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 “Delftia” had an 8.5% increase in median head weight 28 days from transplant (1.8% increase in mean).

[0023] Figure 2 - 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 “Delftia” had an 11.9% increase in median head weight 28 days from transplant (12.0% increase in mean).

[0024] Figure 3 - 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. “Delftia” 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 “Delftia” had an increase in the total number of cucumbers produced by 10.70% and an increase in the total harvested weight of cucumbers by 10.11 %.

[0025] Figure 4 - Results of commercial trial with GrowCoon. Lettuce plants were sown at high density initially in two boxes. Seeds were transplanted twice during the growth cycle. “Delftia” was suspended in sterile water and sprayed onto seeds 48 hours after sowing and again after the final transplant event. Plants treated with “Delftia” had a 9.84% increase in fresh head weight compared to control plants.

[0026] Figure 5 - Results of commercial trial with Crispita II in an Ebb-and-Flow hydroponic system. Lettuce plants were sown at a higher density with two seeds per plug. Seeds were grown to maturity for 9 days before being transplanted into the growth system. Seedlings were propagated in trays at a higher density and were transplanted into a lower density growth system. “Delftia” treated plants had an increase in mean yield of 7.10% compared to the control plants.

[0027] Figure 6 - Results of commercial trial on Beetroot. Beetroot plants were grown for 21 days in inert substrate. “Delftia” treatment was applied to seeds across several trays by spraying. Plants were harvested 21 days after sowing. Plants treated with “Delftia” had a 6.88% increase in mean fresh head weight compared to control plants.

[0028] Figure 7 - Results of commercial trial with multiple crops. Basil, Beetroot and Komatsuna were grown for 21 days in inert substrate. “Delftia” was applied to seeds across several trays by spraying. “Delftia” treatment increased the harvested yield of basil, beetroot and komatsuna by 21 , 31 and 17% respectively.

[0029] Figure 8 - Results of CRO trial examining Lettuce (Mesclita) cultivation using hydroponic, deep water culture ponds. Plants were grown across thirty hydroponic, deep water culture ponds for 28 days before being harvested and weighed. “Delftia” treatments were prepared at a concentration of 1 :10 and applied using a spray bottle to apply two sprays onto the seed after sowing. “Delftia” treated plants exhibited an increased mean fresh head weight of 15.63% compared to control plants.

[0030] Figure 9 - Results of CRO trial examining Lettuce (Verdagio) cultivation in Klasmann- Deilmann Potgrond P peat. All plants were grown across thirty hydroponic, deep water culture ponds for 44 days before being harvested and weighed. “Delftia” treatments were prepared using only the microbe itself suspended in sterile water at a 1 :10 concentration and applied by using a spray bottle to apply two sprays onto the seed after sowing. The treatments were reapplied using the same method by spraying onto the foliage 21 days after sowing, upon transplant. Delftia treated plants exhibited an increased mean fresh head weight (g) of 8.46% compared to control plants.

[0031] Figure 10 - Results of CRO trial examining ‘teenage’ Lettuce cultivation using Klasmann- Deilmann Potgrond P peat. All plants were grown across thirty hydroponic, deep water culture ponds for 28 days before being harvested and weighed. “Delftia” treatments were prepared using only the microbe itself suspended in sterile water at a 1 :10 concentration and applied by using a spray bottle to apply two sprays onto the seed after sowing. Delftia treated plants exhibited an increased mean fresh head weight (g) of 9.01% compared to control plants.

[0032] Detailed Description of the Invention

[0033] 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 Delftia, and particularly Delftia acidovorans. An exemplary microbial strain is the Delftia acidovorans strain deposited under the Budapest Treaty of 1977 as Accession Number 24050702 at the NCTC Patent Depository (LIKHSA), Culture Collections, UK Health Security Agency, Porton Down, Salisbury, SP4 OJG, United Kingdom. The Delftia acidovorans strain 24050702 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 “Delfita acidovorans” [sic].

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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 24050702 at the NCTC Patent Depository (LIKHSA) and having a 16S rRNA gene sequence SEQ ID NO:3, or the progeny thereof.

[0039] 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).

[0040] 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.

[0041] 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 24050702 and having a genome sequence according to SEQ ID NO:1 , or the progeny thereof.

[0042] 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.

[0043] 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).

[0044] 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.

[0045] 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:

[0046] 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.

[0047] 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.

[0048] 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).

[0049] 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.

[0050] 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.

[0051] 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. In some embodiments, the strain is not a nitrogen-fixing bacterium, or not a legume nodulecolonising bacteria.

[0052] The present compositions and methods are applicable to any suitable plants. These may be plants grown ornamental, comestible, textile, constructional, or any other purposes.

[0053] In some embodiments, the plant is a crop plant. As used herein, a “crop plant” is one cultivated primarily for human consumption as food.

[0054] 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.

[0055] 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).

[0056] 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 (Enza Zaden), Nunhems 09185 LTL, Nunhems Nitaflash.

[0057] The lettuce varieties Crispita II (Syngenta), Lalique (Rijk Zwaan), Verdagio (Syngenta) and Mesclita (Syngenta) are also contemplated to benefit from treatment with the compositions of the invention, such as exemplified herein.

[0058] Komatsuna (Brassica rapa var. perviridis) is also contemplated to benefit from treatment with the compositions of the invention, such as exemplified herein.

[0059] 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, 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 compositions of the invention, for example, strawberry plants (Fragaria x ananassa).

[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] Beetroot is particularly contemplated as benefitting from treatment with the compositions of the invention, such as exemplified herein. In some embodiments, the beetroot variety is Soldier (Bull’s Blood).

[0062] 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.

[0063] The compositions containing the bacteria of the present invention are effective in improving plant growth and increasing plant yield.

[0064] 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:

[0065] • Mass (weight) of plant;

[0066] • Total leaf number, area, and / or weight;

[0067] • Average leaf area or weight;

[0068] • Total or average leaf protein content;

[0069] • Height of aerial parts of plant; Length or weight of roots;

[0070] Survival of plant, in particular average rate of survival of a population of plants.

[0071] 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.

[0072] Increases in desirable characteristics of harvested plant parts or products include:

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

[0074] • Total or average weight of harvested plant parts or product per plant, or per square metre used to grow plants.

[0075] • Total or average time required to produce harvestable plant parts or products, e.g. time from sowing or transplant to harvest.

[0076] • 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.

[0077] • 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.

[0078] • 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).

[0079] • 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).

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

[0081] • Total or average sugar content of harvested plant parts or product per plant, or per square metre used to grow plants.

[0082] • 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. • 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).

[0084] • Total or average germination rate (e.g. successful germination per 100 seeds).

[0085] • Total or average rate of survival to first, second, or third true leaf / pair stage (e.g. per 100 seeds).

[0086] • Shelf life of harvested plants, part plants, or products.

[0087] • Homogeneity of appearance (e.g. as measured through any known horticultural or agricultural grading methods).

[0088] 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.

[0089] 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.

[0090] 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. 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 growth media. Growth media may include nutrients, additives, growth regulators, or other agents required by the microbe for growth. Growth 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 growth 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. The skilled person will also understand that an agricultural composition may be described as a composition suitable for use in agriculture. 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.

[0095] 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 10A7, at least 1.5 x 10A7, at least 2 x 10A7, at least 2.1 x 10A7, at least 2.2 x 10A7, at least 2.3 x 10A7, at least 2.4 x 10A7, at least 2.5 x 10A7, at least 2.6 x 10A7, at least 2.7 x 10A7, at least 2.8 x 10A7, at least 2.9 x 10A7, at least 3 x 10A7, at least 3.1 x 10A7, at least 3.2 x 10A7, at least 3.3 x 10A7, at least 3.4 x 10A7, at least 3.5 x 10A7, at least 3.6 x 10A7, at least 3.7 x 10A7, at least 3.8 x 10A7, at least 3.9 x 10A7, at least 4 x 10A7, at least 4.1 x 10A7, at least 4.2 x 10A7, at least 4.3 x 10A7, at least 4.4 x 10A7, at least 4.5 x 10A7, at least 4.6 x 10A7, at least 4.7 x 10A7, at least 4.8 x 10A7, at least 4.9 x 10A7, or at least 5 x 10A7 CFU per unit dose. In some embodiments, an effective dose delivers at least about 3 x 10A7 CFU per unit dose, such as at least 2.9 x 10A7 CFU per unit dose.

[0098] 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 2 x 10A7, at least 2.1 x 10A7, at least 2.2 x 10A7, at least 2.3 x 10A7, at least 2.4 x 10A7, at least 2.5 x 10A7, at least 2.6 x 10A7, at least 2.7 x 10A7, at least 2.8 x 10A7, at least 2.9 x 10A7, at least 3 x 10A7, at least 3.1 x 10A7, at least 3.2 x 10A7, at least 3.3 x 10A7, at least 3.4 x 10A7, at least 3.5 x 10A7, at least 3.6 x 10A7, at least 3.7 x 10A7, at least 3.8 x 10A7, at least 3.9 x 10A7, at least 4 x 10A7, at least 4.1 x 10A7, at least 4.2 x 10A7, at least 4.3 x 10A7, at least 4.4 x 10A7, at least 4.5 x 10A7, at least 4.6 x 10A7, at least 4.7 x 10A7, at least 4.8 x 10A7, at least 4.9 x 10A7, at least 5 x 10A7, at least 5.1 x 10A7, at least 5.2 x 10A7, at least 5.3 x 10A7, at least 5.4 x 10A7, at least 5.5 x 10A7, at least 5.6 x 10A7, at least 5.7 x 10A7, at least 5.8 x 10A7, at least 5.9 x 10A7, or at least 6 x 10A7 CFU per mL.

[0099] In some embodiments, an agricultural composition contains at least 5.8 x 10A7 CFU per mL. In some embodiments, the effective dose per plant delivers at least 1 x 10A4, 2 x 10A4, 3 x 10A4, at least 4 x 10A4, at least 5 x 10A4, at least 6 x 10A4, at least 7 x 10A4, at least 8 x 10A4, at least 9 x 10A4, at least 1 x 10A5, at least 2 x 10A5, at least 3 x 10A5, at least 4 x

[0100] 10A5, at least 5 x 10A5, at least 6 x 10A5, at least 7 x 10A5, at least 8 x 10A5, at least 9 x

[0101] 10A5, at least 1 x 10A6, at least 2 x 10A6, at least 3 x 10A6, at least 4 x 10A6, at least 5 x

[0102] 10A6, at least 6 x 10A6, at least 7 x 10A6, at least 8 x 10A6, at least 9 x 10A6, at least 1 x

[0103] 10A7, at least 2 x 10A7, at least 3 x 10A7, at least 4 x 10A7, at least 5 x 10A7, at least 6 x

[0104] 10A7, at least 7 x 10A7, at least 8 x 10A7, or at least 9 x 10A7 CFU per unit dose.

[0105] 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 10A7 and 5 x 10A7 CFU per unit dose. Suitably, an effective dose per plant for a vine crop may be about 3 x 10A7 CFU per unit dose, such as 2.9 x 10A7 CFU per unit dose. A typical leafy green crop may require a reduced dose compared to a vine crop, for example, between 1 x 10A6 and 5 x 10A6 CFU per unit dose. Suitably, an effective dose for a leafy green crop may be about 3 x 10A6 CFU per unit dose, such as 2.9 x 10A6 CFU per unit dose.

[0106] 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 1 x 10A4 and 4 x 10A4 CFU per unit dose. In one particular embodiment, the minimum effective dose may be about 3 x 10A4 CFU per unit dose, such as 2.9 x 10A4 CFU per unit dose.

[0107] In some embodiments, an agricultural composition contains between 4 x 10A7 and 8 x 10A7 CFU per mL. In some embodiments, an agricultural composition contains between 5 x 10A7 and 7 x 10A7 CFU per mL. In some embodiments, an agricultural composition contains between 5.5 x 10A7 and 6.5 x 10A7 CFU per mL. In some embodiments, an agricultural composition contains between 5.5 x 10A7 and 6 x 10A7 CFU per mL.

[0108] 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 macronutrients and / or plant micronutrients), a herbicide (especially a selective herbicide), a pesticide, a fungicide, a bactericide, an antiviral, a plant hormone, and / or a plant growth regulator.

[0109] 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”.

[0110] 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.

[0111] 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.

[0112] In some embodiments, agricultural compositions are applied to plants multiple times.

[0113] 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.

[0114] The strains and compositions herein can be applied to plants in any growth context.

[0115] 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. 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 as the growth medium, 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 deep-water 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.

[0116] 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.

[0117] 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.

[0118] 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. 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.

[0119] As used herein “average” may refer to mean or median average.

[0120] 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.

[0121] 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.

[0122] 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.

[0123] 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.

[0124] 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.

[0125] Ranges provided herein are understood to include all individual integer values and all subranges within the ranges.

[0126] 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.

[0127] 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.

[0128] 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.

[0129] Examples

[0130] Example 1 - Isolation and characterisation of strain “Delftia”

[0131] The microbial strain given the internal designation “Delftia” was isolated by Concert Bio UK, from the nutrient solution of our internal hydroponic plant growth system.

[0132] 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. This identified 'Delftia acidovorans' closest match.

[0133] 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. The microbial strain was identified as a strain of Delftia acidovorans.

[0134] The full genome sequence is provided as SEQ ID NO:1.

[0135] 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).

[0136] Example 2 - Proof of concept trial

[0137] Analysis of Delftia’s ability to promote plant growth was performed at Concert Bio’s plant research facility in Penryn, UK.

[0138] Lettuce plants were grown for seven days in a hydroponic nursery set-up before transplanting into the main DWC hydroponic growth system. The commercial lettuce variety used for the experiment was Skilton (Enza Zaden). All plants were grown in inert, foam substrate.

[0139] 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.

[0140] 2.9 x 10A7 CFU of microbe was applied per application of treatment.

[0141] 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.

[0142] 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

[0143] All plants were harvested and weighed 28 days after transplanting into the growth system.

[0144] As is standard in the field for assessing yield, fresh head weight is used throughout. 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.

[0145] The results of the experiment are provided in Figure 1 and Table 1 below:

[0146] Table 1 - Results of proof of concept trial

[0147] This data shows that Delftia treatment increases both median and mean head weight by a commercially useful amount in a proof-of-concept trial.

[0148] Moreover, as can be seen in Figure 1 , the mean average was affected by a large number of lower weight plants in the Delftia treated sample set. This may be due to multiple factors, such as favourability of their positioning within the experimental hydroponic growth setup. However, the Delftia treated samples included multiple large heads, and one exceptionally so (>300 g). In a larger trial, we expect that this variance would be reduced. Regardless, the increase in media head weight (8.5%) for treated plants grown under the same growth conditions represents a potentially valuable increase in plant profitability.

[0149] Example 3 - Commercial trial

[0150] 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.

[0151] 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. 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

[0152] 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

[0153] Fresh head weight is used throughout and was independently recorded by the grower

[0154] The results of the experiment are provided in Figure 2 and Table 2 below:

[0155] Table 2 - Results of commercial-scale trial

[0156] This data confirms that Delftia treatment increases both median and mean head weight by a commercially useful margin when grown in a commercial setting.

[0157] This data further demonstrates that the Delftia treatment group showed consistent increases in head weight, including fewer <100g heads (see Figure 2), and this effect is visible in the 12% increase in mean head weight. This is a desirable characteristic for growers, demonstrating a reliable and consistent increase in growth.

[0158] Example 4 - Internal Trial - Cucumbers

[0159] 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 208 plants in each treatment group were used. Seeds were sprayed with “Delftia” treatment at a plant propagator (Delfland Nurseries). Shortly after the crop was delivered to the polytunnels, the plants were treated again with “Delftia” 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.

[0160] The dosage applied was 5.8 x 10A7 CFUs per plant.

[0161] The results can be seen in Figures 3A and 3B and Tables 3 and 4 below. Treatment with “Delftia” increased the total yield of cucumbers by 10.7% and the total cucumber weight by 10.11 % compared to control plants.

[0162] Table 3 - Results of cucumber trial - total number of cucumbers produced

[0163] Table 4 - Results of cucumber trial - total weight of cucumbers produced

[0164] Example 5 - Commercial Trial - Testing Delftia treatment with GrowCoon as a substrate

[0165] 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. “Delftia” 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.

[0166] The dosage applied was 2.9 x 10A7 CFUs per plant.

[0167] Fresh head weight is used throughout and was independently recorded by the grower.

[0168] The results can be seen in Figure 4 and in Table 5 below. Plants were harvested after 48 days after sowing (DAS). “Delftia” treated plants grown in GrowCoon exhibited a commercially-relevant increase in mean yield of 9.84% compared to the control plants.

[0169] Table 5 - Results of commercial Trial with GrowCoon

[0170] Example 6 - Commercial Trial - ebb and flow benches

[0171] This experiment was carried out in a Netherlands-based hydroponic lettuce producer. The commercial lettuce variety used for the experiment was Crispita II.

[0172] All plants were grown in twenty-four ebb and flow benches, for 20 days before being harvested and weighed. Plants were grown in peat. Seeds were sown at a higher density with two seeds per plug, this is lower than their usual five seeds per plug. Seeds were grown to maturity for 9 days before being transplanted into the growth system. Seedlings were propagated in trays at a higher density and were transplanted into a lower density growth system.

[0173] “Delftia” treatments were prepared using only the microbe itself suspended in sterile water. The treatments were then applied as two sprays of the treatment onto the seed after sowing. The treatments were reapplied before transplanting on day 9, at the same dosage per plant. The plants were left to grow for 11 days while being supplied with a continuous flow of nutrient water. Fresh head weight is used throughout and was independently recorded by the grower.

[0174] The dosage applied was 2.9 x 10A7 CFUs per plant.

[0175] The results can be seen in Figure 5 and in Table 6 below. Plants were harvested after 20 days after sowing (DAS). “Delftia” treated plants exhibited a commercially-relevant increase in mean fresh head weight of 7.1% compared to the control plants.

[0176] Table 6 - Results of Crispita II trial

[0177] Example 7 - Commercial Trial - Beetroot

[0178] This experiment was carried out at a large nutrient film technique (NFT) vertical farm in the UK. The commercial variety of beetroot used was Soldier (Bull’s Blood).

[0179] All plants were grown for 21 days in inert substrate (fabric cutoffs). “Delftia” was applied to seeds across several trays by spraying. No transplant step was used, only a single seed application was made.

[0180] The dosage applied was 2.9 x 10A7 CFUs per plant.

[0181] All data was collected independently by the grower.

[0182] The results can be seen in Figure 6 and in Table 7 below. Plants were harvested after 21 days after sowing (DAS). “Delftia” treated plants exhibited a commercially-relevant increase in mean yield of 6.9% compared to the control plants. Table 7 - Results of Beetroot NFT trial

[0183] Example 8 - Commercial Trial - NFT with multiple crop types: Basil, Beetroot, and komatsuna

[0184] The experiment was carried out at a large nutrient film technique (NFT) vertical farm in the UK. The commercial variety of beetroot used was Soldier (Bull’s Blood).

[0185] All plants were grown for 21 days in inert substrate (fabric cutoffs). “Delftia” was applied to seeds across several trays by spraying. No transplant step was used, only a single seed application was made.

[0186] The dosage applied was 2.9 x 10A6 CFUs per plant.

[0187] All data was collected independently by the grower.

[0188] The results can be seen in Figure 7 and in Table 8 below. Plants were harvested after 21 days after sowing (DAS).

[0189] “Delftia” treatment increased the yield of basil, beetroot and Komatsuna by 21 , 31 and 17% respectively. These results show that “Delftia” treatment can improve the yield of vine and leafy crops.

[0190] Table 8 - Results of multi-crop NFT trial

[0191] Example 9 - CRO Trial - Lettuce (Mesclita) cultivation using hydroponic, deep water culture ponds

[0192] The 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 Mesclita.

[0193] All plants were grown across thirty hydroponic, deep water culture ponds for 28 days before being harvested and weighed. Plants were grown in Growfoam provided by Concert Bio. Seeds were sown at a higher density and grown to maturity for 15 days before being transplanted into a lower density growth system. Seedlings were propagated in trays and were transplanted into deep water tanks once mature.

[0194] Delftia treatments were 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. Fresh head weight is used throughout and was independently recorded by the grower.

[0195] The dosage applied was 2.9 x 10A7 CFUs per plant.

[0196] The results can be seen in Figure 8 and in Table 9 below. Delftia treated plants exhibited an increased mean fresh head weight (g) of 15.63% compared to untreated plants. Table 9 - Results of Lettuce (Mesclita) trial

[0197] Example 10 - CRO Trial - Lettuce (Verdaqio (Syngenta)) grown in Klasmann-Deilmann Potgrond P peat

[0198] 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 Verdagio (Syngenta).

[0199] All plants were grown across thirty hydroponic, deep water culture ponds for 44 days before being harvested and weighed. Plants were grown in Klasmann-Deilmann Potgrond P peat provided by Concert Bio. Seeds were sown at a higher density and grown to maturity for 21 days before being transplanted into a lower density growth system. • Seedlings were propagated in trays and were transplanted into deep water tanks once mature.

[0200] “Delftia” treatments were 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 using the same method by spraying onto the foliage 21 days after sowing, upon transplant.

[0201] Fresh head weight is used throughout and was independently recorded by the grower. The dosage applied was 2.9 x 10A6 CFUs per plant.

[0202] The results are shown in Figure 9 and Table 10 below. Delftia treated plants exhibited an increased mean fresh head weight (g) of 8.464% compared to untreated plants. Table 10 - Results of Lettuce (Verdagio) trial with Klasmann-Deilmann Potgrond P peat

[0203] Example 11 - CRO Trial - Lettuce grown in Klasmann-Deilmann Potgrond P peat

[0204] The experiment was carried out at the R&D greenhouse facility with UKATC at Stockbridge Technology Centre (STC). The commercial lettuce variety used for the experiment was unknown and provided by a commercial grower.

[0205] All plants were grown across thirty hydroponic, deep water culture ponds for 28 days before being harvested and weighed. Plants were grown in Klasmann-Deilmann Potgrond P peat provided by Concert Bio. Seeds were sown at a higher density and grown to maturity for 12 days before being transplanted into a lower density growth system. Seedlings were propagated in trays and were transplanted into deep water tanks once mature.

[0206] “Delftia” treatments were 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.

[0207] Fresh head weight is used throughout and was independently recorded by the grower. The dosage applied was 2.9 x 10A6 CFUs per plant.

[0208] The results are shown in Figure 10 and Table 11 below. Delftia treated plants exhibited an increased mean fresh head weight (g) of 9.012% compared to untreated plants. Table 11 - Results of Lettuce trial with Klasmann-Deilmann Potgrond P peat

[0209] Example 12 - Delftia acidovorans - genome analysis

[0210] Using whole-genome sequencing of the Delftia acidovorans strain (Accession No. 24050702), the inventors surprisingly found that the strain contained several unique genes absent in a reference Delftia acidovorans strain. These unique genes may contribute to the strain’s ability to act as a specialised biostimulant, beneficial in hydroponic systems. The inventors theorise that these genes may contribute to enhanced microbial adaptability and metabolism, and heavy metal detoxification and stress tolerance.

[0211] Enhanced Microbial Adaptability and Metabolism

[0212] • Alcohol Dehydrogenases (AdhP, NAD(P)-dependent dehydrogenase): These enzymes have been implicated in supporting efficient microbial metabolism and detoxification of root-derived ethanol, a common stress metabolite under hypoxic conditions typical of hydroponic systems. This metabolic capability may improve microbial establishment and promote root health, potentially enhancing overall plant vigor (Chadha et al., 2024).

[0213] • AbiEi-type Transcriptional Regulator: This regulator may enhance microbial adaptability to environmental stress factors such as nutrient fluctuation, oxidative stress, or temperature variations frequently encountered in hydroponics. The inventors hypothesise that increased microbial resilience may indirectly support plant health and productivity by maintaining stable root microbiome interactions (Chen et al., 2021). • Aldolase (araD): The presence of aldolase may allow for efficient utilisation of diverse carbohydrate substrates from plant root exudates, promoting robust microbial colonization and nutrient cycling. This trait could significantly boost microbial effectiveness in hydroponic environments, facilitating rapid and sustained beneficial interactions with plant roots (Vives-Peris et al., 2020).

[0214] Heavy Metal Detoxification and Stress Tolerance

[0215] • copZ (Heavy Metal Transport / Detoxification Protein): CopZ may augment microbial resilience against copper and other heavy metal accumulations common in hydroponic nutrient solutions. By mitigating heavy metal stress, this gene could support microbial survival and reduces metal-induced plant stress, thereby improving overall plant performance and safety (Nnaji et al., 2024).

[0216] • frmR (Metal / Formaldehyde-sensitive Transcriptional Regulator): This regulator may contribute to robust responses to metal and formaldehyde toxicity, enhancing microbial adaptability and stress tolerance. Such resilience could be crucial for maintaining a stable microbial community within the highly controlled and occasionally stress-prone hydroponic environment (Chen et al., 2016).

[0217] • zntA (Heavy Metal Translocating P-type ATPase): ZntA acts as an efficient efflux mechanism for metals such as zinc, cadmium, and lead, conferring significant protection against metal toxicity. This function may be particularly relevant in hydroponic systems where metal accumulation can impair microbial activity and plant health, ensuring continued microbial support for plant productivity (Hanikenne & Baurain, 2014).

[0218] Deposits and Expert Solution

[0219] The applicant requests that a sample of the deposited microorganism stated in Table 12 below may only be made available to an expert, until the date on which the patent is granted.

[0220] 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.

[0221] 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).

[0222] 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.

[0223] Table 12. Deposited strain made at a depositary institution.

[0224] Sequences

[0225] SEQ ID NO:1 - “Delftia” full genome sequence

[0226] SEQ ID NO:2 - “Delftia” 16S translated rRNA sequence

[0227] SEQ ID NO:3 - “Delftia” 16S rDNA (i.e. genomic) sequence

[0228] References

[0229] 1. Weisburg WG, Barns SM, Pelletier DA, Lane DJ. 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol. 1991 ;173(2) :697-703

[0230] 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.

[0231] 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. 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.

[0232] 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.

[0233] 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.

[0234] 7. Chadha, A., Padhi, S.K., Stella, S., Venkataraman, S., & Saravanan, T. (2024). "Microbial alcohol dehydrogenases: recent developments and applications in asymmetric synthesis." Organic & Biomolecular Chemistry, 22(2), 228-251.

[0235] 8. Chen, X., et al. (2021). "The Mesorhizobium huakuii transcriptional regulator AbiEi plays a critical role in nodulation and is important for bacterial stress response." BMC Microbiology, 21 , Article 245.

[0236] 9. Nnaji, N.D., Anyanwu, C.U., Miri, T., & Onyeaka, H. (2024). "Mechanisms of Heavy Metal Tolerance in Bacteria: A Review." Sustainability, 16(24), 11124.

[0237] 10. Chen, N. H., Djoko, K. Y., Veyrier, F. J., & McEwan, A. G. (2016). "Formaldehyde stress responses in bacterial pathogens." Frontiers in Microbiology, 7, 257.

[0238] 11. Hanikenne, M., & Baurain, D. (2014). "Origin and evolution of metal P-type ATPases in Plantae (Archaeplastida)." Frontiers in Plant Science, 4, 544.

[0239] 12. Vives-Peris, V., de Ollas, C., Gomez-Cadenas, A., & Perez-Clemente, R. M. (2020). "Root exudates: From plant to rhizosphere and beyond." Plant Cell Reports, 39(1), 3-17.

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 24050702, 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, inert substrates (e.g. GrowFoam®) 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 10A4 CFU to about 5 x 10A7 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 2.9 x 10A4 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 2.9 x 10A7 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 2.9 x 10A6 CFU.

Images