EA037750B1 - Integrative solutions for protecting bees using fungi - Google Patents

Abstract

The invention provides a plurality of benefits from the extracts of mycelia of individual fungal species and mixtures of species to provide an armamentarium of defenses from multiple stressors in order to help bees survive a complex of symptoms collectively called colony collapse disorder (CCD). More particularly, the invention utilizes specific concentrations of extracts from pure cultured mycelium from mushroom forming fungi to reduce harmful viruses in bees and to increase the longevity of bees.

Description

This invention claims priority from US Provisional Application No. 62/074023, filed 02/11/2014, currently pending, incorporated herein by reference in its entirety. Also, the application is a partial continuation of US patent application No. 14/247207, filed 04/07/2014, currently pending, which claims the priority of US provisional patent application No. 61/967117, filed 03/10/2014, now pending, both of which are incorporated herein by reference in their entirety.

Technology area

The present invention relates to compositions containing extracts of mycelium species of fungi and mixtures thereof to provide a multi-stress protection toolkit to help bees cope with a complex of symptoms, collectively referred to as colony collapse syndrome (CCD). More specifically, within the framework of the present invention, specific concentrations of usable extracts of pure cultured mycelium of fruiting body-forming fungi are used to reduce the level of harmful viruses in bees and to increase the lifespan of the bees.

State of the art

Approximately 100,000 species of insects, birds and mammals are involved in the pollination of flowering plants. They include approximately 20,000 known species of bees. The Food and Agriculture Organization of the United Nations estimates that of just over 100 crops that provide 90% of the food supply for 146 countries, 71 are pollinated by bees (mostly wild bees), and some others are pollinated by thrips, wasps, flies, beetles, moths and other insects. The annual monetary value of pollination services in global agriculture can be as high as $ 200 billion. Protecting the Pollinators, Food and Agriculture Organization of the United Nations. The co-evolution of plants and bees (Apis species) is fundamental to their mutual survival. The bees distribute pollen, and many plants produce abundant nectar in response.

Approximately 4,000 bee species are native to North America. Following the introduction of European (or Western) honeybees (Apis mellifera) into North America by colonists, commercial orchards and farms that were previously unable to survive began to flourish, although many New World crops and native flowering plants are largely dependent on pollination by native species. bees. Agriculture in Asia also depends on Asian (or Oriental) honey bees (Apis cerana), although generally on a smaller and more regional scale (A. mellifera has also been introduced). In general, in agriculture, the number of fruit, nut and vegetable crops that benefit from bee pollination is astounding, as is the number of flowering trees, shrubs and wild flowers. Indeed, it is difficult to exaggerate the role of bees in commercial food production. The current extinction of bees is unprecedented and poses a huge threat to food security around the world. In some areas of China, for example, the extinction of bees has required manual pollination to conserve crops, which is incredibly difficult.

A honeybee hive is a warm, humid, densely populated environment in which closely related individuals live - ideal conditions for viruses, bacteria, fungi, protozoa and ticks. Bees have successfully defended themselves for millions of years against such threats through unique colony and individual defense systems and immune responses, but these defenses may have been compromised by the intense domestication of the European honeybee and a variety of threats including new anthropogenic stressors. led to a precipitous decline in the number of wild honey bees and native bees in regions including North America, Europe and China from 1972 to 2006, and to the onset of colony collapse syndrome (CCD) in honey bees in 2006.

The domestic beekeeping industry depends on the hatching of queens, a selection process that provides lines to reproduce, and rearing of queens, the process of obtaining and culling queens of honeybees. A significant part of beekeeping in the United States is carried out by 10-15 large queen-producing companies that exchange genetic information from about 500 breeding queens. This limited genetic diversity can lead to susceptibility to various diseases, parasites, or colony collapse syndrome. In particular, queen rearing is disrupted by viruses, especially queen cells and other viruses, including wing warp virus, Israeli acute bee paralysis virus, and about two dozen others. Other viruses are expected to be discovered that cause disease in bees, including queens, their offspring, worker bees, nurse bees and drones.

Colony extinctions and extinctions of bees have occurred throughout the history of beekeeping (apidology), including various honeybee syndromes in the 1880s, from the 1900s to the 1920s, 1960s and 1990s, such as the extinction disease, spring syndrome emaciation, autumn wasting syndrome, fall collapse disease and mysterious disease. In 2006, some beekeepers began to report unusually high losses, accounting for 30-90% of their swarms. This disease of disappearing bees

- 1,037750 has been renamed Colony Collapse Syndrome (CCD, sometimes called spontaneous swarm collapse or Mary Celeste Syndrome in the UK). CCD may or may not be associated with antecedent colony extinction syndromes; it may be a truly new violation or a known violation that previously had only minor impact.

CCD currently reaches 30-40% in many beekeepers; in the case of large farms with up to 84,000 hives in a single location, CCD can kill over 60%. This increased the cost of pollinating almond trees, for example from $ 25-30 per colony of bees for 1/2 to 1 acre of almond tree orchard for 3 weeks to over $ 250. More than one-third of all non-animal food consumption by Americans is dependent on bee pollination. If this upward trend in colony extinction continues, economic and social costs could reach hundreds of billions of dollars.

The loss of functions performed by bees has other far-reaching consequences. For example, it is the source of thousands of popular health products, beauty products, and insecticidal products and relies on pollination by non-adversely affected bees. Interestingly, neem products that contain the active ingredient azadiractin are useful in controlling or killing mites, including Varroa mites, which transmit diseases to bees, and including mites, which transmit diseases to other animals and plants. If the bees become extinct, this vast source of health products and natural insecticide will also be lost.

The main symptoms of CCD are the disappearance of the working class (resulting in few or no adult worker bees in the hive), a live queen, and few to no dead bees around the colony. Often there is still honey in the hive, immature bees of the sealed brood are present (bees usually do not leave the hive until they have fully hatched from the sealed brood), and the hive contains honey and bee pollen that were not immediately plundered by neighboring bees. Also, such a swarm is slow and is plundered by parasites of the colony, such as wax moths or small hive beetles. Varroa mites, which transmit the virus, a parasite of honeybees, are often found in hives infected with CCD. Collapsing colonies usually do not have enough bees to support the offspring of the colony and have worker bees, which are composed of younger adult bees; the progression of symptoms can be rapid or slow (up to two years). The colony may have abundant food supplies and may refuse to consume food provided by the beekeeper. See, for example, Honey Bees and Colony Collapse Disorder, United States Department of Agriculture Agricultural Research Service (2013).

The reasons for the increased collapse of colonies are complex and appear to be the result of many factors. Supposed causes include increased urbanization and loss of biodiversity, in particular the loss of wild meadows and weeds that provided a high-quality food base for bees, poor nutrition and malnutrition, immunodeficiencies, microbial pathogens including viruses, bacteria, fungi and protozoa, both lethal and sublethal exposure to insecticides, fungicides and herbicides, miticides and antibiotics used by the beekeeper, parasitic mites (Varroa destructor and V. jacobsoni mites and Acarapis woodi tracheal mites), Nosema ceranae and N. apis fungi, heavy metals, toxic pollutants, natural plant toxins, , selective breeding in beekeeping and loss of genetic diversity, climate change, concentration of hives and increased environmental stressors due to drought and sudden cold snaps, and a combination of these factors. Another factor is the new nature of commercial beekeeping and the changing practice of beekeeping. In the United States, there are few or no wild bees in many regions, and domesticated bee colonies are often shipped a hundred miles from bee production bees, adding additional stressors to colony health and increasing the spread of infections and parasites among bee populations.

Although the exact cause (s) and mechanisms of CCD remain to be elucidated, the combination of stressors appears to be an important combination, specifically 1) microbial viral and fungal pathogens such as Israeli acute paralysis virus (IAPV), black queen cells virus (BQCV) and wing strain virus (DWV) and Nosema (pathogenic fungi); 2) parasitic mites (in particular, Varroa mites); 3) pesticides in lethal or sublethal doses, including neonicotinoid insecticides (such as clothianidin, thiamethoxam and imidacloprid) and beekeepers' miticides (BAM) and other environmental stressors; 4) stress factors of beekeeping, including an increase in viral exchange from transported bees (in particular, during the migration of pollination of almonds in the middle winter to California); and 5) diets of honey bees that include the use of honey substitutes and exposure to pollen of low nutritional value as opposed to the native varied pollen and nectar of high nutritional value. Research indicates that honeybee diets, parasites, diseases, and a variety of pesticides interact to produce more pronounced negative effects on controlled honeybee colonies, while nutrient restriction and exposure to sublethal doses of pesticides, in particular, may change.

- 2 037750 nmean sensitivity to or severity of parasites and pathogens of bees. Pettis et al., Crop Pollination Exposes Honey Bees to Pesticides Which Alters Their Susceptibility to the Gut Pathogen Nosema ceranae, PLOS ONE, published July 24, 2013, DOI: 10.1371 / journal.pone.0070182.

Defense and immune systems of honey bees.

Colonies of bees can become infected with several types of parasites or diseases at any time, however, the immune systems at the colony and individual levels usually cope with infections (with the possible exception of the parasitic mite Varroa destructor), provided that the environmental conditions are favorable. In the event of a collapse of colonies, this usually effective immune function is clearly malfunctioning. Since the introduction of the parasitic non-native mite Varroa destructor in 1987 in the United States and its abundant distribution in apiary populations, bees are now faced with unprecedented threats from these virus-delivering arthropods - the fight against viruses that they introduce through the immune system is weakened by the effects of complex cocktails of xenobiotic toxins ... This confluence of stressors is a formula for disaster and is evolutionarily unprecedented. Additional stressors are the loss of plant biodiversity as forests are cleared, weeds are removed and monoculture agro-industrial farms smooth out native landscapes. Bees, both domesticated and wild, which are the main pollinators, are under attack from many directions. The extinction of bees in parts of China has already been reported and is expected to occur with increasing frequency around the world.

Honeybees have numerous physical, chemical and behavioral defenses at the local population, hive colony, cage, and individual level. The first line of defense at the colony and individual level is to prevent parasites from becoming entrenched - bees spend large amounts of energy on cooperative behavior of social immunity, including cleaning their body surfaces (both their own cleaning and cleaning other individuals of the hive), cooperative hygienic behavior to detect and eliminate diseased offspring and corpses of adult bees from the hive, cleaning the inner cavity of the nest and sterilizing all surfaces with antimicrobial secretions of their saliva (such as glucose oxidase) and the use (sometimes called theft) of components of the plant's immune system by collecting a high degree of antimicrobial resins found on the kidneys and in leaf wounds, incorporating them into propolis, and using propolis to form an antimicrobial barrier around the colony, including intensive use at the entrance, coating the inner surfaces of the cavity and honeycomb surfaces, and sealing cracks and crevices.

Individual systemic immune response.

Insects have an innate immunity that is characterized by non-specific immune responses against invading pathogens, while they lack complex adaptive or acquired immunity, such as the 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 main hemocytes that react with foreign invading organisms through either phagocytosis and / or encapsulation. The hallmarks of humoral reactions are the synthesis and secretion of antimicrobial peptides (AMP), which accumulate in the hemolymph and directly lyse foreign microbial cells and inhibit the activity of enzymes necessary for the replication of pathogens. See Yoshiyama, Innate immune system in the honey bee, Honey Bee Research Group, National Institute of Livestock and Grassland Science. This induced response of antimicrobial peptides can last for weeks, and it appears that these peptides can be transferred to other members of the nest, imparting resistance prior to infection. Oliver, Sick Bees - Part 3: The Bee Immune System, American Bee Journal, October 2010.

The antiviral response of bees is based on RNA interference (RNA-i). RNA-i suppresses gene expression between the transcription of the genetic code and its translation into functional proteins. MicroRNAs (miRNAs, small non-coding RNAs that function in protein-coding gene networks and cellular physiological processes through transcriptional and post-transcriptional regulation of gene expression) and small interfering RNA (miRNAs, short double-stranded fragments) bind to specific messenger RNA (or mRNA) molecules and increase reduce their activity, such as protein production or the protection of cells from viral nucleotide sequences. miRNAs are a highly conserved evolutionarily ancient component of genetic regulation found in many eukaryotic organisms.

RNA-i is initiated by the Dicer enzyme, which cleaves long double-stranded molecules (dsRNAs) into short double-stranded siRNA fragments. Each siRNA is untwisted into two single-stranded ssRNAs: a passenger strand and a guide strand. The guide strand is incorporated into the RNA-induced silencing complex (RISC). Once inserted into RISC, siRNAs pair up with their target mRNA and cleave it, thereby preventing it from being used as a translation template. When the dsRNA is exogenous (eg, the result of a virus infection), the RNA is imported directly into the cytoplasm and cleaved into short fragments by Dicer.

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Bees possess a greater number of components of the RNA-i cascade than flies, and, apparently, a systemic RNA-i response is more easily induced in them than in flies. It follows that bees should be quite capable of fighting viruses and probably other pathogens through knockdown based on double-stranded RNAs expressed by pathogenic genes (Evans / Spivak 2009). It should be noted that this form of response to viral attack provides long-term memory similar to that resulting from antibodies produced in mammals. Oliver, Sick Bees - Part 4: Immune Response to Viruses, American Bee Journal, November 2010.

Viruses, nozems and microbial pathogens.

Bees are hosts for at least 18 viruses, and virtually all of them are single-stranded RNA viruses. Some are emerging pathogens such as Wing Deform Virus and Acute Bee Paralysis Virus, which were previously considered economically insignificant (Genersch 2010), and then with the advent of Varroa as a vector, began to devastate colonies significantly. Oliver, Sick Bees - Part 4: Immune Response to Viruses, American Bee Journal, November 2010.

Viral diseases include Chronic Paralysis Virus (CPV), Bee Acute Paralysis Virus (ABPV), Israeli Acute Paralysis Virus (IAPV), Kashmir Bee Virus (KBV), Black Queen Virus (BQCV), Black Wing Virus (CWV), Saccular Brood Virus (SBV), Wing Deformation Virus (DWV), Kakugo Virus, Type 6 Rainbow Invertebrate Virus (IIV-6), Lake Senai Viruses (LSV1 and LSV2), and Tobacco Ring Spot Virus (TRSV). Among these viruses, there are many subtypes, the virulence of which against bees is currently being investigated. More pathogenic viruses are likely to be discovered. The coexistence of more than one internalized virus further degrades the immunological health of the bees. I am not aware of any effective antiviral treatment on the market to combat the spectrum of viruses known to be found in bees - Dr. Walter Steve Sheppard, Chair, Department of Entomology, Washington State University, Pullman, Washington (personal communication). Thus, there is a need for promising therapies that are non-toxic yet active against more than one virus.

Bees are also susceptible to host switching of pathogens. Tobacco ring spot virus can replicate and produce virions in the honeybee Apis mellifera, leading to infections throughout the body, including extensive infection of the nervous system, and likely affecting colony survival. TRSV has also been found in the caecum of the midgut of Varroa mites, indicating that Varroa mites can promote 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 discovered in infected tobacco, spreads through the infected pollen of many plant species, including soybeans and numerous crops, weeds and ornamental plants.

Nosema apis is a microsporidium recently reclassified as a fungus that invades the intestinal tract of adult bees and causes Nosema disease, also known as nosematosis. Nosema infection has also been associated with black queen cells and Kashmiri bee viruses. Nosema ceranae is becoming a growing problem for both Asian Apis cerana honeybees and Western honeybees.

Some honeybee viruses (DWV and KBV) and Nosema ceranae can infect other bee and wasp species and possibly Varroa intestinal cells; probably honey bees are the source of 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 vector from bee to bee could be a tipping point causing widespread collapse of many native bee species, the consequences of which are beyond control or imagination. From a historical and biological perspective, this is an emergency. What evolution has provided for millions of years can be lost in decades due to human intervention, whose motives have a short-term perspective at the expense of a long-term one.

Bacterial bee diseases include American bee foulbrood (AFB) caused by Paenibacillus larvae and European bee foulbrood (EFB) caused by the bacteria Melissococcus plutonius. Fungal diseases include Ascosphaera apis and stone brood, a fungal disease caused by Aspergillus fumigatus, Aspergillus flavus and Aspergillus niger. It is also expected that new, as yet unidentified fungal pathogens may also be present at the same time or become the main cause of bee diseases in the future, as humans further modify their natural environment and cause unintended consequences of the use of transgenic crops, more commonly known as GMO-genetically modified crops. organisms. Such potential fungal pathogens include Candida, Cryptococcus, Coccidiodes, and other yeast-like organisms. However, many of these so-called pathogens, especially, for example, presporulatory forms of entomopathogenic fungi, have properties that can provide benefits to insects, including bees, provided that their endogenous toxins are removed, reduced in number or altered so that do not harm the bees, thereby reducing the threat to the bees from causing the disease by transferring

- 4,037,750 disease-spreading or disease-spreading organisms.

All honey bees are infected with more than one species of bacteria, including beneficial endosymbionts, which provide protection against yeast, ascospherosis, and foulbrood. Obviously, healthy bees can also be infected with more than one type of virus. The dynamics of the interactions between bee-bacteria, bee-virus and virus-virus are complex and poorly understood. Certain bee viruses can increase the virulence of other viruses, while some bee viruses can competitively suppress the replication of others. It is also likely that there are still bacteria-bacteria, bacteriophage-bacteria, fungus-bacteria and fungus-virus relationships that scientists have yet to identify. Many virulent bee viruses can exist as an implicit infection - the presence of the virus can be detected in bees, but there are no noticeable negative effects due to infection. Infection with a second virus or other stressor can cause the quiescent virus to replicate. A number of researchers have found that the action of Varroa mites alone, feeding on bees (which includes injection of immunosuppressive drugs by the mite) can induce or activate the replication of subtle or usually non-pathogenic viral infections. Studies of immune responses have also shown that ticks and viruses can alter the transcript levels of immunity-related genes in their respective hosts. For collapsing colonies, simultaneous infection with three or four viruses, Varroa ticks, Nosema ticks (ceranae and especially apis) and trypanosomes is common. See Oliver, Sick Bees - Part 3: The Bee immune System, American Bee Journal, October 2010.

Crithidia bombi is the simplest trypanosomatid parasite of bees known to have serious effects on bumblebees, particularly under starvation conditions. The related Crithidia mellificae may contribute to mortality in honey bees. 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.

Varroa mites and other parasites.

Varroa destructor and Varroa jacobsoni are parasitic mites that feed on the body fluids of adult bees, pupae and larvae. Acarapis woodi is a tracheal mite that infects the respiratory tract of the honey bee. The Asian parasitic nest mites Tropilaelaps clareae and T. mercedesae are considered a serious potential threat to honeybees, although they are not currently found in the United States or Canada.

The Asian honeybee Apis cerana is a natural host for the Varroa jacobsoni mite and the Nosema ceranae parasite. Because they co-evolved with these parasites, A. cerana cleans more thoroughly than A. mellifera, and thus has a more effective defense mechanism against Varroa and Nosema, which are increasingly serious parasites of the western honeybee.

Varroa mites, destroying the hygienic, mechanical and physiological barriers of bees against invasion, increasingly act as a vector for viruses, also causing significant stress in bees. Widespread colony extinction has only been described for countries where Varroa is a problem (Neumann 2010). Tick-free colonies may be virus-free (Highfield 2009), but up to 100% of Varroa colonies may be infected with one or more viruses, even if there are no noticeable symptoms (Tentcheva 2004). Oliver, Sick Bees Part 1, American Bee Journal, Aug 2010.

Varroa mites have been found to be significantly more acid sensitive than honey bees. Organic acids such as oxalic acid, formic acid and lactic acid can be used as natural miticides or anti-mite treatments in the hive, as they are all naturally found in honey. Oxalic acid is typically mixed with distilled water to prevent salt formation, which provides an acidic solution with a pH often <1. It matters that bees can tolerate such low pH values while mites cannot. Oxalic acid captures calcium and other minerals from the exoskeleton of the ticks to form oxalates. When direct contact of oxalic or formic acid with a tick's chitin-like exoskeleton draws out calcium, the exoskeleton weakens, thus making the tick susceptible to other stressors, including, but not limited to, infection or exposure to toxins from entomopathogenic fungi.

In addition to the well-known insect parasites such as the large and small wax moth and the small hive beetle, humpbacks, previously known to be parasites of bumblebees, can also pose a threat to honeybees. 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; Ravoet, above.

Pesticides.

Pesticides cause many forms of stress in bees. Agricultural spraying can affect honey bees, and large-scale spraying programs for mosquitoes, gypsies, spruce leafworms and other insect parasites cause direct or indirect extermination of bees, including native bumblebees and solitary bees. The used types are also switched.

- 5,037,750 pesticides - many, such as neonycytinoids, are less toxic to vertebrates and the need for their repeated use is reduced, however, they act systemically and are absorbed and distributed throughout the plant during seed and soil treatment, including distribution into pollen and nectar.

Sublethal exposures to pesticides, including exposure to cholinergic neonicitinoid insecticides (nicotinic receptor agonists) and / or organophosphorus-based cholinergic miticides (acetylcholinesterase inhibitors), have been found to alter activity, development, egg production, behavior, sex-to-offspring ratio, mobility, ability to navigate and orientate, feeding behavior, learning, memory and immune function, population dynamics and an increase in the predisposition and mortality of bees 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, up 1. by 4. Often, bees are exposed to several pesticides, which may interact. See, for example, Di Prisco et al., Neonicitinoid clothianidin adversely affects insect immunity and promotes replication of a viral pathogen in honey bees, PNAS vol. 110, no. 46, Nov. 12, 2013, 18466-18471; Pettis et al., Crop Pollination Exposes Honey Bees to Pesticides Which Alters Their Susceptibility to the Gut Pathogen Nosema ceranae, PLoS ONE (2013); Palmer et al., Cholinergic pesticides cause mushroom body neuronal inactivation in honeybees, Nature Communications, 4: 1634, (2013); Williamson et al., Exposure to multiple cholinergic pesticides impairs olfactory learning and memory in honeybees, The Journal of Experimental Biology 216, 1799-1807 (2013); Derecka et al., Transient Exposure to Low Levels of Insecticide Affects Metabolic Networks of Honeybee Larvae, PLoS ONE 8 (7), e68191 (2013), doi: l0.1371 / journal.pone.0068191.

Exposure to fungicides also destroys or reduces the beneficial fungi found on the pollen - possibly resulting in a higher incidence of diseases in honey bees, including Nosema infections and ascospherosis (ironically, fungal disease).

The bee genome has relatively few genes that are associated with detoxification compared to single insects such as flies and mosquitoes. Some of the most pronounced differences between bees and other insects are found in the three superfamilies that code for xenobiotic detoxifying pharmaceuticals. While most other insect genomes contain 80 or more cytochrome P450 (CYP) genes, A. mellifera only have 46 cytochrome P450 genes, while humans have 60 CYP genes. Honeybees have only about half of glutathione S-transferases (GST) and carboxyl / cholinesterases (CCE) compared to most insect genomes. This includes a 10-fold or more deficiency of delta and epsilon GST and CYP4 P450, with these clades being associated with 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).

While bees have evolved to deal with plant phytochemicals and natural toxins, they now also have to metabolize and detoxify anthropogenic insecticides, miticides, herbicides, fungicides and environmental pollutants, an unprecedented evolutionary burden.

Control of stress factors in beekeeping.

Use of honey or pollen substitutes (such as sugar syrup; high fructose corn syrup; bee candy; fat cakes containing fat, sugar, and optionally salt or essential oils; or pollen cakes containing soy, yeast, and skim milk powder, to which pollen may be added, possibly from areas contaminated with pesticides) may be a contributing factor to the decline in bee and CCD populations for several reasons. Malnutrition is probably the main factor behind the decline in bee populations. Synthesized bee diets simply do not provide the nutritional value that bees get from a mixture of quality pollen. Although quality proteins, carbohydrates and vitamins can be provided to honeybees in the laboratory, it is still impossible to keep them alive in isolation for more than two months on the very best diets; in captivity, they usually live an average of about 30 days. Garvey, About Bee Nutrition ..., Posts Tagged: from the UC Apiaries newsletter-The California Backyard Orchard.

Honey contains several substances that activate nutrient recognition, metabolism, detoxification and immune processes in European honeybees Apis mellifera, plus other chemicals that are beneficial to the health of honeybees. Enzymes are found on the walls of the pollinated flowers, and they enter the honey by adhering to the limbs of the bees. It is also known that the consumption of tree resins, resinous plant liquids and tree saps by inclusion in propolis or bee glue reduces the sensitivity of bees to both insecticides and microbial pathogens and increases the transcription of genes responsible for detoxification. Honey substitutes or pollen cakes that do not contain these chemicals may thus contribute to colony collapse syndrome. See Mao, Wenfru, Schuler, Mary A. and Berenbaum, May R., Honey constituents.

- 6 037750 up-regulate detoxification and immunity genes in the western honey bee Apis mellifera, Proceedings of the National Academy of Sciences of the United States, 110 (22), 8842-8846 (2013). Mao et al. found that honey components derived from tree pollen and exudates, including p-coumaric acid (= 4-hydroxycinnamic acid), pinocembrin, pinobankin, and pinobankin 5-methyl ester, are potent inducers of cytochrome P450 genes that detoxify through a number of members of the CYP6 families and CYP9. Mass-parallel RNA sequencing and RNA-seq analysis showed that p-coumaric acid specifically activates all classes of detoxification genes, as well as individual genes for antimicrobial peptides required to protect against pesticides and pathogens.

Those honeybee species that nest in tree hollows use propolis to close cracks in their hives, just like bees in domesticated bee hives, although wild honeybees cover the entire inner surface in their nesting hollow, while domesticated honeybees lay relatively little resin in beekeeping hives. The propolis coating has been shown to inhibit AFB (Antύnez 2008), fungi and wax moths; Spivak has demonstrated that propolis from several regions is effective against Varroa and is currently studying its effect on viruses. Of considerable interest is the evidence (Simone 2009) that the abundance of propolis appears to reduce the necessary efforts for the immune function of bees - thus, a colony of bees, through self-treatment with antimicrobial chemicals from plants, incurs lower metabolic costs to combat pathogens. Oliver, Sick Bees - Part 3: The Bee Immune System, American Bee Journal, October 2010.

Bears, mushrooms and bees.

The inventor noticed, during one of his many visits to the relict forests of Olympic Peninsula, Washington State, a Christmas tree scratched by a bear (pictured in the book he authored: Mycelium Running: How Mushrooms Can Help Save the World, 2005, pg. 70 , figure 75. Ten Speed Press, Berkeley). The scientific literature on interactions between bears and fungi suggests that Fomitopsis spp., Brown rot woody polypores, including the common Fomitopsis pinicola and the rare Fomitopsis officinalis, were the most common fungi to grow after bear scratching in the coniferous forests of the Pacific North. -west and elsewhere. Forest scientists have shown that when bears scratch a living tree, they leave an open wound and Fomitopsis species opportunistically gain an area for infection. After scratching, the sugar-rich resin often oozes out as droplets, attractive to bears and bees. Indeed, when the author returned a few years later to the same bear-scratched tree deep in the relict forests along the southern branch of the Hoh River, Fomitopsis pinicola was bearing fruit from the now fallen tree.

On young conifers, such as California fir, bears peel off strips of bark with their teeth to reach the insects or sweet sap inside. The bear's teeth leave long, vertical grooves in the sapwood, and large strips of bark are found around the base of the trees they peel off. These marks are usually applied from April to July, but the results can be seen all year round. This harvesting activity is common in tree plantations where large groups of trees are of similar age and belong to the same species. Link, Living with Wildlife: Black Bears, Washington State Dept. of Fish and Wildlife.

For this reason, the bears were rewarded by stakeholders in the forest, as bears were believed to reduce the profitability of timber production from forests. Tens of thousands of bears have been killed by hunters hired by lumber companies. In the 1990s. it was discovered that bears actually benefit forests by supplying marine minerals, in particular phosphorus and nitrogen, from their salmon and trout harvest in rivers adjacent to forests. Part of the reason that lowland relict forests are significantly larger than relict forests several thousand feet above the reach of migratory fish is that bears bring fish remains to the shore, delivering beneficial phosphorus and other minerals to nearby trees that affect growth of trees. Humans are often prone to making decisions that are contrary to their long-term greatest interests due to a fundamental misunderstanding of interconnectedness in nature.

In Stamets, Growing Gourmet and Medicinal Mushrooms, 1993, p. 42-43, the present inventor states: During 6 weeks of one summer, the bees attacked the mycelium of royal stropharia, exposing the mycelium for exposure to air, and sucking the sugar-enriched cytoplasm from the wounds. It was possible to trace a continuous convoy of bees from morning to evening from our hives to the mushroom site, until the mycelium of the royal stropharia was literally destroyed. When this phenomenon was reported in Harrowsmith Magazine (Ingle, 1988), beekeepers in North America wrote to me to inform them that they had long been perplexed by the bees' attraction to sawdust piles. While it may not be obvious to a person skilled in the art whether the bees were attracted by the mycelium, or the lignin in the sawdust or the resin in the sawdust, the inventor concluded: It is now obvious that the bees were looking for the sweet mushroom mycelium below.

An urgent solution is required to the problems of deteriorating bee health and colony collapse syndrome.

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Description of the invention

The inventor of the present invention believes that the combination and interaction of several mycological methods and compositions is a possible comprehensive solution to the problem of CCD. Each of these elements may be sufficient to provide an effect leading to the prevention or reduction of CCD. As an integrated platform of partial solutions, the combination of these methods will achieve synergistic benefits. More specifically, the present invention focuses on the antiviral and longevity-promoting effects of pure cultured mycelium extracts diluted to specific ranges that are beneficial to bees.

The bases of these compositions and methods include extracellular exudates and extracts obtained therefrom of pure cultured mycelium prior to fruiting body formation in the species Agaricales, Polyporales and Hymenochaetales in combination or independently. Preconidial mycelium and preconidial mycelium extracts from entomopathogenic fungi can optionally be used to control mites and other parasites in bees and hives. Blends of these extracts and bee products such as bee food and bee spray treatments provide a variety of solutions to help prevent CCD or help bees overcome CCD. Sustainable solutions to problems that harm bees are the result of stimulating their natural defenses by improving living conditions with beneficial fungi, for example, by introducing fungi that form the fruiting body, which have antiviral properties, into the wood, ensuring rotting and ultimately moisturizing the nesting holes. which can be good for bees.

The inventor has isolated various strains of fruiting body-forming fungi, including Pleurotus ostreatus, Trametes versicolor, and Psilocybe azurescens, which have demonstrated improved bioremediation or micremediation of various toxins including oil, pesticides and nerve gases such as sarin, soman and VX (dimethylmethylphosphonate) working with Battelle Laboratories, and a public report on this topic was published in Jane's Defense Weekly (Fungi could combat chemical weapons, Jane's Defense Weekly, 1999, 32 (7): 37.)

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, Phellinus linteus, Piptoporus communatus ovaryus, Phellinus urellus, Phellinus uvus, Piptoporus betulinus orereatus. which have demonstrated improved antiviral, antibacterial, antifungal and antiprotozoal properties.

Without wishing to be bound by theory, the present inventor believes that these types of fungi are enriched in compounds that activate genes to detoxify and protect against pollutants, pesticides and pathogens in animals, including humans and bees. Through repeated cultivation and expansion of the non-sporulating sectors of the enthemopathogenic fungi, the inventor also discovered that such presporulatory or preconidial mycelium and preconidial mycelium extracts emit odors and aromas (ranging from toffee, Metarhizium anisopliae and Aspergillus bassau and their vanilla bean) the tastes are attractive to animals, including humans and both non-social and social insects, providing benefits for controlling parasites such as Varroa mites.

Further, the author of the invention believes that the colonization of wood by Fomitopsis due to the capture of bears and their entry into the wound area (see above) leads to the production of enzymes (laccase, lignide peroxidase, cellulase), ergosterols and other sterols, mycoflavonoids and especially a number of nutritional complex polysaccharides, which soften wood, provide water, nutrients and emit aromas, all of which attract bees, while the extracellular exudates secreted by the mycelium can be enriched with p-coumaric acid and coumarins and glycosides of unsubstituted and substituted benzoic, cinnamic and coumaric acids, all of which stimulate the activation of genes and enzymes of the natural cytochrome p450 and also provide antiviral and antibacterial agents, all of which are expressed during the decomposition of the infected tree. The complex mushroom nectar is exudated, which provides physiological benefits and enhances the innate immunity of bees through numerous cascades as the trees decay. In some cases, bees nest in these logs or in the soil beneath them, gaining the benefit of long-term contact. The bees can then incorporate these nutrients into their honey, propolis and combs to protect the offspring, the queen, and eventually the colony.

Also, the inventor believes that combinations of the types of fungi described above and below, including but not limited to phenols found in them, can have additive or even synergistic effects, including the regulation and activation of nutrient recognition, metabolism, detoxification, immunity and genes. and antimicrobial peptide systems. The present invention directly supports the link between honeybee contacts with fungi, which are beneficial, not only in terms of nutritional value, but especially in terms of activating cytochrome P450 cascades for the deactivation and metabolism of xenobiotics and anthropogenic toxins.

The present invention relates to a variety of partial solutions for providing scientists, farmers, biotechnologists, politicians and thought leaders with biological tools in

- 8,037,750 as practical and scalable medicines before environmental disaster puts humanity on a limb more than ever as biodiversity declines. Combinations of these partial solutions will cumulatively and synergistically provide what bees need to overcome CCD.

Extracts of Fomitopsis pinicola, Fomes fomentarius, Inonotus obliquus Ganoderma resinaceum (Ganoderma lucidum var. Resinaceum) and Schizophyllum commune have been found to be effective in reducing viral load in honey bees and prolonging the life of worker bees. As an entomologist with 39 years of bee research experience, I am not aware of any reports of materials that prolong the life of worker bees more than this - Dr. Walter Steve Sheppard, Chair, Department of Entomology, Washington State University, Pullman, Washington (personal communication). The inventor now suggests, as a consequence of the invention, that other forest tinder fungi such as birch tinder fungus, Piptoporus betulinus, and numerous other forest species may have higher or lower antiviral and longevity-promoting effects on bee health when pure cultured mycelium extracts are obtained and diluted to an optimal range, and provided as food, in drinking water, in honey, in pollen cakes, propolis, or even enclosed in wood frames used to construct bee hives or enclosed in sticky strips glued to bee hives. The predominant viruses of interest are wing warp virus, sac brood virus, Israeli acute paralysis virus and queen cells virus, each of which can enhance the activity of other viruses and pathogens because immunity cannot withstand the harmful cumulative effects of these and other multiple stressors.

As Albert Einstein pointed out, we cannot solve our problems by thinking in the same way that we created them. This patent follows this philosophy by offering a comprehensive platform of synergistic solutions addressing a multitude of problems that will ultimately help bees overcome colony collapse syndrome.

Brief Description of Drawings

FIG. 1 is a line graph showing the percentage survival of bees over time that were fed Inonotus obliquus mycelium extracts (0.1, 1 and 10%) with sugar water compared to a control population fed only with sugar water.

FIG. 2 is a line graph showing the percentage survival of bees over time that were fed with Ganoderma resinaceum mycelium extracts (0.1, 1 and 10%) with sugar water compared to a control population fed only with sugar water.

FIG. 3 is a line graph showing the percentage survival of bees over time that were fed Fomitopsis pinicola mycelium extracts (0.1, 1 and 10%) with sugar water compared to a control population fed only with sugar water.

FIG. 4 is a line graph showing the percentage survival of bees over time that were fed Fomes fomentarius mycelium extracts (1%) with sugar water compared to a control population fed only with sugar water.

FIG. 5 presents an estimate of survival using a Kaplan-Meier plot (product limitation), showing the proportion of bees that survived over time when they were given Fomes fomentarius extracts (0.1, 1 and 10%) with water with sugar compared to a control population. which was fed only with sugar and water.

FIG. 6 is a bar graph showing the total level of viral particles in the control population and in bees that were fed with Inonotus obliquus mycelium extracts (0.1, 1 and 10%) with water with sugar, compared to a control population that was fed only with water and sugar in time zero, one week and two weeks later.

FIG. 7 is a bar graph showing the total level of viral particles in a control population fed only with water with sugar and in bees that were fed extracts of Ganoderma resinaceum mycelium (0.1, 1 and 10%) with water and sugar compared to a control population. at time zero, one week later, and two weeks later.

FIG. 8 is a bar graph showing the total level of viral particles in bees that were fed Fomitopsis pinicola mycelium extracts (0.1, 1 and 10%) with sugar water compared to a control population fed only sugar water at time zero and in one week.

FIG. 9 is a bar graph showing the total level of viral particles in bees that were fed with Schizophyllum commune mycelium extracts (0.1, 1 and 10%) with water with sugar compared to a control population fed only with water and sugar at time zero and in two weeks.

FIG. 10 is a line graph showing the cut-off value for queen cell virus cycles over time in a control population and in bees that were treated with Inonotus obliquus (1%) and Ganoderma resinaceum (1%) mycelium extracts with water and sugar compared to a control population. fed only with sugar and water.

- 9 037750

FIG. 11 is a line graph showing the cutoff for queen cell virus cycles over time in a control population and in bees that were given Inonotus obliquus (1%) and Ganoderma resinaceum (1%) mycelium extracts with water and sugar compared to a control population. fed only with sugar and water.

Best Mode (s) for Carrying Out the Invention and Industrial Applicability

Bees are increasingly faced with new anthropogenic stressors. For hundreds of millions of years, fungi have evolved to fight viruses, bacteria, and other fungi; evolved to infect parasites, including insects; their enzymes evolved to destroy toxins; and their substances evolved to activate such processes. This means they provide a potential food treasure of compounds useful in protecting bees and other pollinators from such threats, including many antiviral, antibacterial, antifungal and antiprotozoal compounds and compounds useful for activating the digestive system, detoxification system, and the immune system of bees.

Without wishing to be bound by theory, the inventor believes that fungal mycelium extracts specifically modulate, induce and increase the expression of detoxifying and xenobiotic metabolizing genes, in particular by activating all classes of detoxification genes, increasing pesticide metabolism in the midgut, functioning as nutrients, regulating immune and detoxification processes, activating immune, metabolic and nutritional cascades (cascades of lipid and glucose metabolism) and activating genes encoding antimicrobial peptides. Moreover, certain types of fungi support the microbiome of beneficial microorganisms in the digestive system of bees, and their compatibility is an important bridge between species, balancing the action of beneficial wood rotting fungi with beneficial microbes found in the hind gut of the bees. The extracts of the present invention are expected to be prebiotics for the natural microbiome in the organs of the digestive system of bees, as well as to impart antiviral benefits that will contribute to increasing the lifespan of bees and their colonies and their overall functionality. Another embodiment of the present invention is an enzyme of the mycelium of medicinal fungi Bifidobacterium bifidum, Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus sakei, Leuconostoc lactis, Streptococcus thermophiles and bacteriophages for the production of bee-friendly and environmentally friendly animal compositions, ...

As bees are attacked by a variety of pathogens - ticks, viruses, microsporidia, protozoa, humpbacks, and airborne pollutants - finding a reliable, universal defense platform to help maintain the bees' immune defenses is of paramount importance. For example, the development of methods for obtaining compositions using extracellular exudates of the mycelium of certain types of fungi, including, but not limited to, Stropharia rugoso-annulata, and other representatives of Strophariaceae, Fomitopsis pinicola, and other representatives of Fomitopsidaceae, and Metarhizium anisopliae, and other representatives, Clavicipitaceae can help prevent colony collapse syndrome. It is also expected that many other species of Basidiomycetes and Ascomycetes will provide similar benefits while investigating the benefits of bee-beneficial exudates secreted from laboratory-grown pure cultured mycelium.

With regard to mushroom extracts, mycelium extracts are preferred over fruiting body extracts because the hypha produces extracellular exudates that are enriched with available water, oils, polysaccharides, amino acids, B vitamins, coumarins, p-coumaric acid, phenols and polyphenols, as well as ergosterols. enzymes, acids, including fatty acids, antibacterial agents and antiviral agents. Individual hyphal filaments of mycelium give off complex odors that escape into the air, while fruiting bodies tend to have nutritional value, but do not have extensive exposed areas of the cell surface, as does the same mass of mycelium. The fruiting body of the fungus consists of a hyphae with compacted cells layered on top of each other, so that only a small part of the mycelium mass in the fruiting body is exposed to the atmosphere. Thus, the fruiting bodies of mushrooms are devoid of the mycelium odor from which they are formed. Since these extracellular exudates can be easily dissolved into solution, these exudates can be more effectively incorporated into enhancers such as pollen cakes, sugar solutions or water, bee spray or leaf spray sprays, and are the best attractants for bees and other insects than the fruiting bodies of fungi. At present, this is not obvious to specialists in the fields of mycology and entomology, whose attention has been focused more on the fruiting bodies and the spores of the fruit bodies, and not on the mycelium.

While bees may look for sugar-rich droplets exuding from the mycelium of a rotting tree, 100% of the extracts - what bees collect - are overly concentrated and toxic in most species in their natural form to be beneficial. Even at 10% concentration, most of the investigated extracts were toxic. Thus, if bees were to absorb these droplets in nature, they would probably get sick, die prematurely, and receive no benefit. Author

- 10 037750 Invention and his colleagues discovered that when laboratory-grade culture extracts were diluted to a high degree to 10%, some toxicity persisted, but when they were diluted to 1 or 0.1% or less, lifespan increased significantly, especially middle age, when worker bees are at their peak of activity and are most productive in their extraction and collection of pollen. Likewise, when the extracts were diluted, antiviral benefits were observed, while at the same time there was an increase in lifespan, in the case of several species of fungi studied. This is especially important as reducing pathogen load has an overall net benefit for the overall health and performance of the hive. By combining extracts optimized for antiviral activity with extracts optimized for longevity, greater benefits are expected than in each case alone. As a result of the combination with the benefits to longevity, bees can be more productive as gatherers, as nursing bees, caring for offspring and as hygiene controllers, with less morbidity and improved ability to withstand exogenous stressors. In fact, the functions that bees perform inside the hive and outside in the external environment are greatly enhanced by using the methods and compositions described in the framework of the present invention.

The extract of a pure culture of laboratory mycelium on sterilized substrates differs significantly from the naturally occurring form of mycelium - structurally, quantitatively and qualitatively. Moreover, the cultivation of a pure culture of mycelium, for example, on rice, which is a non-native substrate, without numerous other co-occurring microbes that live on naturally decaying wood, may provide a different substance than exudates from decaying wood, in which many other organisms are present ( 1 g of decaying wood in nature contains hundreds of other microorganisms, including bacteria, protozoa, and other fungi that cohabit with or on wood-decomposing mycelium). Thus, the exudates of common mycelium in nature, containing many organisms, are significantly different and are unlikely to provide bees with the same antiviral and longevity benefits as the benefits that are observed in the case of specially diluted pure culture extracts obtained from mycelium. as described in this document. In other words, some of the changes and manipulations carried out by the inventor of the present invention outside of nature are beneficial to bees: exudates from the mycelium of a pure culture must be significantly diluted to certain concentrations in order for them to demonstrate benefits. After discovering that the original extracts were toxic, most researchers would abandon this line of research. Indeed, when the inventor proposed the idea to entomologists and mycologists skilled in the art, they were in no hurry to maintain contact with the inventor because they expected toxicity and did not want to harm the bees. Initial results, ironically, confirmed their assumptions. Some even called the ideas of the author of the invention meaningless and did not want to consider them further.

As in botany, one would expect mushroom extracts to be more effective than single ingredients or drugs. See, for example, Elfawal et al., Dried whole-plant Artemisia annua slows evolution of malaria drug resistance and overcomes resistance to artemisinin, Proc. Natl. Acad. Sci. USA 112 (3): 821-6 (2015).

It was only recently in a study that it was discovered that the mycelium has more genes turned on than the fruiting bodies of fungi that eventually form from it. As noted by Li et al., 2013, Protein-encoding genes were expressed at a higher level in the mycelium or at initial stages compared to genes in fruiting bodies, Li et al., Complete mitochondrial genome of the medicinal mushroom Ganoderma lucidum. PLoS ONE, 8 (8): e72038 (2013). doi: 10.1371 / journal.pone.0072038.

Moreover, the network-like structure of the mycelium allows for the epigenetic evolution of strains that can be converted to emit substances specifically designed to benefit bees. Such improvements are anticipated by the present inventor as a method of increasing the attractiveness of the strains and compositions to bees and increasing the suitability for helping bees overcome CCD.

In essence, the inventor has discovered a new nutrient that is fortified with a wide variety of coumarins, phenols and polyphenols; and antiviral, antifungal, antibacterial and antiprotozoal agents and a wide variety of specialized metabolites such as antioxidants and antimutagens, which are formed by the breakdown of grains or wood by mycelium and are attractive to bees and support their protection from stress factors and diseases. Mushroom extracts used medicinally for human health have unexpected health benefits for bees, including lower numbers of viruses and longer lifespan. Indeed, the contribution of fungi to propolis and honey, as well as to pollen, strengthens the immune system of bees and, moreover, humans at certain, fundamental, complex levels. The inventor notes that mycelium extracts grown on grain-inoculated wood are expected to contain more polyphenols, coumarins and compounds that activate

- 11 037750 contain genes for detoxification and immunity in bees, in contrast to mycelium extracts grown by liquid fermentation.

Since nature can take decades and even millennia to establish new beneficial associations, due to the fact that bees are unable to react quickly enough to the latest introduction of new herbicides, pesticides, fungicides and miticides, it is possible to start - going ahead of evolution - this process, giving these beneficial species fungi play a major role in the pathways of biology and biochemistry of bees to maintain their immune defenses and prevent CCD. The chemical composition of fungal mycelium is complex and variable among and among different types, families and genera of fungi, which is a trait that makes mushroom extracts a good defense against rapidly evolving parasites and pathogens.

Attractants.

One component of the invention is the use of mushroom extracts, the extracts being obtained from the mycelium of tinder fungus species, basidiomycetes and ascomycetes, to attract bees. Bees are attracted by polysaccharide-rich extracellular and intracellular metabolites secreted by the mycelium. These exudates contain compounds that attract bees, provide them with sugar and other nutrients, provide antiviral, antifungal and antibacterial protection, while maintaining their resistance to pesticides and improving colony health and honey production. In fact, honey containing these fungal components can provide medical benefits for bees and other animals, including humans.

These extracellular exudates, such as those from royal stropharia or wrinkled ring stropharia (Stropharia rugoso-annulata), have the effect of attracting bees, especially when their preferred flowering plants are limited. Bees are attracted to both extracellular extracts and live mycelium. Other non-toxic mushroom species that may or may not have antiviral and longevity properties, including gourmet and medicinal mushrooms, are expected to attract bees to varying degrees in a similar manner.

The pleasant scent of Stropharia rugoso-annulata emitted by the mycelium may attract bees, although no other scientist, as far as the inventor knows, has ever discovered or claimed this. The inventor has discovered that the mycelium of Stropharia rugoso-annulata emits a rich, attractive, floral-like aroma. Oyster mushrooms of the Pleurotus genus, especially Pleurotus ostreatus, P. pulmonarius, P. lignatilis, P. sapidus, P. eryngii, P. populinus and other related species, emit a pleasant anise-like aroma, just like Clitocybe odora. Another candidate is the Split gill tinder fungus, Schizophyllum commune, which is one of the most common forest basidiomycetes, which has a strong sweet aroma in culture, at times oversaturated the olfactory senses of laboratory personnel, and is a source of coumarins and coumaric acids. Interestingly, only those who grow Schizophyllum commune in large quantities in vitro on cereal grains or wood are aware of this strong scent emitted. The inventor is not aware of anyone in his 40 years of experience who mentions or reports this phenomenon of emitting a scent from this species. Schizophyllum commune is one of the best known forest white rot agents among the temperate and tropical regions of the world, and it forms a softened sweet wood that can be beneficial to bees. It is possible that many other species give off attractive odors to bees that are not detectable by humans or are not noticeably attractive.

Mycelium Agaricomycetes and extracts obtained from pure culture of mycelium can be sources of new attractants for bees. Agaricomycetes are the only fungi that degrade lignin, and they include lamellar fungi such as Stropharia rugoso-annulata and polypore fungi such as those related to the Fomitopsis species. Agaricomycetes encompass ~ 16,000 described species. Many Agaricomycetes degrade cellulose and lignin in a dual way. Native bees use rotten logs for nesting, as discussed above under Bears, Mushrooms and Bees, which the inventor believes provide the bees with sugar-rich and cytochrome P450-encoding and activating compounds via water droplets and nectar secreted by the mycelium of Agaricomycetes.

Regenerative forests currently have approximately 10-15% woody residues relative to native forests! This relatively recent loss of degradable woody debris limits the availability of these beneficial fungi to native and imported bees, providing a previously undescribed additional stressor. The continued limitation of areas of debris further disrupts food chains not only for bees, but also for most organisms that depend on healthy and sustained ecosystems.

For example, the inventor has shown that extracts of preconidial (presporulatory) mycelium fungi of non-Agaricomycetes, including Metarhizium anisopliae and Aspergillus flavus, attract humpback beetles (and other insects) (see US Pat. Nos. 6,660,290, 7122176, 791389 and 850 ), stopping their migration and, thus, preventing the transfer of these

- 12 037750 fly diseases. Moreover, the use of these mycelium-based extracts also recruits pathogen-carrying mites and prevents them from moving into bee colonies, thereby reducing not only the pathogen burden borne by the mites, but also reducing the number of mites that might otherwise infect bees. If necessary, similar approaches can be used to control parasites in bee hives such as large and small wax moth and small hive beetle. Moreover, strains of these presporulatory entomopathogenic fungi can be selected due to their high thermal stability and their ability to attract and destroy ticks and flies that harm the bees or the pathogens they carry. Research on post-sporulation and spore-based Metarhizium anisopliae technologies (which may have a lack of mite and / or insect repellency compared to the attractiveness of preconidial mycelium) has demonstrated the relative ease with which strains can be selected for thermal resistance to high hive temperatures and high pathogenicity, and / or mortality for Varroa ticks. 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, New Brunswick Department of Agriculture, Aquaculture and Fisheries, Integrated Pest Management - Compatible Biological Control of Varroa Mite of Honey Bee; Fungi help combat honeybee killer, BBC News Science / Nature, August 9, 2002.

Moreover, the inventor has clearly shown that the preconidial mycelium of entomopathogenic fungi, such as, but not limited to, Metarhizium anisopliae, Beauveria bassiana and Cordyceps species, induces a stimulating food response (phage stimulation) in many insects and other arthropods after smelling and subsequent consumption extracts obtained from the preconidial mycelium. However, bees exhibit a unique tolerance to toxins from the spores and mycelium of Metarhizium anisopliae, which is harmful to mites and humpbacks. Thus, the presence of a mixture of entomopathogenic fungi prior to sporulation or their extracts mixed with the spores (conidia) of these fungi can stimulate bees to consume more mycelium and its extracts, including those from useful polypores, which leads to a unique set of synergistic benefits that includes factors of life expectancy, antiviral, antibacterial and antifungal effects, activation of cytochrome (p450) detoxification cascades, provision of complex sugars, vitamins and nutrients, while reducing the toxicity of anthropogenic insecticides, herbicides, fungicides, anthropogenic toxins and also reducing the population, mites and mites contributing types of fungi that support a healthy bacterial microbial gut in bees. Each of these factors helps the bees to reduce the stressors of the disturbances that cause colony collapse. Combining these beneficial factors in one delivery system — as a composition or as a method — is an unprecedented approach to the knowledge of the present inventor. Methods for selecting and optimizing strains in each species are likely to lead to improvements as each variable is explored and combined.

Humans are limited in their perception of color wavelengths from approximately 390 to 750 nm. Bees, like many insects, see colors from about 300 to 650 nm. Many species of fungi, such as oyster mushrooms (Pleurotus ostreatus), are initiated to fruiting at about 360 nm, which is beyond what humans can detect (see Action spectra for Hyphal Aggregation, the first stage of fruiting, in the basidiomycete Pleurotus ostreatus , Richartz and Maclellan in Photochemistry and Photobiology pages 815-820, May 1987). Fungal mycelium absorbs some of this light and reflects a significant part of it due to absorption restrictions through the transparent cell walls of the mycelium.

When a mycelium growing deep in wood or in the ground reaches the surface of the ground or wood and is exposed to light, a phase change occurs in the life cycle of the fungus, from the mycelium to the first stage of fruiting body formation, hyphal aggregation, and primordium (mushroom pups) formation. The mycelium of many species does not form primordium unless exposure to near-ultraviolet light or 360 nm or less light occurs. This is within the range that bees can detect, but it is beyond what humans can see.

The attractiveness of mycelium stimulated by blue light, invisible to humans but visible to bees, is a discovery of high importance, as bees are most easily trained to associate food with ultraviolet wavelengths of color. As Menzel and Backhaus identified in 1989, bees can learn faster when food is associated with purple compared to all other colors. Menzel, R. and Backhaus, W. 1989. Color vision in honey bees: Phenomena and physiological mechanisms. D. Stavenga and R. Hardie (eds.): Facets of vision, Berlin-Heidelberg-New York: 281-297.

Thus, bees seek surface mycelium when nutrients are transported upward into the preprimordium or primordium-forming mycelium in response to violet wavelengths, and when this length is key to stimulating the mycelium to switch to fruiting body formation, such detection by bees may be an appropriate time for search surface

- 13,037,750 mycelium and assimilation of saturated nutrients when the mycelium is so metabolically active. Although this is a hypothetical and speculative opinion of the inventor, this interaction deserves further investigation, since bees can be trained to search for food based on associations with light spectra. This additional element of the present invention can accelerate the learning process of bees to find new food sources using mycelium traits. As a result, embodiments of the present invention also provide the benefit of enhancing the usefulness and attractiveness of other forms of food for promoting the health of bees using these aforementioned properties of the mycelium, in particular, facilitating the detection of mycelium by bees during primordium formation stages.

Surface mycelium gives off carbon dioxide and gives off odors, and the present inventor believes that bees can detect mycelium not only by smell, but are also attracted to the mycelium in response to this blue spectrum light when the mushroom mycelium begins to pack proteins, vitamins and rich sugar, the nutrients on the surface between the high carbon dioxide content of the substrates and the highly oxygenated environment immediately above them, and thereby constructs the nutrient-rich but accessible primordium, the first stage of fruiting body formation or basidiospore formation (in the case of resupinata tinder fungi such as Inonotus spp. 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 the yellowish ones that the mycelium possesses, can be composed of fungal bioflavonoids, many of which are polyphenols. The inventor of the present invention believes that when examining this rich environment, the contact surfaces - the surfaces of the yellowish membranes of fungal mycelium exposed to the atmosphere - will prove to be a rich reservoir for bees to collect extracellular and intracellular metabolites containing nutrients and compounds that support the immune system, including mycoflavonoids and mycosterols. including phenols and polyphenols not limited to coumarins and benzoic and cinnamic acid derivatives, including coumaric acids and their glycosides.

As a non-limiting example, the mycelium of some species, especially the genus Phellinus and Inonotus, produce brightly colored yellowish pigments in their mycelium, including polyphenols such as hispolones such as 6- (3,4-dihydroxyphenyl) -4-hydroxyhexa-3,5-diene -2-one (C ₁₂ H ₁₂ O ₄ ), a light yellow biologically active group of compounds with antioxidant and immunity-enhancing properties, derived from tinder species such as Inonotus hisipidus and Phellinus linteus. The inventor believes that these light yellow colored mycelium can also attract bees collecting sugars, polyphenols, moisture, nutrients and other secrets that have immunity-enhancing antiviral, antibacterial, antifungal and antiprotozoal properties. Because bees are especially attracted to yellow, those species of fungi such as Phellinus and Inonotus, which are light yellow, will preferentially attract bees and are also directly associated with yellowish polyphenols containing coumarins, helping bees activate their cytochrome P450 enzyme cascades. The inventor of the present invention believes that the cultivation of these wood-decomposing species, which produce brightly pigmented mycelium, is preferred to create mycelium-based platforms and extracts to assist bees. Therefore, mycelium-forming primordium extracts and colored mycelium extracts are preferred attractants of bees.

The mycelium of many fungal species does not form sporulating structures, including, but not limited to, fruiting body formation; such mushrooms are also preferred for researching their mycelium extracts for the attraction and health of bees.

Viruses, fungi, bacteria and protozoa.

Bees infected with viruses can lose immune function as well as the ability to perform other metabolic functions as a result of viruses abducting the ribosomal apparatus for their benefit, chemically interfering with a key phenol oxidase cascade, suppressing immune responses before they start, manipulating the host's immune signaling network, blocking host antimicrobial peptides, interfering with the RNA-i response and / or creating superantigens that can suppress the host's immune system and otherwise adversely affect the health of bees.

The exclusive dependence of viruses on the host cellular apparatus for their reproduction and survival makes them highly sensitive to characteristics of the cellular environment, such as short RNA-mediated interference. It also gives the virus the ability to fight and / or modulate the host to suit its needs. Thus, the range of interactions possible through microRNA-mRNA interactions at the host-pathogen contact surface is large. These interactions can be further adjusted in the host through changes in gene expression, mutations and polymorphisms. In a pathogen, a high mutation rate contributes to a complex network of interactions. Viruses either produce microRNAs or target the host's microRNAs, which are necessary for the host's immune system. Scaria et al. (2006), Host-virus interaction: a new role for microRNAs, Retrovirology, 2006, 3:68; Oliver, Sick Bees - Part 4: Immune Response to Viruses, American Bee Journal, November 2010.

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Fungal mycelium produces a wide range of compounds that can be antibacterial or antiviral. US Pat. No. 8,765,138 to the inventor describes the antiviral activity of Fomitopsis officinalis, which includes activity against avian influenza virus and herpes simplex viruses I and II. Other viruses are expected to be sensitive to the antiviral compounds encoded and expressed by the mycelium of Fomitopsis officinalis, and indeed many species of tinder fungi and basidiomycetes. Mycelium extracts are active against numerous viruses that harm bees, in particular, but not limited to, BQCV (Black Queen Virus), IAPV (Israeli Acute Paralysis Virus), DWV (Wing Deformation Virus), TRV (Tobacco Ring Spot Virus) and related viruses. The virus-limiting active ingredients in the extracts vary, but the two groups are polyphenols, including coumarins, and sterols, including dehydrosulferic acids, eburic acids, and related compounds. The synergistic benefits of these polyphenols and sterols can further enhance the immune defenses of bees. These compounds are found in complexes that include fatty acids, lipids, and sterols. As such, many other active ingredients related to fatty acids, lipids and sterols with antiviral properties are expected to be beneficial to bees. Many of these aforementioned compounds are known as bioflavonoids, and the species producing them are of interest as some of these species produce bright yellow mycelium, which can also serve to attract bees. Very little, if not even done, work has been done by mycologists to detect the colors of mycelial wood visible to bees but invisible or virtually invisible to the human eye, especially light reflected in the ultraviolet range.

The inventor has also discovered the antibacterial properties of Fomitopsis officinalis mycelium extracts against staphylococci, tuberculosis bacteria and E. coli. This antibacterial activity likely confers an additional layer of protection against diseases carried by other organisms. These extracts, likewise, will have a positive effect by limiting the harmful effects of known and as yet undiscovered bacteria that harm bees, animals and plants. See US patent application Serial No. 13/998914 and related applications described above.

It is expected that substances from medicinal fungi suitable for humans will similarly prove beneficial in terms of activation of immune genes and benefits for the immune system of bees. Since many of these genes are evolutionarily conserved or similar, it is expected that extracts of the mycelium of such fungi can similarly be used to activate genes and systems in bees that degrade and fight infections.

The preferred effective dose varies from species to species, in part because the extracts can be, like most drugs, therapeutic at low doses and toxic at high doses. In addition, some species, such as Fomitopsis officinalis, can have pronounced antiviral effects and a lower toxic threshold compared to other medicinal species. As a rule, for all medicinal species of fungi mentioned in the present description by the author of the present invention, the preferred dosages are in the range from 0.0001 to 50%, with the more preferred being the range 0.001-25% and the most preferred being the range from 0.01 to 15 %. In the case of many of the tinder fungus extracts in particular, the results tend to indicate that the extracts must be diluted to less than 10%, or less than 1%, or less than 0.1% for many of these tinder fungus extracts in order to provide antiviral benefit and lifespan extension benefit to bees. The preferred dosage added to liquid or solid food substances for bees, in the case of Fomitopsis officinalis, is 0.0001-0.1%; the preferred dose for Trametes versicolor or Fomes fomentarius and F. pinicoia is 0.1 to 10% based on results that show both an increase in survival and an improvement in viral load reduction at 10% concentrations. With the exception of Trametes versicolor and Fomes fomentarius, usually 10% concentration did not help to increase the lifespan of the bees. Accordingly, higher concentrations in excess of 10% had adverse effects on overall life expectancy.

Medicinal mushrooms and mycelium of medicinal mushrooms are defined as mushrooms and mycelium that support health and nutrient absorption. In the context of bees, they include fungi and mycelium, which have the effect of increasing longevity, increasing gathering ability, increasing resistance to disease, increasing the ability to detoxify anthropogenic toxins, increasing resistance to parasites, having antiviral, antibacterial and / or antifungal activity, and increasing the ability of bees. better withstand the stressors associated with a complex, collectively called colony collapse syndrome.

Suitable and preferred genera of fungi include, by way of example, but not limited to: lamellar mushrooms (Agaricales) Agaricus, Agrocybe, Armillaria, Clitocybe, Collybia, Conocybe, Coprinus, Coprinopsis, Flammulina, Giganopanus, Gymnopilus, Hybepholoma, Incyinus , Lentinus, Lenzites, Lepiota, Lepista, Lyophyllum, Macrocybe, Marasmius, Mycena, Omphalotus, Panellus, Panaeo

- 15 037 750 lus, Sarcomyxa, Pholiota, Pleurotus, Pluteus, Psathyrella, Psilocybe, Schizophyllum, Stropharia, Termitomyces, Tricholoma, Volvariella, etc .; tinder fungi (Polyporaceae) Albatrellus, Antrodia, Bjerkandera, Bondarzewia, Bridgeoporus, Ceriporia, Coltricia, Coriolus, Daedalea, Dentocorticium, Echinodontium, Fistulina, Flavodon, Fomes, Fomit Gloobise, Ganoderlluma, Inexpensive , Oligoporus, Oxyporus, Phaeolus, Phellinus, Piptoporus, Polyporus, Poria, Schizophyllum, Schizopora, Trametes, Wolfiporia; needle 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 mushrooms such as Tremella; mycorrhizal fungi such as Phanerochaete (including fungi such as P. chrysosporium imperfect and P. sordida).

Suitable fungal species and genera include, by way of non-limiting example only: 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 gray dung beetle; 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, Fomitopsis cana, F. subtropica and F. pinicola; G. resinaceum, annularis, G. australe, G. atrum, G. brownii, G. collosum, G. sinensis, G. lingzhi, G. curtisii, G. japonicum, G. lucidum, G. lucidum var. resinaceum, G. neo-japonicum, G. oregonense, G. sinense, G. tornatum and G. tsugae; Gigaspora gigantia, G. gilmorei, G. heterogama, G. margarita; Gliocladium virens; Gloeophyllum saeparium; Glomus aggregatum, G. caledonius, 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 incarnatus, 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. cornucopiae 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, S. rugoso-annulata; Suillus cothurnatus; 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 useful mushrooms.

Preferred strains that have demonstrated exceptional characteristics suitable for the practice of the present invention include, as a non-limiting example, Fomes fomentarius (NY state), Ganoderma applanatum (Duckabush strain), Fomitopsis officinalis (I, VI, X strains), Fomitopsis pinicoia (strain I), Ganoderma oregonense (Meadow Lake), Heterobasidion annosum (Dosewalips), Pleurotus ostreatus (PW-OST, Nisqually strains), Psilocybe azurescens (Stamets strain), Stropharia rugoso Kamiltes-annulata (F strain), Pointicolor Inonotus obliquus (Stamets NY).

Additional suitable genera and species of fungi can be found in standard mycology manuals such as Mushrooms Demystified (1979, 1986), David Arora, The Audubon Society Field

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Guide to North American Mushrooms (1981, 1995), Gary Lincoff, and Psilocybin Mushrooms of the World (1996), Paul Stamets. Continuously updated lists of suitable species based on the latest DNA analyzes can be found on the Tree of Life and Encyclopedia of Life (EOL) Internet projects.

Extracts of mycelium of Fomitopsis officinalis, in particular Inonotus obliquus, Fomes fomentarius, Ganoderma resinaceum, and other Polyporaceae species, as a rule, reduce the pathogenicity of viruses to bees by directly reducing populations of viral particles, also strengthening the immune system of bees, thus limiting their virulence and transmission ... Moreover, bees benefit from the combination of a mixture of antiviral components produced by the mycelium with the antimicrobial properties of coumarins and other compounds produced by the mycelium of Fomitopsis officinalis. The extracellular exudates secreted by the mycelium of the beneficial fungi described herein have a combination of these components, but are balanced so that they have the overall benefit of attracting bees to be strengthened with immune-enhancing and nutritionally beneficial components. This multifaceted effect strengthens the immune system of bees and their colonies, making them less susceptible to viral, bacterial, protozoan and fungal mitigated diseases.

The present inventor has found that Ganoderma, Fomes, Fomitopsis, Fomitoporia, Ganoderma, Antrodia, Inonotus, Irpex, Lenzites, Phellinus, Sparassis, Hypholoma, Pleurotus, Schizophyllum and Stropharia species exhibit significant antifungal properties, and are expected to be beneficial for control of fungal pathogens affecting bees, including, but not limited to, Nosema species and other pathogenic microsporidia, ascospherosis and stone brood.

The first antiviral compound to be discovered from a fungus was from the tinder fungus Ice Man Fomes fomentarius against tobacco mosaic virus, the first virus to be discovered and related to tobacco ring spot virus. This tinder fungus is a saprophyte on birch, beech and other deciduous trees of temperate latitudes. As it grows, the wood softens, releasing moisture, odors that attract insects, and is sweetened with rich complex polysaccharides, as well as proteins and other substances produced by the mycelium of this fungus. This fungus attracts beetles, whose burrows can later be occupied by native bees. In essence, this is one example of what the inventor envisions will have many examples of the role that tinder fungi and other Basidiomycetes play in providing nutrients to bees. Interestingly, Fomes fomentarius is a well-known endophyte of birch trees, which means they are part of the trees' natural immune system. The inventor believes that many of these endophytic fungi impart antiviral properties to plants and bees if they are found in a certain diluted concentration window, as well as other insects if they collect or nest in wood containing these fungi. However, if they are found in their pure form, many of them can actually be toxic. It is in this case that human intervention can help develop a bridge for antiviral benefit that is otherwise unlikely to occur in nature. The inventor believes that interconnected formats in which the biology of bees, fungi and decaying trees and plants intersect will be a fruitful area of scientific research to help ecosystems and their development, which would take decades.

The aggressive wood rotting fungi described herein compete with many other fungi to dominate ecological niches. Tinder fungus species, in particular Antrodia, Fomes, Fomitopsis, Ganoderma, Grifola, Heterobasidion, Inonotus, Stereum and Trametes species, have antifungal properties present in extracts that the present inventor believes will be effective against Nosema, the fungal microsporid parasite causing harm to bees around the world.

Of course, bears are not the only way to spread Fomitopsis and other fungi to trees that can improve the health of bees. Any activity that results in the creation of wounds in trees or the provision of dead trees creates a potential mushroom platform for the benefit of bees. Human use of wood chips as ornamental bark or to form trails or as a top layer around ornamental plants can also serve to create a mycelial platform for the benefit of bees. Ultimately, this means that it is possible to grow the mycelium of these fungi en masse in a presporulating or preconidial state, create mycelial landing sites for bees or extract extracts and thereby create a new generation of bee attractants and nutrients adjusted accordingly.

Taking into account this hypothesis, the author of the present invention envisions the use of a wide range of Basidiomycetes, decomposing the wood of fungi, for the development of fungal bioprotection, bioprotection of bees, for protection against stress factors leading to the colony collapse syndrome.

Moreover, the antibiotic effect of these extracts on microsporidial parasites of bees, in particular Nosema apis, which are the cause of Nosema, which have recently been reclassified as simple fungus, would prove to be a useful co-factor.

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Another advantage of the present invention is a wide range of antiviral, antibacterial and antifungal properties of mycelium. Many of the fractions of mycelium extracts obtained by the inventor show antiviral activity even when the biologically directed fractionation pathway provided antibacterial properties. It is often believed that antimicrobial agents are specific to types of microbes (there is some overlap between antibacterial and antiparasitic agents, and at present there may even be at least one class with both antibacterial and antifungal activity), however, given how difficult it is to achieve protiviral specificity alone, and the absence of known common molecular targets between bacteria and viruses, which also exhibit any degree of host specificity, broad antimicrobial activity is rare. Without wishing to be bound by theory, the inventor believes that the extracts act as immunostimulants, immune system enhancers and immunoregulators with antiviral, antibacterial and antifungal effects.

It is believed that the filamentous components described above and / or other known and unknown compounds are antibacterial and antifungal, assisting immunity, and thus the interaction between bees and pure cultured mycelium extracts at certain concentrations is an unexpected advantage of the present invention.

Hyphodermella corrugata, Polyporus umbellatus and Piptoporus betulinus are polypore species that the inventor of the present invention knows from his research to exhibit pronounced antiprotozoal properties. Agaricic acid is believed to be one of the agents responsible for the antiprotozoal activity of Piptoporus betulinus. Agaric acid is also produced by Fomitopsis officinalis, and possibly other species of polypore fungi. The production of acanthocytes in Stropharia rugoso-annulata, which are known to kill roundworms, may also provide antiprotozoal and acaricidal benefits to bees. As such, these species, their related species, may be preferable for research in relation to antiprotozoal activity and activation of antiprotozoal genes in bees.

Pesticides.

Since bees are limited in the number and variety of enzymes required to destroy natural and anthropogenic toxins, these toxins disrupt their basic immunity, which makes them more susceptible to pathogens of various directions - Varroa, Nosema mites and microsporidia fungi, humpbacks, and viruses and bacteria that they contain. By enhancing the bees 'ability to degrade these toxins and by activating more cytochrome P450 genes, GST genes and / or CCE genes, the bees' immune state is improved to better resist these attacks and other stressors. Moreover, by providing bees with a mixture of mushroom extracts that specifically limit the severity of attacks by humpback bees, Varroa mites, Nosema fungi and viruses, the health of bee colonies can be enhanced for the long-term health of offspring, worker bees, the queen and her drones. These mushroom components are naturally incorporated into honey and propolis, thus providing an advantage for developing generations. Ultimately, it is expected that not only will bees become protected, but honey production will also increase, and the quality of honey will better support the health of future generations and their ability to survive.

It is expected that those types of fungi that are suitable for bioremediation (microremediation) of toxins, pollutants and pesticides and their mycelium extracts contain various substances suitable for turning on, activating and modulating genes necessary for the biodegradation of pesticides. Since many such genes or systems, such as the cytochrome system, are evolutionarily conserved or similar, it is expected that extracts of mycelium from such fungi can similarly be used to activate genes and systems in bees to degrade and control such pesticides. Useful and preferred species include the saprophytic fungi 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 spp., Phaeolus alatus mirabymis, Boston aurantiadisca, M. haematopus, Psilocybe azurescens, P. allenii, P. subaeruginosa, P. ovoideocystidiata, P. cubensis, P. cyanescens, Panaeolus cyanescens, Stropharia ambigua, Stropharia rugoso-annoma, Stropharia fasolides, Hypotharia H. aurantiaca and other species of Strophariodeae and Strophariaceae, Lenzites betulinus, Pholiota adiposa, Pholiota terrestris, Pholiota nameko, Agrocybe aegerita, A. praecox, A. arvalis, Collybia tuberosa, Collybia, Psathyrella hydrophila, P. epimyces, associated with them numerous satellite genera, as well as other lamellar and drone genera and species known to mycological science as primary and secondary decomposing cells julose and lignin.

The mycelium of the species Stropharia rugoso-annulata, Fomitopsis officinalis, Fomitopsis pinicola, Schizophyllum commune, Trametes elegans, Trametes versicolor and many tinder fungus and lamellar basidiomycetes produces bioflavonoids, phenols and polyphenols, including

- 18 037750 and cis-, o- and p-coumaric acids), which activate genes in bees that encode cytochrome P450 enzymes, as well as other enzymes important for digestion, metabolism and the destruction of toxins. The effect of these mycelium components, such as coumarins, p-coumaric acid, o-coumaric acid or their glycosides, is that they include more genes in bees that allow bees to detoxify a wide range of toxins, in particular insecticides, miticides, herbicides , fungicides and pesticides, and enhance the innate immunity of bees.

P-coumaric acid, found in both grain and lignin, is a monomer of sporopollenin, a major component of the cell walls of pollen and propolis, which is a resinous compound collected and processed by bees to cover wax combs. P-coumaric acid is necessary to increase the level of laccase in fungi that cause rotting wood, which is a cellulase enzyme that breaks down lignin in wood, creating derivatives that are attractive to insects as food, as well as creating a habitat (bees can occupy places in tunnels drilled by mycophagous beetles). As the fungi rotting wood, breaking down lignin, they also release water enriched with this p-coumaric acid and nutrients that are beneficial to bees. The more p-coumaric acid, the more laccases are expressed by the mycelium, the more the wood rots, the more mushroom polysaccharides (sugars) and, ultimately, more of these compounds will be in the exudates of the fungi that bees are looking for and which can be useful to them. Another advantage of the present invention is that wood rotting fungi produce p-coumaric acids and coumarins, which can be biologically converted to p-coumaric acids.

As noted by Terron et al., Structurally closely related aromatic compounds have different effects on laccase activity and on 1cc gene expression in the ligninolytic fungus Trametes sp. 1-62, Fungal Genet. Biol., Oct. 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 investigated for their ability to increase laccase production in lignolithic basidiomycetes Trametes sp. I-62. All these compounds led to an increase in laccase activity, with the highest levels being found in the presence of p-coumaric acid (273 times) and guaiacol (73 times).

Interestingly, many grains preferred for mycelial mycelium production in the mushroom industry (see Growing Gourmet & Medicinal Mushrooms of the present inventor, Paul Stamets, 1993, 2000, Ten Speed Press, Berkeley) are also rich sources of p-coumaric acids and can be useful in attractant compositions for bees. The main phenolic acids in rice grains were identified as p-coumaric acid, ferulic acid, and sinapic acid.

P-coumaric acid is not only found in grains that are preferred for mycelium production, but it is also formed during the normal life cycle of fungi, especially before primordium formation. P-coumaric acid is a potent inhibitor of tyrosinase, an enzyme essential for melaninization. The presence and abundance of p-coumaric acid inhibits the production of dark pigments. Ultraviolet light stimulates the photodegradation of p-coumaric acids, providing melaninization and triggering primordium formation. After the formation of primordium, pcumaric acids are degraded to 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. By way of non-limiting example, the mycelium of Auricularia auricula (A. auricularia-judae), when grown in culture, is whitish and devoid of melanin but contains p-coumaric acids. When the fungal mycelium is exposed to light, the mycelium is biologically transformed into dark brown fruiting bodies that have a higher melanin content as they mature, while simultaneously decreasing the amount of pcumaric acids, a melanin inhibitor. This is one example and compelling evidence of the benefits of using light-colored mycelium prior to melaninization as a mycelium source for extracts beneficial to bees due to their high inherent pcumaric acid content, enhanced by the native p-coumaric acid content of the grains used to obtain mycelium for growing mycelium. Interestingly, the ideal time to get the most benefit from the mycelium due to its nutrient and p-coumaric acid content is a short window, often only a few days long, before and immediately after exposure to light, but before the dark fruiting body develops beyond the primordial white. stages.

Given that Ganoderma lucidum, Trametes versicolor and Pleurotus ostreatus are some of the most intense laccase producers still under investigation to date, these species are particularly preferred for making mixtures beneficial to bees.

When bees are not immunologically suppressed by man-made and natural toxins, the natural immune defenses of bees can better protect bees from other harmful agents, including viruses and pathogens transmitted by Varroa mites.

The author of the present invention believes that as knowledge of many derivatives

- 19 037750 of the present overarching invention, certain types of fungi will provide a unique set of beneficial factors. Some will be more antiviral. Some will activate bee detoxification cascades better than others against various toxins. Some will emit higher attractiveness aromas. As such, certain types of mixtures or mushroom cocktails can be tailored to the needs of bees, beekeepers, based on their desired intended benefits, specific ecosystem factors, and conditioned when basic materials are available.

For example, it is important for the beekeeping industry to protect and obtain new queens. Queens are bred and reared by dedicated breeders and are at risk of infection with black queen virus (BQCV) ticks. The search for a selective antiviral agent for protecting the uterus is another significant advantage of the present invention. For breeding and rearing queens, both Inonotus obliquus and Ganoderma resinaceum are very active antiviral additives for reducing the level of black queen cells virus (BQCV), but are not as active against wing deformity virus (DWV), while other species are more active against DWV. Thus, a mixture of two or more species of fungi is preferable to provide broad bio-protection to bees in the form of antiviral activity.

Varroa mites and parasitic insects.

The present inventor has obtained several patents for compositions and methods using the presporulatory mycelium of entomopathogenic fungi as an attractant and insect control treatment, and broader patents are pending (US patent application No. 13/986978) in relation to arthropods and disease-causing insects and arthropod vectors (US patent applications No. 13/317613 and 13/373719). Varroa mites are known as vectors for the Israeli acute paralysis virus and tobacco ring spot viruses. Varroa mites, which are mites that bite both plants and insects, contain more than one viral or bacterial pathogen, which means that mites are one, albeit significant, vector carrying and contributing many pathogens in an attack that threatens the health of the hive. Because bees are weakened by the virus, for example, they are less able to get rid of the adhering Varroa mites. However, the mycelium of entomopathogenic fungi, in particular Aspergillus flaws, Metarhizium anisopliae and Beauveria bassiana, can be used to attract, deplete or destroy Varroa mites, reducing their activity, delivery of pathogenic loads and quantities of pathogens, thus tilting the balance towards improving the immune defense of the colony against CCD. Likewise, spores of entomopathogenic fungi, including Metarhizium, Beauveria, and Entomophthorales, can be used to deplete or kill Varroa mites, although mites may find spores repulsive compared to preconidial mycelium.

Moreover, extracts of Metarhizium anisopliae can be obtained 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 their or other toxins or reduced virulence and pathogenicity. There is a variability in toxins compared to many strains of Aspergillus flavus, a known entomopathogenic fungus mainly toxic due to its aflatoxin content. Aflatoxin-free strains of Aspergillus flavus are currently available that are naturally occurring or can be obtained through crop selection or genetic modification. You can also create destructin-free strains of Metarhizium anisopliae, subject to selection or derived from natural genomes. It is also possible to obtain strains that are not completely free of destructins or aflatoxins, but produce levels so low that they can be toxic to mites, but not very toxic to bees due to the fact that levels and cascades of cytochrome P450 in bees are enhanced by coumarins. and other polyphenols presented by the mycelium. As such, activation of cytochromes p450 (CYP) can help bees better tolerate or detoxify destructins or aflatoxins that bees are exposed to from Metarhizium anisopliae and Aspergillus flavus and other toxins produced by entomopathogenic fungi.

The advantage of a destructin-free or destructin-reduced strain Metarhizium anisopliae is that mycelium extracts with a high sugar and terpene content can be obtained, which can simultaneously attract bees and mites. The use of an appropriately sized grid or barrier or other selection means allows the separation of mites from the bees so that both bees and mites can initially be attracted to the same area with the extracts (or similarly attracted to the preconidial mycelium). The proportion of endemic entomopathogenic toxins can be balanced to deplete mites, but not bees. The use of one or more mushroom extracts, as described herein, provides the freedom and flexibility of customization so that numerous devices, delivery systems, compositions and methods can be made available for the first time to improve the health of bees and reduce CCD. Humpbacks, mosquitoes and mites feeding on mushrooms are well known in the mushroom industry. It was not known that extracts of entomogenic fungi are

- 20 037750 are attractive to these insects and arthropods. The inventor believes that aqueous ethanol extracts of fungi or fungal mycelium with these attractant properties were not known in the mushroom industry prior to the inventor's disclosure in co-pending and approved patents.

The combination of oxalic acid with sugar-rich water loaded with spores or preconidial mycelium of entomopathogenic fungi such as Metarhizium anisopliae and Beauveria bassiana will improve the miticidal effect of the combination of oxalic acid and entomopathogenic fungi and the miticidal properties of other components present or added to water, for example, pollen cakes or bee spray. However, oxalic acid reacts with the minerals in mushroom extracts and this is an obstacle to effective formulation.

When oxalic acid is combined with extracts of filamentous fungi basidiomycetes, the minerals contained in them (calcium, phosphorus, iron) can bind with oxalic acid, thus reducing the leaching of minerals, the miticidal potential of oxalic acid. Thus, an embodiment of the present invention is to demineralize mushroom extracts prior to combining oxalic acid with mushroom extracts. For demineralization, any of a variety of methods suitable for demineralizing mushroom extracts can be used to prevent the conversion of reactive oxalic acid to water-insoluble salts by eliminating calcium and other minerals found in the mushroom extracts. One of the many methods available is the use of ion exchange resin technology. Mushroom extracts can be added to distilled water preferably in a ratio of 1:10, with ranges from 1: 1, which is most concentrated, to 1: 100, which is most diluted, but are less preferred. Once completed, the minerals in the mushroom extracts that might otherwise neutralize the miticidal properties of oxalic acid can be largely, or even completely, removed. After that, oxalic acid can be added to mushroom extracts with a reduced mineral content in a sufficient amount to have a miticidal effect, in the range of 1-10% oxalic acid by weight of the solution, reaching a low pH value in the pH range of 0.5-3.5 s the optimal pH range is 0.5-2.0.

We are trying to understand the botanical sources and biological activities of resins in the field and how resin harvesting behavior changes in response to environmental factors such as infection and other biological stressors. If we could discover plants with preferred and more antimicrobial resins in different areas, it would be possible to create an environment that improves the health of bees by maintaining behaviors and organizational 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 inventor believes that pure cultivated fungi and fungal mycelium, including fungal attractants, fungal entomopathogens, fungal immunostimulants, antiviral compounds, antibacterial compounds and antifungal compounds of fungi, can similarly support health bees and lead to natural resistance to diseases and pesticides. Bees are beneficial to ecosystems and economies that would otherwise suffer without these mushroom medicines.

The present inventor also expects that the effects of the invention will also be beneficial to pollinating insects and animals (bats). Similarly to birds, it is also expected to benefit from similar mushroom-based complete solutions by adding nectar-eating animals and birds to feed due to the activation of immunological and detoxification genes, as well as antiviral benefits, thus extending life span.

Filamentous basidiomycetes are also sources of neurodegenerative compounds. Hericium species (including but not limited to Hericium erinaceus, Hericium corralloides, and Hericium abietis) produce powerful nerve growth factors that induce myelin regeneration on nerve axons and nerve regeneration. See Stamets, Lion's Mane: A Mushroom That Improves Your Memory and Mood ?, The Blog, Huffington Post Healthy Living, 08/08/2012. Psilocybin and psilocybin-containing mushrooms, 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, Gymnopiius luteofolius, Gymnopiius 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 psilocybin-containing mushrooms and fruit bodies of Hericium fungi, or more preferably extracts of their mycelium, can aid in the regeneration of neurons damaged by toxins, cholinergic pesticides, oxidation, aging, or other sources of neurotoxins.

The combined effect of consuming these mixtures of nerve regenerating Hericium mushrooms and psilocybin species can improve the neurological health of bees through neurogenesis and remyelin.

- 21 037750 zations and, of course, animals, including humans. Another improved form of mycological honey may include these elements for the benefit of bees and humans, enhancing cognitive abilities, preventing or recovering from neuropathies that manifest as diseases in humans that fall within the definitions of Alzheimer's disease, Parkinson's disease, Parkinson's disease, MS (multiple sclerosis) or yet uncategorized forms of neurological disorders.

Indeed, such combinations can improve mental ability, sensation ability, memory, reflexes, reaction time, and problem-solving ability. As such, such intelligent mycological honey is contemplated to be within the scope of the present invention.

Ganoderma lucidum is one of the species of particular interest (together with Ganoderma resinaceum, Ganoderma applanatum, Ganoderma brownii, Ganoderma curtisii, Ganoderma oregonense, Ganoderma tsugae, Ganoderma lingzhi, Ganoderma capense, Ganoderma annularis only, since they are not the author's inventions) have strong antiviral properties, but also have sugar complexes that provide a mycelium producing a viscous syrup-like mycological honey that can be used to aid the survival of bees with CCD. The inventor and colleagues at Fungi Perfecti, LLC also noted that Ganoderma resinaceum extracts do not freeze even when freeze dryers reach temperatures of less than -50 ° C under high vacuum, while extracts from investigated species outside of the Ganoderma genus quickly freeze into dry condition under the same conditions. The inventor believes that the extract of mycelium of Ganoderma resinaceum and, probably, extracts of related species of Ganoderma, retain a liquid state even under cryogenic conditions due to their unique range of complex sugars, sterols and glycoproteins that bind to form a unique liquid matrix, significantly different from any other species studied. ... This extract has potential as an anti-freeze agent with wide applications in medicine, avionics, space flight, and can be used under extreme temperature conditions for the chemistry of lubricants, preservatives, and extremophiles.

In all of the examples presented below, the inventor expects, as a consequence of his discovery, that biologically directed fractionation methods will provide increased activity, increased efficiency and reduced cost of obtaining, producing and implementing said inventions and its many variations that will become apparent after this paradigm shifting discoveries.

Example 1.

Fomes fomentarius, Fomitopsis officinalis, Fomitopsis pinicola, Ganoderma resinaceum, Inonotus obliquus, Piptoporus betulinus, Trametes versicolor, Schizophyllum commune and other types of fungi are cultivated and mycelium is grown on rice, barley, flaxseeds, forestry seeds and other grains sawdust or wood chips (for a list of substrates, see Stamets, Growing Gourmet and Medicinal Mushrooms, 1993, Ten Speed Press, Berkeley, Ca. and Stamets & Chilton, The Mushroom Cultivator, Agarikon Press, Olympia, WA). When the mycelium reaches the mass of growth (preferably after cultivation for 5-60 days in fermentation or solid phase fermentation after inoculation, but long before the formation of the fruiting body) the mycelial mass can be extracted by simply washing the substrate with water, a mixture of water / ethanol (both of which are preferred) or ethanol or by compressing the substrate, all of which result in a liquid flowable or absorbable extract including extracellular exudates. These extracts can be used as is, or alcohol (25-50% by volume) can be added to the aqueous extracts as a preservative and solvent (which will precipitate the water-soluble polysaccharides).

The water-ethanol extract can be evaporated or removed, or the alcohol and water can be evaporated or removed separately. The crude extract can be filtered to remove cells using a 0.12-0.20 μm filter. This extract can be frozen or dried for later use. Alternatively, extracts can be used in non-aqueous or non-ethanol solvents such as DMSO, ethyl acetate, ether, and other solvents or solvent combinations known in the art, or subcritical or supercritical liquid extracts can be used using, for example, carbon dioxide or water and optional cosolvents such as alcohols, or extracts obtained by microwave treatment can be used.

The extract can be added to any form of food raw material for consumption by bees. The original extract can be used directly or diluted and added to their drinking water, sugar water, bee candy, honey, propolis, pollen cake, fat cake, and protein supplements to make improved bee feed and nutritional products and improved pollen supplements. food additives, feed additives and nutritional supplements. The extracts can also be incorporated into sprays used to spray beehives, hive components, sticky strips, bees and plants, and included in wax used to make honeycombs and shops. Eating and contacting bees with them improves the ability of bees to form immunity through the activation of enzymes, degradation

- 22,037,750 toxin reducing pathogen load and providing a healthy source of a variety of sugars, amino acids, B vitamins and nutrients. Moreover, the precipitate, although separated from the supernatant, is nutrient-rich and has antiviral health-promoting properties, and can also be used as raw material for the benefit of bees. Both the supernatant and the precipitate can be combined and enzymatically converted using amylase and other enzymes to further transform starches and other ingredients into a more effective composition.

Example 2.

The mycelium of medicinal mushrooms is grown using liquid culture methods. While growing on rice can provide 30-40% conversion of rice to mycelium, a liquid culture in a container can have a substantially complete conversion with> 3x more mycelium per unit weight. Thus, the liquid culture of mycelium in the vessel and its extracellular metabolites are easier to use for the development of the present invention, since the process using the culture in the vessel eliminates the need to remove non-metabolized substrate ingredients.

Example 3.

To obtain a mycelium extract, equal volumes of mycelium grown on grain (barley, flaxseed, rice, rye, millet, millet, rye, corn), seeds, including nuts, sawdust or wood chips (California fir, pines, oaks , birches, cotton trees, olives), and immersed in a 50:50 water-ethanol solution. It is allowed to stand at room temperature for two weeks and then compressed to expel a liquid extract. Within a few days, a precipitate falls out of the water-ethanol solution. The water-ethanol supernatant is decanted from the pasty precipitate. After a few additional weeks using a centrifuge, the precipitate is further concentrated to a semi-solid state. These wet semi-solids are removed and heated to 50 ° C for 6-8 hours with stirring. The wet volume of the semi-solids is reduced to about 40% of the original wet semi-solids. Drying the semi-solids to a caramel-like honey-like substance yields a yield of approximately 16% of the original wet solids weight. Thus, using 1000 ml of wet solid (which is 40% of the original extract) yields approximately 170 ml of a thick, syrup-like caramel-like substance. Continued heating and stirring will concentrate this substance into a noticeably sweeter substance. The extract can be crystallized, powdered, and used as a replacement for other processing methods. Liquid, semi-solid, and crystallized forms have a noticeably sweet taste and can be considered a medicinal candy-like substance useful to both bees and humans for a wide variety of uses.

Example 4.

Mycelium extract is obtained by extracting the fruiting bodies or mycelium of basidiomycete fungi, including Ganoderma resinaceum, in hot water (80-100 ° C) for several hours and combined with an aqueous extract of room temperature (10-30 ° C) mycelium Fomes fomentarius, Fomitopsis officinalis , Fomitopsis pinicola, Ganoderma resinaceum, Inonotus obliquus, Piptoporus betulinus, Trametes versicolor and / or Schizophyllum commune grown on grain or wood. Ethanol is added to these aqueous extracts to obtain a solution with more than 22% EtOH (ethanol), preferably 35-45% EtOH. After the addition of ethanol, the polysaccharides precipitate from the solution and settle to the bottom of the extraction vessel. After removing the supernatant, the precipitated polysaccharides enriched in glycosides, glycoproteins and other nectar-like nutrients are collected and heated at 50-70 ° C for several hours, resulting in a sweet residue that is attractive and beneficial to bees. Alternatively, the supernatant can be stored for several days, which additionally provides useful precipitated polysaccharides. These precipitates contain complex sugars, antiviral compounds, antibacterial compounds that activate cytochrome p450 coumaric acids and coumarins, and can be combined with other ingredients used in drinking water, pollen lozenges, propolis, beeswax, sprays or any delivery system, when by which the bees can contact these precipitates, which helps the bees to overcome the stress factors associated with colony collapse syndrome.

Example 5.

The mycelium extract obtained by extraction of the fruiting bodies or mycelium of basidiomycete fungi, including Ganoderma resinaceum, is first placed in 100% ethanol (1: 1 ratio by weight) for 1-7 days. After draining ethanol, the mushroom or mycelial pomace is immersed in hot water (80-100 ° C) for several hours and combined with the resulting placement in water at room temperature (10-30 ° C) extract of the mycelium Fomes fomentarius, Fomitopsis officinalis, Fomitopsis pinicola, Ganoderma resinaceum , Inonotus obliquus, Piptoporus betulinus, Trametes versicolor and / or Schizophyllum commune grown on grain or wood. To these aqueous extracts are added the ethanol extracts described above to give a total combined solution with greater than 22% EtOH, preferably 35-45% EtOH. After adding the ethanol fraction, polysaccharides precipitate from the races

- 23 037750 solution and settle at the bottom of the extraction vessel. P-coumaric acid, which is more soluble in ethanol than in water, is found in a greater amount in the supernatant extracted with ethanol (the ethanol supernatant with concentrated p-coumaric acids is a reservoir of p450-encoding compounds useful for bees). This water-ethanol supernatant can be stored for several days, which then results in a polysaccharide mixture with proportionally more pcumaric acid than the hot water fractions alone. The precipitate also contains p-coumaric acids and, in addition, other nutrients that can be used to feed the bees. These p-coumaric acid-fortified precipitates also contain complex sugars, antiviral compounds, antibacterial compounds, and the coumarin families, and can be combined with other ingredients such as water-soluble mushroom polysaccharides, corn syrup or sugars used to sweeten drinking water, or, additionally included as an ingredient in pollen cakes, propolis, beeswax, sprays, or any delivery system by which bees contact these precipitates to help bees overcome the stressors associated with colony collapse syndrome.

Example 6.

For each type of mycelium extract (fungal species), honeybees of mixed age from one hive were collected on one day and distributed randomly into 16 cages of approximately 100 bees each. Each set of 16 cells consisted of four control cells (feeding with sugar syrup), four cells of low concentration (feeding with mycelium extract in sugar syrup at a concentration of 0.1% v / v), four cells of medium concentration (feeding with mycelium extract in sugar syrup at 1% v / v) and four cells of high concentration (feeding with mycelium extract in sugar syrup at 10% v / v). In each group of four cells, three cells were used for lifespan tests and the remaining replica cell was used to study the total viral particle count. A separate experiment was performed to evaluate the effect of mushroom extracts on specific types of viruses.

Increased life expectancy.

To study the lifespan (survival), each replica was monitored every day (three cells for each concentration in the food and the control group) and the dead bees were counted. For each day of the experiment, the total number of bees that died on that day was tabulated for each replica cell for each mushroom extract and for the control groups. These tables of dead bees for each day were then used to calculate the percentage of bees that were still alive for each day of the experiment, based on the original number. Then, based on data from three replica cells for each mushroom extract and for the control group, the percentage survival rate was calculated.

Survival plots were constructed using time measured in days as the independent variable (x-axis) and the average percentage of bees surviving at any point in time in the experiment as the dependent variable (y-axis); the lifespan plots show the% surviving individuals of the original population at different points in time. See fig. 1-4.

FIG. 1 is a line graph showing the percentage survival of bees over time that were given Inonotus obliquus mycelium extracts (0.1, 1 and 10%, as indicated by the dotted, dotted and double-dotted lines, respectively) with water with sugar compared to a control population, which was fed only with water and sugar (shown by the solid line).

FIG. 2 is a line graph showing the percentage survival of bees over time that were given extracts of Ganoderma resinaceum mycelium (0.1, 1 and 10%, as indicated by the dotted, dotted and double-dotted lines, respectively) with water with sugar compared to the control population, which was fed only with water and sugar (shown by the solid line).

FIG. 3 is a line graph showing the percentage survival of bees over time that were given Fomitopsis pinicola mycelium extracts (0.1, 1 and 10%, as indicated by the dotted, dotted and double-dotted lines, respectively) with water with sugar compared to the control population, which was fed only with water and sugar (shown by the solid line).

FIG. 4 is a line graph showing the percentage survival of bees over time that were fed Fomes fomentarius mycelium extracts (1%, shown by the light solid line) with water with sugar, compared to a control population fed only with water and sugar (shown by the dark solid line) ... These and similar approaches can be used in the practice of the present invention to demonstrate the effect of the invention on the health and longevity of bees.

The figures show the average values in the experiments; standard error bars have been removed for clarity.

Some, but not all, of the results from these preliminary experiments were statistically significant; improved results are expected after continuing experiments with more replicas. Statistical significance was assessed using the Kaplan-Meier Survival Assessment (product limitation) performed using statistical software.

- 24 037750 JMP® Research Cookies from SAS Institute, Inc. See fig. 5, Kaplan-Meier survival rate graph (product limitation) showing the proportion of bees that survived over time in days when they were given Fomes fomentarius mycelium extracts (0.1, 1 and 10%, as indicated by the dotted, dotted and double-dotted lines, respectively. ) with water with sugar compared to the control population fed only with water with sugar (shown by the solid line). This analysis compared the median survival time for the administration group with the median survival time for the control (fed only with water and sugar), and used the Wilcoxon test to assess whether survival was statistically different from random variation in the survival time of the bees.

FIG. 5 illustrates this embodiment of the invention. In this assay, the composition of Fomes fomentarius when fed at a concentration of 1% v / v. increased the average survival rate of bees by 9.7% (p <0.0006).

As shown in FIG. 1-5, the lifespan of bees fed with extracts of various types of fungi increased, and the increase in lifespan depended both on the type of fungus and on the concentration consumed by the bees. In the practice of the present invention, in most cases, this increase in life expectancy is possibly due to a decrease in viral load (as described below), but also due to other aspects of the invention in cases where life expectancy was increased, but the number of viruses did not decrease.

Increased longevity can be demonstrated by practicing the present invention using survival charts such as, but not limited to, those described above. The increase in longevity can be measured numerically in the practice of the present invention by calculating differences in survival. One such method is based on the average of the function:

T ^s favg U ^{I f (_X) dX b ~} a Ja value where a and b denote the initial and final day, during which the measured effect of the invention; and f (x) denotes a survival graph function as described above.

The difference in these numbers over the specified time interval corresponds to the average percentage increase in life expectancy achieved by applying the present invention to the practice of the invention during the specified time interval.

Other methods for measuring differences in longevity, survival, or population growth, including statistical methods such as Kaplan-Meier analysis, Nelson-Aalen method, and other methods known to those skilled in the art, are alternatives for practicing the present invention.

Similarly, for use in the present invention, the practice can use various quantitative methods for assessing the amounts of viruses in bees, including reverse transcription polymerase chain reaction (RT-PCR) and real-time RT-PCR based on amplification of cDNA by PCR, ELISA (enzyme-linked immunosorbent assay ), including both conventional and sandwich ELISA with various blocking agents, primary / secondary antibodies, reporter enzymes and solutions of their specific colorimetric substrates for detection and quantification, multiplex microarrays using molecular probes for various RNA targets or AGID DNA targets ( agarose gel immunodiffusion), serological methods based on protein profiles or polyclonal and monoclonal antibodies, and a wide variety of other molecular biology methods such as high-throughput sequencing technologies, pyrophosphate-based sequencing technologies, sequencing by Sanger's method (also called chain termination method) and Integrated Virus Detection Systems (IVDS). See, for example, De Miranda, Diagnostic techniques for virus detection in honey bees, Aubert et al. (Eds.), Virology and the honey bee, EEC Publications (2008), p. 121-232 and Evans et al., Standard methodologies for molecular research in Apis mellifera, Journal of Apicultural Research, 52 (4), (2013).

Using this method to measure differences in longevity, the inventor defines an improvement in longevity as an embodiment of the present invention. See table. II, Average percentage increase in the lifespan of bees. The table shows the difference between the average values of the% surviving bees assessed over different time intervals. This difference is given as a numerical difference between these percentages, with the average percent survivors over different time intervals calculated as previously described:

Increased longevity = average% survivors _{fed with} mushroom extract ^- AVERAGE b SURVIVALS control

The increase in longevity increases the number of bee days when workers or other classes of bees are available to collect pollen and maintain the hive and perform other work,

- 25 037750 while improving health status and increasing the survival rate of individuals leads to an improvement in the health and survival of the colony.

Decrease in the overall level of the virus.

To study the antiviral properties of each type of mycelium extract (fungal species), honeybees of mixed age from one hive were collected on one day and distributed randomly into four cages of approximately 100 bees each. This test was carried out in parallel with the life span test described above using bees from the same hives over the same time interval. The set of cells for each type of fungi consisted of a control cell (feeding with sugar syrup), cells of low concentration (feeding with mycelium extract in sugar syrup at a concentration of 0.1% w / v), cells of medium concentration (feeding with mycelium extract in sugar syrup in concentration 1% v / v) and cells of high concentration (feeding with mycelium extract in sugar syrup at a concentration of 10% v / v).

Samples of bees were removed from the cages and frozen on days 0, 7 and 14. The analysis of the total number of viral particles, regardless of the type of virus, was carried out by Dr. David Wick of BVS, Inc. using IVDS technology; see U.S. Patent Nos. 8524155, 8309029, 8146446, 8021884, 7850908, 7250138, 6491872, 6485686 and 6051189 (all Charles Wick) and Charles H. Wick, Integrated Virus Detection, CRC Press (2014). For the analysis of each sample, 6.0 g of bees were mixed with 100 ml of reverse osmosis (RO) purified water and coarsely filtered through a two-layer gauze. Then a 90 ml sub-sample was centrifuged for 60 min at 20,000 xg. The supernatant was isolated and ultrafiltered through a hollow fiber filtration system with a threshold of 500,000 Da, and then washed with 200 ml of RO water and reduced in volume to approximately 2 ml. The solution was prepared for analysis using an Integrated Virus Detection System (IVDS) using a 1:10 dilution with ammonium acetate (AA). Each sample was filtered through a 20 μm w-41 paper or a 0.45 μm PTFE filter. Samples were scanned five times using IVDS and the average virus levels were recorded.

As shown in FIG. 6-9, the total viral load on bees fed with extracts of various types of fungi was reduced, and the decrease in virus levels depended on both the type of fungus and the concentration consumed by the bees.

FIG. 6 is a bar graph showing the total level of viral particles in bees that were given extracts of Inonotus obliquus mycelium (0.1, 1 and 10%, as indicated by the dotted, dotted and double-dotted lines, respectively) with water with sugar compared to the control population. which was fed only water with sugar (shown by the solid line) at time zero, after one week and two weeks.

FIG. 7 is a bar graph showing the total level of viral particles in bees that were given extracts of Ganoderma resinaceum mycelium (0.1, 1 and 10%, as indicated by the dotted, dotted and double-dotted lines, respectively) with water with sugar compared to the control population. which was fed only water with sugar (shown by the solid line) at time zero, after one week and two weeks.

FIG. 8 is a bar graph showing the total level of viral particles in bees that were given extracts of Fomitopsis pinicola mycelium (0.1, 1 and 10%, as indicated by the dotted, dotted and double-dotted lines, respectively) with water with sugar compared to the control population. which was fed only water with sugar (shown by the solid line) at time zero and after one week.

FIG. 9 is a bar graph showing the total level of viral particles in bees that were given Schizophyllum commune mycelium extracts (0.1, 1 and 10%, as indicated by the dotted, dashed and double dashed lines, respectively) with water with sugar compared to the control population. which was fed only water with sugar (shown by the solid line) at time zero and after two weeks.

These and similar figures can be used in the practice of the present invention to demonstrate the effect of the invention on the health of bees. In the practice of the present invention, most, but not all, of the species that provide extended lifespan also reduce viral load. This implies that a decrease in viral levels may contribute to an increase in longevity, however, this improvement in longevity could be observed without a decrease in viral levels due to other beneficial aspects of the invention, such as general stimulation of swarm immunity and antibiotic activity against non-viral pathogens such as Nosema. In the practice of the present invention, there are many possible reasons for the increase in longevity, since different types of fungi appear to have different and specific modes of action against different pathogens of bees, as described below by way of non-limiting example.

The reduction in total viral load can be measured in the practice of the present invention by calculating the difference in detected virus between the bees to which the invention was applied and the bees that were not exposed to the present invention. One such way of quantifying this difference is based on the mean of the function:

26 037750 f ^b favg & _ J f (x) dx where the values a and b denote the starting and ending days during which the effect of the invention was measured; and 'T (x) denotes virus detection rate versus sample collection time.

The percentage difference between these values over the specified time interval corresponds to the average percentage reduction in virus levels achieved by the practice of the present invention over the specified time interval. Other methods of measuring differences in virus levels over time, including percentage differences at individual sampling points, mean differences, and statistical methods such as Kaplan-Meier analysis are acceptable alternatives for practicing the present invention and are incorporated by reference. Using the method described above for measuring the difference between virus levels over different time intervals, the inventor defines virus reduction as an embodiment of the present invention. See table. I, Average percentage reduction in total viral load.

The table shows the difference between the average values of the% surviving bees assessed at different time intervals. This difference is not shown as a numerical difference between these percentages, but instead as a percentage reduction:

% decrease in viral load _ average titer of viruses _{contamination with fungal extract} - average titer of viruses _control average titer of viruses _countryrol

Fungi species / disease specificity.

Specific types of mycelium extract (a species of fungus) fed to mixed-age bees can reduce the level of etiological factors of diseases, such as viral particles, in a species-specific manner. This embodiment of the invention has been demonstrated by feeding mushroom extracts to bees in cages and measuring the levels of specific types of viruses in bees over time. For this analysis, bees of mixed ages were collected from one hive on one day and evenly distributed randomly in 12 cages. Four cells were fed with Ganoderma lucidum var. Mycelium extract. resinaceum at a concentration of 1% v / v in sugar syrup, four cells were fed with Inonotus obliquus mycelium extract at a concentration of 1% v / v. in sugar syrup and four cells were used as control and they were fed with sugar syrup only.

Bee samples were removed from the cage and frozen on days 0, 3, 7 and 14. The bees were sent by Dr. Yanping (Judy) Chen in the United States Department of Agriculture Agricultural Research Service for real-time RT-PCR analysis as described in Chen et al., Quantitative real-time reverse transcription-PCR analysis of Deformed Wing Virus infection in the honeybee (Apis mellifera L.), Appl. Environ. Microbiol., Vol. 71 (2005), p. 436-441 and Khongphinitbunjong et al., Differential viral levels and immune gene expression in three stocks of Apis mellifera induced by different numbers of Varroa destructor, Journal of Insect Physiology, Vol. 72 (2015), p. 28-34.

In this assay, virus levels were quantified based on the accumulation of a fluorescent signal as the virus DNA was amplified in a PCR reaction. The cycle count threshold is defined as the number of cycles required for the fluorescent signal to exceed the background fluorescence level. Cycle threshold levels are thus inversely proportional to the amount of target viral nucleic acid (eg, viral titer) in the sample (ie, the lower the CT level, the greater the amount of target nucleic acid in the sample).

As shown in FIG. 10, in bees fed with extracts of mycelium Ganoderma lucidum var. resinaceum and Inonotus obliquus, the increase in queen cell virus levels (as quantified based on the cycle count threshold) was prevented. See fig. 10, a line graph showing the threshold for the number of cycles for queen cell virus over time in a control population fed only with sugar water (shown by solid line 111) compared to bees fed with Inonotus obliquus mycelium extracts (1%) (shown dotted line 112) and Ganoderma resinaceum (1%) (shown by dotted line 113) with water and sugar. In contrast, the same extracts did not have a significant effect on Wing Deformity Virus levels. See fig. 11, a line graph showing the threshold number of cycles for wing strain virus over time in a control population fed only with sugar water (shown by solid line 121) and bees fed with Inonotus obliquus mycelium extracts (1%) (shown by a dotted line 122) and Ganoderma resinaceum (1%) (shown by the dotted line 113) with water and sugar.

This example of an extract of a fungal species having specificity against one viral pathogen but not others is an antiviral embodiment of the invention. It also supports the argument that particular fungal extract compositions may similarly be specific for other bee pathogens (which reduce lifespan) such as Nosema and Buck

- 27 037750 terii, and / or can mainly activate the metabolic, immune and detoxification systems of bees. Such effects against non-viral pathogens or general metabolic and immunity-enhancing effects may be responsible for cases where life expectancy increased but viral load remained unchanged.

Generalization, Benefits and Significance.

To date, the inventor of the present invention has data (both a lifespan experiment and an experiment to reduce the total viral load) for 8 species of medicinal fungi. The inventor also conducted a third experiment to reduce the level of a specific virus, the black queen cell virus, only for Ganoderma resinaceum and Inonotus obliquus.

The present inventor defines in the present description a measure, the LV index, which is: LV index = average percentage increase in bee lifespan multiplied by the average percentage reduction in total viral load.

This calculation yields a number that assigns equal weight to both aspects to measure the improvement in colony health. Empty boxes in the tables below for antiviral activity, lifespan, or LV indicate that either lifespan or viral load reduction were negative or zero in one or both datasets.

There are many other possible mathematical representations that can infer the relationship between these datasets, such as the percentage increase in life expectancy divided by the percentage reduction in viral load. This calculation focuses on the lifespan portion that could theoretically be associated with a reduction in viral load if the match was 1: 1. Numerous measurement possibilities will be apparent to those skilled in the art, and all such measurement methods to improve the health of bees should be considered to be within the scope of the invention.

The general approach used in the present description is to compare the area under the curve for life expectancy measurements and the results of measuring the total reduction in viral load, as described above. The difference between the areas under the curve over a given time interval is equal to the numbers in the tables for life expectancy. The difference in area under the curve over a given time interval, expressed as a percentage improvement, is equal to the numbers in the tables for the total viral load reduction. See table. I Average percentage reduction in total viral load and tab. II Average percentage increase in the lifespan of bees. These values can then be mathematically correlated to illustrate features of interest when practicing the present invention, such as compositions that are most preferred for increasing longevity and reducing overall viral load in bees. See table. III, Average percentage increase in bee lifespan x average percentage decrease in total viral load (LV index).

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Table I

Average percentage decrease in total viral load Concentration View Time frame 0.1% one% 10% Trametes versicolor 0-7 days 7.5 9.0 0-14 days 4.8 3.6 Fomitopsis pinicola 0-7 days 20.4 22.8 0-14 days 25.5 32.2 2.1 Fomitopsis officinalis 0-7 days 0-14 days 4.5 Schizophyllum commune 0-7 days 3.8 0-14 days 19.5 20.8 26.7 Inonotus obliquus 0-7 days 47.5 41.6 42.2 0-14 days Fomes fomentarius 0-7 days 9, 6 100 0-14 days 9.3 Ganoderma applanatum 0-7 days 2.5 3.6 1, o 0-14 days 4.5 14.2 Ganoderma lucidum var. 0-7 days 73.4 64.4 76, 7 resinaceum 0-14 days 65.4 58.3 85.9 Preferred 1-25% or more reduction in virus levels More preferred 15-25% or more reduction in virus levels Most preferred > 25% level reduction [virus

- 29 037750

Table II

Average percentage increase in bee lifespan _____

Concentration

View 0.1% one% 10% Time frame Trametes versicolor 0-7 days 4.1 0.2 5.0 0-14 days 3, 8 7.8 0-28 days sixteen 3.7 Fomitopsis pinicola 0-7 days 9.2 14.6 0-14 days 13.5 14.1 0-28 days 8.5 3.7 Fomitopsis officinalis 0-7 days 5.2 3.8 0-14 days 2,3 1.0 0-28 days 1, o Schizophyllum commune 0-7 days 0, 1 0.8 0-14 days 3.7 0.5 0-28 days 0, 1 Inonotus obliquus 0-7 days 1.6 1,2 0-14 days 4.1 2.1 0-28 days 3.7 Fomes fomentarius 0-7 days 1.7 22.1 13.5 0-14 days 16, 1 0-28 days 11.2 Ganoderma lucidum var. resinaceum 0-7 days 0-14 days 2.2 3, 7 0-28 days 3, 7 9, 5 Preferred 1-5% or more increase life expectancy More preferred 3-5% or more increase life expectancy Most preferred > 5% increase in life expectancy

Table III

Average percentage increase in bee lifespan x average percentage decrease in total viral load (LV index)

Concentration View 0.1% one% 10% Time frame Trametes versicolor 0-7 days 30, 9 44.8 0-14 days 18.1 27,7 Fomitopsis pinicola 0-7 days 187.5 332.7 0-14 days 344.1 453, 1 Fomitopsis officinalis 0-7 days

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0-14 days 10.4 Schizophyllum commune 0-7 days 3.1 0-14 days 72.6 9.6 Inonotus obliquus 0-7 days 67.0 51.2 0-14 days Fomes fomentarius 0-7 days 16.2 135.2 0-14 days Ganoderma applanatum 0-7 days 0-14 days Ganoderma lucidum var. resinaceum 0-7 days 0-14 days 147.1 212.9 Preferred index LV 1-200 + More preferred index LV 50-200 + Most preferred index LV 200+

In the practice of the present invention, mushroom extract compositions can be ranked differently in terms of preference depending on the intended use of the composition. Examples include, but are not limited to, ranking for improved lifespan, ranking for overall viral load reduction, ranking for lifespan extension, and viral load reduction. It is noteworthy that preference can also be given to compositions that increase lifespan but do not decrease viral load. Such compositions are expected to increase longevity by acting on stressors in bees that are not associated with viruses (examples include Nosema infection, exposure to pesticides, stress exposure to cold temperatures, etc.).

Example 7.

Ganoderma resinaceum extract after 14 days resulted in almost 20% increase in bee survival relative to controls. See also FIG. 5. This difference can be of great importance in promoting colony survival, as the lifespan of worker bees during this critical time results in the host bees not being recruited prematurely, allowing them to better care for the next generation of healthy offspring.

The addition of mycelium extracts from Ganoderma resinaceum led to a sharp decrease in the total viral pathogen load in bees (from a variety of viruses), while control in the form of sugar without mycelium extracts led to an increase in the total virus population. Since many entomologists believe that viruses are the most significant health problem, often contributing to subsequent infection with other types of bacteria (i.e., foulbrood) and fungi, reducing the level of viruses can be a key benefit in protecting bees from Colony Collapse Syndrome and many other associated diseases. stress factors.

In terms of increasing longevity, the addition of 1% extracts of mycelium Fomes fomentarius and Ganoderma resinaceum to water with sugar (water - 50 g, sugar - 49.5 g, mycelium extract - 0.5 g), statistically significantly increased the lifespan of bees - in terms of the number of days in the life of bees - by 17.6 and 8.9%, respectively. The increased life expectancy results in more worker bees available for work tasks, which is a significant advantage for stressed bee colonies with very low working capacity and stressed colonies on the brink of collapse. When there are more bees at any given time, it is important for collecting pollen and maintaining the hive. Moreover, significantly more hives can be saved by feeding bees with mycelium extracts in their sugar water relative to bees fed only sugar water without mycelium extracts. Until field trials, it is not known how much more bee days will shift the balance towards helping to overcome CCD, as there are many challenges. However, there is agreement among scientists studying bees that increasing the lifespan of worker bees under stressful conditions is a significant benefit. Moreover, when extracts are obtained, as in these cases, from birch wood (Betula species), which is the same tree species in which tinder fungi live and the same tree species in which bees nest, the extracts can become more effective with less costs of receiving. That such a high expressed activity of mycelium grown on rice can be shown supports the argument that mycelium is a causal benefit factor (rice controls showed no activity). The use of mycelium grown on rice as a source for inoculation of 10-100x greater mass in the form of birch sawdust results in an exponential expansion of the mycelium relative to the extracts of mycelium on rice described herein. The mycelium grows in more branched and compact mycelial networks on birch sawdust compared to rice, which means that higher

- 31,037,750 surface areas for the expression of extracellular components. Thus, the present inventor believes that birch sawdust mycelium extracts will be the preferred embodiment of the present invention.

Many Ganoderma and other tinder fungi are also expected to provide similar biosecurity. It is possible that lamellar fungi exist, due to their close evolutionary relationship with tinder fungi, which will provide similar benefits.

Example 8.

A liquid mycelium extract, or a precipitate of such an extract, or a concentrated extract from which all or part of the solvent has been removed, containing these active substances, can be added to honey, honey-enriched water, sugar water or bee candy, pollen, pollen substitutes, or to other substances in a different way, obvious to experts in the field of beekeeping science or commercial practice. The extract can be used as a supplement to other medicines, making them more effective. Extracts can be in liquid, frozen, lyophilized, air dried, vacuum dried, dehydrated using Refractance window technology, dehydrated by ultrasonic treatment, or partially purified form in quantities sufficient to have a bee attraction effect and / or bee health benefits, products honey and pollination. Moreover, these derived forms of the extracts may be beneficial for human consumption as they taste good, have high antioxidant content, and other properties that are beneficial to humans and other animals, including bees.

Example 9.

A preferred delivery system is the incorporation of the mycelium extracts into pollen cakes or fat cakes. Pollen cakes are made by beekeepers and placed above the nest box as a food source. They can be made from a wide range of materials, including soy, brewer's yeast, sugar syrup, and optionally can include organically grown pollen. These pollen cakes serve as a nutritional supplement for the bees. Because they are widely used in the fall, they help the bees survive until the next year. These extracts also contain digestive enzymes that help bees better metabolize food sources and help degrade toxins and boost basic immunity.

Example 10.

A mixture of compositions containing extracts of Stropharia rugoso-annulata, Fomes fomentarius, Fomitopsis officinalis, Fomitopsis pinicola, Ganoderma resinaceum, Inonotus obliquus, Piptoporus betulinus, Trametes versicolor and / or Schizophyllum commune, which together provide a variety of beneficial factors. Stropharia rugoso-annulata attracts bees, has a flower-like aroma and provides a source of nutrients rich in sugars (up to 75 polysaccharides). Fomes fomentarius and Fomitopsis officinalis extracts provide antiviral benefits, plus additional beneficial factors mentioned for Stropharia rugoso-annulata. All three extracts contain polyphenols, and more specifically coumarins, which help to activate the p450 enzyme cascades, which helps bees detoxify endogenous, natural, alien and anthropogenic toxins and their associated harmful effects. The mixture of these extracts can be given to bees through their drinking water, their enriched water, honey, propolis, pollen cakes, or even wax used to make preformed honeycombs when setting up honey production shops.

Example 11.

Adding extracts of mycelium of Fomitopsis officinalis, Fomitopsis pinicola, Fomes fomentarius, Inonotus obliquus, Schizophyllum commune, Ganoderma resinaceum, Piptoporus betulinus, Trametes versicolor and / or Inonotus obliquus to water with sugar, which can usually be fed to bees in the early spring, can help reduce levels in the spring load resident viruses early in the season, preventing them from increasing to the point where they become behavior-changing diseases or cause pathogen transmission between bees. The extracts can simply be mixed with water and sugar at a level sufficient for a beneficial effect. The range may preferably be 0.01-20%, or more preferably 0.1-10%, based on the volume of sugar-in-water compositions used by beekeepers. The extracts can first be mixed with water and then added to the sugar to form a typical syrup. The high sugar content can act as a preservative, providing long lasting antiviral and antibacterial properties.

Example 12.

Extracts of mycelium of medicinal mushrooms can be impregnated with paper strips. These paper tapes can be combined with adhesive. The low pH of many medicinal mushroom mycelium extracts, in the pH range of 0.5-4, is toxic to mites, but harmless to bees on contact. Optionally, an oxalic acid solution can be added in effective amounts.

Example 13.

The use of extracts of mycelium or fruit bodies of Ganoderma lucidum, Ganoderma resinaceum, Fomitopsis pinicola, Fomitopsis officinalis, Inonotus obliquus, Piptoporus betulinus, Trametes versicolor and

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Schizophyllum commune, where the extracts are concentrated to a form attractive to bees and sufficient on contact to have the effect of reducing the level of tobacco ring spot virus, Israel acute paralysis virus, queen cells virus, iridescent invertebrate virus, or IIV6, and Nosema microsporidium, results in that bees become better able to overcome colony collapse syndrome.

Example 14.

Application of extracts of mycelium or fruiting bodies of Ganoderma lucidum, Ganoderma resinaceum, Fomes fomentarius, Fomitopsis pinicola, Fomitopsis officinalis, Schizophyllum commune, Inonotus obliquus and Stropharia rugoso-annulata, where the extracts are concentrated in a form that resembles texture, texture for bees and sufficient on contact to have the effect of reducing the levels of viruses, including, but not limited to, tobacco ring spot virus, Israeli acute paralysis virus, black queen cells and Nosema microsporidia virus, and causing activation of cytochrome p450 enzyme cascades, increasing overall immune function, foraging ability, overwintering ability, drought tolerance, ability to survive the extinction of nectar-producing plants lead to increased health of the bees, so that there are measurable benefits for the survival and overcoming of CCD in hives and the emergence of subsequent generations. This mycological honey can be used alone or can be mixed with bee honey to attract and provide benefits to bees. Moreover, mycological honey can be partially dissolved in water as a plant leaf spray, or it can be applied directly to bees. In addition, this mycological honey can be marketed as a nutritional supplement for human consumption.

Example 15.

Bees flying to or from the water with sugar hang at the entrance to the hive and shake their bodies to eliminate ticks. If ticks fall through the net, they contact or become attracted to an entomopathogenic mycoattractant, which itself can be lethal, or to an insecticidal mycelium, where the ticks are depleted or killed, which reduces the tick's ability to move and become infected, thus reducing their threat to bees. Moreover, if bees are sprayed with a spray enriched with oxalic acid, parasitic mites become more susceptible to the infectious or lethal properties of entomopathogenic fungi.

Example 16.

Extracts, hyphae fragments or spores of beneficial fungi such as Stropharia rugoso-annulata and spores of entomopathogenic fungi such as Entomophthorales can be incorporated as a mixture into extract-enriched sugar water, bee food or honey, allowing transfer to the honey production system providing benefits to the offspring, drones, the queen and the hive in general.

Example 17.

Extracts of mycelium or spores of hyphae or tissue fragments of Stropharia rugoso-annulata can be provided on paper strips or in water available to bees. The Stropharia rugoso-annulata scent, to which bees can be accustomed, helps collecting bees to return to their colonies if these scent is in the vicinity or in the hives. Such fragrances can be emitted by any method known in the art of delivering fragrances using sprays, sprays, or aerosol dispensers.

It is hypothesized that Stropharia rugoso-annulata mycelium extracts and other fungal mycelium extracts will induce track-following or navigational behavior through dance language and scent traces.

Example 18.

Metarhizium anisopliae spores and hyphae can be mixed with Fomes fomentarius mycelium to produce Varroa mite sprays and fumes to promote resistance of bees to mites, viruses, etc. to overcome CCD. Many strains of Metarhizium are relatively non-toxic; No harm to humans is expected from exposure to Metarhizium anisopliae strain F52 due to ingestion, inhalation or touching of products containing this active ingredient. Metarhizium anisopliae strain F52 (029056), Biopesticide Fact Sheet.

Example 19.

A blend of extract formulations of Stropharia rugoso-annulata, Fomitopsis officinalis, Fomitopsis pinicola, Fomes fomentarius, Ganoderma resinaceum, Inonotus obliquus, Piptoporus betulinus, Trametes versicolor and / or Schizophyllum commune and Metarhizium anisopliae together provide a variety of beneficial factors that can be added to water together. Stropharia rugoso-annulata attracts bees, has a flower-like aroma and provides a source of nutrients rich in sugars (up to 75 polysaccharides). Various extracts provide antiviral and antibacterial benefits and a longer lifespan, plus the ability to attract Stropharia rugoso-annulata. Extracts of Metarhizium anisopliae may be provided on sticky strips or films, or in any sticky mite or humpback trapping substance, or in water available to them, to attract mites and humpback beetles, where upon contact they are depleted or destroyed, reducing their ability to vector disease; Varroa mite populations can be reduced using Metarhizium extract

- 33 037750 anisopliae before isolating the nest chamber, which reduces the death of bees from the effects of ticks and the diseases they carry. All three extracts contain polyphenols and, more specifically, coumarins, which promote the activation of the p450 enzyme cascades, which help detoxify endogenous, foreign, natural and anthropogenic toxins and reduce the harmful effects associated with them. A solution of these mixed extracts can be provided to the bees through nectar feeders containing their drinking water or their sugar or fructose enriched water, by mixing in bee candy, honey, propolis, pollen cakes, or even by mixing with wax used to obtain preformed honeycomb when creating stores for the production of honey.

Example 20.

Extracts of the preconidial mycelium of Metarhizium anisopliae pathogenic to ticks and / or flies can be mixed with spores or hyphae fragments and presented on sticky strips or plates, or in any sticky tick or humpback trapping substance, or in water available to ticks. This combination attracts ticks or flies and upon contact infects them with an entomopathogenic fungus or exposes them to lethal doses of entomopathogenic toxins.

Example 21.

Extracts of Metarhizium anisopliae preconidial mycelium mixed with extracts, spores or fragments of Stropharia rugoso-annulata hyphae can be provided on paper strips or in water available to bees. This combination attracts mites or flies and bees and, on contact, harms mites and flies, but not bees.

Example 22.

Preconidial mycelium extracts of Aspergillus flavus, Aspergillus niger and Aspergillus fumigatus can be mixed with spores or hyphae fragments of Stropharia rugoso-annulata and provided on paper strips or in water available to bees. This combination attracts mites or flies and bees and, on contact, harms mites and flies, but not bees. Optionally, strains of Aspergillus flavus, Aspergillus niger and Aspergillus fumigatus can be used that have reduced levels of aflatoxin and neurotoxin below levels that are harmful to bees, but above levels that are harmful to mites and flies, thus providing the overall health benefits of the bee colony.

Example 23.

Metarhizium anisopliae preconidial mycelium extracts can be mixed with spores or hyphae fragments of Stropharia rugoso-annulata and provided on paper strips or in water available to bees. This combination attracts mites or flies and bees, and on contact harms mites and flies, but not bees. Optionally, strains of Metarhizium anisopliae can be used that have reduced levels of destructin below levels that are harmful to bees, but above levels that are harmful to mites and flies, thus providing the overall health benefits of the bee colony.

Example 24.

Fruiting body mycelium extracts and / or Metarhizium anisopliae preconidial mycelium extracts can be mixed with extracts and derivatives from neem indianus and provided on paper strips, in water available to bees, or in topical sprays. This combination attracts mites or flies and bees and, on contact, harms mites and flies, but not bees. Optionally, strains of Metarhizium anisopliae can be used that have reduced levels of destructin below levels that are harmful to bees, but above levels that are harmful to mites and flies, thus providing the overall health benefits of the bee colony. Optionally, the concentration of neem indianus extracts (or the active ingredient azadiractin) and sugars can be balanced to optimize the benefit to the bees by reducing the number of mites and their gathering capacity and their pathogenic load. Moreover, this combination can be further enhanced by the addition of extracts of basidiomycete fungi from agaricoid and polyporoid fungi, which not only provide mite-killing oxalic acid and degrading toxin enzymes, but also activate innate enzymatic cascades of cytochrome p450 to destroy anthropomorphic toxins, in addition the level of pathogens associated with viruses, bacteria and fungi affecting bees. Such synergistic effects of multiple components have the cumulative effect of helping bees cope better with colony collapse syndrome. A combination of Metarhizium anisopliae preconidial mycelium, Fomitopsis officinalis and Fomitopsis pinicola extracts, Indian neem extracts, Ganoderma lucidum extracts, Ganoderma resinaceum, Ganoderma applanatum, Pleurotus ostreatus, an effective composition and method for preparing a delivery effective bee spray or ingredient in pollen lozenges or drinking water. Similar compositions can be sprayed on plants or trees that the bees pollinate to benefit both the plant and the bees.

Example 25.

Methods and compositions of oxalic acid fortified with water sugars (or polysaccharides), and

- 34,037,750 fragments of preconidial hyphae from Metarhizium anisopliae selectively harm mites when in contact with bees, while providing total benefits for bees. This composition can optionally be combined with extracts of medicinal fungus mycelium with antiviral and longevity-enhancing properties and included in bee food.

Example 26.

Combination of extracts Fomes fomentarius, Fomitopsis pinicola, Fomitopsis officinalis, Ganoderma lucidum, Ganoderma applanatum, Ganoderma resinaceum, Schizophyllum commune, Stropharia rugoso-annulata and Ganoderma resinaceum in combination with the extracts of preconidial mites and bee extracts with the use of preconidial extracts of Methylus mite, whereby this combination of bee extracts of preconidial mite damage to Varnrnoa mites, by reducing the number of viruses, pathogenic fungi and bacteria, provides a net benefit for bees to overcome colony collapse syndrome.

Example 27.

Combination of preconidial mycelium of Metarhizium anisopliae with polysaccharides Fomitopsis pinicola, Fomitopsis officinalis Ganoderma resinaceum, Ganoderma lucidum, Inonotus obliquus and Schizophyllum communes to attract bees and mites, where contact with this combination of bacteria Varroa causes harm to mites, causing damage to mites and bacteria caused by Varroa harm to bees, has a net benefit for bees to overcome colony collapse syndrome.

Example 28.

The use of extracts of mycelium or fruiting bodies devoid of melanin, such as from the so-called albino fruiting bodies Agaricus blazei, Fomitopsis officinalis, Fomitopsis pinicola, Fomes fomentarius, Schizophyllum commune, Trametes elegans and Stropharia rugoso-annulata, where the extracts are concentrated in a form that is resembles the texture and consistency of honey, in a form that is attractive to bees and sufficient on contact to have the effect of reducing the level of tobacco ring spot virus, israeli acute paralysis virus, queen cells virus, iridescent invertebrate virus, or IIV6, and microsporidium Nosema, and causing activation of cascades cytochrome p450 enzyme, increasing overall immune function, gathering ability, overwintering ability, drought tolerance, ability to survive the extinction of nectar-producing plants, leads to improved health of the bees, so that there are measurable benefits for hive survival and overcoming KS syndrome. racha of the colony and the appearance of subsequent generations. This mycological honey can be used alone or can be mixed with bee honey to attract and provide benefits to bees. Moreover, mycological honey can be partially dissolved in water as a plant leaf spray, or it can be applied directly to bees. In addition, this mycological honey can be marketed as a nutritional supplement for human consumption.

Example 29.

Cultivation of medicinal fungus mycelium on plant materials that are active against viruses, including Ficus bengalensis (Vad), Ficus religiosa (Pimpal), Jasminum aurniculaturn (Jaai), Acacia catechu (Khaim), Azadirachta indica (nem), Curcuma longa (turmeric) , Withania somnifera (ashwagandha) and Silybum marianum (milk thistle). See Deshpande et al., Antiviral activity of plant extracts against sac brood virus in vitro - a preliminary report, International Journal of Institutional Pharmacy and life Sciences 3 (6): November-December 2013, p. 1-22.

Example 30.

Structural materials treated with fungi to help protect bee colonies.

The hives are typically made from wood fibers and stacked shop frames where the bees create honey-rich combs above the chambers for the offspring. These shops and other wood-based frames used by beekeepers end up being destroyed by frequently growing molds, which can be pathogenic to bees and can contribute to insect and arthropod infestations. As a result, beekeepers tend to replace them with stores, offspring chambers and bottoms, etc. every few years. The base of the bee hives tends to last longer, but ultimately all the chambers of the bee hive are destroyed by opportunistic, often pathogenic, fungi, and this can adversely affect the health of the bee colonies as a result.

The present invention relates to the addition, inclusion, impregnation, coating, implantation or attachment of spores or mycelium fragments or extracts thereof, beneficial fungi, more particularly fruiting body-forming fungi of the class Basidiomycetes / Basidiomycota or Ascomycetes, into hive frames that provide health benefits to bees by providing immune protection with one or more synergistic beneficial factors. For example, but not limited to, shop frames can be made from sheets of wood, wood fiber, and a wide range of agricultural products that can be associated with mycelium, with or without spores. Such mushroom-impregnated panels not only provide structural benefits, but fungi can also have antiviral, miticidal, antibacterial, antifungal, anti-insecticidal properties that help bees live on and in the materials used to construct bees.

- 35 037750 hives.

In addition to hives, all structures containing animals can advantageously be treated with extracts or mycelium, including bird cages, stalls and structures that contain bats.

Materials that can be used as a carrier for these beneficial mushrooms can be made from wood, pressed wood, biodegradable structured panels, panel substitutes for wood grown using mushrooms, non-biodegradable materials, and they can also be included in beeswax used for frames, or even propolis, which bees use to close cracks. The compositions and methods of the present invention can be readily incorporated into such open source beehive production.

In fact, as the bee hives age, they gradually, sporadically, or increasingly provide health benefits to the bees due to the addition of the aforementioned fungi to mitigate the increasing stressors that occur as the hive boxes get older. All of the antiviral properties of these fungi, their creation of a wide range of complex and simple sugars, the production of p-coumaric acid and other molecules that activate cytochrome p450, the formation of spores of entomopathogenic fungi such as Metarhizium anisopliae, which harm ticks, but not bees, act synergistically. counteracting the many stressors that bees suffer from throughout the life of the hive colonies from year to year.

One of the many examples is the cultivation of mycelium, for example, tinder fungi Antrodia cinnomonea, Fomes fomentarius, Inonotus obliquus, Inonotus pini, Fomitopsis officinalis, Fomitopsis pinicola, Fomitiporia robusta, Ganoderma, Ganoderma applanatum, Ganoderma atrale, Ganoderma annare Ganoderma lucidum, Ganoderma resinaceum, Ganoderma tsugae, Lenzites betulina, Phellinus igniarius, Phellinus pini, Phellinus weirii, Piptoporus betulinus, Trametes elegans, Trametes versicolor and lamellar mushrooms Schizophyllum commune, Strophainus mycelium could be shaped into structural panels used to construct hive boxes for honey bees. In addition, the tick-killing fungus Metarhizium anisopliae can be grown in a preconidial (presporulating) or post-conidial (postsporulating) state - or mixtures thereof - and can be enclosed directly in structural panels, regardless of their composition, in combination with the fungi described herein. as well as other species that the present inventor believes can help bees, such as Basidiomycetes, Ascomycetes or Entomophthorales, Hypocreales or other fungi that attack mites and humpbacks.

Likewise, chicken coops, bird feeders, plant trellises, etc. can be constructed from cellulosic products impregnated with mycelium or spores.

Moreover, the aforementioned species and many related species contain and secrete toxin-degrading enzymes that can contribute to the degradation of insecticides (including neonicitinoids, neocins), fungicides, pesticides, formaldehydes, tannins, dyes, endemic toxins, and a wide variety of anthropogenic toxins.

The preferred wood-based substrate consists of birch panels, planks or sawdust (Betula species) and preferred species are birch tinder fungi such as, but not limited to, Fomes fomentarius, Inonotus obliquus, Ganoderma resinaceum, and Piptoporus betulinus. If it is made from coniferous wood, then tinder fungi such as Fomitopsis pinicola, Fomitopsis officinalis, Laetiporus suiphureus, Phellinus igniarius, P. pini, P. linteus and P. weirii are ideal candidates; or lamellar mushrooms such as Pleurotus ostreatus, Lentinus ponderosus. Trametes versicolor (= Coriolus versicolor) is a tinder fungus growing on deciduous and coniferous trees and is also a preferred species for use in the context of the present invention. In the case of each of the listed species, they should be taken in the broadest sense of the species, i.e. in a broad sense, and closely related species are also expected to be useful in helping bees. Essentially, when describing Fomitopsis officinalis, Ganoderma applanatum, Ganoderma lucidum, Ganoderma resinaceum, Inonotus obliquus, Trametes versicolor, or any other species of mushrooms, this means Fomitopsis officinalis in a broad sense, Ganoderma applanatum in a broad sense, Ganoderma lucidum in a broad sense resinaceum in a broad sense, Inonotus obliquus in a broad sense, Trametes versicolor in a broad sense and a similar broad description of any other species, each of which means that it refers to the concept of a species, as described in the broadest taxonomic interpretation, covering all historical and modern synonyms , varieties, forms and species that have separated or will separate in the future from these species after publication. As is known in the art, naming changes as new species concepts are created. It is expected that the view can be used in an infinitely wide sense, and many of them are shown in the previously approved 8 US patents of the present inventor and in co-pending patent applications filed to date. However, these views, not previously described, have now become apparent after the discovery of the present inventor.

- 36 037750

The fact that the polypore fungi Fomes fomentarius, Inonotus obliquus and Ganoderma resinaceum are active against viruses that harm bees and humans is significant and, as far as the inventor of the present invention knows, unprecedented from a medical point of view. Moreover, if these beneficial factors for cross-species animal mycelium extracts of these tinder fungi and, in fact, many fungi can be achieved, then more than one animal species can benefit from the broad antiviral properties of the mycelium of these species. Thus, these crops, their extracts or their spores can be added to birdhouses, chicken coops, stalls and animal housing of any type for immunological and community protection benefits, preventing the escalation of disease vectors and even curing diseases of all their inhabitants. Potentially, mycelium and fungal homes can protect inhabitants from viruses, bacteria, insects, arthropods, toxins, environmental stressors, disease vectors, and unexpectedly can impart pleasant aromas specific to the fungi used.

Extracts that can be used for the invention described above can be provided as a by-product of products that use mycelium to fill molds or templates to create structured materials with growing mycelium, such as insulating coatings, transportation materials for foam replacement, construction materials, packaging materials, filter layers, filtration membranes, fabrics, framework for computer chips and processors based on growing mycelium, nanowires based on mycobacteria, etc. In addition, these useful extracts can be harvested by expressing the liquid components of the substrates used in all stages of mushroom production, as well as mushroom fermentation methods used to produce tempeh, koji, enzymes, antibiotics, plant growth enhancers, and pharmaceuticals. In fact, as the mycelium grows, the materials made from the mycelium often dry out. In this case, extracellular and intracellular metabolites and other fluids must be removed. When growing structured mycelium-based materials, excess fluid is removed and is generally not highly valued. The present invention changes the purpose of this residual liquid product into an unexpectedly high value product range that can be enriched with antiviral agents, antimicrobial agents, enzymes, acids, active ingredients, and other chemicals useful in the context of the present invention to help bees, and for many other applications in medicine, chemical engineering, degradation and bioremediation (microremediation) practice. Moreover, the now dried mycelial product can be designed so that the latent population of fungal cells survives the drying process, to reactivate only when the bee hives age, causing the mycelium and its heat-resistant sclerotia and chlamydospores to survive and regrow for unusual benefits - as they age. hives included beneficial fungi compete with fungal pathogens, provide nutrients, increase the overall lifespan of the bee colony. Beneficial fungi can be selected specifically for the survival of thermotolerant chlamydospores and sclerotia after making mycelium-grown structural materials. Redesigned liquid from the mycelium pressing process, as well as heat-resistant mycelium residing on structural materials, can be combined for synergistic health benefits for the bees.

Example 31.

Honey is collected from bees fed with mycelium extracts as described above. This medicinal honey helps bees and humans by activating toxin degradation pathways through the cytochrome p450 cascades. Since honey is more than food for bees and humans, such medicinal honey is expected to have a wide range of uses. The preservative properties of honey can keep these medically active compounds more stable.

Example 32.

Α-amylase, amyloglucosidase, betulinic acid, caffeic acid, protocatechuic acid, transcinnamic acid, ferulic acid, gallic acid, ellagic acid, lanosterol, inotodiol, trametonolic acid, hispolones, ergosterols, chrysin, cordycepse -o-coumaric acid, trans-p-coumaric acid, ellagic acid dihydrate, ergosterol, linoleic acid, trans-ferulic acid, gallic acid hydrate, hexanal, hispolone, 4-hydroxybenzoic acid, quercetin hydrate, rutin hydrate, syringic acid, vanillic acid, sulferic acid, dehydrosulferic acid, eburic acid, 6-chloro-4-phenyl-2H-chromen-2-one, ethyl 6-chloro-2-oxo-4-phenyl-2H-chromene-3-carboxylate, 7- chloro4-phenyl-2H-chromen-2-one, ethyl 7-chloro-2-oxo-4-phenyl-2H-chromene-3-carboxylate, psilocybin, psilocin and related compounds, isomers, structural analogs and substantially similar connections. The compounds are also expected to be beneficial to other animals, including humans.

Example 33.

Since protocatechuic acid, vanillic acid, cinnamic acid, caffeic acid and related compounds, isomers, structural analogs and substantially similar compounds are widespread and present in many food plants, and protocatechuic acid is naturally found at high levels in the brain and grains of brown rice,

37 037750 growing the bee beneficial fungi species mentioned herein on a substrate already containing protocatechuic acid, its precursors and analogs is likely to increase the total amount of these bee beneficial compounds, and is another embodiment to improve the present invention.

Example 33.

Extracts of spores, mycelium and fragments of hyphae or tissue of the fruiting body of the polypore fungi Fomes fomentarius, Fomitopsis pinicola, Fomitopsis officinalis, Piptoporus betulinus, Ganoderma resinaceum, Ganoderma lucidum, Schizophyllum commune and Inonotus obiiquus can be sprayed into droplets or sprayed into clouds a mixture of spores-mycelium Metarhizium anisopliae. Such a mixture can reduce the level of viral pathogens, activate detoxification cascades in bees, provide a wide range of complex and simple sugars, vitamins to enhance immunity and the longevity of the hive. Moreover, the addition of Metarhizium anisopliae spores and mycelium can infect, control and prevent Varroa and humpback mites. The endogenous oxalic acid in all of these fungi can help control these pathogens. Individual use or combinations of birch polypore fungi can limit the growth of microsporidia Nosema and foulbrood bacteria (such as Melissococcus plutonius) that damage the bee colony. Such aerosol sprays can be delivered using hand-held or knapsack sprays. Alternatively, absorbent tapes impregnated with the beneficial agents described above can be used. The materials of construction used to construct hive bodies can also be impregnated and impregnated with these useful agents.

Claims (18)

CLAIM 1. Composition for improving the health of bees, containing an effective dose of 1% or less by volume of one or more extracts of the mycelium of Inonotus obliquus, Ganoderma resinaceum, Fomitopsis pinicola, Fomes fomentarius, Schizophyllum commune, Trametes versicolor, Fomitopsis officinalis, Ganoderma applanatum combinations thereof and one or more bee feed additives. 2. The composition of claim 1, wherein the bee feed additive comprises one or more of water, sugars, sugar syrup, water with high fructose corn syrup, bee candy, nectar, pollen, pollen cakes, fat cakes, propolis, beeswax, bee sprays, bee feed, protein supplements, and combinations thereof. 3. The composition of claim 1, wherein the mycelium is cultured on solid substrates or liquid substrates. 4. The composition of claim 1, wherein the mycelium extracts comprise aqueous mycelium extracts; water-ethanol extracts of mycelium; supernatant remaining after precipitation of the aqueous mycelium extract with ethanol; the supernatant of the aqueous ethanol extract of the mycelium having a portion of the solvent removed; the supernatant of the aqueous ethanol extract of the mycelium in which the solvent has been removed; the supernatant of the water-ethanol extract of the mycelium, in which part of the solvent and all the sediment have been removed; the supernatant of the aqueous ethanol extract of the mycelium, in which the solvent and sediment have been removed; steam distillation extracts; extracts cooked in a microwave oven; or combinations thereof. 5. The composition of claim 1, wherein the composition further comprises one or more extracts of neem, oxalic acid, formic acid, lactic acid, or a combination thereof. 6. The composition of claim 1, wherein the composition improves the health of the bees by increasing the lifespan of the bees by more than 3%. 7. The composition of claim 1, wherein the composition improves the health of the bees by reducing viral load by more than 15%. 8. The composition of claim 1, wherein the composition improves the health conditions of the bees by increasing longevity and decreasing viral load. 9. The composition according to claim 1, additionally containing one or more extracts of Antrodia cinnomonea, Ganoderma atrum, Ganoderma brownii, Ganoderma curtisii, Ganoderma lucidum, Ganoderma lingzhi, Ganoderma oregonense, Ganoderma tsugae, Fomitopsis officinalis officinalis (Lariticiforme) , Inonotus hispidus, Inonotus andersonii, Inonotus dryadeus, Laetiporus cincinnatus, Laetiporus sulphureus, Laetiporus conifericola, Lenzites betulina, Phellinus igniarius, Phellinus linteus, Phellinus polytreum pini, Piptoporus ole complica gibbosa, Trametes hirsuta, Trametes villosa, Trametes cingulata, Trametes ochracea, Trametes pubescens, Trametes ectypa, Trametes aesculi, Wolfiporia cocos, Agaricus augustus, Agaricus blazei, Agaricus symparicus, Agaricus, campicus, campicus Agrocybe pediades, Agrocybe ae gerita, Agrocybe arvalis, Agrocybe praecox, Clitocybe odora, Conocybe cyanopus, Conocybe lacteus, Conocybe rickenii, Conocybe smithii, Conocybe tenera, Coprinopsis nivea, Coprinopsis hydroymaceus lagopus, Coprinoma autopres, Coprinus micronus , Hypholoma capnoides, Hypholoma sublateritium, Hypsizygus marmoreus, Hypsizygus tessulatus, Hypsizygus ulmarius, Lentinus - 38 037750 ponderosus, Lepiota procera (Macrolepiota procera), Lepiota rachodes (Chlorophyllum rachodes), Lepista nuda, Mycena alcalina, Mycena pura, Mycena aurantiadisca, Panellus serotinus, Panaeolus foenisecii, Pteanaeolus orereatus pulmonarius, Pleurotus sapidus, Pleurotus tuberregium, Panellus stipticus, Panellus serotinus, Pluteus cervinus, Psathyrella aquatica, Psathyrella condolleana, Psathyrella hydrophila, Psilocybe allenii, Psilocybe azurescens, Psilocybe caerulescens, Psilocybe coprophila, Psilocybe cubensis, Psilocybe cyanescens, Psilocybe ovoideocystidiata, Psilocybe stuntzii, Psilocybe subaeruginosa, Stropharia aeruginosa, Stropharia cyanea, Stropharia rugoso-annulata, Stropharia semiglobata, Stropharia semigloboides, Stropharia squamosa, Stropharia thrausta, Stropharia volbya umbonotescens, Termitomyina com an amount of 1% or less by volume of the mycelium extract. 10. Composition for improving the health of bees, containing an effective dose of 1% by volume of one or more extracts of Inonotus obliquus, Ganoderma resinaceum, Fomitopsis pinicola, Fomes fomentarius, Schizophyllum commune, Trametes versicolor, Fomitopsis officinalis, Ganoderma applanatum, Ganoder var. resinaceum or combinations thereof. 11. The composition of claim 10 further comprising one or more extracts of neem, oxalic acid, formic acid, lactic acid, or combinations thereof. 12. The composition of claim 10, wherein the mycelium is cultured on solid substrates or liquid substrates. 13. The composition according to claim 9, where the extracts include aqueous extracts of mycelium, water-ethanol extracts of mycelium, supernatant remaining after precipitation of the aqueous extract of mycelium with ethanol; a supernatant from a water-ethanol extract of mycelium having a portion of the solvent removed; the supernatant from the aqueous ethanol extract of the mycelium, from which the solvent has been removed; the supernatant of the aqueous ethanol extract of the mycelium, from which part of the solvent and all the precipitate have been removed; the supernatant of the aqueous ethanol extract of the mycelium, from which the solvent and sediment have been removed; steam distillation extracts; extracts cooked in a microwave oven; or combinations thereof. 14. The use of a composition comprising an effective dose of 1% or less by volume of one or more extracts of mycelium Inonotus obliquus, Ganoderma resinaceum, Fomitopsis pinicola, Fomes fomentarius, Schizophyllum commune, Trametes versicolor, Fomitopsis officinalis, Ganoderma applanatum, Ganoderma lucidma. resinaceum or combinations thereof to improve the health of bees. 15. Use according to claim 14, wherein the composition further comprises extracts of neem, oxalic acid, formic acid, lactic acid, or combinations thereof. 16. Use according to claim 14, wherein the mycelium is cultured on a substrate selected from the group consisting of solid substrates and liquid substrates. 17. The use according to claim 14, where the extracts include aqueous extracts of mycelium; water-ethanol extracts of mycelium; supernatant remaining after precipitation of the aqueous mycelium extract with ethanol; a supernatant from a water-ethanol extract of mycelium having a portion of the solvent removed; the supernatant of the aqueous ethanol extract of the mycelium from which the solvent has been removed; the supernatant of the water-ethanol extract of the mycelium, from which part of the solvent and all the sediment have been removed; the supernatant of the water-ethanol extract of the mycelium, from which the solvent and sediment have been removed; steam distillation extracts; extracts cooked in a microwave oven; or their combination. 18. A composition for improving the health of bees, comprising an effective dose of 1% or less by volume of Fomes fomentarius, Trametes versicolor mycelium extract, or a combination thereof.