Bioprotectant microbes

Abstract

The present invention provides a bioprotectant microbe, wherein the microbe is capable of conferring a bioprotection phenotype to a plant into which it is inoculated or otherwise infected. The present invention also relates to compositions including the bioprotectant microbes, seeds, plants, and parts thereof infected with the bioprotectant microbes; and related methods, including methods for conferring a bioprotection phenotype and for protecting a forage or crop from pests and / or diseases.

Description

[0001] BIOPROTECTANT MICROBES Related Application This application claims convention priority from Australian provisional patent application no 2024902003 filed on 28 June 2024, the entire disclosure of which is incorporated herein by reference. Field of the Invention The present invention relates to bioprotectant microbes, which are capable of conferring a bioprotection phenotype to plants into which they are inoculated or otherwise infected. The present invention also relates to compositions including the bioprotectant microbes, seeds, plants, and parts thereof infected with the bioprotectant microbes; and related methods, including methods for conferring a bioprotection phenotype and for protecting a forage or crop from pests and / or diseases. Background of the Invention Fusarium are soil borne fungi that can cause disease in a variety of horticultural, cropping and forage plants. Fusarium fungi can survive in soil a long time (5-10 years) and can be spread easily by contaminated soil and equipment and infected seed. While some species may be beneficial, some species are pathogenic to plants and may cause root rot, stem rot, wilt and leaf spot. Stunting, wilting, yellowing and necrosis of vascular tissues of plants are also symptoms of Fusarium wilt infections. Fusarium oxysporum is a fungal species known to have significant pathogenic effects on a wide variety of plants including forages such as alfalfa and perennial rye grass; crops such as rice; flowers such as aster, carnations, chrysanthemums, gladiolus, lilies, gerberas and heliconia; palms such as banana palm and date palm and vegetables such as tomatoes, capsicums and curcurbits as well as many other crops. While there are some plant species that have cultivars bred for resistance to Fusarium infection, including alfalfa, other crop host species may have no cultivars with resistance. Furthermore, the breeding of Fusarium resistant cultivars often occurs with a concomitant reduction in yield. This leads to some Fusarium susceptible cultivars with good yield and agronomic properties being passed over in favour of a resistant variety to avoid loss of crop to Fusarium infection. The economic impact of Fusarium pathogens includes yield losses, reduced quality of produce, increased costs of disease management and potential trade restrictions. Alfalfa (Medicago sativa) is a significant world-wide forage plant, known as the Queen of Forage. It has become an important forage plant globally because it has high yield, high quality, good palatability and adaptation to different environments. It is adapted to future climate scenarios predicted to appear in southern Australia’s grazing zones. However, alfalfa is susceptible to fungal pathogens, particularly Fusarium spp that cause root rot. Fusarium oxysporum f sp medicagensis causes an estimated annual yield loss in alfalfa of 20–40% in the world. Increasing restrictions in the use of fungicides, and growing fungal resistance to fungicides available to farmers, renders the availability of alternatives an increasingly important tool for agriculture. There exists a need to overcome, or at least alleviate, one or more of the difficulties or deficiencies associated with the prior art. Summary of the Invention The present invention provides microbes that may be inoculated onto or otherwise infected into seeds that provide the resulting plant with resistance to pests and diseases such as Fusarium infection, for example as an alternative to the use of a Fusarium resistant cultivar or as an alternative to a chemical fungicide. This may allow a wider variety of cultivars to be available to farmers to provide high yield and Fusarium resistance. In addition, the microbes may be useful in protecting plant species for which no Fusarium resistant cultivars are available. In one aspect, the present invention provides substantially purified or isolated bioprotectant microbe comprising a 16S rDNA sequence that is at least 95% identical to any one of SEQ ID Nos 1 to 15. In particular embodiments, the substantially purified or isolated bioprotectant microbe comprises a 16S rDNA sequence that is at least 97% identical to any one of SEQ ID Nos 1 to 15, preferably at least 98% identical to any one of SEQ ID Nos 1 to 15, more preferably at least 99% identical to any one of SEQ ID Nos 1 to 15. In some embodiments, the substantially purified or isolated bioprotectant microbe comprises a 16S rDNA sequence that is 100% identical to any one of SEQ ID Nos 1 to 15. The 16S rDNA sequences of SEQ ID Nos 1 to 15 are DNA sequences from the microbial genome that are transcribed to give the 16S rRNA sequences. In particular embodiments, the microbe is capable of conferring a bioprotection phenotype to a plant into which it is inoculated or otherwise infected. In particular embodiments, the bioprotectant microbe is a microbe of a Paenibacillus or Pseudomonas species. Bioprotection is the use of natural compounds or organisms to help protect plants from pests and / or diseases. As used herein the term “bioprotectant microbe” means that the microbe possesses genetic and / or metabolic characteristics that result in a beneficial phenotype in a plant harbouring, or otherwise associated with, the microbe. Such beneficial properties include improved resistance to pests and / or diseases, improved tolerance to water and / or nutrient stress, enhanced biotic stress tolerance, enhanced drought tolerance, enhanced water use efficiency, reduced toxicity and enhanced vigour in the plant with which the microbe is associated, relative to an organism not harbouring the microbe or harbouring a control microbe. The pests and / or diseases may include, but are not limited to, bacterial and / or fungal pathogens, preferably fungal pathogens. In one embodiment, the microbe may result in the production of a bioprotectant compound in the plant with which it is associated. In a preferred embodiment, the microbe produces a bioprotectant compound and provides bioprotection to the plant against bacterial and / or fungal pathogens, preferably fungal pathogens. As used herein, the term ‘bioprotectant compound’ is meant as a compound that provides bioprotection to the plant or aids the defence of the plant with which it is associated against pests and / or diseases, such as bacterial and / or fungal pathogens, preferably fungal pathogens. Preferably, the bioprotection phenotype is antifungal activity. More preferably the bioprotection phenotype is antifungal activity against the genus Fusarium, for example resistance to or reduction of Fusarium infection, more preferably antifungal activity against the species Fusarium oxysporum, for example resistance to or reduction of Fusarium oxysporum infection. In a preferred embodiment, the bioprotectant microbe is substantially purified or isolated from a plant of the genus Medicago. Preferably, the bioprotectant microbe is substantially purified or isolated from a plant of the species Medicago sativa. Plants of the genus Medicago are commonly known as medick or burclover. Medicago sativa is otherwise known as alfalfa and is an important forage crop. A plant of the Medicago genus includes plant seeds and plant parts thereof. The bioprotectant microbe may be substantially purified or isolated from any particular part of the plant, e.g., an organ. In preferred embodiments, the bioprotectant microbe may be substantially purified or isolated from a flower, flower bract, leaf, petiole, stem, seed, seedpod, or root of the plant, more preferably a seed or seedpod. In a preferred embodiment, the substantially purified or isolated bioprotectant microbe is a Paenibacillus species having a 16S rDNA sequence shown in any one of SEQ ID Nos 1 to 9 (Figure 1), or a sequence having at least approximately 95% sequence identity, more preferably at least approximately 97% identity, even more preferably at least approximately 98% identity, even more preferably at least approximately 99% identity to a 16S rDNA sequence shown in any one of SEQ ID Nos 1 to 9 (Figure 1). Preferably, the substantially purified or isolated bioprotectant microbe is a Paenibacillus species having 16S rDNA sequences with 100% identity to two or more, preferably all, of SEQ ID Nos 1 to 9. In a particularly preferred embodiment, the substantially purified or isolated Paenibacillus species bioprotectant microbe is a strain denoted Paenibacillus sp. Lu_MgY_007, as deposited with the National Measurement Institute of 1 / 153 Bertie St, Port Melbourne, Victoria 3207 Australia on 18 June 2024 with accession number V24 / 010675. Herein, this strain is also referred to as MgY007 and MgY-007. In a preferred embodiment, the substantially purified or isolated bioprotectant microbe is a Pseudomonas species, preferably Pseudomonas orientalis, having a 16S rDNA sequence shown in any one of SEQ ID Nos 10 to 15 (Figure 2), or a sequence having at least approximately 95% sequence identity, more preferably at least approximately 97% identity, even more preferably at least approximately 98% identity, even more preferably at least approximately 99% identity to a 16S rDNA sequence shown in any one of SEQ ID Nos 10 to 15 (Figure 2). Preferably, the substantially purified or isolated bioprotectant microbe is a Pseudomonas species having 16S rDNA sequences with 100% identity to two or more, preferably all, of SEQ ID Nos 10 to 15 In a particularly preferred embodiment, the substantially purified or isolated Pseudomonas species bioprotectant microbe is a strain denoted Pseudomonas orientalis Lu_LA164_018, as deposited with the National Measurement Institute of 1 / 153 Bertie St, Port Melbourne, Victoria 3207 Australia on 18 June 2024 with accession number V24 / 010674. Herein, this strain is also referred to as LA-164_018, LA164-018, LA164_018, La164-018, LA 164-018, LA-164 and La164. The present invention also provides variants of the bioprotectant microbe strains as hereinbefore described. Such variants include naturally occurring allelic variants and non-naturally occurring variants. Non-naturally occurring variants may have artificially introduced genetic variation. The genetic variation may be introduced utilising any standard techniques, e.g., via one or more of random mutagenesis, di / poly-ploidisation, targeted mutagenesis; cisgenesis; transgenesis; intragenesis. Additions, deletions, substitutions and derivatisations of one or more of the nucleotides in the genome of the microbe strains are contemplated, so long as the modifications do not result in loss of functional activity of the variant. In some cases such modifications may increase functional activity of the variant. Preferably the variant has at least approximately 95% sequence identity to the genome of the microbe strain of the invention, more preferably at least approximately 97% identity, even more preferably at least approximately 98% identity, even more preferably at least approximately 99% identity. Such functionally active variants include, for example, those having conservative nucleic acid changes in the genome. By “conservative nucleic acid changes” as used herein, is meant nucleic acid substitutions that result in conservation of the amino acid in the encoded protein owing to the degeneracy of the genetic code. Such functionally active variants also include, for example, those having nucleic acid changes which result in conservative amino acid substitutions of one or more residues in the corresponding amino acid sequence. By “conservative amino acid substitutions”, is meant the substitution of an amino acid by another one of the same class, the classes being as follows: Nonpolar: Ala, Val, Leu, Ile, Pro, Met Phe, Trp Uncharged polar: Gly, Ser, Thr, Cys, Tyr, Asn, Gln Acidic: Asp, Glu Basic: Lys, Arg, His Other conservative amino acid substitutions may also be made as follows: Aromatic: Phe, Tyr, His Proton Donor: Asn, Gln, Lys, Arg, His, Trp Proton Acceptor: Glu, Asp, Thr, Ser, Tyr, Asn, Gln By “substantially purified” is meant that the bioprotectant microbe is free of other organisms. The term includes, for example, a bioprotectant microbe in axenic culture. Preferably, the bioprotectant microbe is at least approximately 90% pure, more preferably at least approximately 95% pure, even more preferably at least approximately 98% pure, even more preferably at least approximately 99% pure. By “isolated” is meant that the bioprotectant microbe is removed from its original environment (e.g., the natural environment if it is naturally occurring; e.g., the plant). For example, a naturally occurring bioprotectant microbe present in nature in a living plant is not isolated, but the same bioprotectant microbe separated from some or all of the coexisting materials in the natural environment is isolated. By “inoculated” is meant that the bioprotectant microbe is placed in association with a plant to confer the bioprotection phenotype, whether that be on, in, or otherwise in close proximity to the plant. In preferred embodiments, the plant or part thereof to which the bioprotectant microbe is inoculated is free of that microbe before inoculation. The bioprotection phenotype which in these embodiments the bioprotectant microbe is capable of conferring may generally be considered as compared to the bioprotection phenotype, or lack thereof as the case may be, of a plant or part thereof that is absent of the bioprotectant microbe (“no bioprotectant microbe control”), and / or as compared to the bioprotection phenotype of a plant that contains a control microbe, which again may be taken from a presented in vitro bioprotectant activity or an observed in planta bioprotectant activity, for example antifungal activity. The seed, plant, or part thereof to which the bioprotectant microbe is capable of conferring a bioprotection phenotype may be any seed, plant or part thereof which contributes to the global food supply as, for example, crop cultivars harvested for human consumption or for use in medicinal and / or food products, or as forage for grazing livestock. As such, this includes plants of many important food and horticultural crops, such as plants of the Fabaceae, Poaceae, Apiaceae, Cruciferae, Solanaceae, Cucurbitaceae, Amaryllidaceae, Malvaceae, Lamiaceae, Rosaceae, Rutaceae or Brassicaceae families. In preferred embodiments, the seed, plant or part thereof to which the bioprotectant microbe is capable of conferring a bioprotection phenotype is or is from a forage plant. In preferred embodiments, the seed, plant or part thereof to which the bioprotectant microbe is capable of conferring a bioprotection phenotype is or is from a plant of the Fabaceae family, preferably a plant of a Medicago spp., for example M. sativa (alfalfa). In a second aspect of the present invention there is provided a composition comprising one or more bioprotectant microbes as described herein together with a suitable carrier or carriers. For example, the composition may be a composition suitable for inoculating or otherwise infecting a seed, plant or plant part or may be suitable for treating soil prior to planting. The carrier may be any carrier that is not detrimental to the bioprotectant microbe and may be solid or liquid. In some embodiments the carrier comprises water. In a preferred embodiment, the composition may be a liquid composition comprising a bioprotectant microbe that is suitable for spraying on soil. In another preferred embodiment, the composition may be a solid composition comprising a bioprotectant microbe that is suitable for mixing with soil, for example, dust, powder or pellets. In another preferred embodiment, the composition may be a seed coatings / treatment, such as a dressing that may be applied to seed before sale or planting. In some embodiments, the bioprotectant microbe may be in a latent state, for example, it may be cryopreserved or lyophilised. The composition may include components that facilitate the viability of the bioprotectant microbe in the composition. For example, the composition may comprise proteins and / or carbohydrates / sugars that facilitate viability of the bioprotectant microbe. Suitable proteins include milk proteins and suitable carbohydrates / sugars include maltose. In some embodiments, the composition may include components that assist with inoculation of the seed, plant or plant part, or transportation or storage of the composition. The composition may further contain components such as a plant growth regulator, encapsulation agent, wetting agent or dispersing agent to enhance the effect of the composition. The bioprotectant microbe may be absorbed onto a granulated carrier that may be planted with a seed or applied to the soil at the time of planting. In some embodiments, the composition is a fermentation broth that is capable of supporting the growth of the bioprotectant microbe, such as Lysogeny broth or Nutrient broth. These broths may for example include nutrients as supplied by components such as tryptone (5-15 g / L), peptone (3-15 g / L) yeast extract (2-10 g / L), beef extract (2-10 g / L), sodium chloride (50 g / L for 5% to 200g / L for 20% and all amounts in between). The broths are aqueous and contain water making up to the required volume. In some embodiments, the bioprotectant microbe may be isolated from the fermentation broth, for example by centrifuge, to isolate the pellet of bioprotectant microbe for resuspension in a suitable buffer, such as phosphate buffered saline. A suitable buffer pH range is approximately 7 to 8, especially about 7.4. In a further aspect of the present invention there is provided a plant seed or embryo coated with a composition comprising the bioprotectant microbe described herein. Plant seeds or embryos isolated from plant seeds may be coated with one or more bioprotectant microbes as disclosed herein in a solid or liquid suspension, directly or in combination with suitable carrier(s), binder(s) and / or filler(s). Suitable carriers, binders and fillers include peat, lime, biochar, wheat bran, vermiculite, clay, talc, bentonite, diatomaceous earth, Fuller’s earth, pasteurised soil, chitosan, methyl cellulose, carboxymethylcellulose, gum arabic, polysaccharide Pelgel®, xanthan gum and alginate. The identity of the carrier, binder and / or filler may depend on the bioprotectant microbe used and the seed being treated. The coating may be a seed dressing, a film, a pellet or an encrustation. The seed or embryo which may be coated with a composition according to the present invention may be from any plant species which contributes to the global food supply as, for example, crop cultivars harvested for human consumption or for use in medicinal and / or food products, or as forage for grazing livestock. As such, this includes plants of many important food and horticultural crops, such as plants of the Fabaceae, Poaceae, Apiaceae, Cruciferae, Solanaceae, Cucurbitaceae, Amaryllidaceae, Malvaceae, Lamiaceae, Rosaceae, Rutaceae or Brassicaceae families. In preferred embodiments, seed or embryo is of a forage plant. In preferred embodiments, the seed or embryo is from a plant of the Fabaceae family, preferably a plant of a Medicago spp., for example M. sativa (alfalfa). In one embodiment, the plant seed or embryo may be treated with an aqueous composition comprising alginate and the bioprotectant microbe followed by treatment with a complexing agent. For example, the bioprotectant microbe may be in a composition comprising sodium alginate and after coating the seed with sufficient composition, a complexing agent such as calcium chloride may be added so solidify the alginate polymer thereby coating the seed. The sodium alginate may be present in the composition at a concentration of 1 to about 10% w / volume in water, especially 3 to 5% w / v. The calcium chloride solution may be at any concentration suitable to result in polymerisation of the alginate, for example, 1-1000 mM, 20- 500 mM or 50-300 mM. The thickness of the seed coating once solidified may be in the range of about 0.1 to about 5 mm, especially about 0.25 to about 1.5 mm. In some embodiments, a second coating may be applied to the seed or embryo, this outer coating not including the bioprotectant microbe. In another aspect, the present invention provides a seed, plant, or part thereof inoculated or otherwise infected with one or more bioprotectant microbes as herein described. The seed, plant, or part thereof may be any plant which contributes to the global food supply as, for example, crop cultivars harvested for human consumption or for use in medicinal and / or food products, or as forage for grazing livestock. As such, this includes plants of many important food and horticultural crops, such as plants of the Fabaceae, Poaceae, Apiaceae, Cruciferae, Solanaceae, Cucurbitaceae, Amaryllidaceae, Malvaceae, Lamiaceae, Rosaceae, Rutaceae or Brassicaceae families. In preferred embodiments, the seed, plant or part thereof is or is from a forage plant. In preferred embodiments, the seed, plant or part thereof is or is from a plant of the Fabaceae family, preferably a plant of a Medicago spp., for example M. sativa (alfalfa). The bioprotectant microbes of the present invention may have the ability to be transferred through propagative material from one plant generation to the next. The bioprotectant microbe may then spread or locate to other tissues as the plant grows, for example, to roots. Alternatively, or in addition, the bioprotectant microbe may be recruited to the plant root, for example from soil, and spread or locate to other tissues. In either case, the bioprotectant microbe may be said to be stably inoculated or infected to the plant. Therefore, the present invention also provides a seed, plant, plant propagative material, or other plant part derived from a plant inoculated with one or more bioprotectant microbes as herein described and infected therewith, preferably stably infected therewith. The present invention provides the use of one or more bioprotectant microbes as described herein to produce a seed, plant, or part thereof infected, preferably stably infected, with said one or more bioprotectant microbes. In a further aspect, the present invention provides a method for conferring a bioprotection phenotype to a seed, plant, or part thereof, the method including inoculating or otherwise infecting the seed, plant, or part thereof with a bioprotectant microbe as herein described. In preferred embodiments, the seed, plant, or plant part inoculated or otherwise infected with the bioprotectant microbe as described herein will exhibit a bioprotection phenotype, or in other words, the bioprotectant microbe will confer thereto a bioprotection phenotype. In a preferred embodiment of this aspect of the present invention, the plant or part thereof may be free of said bioprotectant microbe prior to inoculation and may be stably infected with said bioprotectant microbe. In a further aspect, the present invention provides a method for protecting a forage or crop from pests and / or diseases, said method including exposing seed from which the forage or crop will be grown, the forage or crop itself, or soil in which the forage or crop is planted to a bioprotectant microbe according to the present invention. The forage or crop may be any plant which contributes to the global food supply as, for example, crop cultivars harvested for human consumption or for use in medicinal and / or food products, or as forage for grazing livestock. As such, this includes plants of many important food and horticultural crops, such as plants of the Fabaceae, Poaceae, Apiaceae, Cruciferae, Solanaceae, Cucurbitaceae, Amaryllidaceae, Malvaceae, Lamiaceae, Rosaceae, Rutaceae or Brassicaceae families. In preferred embodiments, the forage or crop is a forage plant. In preferred embodiments, the forage or crop is a plant of the Fabaceae family, preferably a plant of a Medicago spp., for example M. sativa (alfalfa). The pests and / or diseases may include, but are not limited to, bacterial and / or fungal pathogens, preferably fungal pathogens. In one embodiment, the microbe may result in the production of a bioprotectant compound in the plant with which it is associated. In a preferred embodiment, the microbe produces a bioprotectant compound and provides bioprotection to the plant against bacterial and / or fungal pathogens, preferably fungal pathogens. Preferably, the method protects the forage or crop against fungal infections, for example the forage or crop exhibits resistance to or reduction of fungal infections. More preferably the method provides antifungal activity against the genus Fusarium, for example resistance to or reduction of Fusarium infection, more preferably antifungal activity against the species Fusarium oxysporum, for example resistance to or reduction of Fusarium oxysporum infection. In this specification, the term ‘comprises’ and its variants are not intended to exclude the presence of other integers, components or steps. In this specification, reference to any prior art in the specification is not and should not be taken as an acknowledgement or any form of suggestion that this prior art forms part of the common general knowledge in Australia or any other jurisdiction or that this prior art could reasonably expected to be combined by a person skilled in the art. The present invention will now be more fully described with reference to the accompanying Examples and drawings. It should be understood, however, that the description following is illustrative only and should not be taken in any way as a restriction on the generality of the invention described above. Brief Description of the Drawings / Figures In the Figures: Figure 1 shows 16S rDNA sequences of Paenibacillus Lu_MgY_007 (SEQ ID Nos 1 to 9) from which the 16S rRNA sequences are transcribed. Figure 2 shows 16S rDNA sequences of Pseudomonas LA-164_018 (SEQ ID Nos 10 to 15) from which the 16S rRNA sequences are transcribed. Figure 3 shows examples of plate bioprotection assays with various strains of Paenibacillus and Pseudomonas showing activity against Fusarium proliferatum 42191, Fusarium proliferatum 42958, and Fusarium oxysporum 54415. Figure 4 shows a summary of the most active bacterial strains. The most effective were Lu_MgY_00793.98% ANI to Paenibacillus terrae and LA164_01894.85% ANI to Pseudomonas oriantalis (NB Higher numbers are better). Bars represent the following strains (left to right in each panel): F. proliferatum 42191, F. proliferatum 42409, F. proliferatum 42958, F. oxysporum 5189, F. oxysporum 5190a, F. oxysporum 54415. Figure 5 shows petri dishes with millet seedlings inoculated with Fusarium oxysporum strain FS189. Figure 6 shows disease scores and symptoms as per Table 3. Figure 7 shows average disease scores for Alfalfa Grazer and Sequal cultivars inoculated with various microbes. The two strains that performed better in plate based assays produced lower disease scores (with lower numbers being better). Figure 8 shows an in planta assay, with alfalfa seedlings in Fusarium oxysporum fsp medicagensis 5189 inoculated potting mix Figure 9 shows disease scores and symptoms as per Table 4. Figure 10 shows cumulative disease scores for 10 repetitions, with both bioprotectant bacteria inhibiting Fusarium oxysoporum disease symptoms. Non-bioprotectant bacteria show symptoms similar to non-inoculated controls. Repetitions are as follows (from bottom to top): R1, R2, R3, R4, R5, R6, R7, R8, R9, R10. Figure 11 shows shoot and root measurements from bioprotection experiments. While there are high disease scores from the ‘control’ bacteria, they do provide some growth benefit (protection) compared to un-inoculated control plants. Average shoot lengths are shown above the horizontal line and average root lengths below the horizontal line. Figure 12 shows photographs of 5 of the repetitions from the in planta bioprotection assays. The strains are as follows (left to right): Row 1 – F. oxysporum 5189, no F. oxysporum 5189Row 2 – F. oxysporum 5189, F. oxysporum 5189 & Paenibacillus MgY007, F. oxysporum 5189 & Paenibacillus Sv-042 Row 3 - F. oxysporum 5189, F. oxysporum 5189 & Pseudomonas La164, F. oxysporum 5189 & Dugnella TR935-10. Figure 13 shows the experimental set up for testing how the two bioprotectant bacteria respond transcriptionally to co-cultivation with two Fusarium oxysporum fsp. medicagensis strains. Figure 14 shows the experimental set up for RNA isolation. Figure 15 shows Paenibacilus MgY007 differential gene expression and fungal inhibition. The lighter grey line ending at 1494 is MgY-007 Control vs Pathogen F5189+. The darker grey line ending at 699 is MgY-007 Control vs Pathogen FS190+. Figure 16 shows Pseudomonas sp LA164-018 differential gene expression and fungal inhibition. The lighter grey line ending at 795 is LA164-018 Control vs Pathogen FS189+. The darker grey line ending at 0 is LA164-018 Control vs Pathogen FS190+. Detailed Description of the Embodiments Example 1 – In vitro testing of microbial isolates against Fusarium Spp Thirty four bacterial isolates obtained from a Medicago species microbial isolate library were tested against three strains of Fusarium proliferatum and 3 strains of Fusarium oxysporum (Tables 1 and 2).

[0002] Table 1 Bacterial isolates Table 2 Fusarium strains Each bacterial strain was cultured in Nutrient Broth overnight. The bacteria were drop- inoculated (20 μL) onto four equidistant points on a Nutrient Agar plate, which was then incubated overnight at 28°C. Then, a 6 × 6 mm plug of the phytopathogen (actively growing hyphae) was placed at the centre of the plate and incubated at 28°C in dark. The incubation time varied to accommodate the differences in growth rate of the fungal pathogens (6 to 9 days). The diameter of the fungal colony on the plate was measured twice. One reading was taken from the straight line that was defined by two inoculation points and the centre of the plate, and the other reading was taken after rotating the plate for 45 degrees. The average of the two readings was used for statistical analysis. As biological replicates, three plates were prepared for each treatment. Sterile nutrient broth was used as the inactive control to replace bacteria. For statistical analysis, One-way ANOVA and Tukey test was conducted using OriginPro 2020 (version 9.7.0.188 [Academic]) for any significant difference (P< 0.05) among the treatments. Of the thirty four isolates, two bacterial microbes showed anti-fungal activity against more than one Fusarium species. These microbes were a Paenibacillus species designated Lu_MgY_007 and a Pseudomonas species designated LA164_018 (Figure 3). Eight microbial isolates tested had at least some activity against a number of Fusarium strains, particularly LA164_018 which had activity against all strains tested and Lu_MgY_007 which showed significant activity against four of the fungal pathogens tested (Figure 4). Example 2: In Planta assay- Seedlings with no soil A fungal inoculum was prepared by washing and sterilizing millet seeds. The millet seeds were washed three times in sterile distilled water and soaked overnight in sterile distilled water. Water was drained and the soaked millet seeds were transferred to a new Schott bottle. A total of two sterilizations were performed using autoclave for 1 hour at 121° C, by cooling it down in between the sterilizations. A few actively growing Fusarium oxysporum strain FS189 fungal plugs were inserted into the sterile millet seeds. The infected seeds were incubated at RT for ~ 7 days with daily shaking. The Fusarium inoculated millet seeds were stored at RT for up to 2-3 weeks. The seeds were washed by rinsing with autoclaved RO water for three times. Alfalfa seeds were inoculated by soaking in a microbial broth solution containing the test bacteria (bacterial concentration; OD600= 0.3 – 0.4) being tested overnight. The inoculated seeds were allowed to germinate under in vitro conditions, where the seeds were germinated on wet, sterile filter papers in petri dishes sealed with a tape under ample light conditions at room temperature. Healthy seedlings were selected and transferred to new petri dishes, 1 seedling per petri dish of 18 alfalfa cultivars at 5 replicates per cultivar (180 seedlings). The seedlings were kept on sterile, moist filter papers. The roots were covered with the millet seed fungal inoculum (Figure 5). The seedlings were kept at room temperature and monitored for Fusarium wilt symptoms. The seedlings were scored for disease symptoms at day 3, 5, 7, 10, 12 and 14 timepoints. The disease score was based on the following symptoms (see Table 3 and Figure 6). Table 3 Disease scores and symptoms The results are shown in Figure 7. In the two cultivars shown, Paenibacillus Lu-MgY_007 (MgY007) and Pseudomonas LA-164_018 (LA-164) performed better than other microbes including control where Fusarium oxysporum strain 5189 was applied without a microbial bioprotectant. Example 3: In Planta assay- Seedlings with soil Fungal inoculum in the form of millet seed was prepared as given in Example 2. Alfalfa cultivar Grazer seeds were washed by rinsing with autoclaved RO water for three times. Grazer seed was either soaked in bacterial broth overnight (bacterial concentration; OD600= 0.3 – 0.4). The seeds were then sown into seedling trays comprising potting mix and allowed to germinate. At 10 days, the seedling was transferred as a plug from the seedling tray into a plug shaped hole in a potting mix pot lined with Fusarium inoculated millet seed as shown in Figure 8. Control seedlings were planted in pots with no Fusarium inoculant. After 14 days growth in a growth cabinet with 16 h of light and 8 h of darkness at 25◦C and 20◦C respectively, the seedlings were scored (see Table 4 and Figure 9). Table 4 Disease scores and symptoms The cumulative disease scores for 10 repetitions shows that both Paenibacillus Lu_MgY_007 and Pseudomonas LA-164_018 inhibit Fusarium oxysporum disease symptoms with similar results to the non-inoculated control, whereas non-bioprotectant microbes and the inoculated control showed similar disease effects (Figure 10). Shoot and root measurements were taken from the seedlings (Figure 11). While there is some effect of the presence of Paenibacillus Lu_MgY_007 and Pseudomonas LA-164_018 microbes on shoot and root length compared to the non-inoculated control, the shoot and root length of these microbe inoculated seedlings was significantly better than the inoculated control and the non-bioprotectant microbe inoculated seedlings. The plant growth in the plants treated with Paenibacillus Lu_MgY_007 and Pseudomonas LA-164_018 microbes was visually similar to those with no Fusarium infection compared to those with Fusarium infection with and without non-bioprotectant microbes (Figure 12). Example 4: Testing of bioprotective bacteria against grain pathogens The two bioprotective bacteria, Pseudomonas sp La164-018 and Paenibacillus sp 2 MgY002 were tested against the grain pathogens shown in Table 5. Table 5 Testing against grain pathogens Host Pathogen Strain Name PTT20-037 , PTM23 (new isolate Barley Pyrenophora teres f. teres (NFNB) SA) Barley Pyrenophora teres f. maculata (SFNB) PTM21-002 Wheat Septoria tritici (spetoria leaf blotch) 15N Fusarium culmorum (seedling blight, foor rot, Wheat FS22-193 head blight) Wheat Fusarium pseudograminearum (crown rot) FS19-432 Lentil Ascochyta lentis (ascochyta blight) VPRI 32775 Faba bean Botrytis fabae (chocolate spot) L2RK1051-2-7 Didymella pinoides, Didymella pinodella, Field Pea BS20-300 Phoma koolunga (Blackspot) Didymella pinoides (isolate SA)Canola Leptosphaeria maculans (blackleg) D22 Chickpea Ascochyta rabiei (ascochyta blight) AS22-149 Tomato Botrytis cinerea (grey mould) DAR 77536 Tomato Fusarium oxysporum (tomato vascular wilt) VPRI 43900 The results of these assays are shown in Table 6. Active indicates inhibition of the fungal growth. Table 6 Activity against grain pathogens Compared to the other microbes that show an anti-fungal activity a ranking can be applied. Pseudomonas LA164-18 was among the most effective microbes for seven of the pathogens, and Paenibacillus MgY007 was in this most effective cohort for two pathogens (Table 7). Table 7 Activity against grain pathogens Example 5: Response transcriptionally to co-cultivation Experiments were undertaken to investigate how the two bioprotectant bacteria respond transcriptionally to co-cultivation with two Fusarium oxysporum fsp. medicagensis strains. The experimental set up is shown in Figure 13. An overnight liquid culture of the bacteria was grown up, and its optical density was measured at 600nm (OD 600) measured. Twenty microlitres of the overnight culture with an OD600 measurement of approximately 1 was spotted onto an agar plate in 4 spots as shown and then incubated overnight. A small plug of actively growing mycelia was added in the centre of the plate, and the plates grown for 7-9 days. Three replicates of bacteria and fungi were destructively harvested per timepoint and used for RNA isolation (Figure 14). The bacterial total RNA was ribo depleted using an NEBNext® rRNA depletion kit, while the fungal total RNA was treated with the NEBNext® Poly(A) mRNA Magnetic Isolation Module. These processed RNA samples were used as templates in a NEXTFLEX® Rapid Directional RNA Library Prep Kit and the resulting libraries were sequenced on an Illumina Novaseq 6000 platform. The sequenced libraries were trimmed of adapter and for quality using Fastp (-w 8 -3 -5 –detect_adapter_for_pe). Salmon was used to quantify transcripts using filtered RNA-seq reads with parameters: -l A – validateMappings – numBootstraps 1000 –seqBias against genes predicted from the respective bacterial genomes. Percent inhibition of the fungi was also measured at the final timepoint. Differential gene analysis was conducted at each timepoint using Sleuth (significant at q < 0.05 and absolute fold-change ≥ 1.5) through comparing bacterial gene expression when the fungi was present to the no-fungal control at the same timepoint. The results are shown in Figures 15 and 16. For Paenibacilus MgY007 (Figure 15) both fungal strains were equally restricted in growth by the presence of the bacteria. In both cases the number of differentially expressed bacterial genes increased with time, with a larger transcriptional response to fungal strain F1590. For Pseudomonas sp LA164-018 (Figure 16), strain F5189 was less inhibited by the bacterial strain. In contrast to the Paenibacillus bio-protectant, there were differentially expressed (DE) genes seen at day 0 (1 day after co-culturing). After this timepoint the less restricted fungal strain resulted in a 3-fold increase in DE genes at day 5, followed by a turn off of response to the fungi F5189 at day 8. In contrast the fungi showing a more restricted growth, showed a decrease in DE genes at day 5, but an increase in DE genes at day 8. Thus, the two different bioprotectant bacterial species respond differently to the fungi at the level of transcription. The Pseudomonas strain sp LA164-018 has an early response, followed by a different program depending on the fungal strain. For the most repressed strain, there is a day 8 increase in differentially expressed genes, while the other strain F5189 reduces bacterial gene expression. The Paenibacillus strain shows a longer time to respond to the presence of the fungal strain transcriptionally, with no DE genes at day 3 (1 day after co-cultivation) with an increasing number of genes expressed after that timepoint. Again, F5189 seems to induce a lower bacterial transcriptional response. While applicants do not wish to be restricted by theory, these transcriptional responses to Fusarium oxysporum strains indicate the two different bacteria have different transcriptional responses to inhibit the growth of the fungi. Finally, it is to be understood that various alterations, modifications and / or additions may be made without departing from the spirit of the present invention as outlined herein.

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. A substantially purified or isolated bioprotectant microbe comprising a 16S rDNA sequence that is at least 95% identical to any one of SEQ ID Nos 1 to 15.

2. A substantially purified or isolated bioprotectant microbe according to claim 1 comprising a 16S rDNA sequence that is at least 97% identical to any one of SEQ ID Nos 1 to 15.

3. A substantially purified or isolated bioprotectant microbe according to claim 1 or claim 2 comprising a 16S rDNA sequence that is 100% identical to any one of SEQ ID Nos 1 to 15.

4. A substantially purified or isolated bioprotectant microbe according to any one of claims 1 to 3 comprising 16S rDNA sequences that are 100% identical to two or more of SEQ ID Nos 1 to 9.

5. A substantially purified or isolated bioprotectant microbe according to any one of claims 1 to 3 comprising 16S rDNA sequences that are 100% identical to two or more of SEQ ID Nos 10 to 15.

6. A substantially purified or isolated bioprotectant microbe according to any one of claims 1 to 5, wherein the microbe is capable of conferring a bioprotection phenotype to a plant into which it is inoculated or otherwise infected.

7. A microbe according to claim 2, wherein the bioprotection phenotype is antifungal activity.

8. A microbe according to claim 6 or 7, wherein the bioprotection phenotype is antifungal activity against the genus Fusarium.

9. A microbe according to claim 8, wherein the bioprotection phenotype antifungal activity against the species Fusarium oxysporum.

10. A microbe according to any one of claims 1 to 9, wherein the bioprotectant microbe is substantially purified or isolated from a plant of the genus Medicago.

11. A microbe according to claim 10, wherein the bioprotectant microbe is substantially purified or isolated from a plant of the species Medicago sativa.

12. A microbe according to any one of claims 1 to 4 and 6 to 11, wherein the microbe is a strain denoted Paenibacillus Lu_MgY_007, as deposited with the National Measurement Institute of 1 / 153 Bertie St, Port Melbourne, Victoria 3207 Australia on 18 June 2024 with accession number V24 / 010675, or a variant thereof having at least approximately 95% sequence identity to the genome thereof, more preferably at least approximately 97% identity, even more preferably at least approximately 98% identity, even more preferably at least approximately 99% identity.

13. A microbe according to any one of claims 1 to 3 and 5 to 11, wherein the microbe is a strain denoted Pseudomonas LA-164_018, as deposited with the National Measurement Institute of 1 / 153 Bertie St, Port Melbourne, Victoria 3207 Australia on 18 June 2024 with accession number V24 / 010674 , or a variant thereof having at least approximately 95% sequence identity to the genome thereof, more preferably at least approximately 97% identity, even more preferably at least approximately 98% identity, even more preferably at least approximately 99% identity..

14. A composition comprising one or more bioprotectant microbes according to any one of claims 1 to 13, together with a suitable carrier or carriers.

15. A composition according to claim 14, wherein the composition is selected from the group consisting of: (a) a liquid composition suitable for treating soil prior to planting, (b) a solid composition suitable for mixing with soil, and (c) a coating suitable for applying to seed.

16. A plant seed or embryo coated with a microbe according to any one of claims 1 to 13 or a composition according to claim 14 or 15.

17. A plant seed or embryo according to claim 16, wherein the coating is selected from a seed dressing, a film, a pellet or an encrustation.

18. A plant, seed or part thereof inoculated or otherwise infected with one or more microbes according to any one of claims 1 to 13.

19. A seed, plant, plant propagative material, or other plant part derived from a plant inoculated with one or more microbes according to any one of claims 1 to 13 and stably infected therewith.

20. A plant, seed, part thereof, plant propagative material or embryo according to any one of claims 16 to 19, wherein the plant, seed, part thereof, plant propagative material or embryo is from a family selected from the group consisting of Fabaceae, Poaceae, Apiaceae, Cruciferae, Solanaceae, Cucurbitaceae, Amaryllidaceae, Malvaceae, Lamiaceae, Rosaceae, Rutaceae and Brassicaceae.

21. A plant, seed, part thereof, plant propagative material or embryo according to claim 20, wherein the plant, seed, part thereof, plant propagative material or embryo is from the Fabaceae family, preferably a Medicago spp., more preferably M. sativa (alfalfa).

22. Use of one or more microbes according to any one of claims 1 to 13 or use of a composition according to claim 14 or 15 to produce a seed, plant, or part thereof stably infected with said one or more microbes.

23. A method for conferring a bioprotection phenotype to a seed, plant, or part thereof, the method including inoculating or otherwise infecting the seed, plant, or part thereof with a microbe according to any one of claims 1 to 13 or a composition according to claim 14 or 15.

24. A method for protecting a forage or crop from pests and / or diseases, said method including exposing seed from which the forage or crop will be grown, the forage or crop itself, or soil in which the forage or crop is planted to a microbe according to any one of claims 1 to 13 or a composition according to claim 14 or 15.

25. A method according to claim 23 or 24, wherein the seed, plant, or part thereof, forage or crop is from a family selected from the group consisting of Fabaceae, Poaceae, Apiaceae, Cruciferae, Solanaceae, Cucurbitaceae, Amaryllidaceae, Malvaceae, Lamiaceae, Rosaceae, Rutaceae and Brassicaceae.

26. A method according to claim 25, wherein the seed, plant, or part thereof, forage or crop is from the Fabaceae family, preferably a Medicago spp., more preferably M. sativa (alfalfa).

27. A method according to any one of claims 23 to 26, wherein the method protects the seed, plant, part thereof, forage or crop against fungal infection.

28. A method according to claim 27, wherein the method provides antifungal activity against the genus Fusarium, preferably antifungal activity against the species Fusarium oxysporum.

Images