Field of the Invention

The present invention relates to novel Zopfiella sp. fungi and variants thereof, particularly Zopfiella sp. NEM41 and its use in plant growth promotion. The present invention also relates to seeds, plants, and parts inoculated therewith, and biopolymers secreted therefrom. The present invention also relates to compositions comprising the novel fungi, methods of preparing these compositions, apparatus for inoculating plants and seed with the compositions, and methods of using the compositions.

Background of the Invention

Fertilizers are commonly used to increase plant growth, yield, and nutritive value. In particular, inorganic fertilizers deliver essential elements (e.g. nitrogen, phosphate, potassium, calcium, magnesium, etc) for immediate uptake, thereby increasing plant biomass. Secondary benefits resulting from the enhanced ground coverage of nutrient rich farmlands include increased organic matter in the soil, as well as increased microbial, microfauna (e.g. earthworms), and insect activity in and around the treated area.

However, the use of inorganic fertilizers is also accompanied by an undesirable accumulation of heavy metals in the soil that, upon exposure to natural elements (e.g. wind, rain, snowfall, etc.), leads to water, soil, and air pollution. Pollution resulting from the widespread use of inorganic fertilizers in large-scale agricultural operations has detrimental consequences to the environment and human health.

Organic fertilizers (e.g. animal waste and plant refuse) comprise an environmentally friendly alternative to their inorganic counterparts. However, since organic fertilizers must undergo chemical degradation to release the essential elements required for sustained plant growth, the widespread use of organic fertilizers does not constitute a viable means of sustaining the world’s food supply.

Indeed, even as climate change is threatening industrial agricultural productivity, the demand on farmed goods continues to rise. Moreover, consumers have begun to expect higher quality food products grown under organic conditions. Thus, farmers face competing challenges to meet increasing demands. They are challenged to overcome the decline in soil fertility that results from repeated cultivation of a single plot of land, while using sustainable and environmentally friendly methods that limit crop yields. Maintaining food supplies in the face of global warming is an issue of worldwide critical importance.

It is an object of the present invention to overcome, or at least alleviate, one or more of the difficulties or deficiencies associated with the prior art.

Summary of the Invention

In one aspect, the present invention provides a substantially purified or isolated strain of Zopfiella sp., preferably Zopfiella sp. NEM41 , wherein the strain is capable of conferring a biofertilizer property to a seed, plant, or part thereof into which the strain is inoculated.

In a preferred embodiment, the present invention provides a substantially purified or isolated strain of Zopfiella sp., preferably Zopfiella sp. NEM41.

In a preferred embodiment, the present invention provides the strain denoted Zopfiella sp. NEM41 , as deposited with the National Measurement Institute of 1/153 Bertie St, Port Melbourne, Victoria 3207 Australia on 8 September 2021 with accession number V21/018167.

The present invention also provides variants of said fungal strains.

By ‘fungus’ as used herein is meant any member of the kingdom of eukaryotic organisms that includes microorganisms such as yeasts and moulds. The fungus may be an endophyte. The term ‘endophyte’ as used herein is meant an organism that exists in a mutually beneficial relationship with a plant. Endophytes generally live on, in, or otherwise in close proximity to a host plant and rely on the plant for nutrition and environmental protection. In exchange, endophytes confer certain beneficial properties to the plant. Endophytes of Zopfiella sp. are fungal endophytes.

By ‘substantially purified’ as used in the context of a fungus is meant that the fungus is free of other organisms, particularly other fungi. The term includes, for example, a fungus in axenic culture. Preferably, the fungus is at least approximately 90% pure, more preferably at least approximately 95% pure, even more preferably at least approximately 98% pure, even more preferably at least approximately 99% pure. By Isolated’ as used in the context of a fungus is meant that the fungus is removed from its original environment, specifically, the natural environment if it is naturally occurring in the plant or surrounding soil. For example, a naturally occurring fungus that is present in nature living in a plant is not isolated, but the same fungus that is separated from some or all of the coexisting materials of the natural system is isolated.

The strain of Zopfiella sp. may be substantially purified or isolated from any part of the plant, e.g. an organ. In preferred embodiments, the organism is substantially purified or isolated from a flower, flower bract, leaf, petiole, stem, root, soil, or rhizosphere of the plant, more preferably a root, soil, or rhizosphere.

By rhizosphere as used herein is meant the region of soil or other growth medium within sufficient proximity to the roots of a plant to be influenced by the growth, respiration, nutrient exchange, or chemical secretions produced by that plant or endophytes harboured therein.

The present invention arises from the discovery that novel fungi as disclosed herein possess an ability to form mutually beneficial relationships with plants that may be used to confer certain beneficial properties to those plants. In particular, the present invention arises from the surprising discovery that the fungi may be used to confer a biofertilizer property to seeds, plants, or parts thereof, including for example, an enhanced tolerance to abiotic (e.g., water, temperature, salinity) or biotic (e.g., pathogenic microorganisms) stresses.

Generally speaking, a fungus which may confer a biofertilizer property will also possess genetic and/or metabolic characteristics that result in organic fertilization of a plant harbouring or otherwise associated with the fungus. Organic fertilization may improve water, nutrient, or temperature stress tolerance in the plant with which the fungus is associated or otherwise enhance plant growth relative to a plant not associated with the fungus.

By ‘organic fertilization’ as used herein in relation to fungi is meant secretion of one or more biopolymer(s) from the fungus either in isolation or in association with a seed, plant, or part thereof.

By ‘biopolymer’ as used herein is meant an organic compound composed of two or more repeating units, wherein each unit is itself an organic compound and where the molecular weight of the biopolymer is the cumulative molecular weight of the repeat units. Without limiting the generality of the foregoing, the biopolymer may be a polynucleotide, polypeptide or polysaccharide.

By ‘organic compound’ as used herein is meant a chemical compound, the molecules of which contain the element carbon.

In a preferred embodiment, the fungus is capable of conferring a biofertilizer property to the plant or part thereof from which it is substantially purified or isolated and/or may be capable of conferring a biofertilizer property to a seed, plant, or part thereof, into which it is inoculated.

By ‘biofertilizer property’ is meant a preparation that contains live or dormant microorganisms, which when applied or inoculated into a seed, plant, or plant part, assists said seed, plant, or plant part, with accessing nutrient availability in the soil or other growth support medium, or which supplies said seed, plant, or plant part with one or more nutrient-rich biopolymer(s), thus improving fertility and productivity of the plant and/or soil.

In a preferred embodiment of this aspect of the invention, the biofertilizer property may be enhanced growth of the plant or increased crop yields from the plant through the production of one or more biopolymer(s), as compared with a plant that is absent the strain. Preferably, the biopolymer comprises repeating units derived from tyrosine, acetate, malonyl-coenzyme A (malonyl-CoA), or catechol. More preferably, the biopolymer is a pigmented polyphenyl such as eumelanin, pheomelanin, neuromelanin, allomelanin, or pyomelanin.

In another preferred embodiment of this aspect of the present invention, the biofertilizer property may be the enhanced growth of the plant or increased crop yields from the plant under one or more abiotic stress condition(s) as compared with a plant that is absent the strain.

The abiotic stress condition(s) may include nutrient, water, or temperature stress, including nutrient stress comprised of below normal levels of a plant nutrient such as nitrogen, phosphorus or potassium.

By Inoculated’ as used in reference to a fungus is meant to be placed in association with a plant so as to form a mutually beneficial relationship with the plant, whether that be on, in, or otherwise in close proximity to the plant. In preferred embodiments, the seed, plant, or part thereof into which the fungus is inoculated is first free of that fungus. In another preferred embodiment of this aspect of the present invention, the fungus may be isolated from roots, soil, or rhizospheres of the plant. In another preferred embodiment, the fungus may be isolated in a reproductively viable form, including spores and hyphae. By “reproductively viable” with respect to fungi is meant capable of regeneration over consecutive generations, wherein the biofertilizer property is retained in each generation.

In a further preferred embodiment of this aspect of the present invention, the fungus includes a nucleic acid sequence encoding an ITS region of an rRNA gene consisting of sequence(s) shown in Figure 4 hereto (SEQ ID NO 1) a sequence having at least approximately 95%, more preferably 97%, more preferably 98%, more preferably 99% sequence identity to the full length of SEQ ID NO 1.

By ‘nucleic acid’ as used herein is meant a chain of nucleotides encoding genetic information. The term generally refers to genes or functionally active fragments or variants thereof and or other sequences in the genome of the organism that influence its property. The term ‘nucleic acid’ includes DNA (such as cDNA or genomic DNA) and RNA (such as mRNA or microRNA) that is single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases, synthetic nucleic acids, and combinations thereof.

Nucleic acids according to the invention may be full-length genes or part thereof, and are also referred to as “nucleic acid fragments” and “nucleotide sequences” in this specification. For convenience, the expression “nucleic acid or nucleic acid fragment” is used to cover all of these.

The present invention encompasses variants of the nucleic acids of the present invention. Such variants include naturally occurring allelic variants and non-naturally occurring variants. Additions, deletions, substitutions and derivatizations of one or more of the nucleotides are contemplated so long as the modifications do not result in loss of functional activity of the variant.

Preferably the variant has at least approximately 95% sequence identity to the relevant part of the nucleic to which or variant corresponds, more preferably at least approximately 97% identity, even more preferably at least approximately 98% identity, most preferably at least approximately 99% identity. Such functionally active variants include, for example, those having conservative nucleic acid changes. By ‘conservative nucleic acid changes’ as used herein is meant nucleic acid substitutions that result in conservation of the amino acid in the encoded protein owing to the degeneracy of the genetic code. Such functionally active variants also include, for example, those having nucleic acid changes which result in conservative amino acid substitutions of one or more residues in the corresponding amino acid sequence.

By ‘conservative amino acid substitutions’ is meant the substitution of an amino acid by another one of the same class, the classes being as follows:

Nonpolar: Ala, Vai, Leu, lie, Pro, Met Phe, Trp

Uncharged polar: Gly, Ser, Thr, Cys, Tyr, Asn, Gin

Acidic: Asp, Glu

Basic: Lys, Arg, His

Other conservative amino acid substitutions may also be made as follows:

Aromatic: Phe, Tyr, His

Proton Donor: Asn, Gin, Lys, Arg, His, Trp

Proton Acceptor: Glu, Asp, Thr, Ser, Tyr, Asn, Gin

In another aspect, the present invention provides a seed, plant, or part thereof inoculated with one or more strains of Zopfiella sp. or variants thereof as hereinbefore described.

In a preferred embodiment of this aspect of the present invention, the fungus may confer a biofertilizer property to a seed, plant, or part thereof. In a more preferred embodiment of this aspect of the present invention, the biofertilizer property may be enhanced growth of the plant or increased crop yields from the plant, as compared with a plant that is absent the fungus, and optionally under one or more abiotic stress condition(s). The abiotic stress condition(s) may include nutrient, water, or temperature stress, including nutrient stress comprised of below normal levels of a plant nutrient such as nitrogen, phosphorus or potassium. In another preferred embodiment, the biofertilizer property may be associated with enhanced growth or crop yields of a seed, plant, or part thereof, wherein the plant part may be a root or a shoot.

The fungi of the present invention may have the ability to be transferred through propagative material from one plant generation to the next. The fungi may then spread or locate to other tissues as the plant grows, e.g. to roots. Alternatively, or in addition, the fungi may be recruited to the plant root, e.g. from soil, and spread or locate to other tissues. In either regard, the fungi may be said to be stably inoculated and/or infected to the plant.

Therefore, the present invention also provides a seed, plant, or plant propagative material or other plant part derived from a plant inoculated with a fungus as herein described, and inoculated therewith.

In a preferred embodiment of this aspect of the present invention, the seed, plant, or part thereof inoculated with one or more fungi is selected from the group consisting of cereal crops, legumes including annual and perennial legumes, and brassicas.

In a preferred embodiment of this aspect of the present invention, the seed, plant, or part thereof inoculated with one or more fungi is selected from the group consisting of chickpea (C/cer arietinum), faba bean (V/c/a faba), soybean (Glycine max), cotton (Gossypium hirsutum), wheat (Triticum aestivum), barley (Hordeum vulgare), corn (Zea mays), canola (Brassica napus), and broccoli (Brassica oleracea).

The present invention provides the use of a fungus as herein described to produce a seed, plant, or part thereof inoculated, preferably stably inoculated, with the one or more of the described fungi.

The present invention also provides a method for conferring a biofertilizer property to a seed, plant, or part thereof, wherein the method comprises inoculating the seed, plant, or part thereof with a fungus as herein described. In preferred embodiments, the seed, plant, or plant part inoculated or otherwise inoculated with the fungus as herein described will exhibit a biofertilizer property, or in other words, the fungus will confer thereto a biofertilizer property.

The fungi of the present invention may have the ability to secrete a biopolymer which is capable of conferring a biofertilizer property to a seed, plant, or part thereof. Therefore, the present invention also provides a substantially purified or isolated biopolymer synthesized by a fungus as described herein, where the biopolymer is capable of conferring a biofertilizer property to a seed, plant, or part thereof.

Preferably, the biopolymer comprises repeating units derived from tyrosine, acetate, malonylcoenzyme A (malonyl-CoA), or catechol. More preferably, the biopolymer is a pigmented polyphenyl such as eumelanin, pheomelanin, neuromelanin, allomelanin, or pyomelanin. In a preferred embodiment of this aspect of the present invention, the biofertilizer property may be the enhanced growth of the plant or increased crop yields from the plant, as compared with a plant that is absent the fungus, and optionally under one or more abiotic stress condition(s). The abiotic stress condition(s) may include nutrient, water, or temperature stress, including nutrient stress comprised of below normal levels of a plant nutrient such as nitrogen, phosphorus or potassium.

As hereinbefore described, one or more fungi of the present invention may be inoculated into a seed, plant, or part thereof to confer a biofertilizer property to the stably inoculated plant. Alternatively, the one or more fungi may be substantially purified or isolated, and compositions thereof may be manufactured by admixture. For example, spores or hyphae produced by Zopfiella sp., particularly Zopfiella sp. NEM41 or variants thereof, may be isolated from a root of the plant and may be mixed with other components to form a composition. Other components may include, for example, organic compounds or other materials commonly used in compositions, such as binders, carriers, propellants, azeotropes, surfactants, etc., depending on the desired application. These materials and methods of manufacture would be familiar to a skilled worker in the art.

Accordingly, in yet a further aspect, the present invention provides a composition comprising a substantially purified or isolated Zopfiella sp. strain as herein before described, wherein the composition confers a biofertilizer property to a seed, plant, or part thereof into which it is inoculated.

In a preferred embodiment, the composition may include one or more excipients selected from the group consisting of surfactants, including wetting agents, humectants, binders, spreaders, stabilisers, and penetrants.

In another preferred embodiment of this aspect of the present invention, the composition is a liquid formulation. In a more preferred embodiment, the composition may be formulated with a fungal concentration of between about 1x10 ³ to about 1x10 ¹² colony forming units per millilitre (cfu mL ^-1 ), more preferably about 1x10 ⁴ to about 1x10 ¹¹ cfu mL ^-1 , even more preferably about 1x10 ⁵ to about 1x10 ⁸ cfu mL ^-1 , most preferably about 1x10 ⁶ to about 1x10 ⁷ cfu mL ^-1 .

In an alternative preferred embodiment of this aspect of the present invention, the composition is a solid formulation, including a dry powder, granule, pellet, prill, or grain chip including rice chips or corn chips. In a more preferred embodiment, the prill is comprised of one or more powders selected from the group containing bentonite, attapulgite, peat soil, humates, diatomite, and inorganic powders. In a further preferred embodiment of this aspect of the invention, the composition may be formulated with a fungal concentration of between about 1x10 ³ to about 1x10 ¹² colony forming units per gram (cfu g ^-1 ), more preferably about 1x10 ⁴ to about 1x10 ¹¹ cfu g ^-1 , even more preferably about 1x10 ⁵ to about 1x10 ⁸ cfu g ^-1 , most preferably about 1x10 ⁶ to about 1x10 ⁷ cfu g ^-1 .

In a further preferred embodiment of this aspect of the present invention, the biofertilizer property may be the enhanced growth of the plant or increased crop yields from the plant, as compared with a plant that is absent the fungus, and optionally under one or more abiotic stress condition(s). The abiotic stress condition(s) may include nutrient, water, or temperature stress, including nutrient stress comprised of below normal levels of a plant nutrient such as nitrogen, phosphorus or potassium.

Preferably, the seed, plant, or part thereof inoculated with one or more strains of Zopfiella sp., particularly Zopfiella sp. NEM41 , is selected from the group consisting of cereal crops, legumes including annual and perennial legumes, and brassicas. In a preferred embodiment of this aspect of the present invention, the seed, plant, or part thereof inoculated with one or more strains of Zopfiella sp., particularly Zopfiella sp. NEM41 , is selected from the group consisting of chickpea (Cicer arietinum), faba bean (Viola faba), soybean (Glycine max), cotton (Gossypium hirsutum), wheat (Triticum aestivum), barley (Hordeum vulgare), corn (Zea mays), canola (Brassica napus), and broccoli (Brassica oleracea).

In yet a further aspect, the present invention provides a method for preparing a composition for conferring a biofertilizer property to a seed, plant, or part thereof, wherein the method includes: a. culturing one or more fungal strain(s) as described herein, including growing the strain(s) under aerobic or anaerobic conditions in a bioreactor comprising culture medium which contains a source of carbohydrates; b. substantially purifying or isolating the strain(s) in a reproductively viable form; c. preserving the strains(s) in a liquid ferment; d. formulating the strain(s) into a composition for inoculating into seeds, plants, or parts thereof. By a ‘bioreactor’ is meant a device or system that supports a biologically active environment, such as a vessel in which is carried out a chemical process involving fungi of the present invention and/or products thereof. The chemical process may be aerobic or anaerobic. The bioreactor may have a volume ranging in size from milliliters to cubic metres, for example from approximately 50 millilitres to approximately 50,000 litres. The bioreactor may be operated via batch culture, batch feed culture, perfusion culture or continuous culture, for example continuous culture in a stirred-tank bioreactor. Fungi cultured in the bioreactor may be suspended or immobilised.

In a preferred embodiment of this aspect of the invention, the bioreactor is selected from a batch-type, a feed-type, or a continuous-type reactor. More preferably, the bioreactor is a fermenter.

The source of carbohydrates used in culturing the one or more strain(s) may be a starch/sugar-based agar or broth such as potato dextrose agar (PDA), potato dextrose broth or half strength potato dextrose agar (HPDA) or a cereal-based agar or broth such as oatmeal agar (OA) or oatmeal broth. Other sources of carbohydrates can include endophyte agar (ENDO), Murashige and Skoog with 20% sucrose (MS-SUC), half V8 juice/half PDA (V8PDA), water agar (WA) and yeast malt extract agar (YME), molasses, or corn steep liquor.

In a preferred embodiment, the strain(s) may be cultured in a culture medium containing potato dextrose broth, molasses, yeast malt extract, and/or corn steep liquor. In a more preferable embodiment, the strain(s) may be cultured in a medium containing between about 0.05 and about 5 weight percentage (% w/v) malt extract, between about 0.025% and about 2.5% w/v sucrose, between about 0.01% and about 1.0% w/v yeast extract, and between about 0.005% and about 0.5% w/v commercially available brewers’ yeast extract (e.g., vegemite or marmite) in aqueous solution at about pH 4.5.

By weight percentage (% w/v) as used herein is meant mass in grams of a substance per 100 millilitres of solvent.

The fungus may be cultured for a period of approximately 1 to approximately 30 days, more preferably from approximately 1 to approximately 14 days more preferably from approximately 1 to approximately 7 to 10 days. The fungus may also be cultured under conditions of constant temperature, or under conditions of a range of temperatures. Preferably, the fungus is cultured at 28 °C.

In a preferred embodiment, the method may include the further step of substantially purifying or isolating the one or more Zopfiella sp. strain(s) as described herein, particularly Zopfiella sp. NEM41 , by providing samples of a plant or part thereof; preparing extract(s) from the sample(s); and growing fungal colonies from the extract(s) to select fungal colonies capable of conferring a biofertilizer property to seeds, plants, or plant parts inoculated therewith.

Preferably, the sample(s) of plant material is selected from the group comprising flowers, flower bracts, leaves, petioles, stems, roots, and rhizospheres, preferably roots and/or rhizospheres.

In a preferred embodiment of this aspect of the present invention, the biofertilizer property may be the enhanced growth of the plant or increased crop yields from the plant, as compared with a plant that is absent the fungus, and optionally under one or more abiotic stress condition(s). The abiotic stress condition(s) may include nutrient, water, or temperature stress, including nutrient stress comprised of below normal levels of a plant nutrient such as nitrogen, phosphorus or potassium.

In this aspect of the present invention, the step of preserving the strain(s) in a liquid ferment preferably includes the addition of one or more of culture medium, sucrose, maltodextrin, bentonite, guar gum powder, diatomaceous earth, milk powder, soy protein, lecithin, or carboxymethyl cellulose to the substantially purified or isolated strain(s).

Preferably, the liquid ferment contains between about 0.05% and about 5% w/v malt extract, between about 0.025% and about 2.5% w/v sucrose, between about 0.01% and about 1.0% w/v yeast extract, and between about 0.005% and about 0.5% w/v commercially available brewers’ yeast extract (e.g. Vegemite™) in aqueous solution at about pH 4.5. In a further aspect of the present invention, there is provided an apparatus comprising a composition as described herein, wherein the apparatus is used to confer a biofertilizer property to a seed, plant, or part thereof.

In a preferred embodiment of this aspect of the invention, the apparatus may include a seed coating or an aerosolized spray. In yet a further aspect of the present invention there is provided a method to confer a biofertilizer property to a seed, plant, or part thereof wherein the method comprises inoculating a seed, plant, part thereof with one or more Zopfiella sp. strain(s), particularly Zopfiella sp. NEM41 , using a composition as described herein. Preferably, the method includes using an apparatus as described herein.

In a preferred embodiment of this aspect of the invention, the biofertilizer property may be the enhanced growth of the plant or increased crop yields from the plant, as compared with a plant that is absent the fungus, and optionally under one or more abiotic stress condition(s). The abiotic stress condition(s) may include nutrient, water, or temperature stress, including nutrient stress comprised of below normal levels of a plant nutrient such as nitrogen, phosphorus or potassium.

The present invention will now be more fully described with reference to the accompanying Examples and drawings. It should be understood, however, that the description following is illustrative only and should not be taken in any way as a restriction on the generality of the invention described above.

Brief Description of the Drawings/Figures

In the Figures:

Figure 1. Fungal collection Site No. 2. (a) The foreground of the site featuring large perennial rye grass (Lolium perenne), barley grass (Hordeum leporinum), and prairie grass (Bromus catharticus) plants; and (b) an example of one large plant from which root samples were collected.

Figure 2. Examples of the root structure of selected plants from 3 different sampling intervals at Site No. 2. Potential samples for isolation of darkly pigmented endophytic fungi at (a) first collection; (b) second collection; and (c) third collection. In each instance, the upper surface soil contains a layer of sand, under which a discontinuous change in colour is evident within the root structure of the plant.

Figure 3. Zopfiella sp. NEM41 a) grown on potato dextrose agar (PDA) in a petri dish (front and rear sides of the petri dish), and b) NEM41 spores. Figure 4. Nucleotide sequence of the ITS region of the rRNA gene isolated from Zopfiella sp.

NEM41 (SEQ ID NO: 1).

Figure 5. A phylogenetic tree showing the phylogenetic relationships among Zopfiella sp. NEM41 (NEM41 MO-2) and Zopfiella sp. (BJF10, from Quingfen et al. 2018) along with their respective allies. Isolates NEM41 MO-2 and BJF10 are represented by a solid black circle and a solid black square, respectively.

Figure 6. Photographs demonstrating the effect of Zopfiella sp. NEM41 on a) germination and b) plant growth promotion when inoculated into wheat seeds. Wheat seeds inoculated with Zopfiella sp. NEM41 are presented in the left-hand images, and control seeds (no inoculant) are shown in the right-hand images for both a) and b).

Figure 7. Photograph demonstrating the effect of Zopfiella sp. NEM41 on plant growth promotion when inoculated into wheat seeds. Wheat seeds inoculated with Zopfiella sp. NEM41 are shown on the right-hand side and control seeds (no inoculant) are shown on the left-hand side of the image.

Figure 8. Wheat seeds inoculated with Zopfiella sp. NEM41 exhibit enhanced tolerance to water stress. Wheat seeds were germinated and grown for 7 days, during which time the seeds were watered regularly. After day 7, the sprouted wheat seeds were exposed to water deprivation for 21 days. On day 29, wheat seedlings which had been inoculated with Zopfiella sp. NEM41 remained robust and growing (left hand side of the image), while wheat seedlings which were not inoculated with Zopfiella sp. NEM41 appeared severely moisture stressed and dying (right hand side of the image).

Figure 9. Microscopic images (20X and 40X) showing biopolymer deposition by Zopfiella sp. NEM41 cultured on PDA petri plates. The biopolymer (i.e. , the dark pigment) is observed as micro-aggregates at the crossing junction of hyphae.

Figure 10. Mung bean seeds inoculated with Zopfiella sp. NEM41 exhibited enhanced growth upon co-inoculation with nitrogen-fixing rhizobia. Mung bean seeds were germinated and grown in pots as bare seeds, seed coated with rhizobia, or seed coated with rhizobia and inoculated with Zopfiella sp. NEM41. Mung beans grown co-inoculated with Zopfiella sp. NEM41 and rhizobia displayed significant gains in root and shoot dry weights as compared to plants grown without the endophyte. Figure 11. Soybean seeds inoculated with Zopfiella sp. NEM41 exhibited enhanced growth upon co-inoculation with nitrogen-fixing rhizobia. Soybean seeds were germinated and grown in pots as bare seeds (left hand image), seed coated with rhizobia (middle image), or seed coated with rhizobia and inoculated with Zopfiella sp. NEM41 (right hand image). Soybeans grown co-inoculated with Zopfiella sp. NEM41 and rhizobia displayed significant gains in root and shoot dry weights as compared to plants grown from bare seed.

Detailed Description of the Embodiments

Example 1 - Collection of Endophytes

Screening activities were undertaken to identify and isolate fungal endophytes with soil carbon sequestration (SOC) properties. In particular, screening efforts were undertaken to identify fungi which sequester labile carbon from root exudates to store in a more concentrated stable form. The screening protocol was conducted in multiple sites across the southern region of the Australian state of New South Wales. Each site had differing crops, soil types, and local environments which were expected to foster diverse and differing root- associated microfauna.

Using a trowel, approximately 5 to 10 g of visually healthy root material was harvested from a variety of plants at each location. Individual root samples were immediately placed in airtight plastic bags to prevent desiccation and transported under ambient conditions to a laboratory where the samples were stored at 5°C until further use. Isolates of bacteria and fungi were collected, and fungi screened for carbon sequestration and plant growth promotion (PGP).

Example 2 - Isolation of Fungal Endophytes

Root-associated fungi were isolated within 48 h of collection. In total, 119 root-associated fungi were collected (Table 1). For the isolation process, excess soil was shaken from the root system, which was then thoroughly washed with distilled water. Next, the exposed root system was surface sterilized in a laminar flow cabinet by sequentially submerging the roots in 70% v/v ethanol for 10 seconds followed by 2% w/v sodium hypochlorite for 2 minutes. Root systems were then aseptically removed from the hypochlorite solution and washed three times with sterile water. Ten root fragments (1 cm in length) from each freshly washed root system were aseptically sectioned, and each fragment was placed into a sterilised disposable plastic petri dish containing Potato Dextrose Agar (PDA) and an antibacterial agent (i.e. , chloramphenicol). A maximum of five fragments were placed onto a single PDA petri dish. The root fragments were incubated in the dark at 28°C for 14 days. Hyphae emerging from root segments after the incubation period were recorded as root-associated fungi and provided an ID/accession number (Table 1). The effectiveness of the surface sterilisation process was further evaluated by recording imprints of the freshly washed root systems on PDA petri dishes, incubating those imprinted PDA petri dishes in the dark at 28°C for 7 days, and evaluating the PDA petri dishes for epiphytes after the incubation period. Surface sterilisation was deemed effective where no epiphytes were observed.

Selection of Biopolymer-Producing Fungal Endophytes

Selection of root-associated fungi capable of producing a pigmented biopolymer was performed on the basis of qualitative rather than quantitative analysis. Specifically, fungi that produced a dark-coloured pigment were assumed to have accumulated a high concentration of biopolymer in their hyphae (Table 1).

Selection of Non-Pathogenic Fungal Endophytes

Endophytic fungi with darkly pigmented hyphae were repeatedly sub-cultured on PDA until pure cultures of each isolate were obtained. Upon pigmentation, the isolates were further subjected to qualitative pathogenicity testing (Table 1). Non-pathogenic root-associated endophytic fungi that secreted a dark-coloured biopolymer over a period of 4 weeks were then selected and stored in 50% v/v glycerol at 5°C. Working stocks of these isolates were also prepared by inoculating strains on PDA slants in McCartney bottles which were grown in an incubator and then stored at 5°C. Fresh cultures were established from the stock cultures by transferring agar plugs to the centre of potato dextrose agar plates an incubating at 28°C in the dark. A number of non-pathogenic root-associated endophytic fungi which produced a dark-coloured biopolymer were identified and stored for further analysis.

Table 1. Endophytic isolates identified according to their accession number, host plant, and site number, pigmented biopolymer production status, and pathogenicity. Grass hosts include native, introduced, and pasture grass species. Only endophytes with a dark pigmentation (+) were screened for pathogenicity.

Example 3 - Evaluation of Site Number 2

The isolated root-associated endophytic fungi which were non-pathogenic and capable of secreting a dark-coloured biopolymer were all collected at a single site (Table 1, underlined entries). On gross visual inspection, this site exhibits large perennial rye grass (Lolium perenne), barley grass (Hordeum leporinum ), and prairie grass (Bromus catharticus) (Figure

1a). These grasses were observed within a swath of similar grasses, and microbial isolates were collected from large plants, such as that featured in Figure 1b. At that time, the larger plants in the area were distinct from plants observed elsewhere in the swath.

Unsurprisingly, the soil at the site was largely of an alluvial flood plain with one notable exception wherein a small anthropogenic deposit of sand had been made. In areas where the fungal isolates of interest were collected, soil discolouration was also observed. However, the apparent discolouration which is attributed to the KAM41 (i.e. , NEM41) isolate declines rapidly over 2-4 m from the centre of the site (Figure 2).

Top soil samples were also collected at the site to elucidate the source of the aforementioned discolouration observations. Upon evaluation, distinct differences were detected between soil collected spanning a 60 m transect and extending across the site (Table 2).

Table 2. Comparison of soils within the collection site and soils within the 60 m transect near the collection site (0-10 cm sampling depth). Example 4 - Identification of a Novel Fungal Strain

Morphology

The morphologies of the fungal isolates collected at site 2 (and shown in Table 1) were compared. Of these, the isolate which best embodied the collective features (i.e. , KAM41) was selected for further analysis and re-identified as NEM41.

NEM41 is characterised by non-ostiolate ascomata, clavate to cylindrical, usually evanescent asci lacking an apical ring. The ascospores are ellipsoidal, dark brown, transversely septate with a hyaline pedicel which often collapses (Figure 3). This teleomorphic species positions in the family Lasiosphaeriaceae.

Genomic Sequencing

The purified fungal cells of NEM41 were subjected to genomic DNA extraction, and the ITS region of the rRNA gene was sequenced (Figure 4, Table 3) by the Australian Genome Research Facility (AGRF, Adelaide and Melbourne).

Phylogenetic Analysis

A phylogenetic analysis of NEM41 was undertaken by sequence homology comparison of the ITS region of the rRNA gene. Sequence homology searching against the NCBI nucleotide database (BLAST n - type specimen parameter) identified closely related fungi to isolate NEM41 , of which Zopfiella species were the closest match - hence forth called Zopfiella sp. NEM41. These sequences, and sequences of other Zopfiella species (type specimens), were collected and aligned in MEGAX v 6.2.[1] Based on the sequence alignment, phylogenetic relationships were inferred using Maximum Likelihood (ML) analysis (Figure 5). [2] For ML analysis, phenograms were obtained using the nearest-neighbour-interchange method, applying the Tamura-Nei model. Specifically, the heuristic searches were obtained by applying Neighbour-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach and selecting topology with a superior log likelihood value. Confidence of branching was assessed by computing 500 bootstrap replications; branches corresponding to partitions reproduced in less than 50% bootstrap replicates were collapsed from the dataset. [3] A total of 591 partitions remained in the final dataset. Table 3. ITS sequence analysis of Zopfiella sp. NEM41 .

Culture Establishment

Root samples were taken from select plants and washed in clean water. The plant roots were then subjected to a three-stage wash cycle: 1 minute in 1 % w/v calcium hypochlorite, 3 minutes in 70% v/v ethanol, and triplicate rinses with distilled water. The roots were next crushed, and the exudate was filtered and spread onto PDA dilution plates. The plates were incubated at 28°C for 7 days. Growing microbial colonies were selected, isolated from the agar, and purified on PDA dilution plates. These purified cultures were then grown in shaker flasks containing potato dextrose (PD) broth to produce mycelia. The mycelia and broth were then coated onto wheat seed and grown in sterile media. Zopfiella sp. NEM41 that improved the growth of the wheat were isolated and stored for further study.

Pathogenicity

The antagonistic effect of Zopfiella sp. NEM41 was assessed against a number of common plant pathogens and was found to have none (Table 4). The non-pathogenic nature of Zopfiella sp. NEM41 was further evaluated using the quantitative methods discussed below.

Table 4. Qualitative Evaluation of the Pathogenic Nature of Zopfiella sp. NEM41.

To determine the inhibition spectrum of Zopfiella sp. NEM41, the antifungal activity was assessed on PDA using a dual-culture technique. Zopfiella sp. NEM41 and all tested pathogenic fungi were grown separately on PDA. Agar plugs of 5mm size taken from the colony margins of young cultures of Zopfiella sp. NEM41 grown on PDA were inoculated to fresh PDA plates to evaluate inhibition of fungal growth. To each plate one selected isolate and one pathogen of same size agar plug were inoculated to opposite sides of fresh agar plates and incubated for 7 days at 28°C. The experiment was repeated three times with five replicates. At the end of the incubation period, plates were evaluated for inhibition of the growth of the pathogen according to following formula:

The results revealed that Zopfiella sp. NEM41 did not appear to produce any detectible inhibition zone or clear zone against any challenged pathogens whereas all the pathogenic fungi including control completely occupied the Petri plate (Table 4).

Endophytism

Zopfiella sp. NEM41 was evaluated for root endophytism. In particular, root samples were excised from pot cultures of wheat plants and fragmented into 20 to 30 sections which were then surface sterilized with 3% w/v sodium hypochlorite and washed with sterile water. The root fragments were then placed in petri dishes with PDA. Original colonies of Zopfiella sp. NEM41 emerged and were re-isolated from the extremes of the root fragments, confirming the endophytic nature of Zopfiella sp. NEM41 fungi.

Example 5 - Biofertilizer Phenotype of Zopfiella sp. NEM41.

Germination and Plant Growth Promotion

Zopfiella sp. NEM41 was grown on PDA for two weeks at 28°C, at which time it was harvested and prepared for inoculation into wheat seeds. Specifically, Zopfiella sp. NEM41 colonies were macerated in distilled water using a hand blender and subsequently inoculated into wheat seeds at a concentration of approximately 1x10 ⁶ cfu mL ^-1 , as determined by serial dilutions and plate counts. Zopfiella sp. NEM41-inoculated wheat seeds and control wheat seeds (i.e., seeds containing no inoculant) were sown on separate moist paper towels (Figures 6 and 7). After 8 days, germination and plant growth promotion were calculated for the respective plants (Table 5).

Table 5. Effect of Zopfiella sp. NEM41 on germination and plant growth promotion in wheat seeds.

Low Nitrogen Level Tolerance

A single Zopfiella sp. NEM41 colony was grown on PDA and then placed into a petri dish containing a nitrogen-deficient malate medium (NFM) prepared according to Table 6, adjusted to pH 7.2-7.4 with KOH, and supplemented with 50 mg L ^-1 yeast extract. Plates were incubated at 28°C for 14 days. A resulting single colony was then re-streaked onto NFM. The ability of Zopfiella sp. NEM41 to persist under limited nitrogen conditions was qualified relative to a species with known tolerance to low nitrogen levels (i.e. , rhizobium) and to other fungal endophytes (Table 7).

Table 6. Recipe used in the preparation of nitrogen-deficient malate medium (NFM).

Table 7. Qualitative tolerance to low nitrogen levels of Zopfiella sp. NEM41 compared to other endophytic microorganisms. Phosphate Solubilisation Capability

Phosphate solubilisation was evaluated in vitro. Specifically, Zopfiella sp. NEM41 was cultivated on solid National Botanical Research Institute’s phosphate growth medium (NBRIP) prepared according to Table 8. NBRIP provides an accurate means for identifying efficient phosphate solubilisation given that the medium requires cultivated species to utilise inorganic tricalcium phosphate (i.e., Ca ₃ (PO ₄ ) ₂ ) exclusively. The ability of Zopfiella sp. NEM41 to solubilise phosphate under the test conditions was qualified with respect to other endophytic microorganisms, including a known phosphate solubilisation species (i.e., Trichoderma) (Table 9).

Table 8. Recipe used in the preparation of National Botanical Research Institute’s phosphate growth medium (NBRIP).

Table 9. Qualitative phosphate solubilisation capability of Zopfiella sp. NEM41 compared to other endophytic microorganisms. Trought Tolerance

Wheat seeds were inoculated with Zopfiella sp. NEM41 sown on filter paper, germinated, and sustained for 7 days. A control group of wheat seeds containing no Zopfiella sp. NEM41 was also sown on filter paper, germinated, and sustained for 7 days. During the 7-day period, both groups of sprouted seeds were moistened with the same amount of water. After day 7, the wheat seedlings were exposed to water deprivation for 21 days. On day 29, wheat seedlings which had been inoculated with Zopfiella sp. NEM41 remained robust and growing, while wheat seedlings which were not inoculated with Zopfiella sp. NEM41 appeared severely moisture stressed and dying (Figure 8).

Pigmented Biopolymer Production

A Zopfiella sp. NEM41 colony was cultured on PDA at 28°C for three weeks. Black deposits were observed in vitro as micro-aggregates at the crossing junctions of hyphae (Figure 9). Ten fungal mycelial plugs, each with a 5 mm diameter, were collected from the periphery of a growing colony and transferred into a 500 mL Erlenmeyer flask containing 250 mL of sterile liquid broth, pH 4.5. The liquid broth was autoclave sterilised at 121 °C for 20 min prior to use. The mycelial plugs were cultivated in the dark for three weeks at 28°C on a reciprocal shaker (160 rpm). Afterwards, the cultures were centrifuged at 11 ,000 rpm for 15 min. The fungal mycelia were then harvested, desiccated (60°C for 48 h; cooled at room temperature for 20 min), and weighed before storing in the dark at 4°C.

A dark pigment, presumably melanin or melanin-like biopolymer, was extracted from 1 g of desiccated fungal biomass and dissolved in 5 mL of 1M KOH. The solution remained undisturbed under ambient conditions for 48 h, then the solution was autoclave sterilised (20 min at 121 °C). The sterile mixture was centrifuged (11 ,000 rpm for 15 min), and the supernatant was acidified to pH 2.5 with 2M HCI. The acidic solution was then re-centrifuged (11.000 rpm for 15 min), the supernatant was decanted, and the darkly pigmented biopolymer pellet was reserved.

Black cumin seed (Nigella sativa L.) which is known to contain stable forms of melanin, particularly allomelanin, was used as a positive control. Similar to the steps described above, the black cumin seeds were submerged in 2M HCI, and the acidic solution was centrifuged (11 ,000 rpm for 15 min) to pellet the precipitate. The acidic supernatant was decanted, and the darkly pigmented biopolymer pellet was washed reserved. The biopolymers extracted from both the fungal biomass and the black cumin seed were washed three times with deionized water, dialysed, and desiccated (60°C for 48 h). Next, the dehydrated biopolymers were serially washed with chloroform, ethyl acetate, and ethanol and stored at 20°C for further physical and chemical analysis which indicated that the pigmented biopolymer extracted from the fungal biomass is similar to the biopolymer extracted from black cumin seed (Table 10).

Table 10. Comparative analysis of biopolymers extracted from Zopfiella sp. NEM41 fungal biomass and from Nigella sativa L. (black cumin seed), respectively. Example 6- Conferral of Biofertilizer Phenotype (/n planta) by Zopfiella sp. NEM41.

Plant Growth Promotion: Glasshouse Trial

Rhizobia are bacteria that establish mutually beneficial relationships with legumes; the rhizobia fix nitrogen, thereby supplying legumes with an essential nutrient. Here, mung bean seeds were germinated and grown in pots as either bare seeds, seeds coated with rhizobia, or seeds coated with rhizobia and inoculated with Zopfiella sp. NEM41. Under these conditions, mung beans that were both coated with rhizobia and inoculated with Zopfiella sp. NEM41 displayed significant gains in root (P>95%) and shoot (P>68%) dry weights as compared to plants grown without the endophyte (Figure 10). Visual assessment indicated that nodules were larger in plants which received the dual treatment rather than rhizobia alone.

In particular, mung beans co-cultivated with rhizobia and Zopfiella sp. NEM41 exhibited a 90% increase in root dry weight and a 35% increase in shoot dry weight over mung beans cultivated only in the presence of rhizobia (Table 11 , average of 4 replicates).

Table 11. Mung Bean Seeds Co-cultivated with rhizobia and Zopfiella sp. NEM41.

Soybean co-cultivated in the presence of both rhizobia and Zopfiella sp. NEM41 also exhibited significant increases in root and shoot dry weights compared to soybean plants sown from bare seed (Table 12, average of 4 replicates). No significant differences were found between soybean cultivated with dual seed treatment (i.e., rhizobia + Zopfiella sp. NEM41) and single seed treatment (i.e., rhizobia) (Figure 11). Table 12. Soybean Seeds Co-cultivated with rhizobia and Zopfiella sp. NEM41.

Plant Growth Promotion: Small Pot Trial

The plant growth promotion potential of Zopfiella sp. NEM41 was further evaluated in potted plants grown from bare wheat seeds (negative control) or wheat seeds inoculated with Zopfiella sp. NEM41. The wheat seeds for both test conditions were sown in pots (150 mm) that were placed outdoors and watered with same frequency and with approximately equal volumes of water. At harvest (97 days), the above ground biomass was removed and weighed, and all tillers were counted and measured. In addition, all soil was sieved using a 2 mm sieve, and weights were recorded for the soil, the roots, and the small particulate matter (i.e., gravel and small roots).

The data corresponding to all above ground biomass is shown in Table 13. The results indicate that plants grown from seeds inoculated with Zopfiella sp. NEM41 contained significantly more tillers per plant than controls (P>95%) and had larger shoot weight per plant (P>85%). However, no statistical difference in tiller height was detected between the two test variants.

Table 13. Tiller number and height for wheat plants cultivated in pots from seeds sown with or without (control) Zopfiella sp. NEM41 inoculation. Although no significant difference in tiller height was observed, the control treatment contained a higher proportion of larger’ tillers as well as a greater distribution in tiller sizes (i.e., a higher standard deviation) than the Zopfiella sp. NEM41 -inoculated plants. Given that the smaller tillers are unlikely to generate grain heads, the data was further analysed with respect to above ground tillers >20cm (e.g., tiller counts and heights, Table 14). The sorted data continued to show that Zopfiella sp. NEM41 -inoculated plants contain significantly more tillers per plant (P>95%) and also enable a significant advantage in achieving uniform tiller height (P>75%).

Table 14. Tiller number and height for wheat plants with tiller heights >20 cm cultivated in pots from seeds sown with or without (control) Zopfiella sp. NEM41 inoculation.

Promotion of a biofertilizer phenotype by Zopfiella sp. NEM41 was further evidenced by the significantly enhanced root mass in inoculated wheat plants versus control plants (P>99%). Moreover, upon extraction, the root systems and surrounding soil of inoculated plants felt moister than those of control plants, and the soil appeared to hold a more defined aggregate structure akin to that previously observed in relation to melanising fungi. Whole pot root weights and shoot weights are provided in Table 15.

Table 15. Root and shoot masses for wheat plants cultivate in pots from seeds sown with and without (control) Zopfiella sp. NEM41 inoculation. Dry mass was obtained by heating the plant matter to 105°C.

Example 7 - Development of Biofertilizer Composition

Compositions comprising Zopfiella sp. NEM41 were optimized using both solid state fermentation and liquid fermentation processes.

Solid State Fermentation

Endophytic fungal spore production from Zopfiella sp. NEM41 was promoted using a corn grit (2 mm) substrate. In particular, Zopfiella sp. NEM41 was grown on PDA for 2 weeks then macerated using a hand blender. The fungal mixture was then inoculated into a liquid medium formulated according to Table 16. Moisture content was maintained at 80%, and sterile 10% (v/w) inoculum broth (pH 5) was added to the medium. The inoculant was incubated at 28°C for 10 days, at which time all the corn grit was encased in black-coloured

Zopfiella sp. NEM41 fungal biomass. For the optimized solid state fermentation recipe, the fungal spores were produced at a concentration of 1x10 ⁵ cfu g ^-1 . Table 16. Test levels and optimized values for chemical substances, pH levels, and incubation periods used in solid state fermentation of Zopfiella sp. NEM41 on corn grit. All solutions were formulated under aqueous conditions to a final solution volume of 1 L.

Liquid Fermentation

The solid state fermentation method produced good spores but was also susceptible to contamination and problematic to scale up. Consequently, a liquid fermentation protocol was also developed wherein a liquid ferment was optimized according to Table 17.

In particular, the optimized liquid ferment was formulated at about pH 4.5 using citric acid to adjust the pH as necessary. Once formulated, the liquid ferment (400 mL) was autoclave sterilised (121°C for 30 min). Next, a blade was soaked in 70% (v/v) ethanol within a laminar flow hood using aseptic conditions, flame sterilised, and used to excise a 1 x 1 cm sample from the mature mother Zopfiella sp. NEM41 petri plate. The excised fungal sample was inoculated into a flask containing the optimised liquid ferment and incubated on a rotary shaker (160 rpm) at 28°C for 4 days. Table 17. Trial ranges for chemical substances, pH levels, and incubation periods used to optimise the liquid fermentation recipe for Zopfiella sp. NEM41. Aqueous solutions were obtained by combining respective ingredients with water to a final solution volume of 1L.

It is to be understood that various alterations, modifications and/or additions may be made without departing from the spirit of the present invention as outlined herein.

As used herein, except where the context requires otherwise, the term "comprise" and variations of the term, such as "comprising", "comprises", and "comprised", are not intended to be in any way limiting or to exclude further additives, components, integers, or steps.

Reference to any prior art in the specification is not and should not be taken as an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in Australia or any other jurisdiction or that this prior art could reasonably be expected to be ascertained, understood, and/or regarded as relevant by a person skilled in the art. References

1. Kumar S., Stecher G., Li M., Knyaz C., and Tamura K. (2018). MEGA X: Molecular Evolutionary Genetics Analysis across computing platforms. Molecular Biology and Evolution 35:1547-1549.

2. Tamura K. and Nei M. (1993). Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Molecular Biology and Evolution 10:512-526.

3. Felsenstein J. (1985). Confidence limits on phylogenies: An approach using the bootstrap. Evolution 39:783-791.