US20060021470A1

US20060021470A1 - Compositions for enhancing biological functions in organisms

Info

Publication number: US20060021470A1
Application number: US11/128,720
Authority: US
Inventors: Matteo Lorito; Sheridan Woo; Vincenzo Fogliano; Robert Mach
Original assignee: Individual
Current assignee: Individual
Priority date: 2004-05-13
Filing date: 2005-05-13
Publication date: 2006-02-02
Also published as: WO2005112880A3; WO2005112880A2

Abstract

The present invention provides a composition which includes elicitor compounds selected from the group consisting of: (N,N′diacetylhexobiose)_n; (N,N′diacetylhexobiose)_nhaving one or more associated amino acid residues, wherein the amino acid residues are valine or ornthine; (N,N′diacetylhexosamine)_nhaving an associated (dihexobiose)_n; (N,N′diacetylhexosamine)_nhaving an associated (dihexobiose)_nand one or more associated amino acid residues, wherein the amino acid residues are valine or ornithine; or combinations thereof. The (N,N′diacetylhexobiose)_nand (N,N′diacetylhexosamine)_ncomprise N-acetylglucosamine or other amino hexosamines, while the (N,N′diacetylhexobiose)_nand (dihexobiose)_nare any D-hexoaldose or N-acetylamino derivative of D-hexoaldoses. In this aspect of the present invention, n=1 to 5 and the compounds are all 3 kDa or less. Also provided are a method for increasing the rate of fungal growth, a method for increasing extracellular fungal enzyme production, a method for increasing biological control of plant and animal diseases, a method for increasing a method for increasing resistance of plants to diseases, and a method of alleviating pain and increasing resistance to, or recovery from, diseases in animals, using the composition of the present invention.

Description

This application claims benefit of U.S. Provisional Patent Application Ser. No. 60/570,765, filed May 13, 2004, which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to compositions including a series of related carbohydrate moieties or carbohydrate-amino moieties, and the use thereof for enhancing biological functions in a variety of organisms.

BACKGROUND OF THE INVENTION

Cells and tissues of essentially all organisms must respond and adapt to changes in external environmental conditions. In many cases, cells contain specific receptors for particular chemicals. When such chemicals come into contact with cell surfaces, they bind specifically to particular receptors. This binding then triggers a cascade of events within the cells, including up- and down-regulation of genes and activation or repression of specific pathways within the cells. Those processes result in substantial changes in cellular physiology. Thus, these elicitors are triggers of dramatic physiological responses. Moreover, a very small quantity of the elicitor molecule is often sufficient to cause major changes in cellular physiology. Such compounds generally are effective at micromolar concentrations. An example of a class of highly effective physiological elicitors is the group that includes hormones.
An understanding and identification of such elicitors will have a major impact on cellular physiology and permit metabolic engineering to achieve beneficial changes in organismal activity. An example of elicitor-based activity includes induction of immune or resistance responses in plants or animals.
Fungi in the genus Trichoderma are parasitic on other fungi, a property that, in part, allows them to be used in the biological control of plant pathogenic fungi (Chet et al., “Trichoderma—Application, Mode of Action, and Potential as a Biocontrol Agent of Soilborne Plant Pathogenic Fungi,” Innovative Approaches to Plant Disease Control 137-160 (1987); Harman, G. E., “Myths and Dogmas of Biocontrol. Changes in Perceptions Based on Research with Trichoderma harzianum T-22,” Plant Dis. 84,:377-393 (2000); Harman et al., “Trichoderma Species—Opportunistic, Avirulent Plant Symbionts,” Nature Microbiol 2:43-56 (2004)). The events leading to mycoparasitism are complex. First, Trichoderma strains detect other fungi and grow tropically toward them (Chet et al., “Trichoderma Hamatum: Its Hyphal Interactions with Rhizoctonia soloni and Pythium spp,” Microb Ecol 7:29-38 (1981)). Remote sensing is at least partially due to sequential expression of fungal cell wall degrading enzymes (“CWDEs”). These include various classes of chitinases, various classes of glucanases/glucosidases, proteinases, and other enzymes (Chet et al., “Mycoparasitism and Lytic Enzymes,” Trichoderma and Gliocladium 2:153-172 (1998)). This remote sensing system is mediated by elicitors and will be described in the Examples, below. Different strains may follow different patterns of induction, however, the fungi apparently always produce low levels of an extracellular exochitinase. Diffusion of this enzyme catalyzes release of cell wall fragments from target fungi and this, in turn, induces expression of fungitoxic CWDEs (Brunner et al., “The Nag1 N-Acetylglucosaminidase of Trichoderma Atroviride is Essential for Chitinase Induction by Chitin and of Major Relevance to Biocontrol,” Curr Genet 43:289-295 (2003)), that also diffuse and begin the attack on the target fungi before physical contact by the Trichoderma (or other elicitor) is actually made (Viterbo et al., “Expression Regulation of the Endochitinase chit36 from Trichoderma Asperellum (T. Harzianum T-203),” Curr Genet 42:114-122 (2002); Zeilinger et al., “Chitinase Gene Expression During Mycoparasitic Interaction of Trichoderma Harzianum with its Host,” Fung Genet Biol 26:131-140 (1999)). Once the fungi come into contact, Trichoderma spp. attach, and may coil about and form appresoria on the surface of the host.
Attachment is mediated by binding of carbohydrates in the Trichoderma cell wall to lectins on the target fungus (Inbar et al, “Hyphal Interaction Between Trichoderma Harzianum and Sclerotinia Sclerotiorum and its Role in Biological Control,” Soil Biol Biochem 28:757-763 (1996)). The Trichoderma in contact produces fungitoxic CWDEs, and probably also peptaibol antibiotics (i.e., antibiotic antifungal peptides) (Schirmböck et al., “Parallel Formation and Synergism of Hydrolytic Enzymes and Peptaibol Antibiotics, Molecular Mechanisms Involved in the Antagonistic Action of Trichoderma Harzianum Against Phytopathogenic Fungi,” Appl Environ Microbiol 60:4364-4370 (1994)). The combined activity of these materials is necessary for parasitism of the target fungus and dissolution of the cell walls. These products damage the fungal host and make its nutrients available to the attacking fungus. There are at least 20-30 known genes, proteins, or other metabolites directly involved in this process, which is typical of the complex systems employed by these fungi in their interactions with other organisms. Thus, because a wide range of different genes are activated in this process, it is a typical cascade-type response.
Plants also respond to attack by pathogenic microbes by production of a cascade of cellular events; this is a classical case of an elicitation response. Induced resistance systems in plants are complex, but have been partially elucidated in several model plant systems. There are two generally recognized pathways: one that relies upon production of jasmonic acid and another that relies upon salicylic acid produced by the plant as signaling molecules. The compounds or their analogues induce similar responses when applied exogenously. The elicitors described herein probably function upstream of these signal molecules as the initial triggering system. Jasmonate and salicylate probably are evolved as later events in the systems that will be described. There is considerable cross-talk between the two pathways (Bostock et al., “Signal Interactions in Induced Resistance to Pathogens and Insect Herbivores,” Eur J Plant Pathol 107:103-111 (2001)). Detailed information on these pathways can be found in Volume 107, Issue 1 of the European Journal of Plant Pathology (2001), which contains papers from principal workers in the field from a symposium on this topic. The jasmonate- and salicylate-induced pathways are characterized by production of a cascade of pathogenesis-related (PR) proteins. The PR proteins include antifungal enzymes, such as chitinases, glucanases, and thaumatins; oxidative enzymes, such as peroxidases, polyphenol oxidases (PPO, above), lipoxygenases; and others, such as proteinase inhibitors. Small molecular weight compounds with antimicrobial properties (phytoalexins) also may accumulate.
Animals, including humans, also have evolved complex systems involving antibodies to specific pathogens, specialized cells, and defense-related proteins and metabolites. It has been shown that administration of an agent combining a chitin oligosaccharide and a chitosan oligosaccharide (known as a chitin chitosan oligosaccharide, or CCOS), is useful for preventing or treating the common cold, and is an acceptable analgesic for mild to severe pain in humans. U.S. Pat. No. 6,492,350 to Konno et al. Although CCOS have been shown to be useful in initial studies, the active agent is a large molecule, and large molecules are classically difficult to use in human therapeutic applications. Moreover, more purified compounds may prove to be more effective in this application.
What is needed now is the further characterization of specific elicitor molecules responsible for the triggering of these complex systems and methods that utilize such elicitor molecules for the enhancement of cellular functions in a variety of target organism.
The present invention is directed to overcoming these and other deficiencies in the art.

SUMMARY OF THE INVENTION

The present invention relates to a composition, which, when applied to an organism under conditions appropriate for activity, enhances: fungal growth, extracellular enzyme production under repressive or nonrepressive conditions by fungi, biological control of plant pathogens, and resistance to disease in plants and animals. This composition includes an elicitor compound selected from the group consisting of: (N,N′diacetylhexobiose)_n; (N,N′diacetylhexobiose)_nhaving one or more associated amino acid residues, where the amino acid residues comprise valine or ornithine; (N,N′diacetylhexosamine)_nhaving an associated (dihexobiose)_n; (N,N′diacetylhexosamine)_nhaving an associated (dihexobiose)_nand one or more associated amino acid residues, where the amino acid residues comprise valine or ornthine; or combinations thereof. The (N,N′diacetylhexobiose)_nand (N,N′diacetylhexosamine)_ncomprise N-acetylglucosamine or other amino hexosamines, while the (N,N′diacetylhexobiose)_nand (dihexobiose)_ncomprise any D-hexoaldose or a N-acetylamino derivative of D-hexoaldoses. In this aspect of the present invention, n=1 to 5, and each of the elicitor compounds has a molecular weight of 3 kDa or less.
The present invention also relates to a composition, which, when applied to an organism under conditions appropriate for activity, enhances: fungal growth, extracellular enzyme production under repressive or nonrepressive conditions by fungi, biological control of plant pathogens, and resistance to disease in plants and animals. This composition include an elicitor compound selected from the group consisting of: (N,N′diacetylchitobiose)_n; (N,N′diacetylchitobiose)_nhaving one or more associated amino acid residues, where the amino acid residues comprise valine or ornithine; (N,N′diacetylchitobiose)_nhaving an associated (dimeric hexose)_n; (N,N′diacetylchitobiose)_nhaving an associated (dimeric hexose)_nand one or more associated amino acid residues, where the amino acid residues comprise valine or ornthine; or combinations thereof. The (N,N′diacetylchitobiose)_ncomprises N-acetylglucosamine or other amino hexosamines, while the (dimeric hexose)_ncomprises any D-hexoaldose or N-acetylamino derivative of D-hexoaldose. In this aspect of the present invention, n=1 to 5, and each of the elicitor compounds has a molecular weight of 3 kDa or less.
The present invention relates to yet another composition, which, when applied to an organism under conditions appropriate for activity, enhances: fungal growth, extracellular enzyme production under repressive or nonrepressive conditions by fungi, biological control of plant pathogens, and resistance to disease in plants and animals. This composition includes an elicitor compound selected from the group consisting of: N,N′diacetylchitobiose; N,N′diacetylchitobiose having one or more associated amino acid residues, where the amino acid residues comprise valine or ornthine; N,N′diacetylchitobiose having an associated dimeric hexose; N,N′diacetylchitobiose having an associated dimeric hexose and one or more associated amino acid residues, where the amino acid residues comprise valine or ornthine; N,N′diacetylchitobiose having two associated dimeric hexoses; N,N′diacetylchitobiose having two associated dimeric hexoses and one or more associated amino acid residues, where the amino acid residues comprise valine or ornthine; two or more N,N′diacetylchitobiose residues having two or more associated dimeric hexoses; two or more N,N′diacetylchitobiose residues having two or more associated dimeric hexoses and one or more associated amino acid residues, where the amino acid residues comprise valine or ornthine; or combinations thereof. The N,N′diacetylchitobiose comprises N-acetylglucosamine or other amino hexosamines, while the dimeric hexose comprises any D-hexoaldose or N-acetylamino derivative of D-hexoaldose. In this aspect of the present invention, n=1 to 5, and each of the elicitor compounds has a molecular weight of 3 kDa or less.
The present invention also relates to a method of increasing the rate of growth of fungi. This method involves applying a composition of the present invention to fungi under conditions effective to increase growth of the fungi.
Another aspect of the present invention is a method of increasing the production of extracellular enzymes from fungi. This method involves applying a composition of the present invention to fungi under conditions effective to increase production of extracellular enzymes from the fungi.
The present invention also relates to a method of increasing the biological control of plant diseases. This method involves applying a fungal biocontrol organism and a composition of the present invention to plants under conditions effective to increase the biological control of plant diseases.
Yet another aspect of the present invention is a method of increasing resistance of plants to diseases. This method involves applying a composition of the present invention under conditions effective to increase the resistance of the plant to diseases.
Also provided by the present invention is a method of alleviating pain and increasing resistance to, or recovery from, diseases in an animal, including a human. This method involves administering a composition of the present invention under conditions effective to alleviate pain, increase resistance to, or recovery from, diseases in an animal.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the ability of Trichoderma to trigger the release of elicitors from various target fungi, as measured by the induction of green fluorescence protein (“gfp”) (right axis) in the endochitinase promoter labeled strain of P1.
FIG. 2 is a graph showing the release of elicitors from cell walls of some of the same fungi and Oomycetes as in FIG. 1 using various types of enzymes purified from T. atroviride. Elicitor release is measured as fluorescence induction (left hand axis) of a gfp:endochitinase construct in Trichoderma P1. 0=no fluorescence, 3=high fluorescence. The elicitor source organisms are shown on the horizontal axis.
FIG. 3 is a chromatograph showing the activity of fractioned elicitor compounds.
FIG. 4 is a photograph of results of growth assay testing the effect of elicitor compounds on the growth of T. atroviride strain P1. Samples are: Control (no compounds added):(C); undiluted elicitor mixture (I); 10× diluted elicitor mixture (10); 50× diluted elicitor mixture (50); 100× diluted elicitor mixture (100).
FIGS. 5A-B are photographs of germ tubes of T. atroviride produced in culture. FIG. 5A shows germ tubes grown in the absence of elicitor compounds. FIG. 5B shows germ tubes grown in the presence of elicitor compounds. Bars indicate size of germ tubes for comparative purposes.
FIGS. 6A-B are graphs showing extracellular enzyme activity of T. atroviride strain P1 in minimal salts media containing different carbon sources, with and without elicitor compounds added. FIG. 6A shows exochitinase activity (conditions and elicitors shown below FIG. 6B). FIG. 6B shows endochitinase activity under same conditions as “FIG. 6A.
FIGS. 7A-B are photographs showing the appearance of lesions on bean leaves caused by B. cinerea following treatment with T. atroviride alone (FIG. 7A) or with T. atroviride plus elicitors (FIG. 7B).
FIG. 8 is a graph showing the reduction of disease symptoms in plants follow various treatment regimens with or without elicitors.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a composition, which, when applied to an organism under conditions appropriate for activity, enhances: fungal growth, extracellular enzyme production under repressive or nonrepressive conditions by fungi, biological control of plant pathogens, and resistance to disease in plants and animals. This composition includes an elicitor compound selected from the group consisting of: (N,N′diacetylhexobiose)_n; (N,N′diacetylhexobiose)_nhaving one or more associated amino acid residues, where the amino acid residues comprise valine or ornthine; (N,N′diacetylhexosamine)_nhaving an associated (dihexobiose)_n; (N,N′diacetylhexosamine)_nhaving an associated (dihexobiose)_nand one or more associated amino acid residues, where the amino acid residues comprise valine or ornthine; or combinations thereof. The (N,N′diacetylhexobiose)_nand (N,N′diacetylhexosamine)_ncomprise N-acetylglucosamine or other amino hexosamines, while the (N,N′diacetylhexobiose)_nand (dihexobiose)_ncomprise any D-hexoaldose or a N-acetylamino derivative of D-hexoaldoses. In this aspect of the present invention, n=1 to 5, and each of the elicitor compounds has a molecular weight of 3 kDa or less.
The present invention also relates to another composition, which, when applied to an organism under conditions appropriate for activity, enhances: fungal growth, extracellular enzyme production under repressive or nonrepressive conditions by fungi, biological control of plant pathogens, and resistance to disease in plants and animals. This composition includes a compound selected from the group consisting of: (N,N′diacetylchitobiose)_n; (N,N′diacetylchitobiose)_nhaving one or more associated amino acid residues, where the amino acid residues comprise valine or ornithine; (N,N′diacetylchitobiose)_nhaving an associated (dimeric hexose)_n; (N,N′diacetylchitobiose)_nhaving an associated (dimeric hexose)_nand one or more associated amino acid residues, where the amino acid residues comprise valine or ornthine; or combinations thereof. The (N,N′diacetylchitobiose)_ncomprises N-acetylglucosamine or other amino hexosamines, while the (dimeric hexose)_ncomprises any D-hexoaldose or N-acetylamino derivative of D-hexoaldose. In this aspect of the present invention, n=1 to 5, and each of the elicitor compounds has a molecular weight of 3 kDa or less.
The present invention relates to yet another composition, which, when applied to an organism under conditions appropriate for activity, enhances: fungal growth, extracellular enzyme production under repressive or nonrepressive conditions by fungi, biological control of plant pathogens, and resistance to disease in plants and animals. This composition includes an elicitor compound selected from the group consisting of: N,N′diacetylchitobiose; N,N′diacetylchitobiose having one or more associated amino acid residues, where the amino acid residues comprise valine or ornthine; N,N′diacetylchitobiose having an associated dimeric hexose; N,N′diacetylchitobiose having an associated dimeric hexose and one or more attached amino acid residues, where the amino acid residues comprise valine or ornthine; N,N′diacetylchitobiose having two associated dimeric hexoses; N,N′diacetylchitobiose having two associated dimeric hexoses and one or more associated amino acid residues, where the amino acid residues comprise valine or ornithine; two or more N,N′diacetylchitobiose residues having two or more associated dimeric hexoses; two or more N,N′diacetylchitobiose residues having two or more associated dimeric hexoses and one or more associated amino acid residues, where the amino acid residues comprise valine or ornthine; or combinations thereof. The N,N′diacetylchitobiose comprises N-acetylglucosamine or other amino hexosamines, while the dimeric hexose comprises any D-hexoaldose or N-acetylamino derivative of D-hexoaldose. In this aspect of the present invention, n=1 to 5, and each of the elicitor compounds has a molecular weight of 3 kDa or less.
The compounds of the present invention are referred as “elicitors” because of the ability of these compounds to trigger the production of extracellular enzymes in a variety of organisms. The active compounds (elicitors) of the present invention include one or more associated carbohydrate moieties. “Associated” as used herein is meant to encompass a relationship between moieties that includes strong or weak chemical attachment, for example, through covalent (polar or non-polar) or ionic bonds between the moieties, and less formal physical or chemical relationships between the moieties that result in the moieties being identified as a putative individual compound when subjected to high performance liquid chromatography.
In some aspects of the present invention, the compound also includes one or more associated amino acid residues. The amino acid residues may be valine, ornthine, serine, or a combination thereof. The amino acid residues may be associated anywhere on the compound, provided such attachment does not negate the activity of the compound. All compounds of the present invention contain a dimer of chitin, e.g., chitobiose, and some also have one or two dimers of hexoses. The compounds of the present invention have a mass (molecular weight) of 5-3 kDa or less, preferably, 3 kDa or less.
The elicitor compounds of the present invention also include those containing substitutions in the carbohydrate portion of the molecules without loss of activity. The hexose in the dimeric unit can be substituted by any D-hexose sugars, including galactose, mannose, allose, talose, altrose, or idose. Similarly, the N-acetyglucosamine (GlcNac) residues, including those which comprise N,N′-diacetylchitobiose [(GlcNac(1-5)], can be substituted by other amino hexosamines, including the acetylamino derivative of the same sugars listed above.
Sources of the elicitor compounds of the present invention include, without limitation, any fungus, including Basidomycetes, e.g., Rhizoctonia solani, Sclerotinia rolfsii, Pythium ultimum and Phytophthora infestans; any Ascomycetes, e.g., Botrytis cinerea, Colletotrichum acumatum, Penicillium spp., Sclerotium sclerotiorum, Alteraria alternata, and A. longipes. Other suitable sources are any organisms having chitin, glucans, and cellulose in their cell walls that function as active elicitor compounds.
The elicitor compounds of the present invention are purified from their respective source organism using methods well known in the art. “Activity” of a putative elicitor compound, or mixture thereof, is determined by carrying out enzyme assays for the putative extracellular enzyme, such as described in the Examples below, or using any of the enzyme bioassays commonly used in the field. In all aspects of the present invention a “composition” of the present invention encompasses an individual elicitor compound as described herein, or any combination of elicitor compounds, i.e., a mixture of the elicitor compounds of the present invention.
The present invention also relates to a method of increasing the rate of growth of fungi. This method involves applying a composition of the present invention, or a combination thereof, to fungi under conditions effective to increase growth of the fungi. “Applying” as used herein, for this and all other aspects of the present invention, means exposing an organism to an elicitor molecule in a manner that achieves the intended outcome. Thus, applying includes direct application and indirect application. Direct application includes topical application, for examples, spreading, spraying, or rubbing a composition including a compound of the present invention onto the target organism. Indirect application involves putting a sufficient amount of one or more compounds of the present invention in soil, media, food, water, or another matrix or substance that the organism will come into contact with, such that the elicitor compound will trigger a physiological response in the organism to effect the desired outcome. Indirect application encompasses exposure that takes place over time and distance.
“Enhanced” and “increased” as used in all aspects of the present invention refers to an outcome that is enhanced or increased in an organism to which a composition of the present invention has been applied compared to the outcome for an organism to which a composition of the present invention has not been applied.
The organisms to which the elicitors of the present invention may be applied include all species of fungi. Exemplary fungi include, without limitation, fungus from the genera Fusarium, Botrytis, Trichoderma (including but not limited to Trichoderma harzianum stain P1); Rhizoctonia, Sclerotinia, Pythium, Phytophthora, Colletotrichum, Sclerotium, Alteraria, Uncinula, and Ustilago.
As described in greater detail in the Examples, below, micromolar amounts of the composition of the present invention are generally sufficient to trigger the desired cascade of physiological events in the target organism and produce the desired outcome in this and all aspects of the present invention.
Another aspect of the present invention is a method of increasing the production of extracellular enzymes from fungi. This method involves applying a composition of the present invention to fungi under conditions effective to increase production of extracellular enzymes from the fungi. The production of extracellular fungal enzymes that are enhanced in this aspect of the present invention include, without limitation, exochitinases, endochitinases, glucanases, and cellulases. Suitable fungi include, without limitation, those listed herein above or below.
In this aspect of the present invention, the production of extracellular fungal enzymes is enhanced even when the fungi are under repressive conditions. Repressive conditions (as explained in greater detail in Example 5, below) occur when the fungi are in high nitrogen or high glucose media. This leads to catabolic repression of enzyme production. However, the composition of the present invention is capable of inducing enhanced enzyme production whether the fungi are under repressive or nonrepressive conditions.
The present invention also relates to a method of increasing the biological control of plant diseases. This method involves applying a fungal biocontrol organism and a composition, or combination thereof, of the present invention to plants under conditions effective to increase the biological control of plant diseases. Replacement or reduction of chemical application has been achieved through the use of biologically based fungicides. As used herein, biological control means the reduction of the amount of inoculum or disease-producing activity of a pathogen accomplished by or through one or more organisms other than man (Harman et al., “Enzymes, Biological Control and Commercial Applications,” Trichoderma and Gliocladium Vol. 2, Chap. 6 (1998), which is hereby incorporated by reference in its entirety). Biological control strains are known in the art that are effective in a great variety of habitats and against an assortment of pathogens (Harman et al., “Enzymes, Biological Control and Commercial Applications,” Trichoderma and Gliocladium Vol. 2, Chap. 6 (1998), which is hereby incorporated by reference in its entirety). In this aspect of the present invention, the fungal biological control organism may be any of those that have been, or will be, identified as useful for biological control of plant disease, including, without limitation, those from following genera: Trichoderma, Gliocladium, Pythium, Phytophthora, Rhizoctonia, Fusarium, Botrytis, and Sclerotinia (Harman et al., “Enzymes, Biological Control and Commercial Applications,” Trichoderma and Gliocladium Vol. 2, Chap. 6 (1998); U.S. Pat. Nos. 5,474,926 and 6,512,166 to Harman et al., which are both hereby incorporated by reference in its entirety).
Fungal pathogens are a major cause of disease in a wide variety of plants. Thus, this aspect of the present invention is suitable for controlling plant diseases caused by a variety of plant pathogens, including, without limitation, Bipolaris, Botrytis, Colletotrichum, Diplodia, Fusarium, Gliocladium, Gymnosporangium, Rhizoctonia, Trichoderma, Uncinula, Ustilago, Venturia, Erysiphe, Saccharomyces, Sclerotium, and Alternaria.
Plant disease may be controlled in a wide variety of plants or their seeds according this aspect of the present invention. Methods of applying biological control organisms are well known in the art. Some exemplary modes of application for the biological control organism and/or the elicitors of the present invention follow; any others known and used in the art are also suitable.
Incorporation into soils or greenhouse planting mixes. Beneficial microbes suitable for biological control and the desired compound of the present invention are formulated or mixed to prepare granules, dusts or liquid suspensions. These can be mixed directly into soils or planting mixes. The preparations are then mixed into the soil or planting mix volume for greenhouse applications or into the upper volume of field soil (Harman, G. E., “The Dogmas and Myths of Biocontrol. Changes in Perceptions Based on Research with Trichoderma harzianum T-22,” Plant Dis. 84, 377-393 (2000), which is hereby incorporated by reference in its entirety). Equipment and procedures for such applications are well known and used in various agricultural industries. Typical rates for biological control organisms are 0.2 to 10 kg of product containing 10⁷to 10⁹colony forming units (cfu) per cubic meter of planting mix or soil (Harman, G. E., “The Dogmas and Myths of Biocontrol. Changes in Perceptions Based on Research with Trichoderma harzianum T-22,” Plant Dis. 84, 377-393 (2000), which is hereby incorporated by reference in its entirety).
Planter box dust treatments. Formulations for these treatments are prepared as dry powders and usually contain inert ingredients such as graphite to enhance adherence to seeds. These materials are sold directly to farmers who add them to the seeds just at the time of planting. Coatings provided by this method usually are less uniform than those provided by other seed treatment processes.
Drenches for greenhouse or nursery soils and soil mixes. Liquid suspensions of the beneficial microorganisms can be prepared by mixing dry powder formulations into water or other aqueous carrier, including fertilizer solutions, or by diluting a liquid formulation containing the biological control organism in water or other aqueous solutions, including those containing fertilizers. The desired composition is added to the solution at any point during preparation. Such solutions can then be used to water planting mixes either prior to planting or else when plants are actively growing. Typically 10 to 400 ml of product (typically 150 μm or smaller in particle size) containing 10⁷to 10⁹cfu are mixed with 100 L of water for such applications (Harman, G. E., “The Dogmas and Myths of Biocontrol. Changes in Perceptions Based on Research with Trichoderma harzianum T-22,” Plant Dis. 84, 377-393 (2000), which is hereby incorporated by reference in its entirety).
Slurry, film-coated or pelleted seeds. Seeds are commonly treated using slurry, film-coating or pelleting by processes well known in the trade (Harman et al., “Factors Affecting Trichoderma hamatum Applied to Seeds As a Biocontrol Agent,” Phytopathology 71: 569-572 (1981); Taylor et al., “Concepts and Technologies of Selected Seed Treatments,” Ann. Rev. Phytopathol. 28: 321-339 (1990), which are hereby incorporated by reference in their entirety). The beneficial microbial agents of the present invention can effectively be added to any such treatment, providing that the formulations do not contain materials injurious to the beneficial organism. Depending on the microbe in question, this may include chemical fungicides. Typically, powder or liquid formulations (10⁷to 10¹⁰cfu/g) of the organism are suspended in aqueous suspensions to give a bioactive level of the microbe. Harman, G. E., “The Dogmas and Myths of Biocontrol. Changes in Perceptions Based on Research with Trichoderma harzianum T-22,” Plant Dis. 84, 377-393 (2000), which is hereby incorporated by reference in its entirety). The liquid typically contains adhesives and other materials to provide a good level of coverage of the seeds and may also improve its shape for planting or its cosmetic appeal.
Alternatively, an effective amount of the desired biological control organism and a sufficient amount of the desired composition of the present invention are directly applied to the flowers, foliage, or roots of a plant.
The application of the biocontrol agent and the elicitor composition may be made in combination, or each may be applied individually.
In yet another alternative, the biological control organism and the desired composition of the present invention is applied in the form of a spray, or as a solid, wherein the elicitor is present together with an agriculturally acceptable adhesive carrier, e.g., methyl cellulose or gum arabic, and applied as a powder.
Suitable plants for this aspect of the present invention include dicots and monocots. More particularly, useful crop plants can include: alfalfa, rice, wheat, barley, rye, cotton, sunflower, peanut, corn, potato, sweet potato, bean, pea, chicory, lettuce, endive, cabbage, brussel sprout, beet, parsnip, turnip, cauliflower, broccoli, radish, spinach, onion, garlic, eggplant, pepper, celery, carrot, squash, pumpkin, zucchini, cucumber, apple, pear, melon, citrus, strawberry, grape, raspberry, pineapple, soybean, tobacco, tomato, sorghum, and sugarcane. Examples of suitable ornamental plants are: Arabidopsis thaliana, Saintpaulia, petunia, pelargonium, poinsettia, chrysanthemum, carnation, zinnia, and turfgrasses.
Yet another aspect of the present invention is a method of increasing resistance of plants to diseases. This method involves applying a composition of the present invention to plants under conditions effective to increase the resistance of the plant to diseases. Application of the desired composition of the present invention is carried out as described above, using a sufficient amount of the desired composition of the present invention appropriately prepared for the selected mode of application.
In this aspect of the present invention, suitable plants include all those listed above, and resistance to disease includes any disease caused by a plant pathogen, including, without limitation, those listed above.
Also provided by the present invention is a method of alleviating pain and increasing resistance to, or recovery from, diseases in an animal, including a human. This method involves administering a composition of the present invention, or combination thereof, under conditions effective to alleviate pain, increase resistance to, or recovery from, diseases in an animal. In this aspect of the present invention the elicitor compound is purified from any of the sources listed above, and administered in an effective amount to animal for the treatment or prevention of diseases, including, without limitation, the common cold. Alleviation of pain includes acute and chronic pain, for example, pain associated with cancer, fibromyalgia, toothache, or headache.
The compositions of the present invention can be administered orally, parenterally, for example, subcutaneously, intravenously, intramuscularly, intraperitoneally, by intranasal instillation, dermally, or by application to mucous membranes, such as that of the nose, throat, and bronchial tubes.
The compositions of the present invention may be administered alone or with pharmaceutically or physiologically acceptable carriers, excipients, or stabilizers, and can be in solid or liquid form such as, tablets, capsules, powders, solutions, suspensions, or emulsions.
The solid unit dosage forms can be of the conventional type. The solid form can be a capsule, such as an ordinary gelatin type containing the desired composition of the present invention and a carrier, for example, lubricants and inert fillers such as, lactose, sucrose, or cornstarch. In another embodiment, the compound is tableted with conventional tablet bases such as lactose, sucrose, or cornstarch in combination with binders like acacia, cornstarch, or gelatin, disintegrating agents, such as cornstarch, potato starch, or alginic acid, and a lubricant, like stearic acid or magnesium stearate.
The compositions of the present invention may also be administered in injectable dosages by solution or suspension of the composition in a physiologically acceptable diluent with a pharmaceutical carrier. Such carriers include sterile liquids, such as water and oils, with or without the addition of a surfactant and other pharmaceutically and physiologically acceptable carrier, including adjuvants, excipients or stabilizers. Illustrative oils are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, or mineral oil. In general, water, saline, aqueous dextrose and related sugar solution, and glycols, such as propylene glycol or polyethylene glycol, are preferred liquid carriers, particularly for injectable solutions.
For use as aerosols, the compositions of the present invention in solution or suspension may be packaged in a pressurized aerosol container together with suitable propellants, for example, hydrocarbon propellants like propane, butane, or isobutane with conventional adjuvants. The compositions of the present invention also may be administered in a non-pressurized form such as in a nebulizer or atomizer.
The following examples are provided to illustrate embodiments of the present invention, but they are by no means intended to limit its scope.

EXAMPLES

Example 1

Remote Sensing by Elicitors

The discovery of the eliciting compounds of the present invention occurred primarily by defining the remote sensing system describe herein above. Past research demonstrated (a) that Trichoderma strains sense fungi that it can parasitize at a distance and grow tropically toward the target fungus (Chet et al., “Trichoderma Hamatum: Its Hyphal Interactions with Rhizoctonia Solani and Pythium spp,” Microb Ecol 7:29-38 (1981), which is hereby incorporated by reference in its entirety) and (b) that endochitinase, but not exochitinase (N-acetylglucosaminidase) is stimulated to be produced by Trichoderma spp. prior to contact with the fungus (Zeilinger et al., “Chitinase Gene Expression During Mycoparasitic Interaction of Trichoderma Harzianum with its Host,” Fung Genet Biol 26:131 -140 (1999), which is hereby incorporated by reference in its entirety). Both enzymes are important in the parasitic process along with various glucanases and other enzymes and antibiotics (Chet et al., “Mycoparasitism and Lytic Enzymes,” Trichoderma and Gliocladium 2:153-172 (1998); Schirmböck et al., “Parallel Formation and Synergism of Hydrolytic Enzymes and Peptaibol Antibiotics, Molecular Mechanisms Involved in the Antagonistic Action of Trichoderma Harzianum Against Phytopathogenic Fungi,” Appl Environ Microbiol 60:4364-4370 (1994), which are hereby incorporated by reference in their entirety).
To monitor chitinase expression during mycoparasitism of Trichoderma atroviride (formerly T. harzianum) strain P1 in situ, the strain was transformed with plasmids containing fusions of green fluorescent protein (“gfp”) to the 5-regulatory sequences of the T. atroviride nag1 gene (N-acetyl-beta-D-glucosaminidase-encoding) or ech42 (42-kDa endochitinase-encoding) genes. Confronting these strains with the target Rhizoctonia solani led to induction of gene expression before (ech42) or after (nag 1) physical contact. A 12-kDa cut-off membrane separating the two fungi abolished ech42 expression, indicating that macromolecules are involved in precontact activation. No ech42 expression was triggered by culture filtrates of R. solani or by placing T. atroviride onto plates previously colonized by R. solani. Instead, high expression occurred upon incubation of T. atroviride with the supernatant of R. solani cell walls digested with culture filtrates or purified endochitinase 42 (CHIT42, encoded by ech42) from T. atroviride. The chitinase inhibitor allosamidin blocked ech42 expression and reduced inhibition of R. solani growth during confrontation. The results indicate that ech42 is expressed before contact of T. atroviride with R. solani (Zeilinger et al., “Chitinase Gene Expression During Mycoparasitic Interaction of Trichoderma Harzianum with its Host,” Fung Genet Biol 26:131-140 (1999), which is hereby incorporated by reference in its entirety) and its induction is triggered by soluble materials of less than 3 kDa. Subsequent work has lead to the following description of the activation process.

1. T. atroviride, T. harzianum, and any other species of Trichoderma produce small quantities of particular enzymes constitutively. At least one of these may be an N-acetylgluocosaminidase (Brunner et al., “The Nag1 N-acetylglucosaminidase of Trichoderma Atroviride is Essential for Chitinase Induction by Chitin and of Major Relevance to Biocontrol,” Curr Genet 43:289-295 (2003), which is hereby incorporated by reference in its entirety).
2. When these enzymes come into contact with a suitable host fungus, certain small molecular compounds are released. These diffuse back to the Trichoderma strain, where they elicit responses. These responses presumably result from a cascade of events initiated by attaching to specific receptor sites on the surface of the Trichoderma strain. These small molecular weight compounds (preferably <3 kDa) are the elicitors of the present invention.

Example 2

Sources and Release of Elicitors

The ability of Trichoderma to release elicitor compounds from the various target fungi was measured by induction of gfp in the endochitinase promoter labeled P1 strain of Trichoderma. Trichoderma strain P1, transformed to contain an endochitinase promoter:gfp construct, was placed 15 mm or 5 mm distance from the elicitor compound source organism, and, finally in direct contact with various fungi and filamentous Oomycetes. The results are shown in FIG. 1.
In FIG. 1, the level of endochitinase induction is shown on the right hand axis. Gfp gene induction is measured as intensity of gfp fluorescence produced, with 0 being no fluorescence and 4 being the highest level of fluorescence. The graph provides information regarding the distance between the Trichoderma and the target fungus when gene induction (gfp fluorescence) was measured. The elicitor source organisms are shown on the horizontal axis. R(hizoctonia) solani and S(clerotinia) rolfsii are Basidomycetes. B(otrytis) cinerea, C(olletotrichum) acumatum, Penicillium spp., S(clerotium) sclerotiorum, A(lteraria) alternata and A. longipes are Ascomycetes. All of these organisms have primarily chitin and glucans in their cell walls. P(ythium) ultimum and P(hytophthora) infestans are Oomycetes and contain primarily cellulose and glucans. All of the chitin-containing fungi except Trichoderma itself and A. longipes released elicitors that enhanced endochitinase expression at a distance. This result demonstrates that the elicitation reaction between Trichoderma and target fungi is widespread, and not directly contact dependent. The activity demonstrated in FIG. 1 indicated suitable organisms for use in isolating the elicitor compounds of the present invention.
In subsequent experiments, the release of elicitors from cell walls of some of the same fungi and Oomycetes tested above using enzymes (endochitinase, glucanase, exochitinase, mixed cellulase) purified from T. atroviride was assessed. The results are shown in FIG. 2. Again, the elicitation abilities of the materials were assessed with Trichoderma strain P1 transformed to contain the endochitinase promoter:gfp construct. Fluorescence induction is shown on the left hand axis, with the elicitor source organisms on the horizontal axis: Trichoderma atroviride (Tricho), Botrytis cinerea (Botrytis); Pythium ultimum (Pythium), Rhizoctonia solani (Rhizoc) were the organisms tested. The results shown in FIG. 2 demonstrate that, with appropriate enzymes for the cell wall components tested, elicitors were released from all cell walls by enzymes from Trichoderma except when cell walls of Trichoderma itself were used.
Other materials, including extracts from various plants, colloidal chitin derived from crustacean sources, and fungal cells walls without enzyme action were also tested and found to lack elicitor activity, as shown in FIG. 2.

Example 3

Identification of the Elicitor Compounds

Samples with known elicitor activity were fractionated to contain only those compounds with a mass of 3 kDa or less. Compounds in these samples were subjected to high performance liquid chromatography and fractions with elicitor activity, measured with the T. atroviride:gfp system described in Example 1, were identified. These active fractions were then subjected to electrospray mass spectrometry and compounds were identified. FIG. 3 shows the active fractions and their identity. Compounds with elicitor activity are listed in herein above. In summary, the active molecules can be described generally as follows:

(diacetylchitobiose)_n, where n=1-5, and having either no additional amino acid moiety, or having one or more amino acid moieties (valine, L-ornthine, and/or serine) associated with, or attached thereto.
2. (diacetylchitobiose)_n, having a (dimeric hexose)_n, associated with or attached thereto, where n=1-5, and having either no additional amino acid moiety, or having one or more amino acid moieties (valine, L-ornithine, and/or serine) associated with, or attached thereto.

Example 4

Enhancement of Fungal Growth With Elicitors

One aspect of the present invention, the elicitors of the present invention are used to enhance growth of fungi. For example, Trichoderma, Aspergillus, and other fungi are widely used in the commercial production of enzymes for a variety of purposes (as a reference for a compilation of uses and technologies relating to Trichoderma for this purpose see Harman et al., “Enzymes, Biological Control and Commercial Applications,” Trichoderma and Gliocladium Vol. 2 (1998), which is hereby incorporated by reference in its entirety). This multi-billion dollar industry depends upon rapid growth of enzyme producing fungi, so an increase in growth of the fungi is a critical component. Moreover, another growing market for Trichoderma and other fungi is as biological control agents for plant disease and to enhance plant growth and yield (Harman, G. E., “Myths and Dogmas of Biocontrol,” Trichoderma Harzianum T-22 84:377-393 (2000), which is hereby incorporated by reference in its entirety). A method to specifically enhance growth of beneficial fungi is of great value in both markets.
As noted above, binding of elicitors to specific receptors results in a cascade of events. This leads to enhanced growth of fungi as demonstrated here. In FIG. 4, elicitor mixtures were added to the growth media of T. atroviride strain P1 to test the relative effect of the elicitor compounds of the present invention on fungal growth. FIG. 4 shows growth of T. atroviride strain P1 under the same conditions on medium amended with nothing (C), undiluted elicitor mixture (I), 10× diluted mixture (10), to 50× diluted mixture (50) and 100× diluted mixture (100). FIG. 4 clearly shows that the elicitors enhance fungal growth even at the low micromolar concentrations used in this demonstration.
For many applications, speed of spore germination and subsequent fungal growth also is extremely important. For example, in biocontrol applications, the biocontrol agent must occupy the potential court of infection before the pathogen grows. In enzyme applications, rate of growth is essential because reactor time is a major expense. The elicitors of the present invention dramatically speed fungal spore germination and germ tube growth as shown in FIGS. 5A-B.
FIG. 5A shows germ tubes from T. atroviride produced in the absence of any elicitors. FIG. 5B shows germ tubes from the same strain and under the same conditions in the presence of elicitors of the present invention. The dark bars were placed to give sizes of three germ tubes on each side as an aid to visualization of germ tube length. The small circular structure on each germ tube is the spore from which the germ tubes arose. As shown in FIG. 5B compared to FIG. 5A, the presence of the elicitors increased the rate and growth of germinating spores and germ tubes.

Example 5

Enhancement of Enzyme Production From Fungi

A major boon to the enzyme production industry would be a major increase in production efficiency. A major emphasis in the enzyme industry is the development of microbial strains that hyperproduce the product of interest and fermentation conditions optimized to give maximum production for elite strains. Moreover, a perennial problem is catabolite repression. Maximum growth of the producing organism usually occurs in highly nutritious media, but such media are also strongly inhibitory to enzyme production. The presence of glucose and similar materials, frequently together with high nitrogen levels, represses the enzymatic production machinery (Donzelli et al., “Enhanced Enzymatic Hydrolysis of Langostino Shell Chitin with Mixtures of Enzymes from Bacterial and Fungal Sources,” Carbon Res 338:1823-1833 (2003), which is hereby incorporated by reference in its entirety). Consequently, a substantial effort is made to alter strains in ways that abolish the catabolic repression systems (e.g., see Kubicek et al., “Regulatory Aspects of Formation and Secretion of Cellulases by Trichoderma Reesei,” Trichoderma reesei Cellulases, ed 81-102 (1990), which is hereby incorporated by reference in its entirety, and the discussion of Rut C30, which is depressed strain of T. reesei). Such genetic manipulation is time and resource intensive and may result in strains impaired in ways other than catabolic repression. Therefore, there is substantial advantage in overcoming catabolite repression for enzyme production without genetically modifying producing strains.
The graphs shown in FIGS. 6A-B demonstrate that the elicitors of the present invention enhance extracellular enzyme production and also permit high levels of enzymes to be produced even in the presence of catabolic repression.
FIG. 6A shows extracellular exochitinase activity from T. atroviride in minimal salts media with two different carbon sources. FIG. 6B shows endochitinase activity from T. atroviride under conditions described for FIG. 6A. The first three bars are in media without a carbon source, so fungal growth and, therefore, enzyme production, is very limited. In the next series of bars, the medium was augmented with glucose (gluc.), sucrose (sacc.) or cell walls from Botyrtis cinerea. Clearly, more enzyme was produced both under repressed (glucose) and derepressed (cell wall) conditions. However, with fungal cell walls, elicitors would be expected to be released by the activity of the enzymes released from T. atroviride so that the differences in enzyme activity with and without inducers are less than in the presence of glucose. The composition of the two inducers used is as follows: inducer 1 is a larger molecular weight fraction consisting of N-acetylglucosamine associated with dimeric hexose and amino acids, while inducer 2 is a smaller molecular fraction consisting of N-acetylglucosamine or dimeric hexose and the amino acids.
The time course of enzyme production was also investigated. In the presence of elicitors, even in the presence of cell walls, the same levels of enzymes are produced in about half the incubation time as in their absence. Thus, even though addition of fungal cell walls will result in release of elicitor molecules, and, therefore, increase the level of enzyme production, addition of the elicitor molecules at the start of the cultivation will result in more rapid production of enzymes.
Thus, the elicitors of the present invention (a) result in high levels of enzyme production even under repressive (catabolic repression caused by the presence of glucose), (b) result in higher levels of enzymes, and (c) increase the rate of production of enzymes. All of these features are of very high value to the enzyme industry.

Example 6

Control of Plant and Animal Diseases

Plant diseases cause substantial crop loss worldwide. Large quantities of pesticides are sold and applied to prevent these losses. Pesticides, however, are toxic to a range of organisms and may cause environmental pollution. Biological control of plant diseases is becoming an important method of disease control that is much more environmentally friendly. However, biological control tends to be more variable and less effective than the use of synthetic pesticides. Greater efficacy of biological control products can enhance the use of biological solutions to pest control and decrease the current over-reliance on chemical pesticides.
The present invention provides a means to increase biological control efficacy. FIGS. 7A-B demonstrate this improvement in efficacy of bean leaves treated with for biological control in the presence or absence of elicitors. The procedures used to test and assay these responses are described elsewhere (Woo et al., “Disruption of the ech42 (Endochitinase-encoding) Gene Affects Biocontrol Activity in Trichoderma Harzianum P1,” Molec Plant-Microbe Interact 12:419-429 (1999), which is hereby incorporated by reference in its entirety). Briefly, resistance was determined as earlier described (Woo et al., “Disruption of the ech42 (Endochitinase-encoding) Gene Affects Biocontrol Activity in Trichoderma Harzianum P1,” Molec Plant-Microbe Interact 12:419-429 (1999), which is hereby incorporated by reference in its entirety) by measuring the lesion size of at least three leaves different from the leaf into which the inducer was injected. The inducer was injected by using an insulin needle in one leaf main vase and left to act overnight before inoculation with B. cinerea. Micromolar levels of elicitors were used in these experiments.
FIGS. 7A-B show the appearance of lesions on bean leaves caused by B. cinerea following treatment with T. atroviride alone or with T. atroviride plus elicitors. Leaves were inoculated with droplets of water containing B. cinerea spores and then assessed to determine whether or not disease occurred, either as a necrotic (dead) spot or a less serious chlorotic (yellow) spot. FIG. 8 shows the level of decrease in disease by the various treatments. The inducer alone had little or no effect. Strain P1 reduced disease by about 40%, as did a treatment with P1 plus inactivated inducer. However, the combination resulted in about 70% reduction in disease. Thus, the elicitor/biocontrol combination provides a marked enhancement in biocontrol efficacy.
The graph shown in FIG. 8 indicates that a topical application of elicitors does not result in a decrease in disease. However, in other research, the elicitor was injected into plant leaves. This resulted in a decrease in disease in the assay noted above. This difference cannot be attributed to an increase in activity of the Trichoderma biocontrol agent since the agent was not applied to the leaves. Instead, the effect of the injected elicitor must be due to a change in the resistance of plants. Thus, the result of the injection demonstrates that elicitors increase the innate disease resistance of plants, the general physiological changes in plants required for such changes are a consequence of the elicitor-induced cascades of gene activation and protein expression as described earlier.
The improvement in disease control with the combination of the two treatments is attributable both to enhanced activity of the T. atroviride in the presence of the elicitor and to enhancement of the natural resistance mechanisms of the plant. This was confirmed by injection of elicitor into plants. The injection resulted in activation of plant resistance mechanisms.
It is anticipated that the elicitors described herein will also enhance resistance to disease in animals, including humans. This expectation is made more likely by consideration of U.S. Pat. No. 6,492,350, which is hereby incorporated by reference in its entirety. This patent uses chito-oligosaccharides, including chitobiose, as a method of treatment for viral disease, such as the common cold, and for alleviation of pain in humans. Chitobiose is one of the elicitors identified herein, but it is the least effective. It is likely that the other elicitors identified here will be more effective in this and similar human and animal applications than the simple chito-oligomers identified in U.S. Pat. No. 6,492,350.

Claims

1. A composition, which, when applied to an organism under conditions appropriate for activity, enhances: fungal growth, extracellular enzyme production under repressive or nonrepressive conditions by fungi, biological control of plant pathogens, and resistance to disease in plants and animals, wherein said composition comprises:

an elicitor compound selected from the group consisting of: (N,N′diacetylhexobiose)_n; (N,N′diacetylhexobiose)_nhaving one or more associated amino acid residues, wherein the amino acid residues comprise valine or ornthine; (N,N′diacetylhexosamine)_nhaving an attached (dihexobiose)_n; (N,N′diacetylhexosamine)_nhaving an associated (dihexobiose)_nand one or more associated amino acid residues, wherein the amino acid residues comprise valine or ornithine; or combinations thereof;

wherein (N,N′diacetylhexobiose)_nand (N,N′diacetylhexosamine)n comprise N-acetylglucosamine or other amino hexosamines; wherein (N,N′diacetylhexobiose)_nand (dihexobiose)_ncomprise any D-hexoaldose or a N-acetylamino derivative of D-hexoaldoses, wherein n=1 to 5, and wherein each of the elicitor compounds has a molecular weight of 3 kDa or less.

2. A composition, which, when applied to an organism under conditions appropriate for activity, enhances: fungal growth, extracellular enzyme production under repressive or nonrepressive conditions by fungi, biological control of plant pathogens, and resistance to disease in plants and animals, wherein said composition comprises:

an elicitor compound selected from the group consisting of: (N,N′diacetylchitobiose)_n; (N,N′diacetylchitobiose)_nhaving one or more associated amino acid residues, wherein the amino acid residues comprise valine or ornthine; (N,N′diacetylchitobiose)_nhaving an associated (dimeric hexose)_n; (N,N′diacetylchitobiose)_nhaving an associated (dimeric hexose)_nand one or more associated amino acid residues, wherein the amino acid residues comprise valine or ornthine; or combinations thereof;

wherein (N,N′diacetylchitobiose)_ncomprises N-acetylglucosamine or other amino hexosamines; wherein (dimeric hexose)_ncomprises any D-hexoaldose or N-acetylamino derivative of D-hexoaldose, wherein n=1 to 5, and wherein each of the elicitor compounds has a molecular weight of 3 kDa or less.

3. A composition, which, when applied to an organism under conditions appropriate for activity, enhances: fungal growth, extracellular enzyme production under repressive or nonrepressive conditions by fungi, biological control of plant pathogens, and resistance to disease in plants and animals, wherein said composition comprises:

an elicitor compound selected from the group consisting of: N,N′diacetylchitobiose; N,N′diacetylchitobiose having one or more associated amino acid residues, wherein the amino acid residues comprise valine or ornithine; N,N′diacetylchitobiose having an associated dimeric hexose; N,N′diacetylchitobiose having an associated dimeric hexose and one or more associated amino acid residues, wherein the amino acid residues comprise valine or ornithine; N,N′diacetylchitobiose having two associated dimeric hexoses; N,N′diacetylchitobiose having two associated dimeric hexoses and one or more associated amino acid residues, wherein the amino acid residues comprise valine or ornthine; two or more N,N′diacetylchitobiose residues having two or more associated dimeric hexoses; two or more N,N′diacetylchitobiose residues having two or more associated dimeric hexoses and one or more associated amino acid residues, wherein the amino acid residues comprise valine or ornithine; or combinations thereof;

wherein N,N′diacetylchitobiose comprises N-acetylglucosamine or other amino hexosamines; wherein dimeric hexose comprises any D-hexoaldose or N-acetylamino derivative of D-hexoaldose, wherein n=1 to 5, and wherein each of the elicitor compounds has a molecular weight of 3 kDa or less.

4. A method of increasing the rate of growth of fungi comprising:

applying the composition according to claims 1, 2, or 3 to fungi under conditions effective to increase growth of the fungi.

5. A method of increasing the production of extracellular enzymes from fungi comprising:

applying the composition according to claims 1, 2, or 3 to fungi under conditions effective to increase production of extracellular enzymes from the fungi.

6. A method of increasing the biological control of plant diseases comprising:

applying a fungal biocontrol organism and the composition according to claims 1, 2, or 3 to plants under conditions effective to increase the biological control of plant diseases.

7. A method of increasing resistance of plants to diseases comprising:

applying the composition according to claims 1, 2, or 3 to plants under conditions effective to increase the resistance of the plant to diseases.

8. A method of alleviating pain and increasing resistance to, or recovery from, diseases in an animal, including a human, comprising:

administering the composition according to claims 1, 2, or 3 under conditions effective to alleviate pain, increase resistance to, or recovery from, diseases in an animal.