveus and `Inky Caps`; Cordyceps bassiana, C.
variabilis, C. facis, C. subsessilis, C. myrmecophila, C.
sphecocephala, C. entomorrhiza, C. gracilis, C. militaris, C.
washingtonensis, C. melolanthae, C. ravenelii, C. unilateralis, C.
clavulata and C. sinensis; Cyathus stercoreus; Daedalea quercina;
Dentocorticium sulphurellum; Echinodontium tinctorium; Fistulina
hepatica; Flammulina velutipes and F. populicola; Flavodon flavus;
Fomes fomentarius, F. lignosus; Fomitopsis officinalis and F.
pinicola; G. resinaceum, annularis, G. australe, G. brownii, G.
collosum, G. sinensis, G. lingzhi, G. curtisii, G. japonicum, G.
lucidum, G. neo-japonicum, G. oregonense, G. sinense, G. tomatum
and G. tsugae; Gigaspora gigantia, G. gilmorei, G. heterogama, G.
margarita; Gliocladium virens; Gloeophyllum saeparium; Glomus
aggregatum, G. calcdonius, G. clarus, G. fasciculatum, G.
fasiculatus, G. lamellosum, G. macrocarpum and G. mosseae; Grifola
frondosa; Gymnopus dryophilus, Gymnopus peronatus, Hebeloma
anthracophilum and H. crustuliniforme; Hericium abietis, H.
coralloides, H. erinaceus and H. capnoides; Heterobasidion annosum;
Hypholoma capnoides and H. sublateritium; Hypsizygus ulmarius and
H. tessulatus (=H. marmoreus); Inonotus hispidus and I. obliquus;
Irpex lacteus; Lactarius deliciosus; Laetiporus sulphureus
(=Polyporus sulphureus), L. conifercola, L. cinncinatus; Lentinula
edodes; Lentinus lepideus, L. giganteus, L. ponderosa, L.
squarrosulus and L. tigrinus; Lentinula species; Lenzites betulina;
Lepiota rachodes and L. procera; Lepista nuda (=Clitocybe nuda);
Lycoperdon lilacinum and L. perlatum; Lyophyllum decastes;
Macrocybe crassa; Marasmius oreades; Meripilus giganteus; Merulius
incamatus, M. incrassata and M. tremellosus; Morchella angusticeps,
M. crassipes and M. esculenta; Mycena citricolor, M. alcalina and
M. chlorophos; Omphalotus olearius; Panellus stypticus, P.
serotinus; Paxillus involutus; Phaeolus schweinitzii; Phellinus
igniarius, P. pini, P. linteus and P. weirii; Pholiota nameko, P.
squarrosa, Piloderma bicolor, Piptoporus betulinus; Pisolithus
tinctorius; Pleurotus citrinopileatus (=P. comucopiae var.
citrinopileatus), P. cystidiosus, (=P. abalonus, P. smithii (?)),
P. djamor (=P. flabellatus, P. salmoneo-stramineus), P. dryinus, P.
eryngii, P. lignatils, P. euosmus, P. nebrodensis, P. ostreatus, P.
pulmonarius (=P. sajor-caju) and P. tuberregium; Pluteus cervinus;
Polyporus indigenus, P. saporema, P. squamosus, P. tuberaster and
P. umbellatus (=Grifola umbellata); Psathyrella hydrophila,
Psilocybe allenii, aztecorum, P. azurescens, P. baeocystis, P.
bohemica, P. caerulescens, P. coprophila, P. cubensis, P.
cyanescens, P. hoogshagenii, P. mexicana, P. ovoideocystidiata, P.
pelliculosa, P. semilanceata, P. serbica, P. subaeruginosa, P.
tampanensis and P. weilii; Rhizopogon nigrescens, R. roseolus and
R. tenuis (=Glomus tenuis); Schizophyllum commune; Schizopora
paradoxa; Sclerocytis sisuosa; Serpula lacrymans and S.
himantioides; Scleroderma albidum, S. aurantium and S. polyrhizum;
Scutellospora calospora; Sparassis crispa and S. herbstii; Stereum
complicatum and S. ostrea; Stropharia ambigua, S. aeruginosa, S.
cyanea, S. albocyanea, S. caerulea, S. semiglobata, S.
semigloboides, and S. rugoso-annulata; Suillus cothumatus;
Talaromyces flavus; Termitomyces robustus; Trametes elegans,
Trametes T. gibbosa, T. villosa, T. cingulata, T. hirsuta, T.
suaveolens and T. versicolor, Trichoderma viride, T. harmatum;
Tricholoma giganteum and T. magnivelare (Matsutake); Tremella
aurantia, T. fuciformis and T. mesenterica; Volvariella volvacea;
and numerous other beneficial fungi.
[0085] Preferred strains which have shown exceptional
characteristics useful for the practice of this invention, include,
by way of example but not of limitation, Ganoderma applanatum
(Strain Duckabush), Fomitopsis officinalis (Strains I, VI, X),
Fomitopsis pinicola (Strain I), Ganoderma oregonense (Meadow Lake),
Heterobasidion annosum (Dosewalips), Pleurotus ostreatus (Strains
PW-OST, Nisqually), Psilocybe azurescens (Stamets strain),
Stropharia rugoso-annulata (Strain F) and Trametes versicolor
(Strain Kamilche Point).
[0086] Additional suitable mushroom genera and species can be found
in standard mycological field guides such as Mushrooms Demystified
(1979, 1986) by David Arora, The Audubon Society Field Guide to
North American Mushrooms (1981, 1995) by Gary Lincoff, and
Psilocybin Mushrooms of the World (1996) by Paul Stamets.
Continually updated lists of suitable species based on the most
recent DNA analysis can be found at
http://tolweb.org/Agaricomycetes/20535 and http://aftol.org/.
[0087] The extracts from the mycelium of Fomitopsis officinalis
particularly, and other species in the Fomitopsidae and
Polyporaceae generally, reduce the pathogenicity of viruses to bees
by directly reducing the viral particle populations while also
fortifying the immune systems of bees, thus limiting their
virulence and transmissibility. Moreover, bees better benefit from
a combination of a mixture of the antiviral components generated by
the mycelium with the antimicrobial properties of coumarins and
other compounds produced by the Fomitopsis officinalis mycelium.
The extracellular exudates secreted by the mycelium of the
beneficial fungi described herein have a combination of these
constituents, but balanced to have the net benefit of attracting
bees so they are fortified with immune enhancing, and nutritionally
beneficial constituents. This multifaceted effect results in
fortifying the immune systems of bees and their colonies, making
them less susceptible to viral, bacterial, protozoal and fungal
mitigated diseases.
[0088] The present inventor has found that Ganoderma, Fomitopsis,
Fomitoporia, Ganoderma, Antrodia, Inonotus, Irpex, Lenzites,
Phellinus, Sparassis, Hypholoma, Pleurotus, and Stropharia species
demonstrate strong anti-fungal properties and expects these will
also be useful for controlling fungal pathogens afflicting bees,
including but not limited to Nosema species and other pathogenic
microsporidia, Chalkbrood and Stonebrood.
[0089] The first antiviral from a mushroom ever discovered was from
the "Ice Man" polypore, Fomes fomentarius, against the Tobacco
Mosaic Virus, the first virus ever to be discovered, and related to
the Tobacco Ringspot Virus. This polypore mushroom is a saprophyte
on birch, beech and other temperate deciduous hardwoods. When it
grows, the wood is softened, releasing moisture, insect-attracting
fragrances and sweetened with the rich, complex polysaccharides, as
well as proteins and other substances generated by the mycelium of
this fungus. This fungus attracts beetles whose burrows
subsequently can be occupied by native bees. In essence, this is
one example of what the inventor anticipates to be many examples of
the role polypore and other Basidiomycetes fungi play in providing
bees with nutrients and antiviral benefits. Interestingly, Fomes
fomentarius is a known endophyte of birch trees--meaning that they
are part of the tree's natural immune system. The inventor
hypothesizes that many of these endophytic fungi confer antiviral
properties on plants and bees, as well as other insects, as they
forage or nest in wood hosting these fungi. The inventor believes
the inter-relational dimensions wherein the biology of bees, fungi
and decomposing trees and plants all intersect will become a
fertile area of scientific research for helping and evolving
ecosystems for decades to come.
[0090] Recent unpublished research funded by the inventor uses
state-of-the-art Next-Generation ("NextGen") sequencing to show
that the consortium of bacterial species selected by Stropharia
rugoso-annulata mycelium on fermented woodchips is several orders
of separation, taxonomically, from the associated bacterial species
of, for instance, Irpex lacteus. Both Stropharia rugoso-annulata
and Irpex lacteus were inoculated into separate containers holding
the same fermented woodchips. The tests proved the mycelium of
mushroom species influences the bacterial genome in close contact
with the mycelial mycosphere (myco-rhizosphere), selecting subsets
of mixed bacterial populations, and yet the mycelium growth rate,
form and tenacity appears extraordinarily healthy and vigorous for
both fungal species. As such, this inventor anticipates that the
microbiome--or mycobiome, i.e. the mixed matrix of fungally
selected bacteria--will produce healthy mycelial mats productive of
sporulating fruitbodies but whose bacterially endowed mycelium is
also friendly to bees and will also provide a bacterial component
which confers anti-pathogen resistance to, for instance, invading
Nosema, a fungal microsporidium. This idiosyncratic consortium of
fungi and bacteria offers yet another complex bioshield of defense,
protecting all the partners who depend upon each other--bees,
mycelium, bacteria, plants and animals, including humans.
Fungal-bacterial mixtures can be customized to best benefit bees
via a wide variety of compositions and methods of producing new bee
supporting products.
[0091] Of course, bears are not the only way to spread to trees
Fomitopsis and other fungi that may improve bee heath. Any activity
resulting in creating wounds in trees, or in creating dead wood,
creates a potential fungal platform of bee benefit. The human use
of woodchips as `beauty bark` or for making trails, or as a top
dressing around ornamentals, would also serve to create a mycelial
platform of benefit to bees. Ultimately, this means we can grow the
mycelium of these fungi, en masse, in a pre-sporulating or
pre-conidial state, make mycelial `landing pads` for bees, or make
extracts, and in doing so creating a new generation of bee
attractants and nutrition customized accordingly.
[0092] With these hypotheses in mind, the inventor sees use of a
wide array of Basidiomycetes, wood-decomposing fungi to develop a
fungal bioshield, a "bee bioshield" of protection from the
stressors leading to colony collapse disorder.
[0093] Moreover, the antibiotic effect of these extracts on
microsporidium bee parasites, particularly Nosema apis, the cause
of `Nosema,` recently reclassified as a simple fungus, will prove
to be a beneficial co-occurring factor.
[0094] Another advantage of the present invention is the
wide-ranging antiviral, antibacterial and antifungal properties
derived from mycelium. Many of the inventor's mycelium extract
fractions demonstrate antiviral activity even when the bioguided
fractionation pathway led to antibacterials. Microbial agents are
often thought of as microbial-type specific (there is some
cross-over between antibacterials and anti-parasitics and now may
even be at least one class with both anti-bacterial and anti-fungal
activity), but considering how difficult it is to attain anti-viral
specificity alone, and the absence of known shared molecular
targets between bacteria and viruses that also exhibit any degree
of selectivity with respect to the host, broad anti-microbial
activity is rare. Without being bound to any theory, the inventor
would hypothesize that the extracts are acting as
immuno-stimulators, immuno-potentiators and immuno-regulators with
antiviral, antibacterial and antifungal effects.
[0095] It is hypothesized that the mycelial components discussed
above and/or other known and unknown compounds are anti-bacterial
and anti-fungal, helping immunity, and hence the interaction
between bees and mycelium is an unanticipated advantage of the
present invention.
[0096] Hyphodermella corrugata, Polyporus umbellatus, and
Piptoporus betulinus are species of the polyporales known to the
author from his research to exhibit strong antiprotozoal
properties. Agaric acid is thought to be one agent responsible for
Piptoporus betulinus's anti-protozoal activity. Agaric acid is also
produced by Fomitopsis officinalis, and possibly by other species
in the polyporales. The production of acanthocytes by Stropharia
rugoso-annulata, known to kill nematodes, may also provide
antiprotozoal and antimiticidal benefits to bees. As such, these
species and their relatives would be preferred for testing for
antiprotozoal activity and up-regulation of antiprotozoal genes in
bees.
Pesticides:
[0097] As bees are limited in the number and variety of enzymes
needed to denature natural and anthropogenic toxins, these toxins
impair their baseline immunity, making them more susceptible to
pathogens from numerous vectors--from Varroa mites, Nosema and
microsporidia fungi, Phorid flies, and the viruses and bacteria
they carry. By increasing the bees' ability to degrade these toxins
by up-regulation of more cytochrome P450 genes, GST genes and/or
CCE genes, the bees' immune state is improved to better resist
these assaults and other stress factors. Moreover, by providing
bees with a blend of fungal extracts that specifically limit the
severity of assaults from Phorid flies, Varroa mites, Nosema fungi
and viruses, bee colony health can be fortified for the long-term
health of the brood, the queen and her drones. These fungal
components are naturally incorporated into the honey and propolis,
thus imparting an advantage to developing generations. Ultimately,
not only are bees are protected, but honey production is expected
to increase, and the quality of the honey better supports
downstream generational health and survivability.
[0098] Those mushroom species useful in bioremediation
("mycoremediation") of toxins, pollutants and pesticides and
extracts of their mycelium are expected to contain various
substances useful in turning on, up-regulating and modulating the
genes necessary for the biodegradation of pesticides. Since many
such genes, or the systems such as the cytochrome system, are
evolutionarily conserved or similar, it is expected that the
extracts of the mycelium of such mushrooms will similarly be useful
in up-regulating genes and systems in bees to degrade and deal with
such pesticides. Useful and preferred species include the
saprophytic mushrooms Pleurotus ostreatus and other Pleurotus
species, Trametes versicolor, Trametes elegans and other Trametes
species, Fomes fomentarius, Fomitopsis officinalis and F. pinicola,
Ganoderma lucidum, G. annulare, G. brownii, G. collosum, G.
lingzhi, G. curtisii, G. oregonense and G. tsugae; Heterobasidion
annosum, Inonotus obliquus, I. hispidus, Irpex lacteus, Laetiporus
sulphureus, L. conifericola, L. cincinnatus, Polyporus umbellatus,
Polyporus elegans, Polyporus squamosus, Antrodia species, Phaeolus
schweinitzii, Boletus mirabilis, Gymnopus peronatus, Mycena
alcalina, M. aurantiadisca, M. haematopus, Psilocybe azurescens, P.
allenii, P. subaeruginosa, P. ovoideocystidiata, P. cubensis, P.
cyanescens, Panaeolus cyanescens, Stropharia ambigua, Stropharia
rugoso-annulata, Stropharia coronilla, Hypholoma capnoides, H.
fasciulare, H. aurantiaca and other species in the Strophariodeae
and Strophariaceae, Lenzites betulinus, Pholiota adiposa, Pholiota
terrestris, Pholiota nameko, Agrocybe aegerita, A. praecox, A.
arvalis, Collybia tuberosa, Collybia, Psathyrella hydrophila, P.
epimyces, Marasmius oreades, and their associated, numerous
"satellite genera" as well as the other gilled and polypore genera
and species known to the mycological science as primary and
secondary decomposers of cellulose and lignin.
[0099] The mycelium of Stropharia rugoso-annulata, Fomitopsis
officinalis, Fomitopsis pinicola, Schizophyllum commune, Trametes
elegans, Trametes versicolor species and many polyporoid and gilled
basidiomycetes produce bioflavonoids, phenols and polyphenols,
including coumarins and coumaric acids (both trans- and cis- o- and
p-coumaric acids) which up-regulate genes in bees which code for
cytochrome P450 enzymes as well as other enzymes critical for
digestion, metabolism and toxin destruction. The effect of these
mycelial components such as coumarins, p-coumaric acid, o-coumaric
acid or their glycosides, is that they turn on more genes within
bees which allow for the bees to detoxify a wide range of toxins,
particularly insecticides, miticides, herbicides, fungicides and
pesticides, and augment the bee's innate immunity.
[0100] P-coumaric acid, found in both grains and lignin, is a
monomer of sporopollenin, the principal constituent of pollen cell
walls and propolis, the resinous compounds gathered and processed
by bees to line wax cells. P-coumaric acid is essential for
increasing laccase in wood rotting fungi, a cellulase enzyme that
breaks down lignin in wood, creating derivative compounds palatable
to insects as food, as well as creating habitats (bees can take up
residence in tunnels bored by mycophagous beetles). As fungi rot
wood, breaking down lignin, they also weep water, rich in these
p-coumaric and nutraceutical compounds beneficial to bees. The more
p-coumaric acid, the more laccases expressed by the mycelium, the
more the wood rots, the more fungal polysaccharides (sugars) and
ultimately the more these compounds will be in the fungal exudates
that the bees seek and from which they benefit. That wood rotting
fungi produce p-coumaric acids and that coumarins can be
bio-converted into p-coumaric acids is yet another advantage of
this invention.
[0101] As was noted by Terron et al., structurally closely-related
aromatic compounds have different effects on laccase activity and
on 1 cc gene expression in the ligninolytic fungus Trametes sp.
I-62, Fungal Genet. Biol., October 2004; 41(10):954-62: "Nine
phenolic compounds (p-coumaric acid, ferulic acid, guaiacol,
syringol, p-methoxyphenol, pyrocatechol, phloroglucinol,
3,5-dihydroxybenzoic acid, and syringaldazine) were tested for
their ability to increase laccase production in the ligninolytic
basidiomycete Trametes sp. I-62. All these compounds resulted in
increases in laccase activity, with the highest levels being
detected in the presence of p-coumaric acid (273-fold) and guaiacol
(73-fold)."
[0102] Interestingly, many of the grains preferred for mycelial
spawn production for mushroom industry (see Growing Gourmet &
Medicinal Mushrooms by the inventor, Paul Stamets, 1993, 2000, Ten
Speed Press, Berkeley) are also rich sources of p-coumaric acids
and may be useful in bee attractant compositions. The primary
phenolic acids in rice grain were identified as p-coumaric acid,
ferulic acid, and sinapinic acid.
[0103] P-coumaric acid is not only in the grains preferred for
mushroom spawn production but they are also generated during the
normal life cycle of mushrooms, especially prior to primordia
formation. P-coumaric acid is a potent inhibitor of tyrosinase, the
enzyme essential for melaninization. The presence and abundance of
p-coumaric acid interferes with the production of darkly colored
pigments. Ultraviolet light stimulates the photodecomposition of
p-coumaric acids but also triggers primordia formation. Once
primordia forms, p-coumaric acids degrade into p-hydroxybenzoic
acid. Sachan et al., Transforming p-coumaric acid into
p-hydroxybenzoic acid by the mycelial culture of a white rot fungus
Schizophyllum commune, 2010, African Journal of Microbiology
4:267-273. As an example, but not one of limitation, the mycelium
of Auricularia auricula (A. auricularia-judae), when grown in
culture is whitish and lacks melanin but contains p-coumaric acids.
When the mushroom mycelium is exposed to light, the mycelium bio
transforms to create dark brown fruitbodies, which are higher in
melanin as they mature, with p-coumaric acids, an inhibitor of
melanin, concurrently declining. This is one example and a strong
argument for the benefit of using lightly colored mycelium,
pre-melaninization as a source of mycelium for making extracts
beneficial to bees due to its innate p-coumaric acid content
compounded by the native content of p-coumaric acids in the grains
that are used for spawn production for growing mycelium.
Interestingly, the ideal interface for capturing the best benefits
from mycelium for its nutraceutical and p-coumaric acid contents,
is short window, often of just a few days in length, before and
directly after light exposure, but before dark colored fruitbody
development beyond the white primordial stage.
[0104] Given that some of the most abundant laccase producers yet
tested thus far are Ganoderma lucidum, Trametes versicolor and
Pleurotus ostreatus, these species are specifically preferred for
use in creating bee-beneficial mixtures.
[0105] When not immunologically depressed from man-made and natural
toxins, bees natural host defense can better protect bees from
other deleterious agents, including viruses and pathogens
transmitted by Varroa mites.
Varroa Mites and Insect Parasites:
[0106] The inventor has received several patents on compositions
and methods of using the presporulating mycelium of
entomopathogenic fungi as an attractant and treatment for
controlling insects, and more broad patents are pending (U.S.
patent application Ser. No. 13/986,978) on arthropods, and the
diseases insects and arthropods vector (U.S. patent application
Ser. Nos. 13/317,613 and 13/373,719). Varroa mites are known as a
vector of the Israeli Apiary Virus and the Tobacco Ringspot
viruses. Varroa mites, both plant and insect biting mites, carry
more than one virus or bacterial pathogen, meaning that mites are
one, albeit significant, vector carrying and introducing multiple
pathogens in the onslaught threatening beehive health. As bees
weaken from viral exposure, for instance, they are less able to
shed the attaching Varroa mites. However, the mycelium of
entomopathogenic fungi, particularly Aspergillus flavus,
Metarhizium anisopliae and Beauveria bassiana, can be used to
attract, sicken or kill the Varroa mites, reducing their activity,
delivery of pathogen payloads and numbers, thus tilting the balance
in improving the host defense of the colony against CCD. Spores of
entomopathogenic fungi, including Metarhizium, Beauveria and the
Entomophthorales can similarly be used to sicken or kill Varroa
mites, although mites may find spores repellant as compared to
preconidial mycelium.
[0107] Moreover, extracts of Metarhizium anisopliae can be made
specifically to attract, but not kill insects, including bees, by
growing strains of Metarhizium anisopliae that do not contain
destructins, or have reduced levels of these or other toxins, or
reduced virulence and pathogenicity. Variability of toxins is true
when comparing many strains of Aspergillus flavus, a known
entomopathogenic fungus, primarily toxic due to its aflatoxin
content. Aflatoxin-free strains of Aspergillus flavus are available
currently, which are naturally occurring or can be made through
culture selections or genetic modifications. So too can
destructin-free strains of Metarhizium anisopliae strains be
created, selected for, or sourced from natural genomes. Strains can
also be produced which are not entirely free of destructins or
alfatoxins, but produce such low levels that they can be toxic to
mites but not very toxic to bees due to the fact that the bees'
cytochrome P450 levels and pathways have been enhanced from
exposure to the coumarins and other polyphenols presented by the
mycelium. In essence, the up-regulation of cytochrome p450's
(CYP's) may help bees better tolerate or detoxify destructins or
aflatoxins to which the bees are exposed from Metarhizium
anisopliae and Aspergillus flavus and other toxins produced by
entomopathogenic fungi.
[0108] The advantage of a destructin-free or a reduced destructin
strain of Metarhizium anisopliae is that the extracts of the
mycelium could be produced with high sugar and terpene content
which would simultaneously attract bees and mites. Use of an
appropriately sized mesh screen or barrier or other means of
selection allows for mites to be partitioned from bees so both bees
and mites could be initially attracted to the same location of the
extracts (or similarly attracted to preconidial mycelium). The
proportionality of the endemic entomopathogenic toxins can be
balanced to sicken mites but not bees. Using single or multiple
fungal extracts as described herein offers a latitude and
flexibility of customized design, so that numerous devices,
delivery systems, compositions and methods can be made available
for the first time to favor bee health and decrease CCD. Phorid
flies, gnats and mites predating on mushrooms are well known to the
mushroom industry. What was not known is that extracts of
entomopathogenic fungi prior to sporulation are attractive to these
insects and arthropods. The present inventor does not believe that
hydroethanolic extracts of mushrooms or mushroom mycelium with
these attractive properties were known to the mushroom industry
prior to this inventor's disclosures in pending and approved
patents.
[0109] Combining oxalic acids with sugar enriched water loaded with
spores or preconidial mycelia of entomopathogenic fungi such as
Metarhizium anisopliae and Beauveria bassiana will improve the
miticidal actions of the combination of oxalic acids and
entomopathogenic fungi and the anti-miticidal properties of other
components resident or added to sugar water, pollen patties or bee
sprays, for instance. However, oxalic acid is reactive to the
minerals in the fungal extracts, and this poses a hurdle for
effective formulation.
[0110] When combining oxalic acids with the extracts of filamentous
Basidiomycetes fungi, the resident minerals (calcium, phosphorus,
iron) may bind with the oxalic acid thus reducing the mineral
scouring, miticidal potential of the oxalic acids. Therefore,
demineralization of the fungal extracts before combining oxalic
acid to the fungal extracts is an embodiment of this invention.
Demineralization employs any of numerous methods useful for
demineralizing of the fungal extracts so as to prevent conversion
of the reactive oxalic acid into water insoluble salts by
eliminating calcium and other minerals resident within the fungal
extracts. One method of many available is to make use of ion
exchange resin technologies. The fungal extracts can be added to
distilled water at a ratio of 1:10 preferably, with ranges of 1:1
being the most concentrated and 1:100 being most dilute but less
preferable. Upon completion, minerals in the fungal extracts, which
would otherwise neutralize the anti-miticidal properties of oxalic
acid, will be largely if not completely removed. Thereupon, oxalic
acid can be added to the reduced mineral, fungal extracts in a
sufficient quantity to have an anti-miticidal effect, in the range
of 1-10% of oxalic acid to the mass of the solution, resulting in a
low pH in the 0.5-3.5 pH range, with an optimal range in the
0.5-2.0 pH range.
[0111] "We seek to understand the botanical sources and biological
activities of resins in the field and how resin foraging behavior
changes in response to environmental factors, such as infection and
other biological stresses. If we can discover plants with
preferable and more antimicrobial resins in different regions, it
should be possible to better create environments that promote bee
health by supporting behaviors and managerial strategies that lead
to natural disease resistance." Wilson et al., Metabolomics reveals
the origins of antimicrobial resins collected by honey bees. PLoS
One 8(10): e77512, page 11. The present inventor suggests that
fungi and fungal mycelium, including fungal attractants, fungal
entomopathogens, fungal immunostimulators, fungal antivirals,
antibacterials and antifungals can similarly support bee health and
lead to natural resistance to diseases and pesticides. Ecosystems
and economies benefit from bees that would otherwise suffer without
these myco-remedies.
[0112] This inventor also anticipates that pollinating insects and
animals (bats) will also benefit from the effects of this
invention. It is also expected that birds may similarly benefit
from similar integrated fungal solutions via addition to nectar
feeders and bird foods through the up-regulation of immunological
and detoxification genes.
[0113] Filamentous, basidiomycetous fungi are also sources of
neuroregenerative compounds. Species of Hericium, (including but
not limited to Hericium erinaceus, Hericium corralloides and
Hericium abietis) produce potent nerve growth factors causing
regeneration of myelin on the axons of nerves and nerve
regeneration. (See:
http://www.huffingtonpost.com/paul-stamets/mushroom-memory_b.sub.--172558-
3.html). Psilocybin and psilocybin-producing fungi, including but
not limited to species of Psilocybe, Panaeolus, Gymnopilus, Pluteus
and Conocybe such as Psilocybe azurescens, Psilocybe cyanescens,
Psilocybe allenii, Psilocybe cyanofibrillosa, Psilocybe cubensis,
Psilocybe ovoideocystidiata, Psilocybe subaeruginosa, Copelandian
Panaeoli (Copelandia cyanescens, Copelandia tropicalis, Copelandia
bispora), Pluteus salicinus, Gymnopilus luteofolius, Gymnopilus
spectabilis, Conocybe cyanopus and Conocybe smithii can trigger
neurogenesis. (See Catlow et al., Effects of psilocybin on
hippocampal neurogenesis and extinction of trace fear conditioning,
Exp Brain Res (2013) 228:481-491 DOI 10.1007/s00221-013-3579-0).
Individually or in combination, mixtures of extracts of psilocybin
mushroom and Hericium mushroom fruitbodies, or more preferably
their mycelial extracts, could help repair neurons damaged by
toxins, cholinergic pesticides, oxidation, old age, or other
sources of neurotoxins. The net effect of ingesting these mixtures
of nerve regenerating Hericium and psilocybin species would improve
the neurological health of bees through neurogenesis and
re-myelination, and indeed of animals, including humans. Another,
improved form of "mycological honey" might incorporate these
elements for the benefits of bees and people, improving cognition,
preventing or repairing neuropathies presenting themselves as
diseases to humans within scope of the definitions for Alzheimer's,
Parkinson's, Parkisonisms, MS (multiple sclerosis), or as yet
uncategorized forms of neurological impairment. Indeed such
combinations could increase intelligence, sensory abilities,
memory, reflexes, reaction times, and problem solving abilities. As
such a `smart mycological honey` is anticipated to be within the
scope of this invention.
[0114] Ganoderma lucidum is one of the species of particular
interest (along with Ganoderma resinaceum, Ganoderma applanatum,
Ganoderma brownii, Ganoderma curtisii, Ganoderma oregonense,
Ganoderma tsugae, Ganoderma lingzhi, Ganoderma capense, Ganoderma
annularis and Ganoderma collosum) to the inventor as it not only
has strong antiviral properties, but has complexes of sugars that
result in its mycelium producing a viscous syrup-like "mycological
honey" that can be used to help bees survive CCD. The inventor and
his team at Fungi Perfecti, LLC have also noted that the extracts
of Ganoderma resinaceum will not freeze, even when freeze driers
achieve temperatures less than -50 C..degree. under high vacuum,
whereas species tested outside the genus Ganoderma readily freeze
dried into a dried state under the same conditions. The inventor
hypothesizes the mycelial extract of Ganoderma resinaceum, and
likely extracts of related Ganoderma species, maintains a liquid
state even under cryogenic conditions due to its unique assortment
of complex sugars, sterols, and glycoproteins binding to form a
unique liquid matrix far different than any other species tested.
This extract may have potential as an anti-freeze with broad
reaching implications for medicine, avionics, space travel, and
usefulness under extreme temperature conditions for lubricating,
preservation, and extremophile chemistry.
Example 1
[0115] Stropharia rugoso-annulata, Fomitopsis officinalis, Trametes
versicolor, Schizophyllum commune and other mushroom species are
cultured and the mycelium grown on rice, barley, flaxseeds or other
grains or sawdust or wood chips. See Stamets, Growing Gourmet and
Medicinal Mushrooms, 1993, Ten Speed Press, Berkeley, Ca. When the
mycelium reaches a mass of growth (preferably after 5 to 60 days
growth in fermentation or in solid state fermentation subsequent to
inoculation, but well before fruitbody formation) mycelial mass can
be extracted through simple aqueous, water/ethanol (both of which
are preferred) or ethanol washing of the substrate, or from
compression of the substrate, all of which will result in a liquid
fluid or capture-able extract including extracellular exudates.
These extracts can be utilized as they are, or alcohol (25-50% by
volume) may be added to aqueous extracts as a preservative and
solvent (which will precipitate water-soluble polysaccharides). The
hydroethanolic extract can be evaporated or removed, or the alcohol
and water may be evaporated and removed separately. The crude
extract can be cell free filtered using a 0.12-0.20 .mu.m filter.
This extract can be frozen or dried for future use. The extract can
be added to any form of feed stocks for bee consumption. The
original extract can be used directly or diluted to .about.10% and
added to their drinking water, sugar water, bee candy, honey,
propolis, pollen patty, grease patty or even into the wax used for
making combs and supers. Contact by bees improves the bees' ability
to build immunity through up-regulation of toxin degrading enzymes,
reduces pathogen payloads and provide a healthy source of diverse
sugars, amino acids, vitamin B's, and nutrients.
Example 2
[0116] To make the mycelial extract, use equal volumes of mycelium
grown on grain (barley, flaxseed, rice, oats, millet, wheat, rye,
corn), seeds, including nuts, sawdust or wood chips (Douglas fir,
pines, oaks, birches, cottonwoods, olives) and immerse into a 50:50
water-ethanol solution. Allow to sit at room temperature for two
weeks, and then press to expel the liquid extract. Over several
days, a precipitant will fall out of the hydroethanolic solution.
The hydroethanolic supernatant is drawn off above the pasty
precipitant. After several more weeks, or by using a centrifuge,
the precipitant further concentrates into a semisolid state. These
wet semisolids are removed and heated to 50.degree. C. for 6-8
hours while stirring. The wet volume of semisolids is reduced to
about 40% of the original wet semisolids. The drying down of the
semi-solids into the caramel "honey-like" substance yields about
16% of the original wet solids wet. Therefore, using 1000 mL of wet
solids (which was 40% of the initial extract) yields about 170 mL
of thick syrupy caramel like substance. Continued heating and
stirring concentrates this substance with noticeably sweeter
properties. The extract can be crystallized, powdered, and used as
amendment to other treatments. The liquid, semisolid and
crystallized forms are noticeably sweet in taste and could be
considered a medicinal candy-like substance useful to both bees and
people in a wide number of applications.
Example 3
[0117] A mycelial extract is made by extracting fruitbodies or
mycelium of basidiomycetous fungi including Ganoderma resinaceum in
hot water (80-100.degree. C.) for several hours and combined with
the room temperature (10-30.degree. C.) water extraction of
Stropharia rugoso-annulata mycelium grown on grain or wood. To
these water extracts, ethanol is added to make the solution greater
than 22% EtOH, preferably 35-45% EtOH. Upon addition of ethanol,
polysaccharides precipitate out of solution and settle at the
bottom of the extraction vessel. Upon drawing off the supernatant,
the precipitated polysaccharides, rich in glycosides, glycoproteins
and other `nectar-like` nutrients, are collected and heated between
50-70.degree. C. over several hours, resulting in the creation of a
sweet residue attractive to and beneficial to bees. Alternately,
the supernatant can be stored over several days, which further
yields useful precipitating polysaccharides. These precipitants
contain complex sugars, antivirals, antibacterials, cytochrome p450
up-regulating coumaric acids and coumarins, and can be combined
with other ingredients used in the feeding water, pollen patties,
propolis, bees wax, sprays, or in any delivery system whereby bees
make contact with these precipitants, helping bees overcome
stressors associated with Colony Collapse Disorder.
Example 4
[0118] A mycelial extract made from extracting fruitbodies or
mycelium of basidiomycetous fungi including Ganoderma resinaceum is
first soaked in 100% ethanol (1:1 ratio by mass) for 1-7 days. Upon
draining off the ethanol, the mushroom- or mycelial-marc is
immersed into hot water (80-100 C) for several hours and combined
with the room temperature (10-30 C) water immersion and extraction
of Stropharia rugoso-annulata mycelium grown on grain or wood. To
these water extracts, the ethanol extracts previously described are
added to make the total combined solution greater than 22% EtOH,
preferably 35-45% EtOH. Upon addition of ethanol fraction,
polysaccharides precipitate out of solution and settle at the
bottom of the extraction vessel. P-coumaric acid, being more
soluble in ethanol than water, is richer in the ethanolic extracted
supernatant. (The ethanolic supernatant, with concentrated
p-coumaric acids, is a reservoir of bee-beneficial cytochrome p450
coding compounds.) This hydroethanolic supernatant can be stored
over several days, which further yields a mixture of
polysaccharides but which is proportionately higher in p-coumaric
acids than the hot water fractions alone. These p-coumaric enriched
precipitants also contain complex sugars, antivirals,
antibacterials, and families of coumarins, and can be combined with
other ingredients, such as the water soluble mushroom
polysaccharides, corn syrup or sugars used in sweetening the
feeding water, or additionally incorporated as an ingredient in
pollen patties, propolis, bees wax, sprays, or in any delivery
system whereby bees make contact with these precipitants, helping
bees overcome stressors associated with Colony Collapse
Disorder.
Example 5
[0119] A liquid extract of the mycelium, or a precipitate from such
extract, or a concentrated extract from which all or part of the
solvent has been removed, containing these active principles can be
added to the honey, to honey-enriched water, to sugar water or bee
candy, to pollen, to pollen substitutes, or to other substances in
other manners obvious to those skilled in the art of apiary science
or commercial practices. The extract can be used as an adjunct to
other remedies making them more effective. The extracts can be in
liquid, frozen, freeze dried, air dried, vacuum desiccated,
refractance window dehydrated, sonically dehydrated, or partially
purified forms, in amounts sufficient to have the effect of
attracting bees and/or benefitting bee health, honey production and
pollinations.
Example 6
[0120] A preferred delivery system would be to incorporate the
mycelial extracts into pollen patties or grease patties. Pollen
patties are made by beekeepers and placed above the brood chamber
as a source of nutrition. They can be made from a wide range of
materials, including soy, brewers yeast, sugar syrup and may
optionally include organically grown pollen. These pollen patties
supplement the bees nutritionally. Because they are widely used in
the fall, they help the bees survive into the next year. These
extracts also contain digestive enzymes which help the bees better
metabolize food stocks, and help break down toxins and improve
baseline immunity.
Example 7
[0121] A mixture of compositions comprising extracts of Stropharia
rugoso-annulata, Fomes fomentarius and Fomitopsis officinalis,
which together offer a plurality of benefits, can be added to
water. The Stropharia rugoso-annulata attracts bees, has a
flower-like fragrance, and provides sugar-rich (up to 75
polysaccharides) nutrient source. The Fomes fomentarius and
Fomitopsis officinalis extracts confer antiviral benefits, plus
those additional benefits already mentioned for Stropharia
rugoso-annulata. All three extracts contain polyphenols, and more
particularly coumarins, which help activate p450 enzyme pathways,
which help bees detoxify endogenous, natural, foreign and
anthropogenic toxins and their associated deleterious effects. A
mixture of these extracts can be given to the bees via their
drinking water, their enriched water, honey, propolis, pollen
patties or even in the wax used for making preformed combs in the
creation of supers for honey production.
Example 8
[0122] Add the extracts from the mycelium of Fomitopsis officinalis
to the sugar-water typically given to bees in the early spring
before pollen levels rise, to help reduce resident viral loads
early in the season, preventing their escalation to the level of
becoming a behavior-altering disease or for causing bee-to-bee
transfer of pathogens. The extracts can simply be mixed into the
sugar water at a rate sufficient to have a positive effect. The
range could preferably be 0.1-20%, or more preferably 1-10% of the
volume of the sugar water compositions employed by beekeepers. The
extracts would be mixed in the water first and then added to the
sugar to make the typical syrup. The high sugar content would act
as a preservative to keep the antiviral and antibacterial
properties long lasting.
Example 9
[0123] Extracts of Stropharia rugoso-annulata can be soaked into
paper strips. These paper strips can be combined with an adhesive.
The low pH of the Stropharia rugoso-annulata, in the pH 0.5-4
range, is toxic to mites but harmless to bees upon contact. Oxalic
acid solution may optionally be added in effective amounts.
Example 10
[0124] Use extracts of the mycelium or fruitbodies from Ganoderma
lucidum, Ganoderma resinaceum, Fomitopsis pinicola, Fomitopsis
officinalis and Schizophyllum commune whereby the extracts are
concentrated into a form attractive to bees and sufficient, upon
contact, to have the effect of reducing the Tobacco Ringspot Virus,
the Israeli apiary virus, Invertebrate Iridescent Virus, or IIV6,
and Nosema microsporidia, resulting in bees being able to better
overcome Colony Collapse Disorder.
Example 11
[0125] Use extracts of the mycelium or fruitbodies from Ganoderma
lucidum, Ganoderma resinaceum, Fomitopsis pinicola, Fomitopsis
officinalis, Schizophyllum commune and Stropharia rugoso-annulata
whereby the extracts are concentrated into a form that resembles
the texture and consistency of honey, in a form attractive to bees
and sufficient, upon contact, to have the effect of reducing the
Tobacco Ringspot Virus, the Israeli apiary virus, and Nosema
microsporidia, and causing the up-regulation of cytochrome p450
enzyme pathways, improving overall immune function, foraging
ability, overwintering, drought resistance, ability to overcome
losses of nectar providing plants, resulting in an improved health
to bees so that there is a measurable benefit for beehives to
survive and overcome Colony Collapse Disorder and produce
descendent generations. This "mycological honey" can be used
separately, or mixed into bee honey to attract and benefit bees.
Moreover, this "mycological honey" can be partially dissolved into
water as a foliar spray to plants or applied directly onto bees.
Additionally, this `mycological honey` can be marketed as a
nutraceutical for human consumption.
Example 12
[0126] Bees flying to or from the sugar water, upon entering the
beehive, buzz and shake their bodies to dislodge the mites. If the
mites fall through a screen, they are in contact with or attracted
to the entomopathogenic mycoattractant, which in itself may be
lethal, or onto insecticidal mycelium, wherein the mites sicken or
die, reducing the mites' ability to travel and infect, thus
lessening its threat vector to bees. Moreover, if bees are sprayed
with an oxalic acid enriched spray, the parasitic mites become more
susceptible to the infectious or lethal properties of the
entomopathogenic fungi.
Example 13
[0127] The extracts, hyphal fragments or spores of beneficial
fungi, such as Stropharia rugoso-annulata, and the spores of
entomopathogenic fungi such as Entomophthorales, can be
incorporated as a mixture into the extract-enriched sugar water,
bee foods or honey, which allows for transference into the honey
production stream, benefitting the brood, the drones, the queen and
the hive overall.
Example 14
[0128] Extracts of the mycelium of, or spores, hyphal fragments, or
tissue of, Stropharia rugoso-annulata can be presented on paper
strips or in water accessible to the bees. The fragrance of
Stropharia rugoso-annulata, to which bees can be accustomed, helps
foraging bees to return to their colonies if these fragrances are
placed near to or within the hives. Such fragrances can be emitted
via any method known to the art of delivery of fragrances, foggers,
sprays or aerosol dispensers. It is expected that the extracts of
Stropharia rugoso-annulata mycelium and the extracts of other
mushroom mycelia will induce trail following or navigation behavior
via "dance language" and odor plumes.
Example 15
[0129] A mixture of compositions of extracts of Stropharia
rugoso-annulata, Fomitopsis officinalis and Metarhizium anisopliae,
which together offer a plurality of benefits, can be added to
water. The Stropharia rugoso-annulata attracts bees, has a
flower-like fragrance, and provides sugar rich (up to 75
polysaccharides) nutrient source. The Fomitopsis officinalis
extracts confer antiviral and antibacterial benefits, plus those
already mentioned for Stropharia rugoso-annulata. The Metarhizium
anisopliae extracts can be presented in sticky strips or mats, or
into any sticky, mite- or Phorid fly-capturing substance, or in
water accessible to the same to attract mites and Phorid flies,
whereupon contact, they are debilitated or killed, reducing their
ability to be a vector of disease; Varroa mite populations can be
reduced using Metarhizium anisopliae extracts before the brood
chambers are sealed, reducing bee deaths from exposure to mites and
the diseases they carry. All three extracts contain polyphenols,
and more particularly coumarins, which help activate p450 enzyme
pathways, which help bees detoxify endogenous, foreign, natural and
anthropogenic toxins and lessen their associated deleterious
effects. A solution of these mixed extracts can be given to the
bees via nectar feeders containing their drinking water or their
sugar or fructose enriched water, via mixing into bee candy, honey,
propolis, pollen patties or even by mixing into the wax used for
making preformed combs in the creation of supers for honey
production.
Example 16
[0130] Extracts of the preconidial mycelium of Metarhizium
anisopliae pathogenic to mites and/or flies can be mixed with
spores or hyphal fragments of same, and presented in sticky strips
or mats, or into any sticky, mite- or Phorid fly-capturing
substance, or in water accessible to the mites. This combination
attracts mites or flies, which upon contact, infects them with an
entomopathogenic fungus or exposes them to a lethal does of
entomopathogenic toxins.
Example 17
[0131] Extracts of the preconidial mycelium of Metarhizium
anisopliae mixed with the extracts, spores or hyphal fragments of
Stropharia rugoso-annulata can be presented on paper strips or in
water accessible to the bees. This combination attracts mites or
flies, and bees, which upon contact harms the mites and flies but
not bees.
Example 18
[0132] Extracts of the preconidial mycelium of Aspergillus flavus,
Aspergillus niger and Aspergillus fumigatus can be mixed with the
spores or hyphal fragments of Stropharia rugoso-annulata and
presented on paper strips or in water accessible to the bees. This
combination attracts mites or flies, and bees, which upon contact
harms the mites and flies but not bees. Optionally, strains of
Aspergillus flavus, Aspergillus niger and Aspergillus fumigatus can
be used which have reduced aflatoxin and neurotoxin levels, below
the levels which would harm bees but above the levels harming mites
and flies, thus conferring a net benefit to bee colony health.
Example 19
[0133] Extracts of the preconidial mycelium of Metarhizium
anisopliae can be mixed with the spores or hyphal fragments of
Stropharia rugoso-annulata can be presented on paper strips or in
water accessible to the bees. This combination attracts mites or
flies, and bees, which upon contact harms the mites and flies but
not bees. Optionally, strains of Metarhizium anisopliae can be used
which have reduced destructin levels, below the levels which would
harm bees but above the levels harming mites and flies, thus
conferring a net benefit to bee colony health.
Example 20
[0134] Extracts of mushroom mycelium and/or extracts of the
preconidial mycelium of Metarhizium anisopliae can be mixed with
extracts or derivatives from Neem trees and presented on paper
strips, in water accessible to the bees or in topical sprays. This
combination attracts mites or flies, and bees, which upon contact
harms the mites and flies but not bees. Optionally, strains of
Metarhizium anisopliae can be used which have reduced destructin
levels, below the levels which would harm bees but above the levels
harming mites and flies, thus conferring a net benefit to bee
colony health. Optionally, the concentration of Neem tree extracts,
and sugars can be balanced to optimize benefits to bees by reducing
mites and their foraging abilities, and their pathogen payloads.
Furthermore, this combination can be further enhanced with the
addition of extracts of Basidiomycetes fungi from agaricoid and
polyporoid fungi, which not only provide mite-destroying oxalic
acids, and toxin degrading enzymes, but also up-regulates bee's
innate cytochrome p450 enzymatic pathways to break down
anthropomorphic toxins, and additionally reduces virally,
bacterially, and fungally associated pathogens afflicting bees.
Such synergistic effects from multiple constituents have the net
effect of helping bees better survive Colony Collapse Disorder. A
combination of using preconidial mycelium of Metarhizium
anisopliae, the extracts of Fomitopsis officinalis and Fomitopsis
pinicola, the extracts from Neem trees, the extracts of Ganoderma
lucidum, Ganoderma resinaceum, Ganoderma applanatum, Pleurotus
ostreatus, Trametes versicolor and Stropharia rugoso-annulata
immersed and mixed into water is anticipated to be an effective
composition and method for making a deliverable, efficacious bee
spray or ingredient in pollen patties or drinking water. Similar
compositions may be sprayed on plants or trees which bees
pollinate, benefitting both plant and bee.
Example 21
[0135] The composition of oxalic acid, sugar (or polysaccharide)
enriched water, and the preconidial hyphal fragments from
Metarhizium anisopliae which upon contact with bees selectively
harms the mites while having a net benefit to bees. This
composition may optionally be combined with extracts of medicinal
mushroom mycelium and incorporated into bee food.
Example 22
[0136] The combination of the extracts from Fomitopsis pinicola,
Fomitopsis officinalis, Stropharia rugoso-annulata and Ganoderma
resinaceum in combination with the extracts of the preconidial
mycelium of Metarhizium anisopliae to attract bees and mites
whereby contact with this combination harms Varroa mites, reducing
viruses, pathogenic fungi and bacteria, providing a net benefit for
bees overcoming Colony Collapse Disorder.
Example 23
[0137] The combination of the extracts or derivatives from Neem in
combination with the extracts of preconidial mycelium of
Metarhizium anisopliae to attract bees and mites whereby contact
with this combination harms Varroa mites, reducing viruses,
bacteriophages, pathogenic fungi and bacteria that harm bees but
has a net benefit for bees overcoming Colony Collapse Disorder.
Example 24
[0138] The combination of the preconidial mycelium of Metarhizium
anisopliae with polysaccharides of Ganoderma resinaceum or
Ganoderma lucidum to attract bees and mites whereby contact with
this combination harms Varroa mites, and reduces viruses,
bacteriophages, pathogenic fungi and bacteria that harm bees but
has a net benefit for bees overcoming Colony Collapse Disorder.
Example 25
[0139] Honey is collected from bees fed mycelium extracts as above.
This medicinal honey helps both bees and people up-regulate
pathways for denaturing toxins, via cytochrome P450 pathways. Since
honey is a food for more than bees and people, such medicinal
honeys are expected to have a wide range of uses. The preservative
properties of honey can help keep these medicinally active
compounds more stable.
Example 26
[0140] Use extracts of the mycelium or fruitbodies lacking melanin
such as from so called albino fruitbodies of Agaricus blazei,
Schizophyllum commune, Trametes elegans and Stropharia
rugoso-annulata whereby the extracts are concentrated into a form
that resembles the texture and consistency of honey, in a form
attractive to bees and sufficient, upon contact, to have the effect
of reducing the Tobacco Ringspot Virus, the Israeli apiary virus,
Invertebrate Iridescent Virus, or IIV6, and Nosema microsporidia,
and causing the up-regulation of cytochrome p450 enzyme pathways,
improving overall immune function, foraging ability, overwintering,
drought resistance, ability to overcome losses of nectar providing
plants, resulting in an improved health to bees so that there is a
measurable benefit for beehives to survive and overcome Colony
Collapse Disorder and produce descendent generations. This
"mycological honey" can be used separately, or mixed into bee honey
to attract and benefit bees. Moreover, this "mycological honey" can
be partially dissolved into water as a foliar spray to plants or
applied directly onto bees. Additionally, this `mycological honey`
can be marketed as a nutraceutical for human consumption.
* * * * *
References