CROSS-REFERENCE TO RELATED APPLICATIONS

[001] This application claims the benefit of priority of U.S. Provisional Patent Application No. 62/829,104 titled "METHOD FOR IDENTIFYING A FUNGAF SYMBIONT", filed April 4, 2019, the contents of which are incorporated herein by reference in their entirety.

FIELD OF INVENTION

[002] The present invention, in some embodiments, is in the field of microbiology.

BACKGROUND

[003] The root microbiome is important in plant health and it is becoming increasingly clear that the plant is able to control the composition of its microbiome. Evidence is accumulating that plants call for microbial help in time of need. The composition of the surrounding microbial population is greatly determined by soil quality. The high functional redundancy in soil microbial diversity confers protection against soil-borne diseases. The increase in microbial diversity produces a balanced microbiome that does not allow pathogens to flourish.

[004] Fungi are an important component in the plant microbiome as they play a role in plant health and productivity. Trichoderma species (spp.) are non-pathogenic soil-borne (free-living) fungi that colonize the roots of many plants. Members of the genus Trichoderma are known as biocontrol agents of plant pathogens as they are strong mycoparasites and extensive plant colonizers (rhizosphere competent). These plant symbionts provide host fitness enhancements under biotic and abiotic stresses. The broad benefits provided by the plant -Trichoderma interaction include: (a) increased resistance to abiotic stresses, such as drought and salinity, (b) increased plant growth, (c) increased photosynthetic rates (d) enhanced nutrient uptake, and (e) induced host defense responses to pathogens. Many of these plant responses were established with T. virens, T. asperelloides T203 and T. harzianum in maize, cotton, and Arabidopsis .

[005] Fungi have a beneficial role in nutrient supply to plants and confer a plant growth promotion effect in abiotic stressful environments. Nutrient supply is crucial for crop production and plant growth. Recent experimental evidence showed beneficial interaction in plants associated with fungi, and their role in nutrient supply to the plant. For example, the ascomycete fungal endophyte, Colletotrichum tofieldiae. increased Pi translocation into Arabidopsis thaliana plants via root-associated hyphae under phosphate starvation conditions, and Arbuscular mycorrhizal fungi enhanced plant productivity and nutrient uptake, particularly in grassland and in legumes.

[006] Bacteria and fungi co-exist in many distinct environments. Their interaction is often important for the biological activity of either or both organisms and can affect other partners. Bacteria and fungi vary in their physical association, from highly specific symbionts to disordered polymicrobial communities. The presence of intracellular living bacteria of the genus Burkholderia, was reported in the plant pathogenic fungus Rhizopus spp. Apparently, the toxic compound rhizoxin is biosynthesized by the endobacteria, and not by the host. Furthermore, the biosynthesis of rhizoxin depends on the association between the bacteria and the fungus. Another interaction was demonstrated by the ectosymbiosis between cyanobacteria of the genus Nostoc, and the ascomycete fungus Geosiphon pyriformis.

[007] Indeed, the presence of endosymbiotic bacteria in fungal hyphae is not uncommon. About 75 species of endohyphal bacteria were observed in endophytic fungi isolated from foliar tissues. However, only a few cases of bacterial presence on fungal hyphae were reported, comprising methods such as shaking the fungal mycelium, which may cause bacteria to dissociate from the fungal hyphae, and in turn result in biased analysis.

[008] There is still a great need for a method for accurately determining and/or identifying bacterial species in symbiosis with fungi, e.g., residing on fungal hyphae.

SUMMARY

[009] In some embodiments, the present invention is directed to a method for identifying a fungal symbiotic microorganism.

[010] According to a first aspect, there is provided a method for detecting an association of a microorganism with a fungus, comprising the steps of: (1) culturing the fungus on a polysaccharide film comprising a hexose; and (2) contacting the film with a molecule having a specific binding affinity to a biomarker of the microorganism, thereby detecting an association of the microorganism with the fungus.

[Oi l] According to another aspect, there is provided kit for identifying a microorganism associated with a fungus, the kit comprising: (1) a polysaccharide film comprising ahexose; and (2) a molecule having a specific binding affinity to a biomarker of the microorganism.

[012] In some embodiments, the fungus comprises a hypha.

[013] In some embodiments, the microorganism is a bacterium.

[014] In some embodiments, the culturing is for 2 to 7 days.

[015] In some embodiments, the culturing comprises stationary culturing.

[016] In some embodiments, the stationary culturing comprises a step of shaking the culture at a rotational speed or frequency rotation of 1 to 50 revolutions per minute (rpm).

[017] In some embodiments, the method further comprises a step of isolating the fungus from the roots of a plant or a soil sample.

[018] In some embodiments, the polysaccharide is selected from the group consisting of: cellulose, hemicellulose, chitin, starch, and any combination thereof.

[019] In some embodiments, the cellulose is selected from the group consisting of: natural cellulose, regenerated cellulose, and semi- synthetic cellulose.

[020] In some embodiments, the semi- synthetic cellulose is selected from the group consisting of: cellulose acetate, cellulose diacetate, cellulose triacetate, lyocell, modal, and rayon.

[021] In some embodiments, the hemicellulose is selected from the group consisting of: xylan, glucuronoxylan, arabinoxylan, glucomannan, and xyloglucan.

[022] In some embodiments, the hexose is selected from the group consisting of: glucose, N-acetylglucosamine, xylose, mannose, galactose, rhamnose, arabinose, glucuronic acid, galacturonic acid, mannonic acid, and arabinoic acid.

[023] In some embodiments, the polysaccharide film comprises the hexose in the amount of at least 60% (w/w) of the polysaccharide film.

[024] In some embodiments, the polysaccharide film is cellophane. [025] In some embodiments, the molecule is a polynucleotide.

[026] In some embodiments, the polynucleotide hybridizes to the biomarker.

[027] In some embodiments, the method further comprises a step of hybridizing the polynucleotide to the biomarker for a period of 6 to 18 hours.

[028] In some embodiments, the biomarker is 16S ribosomal RNA (16S-rRNA).

[029] In some embodiments, the biomarker is a species-specific biomarker.

[030] In some embodiments, the kit comprises instructions for: (1) culturing the fungus on the polysaccharide film; (2) contacting the film with the molecule; and (3) determining whether the microorganism is present in association with the fungus.

[031] Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

[032] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE FIGURES

[033] Fig. 1 is a work-flow chart of a non-limiting example of the disclosed invention, by which, Trichoderma spp. are isolated from roots (for example maize roots), and hyphae, and are then transferred to a cellophane film, fixed and dehydrated. 16S ribosomal RNA (16S-rRNA) fluorescent in situ hybridization is performed on the cellophane film, which is then visualized and analyzed using confocal microscopy.

[034] Fig. 2 is an image of a Petri dish comprising a Trichoderma harzianum hyphae grown on a cellophane paper. [035] Figs. 3A-3B are fluorescent images of (3A) Trichoderma asperellum and (3B) Trichderma reesei, which were re-isolated from a plant-soil system and grown on a cellophane paper. Bacteria and fungi hyphae were probed using 16S-rRNA FISH probe (TMR red channel) and SYTO-9 (green channel), respectively, and imaged by confocal microscopy. Suspected association of a fungal hyphae and bacteria are encircled. Scale bar = 2 pm.

DETAILED DESCRIPTION

[036] The present invention is directed to a method for identifying a microorganism in symbiosis with a fungus or a part thereof, e.g., a hypha.

[037] The present invention is based, in part, on the finding that a symbiotic microorganism was identified in tight association with fungal hyphae after the fungus was cultured on a cellophane film, and thereafter contacted with a molecule targeting a biomarker of the symbiotic microorganism.

[038] In some embodiments, the present invention provides a method for detecting an association of a microorganism with a fungus, comprising the steps of: culturing said fungus on a polysaccharide film comprising a hexose; and contacting said film with a molecule having a specific binding affinity to a biomarker of said microorganism, thereby detecting an association of the microorganism with the fungus.

[039] The terms "detecting" and "determining" are used herein interchangeably.

[040] As used herein, the term "fungus" refers to any organism that is a member of the fungi kingdom. In some embodiments, a fungus comprises any unicellular or multicellular organism comprising chitin in its cell wall. Non-limiting examples of fungi include, but are not limited to, yeasts, molds and mushrooms.

[041] In some embodiments, the fungi belongs to a genera selected from: Trichoderma, Alternaria, Clonostachys, Fusarium, Gibberella, Sordaria, Rhizopus, Penicillium, Botrytis, and Sclerotinia.

[042] In some embodiments, a fungus cultured according to the method of the present invention comprises a hypha. In one embodiment, a hypha comprises a septate hypha or coenocytic hypha. In some embodiments, the fungus comprises a mycelium. In some embodiments, the fungus comprises a haustoria. In some embodiments, the fungus comprises an arbuscules. In some embodiments, the fungus comprises a mold or colony. In some embodiments, the fungus comprises an apothecium. In some embodiments, the fungus comprises a hymenium. In some embodiments, the fungus comprises a spore bearing cell, a spore, or any combination thereof.

[043] In some embodiments, the fungus is cultured under effective conditions, which allow for maintaining association of the fungus with a symbiotic microorganism. In some embodiments, effective culture conditions include, but are not limited to, effective media, bioreactor, temperature, pH and oxygen conditions that permit for maintaining association of the fungus with the microorganism. In one embodiment, an effective medium refers to any medium in which a fungus is cultured and produces any molecule required to maintain, induce, promote, propagate, or any combination thereof, an association of the fungus with the microorganism. In some embodiments, the medium used according to the present invention comprises an assimilable organic carbon. In some embodiments, a microorganism mixture comprising the fungus and at least one microorganism can be cultured in conventional fermentation bioreactors, incubators, test tubes, microtiter dishes and petri plates. In some embodiments, culturing is carried out at a temperature, pH and oxygen content appropriate for a probiotic microorganism, such as a fungus, a yeast, bacteria, a virus, or any combination thereof. Culturing methods are well within the expertise of one of ordinary skill in the art.

[044] In some embodiments, the fungus is cultured for a period of 1 to 2 days, 1 to 3 days, 1 to 4 days, 1 to 5 days, 1 to 6 days, 1 day to 7 days, 1 to 8 days, 2 to 4 days, 2 to 5 days, 2 to 6 days, 2 to 7 days, 2 to 8 days, 2 to 9 days, 3 to 5 days, 3 to 6 days, 3 to 7 days, 3 to 8 days, 3 to 9 days, 3 to 10 days, or any range therebetween. In some embodiments, the fungus is cultured for a period of hours to days, or days to weeks. Each possibility represents a separate embodiment of the invention.

[045] In some embodiments the fungus is cultured in a temperature ranging from 10 to 50 °C, 15 to 35 °C, 20 to 30 °C, 28 to 37 °C, 22 to 35 °C, 23 to 28 °C, 12 to 25 °C, or 20 to 24 °C, or any range therebetween. Each possibility represents a separate embodiment of the invention.

[046] In some embodiments, culturing comprises or consists of stationary culturing. As used herein, the term "stationary" encompasses static or immobilized culture, referring to that the substrate on which a microorganism is cultured, is not: shacked, mixed, or stirred, or any synonym thereof. In another embodiment, a stationary microorganism culture is shacked, or mixed, or stirred or any synonym thereof, or any combination thereof, at a rotational speed or frequency rotation of : 1-10 revolutions per minute (rpm), 2-12 rpm, 3-14 rpm, 4-8 rpm, 5-15 rpm, 1-20 rpm, 3-30 rpm, 2-7 rpm, 7-13 rpm, 1-50 rpm, or any range therebetween. Each possibility represents a separate embodiment of the invention. In one embodiment, a stationary microorganism culture is: shacked, mixed, or stirred, or any synonym thereof, or any combination thereof, at a rotational speed or frequency rotation of not more than 100 rpm, not more than 75 rpm, not more than 50 rpm, not more than 25 rpm, not more than 15 rpm, not more than 10 rpm, not more than 9 rpm, not more than 7 rpm, not more than 5 rpm, not more than 4 rpm, not more than 3 rpm, not more than 2 rpm, not more than 1 rpm, or any value and range therebetween. Each possibility represents a separate embodiment of the invention.

[047] In some embodiments, the method of the present invention comprises or further comprises a step of isolating a fungus. In one embodiment, the fungus is isolated from a cell culture. In one embodiment, the fungus is isolated from a living organism selected from: animals or plants. In one embodiment, a fungus isolated form a plant's sample is isolated from the plant's root, bark, tip, leaf, flower, pollen, branch, fruit, sap, resin, or any combination thereof. In one embodiment, the fungus is isolated from a water sample. In one embodiment, the fungus is isolated from a soil sample. In one embodiment, the fungus is isolated from an inanimate object (e.g., non-living), for example, the surface of a tool, or an article.

[048] In some embodiments, the substrate is a dry substrate. As used herein, "dry" refers to a substrate comprising water in an amount of not more than 20% (w/w), not more than 15% (w/w), not more than 10% (w/w), not more than 5% (w/w), not more than 2% (w/w), or containing no water, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, a dry substrate comprises water in an amount of 0-5% (w/w), 4-8% (w/w), 3-10% (w/w), or 9-15% (w/w). Each possibility represents a separate embodiment of the invention.

[049] In some embodiments, the method is directed to culturing a fungus on a substrate comprising a polysaccharide.

[050] In some embodiments, the substrate comprises a polysaccharide in an amount of at least 15% (w/w), at least 25% (w/w), at least 35% (w/w), at least 45% (w/w), at least 55% (w/w), at least 65% (w/w), at least 75% (w/w), at least 85% (w/w), at least 95% (w/w), at least 99% (w/w), or is 100% (w/w) cellulose, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, the substrate comprises a polysaccharide in an amount of 5-15% (w/w), 10-25% (w/w), 20-35% (w/w), 30-45% (w/w), 40-55% (w/w), 50-65% (w/w), 60-75% (w/w), 70-85% (w/w), 80-95% (w/w), 90-100% (w/w). Each possibility represents a separate embodiment of the invention.

[051] In some embodiments, polysaccharide is selected from: cellulose, hemicellulose, chitin, starch, or any combination thereof.

[052] As used herein, the term "cellulose" refers to any polysaccharide having a formula (C ₆ HioOs)n, and comprising a linear chain of hundreds to thousands of D- glucose monomers linked to one another via b(1 4) bonds.

[053] As used herein, the term "hemicellulose" refers to any of several heteropolymers (i.e., matrix polysaccharides), comprising hundreds to a few thousands of monomers linked to one another via b(1 4) bonds and a glycosidic bonds, such as a (1 2), or a (1 3). In some embodiments, hemicellulose comprises: xylose, mannose, galactose, rhamnose, arabinose, glucuronic acid, galacturonic acid, mannonic acid, arabinoic acid, or any combination thereof.

[054] As used herein, the term "chitin" refers to any polysaccharide, comprising a linear chain of hundreds to thousands of N-acetylglucosamine monomers linked to one another via b(1 4) bonds.

[055] As used herein, the term "starch" refers to a polysaccharide, comprising linear and branched chains of D-glucose monomers linked to one another via a(l 4) bonds, a(l 6), or both.

[056] As used herein, the term "starch", encompasses both helical amylose and amylopectin.

[057] In some embodiments, cellulose is a natural cellulose. As used herein, the term "natural cellulose" refers to the polymer produced naturally, isolated, obtained, derived, or extracted from plants, such as cotton, wood or hemp.

[058] In some embodiments, cellulose is a regenerated cellulose. As used herein, the term "regenerated cellulose" refers to cellulose produced by converting the naturally produced cellulose to a soluble cellulosic derivative and regenerate it thereafter. [059] In some embodiments, cellulose is a semi- synthetic cellulose. As used herein, term "semi-synthetic cellulose" refers to cellulose produced from naturally long-chain high molecular weight cellulose polymers which were only modified and partially degraded by chemical processes. In some embodiments, semi- synthetic cellulose comprises: cellulose acetate, cellulose diacetate, cellulose triacetate, lyocell, modal, and rayon, or a combination thereof.

[060] In some embodiments, hemicellulose is selected from: xylan, glucuronoxylan, arabinoxylan, glucomannan, xyloglucan, or any combination thereof.

[061] In some embodiments, a polysaccharide comprises or is a polysaccharide fiber. In some embodiments, a polysaccharide substrate comprises or is a polysaccharide film. In some embodiments, a polysaccharide film or polysaccharide fiber comprise cellophane.

[062] In some embodiments, a polysaccharide substrate comprises an assimilable organic carbon source. As used herein, the term "assimilable" denotes that a compound is utilizable by a living cell or an organism. In some embodiments, assimilable, refers to that a compound can be used by a cell or an organism as a building block of other molecules or macromolecules used by the cell or an organism. In some embodiments, the cell or the organism is a cultured fungus. In some embodiments, the polysaccharide substrate comprises at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the assimilable organic carbon source for the cultured fungus, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, the polysaccharide substrate comprises 50-70%, 60-80%, 70-90%, or 80-100% of the assimilable organic carbon source for the cultured fungus. Each possibility represents a separate embodiment of the invention.

[063] In one embodiment, the polysaccharide substrate comprises the only source of assimilable organic carbon available to the cultured fungus. In some embodiments, the cultured fungus utilizes assimilable organic carbon provided only by the polysaccharide substrate. In some embodiments, the polysaccharide substrate is a selective growth medium for culturing fungus, or any part thereof, as disclosed herein.

[064] In some embodiments, a polysaccharide film is pre-treated prior to use, such as in culturing a fungus thereon. In one embodiment, culture pre-treatment comprises: washing, drying, hydrating, rehydrating, cutting, soaking, heating, blocking, cross- linking, or any combination thereof.

[065] As used herein, the terms "polysaccharide substrate", "polysaccharide film", and "polysaccharide fiber" are interchangeable.

[066] In some embodiments, the polysaccharide comprises a hexose or any derivative thereof. As used herein, the term "hexose" refers to any sugar having the formula C6H12O6. As used herein, the term "hexose derivative" comprises any six-carbon monosaccharide comprising a substituted group, thereby deviating from the C MnOe formula. Non-limiting examples of substitutions include, but are not limited to, amination (e.g., addition of Nth), N-acetylation, and others.

[067] In one embodiment, a hexose or a derivative thereof comprises cyclic or acyclic hexose. In some embodiments, a hexose or a derivative thereof comprises D- and L- isomers. Non-limiting examples of hexose or a derivative thereof include, but are not limited to, glucose, fructose, mannose, galactose, sorbose, N-acetylglucosamine, xylose, rhamnose, arabinose, glucuronic acid, galacturonic acid, mannonic acid, and arabinoic acid.

Method for determining microorganism association

[068] In some embodiments, the method is directed to determine the association between a microorganism and a fungus. As used herein, the term "microorganism" comprises any organism selected from: a virus, a bacterium, a fungus, and an archaeon.

[069] In some embodiments, a bacterium comprises gram positive bacterium or gram negative bacterium.

[070] As used herein, the terms "association" and "symbiosis" are interchangeable. In some embodiments, symbiosis comprises any type of biological interaction between two different biological organisms. In some embodiments, and is selected from the group consisting of: commensalism, amensalism, mutualism, and parasitic.

[071] As used herein, the term "microorganism biomarker" refers to any molecule, the detection of which can differentiate or distinguish between different organisms, species, strains, lines, etc.

[072] In some embodiments, the level of a biomarker specificity comprises: kingdom specific, phylum specific, class specific, order specific, family specific, genus specific, species specific, and an individual specific, biomarker. Each possibility represents a separate embodiment of the invention.

[073] In some embodiments, a biomarker is a polynucleotide. In some embodiments, a polynucleotide biomarker comprises DNA, RNA, or both. In some embodiments, a polynucleotide biomarker is an encoding or a non-encoding polynucleotide. Non limiting examples of polynucleotides utilized as biomarkers include, but are not limited to, exon-intron junction, mRNA, tRNA, ribosomal RNA (rRNA), micro RNA (miRNA), single nucleotide polymorphism (SNP), insertion/deletion (InDel), mutations, and others. Detection of exemplary polynucleotide biomarkers would be apparent to one of ordinary skill in the art of molecular biology.

[074] In some embodiments, a microorganism polynucleotide biomarker is 16S ribosomal RNA (16S-rRNA) or the 16S-rRNA encoding gene, i.e., the DNA sequence from which 16S is transcribed.

[075] In some embodiments, a biomarker is a peptide, a polypeptide, or a protein. In some embodiments, a biomarker is a fatty acid. In some embodiments, a biomarker is a metabolite. In some embodiments, a biomarker is a small organic or non-organic compound.

[076] As used herein, the terms "peptide", "polypeptide, or "protein" refer to a polymer of amino acids comprising 2 amino acids, or more.

[077] In some embodiment, the method comprises contacting a polysaccharide film with a molecule having specific binding affinity to a microorganism biomarker. As used herein, specific affinity is binding with a dissociation constant (K _d ) lower than 1 mM, lower than 100 nM, lower than 10 nM, lower than 1 nM, lower than 100 pM, lower than 10 pM, lower than 1 pM, lower than 100 fM, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, specific affinity is binding with a K _d of 100 nM to 1 pM, 10-100 nM, 1-10 nM, 100 pM to 1 nM, 10-100 pM, 1-10 pM, 100 fM to 1 pM, or 10-100 fM. Each possibility represents a separate embodiment of the invention. In some embodiments, specific binding is having a binding affinity at the nanomolar, picomolar, or femtomolar level. Each possibility represents a separate embodiment of the invention.

[078] In some embodiments, specific binding affinity comprises specific nucleic acid base pairing. In some embodiments, specific base pairing comprises at least 70%, 80%, 90%, 95%, 99%, or 100% complementarity, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, the molecule is specific to a microorganism biomarker, e.g., DNA or RNA. In some embodiments, specific comprises base pair specificity, gene specificity, or both. In some embodiments, the molecule is specific to a microorganism biomarker gene. In some embodiments, the molecule is specific to a non-coding microorganism biomarker. In some embodiments, the molecule is specific to a RNA molecule of a microorganism biomarker. In some embodiments, RNA comprises mRNA, or pre-mRNA (polycistronic mRNA). In some embodiments, non-coding microorganism biomarker comprises a non coding DNA sequence (e.g., genomic extra genic region) or a non-coding RNA sequence (e.g., ribosomal RNA, etc.).

[079] The terms "specific binding affinity" and "targeting", as used herein, are interchangeable.

[080] In one embodiment, the method comprises detecting a nucleic acid biomarker molecule of a microorganism. In some embodiments, detection comprises visualization of the nucleic acid molecule. In one embodiment, visualization of a nucleic acid utilizes a probe. In some embodiments, the method is directed to hybridizing a nucleic acid (e.g., polynucleotide) with a microorganism nucleic acid biomarker molecule.

[081] The term“probe” refers to a labeled or unlabeled oligonucleotide capable of selectively hybridizing to a target or template nucleic acid under suitable conditions. Typically, a probe is sufficiently complementary to a specific target sequence contained in a nucleic acid sample to form a stable hybridization duplex with the target sequence under a selected hybridization condition, such as, but not limited to, a stringent hybridization condition. A hybridization assay carried out using the probe under sufficiently stringent hybridization conditions permits the selective detection of a specific target sequence. For use in a hybridization assay for the discrimination of single nucleotide differences in sequence, the hybridizing region is typically from about 8 to about 100 nucleotides in length. Although the hybridizing region generally refers to the entire oligonucleotide, the probe may include additional nucleotide sequences that function, for example, as linker binding sites to provide a site for attaching the probe sequence to a solid support or the like, as sites for hybridization of other oligonucleotides, as restriction enzymes sites or binding sites for other nucleic acid binding enzymes, etc. In certain embodiments, a probe of the invention is included in a nucleic acid that comprises one or more labels (e.g., a reporter dye, a quencher moiety, a fluorescent labeling, etc.), such as a 5 '-nuclease probe, a FRET probe, a molecular beacon, or the like, which can also be utilized to detect hybridization between the probe and target nucleic acids in a sample. In some embodiments, the hybridizing region of the probe is completely complementary to the target sequence. However, in general, complete complementarity is not necessary (i.e., nucleic acids can be partially complementary to one another); stable duplexes may contain mismatched bases or unmatched bases. Modification of the stringent conditions may be necessary to permit a stable hybridization duplex with one or more base pair mismatches or unmatched bases. Sambrook et ah, Molecular Cloning: A Laboratory Manual, 3 ^rd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001), which is incorporated by reference, provides guidance for suitable modification. Stability of the target/probe duplex depends on a number of variables including length of the oligonucleotide, base composition and sequence of the oligonucleotide, temperature, and ionic conditions. One of skill in the art will recognize that, in general, the exact complement of a given probe is similarly useful as a probe. Exemplary probe nucleic acids include 5 '-nuclease probes, molecular beacons, among many others known to persons of skill in the art.

[082] As used herein, “hybridization” refers to a reaction in which at least one polynucleotide reacts to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues. The hydrogen bonding may occur by Watson-Crick base pairing, in any other sequence-specific manner.

[083] In some embodiments, hybridization reactions are performed under conditions of different stringency. In some embodiments, under stringent conditions, nucleic acid molecules at least 60%, at least 65%, at least 70%, or at least 75% identical to each other remain hybridized to each other. A non-limiting example of highly stringent hybridization conditions is hybridization in 6x Sodium chloride/S odium citrate (SSC) at approximately 45 °C, followed by one or more washes in 0.2x SSC and 0.1% SDS at 50 °C, at 55 °C, or at about 60 °C, or more.

[084] In some embodiments, hybridization is performed at a temperature of 30-40 °C, 20-50 °C, 30-60 °C, 45-80 °C, 25-55 °C, 35-42 °C, 40-65 °C, 32-57 °C, 37-47 °C. Each possibility represents a separate embodiment of the invention. In one embodiment, hybridization is performed at a temperature of 46 °C. [085] In some embodiments, hybridization is performed for a period of 2 to 5 hours, 3 to 8 hours, 4 to 12 hours, 10 to 18 hours, 16 to 24 hours, 18 to 36 hours. Each possibility represents a separate embodiment of the invention. In some embodiments, hybridization is performed for a period of at least 3 hours, at least 4 hours, at least 8 hours, at least 12 hours, at least 16 hours, at least 20 hours, at least 24 hours, or any value and range therebetween. Each possibility represents a separate embodiment of the invention.

[086] When hybridization occurs in an anti-parallel configuration between two single- stranded polynucleotides, those polynucleotides are described as complementary.

[087] Hybridization based assays which allow the detection of a biomarker of interest in a biological sample or on a film, as disclosed herein, rely on the use of probe(s) which can be 10, 15, 20, or 30 to 100 nucleotides long, optionally from 10 to 50, or from 40 to 50 nucleotides long.

[088] Thus, the polynucleotides complementary to the microorganism biomarkers utilized by the present invention, according to at least some embodiments, are optionally hybridizable with any of the herein described nucleic acid sequences under moderate to stringent hybridization conditions.

[089] The detection of hybrid duplexes can be carried out by several methods. Typically, hybridization duplexes are separated from unhybridized nucleic acids and the labels bound to the duplexes are then detected. Such labels refer to radioactive, fluorescent, biological or enzymatic tags or labels of standard use in the art. A label can be conjugated to either the oligonucleotide probes or the nucleic acids derived from the biological sample.

[090] Probes can be labeled according to numerous well-known methods. Non-limiting examples of detectable markers include ligands, fluorophores, chemiluminescent agents, enzymes, and antibodies. Other detectable markers for use with probes, which can enable an increase in sensitivity of the method of the invention, include biotin and radio nucleotides. It will become evident to the person of ordinary skill that the choice of a particular label dictates the manner in which it is bound to the probe.

[091] For example, oligonucleotides according to at least some embodiments of the present invention can be labeled subsequently to synthesis, by incorporating biotinylated dNTPs or rNTP, or some similar means (e.g., photo-cross-linking a psoralen derivative of biotin to RNAs), followed by addition of labeled streptavidin (e.g., phycoerythrin- conjugated streptavidin) or the equivalent. Alternatively, when fluorescently-labeled oligonucleotide probes are used, fluorescein, lissamine, phycoerythrin, rhodamine (Perkin Elmer Cetus), Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, Fluor X (Amersham) and others (e.g., Kricka et al. (1992), Academic Press San Diego, Calif.) can be attached to the oligonucleotides. Preferably, detection of the biomarkers of the invention is achieved by using TaqMan assays, preferably by using combined reporter and quencher molecules (Roche Molecular Systems inc.).

[092] Although the present invention is not specifically dependent on the use of a label for the detection of a particular nucleic acid sequence, such a label might be beneficial, by increasing the sensitivity of the detection. Furthermore, it enables automation. Probes can be labeled according to numerous well-known methods.

[093] As commonly known, radioactive nucleotides can be incorporated into probes of the invention by several methods. Non-limiting examples of radioactive labels include ¾, ¹⁴ C, ³² P, and ³⁵ S.

[094] Those skilled in the art will appreciate that wash steps may be employed to wash away excess target polynucleotide or probe as well as unbound conjugate. Further, standard heterogeneous assay formats are suitable for detecting the hybrids using the labels present on the oligonucleotide primers and probes.

[095] It will be appreciated that a variety of controls may be usefully employed to improve accuracy of hybridization assays. For instance, samples may be hybridized to an irrelevant probe and treated with RNase A prior to hybridization, to assess false hybridization.

[096] Probes utilized according to the method of the invention can be naturally occurring sugar-phosphate backbones as well as modified backbones including phosphorothioates, dithionates, alkyl phosphonates and a-nucleotides and the like. Probes of the invention can be constructed of either ribonucleic acid (RNA), deoxyribonucleic acid (DNA), locked nucleic acid (FNA), or combinations thereof.

[097] A nucleic acid test (NAT) known in the art is Fluorescence In Situ Hybridization (FISH). FISH uses fluorescent single- stranded DNA, RNA or FNA probes which are complementary to the nucleotide sequences that are under examination (genes, chromosomes or RNA). These probes hybridize with the complementary nucleotide and allow the identification of the chromosomal location of genomic sequences of DNA or RNA transcripts.

[098] In another embodiment, the method comprises detecting a peptide, a poly peptide, or a protein, detection of which can be performed according to any method utilizing antibodies. A non-limiting example includes but is not limited to immunoassays, such as immunohistochemistry, dot-blot, and the like, all of which would be apparent to one of skill in the art.

[099] In some embodiments, the method of the present invention comprises fixation of the fungus to the polysaccharide film. In some embodiments, fixation is performed after the culturing period of the fungus on the polysaccharide substrate. Any method and agent for fixation of living cells to various substrates are applicable and would be apparent to a skilled artisan. Non-limiting examples of fixation agents or solutions include, but are not limited to: formaldehyde, paraformaldehyde, Carnoy's II solution, Davidson's fixation solution, and Alcohol.

The kit

[0100] According to some embodiments, the present invention is directed to a kit comprising a film comprising 60% (w/w) or more cellulose; and a molecule targeting a microorganism biomarker.

[0101] According to some embodiments, the kit comprises instructions for: (i) culturing a fungus on a film; (ii) contacting the film with a molecule; and (iii) identifying the microorganism associated with the fungus.

[0102] In some embodiments, identifying comprises determining whether the microorganism is present in association with the fungus. In some embodiments, determining, e.g., contacting a fungus cultured on a film with a molecule capable of binding to a microorganism suspected of being in association with the fungus.

[0103] In some embodiments, association comprises binding, attachment, or both. In some embodiments, a microorganism in association with a fungus is bound to or attached to the fungus or any part thereof, e.g., hypha. In some embodiments, association is physical proximity. In some embodiments, association is a distance of not more than 1 nm, 10 nm, 100, 1 pm, 10 pm, 100 pm, or any value and range therebetween. Each possibility represents a separate embodiment thereof. [0104] In some embodiments, the kit comprises a probe molecule or an antibody molecule, or both. In some embodiments, the kit provides and enzyme and a substrate thereof.

[0105] In some embodiments, the kit further provides reagents and/or buffers, such as hybridization or binding buffer, for improving binding of the molecule to the microorganism biomarker. In some embodiments, the kit further provides reagents and/or buffers for detecting binding of the molecule to the microorganism biomarker. In some embodiments, the antibody is conjugated to a dye or label molecule. In some embodiments, the antibody is conjugated to an enzyme. In some embodiments, the enzyme is selected from: alkaline phosphatase and horse radish peroxidase. In some embodiments, a detection buffer comprises an enzyme's specific substrate, co-factors, or any combination thereof. In some embodiments, the enzyme catalyzes a reaction converting the substrate to a detectable product. In some embodiments, the product is soluble or insoluble. Non-limiting examples of substrates include, but are not limited to, CDP star, NPP, NBT-BCIP, and others, all which would be apparent to one of ordinary skill in the art.

[0106] In some embodiments, the components of the kit disclosed herein are sterile. As used herein, the term "sterile" refers to a state of being free from biological contaminants. Any method of sterilization is applicable and would be apparent to one of ordinary skill in the art.

[0107] In some embodiments, the components of the kit are packaged within a container.

[0108] In some embodiments, the container is made of a material selected from the group consisting of thin-walled film or plastic (transparent or opaque), paperboard- based, foil, rigid plastic, metal (e.g., aluminum), glass, etc.

[0109] In some embodiments, the content of the kit is packaged, as described below, to allow for storage of the components until they are needed.

[0110] In some embodiments, some or all components of the kit may be packaged in suitable packaging to maintain sterility.

[0111] In some embodiments, the components of the kit are stored in separate containers within the main kit containment element e.g., box or analogous structure, may or may not be an airtight container, e.g., to further preserve the sterility of some or all of the components of the kit. [0112] In some embodiments, the instructions may be recorded on a suitable recording medium or substrate. For example, the instructions may be printed on a substrate, such as paper or plastic, etc.

[0113] In some embodiments, the instructions may be present in the kit as a package insert, in the labeling of the container of the kit or components thereof (i.e., associated with the packaging or sub-packaging) etc. In other embodiments, the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g. CD-ROM, diskette, etc. In other embodiments, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, e.g. via the internet, are provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and/or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions is recorded on a suitable substrate.

[0114] Any concentration ranges, percentage range, or ratio range recited herein are to be understood to include concentrations, percentages or ratios of any integer within that range and fractions thereof, such as one tenth and one hundredth of an integer, unless otherwise indicated.

[0115] Any number range recited herein relating to any physical feature, are to be understood to include any integer within the recited range, unless otherwise indicated.

[0116] In the discussion unless otherwise stated, adjectives such as“substantially” and “about” modifying a condition or relationship characteristic of a feature or features of an embodiment of the invention, are understood to mean that the condition or characteristic is defined to within tolerances that are acceptable for operation of the embodiment for an application for which it is intended. Unless otherwise indicated, the word“or” in the specification and claims is considered to be the inclusive“or” rather than the exclusive or, and indicates at least one of, or any combination of items it conjoins.

[0117] It should be understood that the terms“a” and“an” as used above and elsewhere herein refer to“one or more” of the enumerated components. It will be clear to one of ordinary skill in the art that the use of the singular includes the plural unless specifically stated otherwise. Therefore, the terms “a,” “an” and “at least one” are used interchangeably in this application. [0118] For purposes of better understanding the present teachings and in no way limiting the scope of the teachings, unless otherwise indicated, all numbers expressing quantities, percentages or proportions, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term“about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0119] In the description and claims of the present application, each of the verbs, “comprise,”“include” and“have” and conjugates thereof, are used to indicate that the object or objects of the verb are not necessarily a complete listing of components, elements or parts of the subject or subjects of the verb.

[0120] Other terms as used herein are meant to be defined by their well-known meanings in the art.

[0121] Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.

[0122] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub -combination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements. EXAMPLES

[0123] Generally, the nomenclature used herein, and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds.) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds); all of which are incorporated by reference. Other general references are provided throughout this document.

Materials and Methods

Identification of associating bacteria on fungal hyphae

[0124] Trichoderma hyphae (about 0.5 cm ² ) grown on agar media were transferred to sterile cellophane (Bio-Rad, Cat. no. 9004-34-6), set on a petri dish 10 cm ² plate, and cultured at 23 °C for 3 to 7 days (about 50% hyphae confluence). A piece of about 1.5 cm x 1.5 cm was taken to fluorescent in situ hybridization (FISH) procedure.

Fixation

[0125] The cellophane piece was placed in a 12-well dish, covered with 1.5 ml of 4% (v/v) paraformaldehyde (PFA; diluted from a stock solution of 16% (v/v) with sterile PBSxl ) and incubated for 3 h in room temperature (R.T) or alternatively, at 4 °C over night (O.N.).

Dehydration

[0126] Serial incubation was performed as follows: 5 min in 50% ethanol (v/v; in PBS) in R.T; 5 min in 70% (v/v) ethanol (in PBS) in R.T; and 5 min in 100% (absolute) ethanol in R.T. All steps were performed under gentle shaking (50 rpm; degree 4 using Belly Dancer shaker).

16S rRNA fluorescence in situ hybridization (16S-FISH)

[0127] Hybridization buffer comprised: 0.9 M NaCl, 0.1% (v/v) SDS, 20 mM Tris-HCl pH 6.8, 1 mM probe EUB338-5 V56-TAMN/GCTGCCTCCCGTAGGAGT-3’(IDT) (SEQ ID NO: 1) and 40% (v/v) formamide. Hybridization buffer (0.5 ml) was added to the cellophane piece set in a well of a 12-well dish, to achieve full coverage. Hybridization was applied in the dark at 46 °C, under gentle agitation (75 rpm) for 3 h up to O.N (16 h).

Wash

[0128] Ready to use wash buffer comprised (50 ml): 460 pi 5 M NaCl, 1 ml Tris HC1 pH 6.8, 50 mΐ 10% SDS and 48.49 ml DEPC-treated water. Hybridization buffer was removed, and 0.5 ml wash buffer was added twice to wash the cellophane.

Stains

[0129] SYTO-9, DAPI and/or Propidium iodide are applicable. Fifty (50) mΐ of SYTO- 9 stain (1:300 in wash buffer or into Fluoromount aqueous mounting Sigma F4680) was loaded on a Superfrost plus Microscopy slide (Thermo-scientific). The cellophane piece was set on stain solution drop and covered with a microscope cover glass. Slides were allowed to dry O.N. at R.T. After the slide was dried, an additional incubation at -20 °C for 24 h was applied prior to microscopic imaging to enhance signal intensity and to improve the specificity. DAPI/Propidium iodide was added, according to the manufacturer's recommendation, immediately before microscopy analysis.

Microscopic analysis

[0130] Slides were viewed using Confocal Zeiss LSM 510 META.

EXAMPLE 1

Identification of bacteria associated with fungal hyphae cultured on cellophane film

[0131] In order to identify bacterial presence on Trichoderma hyphae, Trichoderma sp. were grown on cellophane paper (Fig. 2). 16S-rRNA fluorescence in situ hybridization assay was preformed directly on the cellophane with the fungal hyphae (Fig. 3). Candidate Trichoderma- associated bacteria were identified on the Trichoderma , specifically on Trichoderma asperellum hyphae (TGRO) (Fig. 3A) and T. reesei hyphae

(Fig. 3B).

[0132] While certain features of the invention have been described herein, many modifications, substitutions, changes, and equivalents will now occur to those of ordinary skill in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention.