MUSCODOR ALBUS STRAIN PRODUCING VOLATILE ORGANIC COMPOUNDS

AND METHODS OF USE

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No. 61/705,312, filed September 25, 2012, U.S. Application No. 13/843,755, filed March 15, 2013 and U.S.

Application No. 14/034,353, filed September 23, 2013, all of which are incorporated herein by reference in their entirety.

TECHNICAL FIELD

Disclosed herein is an isolated Muscodor albus strain producing volatile organic compounds (VOCs) as well as cultures of said strain and compositions, and metabolites derived from said strain or culture as well as methods of obtaining said compositions, metabolites and volatiles and their methods of use for controlling pests and phytopathogenic infection. BACKGROUND OF THE INVENTION

Natural products are substances produced by microbes, plants, and other organisms. Microbial natural products offer an abundant source of chemical diversity, and there is a long history of utilizing natural products for pharmaceutical purposes. Despite the emphasis on natural products for human therapeutics, where more than 50% are derived from natural products, only 11% of pesticides are derived from natural sources. Nevertheless, natural product pesticides have a potential to play an important role in controlling pests in both conventional and organic farms. Secondary metabolites produced by microbes (bacteria, actinomycetes and fungi) provide novel chemical compounds which can be used either alone or in combination with known compounds to effectively control insect pests and to reduce the risk for resistance development. In particular endophytic fungi and bacteria, fungi and bacteria living with the tissues of host plants, specifically on the intracellular spaces of plant tissues and coexist with their hosts without any pathogenic symptoms have been found to be a rich source of bioactive natural products.

There are several well-known examples of microbial natural products that are successful as agricultural insecticides (Thompson et al., 2000, Pest Management Science 56: 696-702; Arena et al, 1995, Journal of Parasitology 81: 286-294; Krieg et al. 1983, Z. Angew. Entomol. 96: 500-508). A number of fungal species are known to produce concentrations of volatile antibiotics (see, for example, Strobel, 2006, J. Ind. Microbiol. Biotechnol. 33: 514-22 for review).

Species of endophytic fungi, Muscodor have been disclosed, particularly, Muscodor albus strain CZ 620, Muscodor roseus A3-5 and Muscodor viigenus 2116 ( see, for example, Strobel, 2006, J. Ind. Microbiol. Biotechnol. 33: 514-22; Strobel, 2012, Microbiol. Today 39- 108-109; US Patent No. 6,911,338, US Patent No. 7,267,975). Volatiles produced by these Muscodor strains have been found to possess nematocidal, insecticidal, acaricidal, fungicidal and bactericidal activity (see, for example, Lacey et al., 2008, J. Invertebrate Pathology 97:159-164; Strobel, 2006, J. Ind. Microbiol. Biotechnol. 33: 514-22; Strobel, 2012, Microbiol. Today (May 2012 108-109; Riga et al., 2008, Biological Control 45:380-385; WO2010/132509; US Patent Nos. 6,911,338, 7,267,975, 7,754,203, 8,093,024).

It is an object to provide additional Muscodor strains that have enhanced beneficial biological activity.

SUMMARY OF THE INVENTION

Provided is an isolated Muscodor strain which

(a) produces a product, particularly volatile compounds including but not limited to small alcohols, esters, acids, ketones as well as hydrocarbon and particularly comprising at least one of 3-octanone,(-) aristolene, propanoic acid and/or an ester form, acetic acid ester and in particular, acetic acid, 2-methylpropyl ester and/or acetic acid, 2-phenylethyl ester;

(b) produces volatile compounds that possess fungicidal activity, wherein said culture

produces a product that has at least about 1.5 fold more inhibitory effect on Fusarium and particularly, Fusarium oxysporum, growth than Muscodor albus strain CZ 620;

(c) produces volatile compounds which possess nematicidal activity, wherein said culture produces a product that has at least about 4 fold more of an effect on mortality on Meloidogyne spp. than Muscodor albus strain CZ 620.

(d) produces volatile compounds which exhibit insecticidal activity and in particular with respect to army worm eggs.

In a related aspect, provided is (a) a substantially pure culture or whole cell broth comprising or (b) cell fraction, supernatant, substance, compound, metabolite or volatile derived from the Muscodor strain set forth above. Muscodor culture has at least one of the identifying characteristics of Muscodor albus strain SA-13 (NRRL Accession No. B-50774). In a more particular embodiment, the Muscodor culture or strain has all of the identifying characteristics of Muscodor albus strain SA-13 (NRRL Accession No. B-50774).

In a particular embodiment, provided is a composition comprising the substantially pure culture or whole cell broth comprising said strain or cell fraction, supernatant, substance, compound, metabolite or volatile derived from the said strain. In a specific embodiment the composition comprises a plurality of substances, compounds, metabolites and/or volatiles derived from the culture.

In a related aspect, a method is provided for identifying one or more volatile organic compounds produced by a Muscodor strain. In the method a volatile composition produced by the growing culture, such as the Muscodor strain set forth above, is captured by contacting a gas stream containing the volatile substance or substances with a material or phase capable of removing the volatiles from the gas stream and then recovering the volatiles for analyses. The method may further comprise capturing said volatiles on a nonionic resin that acts as a molecular weight exclusion vehicle and identifying compounds captured on said resin

In another aspect of the invention, a composition comprising a mixture containing the volatile organic compounds (VOCs) produced by the culture or strain and the use of such mixtures to control plant pathogens and infestations are disclosed. The composition may be a reconstituted mixture of products produced by said strain or may be an artificial mixture of VOCs.

In one embodiment, the composition comprises:

Ethanol;

Propanol;

2-Butanone, 4-hydroxy-;

Ethyl Acetate;

Propanoic acid, ethyl ester;

1-Butanol, 3-methyl-;

1-Butanol, 2-methyl-;

Propanoic acid, 2-methyl-, ethyl ester;

Butanoic acid, 2-methyl-, methyl ester;

Butanoic acid, 2-methyl-, ethyl ester;

Propanoic acid, 2-methyl-, butyl ester;

1-Butanol, 3-methyl-, acetate; Ethyl tiglate;

Phenylethyl Alcohol;

Azulene, 1,2,3, 5,6,7, 8, 8a-octahydro-l,4-dimethyl-7-(l- methylethenyl)-, [lS-(l.alpha.,7. alpha., 8a.beta.)]-.

And at least one of: Propanoic acid, 2-methyl-, methyl ester; Acetic acid, 2-methylpropyl ester;

1-Butanol, 2-methyl-, acetate; Propanoic acid, 2-methyl-, butyl ester; Benzene, methoxy-; 3-

Octanone; Propanoic acid, 2-methyl-, 3-methylbutyl ester; Acetic acid, 2-phenylethyl ester; (-) Aristolene; Cyclohexane, l-ethenyl-l-methyl-2,4-bis(l-methylethenyl)-; Azulene,

l,2,3,4,5,6,7,8-octahydro-l,4-dimethyl-7-(l-methylethenyl )-,(lS-(l.alpha.,4.alpha.,7.alpha.)]-;

Bicyclo[5.3.0]decane, 2-methylene-5-(l-methylvinyl)-8-methyl-;

and optionally a carrier, diluent or adjuvant.

In a specific embodiment, the composition comprises

Ethanol;

Propanol;

2- Butanone, 4-hydroxy-;

Ethyl Acetate;

Propanoic acid, 2-methyl-, methyl ester;

Propanoic acid, ethyl ester;

1-Butanol, 3-methyl-;

1-Butanol, 2-methyl-;

Propanoic acid, 2-methyl-, ethyl ester;

Acetic acid, 2-methylpropyl ester;

Butanoic acid, 2-methyl-, methyl ester;

Butanoic acid, 2-methyl-, ethyl ester;

Propanoic acid, 2-methyl-, butyl ester;

1-Butanol, 3-methyl-, acetate;

1-Butanol, 2-methyl-, acetate;

Propanoic acid, 2-methyl-, butyl ester;

Benzene, methoxy-;

Ethyl tiglate;

3- Octanone;

Propanoic acid, 2-methyl-, 3-methylbutyl ester;

Phenylethyl Alcohol;

Acetic acid, 2-phenylethyl ester;

(-)Aristolene;

Cyclohexane, 1 -ethenyl- 1 -methyl-2,4-bis( 1 -methylethenyl)- ;

Azulene, 1,2,3,4,5, 6,7, 8-octahydro-l, 4-dimethyl-7-(l- methylethenyl)-,(lS-(l.alpha.,4.alpha.,7. alpha.)]-;

Bicyclo [5.3.0] decane, 2-methylene-5- ( 1 -methy 1 vinyl)- 8-methyl- ;

and, Azulene, 1,2,3, 5,6,7, 8, 8a-octahydro-l,4-dimethyl-7-(l- methylethenyl)-, [lS-(l.alpha.,7. alpha., 8a.beta.)]-. and optionally a carrier, diluent or adjuvant.

Alternatively, the composition may comprise: ethanol; ethyl acetate; l-Propanol,2- methyl; Propanoic acid, 2-methyl-, methyl ester; 1-Butanol, 3-methyl; 1-Butanol, 2-methyl; and Propanoic acid, 2-methyl-, ethyl ester and optionally at least one of a carrier, diluent, surfactant, and adjuvant.

Further provided is a combination comprising (a) a first substance selected from the group consisting of (i) a substantially pure culture or whole cell broth comprising or (ii) cell fraction, supernatant, metabolite or volatile derived from the culture or Muscodor strain set forth above and (b) at least one of (i) a second substance, wherein said second substance is a chemical or biological pesticide and (ii) at least one of a carrier, diluent, surfactant, adjuvant. The combination may be a composition.

Also provided is a method for modulating pest infestation and/or phytopathogenic infection in a plant comprising applying to the plant and/or seeds, fruits, thereof and/or substrate, such as soil or hydroponic solution, used for growing said plant an amount of the compositions or artificial mixtures or combinations set forth above effective to modulate said pest infestation and/or phytopathogenic infection. The pest may be an insect pest, fungus, virus, bacteria, and nematode. Phytopathogenic infection may be caused by bacteria and/or fungus.

Also provided is a seed, particularly, a barley seed inoculated with said strain.

DESCRIPTION OF THE FIGURES

Figure 1 shows a 20 day old culture of SA-13 growing on a potato dextrose agar (PDA) medium.

Figure 2 shows a Scanning Electron Micrograph (SEM) of Muscodor albus as isolated from Prosopis glandulosa and particularly illustrates the intertwining hyphae.

Figure 3 shows a phylogenetic tree showing genetic relationships among Muscodor spp. The isolate SA-13 is included in the list in the upper right side of the diagram.

Figure 4 shows a chromatographic representation of VOCs produced by Muscodor albus CZ 620 as analyzed using SPME-GCMS.

Figure 5A shows a chromatographic representation of VOCs produced by Muscodor albus SA-13 as analyzed using SPME-GCMS.

Figure 5B shows overlayed chromatograms analyzed using SPME-GCMS of VOCs produced by M. albus SA-13 when grown in potato dextrose broth (PDB) medium. VOCs found in the headspace region are shown in the bottom graph and VOCs found in liquid medium are shown in the top graph.

Figure 6 shows overlayed chromatograms of VOCs produced by Muscodor albus CZ 620 and SA-13 as analyzed using SPME-GCMS.

Figure 7 is a schematic representation of the sampling process for capturing of VOCs produced by Muscodor albus using XAD7 resin.

Figure 8 shows GCMS analyses of XAD7 resin trapped VOCs produced by the M. albus

CZ 620 and SA13 grown on barley grains.

Figure 9 shows inhibition of mycelial growth of Aspergillus niger on PDA plates by Muscodor albus SA-13 (top plates) and growth of A. niger in control PDA plates (bottom plates). DETAILED DESCRIPTION

While the compositions and methods heretofore are susceptible to various modifications and alternative forms, exemplary embodiments will herein be described in detail. It should be understood, however, that there is no intent to limit the invention to the particular forms disclosed, but on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention as defined by the appended claims.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is included therein. Smaller ranges are also included. The upper and lower limits of these smaller ranges are also included therein, subject to any specifically excluded limit in the stated range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described.

It must be noted that as used herein and in the appended claims, the singular forms "a," "and" and "the" include plural references unless the context clearly dictates otherwise.

As defined herein, "derived from" means directly isolated or obtained from a particular source or alternatively having identifying characteristics of a substance or organism isolated or obtained from a particular source. In the event that the "source" is an organism, "derived from" means that it may be isolated or obtained from the organism itself or medium used to culture or grow said organism.

As defined herein, "whole broth culture" refers to a liquid culture containing both cells and media. If bacteria are grown on a plate the cells can be harvested in water or other liquid, whole culture.

The term "supernatant" refers to the liquid remaining when cells that are grown in broth or harvested in another liquid from an agar plate are removed by centrifugation, filtration, sedimentation, or other means well known in the art.

As defined herein, "filtrate" refers to liquid from a whole broth culture that has passed through a membrane.

As defined herein, "extract" refers to liquid substance removed from cells by a solvent (water, detergent, buffer, chemical such as acetone) and separated from the cells by

centrifugation, filtration or other method.

As defined herein, "metabolite" or "volatile" refers to a compound, substance or by product of a fermentation of a microorganism, or supernatant, filtrate, or extract obtained from a microorganism.

As defined herein, an "isolated compound" is essentially free of other compounds or substances, e.g. , at least about 20% pure, preferably at least about 40% pure, more preferably about 60% pure, even more preferably about 80% pure, most preferably about 90% pure, and even most preferably about 95% pure, as determined by analytical methods, including but not limited to chromatographic methods, electrophoretic methods.

A "carrier" as defined herein is an inert, organic or inorganic material, with which the active ingredient is mixed or formulated to facilitate its application to plant or other object to be treated, or its storage, transport and/or handling. The term "modulate" as defined herein is used to mean to alter the amount of pest infestation or rate of spread of pest infestation.

The term "pest infestation" as defined herein, is the presence of a pest in an amount that causes a harmful effect including a disease or infection in a host population or emergence of an undesired weed in a growth system.

A "pesticide" as defined herein, is a substance derived from a biological product or chemical substance that increases mortality or inhibits the growth rate of plant pests and includes but is not limited to nematicides, insecticides, plant fungicides, plant bactericides, and plant viricides.

Methods of Production

As noted above, compounds, metabolites or volatiles may be obtained, are obtainable or derived from an organism having one or more identifying characteristics of the Muscodor strain or culture set forth above. The methods comprise cultivating these organisms and obtaining the compounds and/or compositions of the present invention by isolating these compounds from the culture of these organisms. In particular, the organisms are cultivated in nutrient medium using methods known in the art. The organisms may be cultivated by shake or non-shake cultivation, small scale or large scale fermentation (including but not limited to continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentation apparatus performed in suitable medium and under conditions allowing cell growth or on solid substrates such as agar. The cultivation may take place in suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art. Suitable media are available or may be available from commercial sources or prepared according to published compositions. In a particular embodiment and as set forth in the examples, the Muscodor strain may be cultivated on agar media such as potato dextrose agar (PDA) (D. Ezra et al., 2004. Microbiology,

150:4023) or in various grain media such as barley grains by inoculating the grains with the PDA plugs grown with the strain.

After cultivation, a supernatant, filtrate, volatile and/or extract of or derived from said Muscodor strain (e.g., Muscodor albus SA-13) may be used in formulating a pesticidal composition. Alternatively, after cultivation, the compounds, volatiles and/or metabolites may be extracted from the culture broth.

The extract may be fractionated by chromatography. Chromatographic fractions may be assayed for toxic activity against, for example, fungi Fusarium or nematodes, such as a J2 nematode of Meloidogyne spp. using methods known in the art. This process may be repeated one or more times using the same or different chromatographic methods.

Compositions

Compositions may comprise whole broth cultures, liquid or solid cultures, or suspensions of a Muscodor strain, specifically a Muscodor strain having at least one of the identifying characteristics of Muscodor albus SA-13 strain, as well as supernatant, filtrate and/or extract or one or more and more particularly a plurality of (i) metabolites, (ii) isolated compounds or (iii) volatiles derived from Muscodor albus SA-13 strain of the foregoing which in particular have pesticidal and particularly fungicidal and/or nematicidal activity.

The compositions set forth above can be formulated in any manner. Non-limiting formulation examples include but are not limited to Dried grains such as barley, corn, rye, rice, and wheat, Emulsifiable concentrates (EC), Wettable powders (WP), Soluble liquids (SL), Aerosols, Ultra-low volume concentrate solutions (ULV), Soluble powders (SP),

Microencapsulation, Water dispersed granules (WDG), Flowables (FL), Microemulsions (ME), Nano-emulsions (NE), etc. In any formulation described herein, percent of the active ingredient is within a range of 0.01% to 99.99%.

The compositions may be in the form of a liquid, gel, solid, or biofumigant. A solid composition can be prepared by soaking a solid carrier in a solution of active ingredient(s) and drying the suspension under mild conditions, such as evaporation at room temperature or vacuum evaporation at 65°C or lower. A solid composition can also be dried grains grown with the said strain. The composition may additionally comprise a surfactant to be used for the purpose of emulsification, dispersion, wetting, spreading, integration, disintegration control, stabilization of active ingredients, and improvement of fluidity or rust inhibition. In a particular embodiment, the surfactant is a non-phytotoxic non-ionic surfactant which preferably belongs to EPA List 4B. In another particular embodiment, the nonionic surfactant is polyoxyethylene (20) monolaurate. The concentration of surfactants may range between 0.1-35% of the total formulation, preferred range is 5-25%. The choice of dispersing and emulsifying agents, such as non-ionic, anionic, amphoteric and cationic dispersing and emulsifying agents, and the amount employed is determined by the nature of the composition and the ability of the agent to facilitate the dispersion of the compositions of the present invention.

In an embodiment of the invention, a liquid composition may comprise at least one volatile organic compound produced by a Muscodor strain, which is capable of producing an acetic acid ester, and produces a product that possesses fungicidal, bacterial, nematicidal, and/or insecticidal activity. In a particular embodiment, the liquid compostion comprises acetic acid, 2- methylpropyl ester; 1-Butanol, 2-methyl-, acetate; and/or acetic acid, 2-phenylethyl ester. In yet another embodiment, the liquid composition comprises the following volatile compounds:

Ethanol

Ethyl acetate

1-propanol, 2-methyl

Butanal, 2-methyl

Propanoic acid, 2-methyl-, methyl ester

1-Butanol, 3-methyl-

1-Butanol, 2-methyl-

Acetic acid, 2-methylpropyl ester

1-Butanol, 3-methyl-, acetate

1- Butanol, 2-methyl-, acetate

4-Nonanone

2- Nonanone

Phenylethyl alcohol

Acetic acid, 2-phenylethyl ester

Cyclopeptane, 4-methylene- 1 -methyl-2- [2-methyl- 1 -propene- 1 -y ] - 1 -vinyl

Azulene, 1,2,3,5, 6,7,8, 8a-octahydro-l, 4-dimethyl-7-(l-methylethenyl)-, [1S- (l.alpha.,7. alpha., 8a.beta.)[-; and

lH-2-Benzopyran-l-one, 3,4-dihydro-8-hydroxy-3-methyl-, [R]-. The volatile organic compounds in the liquid composition may be produced by the Muscodor strain or may be synthetic compounds. In another embodiment, the volatile compounds in the liquid composition may be a reconstituted mixture of products produced by the strain or may be an artificial mixture of volatile organic compounds.

In another embodiment, the liquid composition may be a whole cell broth, or the cells of the Muscodor strain may have been removed. The liquid composition of the present invention may be produced by growing the Muscodor strain in a liquid medium and obtaining the liquid medium. The liquid medium may be any suitable liquid nutrient medium comprising carbon and nitrogen sources and inorganic salts. Suitable liquid media are available or may be available from commercial sources or prepared according to published compositions. In a particular embodiment, the Muscodor strain may be grown in liquid media such as potato dextrose broth (PDB).

In an embodiment of the invention, a biofumigant composition comprises a Muscodor strain and/or at least one volatile organic compound produced by the Muscodor strain. A fumigant is a chemical compound that is volatile at ambient temperatures and is often used to control pests in storage bins and buildings, and to control certain pests in the soil. A biofumigant is a fumigant that is produced from natural resources or by natural processes. Many fumigant compositions comprise liquids held in cans or tanks and often comprise mixtures of two or more gases. Alternatively, phosphine or hydrogen phosphide gas can be generated in the presence of moisture from a tablet made up of aluminum phosphide and ammonium carbonate. Fumigants generally more easily access sites that are not easily accessible to other chemicals, due to the penetration and dispersal of the gas. Commonly used fumigants include ethylene dichloride carbon tetrachloride (EDCT), methyl bromide, aluminum phosphide and hydrocyanic acid.

However, fumigants are often not just toxic to pests but may be harmful to the environment. Therefore, fumigants such as methyl bromide are being phased out. In contrast, a biofumigant composition of the present invention may be more advantageous because they are not toxic or pathogenic to humans and are not harmful to the environment.

The composition set forth above may be combined or used with another microorganism and/or pesticide (e.g., nematicide, bactericide, fungicide, insecticide). The microorganism may include but is not limited to an agent derived from Bacillus spp., Paecilomyces spp., Pasteuria spp. Pseudomonas spp., Brevabacillus spp., Lecanicillium spp., non-Ampelomyces spp., Pseudozyma spp., Streptomyces spp, Burkholderia spp, Trichoderma spp, Gliocladium spp. or other Muscodor strains. In a particular embodiment, the additional microorganism is a

Trichoderma spp. Alternatively, the agent may be a natural oil or oil-product having

nematicidal, fungicidal, bactericidal and/or insecticidal activity (e.g., paraffinic oil, tea tree oil, lemongrass oil, clove oil, cinnamon oil, citrus oil, rosemary oil, pyrethrum). The additional microganism, pesticide, or agent may be applied prior to, at the same time, or after application of the Muscodor strain and/or products derived therefrom.

Furthermore, the pesticide may be a single site anti-fungal agent which may include but is not limited to benzimidazole, a demethylation inhibitor (DMI) (e.g., imidazole, piperazine, pyrimidine, triazole), morpholine, hydroxypyrimidine, anilinopyrimidine, phosphorothiolate, quinone outside inhibitor, quinoline, dicarboximide, carboximide, phenylamide,

anilinopyrimidine, phenylpyrrole, aromatic hydrocarbon, cinnamic acid, hydroxyanilide, antibiotic, polyoxin, acylamine, phthalimide, benzenoid (xylylalanine), a demethylation inhibitor selected from the group consisting of imidazole, piperazine, pyrimidine and triazole (e.g.,bitertanol, myclobutanil, penconazole, propiconazole, triadimefon, bromuconazole, cyproconazole, diniconazole, fenbuconazole, hexaconazole, tebuconazole, tetraconazole), myclobutanil, and a quinone outside inhibitor (e.g., strobilurin). The strobilurin may include but is not limited to azoxystrobin, kresoxim-methoyl or trifloxystrobin. In yet another particular embodiment, the anti-fungal agent is a quinone, e.g., quinoxyfen (5,7-dichloro-4-quinolyl 4- fluorophenyl ether). The anti-fungal agent may also be derived from a Reynoutria extract.

The fungicide can also be a multi-site non-inorganic, chemical fungicide selected from the group consisting of chloronitrile, quinoxaline, sulphamide, phosphonate, phosphite, dithiocarbamate, chloralkythios, phenylpyridin-amine, and cyano-acetamide oxime.

As noted above, the composition may further comprise a nematicide. This nematicide may include but is not limited to chemicals such as organophosphates, carbamates, and fumigants, and microbial products such as avermectin, Myrothecium spp., Biome {Bacillus firmus), Pasteuria spp., Paecilomyces spp., and organic products such as saponins and plant oils.

In the case that the composition is applied to a seed, the composition may be applied to the seed as one or more coats prior to planting the seed using one or more seed coating agents including, but are not limited to, ethylene glycol, polyethylene glycol, chitosan, carboxymethyl chitosan, peat moss, resins and waxes or chemical fungicides or bactericides with either single site, multisite or unknown mode of action using methods known in the art.

The composition may be coated on to a conventional seed as noted above. In a particular embodiment, the compostions set forth above may be coated on a barly seed. The coated barley seed may further comprise protein based ingredients such as milk, whey protein, high protein based flour from e.g., rice or wheat to enhance thestorage life of said seeds. Alternatively, the composition may be coated on a genetically modified seed such as Liberty Link (Bayer

CropScience), Roundup Ready seeds (Monsanto), or other herbicide resistant seed, and/or seeds engineered to be insect resistant, or seeds that are "pyrimaded" with more than one genes for herbicide, disease, and insect resistance or other stress, such as drough, cold, salt resistance traits.

Uses

As noted above, the compositions set forth above may be applied using methods known in the art. Specifically, these compositions may be applied to and around plants or plant parts. Plants are to be understood as meaning in the present context all plants and plant populations such as desired and undesired wild plants or crop plants (including naturally occurring crop plants). Crop plants can be plants which can be obtained by conventional plant breeding and optimization methods or by biotechnological and genetic engineering methods or by

combinations of these methods, including the transgenic plants and including the plant cultivars protectable or not protectable by plant breeders' rights. Plant parts are to be understood as meaning all parts and organs of plants above and below the ground, such as shoot, leaf, flower and root, examples which may be mentioned being leaves, needles, stalks, stems, flowers, fruit bodies, fruits, seeds, roots, tubers and rhizomes. The plant parts also include, but are not limited to, harvested material, and vegetative and generative propagation material, for example cuttings, tubers, rhizomes, offshoots and seeds.

Plants that may be treated include but are not limited to: (A) Major edible food crops, which include but are not limited to (1) Cereals (e.g., African rice, barley, durum wheat, einkorn wheat, emmer wheat, finger millet, foxtail millet, hairy crabgrass, Indian barnyard millet, Japanese barnyard millet, maize, nance, oat, pearl millet, proso millet, rice, rye, sorghum, Sorghum spp., rye, spelt wheat); (2) Fruits (e.g., abiu, acerola, achacha, African mangosteen, alpine currant, ambarella, American gooseberry, American persimmon, apple, apricot, araza, Asian palmyra palm, Asian pear, atemoya, Australian desert raisin, avocado, azarole, babaco, bael, banana, Barbados gooseberry, bergamot, betel nut, bignay, bilberry, bilimbi, binjai, biriba, bitter orange, black chokeberry, black mulberry, black sapote, blackberry, blue-berried honeysuckle, borojo, breadfruit, murmese grape, button mangosteen, cacao, calamondin, canistel, cantaloupe, cape gooseberry, cashew nut, cassabanana, cempedak, charichuelo, cherimoya, cherry, cherry of the Rio Grande, cherry plum, Chinese hawthorn, Chinese white pear, chokeberry, citron, cocona, coconut, cocoplum, coffee, coffee Arabica, coffee robusta, Costa Rica pitahaya, currants, custard apple, date, date-plum, dog rose, dragonfruit, durian, elderberry, elephant apple, Ethiopian eggplant, European nettle tree, European wild apple, feijoa, fig, gac, genipapo, giant granadilla, gooseberry, goumi, grape, grapefruit, great morinda, greengage, guava, hardy kiwi, hog plum, horned melon, horse mango, Indian fig, Indian jujube, jabuticaba, jackberry, jackfruit, Japanese persimmon, Japanese wineberry, jocote, jujube, kaffir lime, karanda, kei apple, kepel apple, key lime, kitembilla, kiwi fruit, korlan, kubal vine, kuwini mango, kwai muk, langsat, large cranberry, lemon, Liberian coffee, longan, loquat, lychee, malay apple, mamey sapote, mammee apple, mango, mangosteen, maprang, marang, medlar, melon, Mirabelle plum, miracle fruit, monkey jack, moriche palm, mountain papaya, mountain soursop, mulberry, naranjilla, natal plum, northern highbush blueberry, olive, otaheite gooseberry, oval kumquat, papaya, para guava, passion fruit, pawpaw, peach, peach-palm, pear, pepino, pineapple, pitomba Eugenia luschnathiana, pitomba talisia esculenta, plantain, plum, pomegranate, pomelo, pulasan, purple chokeberry, quince, rambutan, ramontchi, raspberry, red chokeberry, red currant, red mulberry, red-fruited strawberry guava, rhubarb, rose apple, roselle, safou, salak, salmonberry, santol, sapodilla, satsuma, seagrape, soncoya,sour cherry, soursop, Spanish lime, Spanish tamarind, star apple, starfruit, strawberry, strawberry guava, strawberry tree, sugar apple, Surinam cherry, sweet briar, sweet granadilla, sweet lime, tamarillo, tamarind, tangerine, tomatillo, tucuma palm, Vaccinium spp., velvet apple, wampee, watermelon, watery rose apple, wax apple, white currant, white mulberry, white sapote, white star apple, wolfberry (Lyceum barbarum, L.chinense), yellow mombin, yellow pitaya, yellow-fruited strawberry, guava, (3) Vegetables (e.g., ackee, agate, air potato, Amaranthus spp., American groundnut, antroewa, armenian cucumber, arracacha, arrowleaf elephant ear, arrowroot, artichoke, ash gourd, asparagus, avocado, azuki bean, bambara groundnut, bamboo, banana, Barbados gooseberry, beet, beet root, bitter gourd, bitter vetch, bitterleaf, black mustard, black radish, black salsify, blanched celery, breadfruit, broad bean, broccoli, Brussels sprout, Buck's horn plantain, buttercup squash, butternut squash, cabbage, caigua, calabash, caraway seeds, carob, carrot, cassabanana, cassava, catjang, cauliflower, celeriac, celery, celtuce, chard, chayote, chickpea, chicory, chilacayote, chili pepper (Capsicum annuum, C. baccatum, C. chinense, C. frutescens, C. pubescens), Chinese cabbage, Chinese water chestnut, Chinese yam, chives, chufa sedge, cole crops, common bean, common purslane, corn salad, cowpea, cress, cucumber, cushaw pumpkin, drumstick tree, eddoe, eggplant, elephant foot yam, elephant garlic, endive, enset, Ethiopian eggplant, Florence fennel, fluted gourd, gac, garden rocket, garlic, geocarpa groundnut, Good King Henry, grass pea, groundnut, guar bean, horse gram, horseradish, hyacinth bean, ice plant, Indian fig, Indian spinach, ivy gourd, Jerusalem artichoke, jacamar, jute, kale, kohlrabi, konjac, kurrat, leek, lentil, lettuce, Lima bean, lotus, luffa, maca, maize, mangel-wurzel, mashua, moso bamboo, moth bean, mung bean, napa cabbage, neem, oca, okra, Oldham's bamboo, olive, onion, parsnip, pea, pigeon pea, plantain, pointed gourd, potato, pumpkins, squashes, quinoa, radish, rapeseed, red amaranth, rhubarb, ribbed gourd, rice bean, root parsley, runner bean, rutabaga, sago palm, salsify, scallion, sea kale, shallot, snake gourd, snow pea, sorrel, soybean, spilanthes, spinach, spinach beet, sweet potato, taro, tarwi, teasle gourd, tepary bean, tinda, tomato, tuberous pea, turnip, turnip-rooted chervil, urad bean, water caltrop trapa bicornis, water caltrop trapa natans, water morning slory, watercress, welsh onion, west African okra, west Indian gherkin, white goosefoot, white yam, winged bean, winter purslane, yacon, yam, yard-long bean, zucchini); (4) Food crops (e.g., abiu, acerola, achacha, ackee, African mangosteen, African rice, agate, air potato, alpine currant, Amaranthus spp.,

Ambarrella, American gooseberry, American groundnut, American persimmon, antroewa, apple, apricot, araza, Armenian cucumber, arracacha, arrowleaf elephant ear, arrowroot, artichoke, ash gourd, Asian palmyra palm, Asian pear, asparagus, atemoya, Australian desert raisin, avocado, azarole, azuki bean, babaco, bael, bambara groundnut, bamboo, banana, barbados gooseberry, barley, beet, beetroot, bergamot, betel nut, bignay, bilberry, bilimbi, binjai, biriba, bitter gourd, bitter orange, bitter vetch, bitterleaf, black chokeberry, black currant, black mulberry, black mustard, black radish, black salsify, black sapote, blackberry, blanched celery, blue-berried honeysuckle, borojo, breadfruit, broad bean, broccoli, Brussels sprout, Buck's horn plantain, buckwheat, Burmese grape, buttercup squash, butternut squash, button mangosteen, cabbage, cacao, caigua, calabash, calamondin, canistel, cantaloupe, cape gooseberry, caraway seeds, carob, carrot, cashew nut, cassava, catjang, cauliflower, celeriac, celery, celtuce, cempedak, chard, charichuelo, chayote, cherimoya, cherry, cherry of the Rio Grande, cherry plum, chickpea, chicory, chilacayote, chili pepper (Capsicum annuum, C. baccatum, C. chinense, C. frutescens, C. pubescens), Chinese cabbage, Chinese hawthorn, Chinese water chestnut, Chinese white pear, Chinese yam, chives, chokeberry, chufa sedge, citron, cocona, coconut, cocoplum, coffee, coffee (Arabica and Robusta types), cole crops, common bean, common purslane, corn salad, Costa Rica pitahaya, cowpea, cress, cucumber, currants, cushaw pumpkin, custard apple, date, date- plum, dog rose, dragonfruit, drumstick tree, durian, durum wheat, eddoe, eggplant, einkorn wheat, elderberry, elephant apple, elephant foot yam, elephant garlic, emmer wheat, endive, enset, Ethiopian eggplant, European nettle tree, European wild apple, feijoa, fig, finger millet, Florence fennel, fluted gourd, foxtail millet, gac, garden rocket, garlic, genipapo, geocarpa groundnut, giant granadilla, good king henry, gooseberry, goumi, grape, grapefruit, grass pea, great morinda, greengage, groundnut, grumichama, guar bean, guava, hairy crabgrass, hardy kiwi, hog plum, horned melon, horse gram, horse mango, horseradish, hyacinth bean, iceplant, Indian barnyard millet, Indian fig, Indian jujube, Indian spinach, ivy gourd, jabuticaba, jackalberry, jackfruit, jambul, Japanese barnyard millet, Japanese persimmon, Japanese wineberry, Jerusalem artichoke, jocote, jujube, jute, kaffir lime, kale, karanda, kei apple, kepel apple, key lime, kitembilla, kiwifruit, kohlrabi, konjac, korlan, kubal vine, kurrat, kuwini mango, kwai muk, langsat, large cranberry, leek, lemon, lentil, lettuce, Liberian coffee, lima bean, longan, loquat, lotus, luffa, lychee, maca, maize, malay apple, mamey saptoe, mammee apple, mangel-wurzel, mango, mangosteen, maprang, marang, mashua, medlar, melon, Mirabelle plum, miracle fruit, monk fruit, monkey jack, moriche palm, moso bamboo, moth bean, mountain papaya, mountain soursop, mulberry, mung bean, mushrooms, nance, napa cabbage, naranjilla, natal plum, neem, northern highbush blueberry, oat, oca, oil palm, okra, old man's bamboo, olive, onion, orange, otaheite gooseberry, oval kumquat, papaya, para guava, parsnip, passionfruit, pawpaw, pea, peach, peach-palm, pear, pearl millet, pepino, pigeon pea, pineapple, Pitomba (Eugenia luschnathiana, Talisia esculenta), plantain, plum, pointed gourd,

pomegranate, pomelo, potato, proso millet, pulasan, pumpkins and squashes, purple chokeberry, quince, quinoa, radish, rambutan, ramontchi, rapeseed, raspberry, red amaranth, red chokeberry, red currant, red mulberry, red-fruited strawberry guava, rhubarb, ribbed gourd, rice, rice bean, root parsley, rose apple, roselle, runner bean, rutabaga, rye, safou, sago palm, salak,

salmonberry, salsify, santol, sapodilla, Satsuma, scallion, sea kale, seagrape, shallot, snake gourd, snow pea, soncoya, sorghum, Sorghum spp., sorrel, sour cherry, soursop, soybean, Spanish lime, Spanish tamarind, spelt wheat, spilanthes, spinach, spinach beet, star apple, starfruit, strawberry, strawberry guava, strawberry tree, sugar apple, sugar beet, sugarcane, Surinam cherry, sweet briar, sweet granadilla, sweet lime, sweet potato, tamarillo, tamarind, tangerine, taro, tarwi, teasle gourd, tef, tepary bean, tinda, tomatillo, tomato, tuberous pea, tucuma palm, turnip, turnip-rooted chervil, urad bean, Vaccinium spp., velvet apple, wampee, water caltrop (Trapa bicornis, T. natans), water morning glory, watercress, watermelon, watery rose apple, wax apple, welsh onion, west African okra, west Indian gherkin, wheat, white currant, white goosefoot, white mulberry, white sapote, white star apple, white yam, winged bean, winter purslane, wolfberry (Lycium barbarum, L. chinense), yacon, yam, yangmei, yard- long bean, yellow mombin, yellow pitaya, yellow-fruited strawberry guava, zucchini; (B) Other edible crops, which includes but is not limited to (1) Herbs (e.g., Absinthium, alexanders, basil, bay laurel, betel nut, camomile, chervil, chili pepper (Capsicum annuum, C. baccatum, C chinense, C. frutescens, C. pubescens), chili peppers, chives, cicely, common rue, common thyme, coriander, cress, culantro, curly leaf parsley, dill, epazote, fennel, flat leaf parsley, ginseng, gray santolina, herb hyssop, holy basil,hop, jasmine, kaffir lime, lavender, lemon balm, lemon basil, lemon grass, lovage, marjoram, mint, oregano, parsley, peppermint, perilla, pot marigold, rooibos, rosemary, sage, shiny-leaft buckthorn, sorrel, spearmint, summer savory, tarragon, Thai basil, valerian, watercress, wild betel, winter savory, yerba mate); (2) Spices (e.g., ajowan, allspice, anise, bay laurel, black cardamom, black mustard, black pepper, caper, caraway seeds, cardamom, chili pepper (Capsicum annuum, C. baccatum, C. chinense, C. frutescens, C. pubescens), chili peppers, cinnamon, clove, common juniper, coriander, cumin, fennel, fenugreek, garlic, ginger, kaffir lime, liquorice, nutmeg, oregano, pandan, parsley, saffron, star anise, turmeric, vanilla, white mustard); (2) Medicinal plants (e.g., absinthium, alfalfa, aloe vera, anise, artichoke, basil, bay laurel, betel leat, betel nut, bilberry, black cardamom, black mustard, black pepper, blue gum, borojo, chamomile, caper, cardamom, castor bean, chili peppers, Chinese yam, chives, cola nut, common jasmine, common lavender, common myrrh, common rue, cilantro, cumin, dill, dog rose, epazote, fennel, fenugreek, gac, garlic, ginger, gray santolina, gum Arabic, herb hyssop, holy basil, horseradish, incense tree, lavender, lemon grass, liquorice, lovage, marijuana, marjoram, monk fruit, neem, opium, oregano, peppermint, pot marigold, quinine, red acacia, red currant, rooibos, safflower, sage, shiny-leaf buckthorn, sorrel, spilanthes, star anise, tarragon, tea, turmeric, valerian, velvet bean, watercress, white mustard, white sapote, wild betel, wolfberry (Lycium barbarum, L. chinense), yerba mate); (3) Stimulants (e.g., betel leaf, betel nut, cacao, chili pepper (Capsicum annuum, C. baccatum, C. chinense, C. frutescens, C. pubescens), chili peppers, coffee, coffee (Arabica, Robusta), cola nut, khat, Liberian coffee, tea, tobacco, wild betel, yerba mate); (4) Nuts (e.g., almond, betel nut, Brazil nut, cashew nut, chestnut, Chinese water chestnut, coconut, cola nut, common walnut, groundnut, hazelnut, Japanese stone oak, macadamia, nutmeg, paradise nut, pecan nut, pistachio nut, walnut); (5) Edible seeds (e.g., black pepper, Brazil nut, chilacayote, cola nut, fluted gourd, lotus, opium, quinoa, sesame, sunflower, water caltrop (Trapa bicornis, T. natans ); (6) Vegetable oils (e.g., black mustard, camelina, castor bean, coconut, cotton, linseed, maize, neem, Niger seed, oil palm, olive, opium, rapeseed, safflower, sesame, soybean, sunflower, tung tree, turnip); (7) Sugar crops (e.g., Asian palmyra palm, silver date palm, sorghum, sugar beet, sugarcane); (8) Pseudocereals (e.g., Amaranthus spp., buckwheat, quinoa, red amaranth); (9) Aphrodisiacs (e.g., borojo, celery, durian, garden rocket, ginseng, maca, red acacia, velvet bean); (C) Non food categories, including but not limited to (1) forage and dodder crops (e.g., agate, alfalfa, beet, broad bean, camelina, catjang, grass pea, guar bean, horse gram, Indian barnyard millet, Japanese barnyard millet, lespedeza, lupine, maize, mangel-wurzel, mulberry, Niger seed, rapeseed, rice bean, rye); (2) Fiber crops (e.g., coconut, cotton, fique, hemp, henequen, jute, kapok, kenaf, linseed, manila hemp, New Zealand flax, ramie, roselle, sisal, white mulberry); (3) Energy crops (e.g., blue gum, camelina, cassava, maize, rapeseed, sorghum, soybean, Sudan grass, sugar beet, sugarcane, wheat); (4) Alcohol production (e.g., barley, plum, potato, sugarcane, wheat, sorghum); (5) Dye crops (e.g., chay root, henna, indigo, old fustic, safflower, saffron, turmeric); (6) Essential oils (e.g., allspice, bergamot, bitter orange, blue gum, camomile, citronella, clove, common jasmine, common juniper, common lavender, common myrrh, field mint, freesia, gray santolina, herb hyssop, holy basil, incense tree, jasmine, lavender, lemon, marigold, mint, orange, peppermint, pot marigold, spearmint, ylang-ylang tree); (6) Green manures (e.g., alfalfa, clover, lacy Phacelia, sunn hemp, trefoil, velvet bean, vetch); (7) Erosion prevention (e.g., bamboo, cocoplum); (8) Soil improvement (e.g., lupine, vetch); (9) Cover crops (e.g., Alfalfa, lacy Phacelia, radish); (10) Botanical pesticides (e.g., jicama, marigold, neem, pyrethrum); (11) Cut flowers (e.g., carnation, chrysanthemum, daffodil, dahlia, freesia, gerbera, marigold, rose, sunflower, tulip); (12) Ornamental plants (e.g., African mangosteen, aloe vera, alpine currant, aster, black chokeberry, breadfruit, calamondin, carnation, cassabanana, castor bean, cherry plum, chokeberry, chrysanthemum, cocoplum, common lavender, crocus, daffodil, dahlia, freesia, gerbera, hyacinth, Japanese stone oak, Jasmine, lacy Phacelia, lotus, lupine, marigold, New Zealand flax, opium, purple chokeberry, ramie, red chokeberry, rose, sunflower, tulip, white mulberry); (D) Trees which include but are not limited to abelia, almond, apple, apricot, arborvitae nigra American, arborvitae, ash, aspen, azalea, bald cypress, beautybush, beech, birch, black tupelo, blackberry, blueberry, boxwood, buckeye, butterfly bush, butternut, camellia, catalpa, cedar, cherry, chestnut, coffee tree, crab trees, crabapple, crape myrtle, cypress, dogwood, Douglas fir, ebony, elder American, elm, fir, forsythia, ginkgo,

goldenraintree, hackberry, hawthorn, hazelnut, hemlock, hickory, holly, honey locust, horse chestnut, hydrangea, juniper, lilac, linden, magnolia, maple, mock orange, mountain ash, oak, olive, peach, pear, pecan, pine, pistachio, plane tree, plum, poplar, pivet, raspberry, redbud, red cedar, redwood, rhododendron, rose-of-Sharon, sassafras, sequoia, serviceberry, smoke tree, soapberry, sourwood, spruce, strawberry tree, sweet shrub, sycamore, tulip tree, ciborium, walnut, weasel, willow, winterberry, witch-hazel, zelkova; (E) Turf, which includes but is not limited to Kentucky bluegrass, tall fescue, Bermuda grass, zoysia grass, perennial ryegrass, fine fescues (e.g. creeping red, chewings, hard, or sheep fescue).

Treatment of the plants and plant parts with the compositions set forth above may be carried out directly or by allowing the compositions to act on their surroundings, habitat or storage space by, for example, immersion, coating, dipping, spraying, evaporation, fogging, scattering, painting on, injecting.

The compositions may also be applied to the soil using methods known in the art. These include but are not limited to (a) drip irrigation or chemigation; (b) soil incorporation; (c) seed treatment.

The compositions, cultures, supernatants, metabolites and pesticidal compounds set forth above may be used as pesticides and in particular, may be used as insecticides, nematicides, fungicides and bactericides, alone or in combination with one or more pesticidal substances set forth above and applied to plants, plant parts, substrate for growing plants or seeds set forth above.

The compositions, cultures, supernatants, metabolites and pesticidal compounds set forth above may be combined with other enhancing compounds for the said compositions such as, but not limited to, amino acids, chitosan, chitin, starch, hormones, minerals, synergistic microbes to increase efficacy and promote benefits to plants.

Specifically, nematodes that may be controlled using the method set forth above include but are not limited to parasitic nematodes such as root-knot, reniform, cyst, and lesion

nematodes, including but not limited to Aphelenchoides spp., Belonolaimus spp.,

Bursaphalenchus spp., Criconema spp. Globodera spp., Meloidogyne spp., Tylenchorhynchus spp., Helicotylenchus spp., Heterodem spp., Hoplolaimus spp., Pratylenchus spp., Rotylenchulus spp., Trichodorus spp., and Xiphinema spp. In particular, the parasitic nematodes may include but are not limited to seed gall nematodes (Afrina wevelli), bentgrass nematodes (Anguina agrostis), shoot gall nematodes (Anguina spp.), seed gall nematodes (Anguina spp., A.

amsinckiae, A. balsamophila; A. tritici), fescue leaf gall nematodes (A. graminis), ear-cockle (or wheat gall) nematodes (Anguina tritici), bud and leaf (or foliar) nematodes (Aphelenchoides spp., A. subtenuis), begonia leaf (or fern, or spring crimp, or strawberry foliar, or strawberry nematodes, or summer dwarf) nematodes (A. fragariae), fern nematodes (A. olesistus), rice nematodes (A. oryzae), currant nematodes (A. ribes), black currant (or chrysanthemum) nematodes (A. ritzemabosi), chrysanthemum foliar or leaf nematodes (A. ritzemabosi), rice white-tip (or spring dwarf, or strawberry bud) nematodes ( A. besseyi), fungus-feeding

(mushroom) nematodes (Aphelenchoides composticola), Atalodera spp. (Atalodera lonicerae, Atalodera ucri), spine nematodes (Bakernema variabile), sting nematodes (Belonolaimus spp., B. gracilis, B. longicaudatus), pine wood nematodes (Bursaphalenchus spp., B. xylophilus, B.

mucronatus), sessile nematodes (Cacopaurus spp., C. epacris, C.pestis), amaranth cyst nematodes (Cactodera amaranthi), birch cyst nematodes (C betulae), cactus cyst nematodes (C. cacti), estonian cyst nematodes (C estonica), Thome's cyst nematodes (C. thornei), knotweed cyst nematodes (C weissi), ring nematodes (Criconema spp.), spine nematodes (Criconema spp., C. civellae, C. decalineatum, Cspinalineatum), ring nematodes (Criconemella axeste, C curvata, C macrodora, C parva), ring nematodes (Criconemoides spp., C citri, C. simile), spine nematodes (Crossonemafimbriatum), eucalypt cystoid nematodes (Cryphodera eucalypti), bud, stem and bulb nematodes (Ditylenchus spp., D. angustus, D. dipsaci, D.

destructor, D. intermedius), Mushroom spawn nematodes (D. myceliophagus), awl nematodes (Dolichodorus spp., D. heterocephalus, D. heterocephalous), spear nematodes (Dorylaimus spp.), stunt nematodes (Geocenamus superbus), cyst nematodes (Globodera spp.), yarrow cyst nematodes (G. achilleae), milfoil cyst nematodes (G. millefolii), apple cyst nematodes (G. mall), white cyst potato nematodes (G. pallida), golden nematodes (G. rostochiensis), tobacco cyst nematodes (G. tabacum), Osborne's cyst nematodes (G. tabacum solanacearum), horsenettle cyst nematodes (G. tabacum virginiae), pin nematodes (Gracilacus spp., G. idalimus), spiral nematodes (Helicotylenchus spp., H. africanus, H. digonicus, H. dihystera, H. erythrinae, H. multicinctus , H. paragirus, H. pseudorobustus, H. solani, H. spicaudatus), sheathoid nematodes (Hemicriconemoides spp., H. biformis, H. calif ornianus, H chitwoodi, H. floridensis, H.

wessoni), sheath nematodes (Hemicycliophora spp., H. arenaria, H. biosphaera, H.

megalodiscus, H. parvana, H. poranga, H sheri, H similis, H striatula), cyst nematodes (Heterodera spp.), almond cyst nematodes (H. amygdali), oat (or cereal) cyst nematodes (H. avenae), Cajanus (or pigeon pea) cyst nematodes (H. cajani), Bermuda grass (or heart-shaped, or Valentine) cyst nematodes (H. cardiolata), carrot cyst nematodes (H. carotae), cabbage cyst nematodes or brassica root eelworm (H.cruciferae), nutgrass (or sedge) cyst nematodes (H.

cyperi), Japanese cyst nematodes (H. elachista), fig (or ficus, or rubber) cyst nematodes (H. fici), galeopsis cyst nematodes (H. galeopsidis), soybean cyst nematodes (H. glycines), alfalfa root (or pea cyst) nematodes (H. goettingiana), buckwheat cyst nematodes (H. graduni), barley cyst nematodes (H. hordecalis), hop cyst nematodes (H. humuli), Mediterranean cereal (or wheat) cyst nematodes (H. latipons), lespedeza cyst nematodes (H. lespedezae), Kansas cyst nematodes (H. longicolla), cereals root eelworm or oat cyst nematodes (H. major), grass cyst nematodes (H. rnani), lucerne cyst nematodes (H. medicaginis), cyperus (or motha) cyst nematodes (Heterodera mothi), rice cyst nematodes (H. oryzae), Amu-Darya (or camel thorn cyst) nematodes (H.

oxiana), dock cyst nematodes (H. rosii), rumex cyst nemtodes (H. rumicis), sugar beet cyst nematodes (H. schachtii), willow cyst nematodes (H. salixophila), knawel cyst nematodes (H. scleranthii), sowthistle cyst nematodes (H. sonchophila), tadzhik cyst nematodes (H.

tadshikistanica), turkmen cyst nematodes (H. turcomanica), clover cyst nematodes (H. trifolii), nettle cyst nematodes (H. urticae), Ustinov cyst nematodes (H. ustinovi), cowpea cyst nematodes (H. vigni), corn cyst nematodes (H. zeae), rice root nematodes (Hirschmanniella spp., H. belli, H. caudacrena, H. gracilis, H.oryzae), lance nematodes (Hoplolaimus spp.), Columbia nematodes (H. columbus), Cobb's lance nematodes (H. galeatus), crown-headed lance nematodes (H. tylenchiformis), pseudo root-knot nematodes (Hypsoperine graminis), needle nematodes

(Longidorus spp., L. africanus, L. sylphus), ring nematodes (Macroposthonia (=Mesocriconema) xenoplax), cystoid nematodes (Meloidodera spp.), pine cystoid nematodes (M. floridensis), tadzhik cystoid nematodes (M. tadshikistanica), cystoid body nematodes (Meloidoderita spp.), stunt nematodes (Merlinius spp., M. brevidens, M. conicus, M. grandis, M. microdorus), root- knot nematodes (Meloidogyne spp., M. acronea, M. arenaria, M.artiellia, M. brevicauda, M. camelliae, M. carolinensis, M. chitwoodi, M. exigua, M. graminicola, M. hapla, M. hispanica, M. incognita, M. incognita acrita, M. indica, M. inornata, M. javanica, M. kikuyuensis, M.

konaensis, M. mali, M. microtyla, M. naasi, M. ovalis, M. platani, M. querciana, M. sasseri, M. tadshikistanica, M. thamesi), knapweed nematodes (Mesoanguina picridis), Douglas fir nematodes (Nacobbodera chitwoodi), false root-knot nematodes (Nacobbus aberrans, N.

batatiformis , N. dorsalis), sour paste nematodes (Panagrellus redivivus), beer nematodes (P. silusiae), needle nematodes (Paralongidorus microlaimus), spiral nematodes (Pararotylenchus spp.), stubby-root nematodes (Paratrichodorus allius, P. minor, P. porosus, P. renifer), pin nematodes (Paratylenchus spp., P. baldaccii, P. bukowinensis, P. curvitatus, P. dianthus, P. elachistus, P. hamatus, P. holdemani, P. italiensis, P. lepidus, P. nanus, P. neoamplycephalus, P. similis), lesion (or meadow) nematodes (Pratylenchus spp., P. alleni, P. brachyurus, P. cojfeae, P. convallariae, P. crenatus, P.flakkensis, P. goodeyi, P. hexincisus, P. leiocephalus, P. minyus, P. musicola, P. neglectus, P.penetrans, P. pratensis, P. scribneri, P. thornei, P. vulnus, P. zeae), stem gall nematodes (Ptewtylenchus cecidogenus), grass cyst nematodes (Punctodera punctate), stunt nematodes (Quinisulcius acutus, Q. capitatus), burrowing nematodes (Radopholus spp.), banana-root nematodes (R. similis), rice-root nematodes (R. oryzae), red ring (or coconut, or cocopalm) nematodes (Rhadinaphelenchus cocophilus), reniform nematodes (Rotylenchulus spp., R. reniformis, R. parvus), spiral nematodes (Rotylenchus spp., R. buxophilus, R. christiei, R. robustus), Thome's lance nematodes (R. uniformis), Sarisodera hydrophylla, spiral nematodes (Scutellonema spp., S. blaberum, S. brachyurum, S. bradys, S. clathricaudatum, S. christiei, S. conicephalum), grass root-gall nematodes (Subanguina radicicola), round cystoid nematodes (Thecavermiculatus andinus), stubby-root nematodes (Trichodorus spp., T. christiei, T.

kurumeensis, T. pachydermis, T. primitivus), vinegar eels (or nematodes) (Turbatrix aceti), stunt (or stylet) nematodes (Tylenchorhynchus spp., T. agri, T. annulatus, T. aspericutis, T. claytoni, T.ebriensis, T. elegans, T. golden, T.graciliformis, T. martini, T. mashhoodi, T. microconus, T. nudus, T. oleraceae, T. penniseti, T. punensis), citrus nematodes (Tylenchulus semipenetrans), dagger nematodes (Xiphinema spp., X. americanum, X. bakeri, X.brasiliense, X. brevicolle, X. chambersi, X. coxi, X. diver sicaudatum X. index, X. insigne, X. nigeriense, X. radicicola, X.

setariae, X. vulgarae, X. vuittenezi). In a particular embodiment nematodes controlled are member of the Meloidogyne spp, particularly, M. hapla or M. incognita.

Phytopathogenic insects controlled by the method set forth above include but are not limited to non-Culicidae larvae insects from the order (a) Lepidoptera, for example, Acleris spp., Adoxophyes spp., Aegeria spp., Agrotis spp., Alabama argillaceae, Amylois spp., Anticarsia gemmatalis, Archips spp., Argyrotaenia spp., Autographa spp., Busseolafusca, Cadra cautella, Carposina nipponensis, Chilo spp., Choristoneura spp., Clysia ambiguella, Cnaphalocrocis spp., Cnephasia spp., Cochylis spp., Coleophora spp., Crocidolomia binotalis, Cryptophlebia leucotreta, Cydia spp., Diatraea spp., Diparopsis castanea, Earias spp., Ephestia spp., Eucosma spp., Eupoecilia ambiguella, Euproctis spp., Euxoa spp., Grapholita spp., Hedya nubiferana, Heliothis spp., Hellula undalis, Hyphantria cunea, Keiferia lycopersicella, Leucoptera scitella, Lithocollethis spp., Lobesia botrana, Lymantria spp., Lyonetia spp., Malacosoma spp.,

Mamestra brassicae, Manduca sexta, Operophtera spp., Ostrinia nubilalis, Pammene spp., Pandemis spp., Panolis flammea, Pectinophora gossypiella, Phthorimaea operculella, Pieris rapae, Pieris spp., Plutella xylo Stella, Prays spp., Scirpophaga spp., Sesamia spp., Sparganothis spp., Spodoptera spp., Synanthedon spp., Thaumetopoea spp., Tortrix spp., Trichoplusia ni and Yponomeuta spp.; (b) Coleoptera, for example, Agriotes spp., Alphitobius sp., Anomola spp., e.g., Anomala orientalis, Anthonomus spp., Atomaria linearis, Chaetocnema tibialis, Cosmopolites spp., Curculio spp., Cyclocephala spp., e.g., Cyclocephala lurida, Dermestes spp., Diabrotica spp., Epilachna spp., Eremnus spp., Leptinotarsa decemlineata, Lissorhoptrus spp., Melolontha spp., Orycaephilus spp., Otiorhynchus spp., Otiorhynchus sulcatus, Phlyctinus spp., Popillia spp., e.g., Popilla japonica, Psylliodes spp., Rhizopertha spp., e.g., Rhizotrogus majalis,

Sitophilus spp., Sitotroga spp., Tenebrio spp., Tribolium spp. and Trogoderma spp.; (c)

Orthoptera, for example, Blatta spp., Blattella spp., Gryllotalpa spp., Leucophaea maderae, Locusta spp., Periplaneta spp. and Schistocerca spp.; (d) Isoptera, for example, Reticulitermes spp.; (e) Psocoptera, for example, Liposcelis spp.; (f) Anoplura, for example, Haematopinus spp., Linognathus spp., Pediculus spp., Pemphigus spp. and Phylloxera spp.; (g) Mallophaga, for example, Damalinea spp. and Trichodectes spp.; (h) Thysanoptera, for example, Frankliniella spp., Hercinotnrips spp., Taeniothrips spp., Thrips palmi, Thrips tabaci and Scirtothrips aurantii; (i) Hemiptera, for example, Cimex spp., Distantiella theobroma, Dysdercus spp., Euchistus spp., Eurygaster spp., Leptocorisa spp., Nezara spp., Piesma spp., Rhodnius spp., Sahlbergella singularis, Scotinophara spp. and Tniatoma spp.; Aleurothrixus floccosus,

Aleyrodes brassicae, Aonidiella spp., Aphididae, Aphis spp., Aspidiotus spp., Bactericera spp., Bemisia tabaci, Ceroplaster spp., Chrysomphalus aonidium, Chrysomphalus dictyospermi, Coccus hesperidum, Empoasca spp., Eriosoma larigerum, Erythroneura spp., Gascardia spp., Laodelphax spp., Lecanium corni, Lepidosaphes spp., Macrosiphus spp., Myzus spp., Nephotettix spp., Nilaparvata spp., Paratoria spp., Pemphigus spp., Planococcus spp., Pseudaulacaspis spp., Pseudococcus spp., Psylla spp., Pulvinaria aethiopica, Quadraspidiotus spp., Rhopalosiphum spp., Saissetia spp., Scaphoideus spp., Schizaphis spp., Sitobion spp., Trialeurodes

vaporariorum, Triozidae spp., Trioza erytreae and Unaspis citri; (j) Hymenoptera, for example, Acromyrmex, Atta spp., Cephus spp., Diprion spp., Diprionidae, Gilpinia polytoma, Hoplocampa spp., Lasius spp., Monomorium pharaonis, Neodiprion spp., Solenopsis spp. and Vespa spp.; (k) Diptera, for example, Aerfes spp., Antherigona soccata, Bibio hortulanus, Calliphora

erythrocephala, Ceratitis spp., Chrysomyia spp., Cuter ebra spp., Dacus spp., Z)e//a spp., Z)e//a radicum, Drosophila spp., e.g., Drosophila suzukii; Fannia spp., Gastrophilus spp., Glossina spp., Hypoderma spp., Hyppobosca spp., Liriomyza spp., Lucilia spp., Melanagromyza spp., Musca spp., Oestrus spp., Orseolia spp., Oscinella frit, Pegomyia hyoscyami, Phorbia spp., Rhagoletis pomonella, Sciara spp., Stomoxys spp., Tabanus spp., Tannia spp. and Tipula spp.; (1) Siphonaptera, for example, Ceratophyllus spp. and Xenopsylla cheopis; (m) from the order Thysanura, for example, Lepisma saccharina.

Phytopathogenic bacteria includes but is not limited to Agrobacterium spp. (e.g.,

Agrobacterium tumefaciens); Erwinia, Pantoea, Pectobacterium, Serratia, S. marcescens, Acidovorax, Pseudomonas, Ralstonia, Rhizobacter, Rhizomonas, Xanthomonas, Xylophilus, Agrobacterium, Rhizobium, Bacillus, Clostridium, Arthrobacter, Clavibacter, Curtobacterium, Leifsonia, Rhodococcus, Streptomyces, Xanthomonas spp. ( Xanthomonas axonopodis,

Xanthomonas oryzae pv. oryzae, Xanthomonas vesicatoria). In a particular embodiment, phytopathogenic bacteria includes but is not limited to Clavibacter spp., Xanthomonas spp., Pseudomonas (e.g., Pseudomonas syringae), Pectobacterium (e.g., Pectobacterium

carotovorum).

Phytopathogenic fungi includes but is not limited to Alternaria spp. (e.g., Alternaria alternata, Alternaria solani); Aphanomyces spp. (e.g., Aphanomyces euteiches); Aspergillus spp. (e.g., Aspergillus niger, Aspergillus fumigatus); Athelia spp. (e.g., Athelia rolfsii); Aureobasidium spp. (e.g., Aureobasidium pullulans); Bipolaris spp. (e.g. Bipolaris zeicola, Bipolaris maydis); Botrytis spp. (e.g., Botrytis cinerea); Calonectria spp. (e.g., Calonectria kyotensis); Cephalosporium spp. (e.g., Cephalosporium maydis); Cercospora spp. (e.g.,

Cercospora medicaginis, Cercospora sojina, Colletotrichum coccodes, Colletotrichum fragariae, Colletotrichum graminicola); Coniella spp. (e.g., Coniella diplodiella); Coprinopsis spp. (e.g., Coprinopsis psychromorbida); Corynespora spp. (e.g., Corynespora cassiicola;

Curvularia spp. (e.g., Curvularia pallescens); Cylindrocladium spp. (e.g., Cylindrocladium crotalariae); Diplocarpon spp. (e.g., Diplocarpon earlianum); Diplodia spp. (e.g., Diplodia gossyina); Epicoccum spp. (e.g., Epicoccum nigrum); Erysiphe spp. (Erysiphe cichoracearum); Fusarium spp. (e.g., Fusarium graminearum, Fusarium oxysporum f.sp. fragariae, Fusarium oxysporum f.sp. tuberosi, Fusarium proliferatum var. proliferatum, Fusarium solani, Fusarium verticillioides); Ganoderma spp. (e.g., Ganoderma boninense); Geotrichum spp. (e.g.,

Geotrichum candidum); Glomerella spp. (e.g., Glomerella tucumanensis); Guignardia spp. (e.g., Guignardia bidwellii); Kabatiella spp. (e.g., Kabatiella zeae); Leptosphaerulina spp. (e.g., Leptosphaerulina briosiana); Leptotrochila spp. (e.g., Leptotrochila medicaginis);

Macrophomina spp. (e.g., Macrophomina phaseolina); Magnaporthe spp. (e.g., Magnaporthe grisea, Magnaporthe oryzae); Microsphaera spp. (e.g., Microsphaera manshurica); Monilinia spp. (e.g., Monilinia fructicola); Mucor spp.; Mycosphaerella spp. (e.g., Mycosphaerella jijiensis, Mycosphaerella fragariae); Nigrospora spp. (e.g., Nigrospora oryzae); Ophiostoma spp. (e.g., Ophiostoma ulmi); Penicillium spp.; Peronospora spp. (e.g., Peronospora manshurica);

Phakopsora (e.g., Phakopsora pachyrhizi); Phoma spp. (e.g., Phomafoveata, Phoma

medicaginis); Phomopsis spp (e.g. Phomopsis longicolla); Phytophthora spp. (e.g., Phytophthora cinnamomi, Phytophthora erythroseptica, Phytophthora fragariae, Phytophthora infestans, Phytophthora medicaginis, Phytophthora megasperma, Phytophthora palmivora); Podosphaera (e.g., Podosphaera leucotricha); Pseudopeziza spp. (e.g., Pseudopeziza medicaginis); Puccinia spp. (e.g., Puccinia graminis subsp. tritici (UG99), Puccinia striiformis, Puccinia recodita, Puccinia sorghi); Pyricularia spp. (Pyricularia grisea, Pyricularia oryzae); Pythium spp. (e.g., Pythium ultimum); Rhizoctonia spp. (e.g., Rhizoctonia solani, Rhizoctonia zeae); Rosellinia spp., Sclerotinia spp. (e.g., Sclerotinia minor; Sclerotinia sclerotiorum, Sclerotinina trifoliorum); Sclerotium spp. (e.g., Sclerotium rolfsii); Septoria spp. (e.g., Septoria glycines, Septoria lycoperski); Setomelanomma spp. (e.g., Setomelanomma turcica); Sphaerotheca spp. (e.g., Sphaerotheca macularis); Spongospora spp. (e.g., Spongospora subterranean); Stemphylium spp., Synchytrium spp. (e.g., Synchytrium endobioticum), Verticillium spp. (e.g., Verticillium albo-atrum, Verticillium dahliae). In a particular embodiment, the fungus is a member of the Botrytis spp. (e.g., Botrytis cinerea), Sclerotinia spp. (Sclerotinia minor), Sclerotium spp. (e.g., Sclerotium rolfsii), Macrophomina spp. (e.g., Macrophomina phaseolina), Verticillium spp. (e.g., Verticillium dahliae), Fusarium spp. (e.g., Fusarium oxysporum f.sp.fragariae), Rhizoctonia spp. (e.g., Rhizoctonia solani), Pythium spp. (e.g., Pythium ultimum).

EXAMPLES

The example below is presented to describe preferred embodiments and utilities of the invention and is not meant to limit the invention unless otherwise stated in the claims appended hereto. Example 1: Isolation of Muscodor albus strain SA-13

The Muscodor albus strain SA-13 was originally obtained from the host plant Prosopis grandulosa in southern Africa.

The host plant, Prosopis, is a tall shrub or tree of 3-9 m; foliage deciduous; spines axillary, uninodal, 1-4.5 cm long, mostly solitary, sometimes very few, or solitary and geminate alternately on different nodes of the same twig. Leaves glabrous, uni- or bijugate; petiole (with rachis when extant) 2-15 cm long; pinnae 6-17 cm long; leaflets 6 to 17 pairs, ca 7-18 mm distant on the rachis, linear or oblong, obtuse, glabrous, subcoriaceous, prominently veined below, costa frequently of lighter color, (1.5-) 2-6.3 cm long x 1.5-4.5 mm broad, 5 to 15 times as long as broad. Racemes spiciform as usual, ca. 5-14 cm long, multiflorous; petals 2.5-3.5 mm long; ovary stipilate, villous. Legume straight, 8-20 cm long x 0.7-1.3 cm broad, rarely subfalcate, compressed to subterete, submoniliform, glabrous, straw-yellow or tinged with violet, short-stiped, with strong, short, or elongate acumen, ca. 5-18-seeded; joints subquadrate to oval; seeds oblique to longitudinal.

It is native to southern USA (i.e. south-western Kansas, Oklahoma, New Mexico, Texas, Arizona, southern California and southern Nevada) and Mexico. This species is widely naturalized in Australia, but has a scattered distribution. It is present in many parts of Queensland and well as in northern Western Australia and south-western New South Wales. It is also naturalized overseas in southern Africa, western Asia (i.e. Saudi Arabia), the Indian Subcontinent (i.e. India and Pakistan), south-eastern Asia (i.e. Burma) and tropical Southern America.

Example 2: Morphological Characterization of Muscodor albus strain SA-13

Cultures of the organism appear whitish and have an overall greasy tone (Figure 1). Under a stereoscopic microscope the growing hyphae have a spear-like appearance with little or no immediate branching patterns. The organism has never been observed to produce spores in culture or on tissues of its host plant. The mycelia hyphae are intertwined and rope like in appearance and have individual hyphal diameters ranging from 1-3 μΐ (Figure 2). This characteristic is common in all Muscodor spp. (Strobel, G.A. 2006. Current Opinions in

Microbiology. 9: 240-244; Strobel, G.A. 2012. Microbiology Today 39-2: 108-111 and Strobel, G.A. 2011. Phytochemistry Reviews 10:165-172).

Example 3: ITS Sequence Analysis

Phylogenetic analysis of SA-13 was carried out by the acquisition of the ITS-5.8 S ribosomal gene sequence. The fungus was grown on PDA for seven days and DNA templates were prepared by using the Prepman Ultra Sample Preparation Reagent according to the manufacturer's guidelines (Applied Biosystems, USA). The ITS regions of the fungus were amplified with the universal ITS primers ITS1 (5' TCCGTAGGTGAACCTGCGG 3' ) (SEQ ID NO:l)) and ITS4 (5' TCCTCCGCTTATTGATATGC 3' (SEQ ID NO:2)) using the polymerase chain reaction (PCR). The PCR conditions used were as follows: initial denaturation at 94 °C for 3 min followed by 30 cycles of 94 °C for 15 sec, 50 °C for 30 sec, 72 °C for 45 sec, and a final extension at 72 °C for 5 min. The 50 μΐ reaction mixture contained lx PCR buffer, 200 μΜ each dNTP, 1.5 mM MgCl ₂ , 10 pmol of each primer, 1-5 ng of extracted DNA and 2.5 U of Taq DNA polymerase. The amplified product (5 μΐ) was visualized on 1% (w/v) agarose gel to confirm the presence of a single amplified band. The amplified products were purified by Amicon Ultra columns (Millipore, USA) and 20-40 ng were used in a 10 1 sequencing reaction using the Big Dye Terminator sequencing kit (v. 3.1), with 2 pmoles of the forward or the reverse primer in the cycle sequencing reaction. Twenty cycles of 96 °C for 10 sec, 50 °C for 5 sec and 60 °C for 4 min were performed and the extension products were purified by ethanol precipitation, dissolved in 10 1 of HiDi Formamide, incubated at 95 °C for 1 min and loaded on ABI Prism 377 Genetic Analyzer (Perkin-Elmer, USA) for sequencing. All the reagents for sequencing were from Applied Biosystems, USA. The DNA sequence was aligned with the reference sequences in GenBank by BLASTN program as shown in Table 1 below.

Table 1: Comparison of Muscodor albus SA-13 strain rRNA with other Muscodor rRNAs

Max Total Query E Max

Accession Description

score score coverage value ident

Muscodor albus internal transcribed spacer

3 ₂₄ 33 1, partial sequence; 5.8S ribosomal RNA ^ _{1033 100% 0 0 m%} gene, complete sequence; and internal

transcribed spacer 2, partial sequence

Muscodor sp. CMU-WR2 18S ribosomal

RNA gene, partial sequence; internal

JX089321.1 transcribed spacer 1, 5.8S ribosomal RNA ^ ^ _{m% Q Q gg%} gene, and internal transcribed spacer 2,

complete sequence; and 28S ribosomal

RNA gene, partial sequence

Muscodor albus internal transcribed spacer

^^^ 1, partial sequence; 5.8S ribosomal RNA ^ _{1Q24 gg% Q Q gg%} gene, complete sequence; and internal

transcribed spacer 2, partial sequence

Muscodor sp. AB-2011 18S ribosomal

RNA gene, partial sequence; internal

^426^ transcribed spacer 1, 5.8S . ribosomal RNA _{1Ql g m s gg% Q Q gg%} gene, and internal transcribed spacer 2,

complete sequence; and 28S ribosomal

RNA gene, partial sequence

Muscodor sp. A3-5 18S ribosomal RNA

gene, partial sequence; internal transcribed

spacer 1 5.8S ribosomal RNA gene and ^ _{1QU m% Q Q 99%} internal transcribed spacer 2, complete

sequence; and 28S ribosomal RNA gene,

partial sequence

Muscodor cinnanomi strain CMU-Cib 461

internal transcribed spacer 1, partial

GQ848369.1 sequence; 5.8S ribosomal RNA gene, 1013 1013 100% 0.0 99% complete sequence; and internal transcribed

spacer 2, partial sequence

AY244622.1 Muscodor albus 18S ribosomal RNA gene, 1011 1011 100% 0.0 99% Max Total Query E Max

Accession Description

score score coverage value ident partial sequence; internal transcribed spacer

1, 5.8S ribosomal RNA gene, and internal

transcribed spacer 2, complete sequence;

and 28S ribosomal RNA gene, partial

sequence

Fungal endophyte sp. P912B internal

transcribed spacer 1, partial sequence; 5.8S

EU977236.1 ribosomal RNA gene, complete sequence; 1007 1007 98% 0.0 99% and internal transcribed spacer 2, partial

sequence

Fungal endophyte sp. P1509A internal

transcribed spacer 1, partial sequence; 5.8S

EU977187.1 ribosomal RNA gene, complete sequence; 1007 1007 98% 0.0 99% and internal transcribed spacer 2, partial

sequence

Muscodor albus strain GP 206 internal

transcribed spacer 1, 5.8S ribosomal RNA

AY527048.1 1007 1007 99% 0.0 99% gene, and internal transcribed spacer 2,

complete sequence

Muscodor albus strain KN 27 internal

transcribed spacer 1, 5.8S ribosomal RNA

AY527046.1 1007 1007 99% 0.0 99% gene, and internal transcribed spacer 2,

complete sequence

Muscodor albus strain TP 21 internal

transcribed spacer 1, 5.8S ribosomal RNA

AY527045.1 1007 1007 99% 0.0 99% gene, and internal transcribed spacer 2,

complete sequence

Muscodor albus strain KN 26 internal

transcribed spacer 1, 5.8S ribosomal RNA

AY527044.1 1002 1002 99% 0.0 99% gene, and internal transcribed spacer 2,

complete sequence

Fungal endophyte sp. P1907B internal

transcribed spacer 1, partial sequence; 5.8S

EU977281.1 ribosomal RNA gene, complete sequence; 998 998 97% 0.0 99% and internal transcribed spacer 2, partial

sequence

Muscodor sp. CMU-MU3 18S ribosomal

RNA gene, partial sequence; internal

transcribed spacer 1, 5.8S ribosomal RNA

JX089323.1 996 996 98% 0.0 99% gene, and internal transcribed spacer 2,

complete sequence; and 28S ribosomal

RNA gene, partial sequence . · Max Total Query E Max

Accession Description - —— - score score coverage value ident

Sordariomycetes sp. genotype 322 isolate

FL0969 internal transcribed spacer 1, partial

JO760598.1 ^quence; 5.8S ribosomal RNA gene and _{9% 9% %% Q Q gg%} internal transcribed spacer 2, complete

sequence; and 28S ribosomal RNA gene,

partial sequence

Muscodor albus isolate 9-6 internal

transcribed spacer 1, partial sequence; 5.8S

HM034857.1 ribosomal RNA gene, complete sequence; 985 985 95% 0.0 100% and internal transcribed spacer 2, partial

sequence

Muscodor albus strain GP 115 internal

AY5 ₂₂ 0 _4L1 transcribed spacer 1, 5.8S . ribosomal RNA ^ ^ _{99% Q Q gg%} gene, and internal transcribed spacer 2,

complete sequence

Sordariomycetes sp. genotype 322 isolate

FL0502 internal transcribed spacer 1, partial

JO760221.1 ^quence; 5.8S ribosomal RNA gene and ^ ^ _{¾% Q Q gg%} internal transcribed spacer 2, complete

sequence; and 28S ribosomal RNA gene,

partial sequence

Fungal sp. ZH S13-1-2 internal transcribed

spacer 1, partial sequence; 5.8S ribosomal

GO220337.1 RNA gene and internal transcribed spacer 2, 965 965 93% 0.0 100% complete sequence; and 28S ribosomal

RNA gene, partial sequence

Sordariomycetes sp. genotype 380 isolate

FL1272 internal transcribed spacer 1, partial

JO760887.1 sequence; 5.8S ribosomal RNA gene and ^ ^ _{m Q Q gs%} internal transcribed spacer 2, complete

sequence; and 28S ribosomal RNA gene,

partial sequence

Muscodor sp. CMU20 18S ribosomal RNA

gene, partial sequence; internal transcribed

spacer 1, 5.8S ribosomal RNA gene, and ^ ^ _{m Q Q gg%} internal transcribed spacer 2, complete

sequence; and 28S ribosomal RNA gene,

partial sequence

Muscodor crispans isolate B-23 internal

EU195297.1 ^{ti sc} ^ ^d■ P ^{artial 5} - ^8S ₉ 55 955 92% 0.0 100% ribosomal RNA gene, complete sequence;

and internal transcribed spacer 2, partial Max Total Query E Max

Accession Description

score score coverage value ident sequence

Muscodor albus isolate E-6 internal

transcribed spacer 1, partial sequence; 5.8S

EF183509.1 ribosomal RNA gene, complete sequence; 953 953 96% 0.0 99% and internal transcribed spacer 2, partial

sequence

Sordariomycetes sp. genotype 380 isolate

FL0763 internal transcribed spacer 1, partial

sequence; 5.8S ribosomal RNA gene and

internal transcribed spacer 2, complete

sequence; and 28S ribosomal RNA gene,

partial sequence >gblJQ760617.1l

JO760423.1 946 946 96% 0.0 98%

Sordariomycetes sp. genotype 380 isolate

FL0989 internal transcribed spacer 1, partial

sequence; 5.8S ribosomal RNA gene and

internal transcribed spacer 2, complete

sequence; and 28S ribosomal RNA gene,

partial sequence

Fungal endophyte sp. P913A internal

transcribed spacer 1, partial sequence; 5.8S

EU977208.1 ribosomal RNA gene, complete sequence; 937 937 91% 0.0 99% and internal transcribed spacer 2, partial

sequence

Muscodor albus strain GP 100 internal

transcribed spacer 1, 5.8S ribosomal RNA

AY555731.1 929 929 99% 0.0 97% gene, and internal transcribed spacer 2,

complete sequence

Muscodor sp. CMU462 18S ribosomal

RNA gene, partial sequence; internal

transcribed spacer 1, 5.8S ribosomal RNA

JN558830.1 909 909 96% 0.0 97% gene, and internal transcribed spacer 2,

complete sequence; and 28S ribosomal

RNA gene, partial sequence

Muscodor albus strain CMU44 18S

ribosomal RNA gene, partial sequence;

internal transcribed spacer 1, 5.8S

HM473081.1 ribosomal RNA gene, and internal 909 909 88% 0.0 100% transcribed spacer 2, complete sequence;

and 28S ribosomal RNA gene, partial

sequence

Sordariomycetes sp. genotype 475 isolate

JO761048.1 874 874 96% 0.0 96%

FL1438 internal transcribed spacer 1, partial Max Total Query E Max

Accession Description

score score coverage value ident sequence; 5.8S ribosomal RNA gene and

internal transcribed spacer 2, complete

sequence; and 28S ribosomal RNA gene,

partial sequence

Muscodor sp. GBA internal transcribed

spacer 1, partial sequence; 5.8S ribosomal

GU797134.1 874 874 84% 0.0 100%

RNA gene, complete sequence; and internal

transcribed spacer 2, partial sequence

Muscodor sp. 1CCSTITD internal

transcribed spacer 1, partial sequence; 5.8S

JO409997.1 ribosomal RNA gene, complete sequence; 784 784 81% 0.0 97% and internal transcribed spacer 2, partial

sequence

Muscodor sp. 2CCSTITD internal

transcribed spacer 1, partial sequence; 5.8S

JO409998.1 ribosomal RNA gene, complete sequence; 773 773 81% 0.0 98% and internal transcribed spacer 2, partial

sequence

Muscodor sp. 6610CMSTITBRT internal

transcribed spacer 1, partial sequence; 5.8S

JO409999.1 ribosomal RNA gene, complete sequence; 706 706 81% 0.0 94% and internal transcribed spacer 2, partial

sequence

Muscodor yucatanensis strain B110 18S

ribosomal RNA gene, partial sequence;

internal transcribed spacer 1, 5.8S

FJ917287.1 ribosomal RNA gene, and internal 702 702 99% 0.0 90% transcribed spacer 2, complete sequence;

and 28S ribosomal RNA gene, partial

sequence

Muscodor sp. WG-2009a internal

transcribed spacer 1, 5.8S ribosomal RNA

FJ664551.1 693 693 99% 0.0 90% gene, and internal transcribed spacer 2,

complete sequence

Muscodor sp. CMU-M2 18S ribosomal

RNA gene, partial sequence; internal

transcribed spacer 1, 5.8S ribosomal RNA

JX089322.1 680 680 99% 0.0 90% gene, and internal transcribed spacer 2,

complete sequence; and 28S ribosomal

RNA gene, partial sequence

Fungal sp. ARIZ B342 18S ribosomal RNA

FJ612989.1 680 680 99% 0.0 90% gene, partial sequence; internal transcribed Max Total Query E Max

Accession Description

score score coverage value ident spacer 1, 5.8S ribosomal RNA gene, and

internal transcribed spacer 2, complete

sequence; and 28S ribosomal RNA gene,

partial sequence

Sordariomycetes sp. genotype 264 isolate

FL1234 internal transcribed spacer 1, partial

sequence; 5.8S ribosomal RNA gene and

JO760849.1 671 671 96% 0.0 90% internal transcribed spacer 2, complete

sequence; and 28S ribosomal RNA gene,

partial sequence

Sordariomycetes sp. genotype 264 isolate

FL0975 internal transcribed spacer 1, partial

sequence; 5.8S ribosomal RNA gene and

JO760604.1 671 671 96% 0.0 90% internal transcribed spacer 2, complete

sequence; and 28S ribosomal RNA gene,

partial sequence

Sordariomycetes sp. genotype 264 isolate

FL0942 internal transcribed spacer 1, partial

sequence; 5.8S ribosomal RNA gene and

JO760574.1 671 96% 0.0 90% internal transcribed spacer 2, complete

sequence; and 28S ribosomal RNA gene,

partial sequence

Fungal endophyte isolate 2161 18S

ribosomal RNA gene, partial sequence;

internal transcribed spacer 1, 5.8S

ribosomal RNA gene, and internal

transcribed spacer 2, complete sequence;

and 28S ribosomal RNA gene, partial

sequence >gbl JQ760022.11 Sordariomycetes

sp. genotype 264 isolate FL0230 internal

transcribed spacer 1, partial sequence; 5.8S

ribosomal RNA gene and internal

EU687035.1 transcribed spacer 2, complete sequence; 671 96% 0.0 90% and 28S ribosomal RNA gene, partial

sequence >gbl JQ760240.11 Sordariomycetes

sp. genotype 264 isolate FL0523 internal

transcribed spacer 1, partial sequence; 5.8S

ribosomal RNA gene and internal

transcribed spacer 2, complete sequence;

and 28S ribosomal RNA gene, partial

sequence >gblJQ760530.1l Sordariomycetes

sp. genotype 264 isolate FL0894 internal

transcribed spacer 1, partial sequence; 5.8S Max Total Query E Max

Accession Description

score score coverage value ident ribosomal RNA gene and internal

transcribed spacer 2, complete sequence;

and 28S ribosomal RNA gene, partial

sequence >gblJQ760814.1l Sordariomycetes

sp. genotype 264 isolate FL1198 internal

transcribed spacer 1, partial sequence; 5.8S

ribosomal RNA gene and internal

transcribed spacer 2, complete sequence;

and 28S ribosomal RNA gene, partial

sequence >gblJQ760833.1l Sordariomycetes

sp. genotype 264 isolate FL1217 internal

transcribed spacer 1, partial sequence; 5.8S

ribosomal RNA gene and internal

transcribed spacer 2, complete sequence;

and 28S ribosomal RNA gene, partial

sequence >gbl JQ760851.il Sordariomycetes

sp. genotype 264 isolate FL1236 internal

transcribed spacer 1, partial sequence; 5.8S

ribosomal RNA gene and internal

transcribed spacer 2, complete sequence;

and 28S ribosomal RNA gene, partial

sequence >gbl JQ760944.11 Sordariomycetes

sp. genotype 264 isolate FL1326 internal

transcribed spacer 1, partial sequence; 5.8S

ribosomal RNA gene and internal

transcribed spacer 2, complete sequence;

and 28S ribosomal RNA gene, partial

sequence

Muscodor vitigenus internal transcribed

spacer 1, 5.8S ribosomal RNA gene and

AY100022.1 internal transcribed spacer 2. complete 671 671 99% 0.0 89% sequence; and 28S ribosomal RNA gene,

partial sequence

Sordariomycetes sp. genotype 524 isolate

NCI 638 internal transcribed spacer 1,

JQ761995.1 Partial sequence; 5.8S ribosomal RNA gene ^ _{669 95% 0} .0 90% and internal transcribed spacer 2, complete

sequence; and 28S ribosomal RNA gene,

partial sequence

Sordariomycetes sp. genotype 524 isolate

JQ761355.1 ^ ^{03 9 internal c} ^ ^d *pacer l, ^ ^ _{g5% Q Q gQ%} partial sequence; 5.8S ribosomal RNA gene

and internal transcribed spacer 2, complete Max Total Query E Max

Accession Description

score score coverage value ident sequence; and 28S ribosomal RNA gene,

partial sequence

Sordariomycetes sp. genotype 514 isolate

NC0275 internal transcribed spacer 1,

partial sequence; 5.8S ribosomal RNA gene

JQ761313.1 669 669 95% 0.0 90% and internal transcribed spacer 2, complete

sequence; and 28S ribosomal RNA gene,

partial sequence

Muscodor sp. E6710b 18S ribosomal RNA

gene, partial sequence; internal transcribed

spacer 1, 5.8S ribosomal RNA gene, and

HM999898.1 669 669 96% 0.0 90% internal transcribed spacer 2, complete

sequence; and 28S ribosomal RNA gene,

partial sequence

Fungal endophyte isolate 1730 18S

ribosomal RNA gene, partial sequence;

internal transcribed spacer 1, 5.8S

EU686946.1 ribosomal RNA gene, and internal 667 667 94% 0.0 90% transcribed spacer 2, complete sequence;

and 28S ribosomal RNA gene, partial

sequence

Sordariomycetes sp. genotype 531 isolate

NC0363 internal transcribed spacer 1,

partial sequence; 5.8S ribosomal RNA gene

JQ761395.1 665 665 96% 0.0 90% and internal transcribed spacer 2, complete

sequence; and 28S ribosomal RNA gene,

partial sequence

Sordariomycetes sp. genotype 264 isolate

FL1245 internal transcribed spacer 1, partial

sequence; 5.8S ribosomal RNA gene and

JO760860.1 665 665 96% 0.0 90% internal transcribed spacer 2, complete

sequence; and 28S ribosomal RNA gene,

partial sequence

Sordariomycetes sp. genotype 264 isolate

FL1075 internal transcribed spacer 1, partial

sequence; 5.8S ribosomal RNA gene and

JO760698.1 665 665 96% 0.0 90% internal transcribed spacer 2, complete

sequence; and 28S ribosomal RNA gene,

partial sequence

Sordariomycetes sp. genotype 264 isolate

JO760692.1 FL1069 internal transcribed spacer 1, partial 665 665 96% 0.0 90% sequence; 5.8S ribosomal RNA gene and Max Total Query E Max

Accession Description

score score coverage value ident internal transcribed spacer 2, complete

sequence; and 28S ribosomal RNA gene,

partial sequence

Sordariomycetes sp. genotype 264 isolate

FL0935 internal transcribed spacer 1, partial

sequence; 5.8S ribosomal RNA gene and

JO760567.1 665 665 96% 0.0 90% internal transcribed spacer 2, complete

sequence; and 28S ribosomal RNA gene,

partial sequence

Sordariomycetes sp. genotype 264 isolate

FL0905 internal transcribed spacer 1, partial

sequence; 5.8S ribosomal RNA gene and

JO760541.1 665 665 95% 0.0 90% internal transcribed spacer 2, complete

sequence; and 28S ribosomal RNA gene,

partial sequence

Sordariomycetes sp. genotype 264 isolate

FL0901 internal transcribed spacer 1, partial

sequence; 5.8S ribosomal RNA gene and

JO760537.1 665 665 96% 0.0 90% internal transcribed spacer 2, complete

sequence; and 28S ribosomal RNA gene,

partial sequence

The ITS rDNA sequence of the strain SA-13 has a high similarity with other isolates of M. albus and M. crispans. And it has 100% identity with many other isolates of Muscodor albus including the CZ 620 isolate, M. crispans and others shown in Figure 3.

Example 4: Analysis of Volatiles Produced by Muscodor albus CZ 620 and SA-13

Prior to use, the fiber (50/30 μπι DVB/CAR/PDMS, Stableflex 24Ga, Supelco Cat. # 57328-U) was conditioned via the injection port at 250 °C for 30 min under a flow of helium gas. Sampling of the gases produced by Muscodor grown on barley grains was done by exposing the fiber to the gas space region of the culturing flask through a small hole of the culturing flask's lid for 30 min at ambient temperature. The syringe was then inserted into the split less injection port of an Agilent 7890A gas chromatograph containing a 20 m x 0.18 mm I.D. DB-VRX column with a film thickness of 1.0 μιη. The column was temperature programmed as follows: 45 °C for 3 min followed to 170 °C at 15 °C/min and then from 170 °C to 225 °C at 35 °C and then hold at 225 °C for 5 min. Ultra high purity Helium was used as carrier gas and ran at a rate of 55 cm/sec (1.5 mL/min) and initial column head pressure of 29 psi. A 15 sec injection time was used to desorb VOCs trapped on the fiber into the GC. The gas chromatograph was interfaced to an Agilent 5975C inert XL MSD with Triple- Axis Detector. The Mass Spectrometer was set to scan at a rate of 2.3 scans per second over a mass range of 16-500 amu. Data acquisition and processing were done using the Agilent ChemStation software. Initial identification of the unknowns produced by Muscodor albus SA-13 was made through library match with available spectra database from NIST.

Study 1. SPME-GCMS analysis of volatiles produced by Muscodor albus CZ 620 grown on barley grains.

Muscodor albus CZ 620 was grown on barley grains for 17 days and the volatile organic compounds (VOCs) produced were sampled and analyzed. A chromatographic representation of the analysis is shown in Figure 4. An identification of the VOCs produced by Muscodor albus CZ 620 was made via a library match with the available NIST database. The results are tabulated in Table 1.

Table 1. SPME-GCMS analysis of volatiles produced by the Muscodor albus CZ 620 strain grown on barley grains.

RT

Entry (min) Possible Compound ID

1 0.76 Ethanol

2 1.53 Propanol

3 2.47 2-Butanone, 4-hydroxy-

4 2.53 Ethyl Acetate

5 3.97 Propanoic acid, 2-methyl-, methyl ester

6 4.48 2-Butanone, 3-hydroxy-

7 4.58 n-Propyl acetate

8 4.91 1-Butanol, 3-methyl-

9 5.01 1-Butanol, 2-methyl-

10 5.44 Propanoic acid, 2-methyl-, ethyl ester

11 5.62 Propanoic acid, 2-methyl

12 5.78 Butanoic acid, 2-methyl-, methyl ester

13 6.16 Butanoic acid, ethyl ester

14 6.96 Butanoic acid, 2-methyl-, ethyl ester

15 7.3 2-Butenoic acid, 2-methyl-, methyl ester

16 7.39 1-Butanol, 3-methyl-, acetate

17 8.3 Ethyl tiglate

18 10.48 Phenylethyl Alcohol 19 13.19 lH-3a,7-methanoazulene, 2,3,4,7,8,8a-hexahydro-3,6,8,8- tetramethyl-, [3R-(3R(3, alpha., 3a.beta.,7.beta.,8a.alpha.)]-

20 14.92 Azulene, 1,2,3,5,6,7,8, 8a-octahydro-l,4-dimethyl-7-(l- methylethenyl)-,(lS-(l.alpha.,7.alpha.,8a.beta.)]-

Study 2. SPME-GCMS analysis of VOCs produced by Muscodor albus SA-13 strain

A. SPME-GCMS analysis of VOCs produced by Muscodor albus SA-13 strain grown on barley grains.

Muscodor albus SA-13 was grown on barley grains for 10 days and the VOCs produced were sampled and analyzed by SPME-GCMS method as detailed in Example 4. A

chromatographic representation of the analysis is shown in Figure 5A. Results are tabulated in Table 2A.

Table 2 A. SPME-GCMS analysis of VOCs produced by the Muscodor albus SA-13 strain grown on barley grains.

RT

Entry (min) Possible Compound ID

1 0.76 Ethanol

2 1.53 Propanol

3 2.47 2-Butanone, 4-hydroxy-

4 2.53 Ethyl Acetate

5 3.97 Propanoic acid, 2-methyl-, methyl ester

6 4.45 Propanoic acid, ethyl ester

7 4.91 1-Butanol, 3-methyl-

8 5.01 1-Butanol, 2-methyl-

9 5.44 Propanoic acid, 2-methyl-, ethyl ester

10 5.73 Acetic acid, 2-methylpropyl ester

11 5.78 Butanoic acid, 2-methyl-, methyl ester

12 6.96 Butanoic acid, 2-methyl-, ethyl ester

13 7.04 Propanoic acid, 2-methyl-, butyl ester

14 7.39 1-Butanol, 3-methyl-, acetate

15 7.42 1-Butanol, 2-methyl-, acetate

16 7.91 Propanoic acid, 2-methyl-, butyl ester

17 8.1 Benzene, methoxy-

18 8.3 Ethyl tiglate

19 8.9 3-Octanone

20 9.2 Propanoic acid, 2-methyl-, 3-methylbutyl ester

21 10.48 Phenylethyl Alcohol

22 11.96 Acetic acid, 2-phenylethyl ester

23 12.78 (-)Aristolene

24 12.95 Cyclohexane, l-ethenyl-l-methyl-2,4-bis(l-methylethenyl) 25 13.2 Azulene, 1, 2,3,4,5,6,7, 8-octahydro-l,4-dimethyl-7-(l- methylethenyl)- ,( 1 S -( 1. alpha. ,4. alpha. ,7. alpha. )] -

26 13.58 Bicyclo[5.3.0]decane, 2-methylene-5-(l-methylvinyl)-8- methyl-

27 13.66 Azulene, 1, 2,3,5, 6,7,8, 8a-octahydro-l,4-dimethyl-7-(l- methylethenyl)- , [ 1 S - ( 1. alpha. ,7.alpha. ,8a.beta. )] -

28 14.92 Azulene, 1,2,3,5, 6,7,8, 8a-octahydro-l, 4-dimethyl-7-(l- methylethenyl)-,(lS-(l.alpha.,7. alpha., 8a.beta.)]-

Identification of the VOCs produced was made via a library match with the available NIST database.

B. SPME-GCMS analysis of VOCs produced by Muscodor albus SA-13 in potato dextrose broth (PDB) medium

A 12 day-old liquid culture of M. albus SA-13 grown in PDB medium at 25-27 °C was analyzed for VOCs produced by SPME-GCMS technology. The VOCs in the headspace region was sample and analyzed as detailed in Example 4. The chemicals produced by the microbe in the PDB liquid medium were sampled and analyzed by submerging the fiber (50/30 μιη

DVB/CAR/PDMS, Stableflex 24Ga) into the liquid culture for 30 min, followed by the insertion of the fiber into the splitless injection port of the gas chromatograph (GC) as detailed in Example 4 to dislodge the VOCs trapped by the fiber onto the GC. Figure 5B depicts the chromatograms of the profiles of the VOCs produced by the microbe in PDB media found in the liquid medium (top graph) and headspace region (middle graph) and are similar to the chemical profile produced by the microbe cultured on barley grain.

Table 2B summarizes the VOCs produced by M. albus SA-13 found in the headspace region and in the liquid medium.

Table 2B. VOCs produced by M. albus SA-13 when grown in PDB medium

VOCs Found in

RT Headspace Liquid

Entry (min) Possible Compound ID Region Medium

1 0.83 Ethanol V V

2 2.42 Ethyl Acetate V V

3 2.51 1-Propanol, 2-methyl V V

4 3.49 Butanal, 2-methyl V V

5 3.97 Propanoic acid, 2-methyl-, methyl ester V V

6 4.41 2-Butanone, 3-hydroxy- V -

7 4.83 1-Butanol, 3-methyl- V V 8 4.90 1-Butanol, 2-methyl- V V

9 5.73 Acetic acid, 2-methylpropyl ester V V

10 7.37 1-Butanol, 3-methyl-, acetate V V

11 7.40 1-Butanol, 2-methyl-, acetate V V

12 9.94 4-Nonanone V V

13 10.18 2-Nonanone V V

14 10.46 Phenylethyl Alcohol V V

15 10.62 Media or column

16 11.93 Acetic acid, 2-phenylethyl ester - V

Cycloheptane, 4-methylene- 1 -methyl-2- [2-

17 13.56 methyl- l-propene-l-yl]-l -vinyl - V

Azulene, 1,2,3,5, 6,7,8, 8a-octahydro- 1,4- dimethyl-7 - ( 1 -methylethenyl) - , [ 1 S -

18 13.63 (l.alpha.,7.alpha.,8a.beta.)]- V V

lH-2-Benzopyran-l-one, 3,4-dihydro-8-

19 14.12 hydroxy-3-methyl-, [R]- - V

20 14.27 Unknown

Azulene, 1,2,3,5, 6,7,8, 8a-octahydro- 1,4- dimethyl-7 - ( 1 -methylethenyl) - , ( 1 S -

21 14.9 (l.alpha.,7.alpha.,8a.beta.)]- V V

22 15.48 Unknown - V

23 16.02 Unknown - V

24 16.75 Unknown - V

Note: "V", compound detected; "-" , compound not detected.

Identification of the VOCs produced was made via a library match with the available NIST database. Study 3. Comparative analysis of VOCs produced by Muscodor albus CZ 620 and SA-13 grown on barley grains.

Differences in the type and quantity of VOCs produced by Muscodor albus CZ 620 and SA-13 can be observed as shown in Figure 6. A direct comparison of VOCs produced by the Muscodor albus SA-13 strain grown on barley grains is summarized in Table 3.

Table 3. Comparison of VOCs produced by the Muscodor albus SA-13 and 620 strains grown on barley grains

Muscodor

RT Strains

Entry (min) Possible Compound ID 620 SA-13

1 0.76 Ethanol V V

2 1.53 Propanol V V

3 2.47 2-Butanone, 4-hydroxy- V V

4 2.53 Ethyl Acetate V V

5 3.97 Propanoic acid, 2-methyl-, methyl ester V V

6 4.45 Propanoic acid, ethyl ester - V

7 4.48 2-Butanone, 3-hydroxy- V -

8 4.58 n-Propyl acetate V -

9 4.91 1-Butanol, 3-methyl- V V

10 5.01 1-Butanol, 2-methyl- V V

11 5.44 Propanoic acid, 2-methyl-, ethyl ester V V

12 5.62 Propanoic acid, 2-methyl V -

13 5.73 Acetic acid, 2-methylpropyl ester - V

14 5.78 Butanoic acid, 2-methyl-, methyl ester V V

15 6.16 Butanoic acid, ethyl ester V -

16 6.96 Butanoic acid, 2-methyl-, ethyl ester V V

17 7.04 Propanoic acid, 2-methyl-, butyl ester - V

18 7.3 2-Butenoic acid, 2-methyl-, methyl ester (Methyl tiglate) V -

19 7.39 1-Butanol, 3-methyl-, acetate V V

20 7.42 1-Butanol, 2-methyl-, acetate - V

21 7.91 Propanoic acid, 2-methyl-, butyl ester - V

22 8.1 Benzene, methoxy- - V

23 8.3 Ethyl tiglate V V

24 8.9 3-Octanone - V

25 9.2 Propanoic acid, 2-methyl-, 3-methylbutyl ester - V

26 10.48 Phenylethyl Alcohol V V

27 11.96 Acetic acid, 2-phenylethyl ester - V

28 12.78 (-)Aristolene - V

Cyclohexane, 1 -ethenyl- 1 -methyl-2,4-bis( 1 -

29 12.95 methylethenyl)- - V lH-3a,7-methanoazulene, 2,3,4,7, 8, 8a-hexahydro-3, 6,8,8-

30 13.19 tetramethyl-, [3R-(3R(3, alpha., 3a.beta.,7.beta.,8a.alpha.)]- V - Azulene, 1, 2,3,4,5, 6,7, 8-octahydro-l, 4-dimethyl-7-(l-

31 13.2 methylethenyl)-,(lS-(l.alpha.,4.alpha.,7.alpha.)]- - V

Bicyclo[5.3.0]decane, 2-methylene-5-(l-methylvinyl)-8-

32 13.58 methyl- - V

Azulene, 1,2,3, 5,6,7, 8, 8a-octahydro-l,4-dimethyl-7-(l-

33 13.66 methylethenyl)-, [lS-(l.alpha.,7. alpha., 8a.beta.)]- - V

Azulene, 1,2,3, 5,6,7, 8, 8a-octahydro-l, 4-dimethyl-7-(l-

34 14.92 methylethenyl)-,(lS-(l.alpha.,7. alpha., 8a.beta.)]- V V

Note: "V", compound detected; compound not detected.

Study 4. GCMS analysis of the XAD7-trapped VOCs produced by M. albus 620 and SA-13 An active culture of Muscodor albus is grown in a French Square Glass Bottle (250 mL) (120) containing autoclave-sterilized barley grains (-65 g). Filtered air (130) is passed over the culture at a steady bubbling rate via the use of an air pump (140). The exhaust gas was allowed to pass through a bed of XAD7 resin (110) and then bubbled into a test-tube (100) containing ethanol (10 mL) for 16 h as shown in Figure 7.

Upon the completion of the sampling process, the resin was washed with MeOH (4 mL) and an aliquot (1 mL) of the washed MeOH solution was used for GCMS analysis using a Agilent 7890A gas chromatography system containing a 20 m x 0.18 mm I.D. DB-VRX column with a film thickness of 1.0 μιη. The column was temperature programmed as follows: 45 °C for 3 min followed to 170 °C at 15 °C/min and then from 170 °C to 225 °C at 35 °C and then hold at 225 °C for 5 min. Ultra high purity helium was used as carrier gas at a rate of 55 cm/sec (1.5 mL/min) with initial column head pressure of 29 psi, inlet temperature of 150 °C and a split ratio of 60:1. The gas chromatograph was interfaced to an Agilent 5975C inert XL MSD with Triple- Axis Detector. The MS was scanned at a rate of 2.3 scans per second over a mass range of 35-360 amu. Data acquisition and processing were performed on the Agilent ChemStation software system and tentative identification of VOCs produced by Muscodor albus 620 and SA- 13 were made by comparing the mass fragmentation pattern of the unknown with the with the available NIST database.

A chromatographic representation of the VOCs produced by Muscodor albus CZ 620 and SA-13and trapped in the XAD-7 resin is shown in Figure 8. Identification of the VOCs produced was made by comparing the mass fragmentation of the unknown with the available NIST database as well as with authentic samples obtained from commercial sources. The identity of the VOCs produced is summarized in Table 4.

Table 4. Possible VOCs produced by Muscodor albus SA-13 and were trapped with XAD7 resin.

Match Artificial

RT MW % of Quality Mix

Peak

# (min) Possible Compound ID (NIST) (m/z) Total (%) (10 mL)

1 1.14 Ethanol 46 30.6 86 3.06

2 2.71 Ethyl acetate 88 7.5 86 0.75

3 2.82 1-Propanol, 2-methyl- 74 8.7 91 0.87

4 4.14 Propanoic acid, 2-methyl-, 102 2.8 91 0.28 methyl ester

5 4.96 1-Butanol, 3-methyl- 88 30.1 90 3.01

6 5.02 1-Butanol, 2-methyl- 88 19.4 83 1.94

Propanoic acid, 2-methyl-, ethyl

7 5.51 ester 116 0.9 81 0.09

Muscodor albus SA-13 produced all of the seven compounds trapped by XAD7, while Muscodor albus CZ 620 produced only five of the compounds listed (See Table 5).

Table 5. Comparison of XAD7 resin trapped VOCs produced by the Muscodor albus SA-13 and

CZ 620 when grow on barley grains.

Muscodor

RT albus

Entry (min) Possible Compound ID 620 SA-13

1 1.14 Ethanol V V

2 2.71 Ethyl acetate V V

3 2.82 1-Propanol, 2-methyl- V V

Propanoic acid, 2-methyl-, methyl

4 4.14 ester - V

5 4.96 1-Butanol, 3-methyl V V

6 5.02 1-Butanol, 2-methyl- V V

7 5.51 Propanoic acid, 2-methyl-, ethyl ester - V

Note: "V", compound detected; "-" , compound not detected.

Study 5. Effect of the mixture of VOCs reconstituted from the above mentioned

components on pathogen growth.

To test different combinations of the seven compounds that make up the XAD7 resin-trap volatile mixture, the 9.5-cm plates were filled with PDA, and about 3-mm plugs of Fusarium oxysporum f.sp.fragariae and Macrophomina phaseolina were used as examples and were placed 1.5 cm away from the outer edges of the plates. Opposite the plug, a little less than half of the PDA was removed from the plate. Autoclaved caps from 2-ml Eppendorf tubes were used to contain the VOCs. The caps were sterilized then placed upside down on the side without the agar. The VOC mixture of 50 μΐ was loaded in the cap (There were two plates per fungus, and two control plates without VOCs per fungus).

All plates were double wrapped with parafilm and placed in a plastic container. The plastic container was kept in the transfer room at about 25 °C in the dark. Once growth of the pathogens in the 0.0 μΐ VOC controls reach the edges of the PDA plates or show adequate growth, observe/measure the growth of each fungus by measuring from the center of the plug to the furthest edge of the colony.

Table 6. Effect of reconstituted VOCs (artificially mixed VOCs) on the growth of Fusarium oxysporum and Macrphomina phaseolina.

: Data with the same letter are not significantly different with Fisher Protected LSD test at p=0.05 level.

The whole mixture containing all the VOCs showed the strongest effect on Fusarium

oxysporium (Table 6). Other mixtures without certain components also showed efficacy and had no significant differences compared to the whole mixture. Similarly, the growth of

Macrophomina phaseolina was similarly or greatly inhibited by the whole mixture and the mixtures without some ingredients. These results demonstrate that various components of the VOCs can be combined for controlling different disease pathogens.

Example 5: Fungicidal and Bactericidal Effect of Muscodor albus SA-13

Study 1. Fungicical effect

A. M. albus SA-13 on inhibiting the growth of Aspergillus.

Petri plates of 010 cm with PDA were used for evaluating the inhibitive effect of the strain against Aspergillus niger. There were two plates for the Muscodor strain, and two without Muscodor as blank control.A 3-mm PDA plug of the M. albus SA-13 strain was placed 1.5 cm away from the outer edge of one side of the PDA plate. The Muscodor strain was grown for 3 days in sealed plates at room temperature (about 25 °C) and then a 3 mm ² plug of Aspergillus niger was placed at 1.5 cm away from the outer edge of the other side of the plate. The growth of the pathogen was visually assessed as normal growth (++), abnormal growth (+-), or no growth (— ). The assessment of mycelial growth is given in Table 7A and shown in Figure 9. Muscodor albus strain SA-13 showed a highly inhibitive effect on the pathogen.

Table 7 A. Inhibition by Muscodor albus SA-13 on the mycelial growth of Aspergillus niger.

B. Comparison of M. albus SA-13 and CZ 620 PDA plugs on inhibiting the growth of funcal pathogens.

Split petri plates of 010 cm with PDA were used for evaluating the inhibitive effect of the strains against plant pathogens. The following pathogens were used for the evaluation: Botrytis cinerea, Fusarium oxysporum f.sp. fragariae, Pythium ultimum, Rhizoctonia solani, Sclerotinia minor, and Verticillium dahliae. There were two plates for each Muscodor strain, and two without Muscodor as the blank controls for each pathogen.

A 5-mm PDA plug of each isolate was placed 2.5 cm away from the outer edge of one side of the split petri plate. The plates were sealed and isolates were allowed to grow for 3 days at room temperature (about 25 °C). A 3-mm ² PDA plug of each pathogen was placed at 1.5 cm away from the outer edge of the other side of the split plate. One sclerotium of Sclerotinia minor was placed on the agar plate instead of a plug.

The growth of each pathogen was measured from the center of the plug to the furthest edge of the colony after their water controls reached the divider in the plate. The percentage inhibition of mycelial growth is given in Table 7B. The strain SA-13 showed superior inhibition on the mycelium growth of the pathogens tested. Comparatively, the strain M. albus CZ 620 isolated from cinnamon tree (see, for example, US Patent No. 6, 911,338) was less effective on Fusarium oxysporum and Pythium ultimum. Table 7B. Inhibition by Muscodor albus strains SA-13 and CZ 620 on the mycelial growth of various plant pathogens (Botrytis cinerea (Bot), Fusarium oxysporum f.sp. fragariae (Pus), Pythium ultimum (Pyth), Rhizoctonia solani (Rhizo), Sclerotinia minor (Scler), and Verticillium dahliae (Vert)).

C. Comparison of M. albus SA-13 and CZ 620 grown on barley grains on inhibiting the growth of fungal pathogens.

Split petri plates of 010 cm with PDA were used for evaluating the inhibitive effect of the strains against plant pathogens. The following pathogens were used for the evaluation: Botrytis cinerea, Fusarium oxysporum f.sp. fragariae, Pythium ultimum, Verticillium dahliae,

Rhizoctonia solani , Sclerotinia minor, Macrophomina phaseolina, and Scler otium rolfsii. There were two plates for each Muscodor strain, and two without Muscodor as the blank controls for each pathogen.

The Muscodor strains were each grown on hulled barley grains for 13 days. The cultures were then broken up and mixed thoroughly. Approximately 11 g of the cultures were placed in one side of the split petri plate and allowed to recover for 2-7 days at room temperature (about 25 °C). A 3-mm ² PDA plug of each pathogen was placed at 1.5 cm away from the outer edge of the other side of the split plate.

The growth of each pathogen was measured from the center of the plug to the furthest edge of the colony after their water controls reached the divider in the plate or showed adequate growth. The percentage inhibition of mycelial growth is given in Table 7C. The strain SA-13 showed superior inhibition on the mycelium growth of the pathogens tested. Comparatively, the strain M. albus CZ 620 isolated from cinnamon tree (see, for example, US Patent No. 6, 911,338) was less effective on Rhizoctonia solani, Pythium ultimum, Verticillium dahliae, Fusarium oxysporum, and Macrophomina phaseolina. Table 7C. Inhibition by Muscodor albus strains SA-13 and CZ 620 on the mycelial growth of various plant pathogens (Botrytis cinerea, Rhizoctonia solani, Pythium ultimum, Verticillium dahliae, Fusarium ox sporum f.sp. fragariae, Sclerotinia minor, Macrophomina phaseolina, and

Sclerotium rolfsii.

Study 2. Selective inhibition of M. albus SA-13 on soilborne fungi.

Two additional plant pathogens, Macrophomina phaseolina, Sclerotium rolfsii, and one non-pathogenic fungus, Trichoderma viride, were used to evaluate the inhibitive effect of M. albus SA-13.

The above mentioned split petri plates with PDA medium were used in the test. There were two plates for the Muscodor strain, and two without Muscodor as blank control.

A 5-mm plug of the M. albus SA-13 strain was placed 2.5 cm away from the outer edge of one side of the PDA plate. The Muscodor strain was grown for 5 days in sealed plates at room temperature (about 25 °C) and then a 3 mm ² plug of each pathogen or Trichoderma viride was placed at 1.5 cm away from the outer edge of the other side of the split plate. The growth of each pathogen was measured as described in Study 1 and results are given in Table 8. Table 8. Inhibition on the mycelial growth of fungi (Macrophomina phaseolina (Mac), Sclerotium rolfsii (Scl rol), and Trichoderma viride (Tricho).) by Muscodor albus SA-13.

Muscodor albus strain SA-13 showed a highly inhibitive effect on all the plant pathogens. However, it did not show any inhibitative effect on the growth of the beneficial fungus

Trichoderma viride.

Study 3. Inhibitative effect of M. albus SA-13 on bacterial plant pathogens.

Muscodor albus strain SA-13 was further evaluated for its inhibitive effect on bacterial plant pathogens with barley grains.

To culture the Muscodor strain, the barley grains were washed more than three times with deionized water, and soaked for 24 hours at 20 °C. The water was drained off before splitting the grains evenly between autoclave bags with foam stoppers. The grains were then autoclaved for 15 minutes at 121 °C twice. Once the grains cooled, several small plugs of Muscodor albus strain SA-13 were added to each bag and leaving one bag non-inoculated as blank control. The fungus was grown in the bags for 11 days at room temperature. The masses of inoculated barley grains were broken up on the day of the test so the grains were less stuck together.

There were four bacteria tested: Pectobacterium carotovorum (Pec), Pseudomonas syringae (Pst), Xanthomonas vesicatoria (Xan), and Clavibacter michiganensis subsp.

michiganensis (Clav). Each bacterium was grown on an agar medium for one day before being washed off. The OD ₆ oo of each bacterium was adjusted to approximately 0.2 using sterile water (for all bacteria tested OD = 0.2 is approximately 10 cfu/ml). Using sterile water, serial dilutions were done for each bacterium up to 10 ⁵ . From the 10 ⁴ and 10 ⁵ dilutions, 15 μΐ of solution was spread onto 35-mm PDA petri plates until dry. The 35-mm plates were then placed without their lids inside 10-cm petri plates next to 11 g of either the SA-13 inoculated grains, or non-inoculated sterile grains. Each plate was wrapped up with two pieces of Parafilm, placed in a sealed container and incubated at room temperature (-25 °C) in the dark.

The effect on each bacterium was determined by counting the colony forming unit (CFU) on each 35 mm plate after 1-3 days. After 3 days, the 35-mm plates were removed from the 95- mm plates, the lids were replaced, and the small plates were placed in a 25 °C incubator in the dark. After five days in the incubator the recoveries of the bacteria were determined by recounting the number of colonies on the SA-13 exposed plates. The results are given in Table 9.

Table 9. CFU for different concentrations of plant pathogenic bacteria after exposure to M. albus SA-13 grains and non-inoculated grains and CFU after 5 days of recovery from exposure to M.

albus SA-13 grains.

Mean CFU/plate

Recovery after

Exposed to isolate Exposed to non- exposure to isolate SA13 inoculated barley

SA13

10 ⁵ 10 ⁴ 10 ⁵ 10 ⁴ 10 ⁵ 10 ⁴

Bacterium dilution dilution dilution dilution dilution dilution

Pec 0.0 0.0 31.0 358.5 0.0 0.0

Pst 0.0 0.0 7.5 17.5 0.0 0.0

Xan 0.0 0.0 18.5 185.5 0.0 0.0

Clav 0.0 0.0 56.5 543.5 22.0 123.5

Muscodor albus strain SA-13 completely inhibited the growth of all four pathogens and none were able to recover except Clavibacter michiganensis subsp. michiganensis, which did not show a full recovery.

Study 4. Evaluation of the reconstituted VOCs on inhibiting plant pathogens and SA-13.

Reconstituted mixes of six volatiles produced (Ethanol, Ethyl acetate, 2-methyl-l- Propanol, 2-methyl-, methyl ester Propanoic acid, 3 -methyl- 1-Butanol, 2-methyl-l-Butanol) by Muscodor albus strain SA-13 captured with the above mentioned resin trap were evaluated for their inhibitive effect on fungi.

The following fungi were used as test organisms for the VOC bioassay: Fusarium oxysporum f.sp.fragariae (Fus), Botrytis cinerea (Bot) and Muscodor albus isolate SA-13 (SA- 13). A small piece of agar was removed from one side of a PDA petri plate. A 3 -mm plug of each pathogen was placed on the agar 1.5 cm away from the outer edge of the plate, opposite the empty hole. Autoclaved caps from 2-ml Eppendorf tubes were sterilized and placed upside down in the empty space. The artificial VOC mixture was placed in the upside down cap at varying volumes. The test also included control plates which contained empty caps. Each plate was wrapped up with two pieces of Parafilm, placed in a sealed container and incubated at room temperature (-25 °C) in the dark.

The % inhibition of each test organism was determined by measuring the mycelial growth from the center of the agar plug to the furthest edge of the colony. The results are given in Table 10.

Table 10. Inhibition of fungal pathogens after exposure to an M. blbus SA-13 artificial VOC mixture with six compounds at different volumes.

Inhibition (%)

Pathogen 5 ul 20 ul 35 ul 50 ul 75 ul

Fus -8.6 5.2 13.8 25.9 43.1

Bot -11.9 6.0 31.3 49.3 76.1

SA-13 4.7 11.6 20.9 27.9 44.2 The 6 compound mixture at 35 μΐ was able to significantly inhibit the growth of all fungi tested. There is also a positive relationship between the dose and the mycelial inhibition.

Study 5. Reepeated evaluation of the reconstituted VOCs on inhibiting plant pathogens

The reconstituted mixture of the seven volatiles produced (Ethanol, Ethyl acetate, 2- methyl-l-Propanol, 2-methyl-, methyl ester Propanoic acid, 3-methyl-l-Butanol, 2-methyl- 1- Butanol, 2-methyl-, ethyl ester- Propanoic acid) by Muscodor albus strain SA-13 captured with the above mentioned resin trap were evaluated for their inhibitive effect on plant pathogens.

The following plant pathogens were used as test organisms for the VOC bioassay:

Verticillium dahliae (Vert), Macrophomina phaseolina (Mac), Rhizoctonia solani (Rhizo), Pythium ultimum (Pyth), Fusarium oxysporum f.sp.fragariae (Fus), and Sclerotium rolfsii (Scl rol). Slightly less than half of the agar was removed from one side of a PDA petri plate. A 3- mm plug of each pathogen was placed on the agar 1.5 cm away from the outer edge of the plate, opposite the empty side. Autoclaved caps from 2-ml Eppendorf tubes were sterilized and placed upside down on the empty side of the plate. The artificial VOC mixture was placed in the upside down cap at varying volumes. The test also included control plates which contained empty caps. Each plate was wrapped up with two pieces of Parafilm, placed in a sealed container and incubated at room temperature (-25 °C) in the dark.

The percentage inhibition of each test organism was determined by measuring the mycelial growth from the center of the agar plug to the furthest edge of the colony. The test was repeated with a higher dose of the VOC mixture for all fungi that were not significantly inhibited in the initial test. The results are given in Table 11.

Table 11. Inhibition of soil-borne pathogens after exposure to an SA-13 artificial VOC mix at different volumes.

Inhibition (%)

Pathogen 35 μΐ 75 μΐ

Vert 9.5 62.5

Mac 7.1 53.5

Rhizo 53.6 -

Pyth 34.7 -

Fus 24.1 -

Scl rol 65.8 -

Rhizo ctoniasolani, Pythium ultimum, Fusarium oxysporum f.sp. fragariae, and Sclerotium rolfsii pathogens were not tested at the 75 microliter volume, since their growth was inhibited by VOCs at lower volume.

The artificial volatile mixture at 35 1 was able to inhibit the growth of Fusarium oxysporum f.sp.fragariae, Pythium ultimum, Rhizoctonia solani, and Sclerotium rolfsii. At 75 ul, the mixture was able to inhibit the growth of Macrophomina phaseolina and Verticillium dahliae.

Study 6. Repeated evaluation on the inhibition of soilborne pathogens by M. albus SA-13.

Muscodor albus strain SA-13 at different doses was further evaluated for its inhibitive effect on plant pathogens.

To culture the Muscodor strain, the barley grains were washed three times with tap water, and soaked for 24 hours at 20 °C. The water was rewashed and drained off before splitting the grains evenly between autoclave bags with foam stoppers. The grains were then autoclaved for 15 minutes at 121 °C two times. Once the grains cooled, each bag was inoculated with 12 ml of a 10-day culture of Muscodor albus strain SA-13 grown on potato dextrose broth. The fungus was grown in the bags for 9 days at 25 °C. The masses of inoculated barley grains were then broken up and allowed to air-dry until seed moisture was < 10%. The dried grains were ground with a coffee grinder and particles that went through a 2- mm sieve and were saved by a 45 μηι sieve were saved for testing. The grains were rehydrated with water and different doses of the grains were placed in a single center well of a 6-well plate to grow for three days. The three wells adjacent to the grain-occupied well contained PDA. The two non-adjacent wells remained empty for the duration of the test.

The following plant pathogens were used as test organisms for the dose bioassay:

Fusarium oxysporum f.sp. fragariae (Fus), Rhizoctonia solani (Rhizo), and Pythium ultimum (Pyth). A 3-mm plug of each pathogen was placed on the agar in the center of the PDA filled well, each plate holding one dose of the grain and one plug of each pathogen. The test also included control plates that contained no barley grain. Each plate was sealed with tape, and incubated until the controls reached the edges of the wells, or showed adequate growth.

The percentage inhibition of each plant pathogen was determined by measuring the mycelial growth from the center of the agar plug to the furthest edge of the colony. The results are given in Table 12.

Table 12. Inhibition of Muscodor albus strain SA-13 at different doses on the mycelial growth of plant pathogens.

Inhibition (%)

M. albus (g) Fus Rhizo Pyth

0.05 -12.5 66.7 100.0

0.1 -12.5 100.0 100.0

0.5 12.5 66.7 100.0

1.0 75.0 100.0 100.0

2.0 75.0 100.0 100.0

3.0 75.0 100.0 100.0

5.0 62.5 100.0 100.0

Muscodor albus strain SA-13 showed inhibition of the mycelial growth of all the pathogens tested.

Study 7. Inhibition of more soilborne pathogens by M. albus SA-13.

A repeated test on Muscodor albus strain SA-13 at different doses was conducted to evaluate its inhibitive effect on additional plant pathogens.

The grains used in Study 6 were also used in this study. The following plant pathogens were used as test organisms for the bioassay: Verticillium dahliae (Vert), Macrophomina phaseolina (Mac), and Sclerotium rolfsii (Scl rol). The barley grain culture and plant pathogens were arranged in the plates as described in Study 6.

The percentage inhibition of each test organism was determined by measuring the mycelial growth from the center of the agar plug to the furthest edge of the colony. The results are given in Table 13.

Table 13. Inhibition of Muscodor albus strain SA-13 at different doses on the mycelial growth of plant pathogens.

Inhibition (%)

M. albus (g) Vert Mac Scl rol

0.05 100.0 100.0 100.0

0.1 100.0 100.0 100.0

0.5 100.0 100.0 100.0

1.0 100.0 100.0 100.0

3.0 100.0 100.0 100.0

Muscodor albus strain SA-13 showed complete inhibition of the mycelial growth of all pathogens at all doses tested.

Study 8. Control of soilborne diseases by incorporation of M. albus SA-13 in soil.

A. Muscodor albus isolate SA-13 grown on barley grains from a plug was evaluated for its efficacy in controlling Rhizoctonia solani on soybean.

To culture the Muscodor strain, the barley grains were washed with deionized water, and soaked for 24 hours. The water was drained off before splitting the grains evenly between autoclave bags with foam stoppers. The grains were then autoclaved for 15 minutes at 121 °C twice. Once the grains cooled, several small plugs of Muscodor albus strain SA-13 were added to each bag. The pathogen was grown in the bags for 12 days at room temperature. The masses of inoculated barley grains were broken up on the day of the test so the grains were less stuck together.

Rhizoctonia inoculum was mixed into artificial soil media at a rate of 1:1200. The inoculated media was placed into plastic boxes at 1 L per box. SA-13 barley seeds were then mixed into the infested soil media at the rates 2 mg/ml, 4 mg/ml, and 6 mg/ml. In separate treatments, SA-13 seeds were scattered over the top of the inoculated soil media at 2.8 mg/cm ,

28 mg/cm 2 , and 56 mg/cm 2. The boxes were watered, closed, and sealed with tape for two days. After two days of treatment, 24 soybean seeds were planted in each box before re-sealing the boxes and placing them under fluorescent lights for ten days.

The soybean emergence after ten days was determined by counting the number of emerged seedlings within each box. The results are given in Table 14A.

Table 14A. Emergence of soybeans planted in Rhizoctonia solani infested soil treated with SA- 13 grains either incorporated into the soil or surface treated.

Treatment Emergence (%)

Non-treated 0.0

2.0 mg/ml soil incorporation 1.4

4.0 mg/ml soil incorporation 9.7

6.0 mg/ml soil incorporation 4.2

2.8 mg/cm ² surface treatment 0.0

28.0 mg/cm surface treatment 33.3

56.0 mg/cm surface treatment 31.3

When incorporated into the soil or surface apllied, M. albus SA-13 grown on barley grains increased emergence of soybean seeds.

B. Muscodor albus isolate SA-13 grown on barley grains from a culture grown in potato dextrose broth was evaluated for its efficacy in controlling Rhizoctonia solani on soybean.

Muscodor albus isolate SA-13 was further evaluated for its efficacy in controlling

Rhizoctonia solani on soybean.

To culture the Muscodor strain, the barley grains were washed with tap water, and soaked for 24 hours. The water was drained off and the grains were rewashed three more times before splitting them evenly between autoclave bags with foam stoppers. The grains were then autoclaved for 15 minutes at 121 °C twice. Once the grains cooled, each bag was inoculated with 5 ml of a 7 day old culture of Muscodor albus strain SA-13 grown in potato dextrose broth. The fungus was grown for 10 days in the bags at 25 °C. The masses of inoculated barley grains were then broken up and allowed to air-dry until seed moisture was < 10%.

Rhizoctonia inoculum was mixed into artificial soil media at a rate of 1:1400. The inoculated media was divided up and the Muscodor albus SA-13 barley grain was mixed into the soil media at the rates 0.74 g/L, 7.36 g/L, and 22.07 g/L. The soil media was placed in pots, with 3 pots per replicate and 3 replicates per treatment, including an inoculated treatment without Muscodor albus SA-13 and a non-inoculated treatment without Muscodor albus SA-13. The pots were watered with 200 ml tap water, randomized in a complete block design, and placed under fluorescent lights for six days. Soybean seeds were then planted 1 cm deep at 9 seeds per pot.

The soybean emergence, above ground height, and above ground fresh weight per rep after ten days was determined by counting the number of emerged seedlings within each rep, cutting off the stems at the soil line, and measuring and weighing them. The results are given in Table 14B. The percent emergence, seedling height, and weight per rep were significantly greater for the highest rate than the inoculated treatment that did not contain Muscodor albus SA-13.

Table 14B. Emergence, height, and fresh weight of soybeans planted in Rhizoctonia solani

(Rhiz. infested soil treated with SA-13 grains.

M. albus Average % Average Average

Rhiz.:soil

SA-13 g/L Emergence Height (cm) Weight/Rep (g)

0.0 0 90.1 A* 15.9 A 25.9 A

0.0 1:1400 25.9 C 7.1 D 4.8 C

0.74 1:1400 1.2 D 5.5 ABC 0.4 D

7.36 1:1400 17.3 CD 5.9 BD 3.7 CD

22.07 1:1400 63.0 B 11.9 C 14.2 B

: Data with the same letter are not significantly different from each other according to Fisher's Protected LSD at p=0.05 level.

Study 9. Use of M. albus SA-13 for controlling strawberry postharvest diseases.

Muscodor albus strain SA-13 was further evaluated for its use in controlling post-harvest disease caused by Botrytis cinerea.

To culture the Muscodor strain, barley grains were washed three times with tap water, and soaked for 24 hours at 20 °C. The grains were rewashed and the water was drained off before splitting the grains evenly between autoclave bags with foam stoppers. The grains were then autoclaved for 15 minutes at 121 °C two times. Once the grains cooled, each bag was inoculated with 10 ml of a 7-day culture of Muscodor albus strain SA-13 grown in potato dextrose broth. The fungus was grown in the bags for 11 days at 25 °C. The masses of inoculated barley grains were then broken up and allowed to air-dry until seed moisture was < 10%. The grains were rehydrated with water and allowed to grow in a humid environment for four days prior to use.

The B. cinerea inoculum was prepared by flooding a mature culture in an agar plate with sterile water, filtering out any mycelia, and adjusting the concentration to approximately 10 ⁵ conidia/ml. Organic strawberries were washed with tap water, surface sterilized, and rinsed three times with sterile water. After allowing the fruit surface to dry, each fruit was dipped in the inoculum for 5 seconds and placed on a rack in a crisper box. After all fruits were inoculated, the rehydrated M. albus SA-13 barley grains were placed inside the crisper boxes, which were then flooded with a small amount of water, sealed with tape, and placed in darkness at -25 °C for 6 days. There were three different doses of M. albus SA-13 grown barley grains and a control box that did not contain any M. albus SA-13.

The severity of the disease was determined by estimating the precentage coverage of the pathogen growth on each strawberry fruit. The results are given in Table 15.

Table 15. Mycelia and rot coverage on strawberries inoculated with Botrytis and treated with

SA-13 grains.

M. albus (g) Mycelia (%) Rot (%)

0.0 65.0 A* 75.5 A

1.0 0.0 B 46.5 B

5.0 0.0 B 9.0 C

10.0 0.0 B 15.0 C

*: Data with the same letter are not significantly difference according to Fisher Protected LSD at p=0.05 level.

When contained in a sealed box, M. albus SA-13 grown on barley grains completely inhibited the development of Botrytis mycelia growth and reduced the development of rot on strawberries.

Study 10. Use of M. albus SA-13 for controlling citrus fruit postharvest diseases

Muscodor albus strain SA-13 was further evaluated for its inhibitive effect on postharvest pathogen Penicillium digitatum.

The grains and method of rehydrating the grains used in Study 9 were also used in this study.

The Penicillium inoculum was prepared by flooding a mature culture in an agar plate with sterile water, filtering out any mycelia, and adjusting the concentration to approximately 10 ⁶ conidia/ml. Using a 5-mm diameter borer, skin deep lesions were made on organic navel oranges. The oranges were then washed with tap water, surface sterilized, and rinsed three more times with sterile water. After allowing the fruit surface to dry, each lesion was inoculated with 15 μΐ of inoculum. The fruit were stored in crisper boxes with grains as described in Study 9. The severity of the disease was determined by measuring the widest diameter of the rotting region on the surface of each orange. The results are given in Table 16A.

Table 16A. Control of fruit rot of citrus caused by the Penicillium sp..

M. albus (g) Lesion (ø mm) ^*

0.0 16.8 A

1.0 8.4 B

5.0 10.6 B

10.0 5.6 B

: Data with the same letter are not significantly different from each other according to Fisher's Protected LSD at p=0.05 level.

When contained in a sealed box, the barley grains grown with M. albus SA-13 reduced the development of Penicillium rot on citrus.

Study 11. Use of M. albus SA-13 for controlling potato postharvest diseases.

Muscodor albus strain SA-13 was further evaluated for its use in controlling post-harvest disease caused by Pectobacterium carotovorum.

To culture the Muscodor strain, barley grains were washed three times with tap water, and soaked for 24 hours at 20 °C. The grains were rewashed and the water was drained off before splitting the grains evenly between autoclave bags with foam stoppers. The grains were then autoclaved for 15 minutes at 121 °C two times. Once the grains cooled, each bag was inoculated with 10 ml of a culture of Muscodor albus strain SA-13 grown in potato dextrose broth. The fungus was thoroughly grown in the bags at 25 °C. The masses of inoculated barley grains were then broken up and allowed to air-dry. The grains were rehydrated with water and allowed to grow in a humid environment for five days prior to use.

The P. carotovorum inoculum was prepared by flooding a one day old culture in an agar plate with sterile water, centrifuging it, resuspending the pellet in new sterile water, and adjusting the OD ₆ oo to 0.1.

Russet potatoes were washed with tap water and cut into 4-5 cm pieces without the skin. They were surface sterilized and rinsed three times with sterile water. After allowing the potato pieces to dry, the pieces were placed on a rack in a crisper box and 5 μΐ of the bacterial inoculum was placed in the center of the top side of each of the pieces. After all fruits were inoculated, the rehydrated M. albus SA-13 barley grains were placed inside the crisper boxes, which were then flooded with a small amount of water, sealed with tape, and placed in darkness at -25 °C for 5 days. There were three different doses of M. albus SA-13 grown barley grains and a control box that did not contain any M. albus SA-13.

The severity of the disease was determined by cutting cross sections of the pieces perpendicular to the flat inoculated surface. Three measurements of the rot were taken: two radii of the flat surface at ninety degrees apart, and the depth. The rot volumes of approximately half ellipsoid were calculated and ranked from lowest rot volume to highest. One-way ANOVA was used to compare the ranks. The results are given in Table 16B. The average rankings were significantly lower for the potatoes treated at the two higher rates with M. albus SA-13 than the control and lowest rate.

Table 16B. Rankings of the rot volumes on potatoes inoculated with Pectobacterium and treated with SA-13 grains.

M. albus (g) Ranked Volumes

0.0 22.1 A*

1.0 21.3 A

5.0 9.2 B

10.0 13.4 B

: Data with the same letter are not significantly different from each other according to Fisher's Protected LSD at p=0.05 level.

Example 6. Nematicidal Effects of Muscodor albus Strains SA-13 and CZ 620

Study 1. Evaluation of nematicidal activity of Muscodor albus SA-13 on PDA.

The 10-cm split petri dishes with PDA were used for evaluating the mortality effect of the strains on plant parasitic nematodes Meloidogyne spp. The nematodes used in the tests were a mixed culture of M. incognita and M. hapla maintained on tomato roots in the growth room. There was one plate for each of Muscodor strain, and one without Muscodor as the blank control for the nematodes.

A 5-mm PDA plug of each isolate was placed 2.5 cm away from the outer edge of one side of the split petri dish plate. The plates were sealed and isolates were allowed to grow for four days at room temperature (about 25 °C). An aliquot of 62 μΐ second stage juveniles (J2s) suspension, obtained by adding water into newly hatched Meloidogyne spp. J2 from eggs and containing about 14-15 J2s per aliquot, was placed at 1.5 cm away from the outer edge of the other side of the split plate. The plates were sealed with parafilm and put in a sealed container. The plates were incubated in darkness at room temperatures before taking any data. The mortality of the J2s was recorded under a dissecting microscope at 24, 48, 72 and

144 hours after incubation. In general, there was a trend with increasing percentage of mortality

(10 to 100%) of nematode J2s after 24 hours of co-incubation with Muscodor strains. The percentage of mortality of nematode J2s with time is shown in Table 17.

Table 17. Mortality effects of Muscodor albus strain SA-13 on plant parasitic nematodes of

Meloidogyne spp.

Muscodor albus SA-13 showed mortality effects on the tested Meloidogyne spp. nematodes.

Study 2. A repeated evaluation of nematicidal activity of M. albus SA-13.

A repeated test of Study 1 was conducted with two plates of each Muscodor strain, two without Muscodor as blank controls and two with 1% Avid 0.15 EC (Syngenta Crop Protection, Inc., Greensboro, NC) as positive controls. The 10-cm split petri dishes with PDA were used for evaluating the mortality effect of the strains on plant parasitic nematodes of Meloidogyne incognita. The nematode was cultured on tomato root with a southern California origin.

A 5-mm PDA plug of each isolate was placed 2.5 cm away from the outer edge of one side of the split petri dish plate. The plates were sealed and isolates was allowed to grow for four days at room temperature (about 25°C). An aliquot of 80 μΐ second stage juveniles (J2s) of M. incognita suspension, obtained by adding water into newly hatched of J2s from eggs and comprised of around 5-20 J2s, was placed at 1.5 cm away from the outer edge of the other side of the split plate. A mixture of an 80 μΐ aliquot of J2 suspension with the same amount of 2% Avid served as positive control in a split plate without any grains. The plates were sealed with parafilm. All plates with the same Muscodor strains were put in a plastic bag and zipped to avoid the potential mixing effects of volatiles released from different strains. Plates in individual bags were put in a sealed container and incubated in darkness at room temperatures before taking any data.

The total number of nematode J2s in each aliquot of the nematode suspension, and the number of the nematodes J2s that showed mortality in each aliquot, was recorded under a dissecting microscopes at 24, 48, 72 and 144 hours after incubation. The percentage of J2 mortality was then calculated. In general, there was a trend with an increasing percentage of mortality (0 to 87%) of nematode J2s after 24 hours of co-incubation with Muscodor strains. The strain SA-13 showed superior mortality effect on the M. incognita J2s when compared with the strain M. albus CZ-620 isolated from cinnamon tree. The effects of Muscodor albus strains SA- 13 and CZ 620 on nematode J2s mortality are shown in Table 18.

Table 18. Mortality effects of Muscodor albus strain SA-13 and CZ 620 on plant parasitic nematodes of Meloidogyne incognita.

Study 3: Evaluation of nematicidal activity of Muscodor albus SA-13 on barley grains.

The nematodes used in the test were a mixed culture of M. incognita and M. hapla maintained on tomato roots. There was one plate for each Muscodor strain, and one without Muscodor strains as a blank control. To culture the Muscodor strains, the barley grains were washed more than three times with deionized water, and soaked for 24 hours at 20 °C. The water was drained off before splitting the grains evenly between autoclave bags with foam stoppers. The grains were then autoclaved for 15 minutes at 121 °C twice. Once the grains cooled, several small plugs of each Muscodor albus strain were added to each bag and leaving one bag non- inoculated as blank control. The foam stoppers were replaced with autoclaved rubber stoppers after putting the Muscodor plugs in. The Muscodor strains were grown in the bags for 13 days at room temperature. The masses of inoculated barley grains were periodically broken up so the grains were less stuck together.

Half of each split plate was filled with nematode J2 suspension and the other half was filled with 20 ml barley grains grown with Muscodor strains or with non-inoculated grains as blank controls. An aliquot of 62 μΐ J2 suspension, obtained by adding water into newly hatched Meloidogyne spp. J2 from eggs and containing around 14-15 J2s per aliquot, was placed at 1.5 cm away from the outer edge of the other side of the split plate. The plates were sealed with parafilm and put in a sealed container. The plates were incubated in darkness at room

temperatures before taking any data.

The mortality of the J2s was recorded under a dissecting microscope at 24, 48, 72 and 144 hours after incubation. In general, there was a trend with increasing percentage of mortality (0 to 100%) of nematode J2s after 24 hours of co-incubation with Muscodor strains. The strain SA-13 showed superior mortality effect on the nematode J2s. Comparatively, the strain M. albus CZ 620 was less effective on killing the nematode J2s even after 144 hours. The percentage of mortality of nematode J2s as a function of time is shown in Table 19.

Table 19. Mortality effects of Muscodor albus strain SA-13 and CZ 620 on plant parasitic nematodes of Meloidogyne spp.

Study 4: A repeated evaluation of nematicidal activity of M. albus SA-13 on barley grains.

A repeated test described in Study 3 was conducted with two plates of each of Muscodor strain, two without Muscodor as blank controls and two with 1% Avid as positive controls.

Muscodor albus strains were further evaluated for their mortality effect on plant parasitic nematodes of Meloidogyne incognita with barley grain medium.

To culture the Muscodor strains, the barley grains were washed more than three times with deionized water, and soaked for 24 hours at 20 °C. The water was drained off before splitting the grains evenly between autoclave bags with foam stoppers. The grains were then autoclaved for 15 minutes at 121 °C twice. Once the grains cooled, several small plugs of each Muscodor albus strain were added to each bag and leaving one bag non-inoculated as blank control. The foam stoppers were replaced with autoclaved rubber stoppers after putting the Muscodor plugs in. The Muscodor strains were grown in the bags for 13 days at room

temperature. The masses of inoculated barley grains were periodically broken up so the grains were less stuck together.

Half of each split plate was filled with nematode J2 suspension and the other half was filled with 20 ml barley grains grown with Muscodor strains or with non-inoculated grains as blank controls. A mixture of 80 μΐ aliquot of J2 suspension with the same amount of 2% Avid served as positive control in a split plate without any grains.

An aliquot of 80 μΐ J2s suspension, obtained by adding water into newly hatched M. incognita J2 from eggs and comprised of around 14-15 J2s per aliquot, was placed at 1.5 cm away from the outer edge of the other side of the split plate. All plates were sealed with parafilm. The plates with the same Muscodor strains were put in one plastic bag and zipped to avoid the potential mixing effects of volatiles released from different strains. The plates were sealed with Parafilm and put in a sealed container. The plates were incubated in darkness at room temperatures before taking any data.

The total number of nematode J2s in each aliquot of the nematode suspension, and the number of the nematodes J2s showing mortality was recorded at 24, 48, 72 and 144 hours after incubation under a dissecting microscope. The percentage of J2 mortality was calculated. In a general trend, nematode J2s showed increasing percentage of mortality (27 to 92%) after 24 hours of co-incubation with Muscodor strains. The strain SA-13 showed superior mortality effect on the M. incognita J2s when compared with the original strain M. albus 620 isolated from cinnamon tree. The percentage of mortality of nematode J2s as a function of time is shown in Table 20.

Table 20. Mortality effects of Muscodor albus strain SA-13 and CZ 620 on plant parasitic nematodes of Meloidogyne incognita

Study 5: Nematicidal activity of the artificial mix of seven VOCs from M. albus SA-13.

The seven volatile organic compounds (VOCs) that were trapped using XAD7 resin were reconstituted. Samples were prepared by artificially mixing the seven compounds. The eggs of root-knot nematodes (Meloidogyne incognita) were extracted from tomato plants that were inoculated and incubated in the greenhouse for about two months. The eggs extracted were set to hatch into second stage juveniles (J2s) on two layers of Kimwipe paper supported by wire mesh on a plastic beaker. The suspension of J2s were collected and diluted to 300 J2s/100 μΐ of deionized water.

Filter paper was cut to fit the petri dish (95 mm x 15 mm) and moistened with deionized water. A single well concavity slide and a cap of 2 ml centrifuge tube were placed in the petri dish. 100 μΐ of the J2s suspension was pipetted onto the well of concavity slide. 0 μ 1, 25 μ 1, 75 μ 1, 100 μΐ, and 125 μΐ of samples were pipetted onto the centrifuge tube cap. A 75 μΐ of deionized water was used as a negative control. Four caps of 15 ml centrifuge tubes (about 11 cm ³ ) in one petri dish were used for holding barley grains that were colonized by M. albus SA-13 as a positive control. Each treatment had two replicates. The petri dishes were capped and sealed with two layers of Parafilm. All petri dishes were placed in a plastic box. The box was sealed with marking tape, covered with aluminum foil, and incubated at around 26.7 °C for 48 hours.

After 48-hour incubation, the nematode drops on concavity slides were observed under a stereoscope. The percentage of immobilized J2s was visually scored on a scale of 0 to 100% (Table 21). Curled or moving J2s were considered mobile; straight J2s were considered immobile.

Table 21. Immobility of Meloidogyne incognita J2s in concavity slide 48 hours after exposure to

the artificial mix of VOCs from M. albus SA-13.

: Data are means + standard deviations (SD) with two replicates. Data without SD were data from one replicate because solution in one of the replicates evaporated and nematodes died prematurely. The values followed by a different letter in the same column indicate significant difference between treatments at p = 0.05 according to Fisher's LSD test.

After the percentage of immobilized J2s in concavity slide was scored, 20 μΐ drops from the concavity slide were pipetted onto the surface of 1.2% water agar in 6-well plates. After the drop dried in a fume hood, a circle was drawn around the boundary of the 20 μΐ drop of the J2s at the bottom of the 6-well plate. The number of J2s in the circle was counted and recorded as the total J2s. The plates were left at room temperature (about 25 °C) for 24 hours. Then the number of J2s still remaining in the circle was counted and recorded as the immobile J2s. The percentage of immobilized J2s was calculated as immobile J2s/total J2s x 100 (Table 22). For treatments with 25 μΐ, 75 μΐ, and 125 μΐ of synthetic compounds, only 0-2 J2s were transferred on the the agar. Any transferred J2s of those samples did not move out of the circle. Table 22. Immobility of Meloidogyne incognita J2s on 6-well plate 24 hours after exposure to the artificial mix of seven VOCs from Muscodor albus SA-13.

1 rcalmcnl I mmobilin (' t )

0.0 μΐ VOC sample 23.6+2.0 c*

25.0 μΐ VOC sample 100.0+0.0 a

75.0 μΐ VOC sample 100.0 ab

100.0 μΐ VOC sample 100.0 ab

125.0 μΐ VOC sample -

75.0 μΐ deionized water 23.0+6.9 c

11.0 cm ³ M.albus SA-13 grown

66.3+23.1 b

barley grains

: Data are means + standard deviations (SD) with two replicates. Data without SD are data from one replicate because one of the replicates had no nematodes successfully transferred onto the 6- well plate. The values followed by a different letter in the same column indicate significant difference between treatments at p=0.05 level according to Fisher' s LSD test.

No nematodes were successfully transferred to evaluate the immobility.

Meloidogyne incognita J2s became straight, characteristic to dead nematodes, in the presence of the reconstituted VOCs for 48 hours. They failed to recover in the absence of the VOC mixture after 24 hours.

Study 6: Nematicidal activity of Muscodor albus SA-13 grown barley grains.

Eggs and J2 of root-knot nematodes M. incognita were extracted and prepared in the same way as described previously in Study 5. Dry ground barley inoculated with M. albus SA13 was rehydrated with water for 24 hours at room temperature before test. Filter paper was cut to fit undivided petri dish (95 mm - 15 mm) or divided petri dish of the same size. The filter paper was moistened with deionized water. A single well concavity slide holding 100 μΐ of J2 suspension and a 35-mm petri dish holding 0.05 g, 0.1 g, and 0.5 g of wet ground barley with M. albus SA-13 were placed in the 95 mm x 15 mm undivided petri dish. A blank 35-mm petri dish without ground barley was set for the negative control. For 1 g and 2 g of wet ground barley grains, divided petri dishes without 35-mm petri dish were used to correlate increasing surface area of ground barley with increasing mass. Each treatment had two replicates. All petri dishes were sealed with two layers of Parafilm and placed in a plastic box. The box was sealed with marking tape, covered with aluminum foil, and incubated at about 26°C for 48 hours. After 48-hour incubation, the nematode drops on the concavity slide were observed under a stereoscope. The percentage of immobilized J2s was visually scored based on a scale of 0 to

100% (Table 23). Curled or moving J2s were considered mobile; straight J2s were considered immobile. One replicate of 0.05 g ground barley did not show any nematicidal activity because the surface of the ground barley showed no indication of growth of M. albus SA-13.

Table 23. Immobility of Meloidogyne incognita J2s on concavity slide 48 hours after exposure to the ground barley grains with Muscodor albus SA-13.

1 rc lmcnl I mmobilin (' t )

Check, barley grains 0.0 + 0.0 c*

0.05 g M. albus SA-13 barley gra dns 27.5 + 31.8 be

0.1 g M. albus SA-13 barley grai ns 72.5 + 3.5 be

0.5 g M. albus SA-13 barley grai ns 60.0 + 14.1 ab

1.0 g M. albus SA-13 barley grai ns 80.0 + 7.1 a

2.0 g M. albus SA-13 barley grai ns 90.0 + 0.0 a

: Data are means + standard deviations (SD) with two replicates. The values followed by a different letter in the same column indicate significant difference between treatments at p=0.05 level according to Fisher' s LSD test.

After the percentage of immobilized J2s in petri dish was scored, 20 μΐ drops from the concavity slide were pipetted onto the surface of 1.2% water agar in 6-well plates. After the drop dried in a fume hood, a circle was drawn around the boundary of the 20 μΐ drop of the J2s at the bottom of the 6-well plate. The number of J2s in the circle was counted and recorded as the total

J2s. The plates were left at room temperature (about 25 °C) for 24 hours. Then the number of J2s still remaining in the circle was counted and recorded as the immobile J2s. The percentage of immobilized J2 was calculated as immobile J2s/total J2s x 100 (Table 24).

Table 24. Immobility of Meloidogyne incognita J2s on 6-well plate with 24-hour exposure to the ground barley grains with Muscodor albus SA-13

1 i Hl menl I mmohilitv C r )

Check, barley grains 20.6 + 1.7 c*

0.05 g M. albus SA-13 barley gn lins 41.0 + 34.1 be

0.1 g M. albus SA-13 barley grai ns 73.1 + 19.5 ab

0.5 g M. albus SA-13 barley grai ns 77.6 + 2.5 ab

1.0 g M. albus SA-13 barley grai ns 80.9 + 2.5 ab

2.0 g M. albus SA-13 barley grai ns 90.7 + 10.9 a : Data are means + standard deviations (SD) with two replicates. The values followed by a different letter in the same column indicate significant difference between treatments at p=0.05 level according to Fisher's LSD test.

In summary, M. incognita J2s became straight, characteristic to dead nematodes, in the presence of M. albus SA-13 on rehydrated ground barley grains. Most J2s failed to move out of the circle in the absence of the barley after 24 hours though the bodies of J2 were curved.

Example 7: Insecticidal Effect of Muscodor albus SA-13

Study 1. Effect of M. albus SA-13 on armyworm eggs.

Two small petri dishes containing approximately 20 g of autoclaved barley grains grown with M. albus were placed in a plastic box (approximately 2800 cm in volume). A companion box was set up at room temperature without the petri dishes of fungus. Then 48- well microtitre plates containing beet armyworm (Spodoptera exigua) eggs that had been overlaid onto artificial diet were introduced into each box. After three days, the eggs in the box without the M. albus

SA-13 began to hatch for 48.0%. The armyworm eggs did not hatch (0.0%) in the box containing the barley culture of M. albus SA-13.

Study 2. Re-evaluation of M. albus SA-13 on armyworm eggs.

Petri dishes containing different amounts (1 g, 5 g, and 10 g) of M. albus SA-13 grown barley grains were placed in plastic boxes (approximately 2800 cm in volume). A separate box was set up at room temperature without the petri dishes of the fungus. Then 48-well microtitre plates containing beet armyworm (Spodoptera exigua) eggs that had been overlaid onto artificial diet were introduced into each box. After three days, the eggs in the box without the M. albus SA-13 began to hatch. After 6 days, each of the 48-well microtitre plates was evaluated for hatching rates.

The number of hatched larvae exposed to 0, 1, 5, and 10 g of M. albus SA-13 barley grains were 82.0, 96.0, 38.0 and 0.0, respectively. The M. albus SA-13 inhibited the hatching of armyworm eggs. Example 8: Enhanced effect by the combination of M. albus SA-13 and Trichoderma spp.

Study 1. Control of soilborne diseases by combined use of M. albus SA-13 and

Trichoderma spp. in soil

Muscodor albus isolate SA-13 was evaluated for enhanced efficacy in controlling Rhizoctonia solani on soybean when combined with Trichoderma spp.

To culture the Muscodor strain, the barley grains were washed with tap water, and soaked for 24 hours. The water was drained off and the grains were rewashed three more times before splitting them evenly between autoclave bags with foam stoppers. The grains were then autoclaved for 15 minutes at 121 °C twice. Once the grains cooled, each bag was inoculated with 5 ml of a 7 day old culture of Muscodor albus strain SA-13 grown in potato dextrose broth. The fungus was grown for 10 days in the bags at 25 °C. The masses of inoculated barley grains were then broken up and allowed to air-dry until seed moisture was < 10%.

Rhizoctonia inoculum was mixed into artificial soil media at a rate of 1:1600. Muscodor albus SA-13 barley grain was mixed into one half of the soil media at 22.07 g/L. The soil media was placed in pots, with 3 pots per replicate and 3 replicates per treatment, including an inoculated treatment without Muscodor albus SA-13 and a non-inoculated treatment without Muscodor albus SA-13. Soybean seeds were then planted 1 cm deep at 9 seeds per pot. The pots were watered with 100 ml tap water, then received either a water drench or a Rootshield ^® Home and Garden (Trichoderma harzianum Rifai strain T-22) drench at 50 ml/500 ml soil. Rootshield ^® Home and Garden was drenched at 15 g/L and 45 g/L alone and in combination with Muscodor albus SA-13. The pots were arranged in a randomized complete block design, and placed under fluorescent lights with a 12 hr photo period for ten days.

The soybean emergence, above ground height, and above ground fresh weight per was determined after ten days by counting the number of emerged seedlings within each rep, cutting off the stems at the soil line, and measuring and weighing them. The results are given in Table 25. When Rootshield ^® Home and Garden was combined with MBI-601, the emergence tended to be greater than the emergence of either product alone and the combinations were similar to the non-inoculated, non-treated treatment. This was a surprising effect because Muscodor has a much slower growth rate and Trichoderma would have been expected to outcompete Muscodor in the soil. Table 25. Emergence, height, and fresh weight of soybeans planted in Rhizoctonia solani (Rhiz.) infested soil treated with SA-13 grains.

M. albus Rootshield Emergence

Weight (g) Height (cm)

SA-13 g/L g/L (%)

Non-inoculated and

non-treated 81 A* 20.3 A 13.9 A

0.0 0.0 33 D 5.8 C 5.6 C

22.07 0.0 40 CD 6.2 C 7.1 C

0.0 15 52 BCD 9.2 BC 6.8 C

0.0 45 67 AB 13.1 B 9.2 B

22.07 15 57 BC 8.9 BC 5.8 C

22.07 45 79 A 12.5 B 7.2 C

: Data with the same letter are not significantly different from each other according to Fisher's Protected LSD at p=0.05 level.

Study 2. Control of soilborne diseases by successional use of M. albus SA-13 and

Trichoderma spp. in soil

Muscodor albus isolate SA-13 was evaluated for enhanced efficacy in controlling Rhizoctonia solani on soybean when used as a pre-treatment to Trichoderma spp application(s).

To culture the Muscodor strain, the barley grains were washed with tap water, and soaked for 24 hours. The water was drained off and the grains were rewashed three more times before splitting them evenly between autoclave bags with foam stoppers. The grains were then autoclaved for 15 minutes at 121 °C twice. Once the grains cooled, each bag was inoculated with 5 ml of a 7 day old culture of Muscodor albus strain SA-13 grown in potato dextrose broth. The fungus was grown for 10 days in the bags at 25 °C. The masses of inoculated barley grains were then broken up and allowed to air-dry until seed moisture was < 10%.

Rhizoctonia inoculum was mixed into artificial soil media at a rate of 1:1600. Muscodor albus SA-13 barley grain was mixed into one half of the soil media at 22.07 g/L. The soil media was placed in pots, with 3 pots per replicate and 3 replicates per treatment, including an inoculated treatment without Muscodor albus SA-13 and a non-inoculated treatment without Muscodor albus SA-13. The pots were each watered with 200 ml tap water, arranged in a randomized complete block design, and placed under fluorescent lights with a 12 hour photo period for six days. Soybean seeds were then planted 1 cm deep at 9 seeds per pot. The pots received either a water drench or a Rootshield ^® Home and Garden {Trichoderma harzianum Rifai strain T-22) drench at 50 ml/500 ml soil. Rootshield Home and Garden was drenched at 15 g/L and 45 g/L alone and in combination with Muscodor albus SA-13. The pots were placed back under the fluorescent lights for eight days.

The soybean emergence, above ground height, and above ground fresh weight per rep after eight days was determined by counting the number of emerged seedlings within each rep, cutting off the stems at the soil line, and measuring and weighing them. The results are given in Table 26. When pretreated with a low rate of Rootshield ^® Home and Garden and MB 1-601, the emergence, fresh weight/rep, and plant height tended to be greater than that of either product alone. The combination of these two products was similar to the non-inoculated, non-treated treatments. This again was a surprising effect because Muscodor has a much slower growth rate and Trichoderma would have been expected to outcompete Muscodor in the soil.

Table 26. Emergence, height, and fresh weight of soybeans planted in Rhizoctonia solani (Rhiz.) infested soil treated with SA-13 grains.

M. albus Rootshield ⁸ Emergence

Weight (g) Height (cm)

SA-13 g/L g/L (%)

Non-inoculated and

non-treated 81 A* 17.9 A 8.8 AB

0.0 0.0 21 D 3.4 E 5.2 C

22.07 0.0 43 CD 6.7 DE 5.5 C

0.0 15 56 BC 10.9 CD 8.0 B

0.0 45 67 AB 12.9 BC 7.7 B

22.07 15 74 AB 16.1 AB 9.3 A

22.07 45 69 AB 14.5 ABC 8.0 B : Data with the same letter are not significantly different from each other according to Fisher's Protected LSD at p=0.05 level.

Example 9: Muscodor albus SA-13 Increases Plant Vigor and Product Yield Muscodor albus SA-13 grown on barley grains was evaluated for phytotocixity, growth promotion, efficacy, yield benefit, and application rates by controlling strawberry charcoal rot.

Trial and Treatment. The field trial was conducted in Arroyo Grande, California. The plots were treated on November 21, 2012, and strawberry bare root plants were planted on November 26, 2012. Each treatment comprised 4 replicates of 25 feet (length) x 24 inch bed top per plot with a randomized complete block design.

Treatments included the following:

1. Untreated control

2. Standard control (Pic-Chlor 60 EC [SOURCE], drip fumigated @25 gallons per acre (GPA)

3. Muscodor albus SA-13 @250 lb/acre

4. Muscodor albus SA-13 @500 lb/acre

5. Muscodor albus SA-13 @ 1000 lb/acre

6. Muscodor albus SA-13 @3000 lb/acre

The Muscodor albus SA-13 product was band applied and incorporated in soil at about 0.5 foot with a rotor tiller.

Disease Inoculation. The strawberry field was known to have Macrophomina infection (charcoal rot).

Trial Maintenance. To control powdery mildew, the fungicide Rally (Dow

AgroSciences, Indianapolis, IN) was applied at 3.0 oz/A on February 20, 2012, and June 5, 2013; Quintec (Dow AgroSciences, Indianapolis, IN) was applied at 4 fl. oz/A on February 6, 2012 and March 10 2013; and Procure (Chemtura USA Corporation, Middlebury, CT) was applied at 5 oz/A on May 8, 2013.Botrytis was controlled by Pristine (BAS Corporation, Research Triangle Park, NC), which was applied at 20 oz/A on April 17, 2013 and June 15, 2013, and Switch (Syngenta Crop Protection, Greensboro, NC), which was applied at 1.0 lb/A on May 20, 2013. Mites were controlled by Oberon (Bayer CropScience, Research Triangle Park, NC), which was applied at 14.0 oz/ A on March 29 and April 17, 2013, and Zeal (Valent U.S.A. Corporation, Walnut Creek, CA), which was applied at 2.0 oz/A on April 7,2013. Thrips were controlled by Radiant (Dow AgroSciences, Indianapolis, IN), which was applied at 8.0 oz/A on April 24, 2013, Provado (Bayer CropScience, Research Triangle Park, NC), which was applied at 8.0 fl. oz/A on May 18, 2013, amd Entrust (Dow AgroSciences, Indianapolis, IN), which was applied at 1.0 lb/A on May 20, 2013. Lygus was controlled by Assail (United Phosphorus, Inc., King of Prussia, PA), which was applied at 1.7 oz/A on June 5, 2013 and Rimon (Chemtura USA Corporation, Middlebury, CT), which was applied at 12.0 oz/A on June 15, 2013. Data Recording. Plant vigor was assessed every 6 weeks for visible plant stress and disease symptoms. Vigor was rated on a scale of 0-10, with 0 as no growth and 10 as exhibiting the best growth, juged by foliar color and size. Fruits were harvested every 3-7 days and yield was assessed by the number and weight of marketable and unmarketable fruit at each harvest (12-18 pickings from March- June 2013). Yield was calculated per acre, marketable fruit from total fruit, and gross return per acre of marketable fruit. Crop safety assessment data were collected at 14 and 28 days after planting and phytotoxicity was rated on a scale of 0-10, with 0 as no injusry and 10 as exhibiting the most severe injury as determined by foliar symptoms. Data was analysized with ARM software (version 8.0, Gylling Data Management, Inc., SD) and means were compared with Fisher's Least Significant Difference (LSD) at p=0.05 level.

Results. Results from the strawberry trial were collected up to June 18, 2013. As shown in Table 27, there was no phytotoxicity at 14 days and 28 days post-planting, even when treated at the highest rate of 3000 lb/a of Muscodor albus SA-13.

Table 27. Phytotocixity (on a scale of 0-10) of strawberry plants treated with Muscodor albus

SA-13 at 14 days and 28 days post-planting

*: Data with the same letter in a column are not significantly different accoding to Fisher's LSD test at p=0.05 level.

As shown in Table 28, initial vigor rating showed somewhat better growth of Muscodor albus SA-13-treated plants. By May 23, 2013, about 25 weeks post-planting, the vigor of Muscodor albus SA-13 was significantly better than the untreated control at all treatment rates. Table 28. Vigor (on a scale of 0-10) of strawberry plants treated with Muscodor albus SA-13 compared to untreated control and Pic-Clor 60 EC in 2013.

*: Data with the same letter umn are not significantly different accoding to Fisher's LSD test at p=0.05 level.

As shown in Table 29, Muscodor albus SA-13 increased the yield of strawberries. However, treatment at the highest rate of 3000 lb/a did not show a statistically significant difference in yield compared to those treated at lower rates.

Table 29. Cumulative marketable yield and their equivalent yield in commercial flats of strawberries from Muscodor albus SA-13 treated plants up to June 18, 2013.

*: Data with the same letter in a column are not significantly different accoding to Fisher's LSD test at p=0.05 level.

DEPOSIT OF BIOLOGICAL MATERIAL

The following biological material has been deposited under the terms of the Budapest Treaty with the Agricultural Research Culture Collection (NRRL), 1815 N. University Street, Peoria, Illinois 61604 USA, and given the following number:

Deposit

Muscodor albus Strain SA-13 NRRL B-50774 August 31, 2012

The strain has been deposited under conditions that assure that access to the culture will be available during the pendency of this patent application to one determined by the Commissioner of Patents and Trademarks to be entitled thereto under 37 C.F.R. §1.14 and 35 U.S.C. §122. The deposit represents a substantially pure culture of the deposited strain. The deposit is available as required by foreign patent laws in countries wherein counterparts of the subject application, or its progeny are filed. However, it should be understood that the availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by government action.

The invention described and claimed herein is not to be limited in scope by the specific aspects herein disclosed, since these aspects are intended as illustrations of several aspects of the invention. Any equivalent aspects are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. In the case of conflict, the present disclosure including definitions will control.

Various references are cited herein, the disclosures of which are incorporated by reference in their entireties.