Microbial composition for enhancing plant growth

Abstract

The present invention relates to microbial compositions, useful as biological amendments to enhance plant growth. Provided are isolated microbial strains, agricultural compositions comprising them, and their use in methods of improving plant growth and / or increasing plant yield.

Description

[0001] MICROBIAL COMPOSITION FOR ENHANCING PLANT GROWTH

[0002] Field of the Invention

[0003] The present invention relates to microbial compositions, useful as biological amendments to enhance plant growth. Provided are isolated microbial strains, agricultural compositions comprising them, and their use in methods of improving plant growth and / or increasing plant yield.

[0004] Background to the Invention

[0005] The role of microorganisms in plant growth and crop yield has long been recognised. Rhizobacteria are known in their roles as nitrogen-fixers and phosphate-solubilizers, as well as other root-associated beneficial bacteria which enhance the availability of macro- and micro-nutrients to the host plant. Such bacteria are commonly referred to as plant growth promoting rhizobacteria (PGPR).

[0006] Various agricultural amendments that aim to use beneficial rhizospheric or endophytic microbes to promote plant health have been developed with the aim of delivering microorganisms to plants as a probiotic to enhance growth and reduce the need for fertilisers. However, with the exception of legume inoculants, their mechanisms of action remain poorly understood, and their effectiveness unproven.

[0007] There remains a need for improved plant probiotic compositions.

[0008] Summary of the Invention

[0009] The present invention relates to microbial strains, and their use as plant growth-promoting probiotics.

[0010] In a first aspect, provided is an isolated microbial strain.

[0011] In a second aspect, provided is a microbial strain comprising the progeny of the isolated microbial strain of the first aspect.

[0012] In a third aspect, provided is a substantially pure culture of an isolated microbial strain according to the first aspect. In a fourth aspect, provided is an agricultural composition comprising an effective amount of the isolated microbial strain of the first aspect, the microbial strain of the second aspect, or the substantially pure culture of the third aspect, and an agriculturally acceptable carrier.

[0013] In a fifth aspect, provided is a method of improving plant growth and / or increasing plant yield, comprising contacting a plant, a plant part, or a growth medium in which said plant is located, with an agricultural composition according to the fourth aspect.

[0014] In a further aspect, provided is a plant, seed, or plant part, treated with an agricultural composition according to the fifth aspect.

[0015] In a further aspect, provided is a synthetic combination of a plant and the agricultural composition of the fifth aspect.

[0016] In a further aspect, provided is a cell-free or inactivated preparation of the isolated microbial strain of the first aspect, the microbial strain of the second aspect, or the substantially pure culture of the third aspect.

[0017] In a further aspect, provided is a metabolite produced by the isolated microbial strain of the first aspect, the microbial strain of the second aspect, or the substantially pure culture of the third aspect.

[0018] In a further aspect, provided is a method of producing a microbial composition comprising culturing the isolated microbial strain of the first aspect, the microbial strain of the second aspect, or the substantially pure culture of the third aspect.

[0019] In a further aspect, provided is a microbial culture produced by the method of producing a microbial composition described in the above aspect.

[0020] In a further aspect, provided is a use of the agricultural composition according to the fourth aspect for promoting plant growth; wherein the use comprises applying the agricultural composition to a plant, a plant part, or a growth medium in which said plant is located.

[0021] Brief Description of the Figures

[0022] Figure 1 - Results of proof-of-concept trial. Lettuce plants were grown for seven days in a hydroponic nursery before transplanting into a hydroponic growth system and grown in inert, foam substrate. Microbial compositions were applied three times by directly pipetting 0.5 mL of the treatment: onto the seed after sowing, when the plant began to emerge from the seed coat (3-4 days after sowing), and at transplant (1 week after sowing). Plants treated with “Pantoea” had a 16.9% increase in median head weight 28 days from transplant (13.3% increase in mean).

[0023] Figure 2 - Results of further internal trial. Lettuce plants were grown for seven days in a hydroponic nursery before transplanting into a hydroponic growth system and grown in inert, foam substrate. Microbial compositions were applied three times by applying 0.5 mL of the treatment: onto the seed after sowing, when the plant began to emerge from the seed coat (3-4 days after sowing), and at transplant (1 week after sowing), by directly pipetting or by spraying. Plants treated with “Pantoea” through pipette had a 10.2% increase in median head weight (12.8% increase in mean) and spray treatment a 13.4% increase in median head weight (21.2% increase in mean), relative to control plants. All measurements were made 28 days from transplant.

[0024] Figure 3 - Results of commercial trial. Lettuce plants were sown and grown for 17 days before transplanting into peat substrate in deep water culture ponds, and grown for 57 days before being harvested and weighed. Microbial compositions were applied two times by directly pipetting 0.5 mL of the treatment: onto the seed after sowing, and reapplied 17 days after sowing. Plants treated with “Pantoea” had a 13.7% increase in average head weight 28 days from transplant (20% increase in mean).

[0025] Figure 4 - Results of commercial trial in Ebb-and-Flow hydroponic system. Lettuce plants were sown and grown for 21 days in an Ebb-and-Flow hydroponic system on inert substrate, before being harvested and weighed. Microbial compositions were applied by spraying Pantoea suspended in sterile water onto the seed after sowing. Plants treated with “Pantoea” had a 6.7% increase in relative yield compared to control plants.

[0026] Figure 5 - Results of internal trial with cucumbers. (A) Total number of cucumber plants harvested. (B) Total weight of cucumbers harvested. Cucumber plants were grown in ETFE polytunnels. “Pantoea” treatment was applied by spraying two times: onto seeds and after transfer to the polytunnel. Cucumbers were continually harvested throughout the growth cycle. Plants treated with “Pantoea” produced 5.65% more cucumbers compared to control plants.

[0027] Figure 6 - Results of commercial trial with GrowCoon. Lettuce plants were sown at high density initially in two boxes with Growcoon. Seeds were transplanted twice during the growth cycle. “Pantoea” suspended in sterile water was sprayed onto seeds 48-hours after sowing and after the final transplant event. Plants treated with “Pantoea” had a 8.730% mean increase in relative yield compared to control plants.

[0028] Figure 7 - Results of CRO trial in a hydroponic, deep-water culture system. Lettuce plants were sown and grown for 50 days before being harvested and weighed. Seeds were sown at a higher density and grown to maturity for 22 days before being transplanted into a lower density growth system. Seedlings were propagated in trays and were transplanted into deep water tanks once mature. “Pantoea” treatment was applied directly by pipetting 0.5 mL of “Pantoea” suspended in sterile water onto the seed after sowing. The treatment was reapplied directly to the tank solution 22 days after sowing, at the same dosage per plant as the pipetted solution. Plants treated with “Pantoea” had a 7.65% increase in relative mean yield compared to control plants.

[0029] Figure 8 - Results of CRO trial using mixed peat in a hydroponic, deep-water culture system. Lettuce plants were sown at a higher density and grown to maturity for 14 days before being transplanted into a lower density growth system. Seedlings were propagated in trays and were transplanted into deep water tanks once mature. “Pantoea” was prepared at a 1 :10 concentration and applied twice using a spray bottle onto the seeds after sowing. The treatment was reapplied directly to the tank after 14 days after sowing at the same dosage per plant as the sprayed solution. Plants treated with “Pantoea” had a 16.6% increase in mean relative yield compared to control plants.

[0030] Figure 9 - Results of CRO trial on tomato plants. Tomato plants were grown in deep water culture trays across five rigs, with an experimental matrix applied to ensure even distribution of the trays throughout the room. “Pantoea” treatment was applied by spraying onto the seeds. A foliar spray at the same dosage was applied at the point of transplant, and thereafter the treatment was dosed into the nutrient solution of the trays every four weeks. Pantoea treated plants exhibited a 10.8% increase in mean fresh weight compared to control plants.

[0031] Detailed Description of the Invention

[0032] The present invention relates to isolated microbial strains, and progeny, derivatives and variants thereof having the same or similar functionality as the isolated microbial strains of the invention, and populations thereof. Preferred strains are members of the genus Pantoea, and particularly Pantoea septica. An exemplary microbial strain is the Pantoea septica strain deposited under the Budapest Treaty of 1977 as Accession Number 24050701 at the NCTC Patent Depository (LIKHSA), Culture Collections, UK Health Security Agency, Porton Down, Salisbury, SP4 OJG, United Kingdom. The Pantoea septica strain 24050701 was deposited on the 7thMay 2024 by Concert Bio Ltd (Address: Translation & Innovation Hub, 84 Wood Lane, London, W12 0BZ, United Kingdom); the identification listed on the deposit documentation is “Pantoea septica”.

[0033] As used herein, an “isolated microbial strain” is meant to mean a microbial strain (or population thereof) that is substantially free from the materials with which the microbial strain is normally associated in nature, or from the materials with which the microbial strain is associated in an artificial culture or production system.

[0034] As used herein, strains have “the same or similar functionality” if they are both capable of improving plant growth and / or increasing plant yield when applied to a plant, a plant part, or a growth medium in which said plant is located. In some embodiments, strains with the same or similar functionality share functional and / or genetic characteristics, such as the presence of particular genetic markers, proteins, cell wall polysaccharides, or enzymatic capabilities. These characteristics may be used to select for the microbial strains.

[0035] As used herein, a “derivative” or “variant” of an isolated microbial strain is meant to mean a microbial strain (or population thereof) which is derived from the isolated microbial strain, but with one or more changes on the provision that these do not abrogate the strain’s capability for improving plant growth and / or increasing plant yield when applied to a plant, a plant part, or a growth medium in which said plant is located. Derivatives or variants may comprise a number of mutations (insertions, deletions, and / or substitutions) at one or more positions within the genome. Derivatives or variants may include additional genes, for example on plasmids or that have been inserted into the bacterial chromosome.

[0036] As used herein, the “progeny” of an isolated microbial strain is meant to mean a microbial strain (or population thereof) which is descended from a parental isolated microbial strain, which shares functional and / or genetic characteristics with the parental strain. Progeny include mutants, whether natural or induced, genetically modified microbes, or gene edited microbes. Progeny may be produced by culturing the isolated microbial strain or its progeny, including repeatedly culturing, optionally further including functional or genetic selection of progeny with the same functional or genetic characteristics as the parental isolated microbial strain. Progeny may undergo genetic modification and / or gene editing.

[0037] In some embodiments, an isolated microbial strain of the invention may be characterised by comparison of the 16S rRNA gene sequence to that of a reference microbial strain, for example the strain deposited as Accession Number 24050701 at the NCTC Patent Depository (LIKHSA) and having a 16S rRNA gene sequence SEQ ID NO:3, or the progeny thereof.

[0038] The prokaryotic 16S rRNA gene is approximately 1500 bp long, with nine variable regions interspersed between conserved regions. Variable regions of the 16S rRNA gene are frequently used for phylogenetic classification of genus or species in diverse microbial populations (Weisburg WG, Barns SM, Pelletier DA, Lane DJ. 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol. 1991 ;173(2):697-703).

[0039] In some embodiments, an isolated microbial strain has a 16S rRNA gene sequence which has sequence similarity or sequence identity with SEQ ID NO:3. In some embodiments, the 16S rRNA gene sequence has at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 96%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% sequence identity or sequence similarity with SEQ ID NO:3.

[0040] 16S rRNA may also be used to characterise strains through DNA reassociation kinetics. In some embodiments, an isolated microbial strain has a 16S rRNA gene sequence which hybridises with SEQ ID NO:3. The skilled person will be familiar with these techniques, for example as described in Janda and Abbot, J Clin Microbiol. 2007 Sep; 45(9): 2761-2764. In some embodiments, the 16S rRNA gene sequence exhibits at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% DNA-DNA relatedness in reciprocal hybridisation reactions with SEQ ID NO:3. Additionally or alternatively, in some embodiments, the isolated microbial strain has a 16S rRNA gene sequence which forms a homoduplex with 5°C or less ATm when compared to a heteroduplex formed between it and SEQ ID NO:3. In some embodiments, ATm is 4°C or less, 3°C or less, 2°C or less, or 1°C or less. In some embodiments, the 16S rRNA gene sequence exhibits at least 70% DNA-DNA relatedness in reciprocal hybridisation reactions with SEQ ID NO:3 and forms a homoduplex with 5°C or less ATm when compared to a heteroduplex formed between it and SEQ ID NO:3. In some embodiments, an isolated microbial strain of the invention may be characterised by comparison of its genome sequence to that of a reference microbial strain, for example the strain deposited as Accession Number 24050701 at the NCTC Patent Depository (LIKHSA) at the NCTC Patent Depository (LIKHSA) and having a genome sequence according to SEQ ID NO:1 , or the progeny thereof.

[0041] In some embodiments, the isolated microbial strain of the invention is characterised by genome sequence homology or identity with SEQ ID NO:1. In some embodiments, the isolated microbial strain of the invention has a genome with at least 70%, at least 75%, at least 80%, at least 85% or at least 90% genome homology or identity with SEQ ID NO:1 , in particular at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.5% genome homology or identity with SEQ ID NO:1.

[0042] By “sequence identity” or “sequence homology” we mean the identical sequence of base pairs in the specific DNA or protein region. For example, in a sequence that has 96% sequence homology or sequence identity to a reference sequence, 95% of the base pairs or amino acids are identical. The percent sequence identity between two polypeptides may be determined using suitable computer programs, for example the GAP program of the University of Wisconsin Genetic Computing Group and it will be appreciated that percent identity is calculated in relation to polypeptides whose sequence has been aligned optimally. The alignment may alternatively be carried out using the Clustal W program (Thompson et al., (1994) Nucleic Acids Res 22, 4673-80). The parameters used may be as follows: Fast pairwise alignment parameters: K-tuple(word) size; 1 , window size; 5, gap penalty; 3, number of top diagonals; 5. Scoring method: x percent. Multiple alignment parameters: gap open penalty; 10, gap extension penalty; 0.05. Scoring matrix: BLOSUM. When determining the sequence identity of multiple sequences compared to a reference, multiple sequence alignment tools can be used, for example the Clustal Omega program which can align up to 4000 sequences at a time (Sievers et al., (2011) Mol. Syst. Biol. 7:539).

[0043] Sequence identity may be calculated through average nucleotide identity (ANI). In some embodiments, the isolated microbial strain of the invention is characterised through average nucleotide identity (ANI) with SEQ ID NO:1.

[0044] ANI analysis is a computational method used to compare the genetic similarity between two bacterial genomes. It works by comparing the nucleotide sequences of orthologous genes or genetic sequences between two bacterial strains. The skilled person may perform ANI analysis through any known method in the art. Regardless of technique, ANI has the following broad steps for the identification of strains of the invention:

[0045] 1. Genome Comparison: ANI analysis starts with the comparison of the entire genomes of two bacterial strains or species. One of these genomes belongs to a reference bacterial strain of the invention, for example as represented by SEQ ID NO:1. Comparison involves aligning the nucleotide sequences of all genes in both genomes to identify orthologous regions.

[0046] 2. Orthologous Gene Identification: Once the genomes are aligned, orthologous regions are identified. These likely encode genetic features that have similar functions and are present in both genomes, although they may have evolved slightly differently due to mutations and genetic drift.

[0047] 3. Calculation of Average Nucleotide Identity: After identifying orthologous regions, ANI calculates the average percentage identity of nucleotides between these orthologous regions in the two genomes, e.g. through use of BLAST (Basic Local Alignment Search Tool) or MUMmer (Maximal Unique Matches).

[0048] 4. Threshold for Strain Identification: A threshold value is set to distinguish between strains. Bacteria with ANI values above this threshold are considered to belong to the same species, while those below the threshold are considered to be of different species.

[0049] 5. Interpretation: If the ANI value between two bacterial genomes is above the threshold, they belong to the same strain. Conversely, if the ANI value is below the threshold, it indicates that the genomes are from different strains or species.

[0050] In some embodiments, the threshold is at least 70%, at least 75%, at least 80%, at least 85% or at least 90%, in particular wherein the threshold is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.5%, such that the isolated microbial strain of the invention has a genome with at least 70%, at least 75%, at least 80%, at least 85% or at least 90%, in particular at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.5% genome homology or identity with SEQ ID NO:1 , as determined by average nucleotide identity (ANI) analysis.

[0051] In some embodiments, the strain is not a nitrogen-fixing bacterium. The present compositions and methods are applicable to any suitable plants. These may be plants grown ornamental, comestible, textile, constructional, or any other purposes.

[0052] In some embodiments, the plant is a crop plant. As used herein, a “crop plant” is one cultivated primarily for human consumption as food.

[0053] As used herein a “plant part” includes a leaf, a stem, a fruit, a root, a tuber, a seed, a grain, an embryo, or a part thereof. Plant parts include isolated cells, and tissue culture samples.

[0054] In some embodiments, the plant is a leaf crop. As used herein, “leaf crops” are any plants cultivated for human consumption where the leaves are a, the primary, or the only article of consumption. They may be distinguished from “root crops”, where swollen roots or tubers are eaten, and fruiting or grain crops, where it is the fruiting bodies or seeds that are eaten. Leaf crops include lettuce (Lactuca sativa), spinach (Spinacia oleracea), chard (Beta vulgaris) endive (Cichorium spp, for example C. intybus and / or C. endive, especially C. endive var. crispum), Brassica spp. (in particular B. oleracea var. and B. rapa), basil (Ocimum basilicum).

[0055] Lettuce is particularly contemplated as benefitting from treatment with the compositions of the invention. In some embodiments, a lettuce is of a specific type selected from babyleaf, multileaf, oakleaf, iceberg, cos, little gem, loose leaf, or butterhead. Exemplary varieties of lettuce include Skilton, Nunhems 09185 LTL, Nunhems Nitaflash.

[0056] Tomatoes are further particularly contemplated for use with the invention. As tomato plants are widely grown in hydroponic settings, they are particularly contemplated for use with the methods and compositions of the invention when grown hydroponically, but are equally suited when grown using in-soil cultivation. In some embodiments, a tomato may be of or related to the Balconi F1 variety, such as exemplified herein.

[0057] In some embodiments, the plant is a fruiting crop, i.e. one where the fruiting bodies or seeds are the primary articles of human consumption for which the plant is cultivated. Exemplary fruiting crops include tomato (Solanum lycopersicum), cucurbits such as cucumber (Cucumis sativus) and melon (Cucumis species, particularly C. melo and its subspecies), and capsicum species (C. annuum, including bell or “sweet” peppers and chilli peppers, as well as C. chinense, and C. frutescens). In particular, hybrid plants may also benefit from the composition of the invention, for example, strawberry (Fragaria x ananassa).

[0058] The skilled person would understand that vine crops, which include vegetables such as cucurbits may also benefit from the invention.

[0059] Cucumbers are also contemplated to benefit from treatment with the compositions of the invention. In some embodiments, a cucumber may be of or related to the Quatro (Rijk Zwann) variety, such as exemplified herein.

[0060] In some embodiments, the plant is a root vegetable, i.e. one where the plant parts for human consumption are typically grown underground. The term covers true roots and nonroots, for example bulbs, corms, rhizomes and stem tubers. Exemplary root vegetables include carrots (Daucus carota subsp. sativus), turnips (Brassica rapa subsp. rapa), celeriac (Apium graveolens (Celeriac group)), kolrabi (Brassica oleracea (Gongylodes group)), beetroot (Beta vulgaris subsp. vulgaris), parsnips (Pastinaca sativa), and rutabaga (Brassica napus (Napobrassica group)).

[0061] In some embodiments, the plant is the source of a medicinal or recreational drug. In particular, in some embodiments, the plant is a Cannabis plant, optionally a Cannabis plant selected from a Cannabis sativa, Cannabis indica, and / or Cannabis ruderalis plant.

[0062] The compositions containing the bacteria of the present invention are effective in improving plant growth and increasing plant yield.

[0063] As used herein, “improving plant growth” means an improvement in one or more growth characteristic, compared to a control plant. Improvements may be at a particular comparison date. Improvements in plant growth may be measured through an increase in one or more characteristic selected from:

[0064] • Mass (weight) of plant;

[0065] • Total leaf number, area, and / or weight;

[0066] • Average leaf area or weight;

[0067] • Total or average leaf protein content;

[0068] • Height of aerial parts of plant;

[0069] • Length or weight of roots; Survival of plant, in particular average rate of survival of a population of plants.

[0070] As used herein, “increasing plant yield” means an improvement in one or more desirable characteristic of a harvested plant part or product, for example a leaf, root, tuber, or fruit. These may accompany improved growth or increased production of a non-harvested plant part or product, as increased leaf area and growth rate will result in increased photosynthetic capacity, and therefore increased ability to produce harvestable parts of interest such as fruits. Harvested plant parts or products may be comestible products.

[0071] Increases in desirable characteristics of harvested plant parts or products include:

[0072] • T otal or average number of harvested plant parts or product per plant, or per square metre used to grow plants.

[0073] • Total or average weight of harvested plant parts or product per plant, or per square metre used to grow plants.

[0074] • Total or average time required to produce harvestable plant parts or products, e.g. time from sowing or transplant to harvest.

[0075] • Total or average time required to produce a given quantity of harvested plant parts or products, e.g. time from sowing or transplant to harvest.

[0076] • Total or average time required to produce a given weight of harvested plant parts or products, e.g. time from sowing or transplant to harvest.

[0077] • Total or average quantity and / or weight of plant parts, or products produced per plant in a given time period (e.g. 1 , 2, 3, 4, 5, or 6 months from sowing or transplant)

[0078] • Total or average quantity and / or weight of plant parts, or products produced per square metre in a given time period (e.g. 1 , 2, 3, 4, 5, or 6 months from sowing or transplant)

[0079] • Total or average protein content of harvested plant parts or product per plant, or per square metre used to grow plants.

[0080] • Total or average sugar content of harvested plant parts or product per plant, or per square metre used to grow plants.

[0081] • Total or average content of a molecule of interest (for example, a plant secondary metabolite) per plant, or per square metre used to grow plants.

[0082] • Total or average quality score of harvested plant part or product per plant, or per square metre used to grow plants, as measured by any industry standard technique.

[0083] • Total or average yield (number of harvested plants). • Total or average germination rate (e.g. successful germination per 100 seeds).

[0084] • Total or average rate of survival to first, second, or third true leaf / pair stage (e.g. per 100 seeds).

[0085] • Shelf life of harvested plants, part plants, or products.

[0086] • Homogeneity of appearance (e.g. as measured through any known horticultural or agricultural grading methods)

[0087] Improvements and increases in a characteristic are measured relative to a control plant. “Control plants” are plants of the same species and variety which differ only in that they were not contacted with the microbial strains and compositions of the invention.

[0088] Improvements and increases in a characteristic may be made at a particular comparison date. Comparison dates may be tied to an agronomically important event - for example, first true leaf stage, second true leaf stage, transplant, first harvest, or final harvest. Comparison dates may be relative to the date the plant was sown. For example, the comparison date may be 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 38, 39, 40, 50, 60, 70, 80, 90, or 100 days or more from the date the plant was sown. Alternatively, the comparison date may be 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, or 26 weeks from the date the plant was sown. The comparison date may be 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , or 12 months or more from the date the plant was sown.

[0089] Comparison dates may be relative to the date the plants were first contacted with the with the microbial strains and compositions of the invention. For example, the comparison date may be 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, or 100 days or more from the date the plants were first contacted with the with the microbial strains and compositions of the invention. Alternatively, the comparison date may be 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, or 26 weeks or more from the date the plants were first contacted with the with the microbial strains and compositions of the invention. The comparison date may be 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , or 12 months or more from the date the plants were first contacted with the with the microbial strains and compositions of the invention.

[0090] Improvements and increases may be quantitatively assessed. In an agricultural setting, even increases in yield of a fraction of a percentage point can be commercially useful, especially where capital costs are high as in hydroponic growing. In some embodiments, an improvement or increase in a characteristic may be by at least 0.5%, 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or more, relative to a control plant or population thereof.

[0091] The isolated microbial strains can be used to generate multiple compositions, including cultures, agricultural compositions, cell-free preparations, and preparations comprising metabolites.

[0092] As used herein, a “culture” refers to a composition produced by culturing an isolated microbial strain in or on an acceptable culture media. Culture media may include nutrients, additives, growth regulators, or other agents required by the microbe for growth. Culture media may include one or more antibiotic and / or antifungal tolerated by the microbes of the invention. Media may be liquid, a film, or a gel, e.g. an agar gel. Cultures may also include one or more further microbial strains, in which case they may be referred to as “cocultures”. Cultures may be formed by combining the microbial strain with a culture media, and incubating under conditions that allow the bacteria to proliferate.

[0093] Cultures may be processed to remove cells, e.g. through cell lysis and / or filtration, to produce a cell-free preparation. Cell-free preparations may be further processed to isolate one or more metabolites, for example through chromatography. Alternatively, cultures may be processed to inactivate cells, e.g. through heating or treating with an agent which kills the microbes such as an antibiotic.

[0094] Cultures and strains of the invention may be used to generate agricultural compositions. As used herein, an “agricultural composition” comprises an effective amount of the microbes or culture of the invention, and an agriculturally acceptable carrier (i.e. a carrier which is non-toxic to plants and / or humans). Exemplary carriers include water, such as distilled water, oils (in particular vegetable or other food-safe oils), and inorganic carriers (e.g. clay, kaolin, sand, perlite, vermiculite, bentonite, or biochar). Agricultural compositions may further include binders (e.g. adhesive compounds), fillers, bulking agent), flow regulators, anti-flocculants, surfactants.

[0095] As used herein, an “effective amount” of the microbes or culture of the invention is the quantity required to achieve an improvement or increase in a characteristic. In some embodiments, an agricultural composition is formulated so as to deliver an effective dose when applied to a plant or plant part.

[0096] Effective dose may therefore be measured in colony former units (CFUs) of the microbes or culture of the invention per unit dose (e.g. where a dose is 0.5 mL).

[0097] In some embodiments, an effective dose delivers at least 1 x 10A8, at least 1.5 x 10A8, at least 2 x 10A8, at least 2.1 x 10A8, at least 2.2 x 10A8, at least 2.3 x 10A8, at least 2.4 x 10A8, at least 2.5 x 10A8, at least 2.6 x 10A8, at least 2.7 x 10A8, at least 2.8 x 10A8, at least 2.9 x 10A8, at least 3 x 10A8, at least 3.1 x 10A8, at least 3.2 x 10A8, at least 3.3 x 10A8, at least 3.4 x 10A8, at least 3.5 x 10A8, at least 3.6 x 10A8, at least 3.7 x 10A8, at least 3.8 x 10A8, at least 3.9 x 10A8, at least 4 x 10A8, at least 4.1 x 10A8, at least 4.2 x 10A8, at least 4.3 x 10A8, at least 4.4 x 10A8, at least 4.5 x 10A8, at least 4.6 x 10A8, at least 4.7 x 10A8, at least 4.8 x 10A8, at least 4.9 x 10A8, or at least 5 x 10A8 CFU per unit dose. In some embodiments, an effective dose delivers at least about 3 x 10A8 CFU per unit dose, such as at least 3.1 x 10A8 CFU per unit dose.

[0098] In some embodiments, an effective dose delivers at least 3 x 10A8, at least 3.01 x 10A8, at least 3.02 x 10A8, at least 3.03 x 10A8, at least 3.04 x 10A8, at least 3.05 x 10A8, at least 3.06 x 10A8, at least 3.07 x 10A8, at least 3.08 x 10A8, at least 3.09 x 10A8, at least 3.1 x 10A8, at least 3.11 x 10A8, at least 3.12 x 10A8, at least 3.13 x 10A8, at least 3.14 x 10A8, at least 3.15 x 10A8, at least 3.16 x 10A8, at least 3.17 x 10A8, at least 3.18 x 10A8, at least 3.19 x 10A8, or at least 3.2 x 10A8 CFU per unit dose. In some embodiments, an effective dose delivers at least 3.16 x 10A8 CFU per unit dose.

[0099] In some embodiments, the effective dose per plant delivers at least 1 x 10A5, at least 2 x 10A5, at least 3 x 10A5, at least 4 x 10A5, at least 5 x 10A5, at least 6 x 10A5, at least 7 x

[0100] 10A5, at least 8 x 10A5, at least 9 x 10A5, at least 1 x 10A6, at least 2 x 10A6, at least 3 x

[0101] 10A6, at least 4 x 10A6, at least 5 x 10A6, at least 6 x 10A6, at least 7 x 10A6, at least 8 x

[0102] 10A6, at least 9 x 10A6, at least 1 x 10A7, at least 2 x 10A7, at least 3 x 10A7, at least 4 x

[0103] 10A7, at least 5 x 10A7, at least 6 x 10A7, at least 7 x 10A7, at least 8 x 10A7, at least 9 x

[0104] 10A7, at least 1 x 10A8, at least 2 x 10A8, at least 3 x 10A8, at least 4 x 10A8, at least 5 x

[0105] 10A8, at least 6 x 10A8, at least 7 x 10A8, at least 8 x 10A8, or at least 9 x 10A8 CFU per unit dose.

[0106] In some instances, the effective dose per plant may be adapted depending on the crop and the system. For example, an effective dose for a vine crop may be between 1 x 10A8 and 5 x 10A8 CFU per unit dose. Suitably, an effective dose per plant for a vine crop may be about 3 x 10A8 CFU per unit dose, such as 3.16 x 10A8 CFU per unit dose. A typical leafy green crop may require a reduced dose compared to a vine crop, for example, between 1 x 10A7 and 5 x 10A7 CFU per unit dose. Suitably, an effective dose for a leafy green crop may be about 3 x 10A7 CFU per unit dose, such as 3.16 x 10A7 CFU per unit dose.

[0107] The skilled person would be aware that the unit dose may be further reduced to a minimum effective dose. As described above, this dose would depend on the crop type and the system. For example, in some embodiments, the minimum effective dose may be between 3 x 10A5 and 4 x 10A5 CFU per unit dose. In one particular embodiment, the minimum effective dose may be about 3 x 10A5 CFU per unit dose, such as 3.16 x 10A5 CFU per unit dose.

[0108] Effective dose may alternatively be measured in CFU per mL. In some embodiments, an agricultural composition containing an effective dose of the microbes or culture contains at least 4 x 10A8, at least 4.1 x 10A8, at least 4.2 x 10A8, at least 4.3 x 10A8, at least 4.4 x 10A8, at least 4.5 x 10A8, at least 4.6 x 10A8, at least 4.7 x 10A8, at least 4.8 x 10A8, at least 4.9 x 10A8, at least 5 x 10A8, at least 5.1 x 10A8, at least 5.2 x 10A8, at least 5.3 x 10A8, at least 5.4 x 10A8, at least 5.5 x 10A8, at least 5.6 x 10A8, at least 5.7 x 10A8, at least 5.8 x 10A8, at least 5.9 x 10A8, at least 6 x 10A8, at least 6.1 x 10A8, at least 6.2 x 10A8, at least 6.3 x 10A8, at least 6.4 x 10A8, at least 6.5 x 10A8, at least 6.6 x 10A8, at least 6.7 x 10A8, at least 6.8 x 10A8, at least 6.9 x 10A8, at least 7 x 10A8, at least 7.1 x 10A8, at least 7.2 x 10A8, at least 7.3 x 10A8, at least 7.4 x 10A8, at least 7.5 x 10A8, at least 7.6 x 10A8, at least 7.7 x 10A8, at least 7.8 x 10A8, at least 7.9 x 10A8, or at least 8 x 10A8 CFU per mL.

[0109] In some embodiments, an agricultural composition contains at least 6.2 x 10A8, at least 6.21 x 10A8, at least 6.22 x 10A8, at least 6.23 x 10A8, at least 6.24 x 10A8, at least 6.25 x 10A8, at least 6.26 x 10A8, at least 6.27 x 10A8, at least 6.28 x 10A8, at least 6.29 x 10A8, at least 6.3 x 10A8, at least 6.31 x 10A8, at least 6.32 x 10A8, at least 6.33 x 10A8, at least 6.34 x 10A8, at least 6.35 x 10A8, at least 6.36 x 10A8, at least 6.37 x 10A8, at least 6.38 x 10A8, at least 6.39 x 10A8, or at least 6.40 x 10A8 CFU per mL.

[0110] In some embodiments, an agricultural composition contains at least 6.2 x 10A8 CFU per mL. In some embodiments, an agricultural composition contains at least 6.3 x 10A8 CFU per mL. In some embodiments, an agricultural composition contains at least 6.32 x 10A8 CFU per mL.

[0111] In some embodiments, an agricultural composition contains between 4 x 10A8 and 8 x 10A8 CFU per mL. In some embodiments, an agricultural composition contains between 5 x 10A8 and 7 x 10A8 CFU per mL. In some embodiments, an agricultural composition contains between 5.5 x 10A8 and 6.5 x 10A8 CFU per mL. In some embodiments, an agricultural composition contains between 6 x 10A8 and 6.5 x 10A8 CFU per mL.

[0112] In some embodiments, the agricultural composition comprises one or more additional components which promote plant growth. For example, an agricultural composition may comprise additional microbial (i.e. bacterial, fungal, or archaea) strains of interest that are capable of increasing plant growth and / or improving plant yield. Alternatively or additionally, an agricultural composition may comprise a fertiliser, a plant nutrient composition (comprising one or more plant macronutrient and / or plant micronutrient), a herbicide (especially a selective herbicide), a pesticide, a fungicide, a bactericide, an antiviral a plant hormone, and / or a plant growth regulator.

[0113] Agricultural compositions may be formulated for any appropriate mode of application to a plant or plant part, or to a plant growth substrate. For example, agricultural compositions may be formulated as a seed coating, as a fertilizer additive, a spray (for example, a foliar spray, a soil spray, a seed spray, or a substrate spray), a drench, a hydroponic nutrient solution, and combinations thereof. Agricultural compositions may be formulated as concentrates or diluted “working stocks”.

[0114] The agricultural compositions may also be formulated in the form of a powder. Powders may be prepared using suitable methods such as lyophilization (freeze-drying) or spray drying. The skilled person would be aware that microbial preparations in the form of powders may be advantageous over liquid preparation, for example, powder preparation may have an extended shelf-life.

[0115] Agricultural compositions may be applied to plants at any point during growth, including to ungerminated seeds, to germinated seeds, to cotyledon stage seedlings, to seedlings at transplant stage, and to mature plants.

[0116] In some embodiments, agricultural compositions are applied to plants multiple times. For example, agricultural compositions may be applied to seeds at sowing and subsequently re-applied one or more times to growing plants. Compositions may be applied 1 , 2, 3, 4 or more times. Applications may be independently separated by 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or more days.

[0117] The strains and compositions herein can be applied to plants in any growth context.

[0118] Plants herein may be grown in soil. “Soil”, as used herein, is meant to refer to any conventional aggregate-based plant growth substrate, including topsoil, compost, sand, loam, peat, coconut coir, and combinations thereof. This encompasses growth in open ground or in containers. Where plants are grown in soil, it is particularly contemplated that agricultural compositions are applied in the form of a drench or a spray, applied directly to the plants and / or the soil. Agricultural compositions may also be applied to a plant directly, for example, using a syringe or pipette. The inventors have found the latter method to be particularly effective in the dosing of vine crops.

[0119] Additionally, plants herein can be grown hydroponically, in a hydroponics system. As described herein, hydroponics means the cultivation of plants in a hydroponic nutrient solution, with or without the mechanical support of a substrate. This method eliminates the need for traditional soil, allowing for precise control over plant nutrition and growth conditions. Plants may be grown in hydroponic systems for all or only some of their lifespan (as with some trees and rice). In some embodiments, the hydroponics system is a deepwater culture (DWC) system, an aeroponics system, a drip system, a wick system, an ebb and flow system, a nutrient film technique (NFT) system, a static system, or an aquaponics system.

[0120] In some embodiments, agricultural compositions are applied to the substrate in a hydroponics system. Suitable substrates for use with hydroponics include inorganic substrates such as mineral wool (e.g. rockwool), expanded clay aggregate, mineral particulates, gravel, sand, or perlite, or a combination thereof. Alternatively, the substrate may comprise an organic, inert and / or biodegradable substrate such as GrowFoam®, Nygaia ®, coconut coir, or peat. Substrates may further comprise hydrogels. The substrate may comprise a blend of multiple organic or inorganic substrates described above. In some embodiments, agricultural compositions are combined with the hydroponic nutrient solution. In some embodiments, the substrate may comprise a biodegradable plug such as GrowCoon. In some embodiments, the agricultural composition is applied to a plant seed. Beneficially, this allows the microbe to contact the plant as early as possible, and throughout, its growth cycle. The seed may be ungerminated and / or have germinated. In some embodiments, the agricultural composition is applied to a plant seed as a seed coating. In such an embodiment, the seed may undergo a drying step to remove water or another carrier from the agricultural composition to produce a viable and shelf-stable seed covered with a coating comprising the microbial composition (e.g. as a pellet, or crust- or film-coated seed). The seed coating may further comprise a carrier, a wax, a gel, an adhesive, or a soluble compound, so as to hold the microbes in contact with the seed until planting.

[0121] Agricultural compositions and bacteria in contact with plants may form a synthetic combination. As used herein, a “synthetic combination” refers to one which does not otherwise exist in nature. The skilled person will readily understand that this is distinctive, for example by the amount of microbes present, and may be distinguished from de minimis levels of the microbes present in combinations which may or may not exist in nature.

[0122] Also disclosed are metabolites produced by isolated microbial strains and cultures described herein. These metabolites may be isolated from microbial strains as discussed above. Metabolites may be present with the microbial cells, in a crude lysate, or in a substantially purified form.

[0123] Also disclosed is a method of applying a probiotic microbe to a plant by spraying an agricultural composition comprising the probiotic microbe. The method may be a method of improving plant growth and / or increasing plant yield by applying the probiotic microbe in this manner. This method is suitable with all probiotic microbes, including those described herein. Surprisingly, the inventors discovered that application through spray was at least as effective as traditional pipetting or drench-based approaches, and indeed even more effective, with increased ease of handling, especially in a hydroponic context. This is avoided because hydroponic growers often avoid spraying any liquid (beside water without additives) to their crops, since ‘no spray’ claims are often seen as desirable by consumers.

[0124] Although the microbes of the invention are particularly contemplated, any plant growth enhancing microbes are applicable for use with spray delivery. Combinations of microbes, including combinations of different strains or species of bacteria, fungi, and / or archaea may be used. Agricultural compositions formulated for spraying may further comprise additional agents (carriers, surfactants, binders, fillers, glow regulators, anti-flocculants, fertilisers, herbicides, pesticides, plant hormones and / or plant growth regulators) as described herein. Spray delivery is suitable for applying agricultural formulations to plants, plant parts, or a growth media in which plants are located, as described herein.

[0125] As used herein “average” may refer to mean or median average.

[0126] For convenience, the meaning of certain terms and phrases used in the specification, examples, and appended claims, are provided herein. If there is an apparent discrepancy between the usage of a term in other parts of this specification and its definition provided in this section, the definition in this section shall prevail.

[0127] The term “about” when referring to a number or a numerical range means that the number or numerical range referred to is an approximation within experimental variability (or within statistical experimental error), and thus the number or numerical range may vary from, for example, between 1% and 15% of the stated number or numerical range.

[0128] The term “at least” prior to a number or series of numbers is understood to include the number adjacent to the term “at least”, and all subsequent numbers or integers that could logically be included, as clear from context. When at least is present before a series of numbers or a range, it is understood that “at least” can modify each of the numbers in the series or range.

[0129] As used herein, “no more than” or “less than” is understood as the value adjacent to the phrase and logical lower values or integers, as logical from context, to zero. When “no more than” is present before a series of numbers or a range, it is understood that “no more than” can modify each of the numbers in the series or range.

[0130] As used herein, “up to” as in “up to 10” is understood as up to and including 10, i.e., 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0131] Ranges provided herein are understood to include all individual integer values and all subranges within the ranges.

[0132] The present disclosure includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided. Throughout this specification, including the claims which follow, unless the context requires otherwise, the word ‘comprise,’ and variations such as ‘comprises’ and ‘comprising,’ will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0133] It must be noted that, as used in the specification and the appended claims, the singular forms ‘a,’ ‘an,’ and ‘the’ include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from ‘about’ one particular value, and / or to ‘about’ another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent ‘about,’ it will be understood that the particular value forms another embodiment.

[0134] Examples embodying certain aspects of the invention shall now be described, with reference to the following figures. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.

[0135] Examples

[0136] Example 1 - Isolation and characterisation of strain “Pantoea’

[0137] The microbial strain given the internal designation Pantoea was isolated by Concert Bio UK from hydroponic substrate.

[0138] A partial 16S rRNA gene sequence was obtained using Sanger sequencing by the third- party services of Genewiz (Azenta). Analysis was done using the Basic Local Alignment Search Tool (BLAST) against the NCBI 16S rRNA gene database identified ‘Pantoea agglomerans’ as the closest match.

[0139] Genomic DNA was extracted using the QIAGEN DNeasy Blood & Tissue Kit, following the manufacturer's instructions. Sequencing libraries were prepared using the Oxford Nanopore Technologies (ONT) Native Barcoding Kit 24 V14 (SQK-NBD114.24). Whole genome sequencing was performed on ONT MinlON devices equipped with R10.4.1 flow cells, with supervised base calling enabled. The sequence data was analysed using Genome Taxonomy Database toolkit (GTDB-Tk), as described in Chaumeil et al 2020 and 2022, to assign the taxonomy of bacterial genomes against the Genome Taxonomy Database (GTDB). Average nucleotide identity (ANI) was calculated and compared, using FASTANI, across whole genomes within the database, using a 95% identity cut-off , as described in Ciufo et al 2018 and Jain et al 2018.

[0140] The microbial strain was identified as a strain of Pantoea septica.

[0141] The full genome sequence is provided as SEQ ID NO:1.

[0142] The genome sequence was annotated using bakta which is described in Schwengers et al, 2021. The 16S rRNA gene sequence is provided as SEQ ID NO:3 (the rRNA transcript is provided as SEQ ID NO:2).

[0143] Example 2 - Proof of concept trial

[0144] Analysis of Pantoea ability to promote plant growth was performed at ExeterConcert Bio’s plant research facility in Penryn, UK.

[0145] Lettuce plants were grown for seven days in a hydroponic nursery set-up before transplanting into the main DWG hydroponic growth system. The commercial lettuce variety used for the experiment was Skilton (Enza Zaden). All plants were grown in inert, foam substrate.

[0146] The microbial treatments were prepared using only the microbe itself suspended in sterile water. The treatments were then applied by directly pipetting 0.5 mL of the treatment onto the seed after sowing.

[0147] 3.16 x 10A8 CFU of microbe was applied per application of treatment.

[0148] The treatments were reapplied to the seedling using the same method 3-4 days after sowing when the plant began to emerge from the seed coat

[0149] The treatments were also reapplied to the plants using the same method 1 week after sowing when the plants were transplanted from the nursery into the growth system All plants were harvested and weighed 28 days after transplanting into the growth system. As is standard in the field for assessing yield, fresh head weight is used throughout.

[0150] Plants that died during the experiment, or plants that were small enough to be deemed non-viable, were excluded from the analysis and in graphical representations.

[0151] The results of the experiment are provided in Figure 1 and Table 1 below:

[0152] Table 1 - Results of proof of concept trial

[0153] This data shows that Pantoea treatment increases both median and mean head weight by a commercially useful amount in a proof-of-concept trial.

[0154] Example 3 - Further internal trial

[0155] A further trial was performed to examine different application methods for the agricultural compositions.

[0156] Using the same plant variety, growth system, and general experimental protocol as in Example 2, plants were grown for seven days in a hydroponic nursery set-up before transplanting into the main hydroponic growth system. Plants were grown in inert foam substrate as outlined above.

[0157] The microbial treatments were prepared using only the microbe itself suspended in sterile water. Two different application methods of the treatment were used for this trial:

[0158] • The first method was the same as Example 2, by directly pipetting 0.5 mL of the treatment.

[0159] • The second method involved spraying the treatment. For both application methods, the treatment was first applied to the seed after sowing.

[0160] The treatments were reapplied to the seedling using the same method 3-4 days after sowing when the plant began to emerge from the seed coat. The treatments were also reapplied to the plants using the same method one week after sowing when the plants were transplanted from the nursery into the growth system.

[0161] All plants were harvested and weighed 28 days after transplanting into the growth system, and fresh head weight is used throughout.

[0162] The dosage applied was 3.16 x 10A8 CFUs per plant.

[0163] The results of the experiment are provided in Figure 2 and Table 2 below:

[0164] Table 2 - Results of further internal trial

[0165] This data shows that Pantoea treatment increases both median and mean head weight by a commercially useful amount when delivered by pipette or by spray.

[0166] Example 4 - Commercial trial

[0167] This experiment was carried out at the R&D greenhouse facility of a UK-based hydroponic lettuce producer. The commercial lettuce variety used for the experiment was Nunhems 09185 LTL All plants were grown across two hydroponic, deep water culture ponds for 57 days before being harvested and weighed. All plants were grown in peat substrate.

[0168] Seeds were sown at a higher density and grown to maturity for 17 days before being transplanted into a lower density growth system. Both seedlings and mature plants were grown on the same ponds

[0169] The microbial treatments were prepared using only the microbe itself suspended in sterile water. The treatments were then applied by directly pipetting 0.5 mL of the treatment onto the seed after sowing. The treatments were reapplied to the seedling through pipetting at the same concentration 17 days after sowing

[0170] Fresh head weight is used throughout and was independently recorded by the grower

[0171] The dosage applied was 3.16 x 10A8 CFUs per plant.

[0172] The results of the experiment are provided in Figure 3 and Table 3 below:

[0173] Table 3 - Results of commercial-scale trial

[0174] This data confirms that Pantoea treatment increases both median and mean head weight by a commercially useful margin when grown in a commercial setting.

[0175] This data further demonstrates that the Pantoea treatment group showed consistent increases in head weight, including fewer <100g heads (see Figure 3), and this effect is visible in the 20% increase in mean head weight. This is a desirable characteristic for growers, demonstrating a reliable and consistent increase in growth.

[0176] Example 5 - Commercial trial - Ebb and Flow culture This experiment was carried out at the R&D facility of a UK-based lettuce vertical farm. The commercial lettuce variety used for the experiment was Nunhems Nitaflash. All plants were grown for 21 days in an Ebb and Flow hydroponic system, on inert substrate. The microbial treatment was prepared using only the microbe itself suspended in sterile water and applied by spraying directly onto the seeds.

[0177] Fresh head weight is used throughout (as relative yield) and was independently recorded by the grower.

[0178] The dosage applied was 3.16 x 10A8 CFUs per plant.

[0179] The results can be seen in Figure 4. Pantoea treated plants exhibited a commerciallyrelevant 6.7% increase in yield.

[0180] Example 6 - Internal Trial - Cucumbers

[0181] This experiment was carried out in an ETFE polytunnel located at the Wellesbourne campus at the University of Warwick. The commercial cucumber variety used was Quatro (Rijk Zwaan). Each treatment had its own set of control plants which were interspersed with the treated plants. A total of 104 plants in each treatment group were used.

[0182] Seeds were sprayed with “Pantoea” treatment at a plant propagator (Delfland Nurseries). Shortly after the crop was delivered to the polytunnels, the plants were treated again with “Pantoea” in the polytunnels. Plants were grown in drip irrigation slab culture on rockwool using a highwire growing protocol. Cucumbers were continually harvested and weight and number recorded throughout the growth cycle.

[0183] The dosage applied was 6.32 x 10A8 CFUs per plant.

[0184] The results can be seen in Figures 5A and 5B, and Tables 4 and 5 below. Treatment with “Pantoea” increased the total yield of cucumbers by 5.65% and the total harvested weight of cucumbers by 5.67% compared to control plants.

[0185] Table 4 - Results of cucumber trial - total number of cucumbers produced

[0186] Table 5 - Results of cucumber trial - total weight of cucumbers produced

[0187] Example 7 - Commercial Trial - Testing Pantoea treatment with GrowCoon as a substrate

[0188] This experiment was carried out in the container lab of a vertical farm in Austria. The commercial lettuce variety used for the experiment was Lalique (Rijk Zwann). All plants were sown at higher density initially in two boxes. Growcoon was selected as the substrate. Seeds were transplanted twice during the growth cycle. A single seed per plug was sown.

[0189] Pantoea treatment was prepared by suspending the microbe itself in sterile water. The treatment was then applied by spraying the microbe onto the seeds at two time points: 48 hours after sowing, and after the final transplant event.

[0190] Fresh head weight is used throughout and was independently recorded by the grower.

[0191] The results can be seen in Figure 6 and in Table 6 below. Plants were harvested after 48 days after sowing (DAS). Pantoea treated plants exhibited a commercially-relevant increase in mean yield of 8.73% compared to the control plants.

[0192] The dosage applied was 3.16 x 10A8 CFUs per plant.

[0193] Table 6 - Results of substrate comparison trial

[0194] Example 8 - CRO Trial - hydroponic, deep-water cultures

[0195] This experiment was carried out at the R&D greenhouse facility with LIKATC at Stockbridge Technology Centre (STC). The commercial lettuce variety used for the experiment was Skilton.

[0196] All plants were grown across thirty hydroponic, deep water culture ponds for 50 days before being harvested and weighed. Plants were grown in peat provided by STC. Seeds were sown at a higher density and grown to maturity for 22 days before being transplanted into a lower density growth system. Seedlings were propagated in trays and were transplanted into deep water tanks once mature.

[0197] The microbial treatments were prepared using only the microbe itself suspended in sterile water. The treatments were then applied by directly pipetting 0.5 mL of the treatment onto the seed after sowing. The treatments were reapplied directly to the tank solution 22 days after sowing, at the same dosage per plant as the pipetted solution.

[0198] Fresh head weight is used throughout and was independently recorded by the grower.

[0199] The dosage applied was 3.16 x 10A8 CFUs per plant.

[0200] The results can be seen in Figure 7 and in Table 7 below. Plants were harvested after 50 days after sowing (DAS). Pantoea treated plants exhibited a commercially relevant increase in mean yield of 7.65% compared to the control plants.

[0201] Table 7 - Results from hydroponic, deep water culture trial

[0202] Example 9 - CRO Trial - mixed peat

[0203] This experiment was carried out at the R&D greenhouse facility with LIKATC at Stockbridge Technology Centre (STC). The commercial “teen leaf” lettuce variety was provided by the grower.

[0204] All plants were grown across thirty hydroponic, deep water culture ponds for 31 days before being harvested and weighed. Plants were grown in mixed peat provided by Concert Bio. Seeds were sown at a higher density and grown to maturity for 14 days before being transplanted into a lower density growth system. Seedlings were propagated in trays and were transplanted into deep water tanks once mature.

[0205] Pantoea treatment was prepared using only the microbe itself suspended in sterile water at a 1 :10 concentration. The treatments were then applied by using a spray bottle to apply two sprays onto the seed after sowing. The treatments were reapplied directly to the tank solution 14 days after sowing, at the same dosage per plant as the sprayed solution.

[0206] The dosage applied was 3.16 x 10A7 CFUs per plant.

[0207] Fresh head weight is used throughout and was independently recorded by the grower.

[0208] The results can be seen in Figure 8 and in Table 8 below. Plants were harvested after 31 days after sowing (DAS). Pantoea treated plants exhibited a commercially relevant increase in mean yield of 16.8% compared to the control plants.

[0209] Table 8 - Results from mixed-peat CRO trial Example 10 - CRO Trial - Effect of Pantoea treatment on Tomatoes

[0210] The experiment was carried out at the R&D indoor facility at the North East Advanced Grow Hub. The commercial tomato variety used for the experiment was Balconi F1

[0211] Plants were grown for 20 weeks after seeding. 48 plants per treatment were used in total Plants were grown in deep water culture trays across five rigs, with an experimental matrix applied to ensure even distribution of the trays throughout the room. The microbial treatment was applied by spraying onto the seeds.

[0212] A foliar spray at the same dosage was applied at the point of transplant, and thereafter the treatment was dosed into the nutrient solution of the trays every four weeks.

[0213] The dosage applied was 3.16 x 10A8 CFUs per plant.

[0214] The results can be seen in Figure 9. Pantoea treated plants exhibited a 10.8% increase in mean fresh weight.

[0215] Example 11 - Pantoea septica - genome analysis

[0216] The inventors were able to identify a number of genes unique to Pantoea septica that are not found a reference genome from a different Pantoea sp. The inventors hypothesize that these genes contribute to the ability of the strain deposited with NCTC accession number 24050701 , and more widely the proposed ability of the species Pantoea septica, to promote plant growth in general and may be especially beneficial under hydroponic conditions. In particular, these include genes that may support microbial persistence in oxidative and chemically complex environments.

[0217] Microbial persistence in oxidative and chemically complex environments

[0218] Hydrogen peroxide (H2O2), widely used in hydroponic systems as a disinfectant, imposes oxidative stress that many microbes cannot withstand. The strain described herein contains the bsmA gene (formerly yjfO), which has been associated with biofilm formation and peroxide stress resistance. This gene may contribute to microbial survival by reinforcing biofilm architecture and reducing oxidative damage, helping the microbe persist on plant roots after disinfection (Weber et al., 2010). Additionally, the strain contains alkB, encoding an oxidative DNA demethylase, which likely supports stress resilience by repairing DNA damage caused by reactive oxygen species (Sedgwick et al., 2007). The strain also comprises the ariR gene (also known as ymgB), which has been implicated in biofilm development and acid resistance, potentially facilitating microbial adaptation in the chemically dynamic environments of treated root zones (Lee et al., 2007).

[0219] Metal detoxification and stress tolerance

[0220] Hydroponic systems also expose microbes to fluctuating concentrations of metal ions introduced through nutrient solutions or released from system components. Metal resistance genes in the microbe may therefore be advantageous. The Pantoea septica strain described herein contains at least three such genes, which include: chrA, which encodes a chromate efflux transporter, is implicated in Cr(VI) efflux, thereby preventing DNA and protein damage (Cervantes et al., 2006). copD, involved in copper homeostasis, which may contribute to safe intracellular copper handling by facilitating regulated uptake and buffering, a key adaptation in copper-enriched or micronutrient-supplemented environments such as those found in commercial hydroponics (Wang et al., 2016). Similarly in connection with metal homeostasis is rcnB, a periplasmic protein, which may play a role in maintaining nickel and cobalt homeostasis by modulating the activity of the RenA efflux pump, thus preventing excessive loss of these essential metals and ensuring proper cellular function (Guiliani et al., 2011).

[0221] While these gene functions remain to be directly validated in hydroponic conditions, their proposed roles in the context of some other bacteria suggest they could collectively enhance microbial survival, promote stable root colonization, and ultimately support plant growth in oxidative and metal-rich systems.

[0222] Sequences

[0223] SEQ ID NO:1 - “Pantoea” full genome sequence

[0224] SEQ ID NO:2 - “Pantoea” 16S translated rRNA sequence

[0225] SEQ ID NO:3 - “Pantoea” 16S rDNA (i.e. genomic) sequence Deposits and Expert Solution

[0226] The applicant requests that a sample of the deposited microorganism stated in Table 9 below may only be made available to an expert, until the date on which the patent is granted.

[0227] The applicant requests that the availability of the deposited microorganism referred to in Rule 33 EPC shall be effected only by the issue of a sample to an independent expert nominated by the requester (Rule 32(1) EPC). If an expert solution has been requested, restrictions concerning the furnishing of samples apply.

[0228] The deposit was made according to the Budapest treaty on the international recognition of the deposit of microorganisms for the purposes of patent procedure at the NCTC Patent Depository (LIKHSA).

[0229] The Budapest T reaty provides that any restriction of public access to samples of deposited biological material must be irrevocably removed as of the date of grant of the relevant patent.

[0230] Table 9. Deposited strain made at a depositary institution.

[0231] References

[0232] 1. Weisburg WG, Barns SM, Pelletier DA, Lane DJ. 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol. 1991 ;173(2) :697-703

[0233] 2. Chaumeil, P.A., Mussig, A. J., Hugenholtz, P., and Parks, D.H., 2020. GTDB-Tk: a toolkit to classify genomes with the Genome Taxonomy Database.

[0234] 3. Chaumeil, P.A., Mussig, A. J., Hugenholtz, P., and Parks, D.H., 2022. GTDB-Tk v2: memory-friendly classification with the genome taxonomy database. Bioinformatics, 38(23), pp.5315-5316.

[0235] 4. Ciufo, S., Kannan, S., Sharma, S., Badretdin, A., Clark, K., Turner, S., Brover, S., Schoch, C.L., Kimchi, A., and DiCuccio, M., 2018. Using average nucleotide identity to improve taxonomic assignments in prokaryotic genomes at the NCBI. International Journal of Systematic and Evolutionary Microbiology, 68(7), pp.2386- 2392. 5. Jain, C., Rodriguez-R, L.M., Phillippy, A.M., Konstantinidis, K.T., and Aluru, S., 2018. High throughput ANI analysis of 90K prokaryotic genomes reveals clear species boundaries. Nature Communications, 9(1), p.5114.

[0236] 6. Schwengers, O., Jelonek, L., Dieckmann, M.A., Beyvers, S., Blom, J. and Goesmann, A., 2021. Bakta: rapid and standardized annotation of bacterial genomes via alignment-free sequence identification. Microbial genomics, 7(11), p.000685.

[0237] 7. Cervantes, C., Campos-Garcia, J., Devars, S., Gutierrez-Corona, F., Loza-Tavera, H., Torres-Guzman, J.C. and Moreno-Sanchez, R., 2006. Interactions of chromium with microorganisms and plants. FEMS Microbiology Letters, 262(2), pp.178-188. https: / / doi.Org / 10.1111 / j.1574-6968.2006.00392.X

[0238] 8. Guiliani, N., Rodriguez, F., Santos, J., Norambuena, J., Jerez, C.A., 2011. RcnB is a periplasmic protein essential for maintaining intracellular nickel and cobalt homeostasis in Escherichia coli. Journal of Bacteriology, 193(16), pp.4254-4261. https: / / doi.org / 10.1128 / JB.05032-11

[0239] 9. Lee, J., Page, R., Garcia-Contreras, R., Palermino, J.M., Zhang, X.S., Doshi, O., Wood, T.K. and Peti, W., 2007. Structure and function of the Escherichia coli protein YmgB: a protein critical for biofilm formation and acid-resistance. Journal of Molecular Biology, 373(1), pp.11-26.

[0240] 10. Sedgwick, B., Bates, P.A., Paik, J., Jacobs, S.C. and Lindahl, T., 2007. Repair of alkylated DNA: recent advances. DNA Repair, 6(4), pp.429-442.

[0241] 11. Wang, Y., Shi, J., Wang, H., Lin, Q., Chen, X. and Chen, Y., 2016. Copper resistance mechanisms of bacteria and fungi in copper-contaminated soil. Environmental Science and Pollution Research, 23, pp.6270-6280.

[0242] 12. Weber, M.M., French, C.L., Barnes, M.B., Siegele, D.A. and McLean, R.J.C., 2010. A previously uncharacterized gene, yjfO (bsmA), influences Escherichia coli biofilm formation and stress response. Microbiology, 156(1), pp.139-147. https: / / d0i.0rg / l 0.1099 / mic.0.031468-0

Claims

CLAIMS1. An isolated microbial strain comprising a microbe which comprises a genome sequence with at least 95% homology to SEQ ID NO:1.

2. An isolated microbial strain comprising a microbe which comprises a 16S rRNA gene sequence with at least 90% homology to SEQ ID NO: 3.

3. The isolated microbial strain of claim 2, wherein the microbe comprises a 16S rRNA gene sequence with at least 99% homology to SEQ ID NO: 3; and / or that exhibits at least 70% DNA-DNA relatedness in reciprocal hybridisation reactions with SEQ ID NO:3 and 5°C or less ATm for the stability of heteroduplex molecules with SEQ ID NO:3.

4. The isolated microbial strain of claim 2 or claim 3, wherein the microbe comprises a genome sequence with at least 70% homology to SEQ ID NO:1, optionally wherein the microbe comprises a genome sequence with at least 95% homology to SEQ ID NO:1.

5. The isolated microbial strain of any previous claim, wherein the microbe is the microbe deposited as NCTC Accession Number 24050701 or the progeny thereof.

6. An isolated microbial strain having substantially similar morphological and physiological characteristics as an isolated microbial strain according to any previous claim.

7. An isolated microbial strain comprising a mutant of an isolated microbial strain according to any previous claim.

8. A microbial strain comprising the progeny of the isolated microbial strain of any previous claim.

9. A substantially pure culture of an isolated microbial strain according to any previous claim.

10. An agricultural composition comprising an effective amount of the isolated microbial strain of any one of claims 1 to 7, the microbial strain of claim 8, or the substantially pure culture of claim 9 and an agriculturally acceptable carrier.

11. A method of improving plant growth and / or increasing plant yield, comprising contacting a plant, a plant part, or a growth medium in which said plant is located, with an agricultural composition according to claim 10.

12. The method of claim 11 , wherein the plant is selected from a leaf crop or a fruiting crop.

13. The method of claim 11, wherein the plant is selected from: lettuce (Lactuca sativa) or tomato (Solanum lycopersicum).

14. The method according to any one of claims 11 to 13, wherein the agricultural composition is applied to a plant seed.

15. The method of any one of claims 11 to 14, wherein the plant growth media is a hydroponic nutrient solution or hydroponic growth substrate, and wherein the plants are grown hydroponically.

16. The method of claim 15, wherein the agricultural composition is combined with the hydroponic nutrient solution, or hydroponic growth substrate.

17. The method of claim 15, wherein the agricultural composition is applied to a hydroponic growth substrate, optionally selected from mineral wool, expanded clay aggregate, coconut coir, gravel, mineral particulates, and peat.

18. The method of any one of claims 11 to 14, wherein the growth media is soil, and the plants are grown in soil.

19. The method of claim 18, wherein the agricultural composition is applied to the soil.

20. A plant, seed, or plant part, treated with an agricultural composition according to claim 10.

21. A synthetic combination of a plant and the agricultural composition of claim 10.

22. A cell-free or inactivated preparation of an isolated microbial strain of any one of claims 1 to 7, a microbial strain of claim 8, or a substantially pure culture of claim 9.

23. A metabolite produced by an isolated microbial strain of any one of claims 1 to 7, a microbial strain of claim 8, or a substantially pure culture of claim 9.

24. A method of producing a microbial composition comprising culturing an isolated microbial strain of any one of claims 1 to 7, a microbial strain of claim 8, or a substantially pure culture of claim 9.

25. A microbial culture produced by the method of claim 24.

26. Use of the agricultural composition according to claim 10 for promoting plant growth; wherein the use comprises applying the agricultural composition to a plant, a plant part, or a growth medium in which said plant is located.

27. The use according to claim 26, wherein the agricultural composition is applied two or more times to a plant, a plant part, or a growth medium in which said plant is located.

28. The use according to claim 26 or 27, wherein the agricultural composition is applied at an effective dose per plant of between about 1 x 10A5 CFU to about 7 x 10A8 CFU.

29. The use according to any of claims 26-28, wherein the agricultural composition is applied at an effective dose per plant of about 3.16 x 10A5 CFU.

30. The use according to any of claims 26-28, wherein the agricultural composition is applied at an effective dose per plant of about 3.16 x 10A8 CFU.

31. The use according to any of claims 26-28, wherein the agricultural composition is applied at an effective dose per plant of about 3.16 x 10A7 CFU.

Images