WO2022174313A1

WO2022174313A1 - Methods for carbon capture and increasing yield of crop plants

Info

Publication number: WO2022174313A1
Application number: PCT/AU2022/050137
Authority: WO
Inventors: Ahsanul HAQUE; Suresh SUBASHCHANDRABOSE; Abed CHAUDHURY; Neeraj PURUSHOTHAM; Tegan Nock; Frank Oly
Original assignee: Loam Bio Pty Ltd
Priority date: 2021-02-22
Filing date: 2022-02-22
Publication date: 2022-08-25
Also published as: AU2022222914A9; AU2022222914A1

Abstract

The present invention relates to enhancement of agricultural soils, mitigating atmospheric carbon dioxide, and providing agronomic benefits to crop plants. More particularly, the present disclosure relates to methods and compositions for increasing soil organic carbon in soil, and increasing crop plant yield.

Description

METHODS FOR CARBON CAPTURE AND INCREASING YIELD OF CROP PLANTS

The present application claims priority from Australian application no. 2021900474 filed 22 February 2021, the entirety of which is incorporated herein by reference.

Technical Field

The present disclosure relates to enhancement of agricultural soils, mitigating atmospheric carbon dioxide, and providing agronomic benefits to crop plants. More particularly, the present disclosure relates to methods and compositions for increasing soil organic carbon in soil, and increasing crop plant yield.

Background

Carbon dioxide and methane absorb and retain heat in the atmosphere, and therefore both gases play a pivotal role in the greenhouse effect. As methane is much more short-lived than carbon dioxide, carbon dioxide is often considered to be more important than that of methane to the greenhouse effect.

The life cycle of carbon includes the removal of carbon dioxide from the atmosphere by plants through photosynthesis. During the process of photosynthesis, the carbon dioxide gets absorbed through stroma of leaves, further converted into sugars and absorbed by the roots. Such sugars become nutrients for plants and microbes present in the soil. Carbon enters back into the atmosphere in the form of carbon dioxide by respiration and combustion. Hence, a balanced amount of release and absorption of the carbon dioxide is an essential step for balancing the ecosystem.

Human activities such as combustion of fuels, overpopulation, forest degradation, soil erosion, etc. have led to an increase in atmospheric carbon dioxide. Approaches for sequestering carbon dioxide from the atmosphere therefore present an important component of a strategy for reducing or controlling atmospheric carbon dioxide. However, for this to be successful, there must also be a reduction in the release of carbon dioxide from soil back into the atmosphere.

Decay of plants, animals, and microbes into the soil can lead to the build-up of soil organic carbon (SOC), which promotes physical stability of the structure of the soil, soil aeration, water drainage and retention, thus reducing soil erosion and nutrient leaching. However, intensive cultivation has also led to a decline in SOC, eventually making the land unsuitable for commercial crop production or even for reclaiming by native growth.

Additionally, farming practices to improve crop yields, such as crop rotation and the use of chemical fertilisers, can result in adverse consequences, such as altered natural landscapes owing to the requirement for greater land areas, degradation of soil quality (e.g., compaction, acidification), and pollution of water systems.

It would be advantageous to develop compositions and methods for increasing soil carbon in a manner that will produce more stable carbon in the soil by sequestering atmospheric carbon, as well as provide agronomic benefit such as increased crop yield.

Summary

The inventors have found that some species of fungi, and in particular endophytic fungi, are capable of fixing carbon in the soil and/or increasing the yield of crop plants, when the plant is inoculated with the fungus.

The present disclosure provides a microbial treatment which comprises at least one fungal strain to be deployed in the soil, wherein the fungus may be deployed in conjunction with, or associated with, a crop plant that is a non-native plant host of the fungus. A first aspect provides a microbial treatment to be deployed in soil and/or associated with a crop plant, the treatment comprising: one or more fungal species from at least one genus selected from Clohesyomyces, Darksidea and Phialocephala fortinii s.l - Acephala applanate species complex (PAC), wherein the deployment of said treatment has one or more desirable effects selected from the group consisting of increasing the sequestration of atmospheric carbon for storage as stable carbon in the soil, providing agronomic benefits to said commercial crops, increasing the levels of stable carbon in the soil, and increasing the soil aggregate stability of the soil.

A second aspect provides a method for sequestering atmospheric carbon for storage as stable carbon in soil, the method comprising deploying a treatment comprising one or more fungal species from at least one genus selected from Clohesyomyces, Darksidea, and Phialocephala fortinii s.l - Acephala applanate species complex, in said soil and/or associating said fungus with a crop plant being cultivated in said soil.

A third aspect provides a method for sequestering atmospheric carbon for storage as stable carbon in soil, the method comprising deploying a treatment comprising one or more fungal species, wherein the one or more fungal species is selected from at least one fungal species having a nuclear ribosomal internal transcribed spacer 2(ITS2) sequence that is at least 90% identical to the nucleotide sequence of SEQ ID No: 1, 2, 3, 4, 5, or 6.

A fourth aspect provides a method of increasing soil organic carbon in a soil and/or increasing yield of a crop plant being cultivated in the soil, the method comprising inoculating the soil and/or the crop plant with an effective amount of one or more fungal species that are heterologous to the crop plant, wherein the one or more fungal species are from at least one genus selected from Clohesyomyces, Darksidea, and Phialocephala fortinii s.l - Acephala applanate species complex. A fifth aspect provides a method of increasing soil organic carbon in a soil and/or increasing yield of a wheat plant being cultivated in the soil, the method comprising inoculating the soil and/or the wheat plant with an effective amount of one or more fungal species that are heterologous to the wheat plant, wherein the one or more fungal species are from at least one genus selected from Clohesyomyces and Darksidea.

A sixth aspect provides a method of increasing soil organic carbon in a soil and/or increasing yield of a canola plant being cultivated in the soil, the method comprising inoculating the soil and/or the canola plant with an effective amount of one or more fungal species that are heterologous to the canola plant, wherein the one or more fungal species are from at least one genus selected from Clohesyomyces, Darksidea, and Phialocephala fortinii s.l - Acephala applanate species complex.

A seventh aspect provides a method of increasing soil organic carbon in a soil and/or increasing yield of a barley plant being cultivated in the soil, the method comprising inoculating the soil and/or the barley plant with an effective amount of one or more fungal species that are heterologous to the barley plant, wherein the one or more fungal species are from at least one genus selected from Darksidea and Phialocephala fortinii s.l - Acephala applanate species complex.

A eighth aspect provides a method of increasing soil organic carbon in a soil and/or increasing yield of a crop plant being cultivated in the soil, the method comprising inoculating the soil and/or the plant with an effective amount of one or more fungal species, wherein the one or more fungal species are heterologous to the crop plant, and are selected from at least one fungal species having a nuclear ribosomal internal transcribed spacer 2 (ITS2) sequence that is at least 90% identical to the nucleotide sequence of SEQ ID No: 1, 2, 3, 4, 5, or 6. A ninth aspect provides a method of increasing soil organic carbon in a soil and/or increasing yield of a crop plant being cultivated in the soil, the method comprising inoculating the soil and/or the plant with an effective amount of one or more fungal strains, wherein the one or more fungal strains are heterologous to the crop plant, and are selected from strain V21/003116, V21/003117, V21/002326, V21/002327 and V21/002328.

A tenth aspect provides a composition for increasing soil organic carbon in a soil and/or increasing yield of a crop plant, comprising one or more fungal species, wherein the one or more fungal species are from at least one genus selected from Clohesyomyces, Darksidea, and Phialocephala fortinii s.l - Acephala applanate species complex.

An eleventh aspect provides a composition when used for increasing soil organic carbon in a soil and/or yield of a crop plant, the composition comprising one or more fungal species that are heterologous to the crop plant, wherein the one or more fungal species are from at least one genus selected from Clohesyomyces, Darksidea, and Phialocephala fortinii s.l - Acephala applanate species complex.

A twelfth aspect provides a composition for increasing soil organic carbon in a soil and/or increasing yield of a wheat plant, comprising one or more fungal species, wherein the one or more fungal species are from at least one genus selected from Clohesyomyces and Darksidea.

A thirteenth aspect provides a composition for increasing soil organic carbon in a soil and/or increasing yield of a canola plant, the composition comprising one or more fungal species, wherein the one or more fungal species are from at least one genus selected from Clohesyomyces, Darksidea, and Phialocephala fortinii s.l - Acephala applanate species complex. A fourteenth aspect provides a composition for increasing soil organic carbon in a soil and/or increasing yield in a barley plant, the composition comprising one or more fungal species, wherein the one or more fungal species are from at least one genus selected from Darksidea and Phialocephala fortinii s.l - Acephala applanate species complex.

A fifteenth aspect provides a composition for increasing soil organic carbon in a soil and/or increasing yield of a crop plant, comprising one or more fungal species that are heterologous to the crop plant, wherein the one or more fungal species is selected from at least one fungal species comprising a nuclear ribosomal internal transcribed spacer 2(ITS2) sequence that is at least 90% identical to the nucleotide sequence of SEQ ID No: 1, 2, 3, 4, 5, or 6.

A sixteenth aspect provides a composition for increasing soil organic carbon in a soil and/or increasing yield of a crop plant, comprising one or more fungal strains, wherein the one or more fungal strains are selected from strains V21/003116, V21/003117,

V21/002326, V21/002327 and V21/002328.

A seventeenth aspect provides a kit for increasing soil organic carbon in a soil and/or increasing yield of a crop plant, the kit comprising one or more fungal species, wherein the one or more fungal species are from at least one genus selected from Clohesyomyces, Darksidea, and Phialocephala fortinii s.l - Acephala applanate species complex, or a composition of any one of the tenth to sixteenth aspects.

An eighteenth aspect provides a kit for increasing soil organic carbon in a soil and/or increasing yield of a wheat plant, the kit comprising one or more fungal species, wherein the one or more fungal species are from at least one genus selected from Clohesyomyces and Darksidea.

A nineteenth aspect provides a kit for increasing soil organic carbon in a soil and/or increasing yield of a canola plant, comprising one or more fungal species, wherein the one or more fungal species are selected from at least one genus selected from Clohesyomyces, Darksidea and Phialocephala fortinii s.l - Acephala applanate species complex.

A twentieth aspect provides a kit for increasing soil organic carbon in a soil and/or increasing yield of a barley plant, comprising one or more fungal species, wherein the one or more fungal species are from at least one genus selected from Darksidea and Phialocephala fortinii s.l - Acephala applanate species complex.

A twenty first aspect provides a kit when used for increasing soil organic carbon in a soil and/or increasing yield of a crop plant, the kit comprising one or more fungal species that are heterologous to the crop plant, wherein the one or more fungal species are selected from species of the genus Clohesyomyces, Darksidea, and Phialocephala fortinii s.l - Acephala applanate species complex, or with a composition of any one of the eleventh to seventeenth aspect.

A twenty second aspect provides a kit for increasing soil organic carbon in a soil and/or increasing yield of a crop plant, the kit comprising one or more fungal species, wherein the one or more fungal species that are heterologous to the crop plant, wherein the one or more fungal species are selected from at least one fungal species having a nuclear ribosomal internal transcribed spacer 2(ITS2) sequence that is at least 90% identical to the nucleotide sequence of SEQ ID No: 1, 2, 3, 4, 5,or 6.

A twenty third aspect provides a kit for increasing soil organic carbon in a soil and/or increasing yield of a crop plant, the kit comprising one or more fungal strains, wherein the one or more fungal strains are selected from strains V21/003116,

V21/003117, V21/002326, V21/002327 and V21/002328. A twenty fourth aspect provides a method for sequestering atmospheric carbon for storage as stable carbon in soil, the method comprising deploying a treatment comprising at least one melanised fungus, wherein said melanised fungus is a species from a genera selected from the group consisting of Clohesyomyces, Darksidea, and Phialocephala fortinii s.l. - Acephala applanata species complex (PAC), in said soil and/or associating said melanised fungus with a commercial crop being cultivated in said soil.

A twenty fifth aspect provides a method for increasing the levels of stable carbon in soil used to cultivate a commercial crop, the method comprising deploying a treatment comprising at least one melanised fungus, wherein said melanised fungus is a species from a genera selected from the group consisting of Clohesyomyces, Darksidea, and Phialocephala fortinii s.l. - Acephala applanata species complex (PAC), in said soil and/or associating said melanised fungus with said commercial crop.

A twenty sixth aspect provides a method for providing agronomic benefits to a commercial crop being cultivated in soil, the method comprising deploying a treatment comprising at least one melanised fungus, wherein said melanised fungus is a species from a genera selected from the group consisting of Clohesyomyces, Darksidea, and Phialocephala fortinii s.l. - Acephala applanata species complex (PAC), in said soil and/or associating said melanised fungus with said commercial crop.

A twenty seventh aspect provides a method for increasing the soil aggregate stability of soil used to cultivate a commercial crop, the method comprising deploying a treatment comprising at least one melanised fungus, wherein said melanised fungus is a species from a genera selected from the group consisting of Clohesyomyces, Darksidea, and Phialocephala fortinii s.l.

- Acephala applanata species complex (PAC), in said soil and/or associating said melanised fungus with said commercial crop.

Detailed Description

The present disclosure relates to a method of increasing soil organic carbon in a soil and/or providing agronomic benefits to a crop plant being cultivated in the soil, such as an increase in yield of the crop plant. The method comprises inoculating the soil and/or the crop plant with an effective amount of one or more fungal species from at least one genus selected from Clohesyomyces, Darksidea, and Phialocephala fortinii s.l - Acephala applanate species complex.

As used herein, a fungus of the genus Phialocephala fortinii s.l - Acephala applanate species complex refers to a species of fungus within the Phialocephala fortinii s.l - Acephala applanate species complex of fungal species. As used herein, a reference to "PAC" or "Phialocephala" or "Phialocephala acephala complex" is a reference to the Phialocephala fortinii s.l - Acephala applanate species complex.

It will be appreciated that the strains of fungi will be fungal strains that are compatible with the crop plant to which they are to be applied. A fungal strain that is compatible with a crop plant, such as a commercial crop plant, is a strain that is not pathogenic to that crop plant. Methods for assessing whether a strain of fungus is non-pathogenic to a particular crop plant are known in the art.

An increase in soil organic carbon is an increase in the amount of organic carbon in soil treated with the one or more fungal species relative to the amount of organic carbon in untreated soil. The soil is treated by inoculating the soil directly with the one or more fungal species, or by inoculating a plant being cultivated in the soil with the one or more fungal species.

As used herein, inoculating a crop plant refers to inoculating a crop plant or any part of a crop plant from which a crop plant could be grown, such as a seed, seedling, shoot, leaf, root, etc, of the crop plant.

In some embodiments, an increase in soil organic carbon comprises an increase in the level of stable carbon in the soil.

In some embodiments, an increase in soil organic carbon comprises an increase in soil aggregate stability of the soil.

An agronomic benefit may be an increase in yield of the crop plant. An increase in yield of a crop plant treated with the one or more fungal species is an increase in fruit, grain or vegetative tissue production of the treated plant relative to that of a crop plant that is the same but which has not been treated with the one or more fungal species described herein when the treated and untreated plant are grown under the same growing conditions. For example, an increase in yield of a treated wheat plant is an increase in the number and/or weight of wheat grains produced by the treated wheat plant relative to that of an untreated wheat plant grown under the same growth conditions. Typically, the increase in yield of a plant treated with the one or more fungal species is an increase in fruit, grain or vegetative tissue production of the treated plant relative to that of a healthy plant of the same type that has not been treated with the one or more fungal species described herein when the treated and untreated plant are grown under the same growing conditions. A healthy plant is a plant that is not infected with, or affected by, a plant pathogen. Typically, a healthy plant is a plant that is not infected with, or affected by, a plant pathogen, and which is grown under conditions for normal growth of that plant (e.g., is not under stress, such as nutrient or drought stress), such as, for example, the conditions under which the crop plant would be grown under during commercial crop production.

As used herein, a crop plant is a crop plant of agronomic importance which is cultivated for food, animal feed, fiber, fuel, and/or industrial purposes. In one embodiment, the crop plant is a commercial crop plant. A commercial crop plant is a plant cultivated to produce a harvested horticultural product that is for sale and/or profit, as well as subsistence crops which may be grown to support other agricultural products, such as livestock.

The inventors have found that growing a crop plant that has been inoculated with a fungal strain that is heterologous to the crop plant, and which is of a species selected from the genus Clohesyomyces, Darksidea, and Phialocephala fortinii s.l - Acephala applanate species complex (PAC), or a combination thereof, results in an increase in soil carbon and/or an increase in yield of the crop plant being cultivated in the soil. As described in the Examples, the inventors have found that agronomic benefits to the crop plants wheat, canola, and barley, such as increased yield, could be provided following growth of the crop plants that had been inoculated with a fungal strain from species of the genus Clohesyomyces, Darksidea, or Phialocephala fortinii s.l - Acephala applanate species complex (PAC), or combinations thereof. The inventors have found that various crop plants inoculated with the fungal species described herein, and in particular endophytic fungal species described herein, exhibit increased yield relative to uninoculated plants. The inventors have further found that the soil in which these plants are grown can have increased organic carbon content relative to soil in which uninoculated plants are grown.

One aspect provides a microbial treatment to be deployed in soil and/or associated with a commercial crop, the treatment comprising: one or more fungal species from at least one genus selected from Clohesyomyces, Darksidea and Phialocephala fortinii s.l - Acephala applanate species complex (PAC), wherein the deployment of said treatment has one or more desirable effects selected from the group consisting of increasing the sequestration of atmospheric carbon for storage as stable carbon in the soil, providing agronomic benefits to said commercial crops, increasing the levels of stable carbon in the soil, and increasing the soil aggregate stability of the soil.

One aspect provides a method of increasing soil organic carbon in a soil and/or increasing yield of a crop plant being cultivated in the soil, the method comprising inoculating the soil and/or the crop plant with an effective amount of one or more fungal species that are heterologous to the crop plant, wherein the one or more fungal species are from at least one genus selected from Clohesyomyces, Darksidea, and Phialocephala fortinii s.l - Acephala applanate species complex (PAC).

In one embodiment, the one or more fungal species is a melanised fungus. The term melanised fungus refers to a fungus that accumulates melanin within its cell walls.

In one embodiment, the one or more fungal species comprise at least about 5% melanin, or at least about 10% melanin, or at least about 15% melanin, or at least about 20% melanin.

In some embodiments, the fungal species are dark septate endophytic (DSE) fungal species. Dark septate endophytic (DSE) fungal species are endophytic fungi which have melanised, septate hyphae and colonise plant roots. Examples of genera which include DSE fungal species include, for example, Clohesyomyces, and Darksidea.

In various embodiments, the one or more fungal species are from at least one genus selected from: a. Clohesyomyces, Darksidea, and Phialocephala; b. Darksidea, and Phialocephala; c. Clohesyomyces and Phialocephala; d. Clohesyomyces and Darksidea.

In one embodiment, at least one of the one or more fungal species is from the genus Clohesyomyces.

In one embodiment, at least one of the one or more fungal species is from the genus Darksidea.

In one embodiment, at least one of the one or more fungal species is from the genus Phialocephala.

In one embodiment, the one or more fungal species is from the genus Phialocephala and Clohesyomyces.

In one embodiment, the one or more fungal species is from the genus Clohesyomyces and Darksidea.

In one embodiment, the one or more fungal species is from the genus Phialocephala and Darksidea. In one embodiment, the one or more fungal species is from the genus Clohesyomyces, Darksidea and Phialocephala.

In one embodiment, the fungal species from the genus Clohesyomyces is Clohesyomyces aquaticus.

In one embodiment, the fungal species from the genus Darksidea is Darksidea delta or Darksidea zeta.

In one embodiment, the fungal species from the genus Darksidea is Darksidea delta.

In one embodiment, the fungal species from the genus Darksidea is Darksidea zeta.

In one embodiment, the fungal species from the genus Darksidea is Darksidea delta and Darksidea zeta.

A fungal species from the genus Phialocephala is a species from the Phialocephala fortinii s.l - Acephala applanate species complex (PAC).

In various embodiments, the soil and/or crop plant is inoculated with: a. Clohesyomyces aquaticus, Darksidea delta, and Darksidea zeta; b. Clohesyomyces aquaticus and Darksidea zeta; c. Clohesyomyces aquaticus and Darksidea delta; d. Clohesyomyces aquaticus, Darksidea delta, Darksidea zeta, and Phialocephala Acephela complex; e. Clohesyomyces aquaticus, Darksidea delta, and Phialocephala Acephala complex; f. Clohesyomyces aquaticus, Darksidea zeta, and Phialocephala Acephala complex; g. Darksidea delta and Darksidea zeta; h. Clohesyomyces aquaticus; i. Darksidea delta; j. Darksidea zeta; k. Phialocephala Acephela complex; or l. Phialocephala Acephela complex and Clohesyomyces aquaticus.

In one embodiment, the one or more fungal species is selected from at least one fungal species having a nuclear ribosomal internal transcribed spacer 2(ITS2) sequence that is at least 90% identical, typically at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or 100% identical, with the nuclear ribosomal internal transcribed spacer 2 (ITS2) sequence of: Phialocephala; Clohesyomyces; or Darksidea.

In some embodiments, the one or more fungal species is selected from at least one fungal species having a nuclear ribosomal internal transcribed spacer 2(ITS2) sequence that is at least 90% identical, typically at least 91%, least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, more typically 100% identical, with the nucleotide sequence of SEQ ID Nos: 1, 2, 3, 4, 5, or 6.

The SEQ ID Nos 1-6 are shown in Table 1.

Fungi from the genus Darksidea suitable for use in the methods described herein may be determined by analysis of the nuclear ribosomal internal transcribed spacer (ITS) sequence of the fungi with the herein defined SEQ ID No. 3, 4, 5 or 6.

In one embodiment, a fungus of the fungal species Darksidea delta has a nuclear ribosomal internal transcribed spacer (ITS) sequence that is at least 97% identical, at least 98% identical, or at least 99% identical, to the nucleotide sequence of SEQ ID NO: 4 or 6.

In one embodiment, a fungus of the fungal species Darksidea zeta has a nuclear ribosomal internal transcribed spacer (ITS) sequence that is at least 97% identical, at least 98% identical, typically at least 99% identical, to the nucleotide sequence of SEQ ID NO: 3 or 5.

Fungi from the genus Phialocephala fortinii s.l - Acephala applanate species complex (PAC) suitable for use in the methods described herein may be determined by analysis of the nuclear ribosomal internal transcribed spacer (ITS) sequence of the fungi with the herein defined SEQ ID No. 1.

In one embodiment, a fungal species of the Phialocephala fortinii s.l - Acephala applanate species complex (PAC) has a nuclear ribosomal internal transcribed spacer (ITS) sequence that is at least 97% identical, at least 98% identical, or at least 99% identical, to the nucleotide sequence of SEQ ID NO: 1.

Fungi from Chiohesyomyces suitable for use in the methods described herein may be determined by analysis of the nuclear ribosomal internal transcribed spacer (ITS) sequence of the fungi with the herein defined SEQ ID Nos. 2.

In one embodiment, a fungus of the fungal species Clohesyomyces aquaticus has a nuclear ribosomal internal transcribed spacer (ITS) sequence that is at least 97% identical, at least 98% identical, typically at least 99% identical, to the nucleotide sequence of SEQ ID NO: 2.

The terms "identical" or "% identical," in the context of two or more nucleic acids refers to two or more sequences that are the same or have a specified percentage of nucleotides that are the same (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection (see, e.g., NCBI web site http://www.ncbi.nlm.nih.gov/BLAST/, or the like).

Algorithms for determining % identity are known in the art.

An example of an algorithm that is suitable for determining percent sequence identity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410 (1990), respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.rtlrn.nih.gov/).

Methods for determining the sequence of ITS2 regions of fungi are known in the art and described in, for example, Ihrmark et al., (2012) FEMS Microbiology Ecology, 82(3):666-677; Yang et al., (2018)

PLoS ONE 13(10): e0206428. As described in the Examples, the inventors have found that soil carbon and/or plant yield can be increased in crop plants by inoculating the crop plant to be cultivated in the soil with the fungi described herein.

In one embodiment, the crop plant is a commercial crop plant. In one embodiment, the commercial crop plant is a compatible crop selected from the group consisting of species of the genus Triticum, Brassica, Gossypium, Zea, Corchorus,

Saccharum, Medicago, Lolium, Coffea, Camellia, Oryza, Hordeum, Boehmeria, Nicotiana and Cannabis.

In one embodiment, the commercial crop is selected from the group consisting of the species Triticum aestivum, Brassica napus, Brassica rapa, Brassica juncea, Gossypium hirsutum, Gossypium barbadense, Gossypium arboretum, Gossypium Herbaceum, Zea mays, Medicago sativa, Lolium multiflorum, Corchorus capsularis, Saccharum officinarum, Cannabis sativa, Coffea Arabica, Coffea Robusta, Camellia sinensis, Oryza sativa, Hordeum vulgare, Boehmeria nivea and Nicotiana tabacum.

In one embodiment, the crop plant is a cereal plant. Cereal plants include, for example, wheat [Triticum), rice [Oryza), barley [Hordeum), corn [Zea).

In one embodiment, the cereal plant is selected from wheat, and barley.

In one embodiment, the cereal plant is a wheat plant.

The inventors have found that inoculating wheat with fungal species from the genus Clohesyomyces or Darksidea, results in: a. an increase in soil organic carbon in the soil in which inoculated wheat plants are grown compared to soil in which uninoculated wheat plants are grown; and/or b. an increase in wheat yield in inoculated wheat plants compared to uninoculated wheat plants grown under the same conditions.

One aspect provides a method of increasing soil organic carbon in a soil and/or increasing yield of a wheat plant being cultivated in the soil, the method comprising inoculating the soil and/or the wheat plant with an effective amount of one or more fungal species from at least one genus selected from Clohesyomyces and Darksidea. One embodiment provides a method of increasing soil organic carbon in a soil and/or increasing yield of a wheat plant being cultivated in the soil, the method comprising inoculating the soil and/or the wheat plant with an effective amount of one or more fungal species selected from Clohesyomyces aquaticus, Darksidea delta, and Darksidea zeta.

In one embodiment, the crop plant is a Brassica. A Brassica is a plant from the genus Brassica. In one embodiment, the plant from the genus Brassica is a canola plant.

The inventors have further found inoculating canola with fungal species from the genus Clohesyomyces, Darksidea, and/or Phialocephala results in: a. an increase in soil organic carbon in the soil in which the inoculated canola plants are grown compared to soil in which uninoculated canola plants is grown; and/or b. an increase in canola yield in inoculated plants compared to yield in uninoculated canola plants grown under the same conditions.

One aspect therefore provides a method of increasing soil organic carbon in a soil and/or increasing yield in canola plant being cultivated in the soil, the method comprising inoculating the soil and/or the canola plant with an effective amount of one or more fungal species from at least one genus selected from Clohesyomyces, Darksidea, and Phialocephala acephala complex, or a combination thereof.

One embodiment provides a method of increasing soil organic carbon in a soil and/or increasing yield in canola plant being cultivated in the soil, the method comprising inoculating the soil and/or the canola plant with an effective amount of one or more fungal species selected from Clohesyomyces aquaticus, Darksidea delta, Darksidea zeta, and Phialocephala acephala complex.

The inventors have further found that rye grass and lucerne can be colonised by the fungi described herein.

One aspect provides a method of increasing soil organic carbon in a soil and/or increasing yield in rye grass being cultivated in the soil, the method comprising inoculating the soil and/or the rye grass with an effective amount of one or more fungal species from at least one genus selected from Clohesyomyces, Darksidea, and Phialocephala acephala complex, or a combination thereof.

One embodiment provides a method of increasing soil organic carbon in a soil and/or increasing yield in rye grass being cultivated in the soil, the method comprising inoculating the soil and/or the canola plant with an effective amount of one or more fungal species selected from Clohesyomyces aquaticus, Darksidea delta, Darksidea zeta, and Phialocephala acephala complex.

One aspect provides a method of increasing soil organic carbon in a soil and/or increasing yield in lucerne being cultivated in the soil, the method comprising inoculating the soil and/or the lucerne with an effective amount of one or more fungal species from at least one genus selected from Clohesyomyces, Darksidea, and Phialocephala acephala complex, or a combination thereof.

One embodiment provides a method of increasing soil organic carbon in a soil and/or increasing yield in lucerne being cultivated in the soil, the method comprising inoculating the soil and/or the lucerne with an effective amount of one or more fungal species selected from Clohesyomyces aquaticus, Darksidea delta, Darksidea zeta, and Phialocephala acephala complex.

It will be appreciated that the soil and/or the crop plant can be inoculated with a combination of the fungal species described herein.

In various embodiments, the soil and/or plant is inoculated with a combination of fungal species selected from the following combinations:

Phialocephala fortinii and Clohesyomyces aqua;

Darksidea delta and Darksidea zeta;

Clohesyomyces aquaticus, Darksidea delta, Darksidea zeta, and Phialocephala acephala;

Darksidea delta, Darksidea zeta, and Phialocephala acephala; Clohesyomyces aquaticus, and Phialocephala acephala;

Clohesyomyces aquaticus, Darksidea zeta, and Phialocephala acephala; Clohesyomyces aquaticus, Darksidea delta, and Phialocephala acephala;

Darksidea zeta, and Phialocephala acephala complex; or Darksidea delta and Phialocephala acephala complex. The inoculation of the plant with the one or more fungal species may be achieved by any suitable means such as direct addition to the soil and/or plant roots and/or to soil proximal to plant roots, or may be achieved by an initial fungal inoculation of the seeds, seedlings and/or immature plants of the crop plant prior to placement of the seed, seedling or immature plant in the soil within which the plant will grow. The inoculation of the one or more fungal species may also be achieved by direct addition to a cultivated soil prior to sowing seeds or planting seedlings that are coated or partially coated with one or more fungal species such that the fungi will become associated with, or grow proximal to, or grow into the roots of a commercial crop as the crop matures.

As used herein, inoculating a soil or plant with a fungus refers to applying the fungus to the soil or to the plant or seed of the crop plant or any other part of the crop plant in a manner that encourages or allows the fungus to become established in the soil and/or grow proximal to, or grow into, the roots ofthe plant (i.e., become associated with the plant).

In some embodiments, the soil is inoculated with the one or more fungal species. The soil may be inoculated with the one or more fungal species prior to planting the plant, for example before, during, or after tilling the soil in preparation for planting. In other embodiments, the soil may be inoculated with the one or more fungal species after the plant has been planted. In some embodiment, the soil is inoculated with the one or more fungal species by planting in the soil plants that have been inoculated with the one or more fungal species.

In some embodiments, the step of inoculating a crop plant comprises applying the one or more fungal species to seeds of the plant prior to planting.

In some embodiments, the step of inoculating a crop plant comprises applying the one or more fungal species to seedlings of the plant. In some embodiments, the step of inoculating soil comprises deploying the one or more fungal species to a plot of soil that is cultivated, such that the fungus is retained by the soil as the crops are rotated, even in the absence of crops for periods of time.

In one embodiment, the plants are inoculated with one or more fungal species as a seed coating before, during or after one or more of the stages of germination of a seed, or as a root inoculant of a seedling. For example, the treatment may be applied as a seed coating to seeds en masse prior to sowing a crop.

The one or more fungal species for inoculation may be in any suitable form, including, for example, as hyphae, mycelia, conidia and/or combinations thereof. In general, the one or more fungal species for inoculating the plant will be in a form that is substantially free of contaminating microorganisms, with the exception that additional desirable microbes may be added for additional benefits.

In inoculating the soil and/or plant with the one or more fungal species, the one or more fungal species are encouraged to become established in the soil and/or grow proximal to the roots, or grow into the roots of the plant (i.e., become associated with), wherein it would be understood the one or more fungal species may exist and grow in the soil or exist within the plant, or in both simultaneously. In embodiments where the methods in which the fungal treatment is in association with a plant, it would be understood the one or more fungal species need only be associated with the plant for parts of the fungus' lifecycle and that the fungus may survive in the soil in the absence of a plant host at all.

In one aspect, there is provided a soil for increasing yield of a crop plant, the soil comprising one or more fungal species from at least one genus selected from Clohesyomyces, Darksidea, and Phialocephala.

Application of the fungi to the plant and/or soil may have one or more desirable effects on the soil and/or associated crops cultivated in the treated soil, including for example, sequestering atmospheric carbon for storage as stable carbon in the soil; and/or increasing the levels of stabilised carbon in the soil. That the inoculation of the soil and/or plants with the fungi may have simultaneous beneficial effects on the soil. For example, sequestration of atmospheric carbon by endophytic fungi as described herein can lead to an increase in the complex polysaccharides in the soil resulting in long-term storage of sequestered atmospheric carbon in a stable form.

In some embodiments, the method may further include addition of one or more preservatives, stabilizers, nutrition enhancers, wetter-spreaders, stickers, penetrants, root promoters, fungicides, urea, fertilisers, pesticides, fulvic acid, humus, nanoparticles/nanomaterials, other components commonly used in agriculture, and/or combinations thereof.

In some embodiments, the soil and/or crop plant may be inoculated with the one or more fungal species without other components, or without nutrition enhancers, root promoters, fungicides, urea, fertilisers, pesticides, fulvic acid, humus, nanoparticles/nanomaterials.

In one embodiment, the method further comprises inoculating the soil and/or crop plant with one or more bacterial species. In one embodiment, the one or more bacterial species is selected from the genus Pseudomonas, Streptomyces, Rhizobium, Bradyrhizobium, Bacillus, Azotobacter, and Azospirillum.

Another aspect provides a composition for increasing soil organic carbon in a soil and/or increasing yield in a crop plant, the composition comprising one or more fungal species from at least one genus selected from Clohesyomyces, Darksidea, and Phialocephala fortinii s.l - Acephala applanate species complex.

The composition may comprise a single fungal species, or a combination of the one or more fungal species.

In various embodiments, there is provided a composition comprising:

(a) Darksidea delta and Darksidea zeta;

(b) Clohesyomyces aquaticus, Darksidea delta, Darksidea zeta, and Phialocephala acephala complex;

(c) Darksidea delta, Darksidea zeta, and Phialocephala acephala complex;

(d) Clohesyomyces aquaticus and Phialocephala acephala complex; (e) Clohesyomyces aquaticus, Darksidea zeta, and Phialocephala acephala complex;

(f) Clohesyomyces aquaticus, Darksidea delta, and Phialocephala acephala complex;

(g) Darksidea zeta, and Phialocephala acephala complex; or

(h) Darksidea delta and Phialocephala acephala complex;

(i) Phialocephala acephala complex;

(j) Clohesyomyces aquaticus;

(k) Darksidea zeta; or (i) Darksidea delta.

In one embodiment, the fungi from the species Clohesyomyces aquaticus is strain V21/002328.

In one embodiment, the fungi from the species Darksidea zeta is strain V21/002326.

In one embodiment, the fungi from the genus Darksidea zeta is strain V21/003117.

In one embodiment, the fungi from the genus Darksidea is strain V21/003116.

In one embodiment, the fungi from Phialocephala acephala complex is strain V21/002327.

In one embodiment, the composition comprises an agriculturally acceptable excipient.

As used herein, an "agriculturally acceptable excipient" refers to an essentially inert substance that can be used as a diluent and/or carrier for the one or more fungal species and is not detrimental to the one or more fungal species or to the crop plant.

In one embodiment, the agriculturally acceptable excipient is a solid or liquid carrier. Suitable solid carriers include mineral earths (e.g., calcium phosphate, calk, clay, diatomaceous earth, dolomite, kaolin, silicates, silica gels, talc, etc), cellulose, and starch. Suitable liquid carriers include water, or any other liquid solvents which are not toxic to the fungus or the plant.

In one embodiment, the composition comprises an agriculturally acceptable additive. As used herein, an "agriculturally acceptable additive" is an additive that may assist in or enhance the performance of the one or more fungal species and/or the crop plant, and is not detrimental to the one or more fungal species or to the crop plant. Examples of agriculturally acceptable additives include preservatives, stabilizers, nutrition enhancers, wetter-spreaders, stickers (adhesives), penetrants, root promoters, fungicides, urea, fertilisers, pesticides, fulvic acid, humus, nanoparticles/nanomaterials.

In some embodiments, the composition may be in the form of a dried powder, a spray, a slurry, a sachet, a liquid, a jelly, a seed coating, an enhancer, and/or combinations thereof.

In some embodiments, the composition is in the form of a seed coating, a foliar spray, granule, powder, soil drench or a root dip.

In one embodiment, the composition is in the form of a seed coating.

In one embodiment, the composition is in the form of a foliar spray.

In one embodiment, the composition is in the form of a root dip.

In one embodiment, the composition is a granule.

In one embodiment, the composition is a powder.

In one embodiment, the composition is a soil drench.

Methods for preparing compositions for agricultural applications are known in the art and described in, for example, US Patent No. 10,492,497.

In one embodiment, the composition comprises suitable solid or liquid carriers and/or an adhesive agent.

In one embodiment, the composition comprises one or more of the fungal species described herein and an adhesive.

In one embodiment, the composition consists essentially of one or more of the fungal species described herein and an adhesive.

In one embodiment, the composition comprises one or more of the fungal species described herein, an adhesive and an agriculturally acceptable excipient.

In one embodiment, the composition consists essentially of one or more of the fungal species described herein, an adhesive and an agriculturally acceptable excipient.

Suitable adhesives include for example, methylcellulose, carboxymethyl cellulose, xanthan gum, gum Arabic, and polysaccharides. In one embodiment, the composition consists essentially of one or more of the fungal species described herein, and an agriculturally acceptable excipient.

In some embodiments, the one or more fungal species are compatible with commonly used agricultural fungicides. By compatible is meant the one or more fungal species in the treatment is not killed or substantially inhibited (growth or germination or otherwise) by the fungicide, thereby allowing the fungi in the treatment to flourish while restricting the growth of undesirable fungal strains that may have a deleterious effect on the soil, the proximal crops or plants, and/or the level of carbon sequestration and stable carbon production. The fungicide may be any synthetic or natural compound that has a fungistatic or fungicidal function and are commonly used in agriculture. Based on their mode of action, they may kill the fungi or inhibit the germination of fungal spores.

The fungal compositions described herein may comprise a plant part to provide a combination of plant part and fungus that can be applied to the soil.

Accordingly, one aspect provides a composition comprising a crop plant or part thereof and one or more fungal species that are heterologous to the crop plant, wherein the one or more fungal species are from at least one genus selected from Clohesyomyces, Darksidea, and Phialocephala.

As used herein, a plant part is a portion of a plant that can be applied to a soil and from which a crop plant can grow. Examples of a plant part include seeds, shoots, leaves, stems, roots, fruit, or any other part of the crop plant from which a crop plant can be grown.

In one embodiment, the composition comprises an adhesive which binds the one or more fungi to the crop plant, seed or other plant part.

One embodiment provides a composition comprising seed from a crop plant and one or more fungal species that are heterologous to the crop plant, wherein the one or more fungal species are from at least one genus selected from Clohesyomyces, Darksidea, and Phialocephala. In one embodiment, the composition comprises an adhesive which binds the one or more fungi to the seed. In one embodiment, the seed is coated with the one or more fungi.

In one embodiment, the composition further comprises one or more bacterial species. In one embodiment, the one or more bacterial species is selected from the genus Pseudomonas, Streptomyces, Rhizobium, Bradyrhizobium, Bacillus, Azotobacter, and Azospirilium.

A further aspect provides a kit comprising the one or more fungi described herein, or the compositions described herein.

The kit may further comprise a container for the one or more fungi. The kit may further comprise instructions for use.

In some embodiments, the one or more fungal species comprises dark septate endophytic (DSE) fungi.

The term "endophytic" relates to a microbe that generally lives within a plant for at least part of its lifecycle, often due to the microbe being able to grow inward into plant tissues in finger-like projections from a superficial site of origin. These fungi can infiltrate plant living tissues for at least a portion of the fungal life cycle often without causing any apparent diseases or harm to the plant that is a native host, in that they are generally not pathogenic to their native hosts. It would be understood that the one or more fungal species of the methods described herein can exist during some portion of the fungal life cycle within the roots of a plant host as an endophyte and in other parts of its life cycle within the soil, and will typically alternate or cycle between a root endophytic phase and a free-living soil phase.

Though some endophytic fungi are known for enriching the organic carbon in the soil, each fungus species will generally behave differently when associated with different, and/or non native, plant hosts and/or soil environments, and stabilize the organic carbon with different efficiency.

Endophytic fungi are known to have preferred hosts and growth conditions, and will not necessarily flourish, and therefore produce the desired stable SOC, in the absence of their typical growth environment or an association with their native hosts. Moreover, when considering the survival of the fungi in non-native plant hosts, it is difficult to anticipate whether the fungi will prove to be pathogenic to the non-native host. Therefore, fungal species may readily be compatible with a non-native crop plant host.

"Heterologous" and "non-native" are used interchangeably herein. A fungus is "heterologous" or "non-native" with respect to a crop plant when the fungal strain was collected from a host other than said crop plant. In one embodiment, an increase in soil organic carbon is an increase in stable carbon. An increase in the sequestration of atmospheric carbon for storage as stable carbon in the soil, and increasing the levels of stable carbon in the soil, is an increase relative to the amount of sequestration of atmospheric carbon for storage as stable carbon in the soil, and levels of stable carbon in the soil, produced by a plant that has not been treated with the methods of the present disclosure.

In some embodiments, the fungi used in the methods and compositions described herein will be capable of sequestering and fixing carbon from atmospheric carbon dioxide and converting this carbon to complex polysaccharides for storage as stable carbon in the soil. The sequestered and fixed carbon may also be converted and stored as a stable carbon source by the fungi in the fungi itself as, for example, melanin, chitin, lignin, suberin and carotenoid compounds, or the fungi may exude these compounds to increase the stable carbon in the soil. The deployed fungal endophyte may also convert simple polysaccharide exudate from a host plant into complex polysaccharides for storage as stable carbon in the soil, or within the fungi itself. Lastly, the stability of organic carbon may be enhanced in soil with more stable soil aggregates.

The methods and treatments and compositions of the present invention may increase the overall levels of carbon in the soil, but even in cases where overall carbon remains the same or is only slightly increased, it would be understood that the levels of stable carbon in the soil may be increased due to the production and exudation in the soil of complex polysaccharides by the disclosed fungal species.

The increase in overall soil carbon and stable soil carbon of a soil that is subjected to the treatments and/or methods of the present disclosure compared to a control may be quantified by any methods known to those skilled in the art. The control would be a soil sample would be a similar soil sample that had not been exposed to an endophytic fungus as claimed herein (i.e., a fungus had not been deployed in the soil or associated with a plant that had been cultivated in said soil). By "similar soil sample" is meant that the soil would be from a proximal area with a similar climate and, if the soil had been cultivated, the control sample would have been cultivated by the same plant as the test soil.

Soil organic carbon (SOC) is the overall soil carbon content of a soil and may also be generally referred to as total organic carbon (TOC) (the terms may be used interchangeably), and this refers only to the carbon component of the organic matter in the soil. However, fluctuations in soil organic carbon may not necessarily correlate to the same fluctuations in stable soil carbon. Indeed, soils subjected to the treatments and methods may demonstrate minimal increases in TOC, but the percentage of said TOC that is captured in a stable form in the soil or in the fungi proliferating in the soil (i.e., complex polysaccharides, melanin, chitin, lignin, suberin and carotenoid compounds) may increase. The skilled addressee would also understand that changes in TOC and stable carbon in soil as a result of the treatments and methods of the present may take weeks, months or years, and therefore appropriate measurement timeframes must be applied. In one embodiment, the increase in soil organic carbon in a soil comprises an increase in stable carbon in the soil.

The soil carbon may be measured by methods including, but not limited to, dry combustion or elemental tests that may be analysed using, for example, the LECO analysis method, and loss on ignition (LOI) tests that may be analysed using the Walkley-Black method (see, for example, Walkley A, and Black IA (1934) An examination of the Degtjareff method for determining soil organic matter, and a proposed modification of the chromic acid titration method. Soil Science 37 , 29-38). To assess the prevalence of different types of carbon on the TOC (i.e. to measure the stable, or "recalcitrant" organic carbon), methods may be employed to fractionate to TOC by, for example, measuring soil respiration or the bulk density of the soil. The increase in soil aggregate stability, or soil aggregation per se, of a soil that is subjected to the treatments and/or methods described herein compared to a control may be quantified by any methods known to those skilled in the art. The control would be a similar soil sample that had not been exposed to the relevant fungus (i.e., a fungus had not been deployed in the soil or associated with a plant that had been cultivated in said soil). By "similar soil sample" is meant that the soil would be from a proximal area with a similar climate and, if the soil had been cultivated, the control sample would have been cultivated by the same plant as the test soil. The soil aggregate stability may be quantified by measurements compared to controls such as, but not limited to, soil mean weight diameter (MWD), geometric mean diameter (GMD), fractal dimension (D), percentage of aggregates destruction (PAD) and water- stable aggregates stability rate (WSAR). An increase in the MWD,

GMD, WSAR and D values are indicative of an increase in soil aggregate stability, while a decrease in PAD value is indicative of an increase in soil aggregate stability.

The methods and treatments and compositions of the present disclosure relate to the provision of agronomic benefits to commercial crops being cultivated in soil wherein the microbial treatment of the invention has been deployed. While the agronomic benefits may be collectively defined as enhanced growth and yield of said plants, it would be understood these benefits include, but are not limited to, increased yields (i.e., of fruit, grain, or vegetative tissue), enhanced growth, enhanced vigour, increased root biomass, increased above-ground biomass, enhanced nutrient uptake, lower production costs through reduced water requirements, enhanced fertility, improved nutrition of the produced crop, improved resistance to drought and pests and enhanced tolerance to abiotic stress. The fungal strains may also contribute to the increased soil aggregation,thereby improving soil fertility, water holding, and water infiltration capacities of the soil to the benefit of the commercial crops. The agronomic benefits of the methods and treatments of the present invention may be quantified by measurements compared to controls such as, but not limited to, above-ground biomass (encompassing measurements of wet biomass or dry biomass), shoot weight, head weight/numbers per plant, plant height at harvest, time taken to reach maturity, nitrogen/phosphorus/potassium/magnesium content of the plant, pod weight/numbers per plant, fruit weight/numbers per plant, grain yield, tiller number, combinations thereof and the like. To quantify the agronomic benefits, the control would be a plant or plants derived from the same or similar batch of seeds or seedlings that were not exposed to a fungus as described in the methods and treatments of the present invention. By "not exposed" includes the limitation that the control would not be grown in soil that was or had been colonised by a melanised fungus of the present invention.

In one embodiment, the methods described herein result in enhanced yield of crop plants. It would be understood these benefits include increased yields (i.e., of fruit, grain, or vegetative tissue).

The one or more fungi described herein are endophytic fungi that are non-pathogenic to crop plants.

Some representative examples of suitable strains of fungi that have been exemplified herein are designated CTR7788, CTR7800, CTR6853, CTR360 and CTR4796. These fungal strains are examples of the one or more fungal species described herein that are not pathogenic to non-native commercial crop hosts that can be used in the methods described herein. It will be appreciated that the deposited strains are representative of species of Clohesyomyces aquaticus, Phialocephala fortinii s.l. - Acephala applanata species complex (PAC) and Darksidea, and other members of the species may be employed in the methods described herein.

Strain CTR7788 is a strain of the Phialocephala fortinii s.l.

- Acephala applanata species complex (PAC). CTR7788 may be deployed as a microbial treatment in conjunction with a commercial crop that is a non-native crop plant as described herein, and the CTR7788 fungal strain is capable of sequestering atmospheric carbon for storage as stable carbon in the soil, thereby increasing the levels of stable carbon in the soil used to cultivate the crop plant; and enhancing the growth and yield of the crop plant. Strain CTR7788 may be deployed in the methods and treatments described herein in conjunction with crop plants that are a non-native host such as species of the genus Triticum, Brassica, Hordeum, Lolium and Medicago. CTR7788 fungal strain is capable of sequestering atmospheric carbon for storage as stable carbon in the soil, thereby increasing the levels of stable carbon in the soil used to cultivate the commercial crop; and enhancing the growth and yield of the commercial crop. Strain CTR7788 has been deposited as an example of the species Phialocephala fortinii s.l. - Acephala applanata species complex (PAC) under the Budapest Treaty at the National Measurement Institute, 1/153 Bertie Street, Port Melbourne, Victoria 3207, Australia, on 8 February 2021 under accession number V21/002327. Therefore, an example of a strain of Phialocephala Acephala complex is V21/002327.

Strain CTR7800 belongs to the species Clohesyomyces aquaticus. The Clohesyomyces aquaticus fungal species are known to be saprobic fungi that generally subsist on dead and decaying matter in aquatic, low oxygen environments. As such, the commercial crops described in the present disclosure would be deemed non-native hosts of Clohesyomyces aquaticus. Strain CTR7800 has been deposited as an example of the species Clohesyomyces aquaticus under the Budapest Treaty at the National Measurement Institute, 1/153 Bertie Street, Port Melbourne, Victoria 3207, Australia, on 8 February 2021 under accession number V21/002328. Therefore, an example of a strain of the fungal species Clohesyomyces aquaticus is strain V21/002328. In various embodiments, CTR7800 is deployed as a microbial treatment in conjunction with a crop plant that is a non-native host, such as species of the genus Triticum, Brassica, Hordeum, Lolium and Medicago, wherein the CTR7800 fungal strain is capable of sequestering atmospheric carbon for storage as stable carbon in the soil, thereby increasing the levels of stable carbon in the soil used to cultivate the commercial crop; enhancing the growth and yield of the commercial crop; and increasing the soil aggregate stability of the soil used to cultivate the commercial crop. Strains CTR6853, CTR360 and CTR4796 belong to the genus Darksidea, with strains CTR6853 and CTR4796 belonging to the species Darksidea zeta. While Darksidea zeta is a known root-colonising fungus, it is generally found associated with stressed plants in semi-arid, sandy conditions and is not known to be associated with commercial crop plants. Strain CTR6853 has been deposited as an example of the species Darksidea zeta under the Budapest Treaty at the National Measurement Institute, 1/153 Bertie Street, Port Melbourne, Victoria 3207, Australia, on 8 February 2021 under accession number V21/002326. Strain CTR360 has been deposited as an example of a species of Darksidea under the Budapest Treaty at the National Measurement Institute, 1/153 Bertie Street, Port Melbourne, Victoria 3207, Australia, on 19 February 2021 under accession number V21/003116. Strain CTR4796 has been deposited as another example of the species Darksidea zeta under the Budapest Treaty at the National Measurement Institute, 1/153 Bertie Street, Port Melbourne, Victoria 3207, Australia, on 19 February 2021 under accession number V21/003117. Therefore, an example of strains of the fungal species Darksidea zeta is V21/002326 and V21/003117, and an example of a fungal species from the genus Darksidea is V21/003116.

In various embodiments, CTR6853, CTR360 and/or CTR4796 is deployed as a microbialtreatment in conjunction with a commercial crop that is a non-native host, such as species of the genus Triticum,

Brassica, Hordeum, Lolium and Medicago, wherein the CTR6853, CTR360 or CTR4796 fungal strains are capable sequestering atmospheric carbon for storage as stabilised carbon in the soil, thereby increasing the levels of stabilised carbon in the soil used to cultivate the commercial crop; and enhancing the growth and yield of the commercial crop.

One aspect provides an isolated fungal strain selected from the group consisting of strain V21/002326, V21/002327, V21/002328, V21/003116, and V21/003117.

In one embodiment, the isolated fungal strain is strain V21/002326. In one embodiment, the isolated fungal strain is strain is V21/002327. In one embodiment, the isolated fungal strain is strain is V21/002328. In one embodiment, the isolated fungal strain is strain is V21/003116. In one embodiment, the isolated fungal strain is strain is V21/003117.

Besides the strains exemplified, additional isolated fungi from the fungal species described herein suitable for use in the treatments and methods of the present inventions may be determined by the methods described herein e.g., melanin content. The melanin content of a melanised fungal strain may be measured using a hyphal mass of said strain grown in laboratory conditions, by, for example, spectrophotometry and expressed as percent of hyphal mass by weight. The fungi employed in the treatments and methods described herein may in some embodiments be strains which accumulate high levels of melanin within their cell walls, resulting in a fungal hyphal mass comprising at least 5% melanin by mass. Melanin is a complex polysaccharide that resists degradation and the presence of melanised fungi that produce high levels of melanin not only leads to overall increased levels of SOC/TOC, but also results in improved soil quality due to the deposition of melanin, a stable carbon compound, in the soil.

While melanin may be measured in an in vitro culture to identify melanised fungal strains for use in treatments and methods described herein, it would also be understood that even higher levels of melanin may be present in melanised strains deployed on plants or in plant soils, as exposure to UV radiation has been shown to increase melanin production in various melanised fungal strains. As such, a melanised fungal strain that comprises at least 5% melanin when the melanin of a laboratory grown hyphal mass of said strain is measured under laboratory conditions, the same strain may comprise, for example, at least 10% melanin when grown in a cultivated soil.

As described herein, the deployment of microbial treatments described herein in conjunction with a non-native commercial crop host can lead to desirable improvements in the growth and yield of the commercial crop along with enhanced sequestration of atmospheric carbon.

It would be understood that fungal strains from the same species as those described herein may have similar desirable attributes and are encompassed by the treatments and methods of the present invention. As used herein, the term "effective amount" means a sufficient quantity of a substance (e.g. fungus) to promote a desired outcome (e.g., an increase in soil carbon and/or yield of a crop plant). This term is not to be construed to limit the disclosure to a specific quantity, e.g., number of fungal cells; rather the present disclosure encompasses any amount of the one or more fungal species that is sufficient to achieve the stated purpose. The amount of the one or more fungal species should not be so large as to cause adverse effects in the plant. Generally, the amount of the one or more fungal species may be varied with the way in which the fungi are applied (e.g., to the soil, to the seed or to the seedling) and can be determined by a person skilled in the art.

Throughout the specification and claims, unless the context requires otherwise, the term "substantially" or "about" will be understood to not be limited to the value for the range qualified by the terms. For example, the term "about" may include a range that is +5%, +2.5% or +1% of the value to which the term is applied.

In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word "comprise" or variations such as "comprises" or "comprising" is used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.

All publications mentioned in this specification are herein incorporated by reference. It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

In various embodiments, there is provided the following items:

1. A microbial treatment to be deployed in soil and/or associated with a commercialcrop, the treatment comprising: at least one melanised fungus, wherein said melanised fungus is a species from a genera selected from the group consisting of Clohesyomyces, Darksidea,and Phialocephala fortinii s.l. - Acephala applanata species complex (PAC), wherein the deployment of said treatment has one or more desirable effects selected from the group consisting of increasing the sequestration of atmosphericcarbon for storage as stable carbon in the soil, providing agronomic benefits to said commercial crops, increasing the levels of stable carbon in the soil, and increasing the soil aggregate stability of the soil.

2.The treatment according to item 1, wherein the melanised fungus comprises at least about 10% melanin.

3.The treatment according to item 1 or item 2, wherein the melanised fungus has a nuclear ribosomal internal transcribed spacer (ITS) sequence having at least about 95% sequence identity with any one of SEQ ID Nos. 1, 2, 3, 4, 5, or 6.

4.The treatment according to any one of items 1 to 3, wherein said melanised fungus belongs to a species selected from the group consisting of Clohesyomyces aquaticus, Darksidea zeta, Darksidea alpha and Phialocephala.

5.The treatment according to item 4, wherein the melanised fungus is selected from the group consisting of strains designated CTR7788, CTR7800, CTR6853, CTR360 and CTR4796.

6.The treatment according to any one of items 1 to 5, further comprising one or more fungi that is not a melanised fungus and/or a one or more microorganisms selected from the group consisting of strains of Bacillus, Pseudomonas, Streptomyces and Rhizobium.

7.The treatment according to any one of items 1 to 6, wherein, when the treatment is deployed in association with a commercial crop, the commercial crop is a compatible crop selected from the group consisting of species of the genus Triticum, Brassica,

Gossypium, Zea, Corchorus, Saccharum, Medicago, Lolium,

Coffea, Camellia, Oryza, Hordeum, Boehmeria, Nicotiana and Cannabis.

8.The treatment according to item 7, wherein the commercial crop is selected from the group consisting of the species Triticum aestivum, Brassica napus, Brassica rapa, Brassica juncea, Gossypium hirsutum, Gossypium barbadense, Gossypium arboretum, Gossypium Herbaceum, Zea mays, Medicago sativa, Lolium multiflorum, Corchorus capsularis, Saccharum officinarum, Cannabis sativa, Coffea Arabica, Coffea Robusta, Camellia sinensis, Oryza sativa,

Hordeum vulgare, Boehmeria nivea and Nicotiana tabacum.

9.The treatment according to item 8, wherein the commercial crop is Triticum aestivum or Brassica napus.

10. The treatment according to any one of claims 1 to 9, wherein the melanised fungus is compatible with one or more fungicidal agents.

11. The treatment according to item 10, wherein fungicidal agent is selected from the group consisting of triazole fungicides, systemic fungicides and SDHI fungicides, and combinations and/or derivatives thereof.

12. The treatment according to any one of items s 1 to 11 wherein, when the treatment is deployed in associated with a commercial crop, the treatment is deployed as a seed coating before, during or after one or more of the stages of germination of the seed of said commercial crop, or as a root inoculant of a seedling of said commercial crop.

13.A method for sequestering atmospheric carbon for storage as stable carbon in soil, the method comprising deploying a treatment comprising at least one melanised fungus, wherein said melanised fungus is a species from a genera selected from the group consisting of Clohesyomyces, Darksidea, and Phialocephala fortinii s.l. - Acephala applanata species complex (PAC), in said soil and/or associating said melanised fungus with a commercial crop being cultivated in said soil.

14. A method for increasing the levels of stable carbon in soil used to cultivate a commercial crop, the method comprising deploying a treatment comprising at least one melanised fungus, wherein said melanised fungus is a species from a genera selected from the group consisting of Clohesyomyces, Darksidea, and Phialocephala fortinii s.l. - Acephala applanata species complex (PAC), in said soil and/or associating said melanised fungus with said commercial crop. A method for providing agronomic benefits to a commercial crop being cultivated in soil, the method comprising deploying a treatment comprising at least one melanised fungus, wherein said melanised fungus is a species from a genera selected from the group consisting of Clohesyomyces, Darksidea, and

Phialocephala fortinii s.l. - Acephala applanata species complex (PAC), in said soil and/or associating said melanised fungus with said commercial crop. A method for increasing the soil aggregate stability of soil used to cultivate a commercial crop, the method comprising deploying a treatment comprising at least one melanised fungus, wherein said melanised fungus is a species from a genera selected from the group consisting of Clohesyomyces, Darksidea, and Phialocephala fortinii s.l. Acephala applanata species complex (PAC), in said soil and/or associating said melanised fungus with said commercial crop. The method according to any one of items 13 to 16, wherein the melanised fungus comprises at least about 10% melanin. The method according to any one of items 13 to 16, wherein the melanised fungus has a nuclear ribosomal internal transcribed spacer (ITS) sequence having at least about 95% sequence identity with any one of SEQ ID Nos. 1, 2, 3,4, 5, or 6. The method according to any one of items 13 to 16, wherein the melanised fungus belongs to a species selected from the group consisting of Clohesyomyces aquaticus, Darksidea zeta, Darksidea alpha and Phialocephala. The method according to any one of items 13 to 16, wherein the melanised fungus is selected from the group consisting of strains designated CTR7788, CTR7800, CTR6853, CTR360 and CTR4796. The method according to any one of items 13 to 16, wherein the commercial crop is selected from the group consisting of the species Triticum aestivum, Brassica napus, Brassica rapa, Brassica juncea, Gossypium hirsutum, Gossypium barbadense, Gossypium arboretum, Gossypium Herbaceum, Zea mays, Medicago sativa, Lolium multiflorum, Corchorus capsularis, Saccharum officinarum, Cannabis sativa, Coffea

Arabica, Coffea Robusta, Camellia sinensis, Oryza sativa, Hordeum vulgare, Boehmeria nivea and Nicotiana tabacum.

In order to exemplify the nature of the present invention such that it may be more clearly understood, the following non-limiting examples are provided.

EXAMPLES Fungal strains

Strains of fungus were isolated from roots of healthy plants and tested for pathogenicity towards various crop plants. Examples of non-pathogenic strains were selected as representatives of various species, and further tested. Examples of non-pathogenic strains of fungal species used in these studies are listed in Table 1.

Table 1

Example 1

Melanised fungal endophytes were isolated from plant samples by growing surface sterilised root samples (2% NaOCl solution) on growth media amended with antibiotics such as Streptomycin Sulfate and or Penicillin G sodium salt. The initial isolated cultures were then incubated at 25°C and subsequently subcultured until a pure culture was observed. Organisms were identified via ITS sequencing and BLAST comparison.

The fungal strain designated CTR7788 was isolated from a plant in the Sydney Basin area and was identified as a species in the Phialocephala fortinii s.l. - Acephala applanata species complex (PAC). The nuclear ribosomal internal transcribed spacer (ITS) sequence of CTR7788 is shown below as SEQ ID NO:1.

AACGTCGCTCGTAGTGACCTGCGGAGGATCATTACAAGTGAAGGCCGTCGATACACCCGTAAAACGGT GGGTAGACGGTTTACACCCACCCGTGTTTATATACCCTTGTTGCTTTGGCGGGCCGTGGCCTCCACTA CGGGCTCCGCTCGTGTGTGCCCGCCAGAGGATCAAACTCTGGATGTTAGTGATGTCTGAGTACTATCT AATAGTTAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCGAAATGCGAT AAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTGTGGTATT CCGCAGGGCATGCCTGTTCGAGCGTCATTTAACCACTCACGCCTGCGTGGGTGTTGGGGCACGCGGTT CCGCGGCCCTCAAAACCAGTGGCGGCGCCGCTGGGCTCTAAGCGTAGTACATACTCCCGCTATAGAGT TCCGTCGGTGGCTCGCCAGAACCCCCTATTTTTACAGGTTGACCTCGGATCAGGTAGGGATACCCGCT GAACTTAAG CATATCAATAAGCGGAGGAA (SEQ ID NO:1)

Melanin testing of a wet fungal biomass of CTR7788 via the Azurea dye method demonstrated the strain comprises at least about 10% melanin. CTR7788 has been shown to be capable of colonising wheat, barley, rye grass, canola and lucerne and is not pathogenic to any of these commercial crops. Analysis of the survival of fungal colonies of CTR7788 in the presence of commonly used crop fungicides and insecticides demonstrated that CTR7788 is compatible with at least Maxim XL (10 mg/L, 5mg/L, 1 mg/L and 0.5 mg/L), Saltro Duo (10 mg/L, 5 mg/L, 1 mg/L and 0.5 mg/L), Poncho Plus and Cruiser Opti.

The fungal strain designated CTR7800 was isolated from a grass from an alluvial soil in Central NSW and belongs to the species Clohesyomyces aquaticus. The nuclear ribosomal internal transcribed spacer (ITS) sequence of CTR7800 is shown below as SEQ ID NO:2.

AATTCGATACGTAGTGACCTGCGGAGGATCATTACAGATTCAAGATATTAGAAGGAGCCAGCAGGGCG CCACCAGCGGTGGGGACAACACGGGGGTAACCCCCCCGCCCCAGCCGGGCGGCGTCACTCTGGCCGAG TGAGAGCATTCGCTTGGGCTTCTTTGTATTCATTTCGAATACCGAGAAATAGGCAGTCGTGCGAGCCA CCGGGGTGTAAGCAATCCCCGGAGGTCTCGGCGGCTGGTACAGCGGTCAAATTCCATGACGACGGAGC ATAGATAGCTAACCCCTCTTGTCGCTTGCCGAAAAAAGAAGTCCTCCGGGACCCCGGCCTTACAGAGG GACGCTTTGCTCTAGAGAGGTCAGCTGCGTACTTGGATGGCCCCCGTTGGGGCAGTCCAGCCTTGATT CTATTGCCTGCACACGCGAGGTCTGGGTTATCAAATACCCACTAGCCTATGTACGCGGAGAGAGGGCG GACGGAAACAGTCAACGTAAGACGAACGCGGGGGACAAAGGCGCCGTAAGCTCTGCGGCGTCCCCCTG CGGGGTTGTAAGCCATCGACTCACCTTCCACACGGCGTAATTCTGCCAGCGGAGTTGGTTTGGATCTT CGGATCAGGTGAGGGCTTGTTCAGCGACCCGTCGATGAGTTGGCGTTGCCATCCCGCCCCCCGCGTGA GGGGCCTCAGCCGAGCCACACCGGTCCCCGCCGTAAGGGGGCACGAGTGCTCGGTGGATGCGTGATGT GACCCCTGGCCGACATACCAGGAGCCATCCGATCCGATTGCCGTGCGCGGGCCCCGTTCCATCGTCCC CTGGGCGCTCTGGGAAGCGATTGTTCCACCCGGTCGGTGACCTGCTTGTTAAATTTCTTCGCGCCGCC GCGTGTCATCCTCGGGTCTCTTGACCTCGAGTGAGGACTGCGGGCTCCACAGGCAACCCCAGCCCATG GACAGTTTGGGCGCAGGAGATCCTCTATACCAACTCCCCGACCCCTTTGTCTATGTGTACTCTCGTTT TCCTCGGCGGGCTCGCCCCGCCAACGGGGACAACAAACCCATTCTTTTGCAGTGTAGTTTCTGTCTGA TATACAACCCTTATTAACTTTCACACGATCCTCTTATTCTGCCATCGATGAGAACGCAACGATGCGAT AGTAGTGTGATTTGCCAGAGTTCAGTTAATTCAATCGCGAATCT (SEQ ID NO:2)

Melanin testing of a wet fungal biomass of CTR7800 via the Azurea dye method demonstrated the strain comprises at least about 20% melanin. CTR7800 was shown to be capable of colonising wheat, barley, rye grass, canola and lucerne and is not pathogenic to any of these commercial crops. Analysis of the survival of fungal colonies of CTR7800 in the presence of commonly used crop fungicides and insecticides demonstrated that CTR7800 is compatible with at least Maxim XL (10 mg/L, 5mg/L, 1 mg/L and 0.5 mg/L), Saltro Duo (10 mg/L, 5 mg/L, 1 mg/L and 0.5 mg/L), Poncho Plus and Cruiser Opti.

The fungal strain designated CTR6853 was isolated from a grass from a sandy soil in Western NSW and belongs to the species Darksidea zeta. The nuclear ribosomal internal transcribed spacer (ITS) sequence of CTR6853 is shown below as SEQ ID NO:3. CTR6853 has been shown to be capable of colonising wheat, barley, rye grass, canola and lucerne and is not pathogenic to any of these commercial crops.

GCATCGATGAAGAACGCAGCGAAATGCGATAAGTAGTGTGAATTGCAGAATTCAGTGAATCATCGAAT CTTTGAACGCACATTGCGCCCCATGGTATTCCGTGGGGCATGCCTGTTCGAGCGTCATTTACCCCCTC AAGCTCCGCTTGGTGTTGGGCGTCTGTCCCGCTTCGCGCGCGGACTCGCCCCAAAGGTATTGGCAGCG GTCGTGCCAGCTTCTCGCGCAGCACATTGCGCTTCTCGAGGCACCGGCGGGCCCGtGTCCATCAAGCT CACCCCCCCAGTTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCATATCAATAAGCGGAG GA (SEQ ID NO:3)

The fungal strain designated CTR360 was isolated from a grass from a clay soil in central NSW and belongs to the species Darksidea zeta. The nuclear ribosomal internal transcribed spacer (ITS) sequence of CTR6853 is shown below as SEQ ID NO:4.

GCATCGATGAAGAACGCAGCGAAATGCGATAAGTAGTGTGAATTGCAGAATTCAGTGAATCATCG AATCTTTGAACGCACATTGCGCCCCATGGTATTCCGTGGGGCATGCCTGTTCGAGCGTCATTTAC CCCCTCAAGCTCCGCTTGGTGTTGGGCGTCTGTCCCGCTTCACGCGCGGACTCGCCCCAAAGGTA TTGGCAGCGGTCGTGCCAGCTTCTCGCGCAGCACATTGCGCTTCTCGAGGCACCGGCGGGCCCGC GTCCATCAAGCTCAACCCCCCCAGTTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGC ATAT CAATAAGCGGAGGA (SEQ ID NO:4)

Melanin testing of a wet fungal biomass of CTR360 via the Azurea dye method demonstrated the strain comprises at least about 10% melanin. CTR360 has been shown to be capable of colonising wheat, barley, rye grass, canola and lucerne and is not pathogenic to any of these commercial crops. Analysis of the survival of fungal colonies of CTR360 in the presence of commonly used crop fungicides and insecticides demonstrated that CTR360 is compatible with at least Maxim XL (10 mg/L, 5mg/L, 1 mg/L and 0.5 mg/L), Poncho Plus and Cruiser Opti.

The fungal strain designated CTR4796 was isolated from a plant from a sandy soil in western NSW and belongs to the species Darksidea alpha. The nuclear ribosomal internal transcribed spacer (ITS) sequence of CTR6853 is shown below as SEQ ID NO:5.

GGATTAGCATCGTAGGTGACCTGCGGAGGATCATTACCTGGCCTTGGGCCGCTCGGGGGAGCCAGTCG

CTTGCGACGACGCTGCCTTGGGCGCTTAGCCCTTGACTATCACCTTGACTACGTGCACCTTTTGTTGT

TTCCTCGGCAGGTCATCTGCCGCCAGGAACCCCCTAAACCTTTTTGCAATAGCATCTAAACTTCTGAA

AACAAACCCAAATCATTTACAACTTTTAACAATGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAG

CGAAATGCGATAAGTAGTGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGC

CCCATGGTATTCCGTGGGGCATGCCTGTTCGAGCGTCATTTACCCCCTCAAGCTCCGCTTGGTGTTGG

GCGTCTGTCCCGCTTCACGCGCGGACTCGCCCCAAAGGTATTGGCAGCGGTCGTGCCAGCTTCTCGCG

CAGCACATTGCGCTTCTCGAGGCACCGGCGGGCCCGCGTCCATCAAGCTCAACCCCCCAGTTTGACCT

CGGATCAGGTAGGGATACCCGCTGAACTTAAGCATATCAAAAAGCCGGAGGAAA

CTR4796 has been shown to be capable of colonising wheat, barley, rye grass, canola and lucerne and is not pathogenic to any of these commercial crops. Analysis of the survival of fungal colonies of CTR4796 in the presence of commonly used crop fungicides and insecticides demonstrated that CTR4796 is compatible with at least Maxim XL (10 mg/L, 5mg/L, 1 mg/L and 0.5 mg/L), Poncho Plus and Cruiser Opti.

Example 2

A glasshouse trail was conducted for eight weeks at a glasshouse facility in NSW in 2020 following a Randomized Complete Block (RCB) design with 6 replicates for treatments comprising the CTR4796, CTR6853, CTR7788 AND CTR7800 strains in association with canola plants. The treatments were prepared as agar plugs of the fungal colonies growing on PDA plates. Blank PDA plug was used as negative control. The canola seeds were pre-germinated and then inoculated with a treatment by placing the treatment plugs in close contact with the pre-germinated canola seeds. The inoculated seeds were planted, and plant height, above-ground fresh biomass, above ground dry biomass, number of pods, pod weight and grain yield were measured after 8 weeks. During the 8 weeks, the root colonisation of the canola plants by the strains in the treatments were also confirmed by testing root sections of the plants for the treatment fungus.

The results showed that after 8 weeks the canola plants that grew in association with the CTR4796 strain had a 52.88% increase in above-ground fresh biomass, a 16.25% increased above-ground dry biomass, a 5.23% decrease in pod count, a 14.21% increase in pod weight, a 0.93% increase in grain yield, when compared to the untreated controls.

The results showed that after 8 weeks the canola plants that grew in association with the CTR6853 strain had a 57.89% increase in above-ground fresh biomass, a 35.56% increased above-ground dry biomass a 20.92 % increase in pod count, a 33.87% increase in pod weight, a 19.43% increase in grain yield, and produced a 0.79% increase in total organic soil carbon, when compared to the untreated controls.

The results showed that after 8 weeks the canola plants that grew in association with the CTR7788 strain had a 19.88% increase in above-ground fresh biomass, a 17.82% increased above-ground dry biomass a 0.33% decrease in pod count, a 21.18% increase in pod weight, a 21.84% increase in grain yield, and produced a 1.74% increase in total organic soil carbon, when compared to the untreated controls.

The results showed that after 8 weeks the canola plants that grew in association with the CTR7800 strain had a 11.74% increase in above-ground fresh biomass, a 17.34% increased above-ground dry biomass, a 9.48% increase in pod count, a 16.27% increase in pod weight, a 11.01% increase in grain yield, and produced a 3.31% decrease in total organic soil carbon, when compared to the untreated controls.

Example 3

A "speed breeding" glasshouse trail was conducted for eight weeks at a glasshouse facility in ACT in 2020. 'Speed breeding' is a method, developed by plant breeders to shorten the time between germination and mature plant stage. Exposure to 22 hours of daylight per day, controlled humidity and temperature allow for a 'seed to seed timeframe' of 6 to 8 weeks. Treatments comprising the CTR360 strain were deployed in association with wheat plants. The plants were allowed to grow to maturity in 6 weeks following germination. During the 6 weeks, the root colonisation of the wheat plants by the strains in the treatments were also confirmed by testing root sections of the plants for the treatment fungus.

The results showed that after 6 weeks the wheat plants that grew in association with the CTR360 strain had a 57.1% increase in shoot weight, and a 57.4% increase in head weight, when compared to the untreated controls.

Example 4

A field trial was conducted for eight weeks in NSW in 2020 following a Randomized Complete Block (RCB) design with 6 replicates for treatments comprising the CTR4796 and CTR7800 strains, both alone and in combination, and in further combination with other microorganisms (Azospirillum, Azotobacter, Bacillus) strains in association with wheat plants.

The results of the soil carbon analysis of the soil associated with the cultivated wheat is summarised below in Table 2.

Table 2:

Example 5 - Canola

Methodology and Materials for inoculation with fungi

Fully replicated glasshouse trials of were conducted following

RCB design where canola seeds were inoculated with colonised agar fungal plugs. Two pre-germinated seeds were put at a depth of 1/2 cm by placing the radicle downward in the centre of the pot containing non-sterile field soil. The seed was inoculated by aseptically transferring a 5 mm colonized agar plug in close contact with the seed using a sterile inoculation loop. The plugs were prepared beforehand by cutting from a fungal colony margin actively growing on a PDA plate using a 5 mm sterile cork borer under a biosafety cabinet.

Fully replicated field Trials were conducted in multiple locations following RCB design where seeds were inoculated with Liquid inoculum of different fungal strains. For each treatment, 16 mL of liquid inoculum consisting of spore and hyphal suspension

(7X10⁷ CFU) and 6 mL of sticker (1% Methyl Cellulose) were added per Kg of seed and mixed thoroughly to ensure uniform seed coating. Coated seeds were mechanically sown using a sower.

At harvest, soil samples were randomly collected from each treatment plot and analysed for TOC following 6B2b Total organic C - Dumas high-temperature combustion followed by infrared/thermal conductivity detection method.

Results

Total Organic Carbon at harvest with various treatments is shown in Table 3.

Table 3:

At harvest, the treatments increased TOC by 3-5% compared to the uninoculated control treatment. An increment of 5.34% was recorded with the Darksidea complex (CTR4796+CTR140+CTR6853) and combination of strains CTR7788+CTR7800. There was high variation among the replicates for all treatments.

Example 6 — Barley

3 field trials were conducted for a full growing season across multiple locations in NSW and W.A (Canowindra, Corowa and Perth). Fungi were grown on PD broth and blended to form a hyphal suspension. Barley seeds were inoculated with the hyphal suspension of fungal strains. For each treatment, 16 mL of suspension and 6 mL of sticker (1% Methyl Cellulose) were added per Kg of seed and mixed thoroughly to ensure uniform seed coating. Coated seeds were mechanically sown.

At harvest, soil samples were randomly collected from each treatment plot and analysed for TOC, measured by high-temperature combustion (Leco analyser). The results of the soil carbon analysis showed that strain 7788 associated with Barley increased TOC by 5.3% when compared to an untreated control. Grain yield, measured in weight per plot, was increased by an average of 6.4% when compared to an untreated control.

Claims

1. A microbial treatment to be deployed in soil and/or associated with a crop plant, the treatment comprising: one or more fungal species from at least one genus selected from Clohesyomyces, Darksidea and Phialocephala fortinii s.l - Acephala applanate species complex (PAC), wherein the deployment of said treatment has one or more desirable effects selected from the group consisting of increasing the sequestration of atmospheric carbon for storage as stable carbon in the soil, providing agronomic benefits to said commercial crops, increasing the levels of stable carbon in the soil, and increasing the soil aggregate stability of the soil.

2. A method for sequestering atmospheric carbon for storage as stable carbon in soil, the method comprising deploying a treatment comprising one or more fungal species from at least one genus selected from Clohesyomyces, Darksidea, and Phialocephala, in said soil and/or associating said fungus with a crop plant being cultivated in said soil.

3. A method for sequestering atmospheric carbon for storage as stable carbon in soil, the method comprising deploying a treatment comprising one or more fungal species, wherein the one or more fungal species is selected from at least one fungal species having a nuclear ribosomal internal transcribed spacer 2(ITS2) sequence that is at least 90% identical to the nucleotide sequence of SEQ ID No:

1, 2, 3, 4, 5, 6 or 7.

4. A method of increasing soil organic carbon in a soil and/or increasing yield of a crop plant being cultivated in the soil, the method comprising inoculating the soil and/or the crop plant with an effective amount of one or more fungal species from at least one genus selected from Clohesyomyces, Darksidea, and Phialocephala.

5. The method of any one of claims 1 to 4, wherein the one or more fungal species is one or more dark septate endophytic fungal species.

6. The method of any one of claims 1 to 5, wherein the crop plant is a cereal plant.

7. The method of claim 6, wherein the cereal plant is selected from wheat and barley.

8. The method of claim 7, wherein the cereal plant is wheat and the one or more fungal species is selected from at least one genus selected from Clohesyomyces and Darksidea.

9. The method of claim 7, wherein the cereal plant is barley and the one or more fungal species is selected from the genus Phialocephala.

10. The method of any one of claims 1 to 5, wherein the crop plant is a brassica.

11. The method of claim 10, wherein the brassica is canola.

12.The method of claim 11, wherein the crop plant is canola and the one or more fungal species is selected from at least one genus selected from Clohesyomyces, Darksidea, and Phialocephala.

13. The method of any one of claims 1 to 12, wherein:

(a) the fungal species of the genus Clohesyomyces comprises a nuclear ribosomal internal transcribed spacer 2(ITS2) sequence that comprises nucleotide sequence that is at least 97% identical to the nucleotide sequence of SEQ ID No: 2;

(b) the fungal species of the genus Darksidea comprises a nuclear ribosomal internal transcribed spacer 2(ITS2) sequence that comprises nucleotide sequence that is at least 97% identical to the nucleotide sequence of SEQ ID No: 3, 4, 5, or 6;

(c) the fungal species of the genus Phialocephala Acephala complex comprises a nuclear ribosomal internal transcribed spacer 2(ITS2) sequence that comprises nucleotide sequence that is at least 97% identical to the nucleotide sequence of SEQ ID NO: 1.

14. The method of any one of claims 1 to 13, wherein:

(a) the fungal species from the genus Clohesyomyces is Clohesyomyces aquaticus;

(b) the fungal species from the genus Darksidea is Darksidea delta or Darksidea zeta;

(c) the fungal species from the genus Phialocephala is a species from the Phialocephala fortinii s.l - Acephala applanate species complex.

15. The method of any one of claims 1 to 14, wherein the one or more fungal species is one or more fungi selected from Clohesyomyces aquaticus strain V21/002328, Phialocephala Acephala complex strain V21/002327, Darksidea zeta strain V21/002326, Darksidea zeta strain V21/003117, and Darksidea strain V21/003116.

16. The method of any one of claims 1 to 15, wherein seed of the crop plant is inoculated with the one or more fungal species.

17. The method of any one of claims 1 to 15, wherein a seedling of the plant is inoculated with the one or more fungal species.

18. A composition for increasing soil organic carbon in a soil and/or increasing yield of a crop plant, comprising one or more fungal species from at least one genus selected from Clohesyomyces, Darksidea, and Phialocephala.

19. The composition of claim 18, wherein the crop plant is wheat and the one or more fungal species is from at least one genus selected from Clohesyomyces, and Darksidea.

20. The composition of claim 18, wherein the crop plant is canola and the one or more fungal species is from at least one genus selected from Clohesyomyces, Darksidea, and Phialocephala.

21. The composition of claim 18, wherein the crop plant is barley, and the one or more fungal species is from at least one genus selected from Phialocephala.

22. The composition of any one of claims 18 to 21, wherein the one or more fungal species is selected from at least one fungi having a nuclear ribosomal internal transcribed spacer 2(ITS2) sequence that is at least 97% identical, with the nucleotide sequence of any one of SEQ ID No: 1 -6.

23. The composition of any one of claims 18 to 22, wherein the one or more fungal species is one or more fungi selected from Clohesyomyces aquaticus strain V21/002328, Phialocephala Acephala complex strain V21/002327, Darksidea zeta strain V21/002326, Darksidea zeta strain V21/003117, and Darksidea strain V21/003116.

24. The composition of any one of claims 18 to 23, wherein the composition is in the form of a seed coating, a foliar spray, a root dip, or soil drench.

25. A method for increasing the levels of stable carbon in soil used to cultivate a crop plant, the method comprising deploying a treatment comprising at least one melanised fungus, wherein said melanised fungus is a species from a genera selected from the group consisting of Clohesyomyces, Darksidea, and Phialocephala fortinii s.l. - Acephala applanata species complex (PAC), in said soil and/or associating said melanised fungus with said crop plant.

26. A method for providing agronomic benefits to a commercial crop being cultivated in soil, the method comprising deploying a treatment comprising at least one melanised fungus, wherein said melanised fungus is a species from a genera selected from the group consisting of Clohesyomyces, Darksidea, and Phialocephala fortinii s.l. - Acephala applanata species complex (PAC), in said soil and/or associating said melanised fungus with said crop plant.

27. A method for increasing the soil aggregate stability of soil used to cultivate a crop plant, the method comprising deploying a treatment comprising at least one melanised fungus, wherein said melanised fungus is a species from a genera selected from the group consisting of Clohesyomyces, Darksidea, and Phialocephala fortinii s.l. - Acephala applanata species complex (PAC), in said soil and/or associating said melanised fungus with said crop plant.

28. The method according to any one of claims 25 to 27, wherein the melanised fungus comprises at least about 10% melanin.

29. The method according to any one of claims 25 to 28, wherein the melanised fungus has a nuclear ribosomal internal transcribed spacer (ITS) sequence having at least about 95% sequence identity with any one of SEQ ID Nos. 1, 2, 3,4, 5, or

6.

30. The method according to any one of claims 25 to 29, wherein the one or more fungal species is one or more fungi selected from Clohesyomyces aquaticus strain V21/002328, Phialocephala Acephala complex strain V21/002327, Darksidea zeta strain V21/002326, Darksidea zeta strain V21/003117, and Darksidea strain V21/003116.

31. A kit comprising one or more fungi selected from Clohesyomyces aquaticus strain V21/002328, Phialocephala Acephala complex strain V21/002327, Darksidea zeta strain V21/002326, Darksidea zeta strain V21/003117, and Darksidea strain V21/003116.