D E S C R I P T I O N PLANT BIOSTIMULANT, METHODS AND USES THEREOF TECHNICAL FIELD [0001] The present disclosure relates to the field of agriculture and horticulture, specifically to biological plant biostimulants (PBs), more specifically to compositions, methods, and uses of a strain of Bacillus velezensis for plant biostimulation and increasing plant tolerance to abiotic stresses. BACKGROUND [0002] Agriculture involves the cultivation of plants for food, biofuels and raw materials. Crops are endangered by pests and diseases as well as decreasing soil quality and abiotic stresses, which can all drastically reduce harvest yields. Pesticides are substances that can repel, eradicate, or mitigate pests and diseases. According to the Food and Agriculture Organisation of the United Nations, four million tonnes of chemical-based pesticides were used globally in 2018, causing a negative impact on soil, air and water quality as well as affecting non-target organisms. [0003] The agricultural sector is also confronted with the concomitant challenge of increasing crop yields to feed the growing global population and increase the use efficiency of valuable resources while reducing the environmental impact on the ecosystems and human health. In recent years, several technological innovations have been proposed to enhance the sustainability of agricultural production systems, through a significant reduction of synthetic agrochemicals like pesticides and fertilisers via replacement by innovative biological plant protection agents (BPPAs). [0004] Another promising and environmentally friendly approach entails the use of naturally occurring biological plant biostimulants (PBs) that can enhance seed germination, flowering, plant growth, crop yield, crop productivity and nutrient use efficiency (NUE), that also have concomitant capacity to increase plant tolerance towards a range of abiotic stresses without being a direct source of nutrients. These PBs improve plant health, optimizing nutrient use, and increasing tolerance to environmental stresses such as drought, salinity, or extreme temperatures. [0005] A biological PB may be classified as any natural substance or microorganism applied to plants or soils with the aim to enhance root growth, plant growth and development, nutrition efficiency, abiotic stress tolerance and/or crop quality traits. [0006] However, there is still a requirement for an improved standalone PB with these combined properties for use across a broad range of plants, especially horticultural plants and cereal crops, which are increasingly vulnerable to environmental stresses that hinder productivity and quality. Furthermore,
with the growing demand for higher agricultural production in areas with less favourable growing conditions, such as regions experiencing high levels of salt in the water, water scarcity, or soil degradation, the necessity for biostimulants that can help plants thrive under such challenges becomes even more pressing. [0007] This evidence, taken together, clearly indicates that there exists a pressing urgency for efficacious, environmentally benign, sustainable and economically viable PB solutions that to mitigate the effects of abiotic stresses while supporting sustainable agricultural practices and increasing yields in less-than-ideal growing environments, and making production both feasible and economically viable. [0008] Several strains of B. velezensis have been recently proposed as ecologically safe BPPAs as well as PBs (Fan, 2018, Reva, 2019, Joly, 2021, Alenezi, 2021, Gharsa, 2021, Myo, 2021, Shin, 2021, Kim, 2021, Wang, 2022). [0009] These facts are disclosed in order to illustrate the technical problem addressed by the present disclosure. GENERAL DESCRIPTION [0010] The present disclosure relates to biological plant biostimulants (PBs), more specifically to compositions, methods, and uses. Particularly, the present disclosure relates to a strain of B. velezensis for plant biostimulation and increasing plant resistance to abiotic stresses, such as drought, salinity, temperature extremes, flooding, nutrient deficiencies, and high light intensity. [0011] The present disclosure relates to an isolated B. velezensis strain, with the deposit number CECT30902 received on 12th July 2023 at the Spanish Type Culture Collection (CECT), Parc Científic de la Universitat de València, Carrer del Catedràtic Agustín Escardino Benlloch, 9, 46980 Paterna, Valencia, Espanha (henceforth termed I21 B. velezensis) that is an effective biological plant protection agent (BPPA) and surprisingly efficacious as a PB whilst also endowed with the capacity to increase plant tolerance towards abiotic stresses. The I21 B. velezensis was isolated from pear plants of the Rocha variety from an orchard located in the Oeste region of Portugal. [0012] The I21 B. velezensis was previously disclosed in patent application PCT/IB2023/057464, which is herein incorporated by reference. Therein, I21 B. velezensis was disclosed as a BPPA based on its unexpectedly strong inhibition of a virulent Erwinia amylovora strain (isolated from Pyrus communis cv. ‘Rocha’) from the Campo Maior region in Portugal and of virulent Magnaporthe oryzae isolates (isolated from different rice-producing regions in Portugal). [0013] The genetic characterisation of I21 B. velezensis was also disclosed in said patent application PCT/IB2023/057464.
[0014] It has since been surprisingly found that I21 B. velezensis possesses additional properties that are also beneficial for plants. Unexpectedly, I21 B. velezensis exhibits powerful plant biostimulant properties, enhancing plant resilience under abiotic stress conditions, particularly salinity, through its rapid and efficient colonization of the plant, mainly by the root system. This unique capability indirectly contributes to improving worldwide food production by enabling plants to thrive in challenging environmental conditions, thereby supporting sustainable agriculture and crop productivity. [0015] In an embodiment for better results, the I21 B. velezensis may be used as a plant biostimulant. [0016] In an embodiment for better results, the I21 B. velezensis may be used as a biostimulant for horticultural plants. Examples of horticultural plants include but are not limited to vegetable crops (roots, tubers, shoots, stems leaves, fruits and flowers of edible mainly annual plants), sugar crops, some oilseed crops, trees, bushes, shrubs, vines, nuts and palms. Examples of horticultural plant products include, but are not limited to: fruits, vegetables, flowers, spices and nuts. [0017] In an embodiment for better results, the horticultural plant may be a tomato plant or lettuce. [0018] In another embodiment for better results, the I21 B. velezensis may be used as a biostimulant for cereal crops. Examples of cereal crops include, but are not limited to, rice, wheat, maize, barley, oats, rye and sorghum. [0019] In an embodiment for better results, the I21 B. velezensis may be used to increase plant tolerance to abiotic stresses. Abiotic stress is the adverse effect of any abiotic factor on a plant in a given environment, negatively impacting plant growth and development. Examples of abiotic stresses include, but are not limited to drought, salinity, low or high temperatures, lack of or excessive light, mechanical stress, lack of or excess of nutrients and metals, amongst others. [0020] In an embodiment for better results, the I21 B. velezensis may be applied by irrigation or spraying. [0021] Another aspect of the present disclosure relates to a composition for plant biostimulation comprising an effective amount of the I21 B. velezensis described in the present disclosure, optionally in the presence of a suitable additive. [0022] In an embodiment for better results, the suitable additive may be selected from a list consisting of: a carrier, a solvent, a buffer, a stabiliser, a preservative, an excipient, a surfactant, an adhesion agent, an emulsifier, an osmoprotectant, a sun protectant, a wetting agent, or mixtures thereof. [0023] In an embodiment for better results, the carrier may be alginate, chitosan, ulvan, fucoidan, carrageenan, xanthan gum, gellan gum, gelatin, arabic gum, lignin, maltodextrin, dextrin, zein, protamine sulphate, lecithin, gelatin matrices, methacrylate-modified lignin, methacrylated gellan gum,
polyvinyl alcohol, low density polyethylene, poly(ε-caprolactone), polyethylene glycol or poly(lactic-co- glycolic acid), or combinations thereof. [0024] In an embodiment for better results, the composition may further comprise an agriculturally acceptable compound; preferably the agriculturally acceptable compound may be selected from: plant strengtheners, other biostimulants, nutrients, fertilisers, vitamins, minerals; or mixtures thereof. [0025] In an embodiment for better results, the composition may further comprise a BPPA, such as, for example, an essential oil. [0026] In an embodiment for better results, the composition may comprise an effective amount of the I21 B. velezensis in the ranges from 1.0 x 103 CFU/mL to 1.0 x 1011 CFU/mL, preferably from 1.0 x 105 CFU/mL to 1.0 x 1010 CFU/mL; more preferably from 1.0 x 106 CFU/mL to 1.0 x 109 CFU/mL. [0027] In an embodiment for better results, the composition may be in the form of a liquid, suspension concentrate, flowables, microencapsulations or nanoencapsulations. [0028] In another embodiment, the composition comprising the I21 B. velezensis may be in solid form, as a powder, wettable powder or granules. Suitable solid supports may include clays such as bentonite, kaolinite, montmorillonite and diatomaceous earth amongst others. These may be mixed with flours and starches and other additives to improve the stability of the microorganism over time, such as soy flour, corn flour or tapioca starch, amongst others. [0029] A method for plant biostimulation and increasing plant tolerance to abiotic stresses comprising applying an effective amount of the I21 B. velezensis or the composition as described in the present disclosure. [0030] An aspect of the present disclosure relates to the use of the Bacillus velezensis strain with the deposit number CECT30902 received on 12th July 2023 at the Spanish Type Culture Collection (CECT) as a plant biostimulant. [0031] Another aspect of the present disclosure relates to the use of the Bacillus velezensis strain with the deposit number CECT30902 received on 12th July 2023 at the Spanish Type Culture Collection (CECT) as an increaser of plant tolerance to abiotic stresses; preferably resistance to salinity stress. [0032] In an embodiment for better results, the Bacillus velezensis of the present disclosure may be used as an increaser of resistance to salinity stress. [0033] In another embodiment, the Bacillus velezensis of the present disclosure may be used as a synergically acting plant biostimulant and increaser of plant tolerance to abiotic stresses. [0034] Even in another embodiment, the Bacillus velezensis of the present disclosure may be used as an increaser of plant growth and resistance to salinity stress. [0035] In an embodiment for better results, the Bacillus velezensis may colonize a plant.
[0036] In an embodiment for better results, the colonization of the plant may occur through the root system. [0037] In an embodiment for better results, the plant may be a horticultural plant or a cereal crop. [0038] In an embodiment for better results, the horticultural plant may be tomato, or lettuce. [0039] In an embodiment for better results, the cereal crop may be rice, or wheat; preferably rice. [0040] An aspect of the present relates to a composition and its use as described in the present disclosure comprising an effective amount of the Bacillus velezensis as a biostimulant and/or as an increaser of plant tolerance to abiotic stresses, and a suitable additive. [0041] In an embodiment for better results, the suitable additive may be selected from a list consisting of: a carrier, water, a solvent, a buffer, a stabiliser, a preservative, an excipient, a surfactant, an adhesion agent, an emulsifier, osmoprotectant, sun protectant, wetting agent, or mixtures thereof. [0042] In an embodiment for better results, the carrier may be selected from a list consisting of: alginate, chitosan, ulvan, fucoidan, carrageenan, xanthan gum, gellan gum, gelatin, arabic gum, lignin, maltodextrin, zein, protamine sulphate, lecithin, gelatin matrices, methacrylate-modified lignin, methacrylated gellan gum, polyvinyl alcohol, low density polyethylene, poly(ε-caprolactone), polyethylene glycol, lignin, dextrin or poly(lactic-co-glycolic acid), or combinations thereof. [0043] In an embodiment for better results, the composition may be in the form of a liquid, emulsion, suspension concentrate, wettable powder, solid, granules, flowables, microencapsulations or nanoencapsulations; preferably solid. [0044] In an embodiment for better results, the effective amount of Bacillus velezensis strain in the composition may range from between 1.0 x 106 CFU/kg substrate and 1.0 x 1011 CFU/kg substrate; more preferably between 1.0 x 107 CFU/kg substrate and 1.0 x 1011 CFU/kg substrate; and even more preferably between 1.0 x 108 CFU/kg substrate and 1.0 x 1011 CFU/kg substrate. [0045] In an embodiment for better results, the administration frequency may range from 1 to 12 applications; preferably 2 to 10 applications; more preferably wherein each application is performing once a week over 12 weeks. [0046] In an embodiment for better results, the composition may be applied at any stage of plant development; preferably at the vegetative growth stage. [0047] In another embodiment for better results, the composition may be applied by spraying onto the plant or by direct application to the soil surrounding the plant. [0048] In another embodiment for better results, the composition may be applied at least once; preferably once a week.
[0049] Another aspect of the present disclosure relates to a method for increasing plant tolerance to abiotic stresses comprising applying an effective amount of the Bacillus velezensis composition described in any of the previous claims. [0050] In an embodiment for better results, the method may further comprise the step of applying an effective amount of Bacillus velezensis to the root of the plant. BRIEF DESCRIPTION OF THE DRAWINGS [0051] The following figures provide preferred embodiments for illustrating the disclosure and should not be seen as limiting the scope of the invention. [0052] Figure 1. Illustration of indole-acetic-acid (IAA) production by I21 B. velezensis. [0053] Figure 2. Illustration of 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase activity of I21 B. velezensis. [0054] Figure 3. Illustration of catalase activity of I21 B. velezensis. [0055] Figure 4. (A) Illustration of the effect of I21 B. velezensis on Solanum lycopersicum var. cerasiforme (cherry tomato) seedling establishment, scale bar 5 cm, with plant morphological and biomass parameters (B), after 4 weeks of development. [0056] Figure 5. (A) Illustration of the effect of I21 B. velezensis on Solanum lycopersicum var. cerasiforme (cherry tomato) on early-stage vegetative growth, scale bar 5 cm, with plant morphological and biomass parameters (B) after 5 weeks of development. [0057] Figure 6. (A) Illustration of the effect of I21 B. velezensis on Solanum lycopersicum var. cerasiforme (cherry tomato) fruit production, scale bar 5 cm. (B) Illustration of fruit production quantification of Solanum lycopersicum var. cerasiforme (cherry tomato) plants treated with I21 B. velezensis strain. [0058] Figure 7. Illustration of the presence of I21 B. velezensis on Solanum lycopersicum var. cerasiforme (cherry tomato) shoot and root after treatment. [0059] Figure 8. (A) Illustration of plant photosynthetic pigments quantification of Solanum lycopersicum var. cerasiforme (cherry tomato) plants treated with I21 B. velezensis. (B) Illustration of plant photosynthetic pigments and flavonols quantification of Solanum lycopersicum var. cerasiforme (cherry tomato) plants treated with I21 B. velezensis. [0060] Figure 9. Illustration of photosynthetic quantum yield quantification of Solanum lycopersicum var. cerasiforme (cherry tomato) plants treated with I21 B. velezensis during vegetative growth stage (A), and until fruit production (B).
[0061] Figure 10. (A) Illustration of the effect of I21 B. velezensis strain on Solanum lycopersicum var. cerasiforme (cherry tomato) at 5 weeks of plant growth under salt stress. (B) Illustration of plant biomass and morphological parameters of Solanum lycopersicum var. cerasiforme (cherry tomato) plants under salt stress treated with I21 B. velezensis. [0062] Figure 11. (A) Illustration of the effect of I21 B. velezensis strain on Lactuca sativa var. Gentilina at 6 weeks of plant growth, scale bar 10 cm. (B) Illustration of plant biomass of Lactuca sativa var. Gentilina plants treated with I21 B. velezensis. [0063] Figure 12. (A) Illustration of the effect of I21 B. velezensis strain on rice (Oryza sativa L. var. Leonardo) at 4 weeks of plant growth, scale bar 10 cm; (B); (B) Illustration of the effect of I21 B. velezensis strain on wheat (Triticum var. Roxo) at 5 weeks of plant growth, scale bar 10 cm; [0064] Figure 13.1 – 13.2. (13.1A) Illustration of the effect of non-formulated I21 B. velezensis strain (treatment) and a solid formulation of that same strain (formulated treatment) on Solanum lycopersicum var. cerasiforme (cherry tomato) at 4 weeks of plant growth, scale bar 15 cm. (B) Illustration of plant biomass (13.1B) and morphological parameters (13.2A) of Solanum lycopersicum var. cerasiforme (cherry tomato) plants treated with non-formulated I21 B. velezensis strain (treatment) and a formulation of that same strain (formulated treatment). [0065] Figure 14. (A) Illustration of the effect of non-formulated I21 B. velezensis strain (treatment) and a solid formulation of that same strain (formulated treatment) on fruit production, scale bar 3 cm. (B) Illustration of fruit production quantification of Solanum lycopersicum var. cerasiforme (cherry tomato) plants treated with non-formulated I21 B. velezensis strain (treatment) and a formulation of that same strain (formulated treatment). [0066] Figure 15.1 – 15.2. (15.1A) Illustration of the effect of non-formulated I21 B. velezensis strain (treatment) and a liquid formulation of that same strain (formulated treatment) on Solanum lycopersicum var. cerasiforme (cherry tomato) at 4 weeks of plant growth, scale bar 15 cm. (B) Illustration of plant biomass (15.1B) and morphological parameters (15.2A) of Solanum lycopersicum var. cerasiforme (cherry tomato) plants treated with non-formulated I21 B. velezensis strain (treatment) and a formulation of that same strain (formulated treatment). [0067] Figure 16. (A) Illustration of the effect of non-formulated I21 B. velezensis strain (treatment) and a liquid formulation of that same strain (formulated treatment) on fruit production, scale bar 3 cm. (B) Illustration of fruit production quantification of Solanum lycopersicum var. cerasiforme (cherry tomato) plants treated with non-formulated I21 B. velezensis strain (treatment) and a formulation of that same strain (formulated treatment).
[0068] Figure 17. (A) Illustration of the effect of I21 B. velezensis strain (treatment) on SODCC.1 gene expression variation; (B) Illustration of the effect of non-formulated I21 B. velezensis strain (treatment) on Pr-1A gene expression variation. DETAILED DESCRIPTION [0069] The present disclosure relates to the use of a B. velezensis strain, I21 B. velezensis, for plant biostimulation and resistance to abiotic stresses, a a composition comprising an effective amount of said I21 B. velezensis and a method for increasing plant tolerance to abiotic stresses comprising applying an effective amount of the said I21 B. velezensis or said composition to horticultural plant or cereal crops. [0070] In an embodiment, the I21 B. velezensis may be used as a plant biostimulant. [0071] In an embodiment, the I21 B. velezensis may be used as a horticultural plant biostimulant. [0072] In an embodiment, the I21 B. velezensis may be used as a cereal crop biostimulant. [0073] In an embodiment, the I21 B. velezensis may exert its effect through several mechanisms, including release of biosynthetic metabolites/peptides with plant biostimulation properties. [0074] In an embodiment, the composition comprising the I21 B. velezensis may contain a solvent, water and/or an acceptable buffer. [0075] In an embodiment, the composition comprising the I21 B. velezensis may be in solid form, as a powder, wettable powder or granules. Suitable solid supports may include clays such as bentonite, kaolinite, montmorillonite and diatomaceous earths, optionally in the presence of other additives. [0076] In a preferred embodiment, the composition comprising the I21 B. velezensis may contain other elements which serve to: i) stabilise the microorganism and allow application in the field via irrigation, spraying or mixing with soil using contemporary agricultural equipment; ii) protect the bacteria from premature degradation due to humidity, temperature and solar ultraviolet light exposure; iii) maintain the viability of the microorganism over time and iv) finally control the release rate over time. Agriculturally acceptable compounds may be added to the composition comprising the I21 B. velezensis to extend cell viability, and shelf-life and optimise the processability and physicochemical characteristics of the formulation. Such agriculturally acceptable compounds can include but are not restricted to, plant strengtheners, other growth promoters, nutrients, wetting agents, solvents, buffers, stabilisers, osmoprotectants, solar protectants, excipients, surfactants, adhesion agents, fertilisers, emulsifiers, vitamins, minerals, additives and/or preservatives and so on. [0077] In another embodiment, the composition allows the I21 B. velezensis to colonise the plant through the root system effectively.
[0078] In a further embodiment, to improve efficacy, onset and duration of action, the composition comprising the I21 B. velezensis may be augmented with at least one other PB, that can comprise, but not restricted to, essential oils, other beneficial microorganisms, extracts from microorganisms or other natural, semi-synthetic or synthetic biodegradable and non-toxic biomolecules. [0079] In another aspect, the composition comprising the I21 B. velezensis may be in the form of a liquid, concentrate, tablet, gel, pellet, wettable powder, granule or flowable, amongst others. In a preferred embodiment, the formulation is in the form of microcapsules (size range 1-999 µm) or nanocapsules (size range 1-999 nm) wherein the microorganism is initially retained within the interior of the capsule shell and released over time. The microcapsules or nanocapsules may be prepared from one or more biodegradable, non-toxic materials that may be selected from natural-origin polymers, proteins or lipids, such as, but not limited to, chitosan, alginate, ulvan, fucoidan, carrageenan, xanthan gum, gellan gum, gelatin, arabic gum, lignin, maltodextrin, dextrin, zein, protamine sulphate, dextrin or lecithin. [0080] In another embodiment, the microcapsules or nanocapsules comprising the I21 B. velezensis may be prepared from one or more non-toxic, biodegradable synthetic or semi-synthetic materials such as, but not limited to, methacrylate-modified lignin, methacrylated gellan gum, polyvinyl alcohol, low density polyethylene, poly(ε-caprolactone), polyethylene glycol or poly(lactic-co-glycolic acid). [0081] Through judicious choice of the encapsulation material(s), variation of the polymer concentration, crosslinking density, interior pore size, capsule size, shape and surface charge, it is possible to tune the loading capacity, entrapment efficiency and release kinetics of the entrapped microorganism and metabolites. Suitable techniques for microencapsulation include ionic gelation, spray-drying and microfluidics, the latter being suitable also for nanoencapsulation. By optimisation of parameters such as air temperature, feed flow rates and so on, it is possible to fine-tune the physicochemical properties of the capsules, which can, if necessary, be further processed downstream by techniques such as ball-milling and drying by conventional means or on a fluid bed dryer. [0082] In a preferred embodiment, the concentration of the I21 B. velezensis in the composition of the present disclosure may be in an effective range, preferably between 1.0 x 106 CFU/kg substrate and 1.0 x 1011 CFU/kg substrate, more preferably between 1.0 x 107 CFU/kg substrate and 1.0 x 1011 CFU/kg substrate and even more preferably between 1.0 x 108 CFU/kg substrate and 1.0 x 1011 CFU/kg substrate. [0083] In a preferred embodiment, the composition may comprise 0.1-10 % (w/w) of the I21 B. velezensis, more preferably between 0.5-8 % (w/w), and even more preferably between 1-3 % (w/w). [0084] In an embodiment, any plant species may be treated with the composition. [0085] In another embodiment, the plant treated with the composition may be a horticultural plant.
[0086] In another embodiment, the plant treated with the composition may be a cereal crop. [0087] In an embodiment, the I21 B. velezensis composition may be applied by irrigation. [0088] In an embodiment, the I21 B. velezensis composition may be applied by spraying. [0089] In an embodiment, the I21 B. velezensis composition may be applied by mixing with the soil. [0090] Another embodiment may comprise the treatment of the plant with a single application or repeated applications of the I21 B. velezensis composition. Preferably, as few applications as possible are administered. [0091] In an embodiment, the application of the composition comprising the I21 B. velezensis is not dependent upon whether the plants have received, or not received prior treatment with other PBs of whatever class. [0092] In an embodiment, the composition comprising the I21 B. velezensis may be applied at any stage of plant development. [0093] In a preferred embodiment, at least a first application of the I21 B. velezensis composition to the plant takes place at the vegetative growth stage. Examples 1. Isolation of I21 B. velezensis from Pyrus communis cultivar Rocha [0094] Secondary shoots from a fire blight symptomatic pear orchard (Pyrus communis cultivar Rocha) were collected from the Oeste region in Portugal. The plant stems were stamped onto Nutrient Agar (NA, Biolife) and King’s B (KB) agar media. Morphologically different bacteria were selected and re- streaked several times to obtain single colonies. Genomic DNA extraction was performed using a quick DNA extraction protocol in which individual bacterial colonies were re-suspended in 500 μL of 0.9 % (w/v) NaCl, followed by centrifugation at 12,000 xg for 3 min at room temperature. The supernatant was discarded, the pellet re-suspended in 100 μL NaOH solution (20 mM) and vortexed for 1 min in a turbo vortex (this step was repeated twice). The extraction solution was incubated at 100 °C for 5 min, with two cycles of turbo vortexing for 2 min, and then sonication on a water bath for 1 min. Stocks for long term storage were prepared in a final volume of 25 % (v/v) glycerol and kept at -80 °C. To molecularly identify the bacterial species isolated, a partial sequence of the 16S rRNA gene (1.5 Kb length) was amplified and sequenced. For that, a volume of 1 μL of gDNA was used for PCR. The PCR amplification step was as follows: 3 min of denaturation at 94 °C, followed by 35 cycles (each 30 sec) of denaturation at 94 °C, 20 sec annealing at 55 °C and 1:10 min of extension at 72 °C, finishing with an extension step of 5 min at 72 °C. A 1 % (v/v) agarose electrophoresis was performed to confirm amplification of the PCR product. PCR products were purified with a NZYGelpure kit (NZYTech, Lisbon) and Sanger sequenced with the same primers used for amplification (StabVida, Caparica). The sequences were manually
trimmed, searched against publicly available databases and identified as Bacillus sp. To further characterise the strain, two Bacillus genes rpoB (SEQ ID No 28) (Rooney, 2009) and gyrA (SEQ ID No 29) (Rooney, 2009) were amplified and sequenced. The database search and phylogenetic analysis revealed that the strain belongs to the genus Bacillus velezensis, having high sequence similarity to strain ZeaDK315Endobac16, with the GenBank accession number CP043809.1. 2. Indole-acetic-acid (IAA) production [0095] Auxins are hormones that enhance plant root growth, hence improving crop growth and productivity. Microbes that produce auxins can help plants improve biotic and abiotic stress tolerance. Fig.1 shows the quantification of auxin production of I21 B. velezensis. The methodology used for auxin quantification was an adapted colorimetric procedure (Gilbert, 2018). I21 was incubated in liquid Luria Broth (LB) media at 28 °C with 180 rpm during 16 h. The OD600 was then adjusted to 0.05 and the inoculum supplemented with L-tryptophan (5 mg/mL). A volume of 200 μL of inoculum was added onto a 96-well plate and incubated at 28 °C at 180 rpm for 24 h. I21 B. velezensis bacterial culture was centrifuged at 3,000 rpm for 30 min and 100 μL of supernatant was transferred into a fresh 96-well plate. A 1:1 (v/v) solution of Salkowski’s reagent (34.3 % (v/v) perchloric acid and 10 mM FeCl3) was added to each well and the plate incubated for 30 min at room temperature and protected from light. The colour change was recorded at 530 nm in a spectrophotometer. An IAA standard curve was produced in a range of (1-0 μg/mL). Sterile Luria broth (LB) supplemented with 5 mg/mL L-tryptophan was used as control and its relative IAA concentration was subtracted from the values obtained for the bacterial isolate. The amount of IAA produced was obtained by comparison with the IAA standard curve. 3.1-Aminocyclopropane-1-carboxylic acid (ACC) deaminase activity [0096] Ethylene is a hormone that mediates plant response to environmental stresses. However, when produced above a certain threshold, ethylene hinders plant growth and development. Bacteria that produce 1-aminocyclopropane-1-carboxylate deaminase (ACCd), an enzyme that breaks down an intermediate of ethylene, have the ability to ameliorate the deleterious effects of environmental stresses. Fig.2 shows the quantification of ACC deaminase activity of I21 B. velezensis. The qualitative assessment of ACCd activity was based on a previously developed methodology (Penrose, 2003). I21 B. velezensis was incubated at 28 °C, 180 rpm in liquid LB for 16 h. The pre-inoculum was adjusted to an OD600 of 0.05 in minimal nutrient medium M9 media using ACC as the sole nitrogen and carbon source (M9; 22 mM KH2PO4, 33.7 mM Na2HPO4, 9.35 mM NH4Cl, 8.55 mM NaCl, 1 mM MgSO4, 0.3 mM CaCl2 and 3 mM ACC). A volume of 100 μL of I21 B. velezensis inoculum was added to a 96-well plate and incubated for 48 h. Bacterial growth indicates ACCd activity and was recorded by spectrophotometric measurement at 600 nm (OD600). A blank control was also used to normalise the data. Bacterial growth
was measured by an increase in optical density measurement at 600 nm over time, thereby confirming ACCd activity. 4. Catalase activity [0097] Catalase is an enzyme that acts as an antioxidant by neutralising the harmful effect of non- radical reactive oxygen species (ROS). It acts by converting ROS, such as the case of hydrogen peroxide, into water and oxygen. Bacteria that produce catalase have a greater chance to survive in environments such as the soil ecosystem due to the capacity to protect themselves against oxidative stress. Also, PBs that produce this enzyme indirectly promote plant growth by protecting the plant against oxidative stresses. Qualitative catalase activity was evaluated by adding fresh single colonies of I21 B. velezensis onto a 3 % (v/v) hydrogen peroxide solution on a sterile slide (Hansen, 1978). Oxygen release observed by effervescence indicated positive catalase activity as shown in Fig.3 for I21 B. velezensis. 5. Irrigation treatment [0098] Fresh LB plates of I21 B. velezensis were used to inoculate 50 mL of LB liquid media. I21 B. velezensis liquid culture was grown overnight at 28 °C with agitation at 220 rpm. The bacterial culture was used as pre-inoculum for a new growth cycle into 2 L baffled Erlenmeyer flasks containing 750 mL of liquid LB. The culture was grown at 28 °C at 150 rpm until reaching an OD600 of 1. I21 B. velezensis bacterial culture was centrifuged at 5000 xg for 10 min. Supernatant was discarded and the bacterial pellet resuspended in the same initial inoculum volume. The mixture was gently homogenised. The treatment applied to the tomato plants, henceforth called “Treatment”, consisted of a solution containing approximately 1 x 109 CFU/mL of I21 B. velezensis bacterial cells. 6. Plant growth [0099] Seeds of Solanum lycopersicum var. cerasiforme, henceforth termed “cherry tomato”, were pre-germinated in distilled water for 4 days in the dark. For the vegetative plant growth stage, seedlings were planted in alveoli filled with Siro substrate. Plants were placed in a climatic chamber at 25 °C under a 16 h light/8 h dark photoperiod. For seedling establishment each alveolus of 100 mL was irrigated with a specific volume of tap water whenever necessary. Treatment, I21 B. velezensis bacterial cells at 1 x 108 – 1 x 1011 CFU/kg substrate, was applied once a week by irrigation with 10 mL of the irrigation treatment. For early vegetative stages, 2-week-old plants grown in alveoli were transplanted to pots of 1L filled with Siro substrate and placed in the greenhouse. Each pot was irrigated with a specific volume of tap water whenever necessary. Treatment, 12.5x108 CFU/kg substrate was applied twice (at the second and fifth week of plant development) by irrigation with a volume of 50 mL. For fruit production, plants were transplanted to pots of 3.5 L filled with Siro substrate and placed in a greenhouse. Each pot was irrigated with a specific volume of tap water whenever necessary. Treatment, I21 at 12.5x108
CFU/kg substrate, was applied twice (at the second and fifth week of development) and once a week (starting from the second week of development) by irrigation with a volume of 100 mL. Fig.4 shows seedling establishment in alveoli after 4 weeks, and Fig. 5 shows plant growth during early vegetative stages (5 weeks). [00100] Seeds of Lactuca sativa var. Gentilina, henceforth termed “lettuce”, were pre-germinated in distilled water for 4 days in the 16h light/8h dark photoperiod. For the vegetative plant growth stage, seedlings were planted in 1 L pots filled with Siro substrate. Treatment, I21 B. velezensis bacterial cells at 1.25 x 1011 CFU/kg substrate, was applied once a week (on the 2nd, 3rd, 4th and 5th week) by irrigation with 50 mL of the irrigation treatment. [00101] Seeds of Oryza sativa L. var. Leonardo, henceforth termed “rice”, were pre-germinated in distilled water for 3 days in the 16h light/8h dark photoperiod. For the vegetative plant growth stage, seedlings were planted in 1 L pots filled with Siro substrate. Treatment, I21 B. velezensis bacterial cells at 1 x 1011 CFU/kg substrate, was applied once a week (on the 1st week or on the 1st and 2nd week) by irrigation with 10 mL of the irrigation treatment. [00102] Seeds of Triticum var. Roxo, henceforth termed “wheat”, were pre-germinated in distilled water for 1 day in the dark and 3 days in the 16h light/8h dark photoperiod. For the vegetative plant growth stage, seedlings were planted in 1 L pots filled with Siro substrate. Treatment, I21 B. velezensis bacterial cells at 1 x 1011 CFU/kg substrate, was applied once a week (on the 1st, 2nd and 3rd week) by irrigation with 10 mL of the irrigation treatment. 7. Colonization of cherry tomato plants with I21 B.velezensis [00103] Tomato plants were collected. Roots were gently washed with water to remove soil particles. Shoots and roots were separated, weighed and surface sterilised by immersion in 75 % ethanol for 1 min with constant shaking. The ethanol was discarded followed by 3 successive washes during 1 min with constant shaking with sterile distilled water. Plant material was ground with a mortar and pestle in 0.45 % NaCl. Several dilutions of the mixture were done and 100 µL of each were spread in LB agar plates. Technical replicates were done for each dilution. LB agar plates were incubated at 28 °C for 24 h to 48 h. Bacillus-like colonies were counted and the colony forming units (CFUs) were calculated using the following formula . As shown in Fig. 7, plants treated with I21 B. velezensis contain viable bacteria in both shoots and roots, indicating colonisation ability in horticultural plants. 8. Plant growth evaluation [00104] Plant biomass is the weight of living plant material. Plant biomass and morphological parameters are indicators of plant growth and development. Shoot dry weight, root dry weight and
shoot height were measured. Shoots were separated from the roots and dried at 60 °C for at least 24 h. After that period, shoot dry weight was evaluated. Statistical analyses were performed using R version 4.3.2. Continuous variables were subjected to hypothesis testing using either One-way analysis of variance (ANOVA), Welch's ANOVA, or the Kruskal-Wallis test, based on the assumptions of normality (Shapiro's test) and heteroscedasticity (Levene's test). For count variables, Quasi-Poisson generalised linear models were employed to compare means between treatments. The null hypothesis of equal means was rejected when the p-values were below the significance thresholds: • p-value ≤ 0.10, * p- value ≤ 0.05, ** p-value ≤ 0.01, *** p-value ≤ 0.001. Fig.4 and Fig.5 show the growth and development of tomato plants treated with I21 B. velezensis demonstrated by the shoot height and shoot and root dry weights, normalized by the control. [00105] Lettuce plants were cultivated to produce fresh leaves biomass. Shoot fresh weight was measured as an indicator of plant growth and development. Statistical analyses were performed using R version 4.3.2. Continuous variables were subjected to hypothesis testing using either One-way analysis of variance (ANOVA), Welch's ANOVA, or the Kruskal-Wallis test, based on the assumptions of normality (Shapiro's test) and heteroscedasticity (Levene's test). For count variables, Quasi-Poisson generalised linear models were employed to compare means between treatments. The null hypothesis of equal means was rejected when the p-values were below the significance thresholds: • p-value ≤ 0.10, * p- value ≤ 0.05, ** p-value ≤ 0.01, *** p-value ≤ 0.001. Fig.9 shows the shoot fresh weight increment of lettuce plants treated with I21 B. velezensis, normalized by the control. [00106] For cereals (rice and wheat), shoot dry weight, shoot height and the number of tillers (for wheat) were measured. Shoots were separated from the roots and dried at 60 °C for at least 24 h. After that period, shoot dry weight was evaluated. Statistical analyses were performed using R version 4.3.2. Continuous variables were subjected to hypothesis testing using either One-way analysis of variance (ANOVA), Welch's ANOVA, or the Kruskal-Wallis test, based on the assumptions of normality (Shapiro's test) and heteroscedasticity (Levene's test). For count variables, Quasi-Poisson generalised linear models were employed to compare means between treatments. The null hypothesis of equal means was rejected when the p-values were below the significance thresholds: • p-value ≤ 0.10, * p-value ≤ 0.05, ** p-value ≤ 0.01, *** p-value ≤ 0.001. Fig.10 shows the growth and development of rice and wheat plants treated with I21 B. velezensis demonstrated by the shoot height and shoot and root dry weights, normalized by the control. [00107] In lettuce treated with I21 B. velezensis, plant growth was increased by 19.33%; while a commercially available reference product NOVASTIM® provided a significantly lower increase of 9.3% when applied under the recommended conditions. 9. Fruit production evaluation
[00108] Tomato plants were cultivated to produce edible fruits. After collecting the fruits, the total fruit mass per plant was measured. The number of fruits in each plant was also recorded. Statistical analyses were performed using R version 4.3.2. Continuous variables were subjected to hypothesis testing using either One-way analysis of variance (ANOVA), Welch's ANOVA, or the Kruskal-Wallis test, based on the assumptions of normality (Shapiro's test) and heteroscedasticity (Levene's test). For count variables, Quasi-Poisson generalised linear models were employed to compare means between treatments. The null hypothesis of equal means was rejected when the p-values were below the significance thresholds: • p-value ≤ 0.10, * p-value ≤ 0.05, ** p-value ≤ 0.01, *** p-value ≤ 0.001. Fig.6 shows the number of fruits and total fruit mass per plant in cherry tomato plants treated with I21 B. velezensis, normalized by the control. 10. Plant photosynthetic pigments and flavonols quantification [00109] Pigments may have physiological and/or biological functions in plants. Chlorophyll a (henceforth termed ‘Ca’), Chlorophyll b (henceforth termed ‘Cb’), Chlorophyll a+b (henceforth termed ‘Ca+b’) and carotenoids were quantified. Two leaf disks of 1 cm diameter were placed in 95 % (v/v) ethanol in the dark at 4 °C for at least 24 h, then a volume of 150 μL was added to a 96-well plate and chlorophyll and carotenoid content was recorded by spectrophotometric measurements at 470, 649 and 664 nm (Lichtenthaler and Buschmann, 2001). Fig. 8 shows the photosynthetic pigments, Ca, Cb, Ca+b and carotenoids, of cherry tomato plants treated with I21 B. velezensis strain during the vegetative growth stage, normalized by the control. The chlorophyll content index (henceforth termed “CCI”), anthocyanins (henceforth termed “AntM”) and flavonols (henceforth termed “FlavM”) were quantified using the MPM-100 Multi-Pigment-Meter (Opti-Sciences). Fig.9 shows the CCI, AntM and FlavM content of cherry tomato plants treated with I21 B. velezensis until fruit production. Statistical analyses were performed using R version 4.3.2. Continuous variables were subjected to hypothesis testing using either One-way analysis of variance (ANOVA), Welch's ANOVA, or the Kruskal-Wallis test, based on the assumptions of normality (Shapiro's test) and heteroscedasticity (Levene's test). For count variables, Quasi-Poisson generalised linear models were employed to compare means between treatments. The null hypothesis of equal means was rejected when the p-values were below the significance thresholds: • p-value ≤ 0.10, * p-value ≤ 0.05, ** p-value ≤ 0.01, *** p-value ≤ 0.001. 11. Photosynthetic quantum yield quantification [00110] Photosynthesis is the process by which plants use light as a source of energy. The photosynthetic efficiency defined by the ratio of the number of photons emitted to the number of photons absorbed was quantified through photosynthetic quantum yield, using a FluorPen (Photon Systems Instruments). Measurements were performed in the terminal leaflet of the 3rd youngest leaf. Statistical analyses were performed using R version 4.3.2. Continuous variables were subjected to
hypothesis testing using either One-way analysis of variance (ANOVA), Welch's ANOVA, or the Kruskal- Wallis test, based on the assumptions of normality (Shapiro's test) and heteroscedasticity (Levene's test). For count variables, Quasi-Poisson generalised linear models were employed to compare means between treatments. The null hypothesis of equal means was rejected when the p-values were below the significance thresholds: • p-value ≤ 0.10, * p-value ≤ 0.05, ** p-value ≤ 0.01, *** p-value ≤ 0.001. As shown in Fig.10 and Fig.11, the photosynthetic quantum yield of cherry tomato plants treated with I21 B. velezensis, normalized by the control, was greater in the vegetative stage and in the fruit production stage. 12. Effect on abiotic stress [00111] Biostimulant activity can enhance abiotic stress effects mitigation, by improving plant growth under unfavourable conditions. Salt stress treatment (30 g/L NaCl during the 3rd week of growth) was applied to tomato plants, growing in pots of 1L filled with Siro substrate placed on the greenhouse. Treatment, I21 at 12.5x1011 CFU/kg substrate, was applied twice (at the second and third week of development) with a volume of 40 mL. Fig.8 shows tomato plants recovery after salt stress treatment. [00112] Biostimulant activity can promote the upregulation of specific genes, including SODCC.1 (SUPEROXIDE DISMUTASE COPPER CHAPERONE 1) (Acession number: Solyc01g067740; SGN - Solanaceae Genomics Network) and Pr-1A (PATHOGENESIS-RELATED PROTEIN 1A) (Acession number: AJ011520; GenBank) under conditions of stress. The transcriptional activation of SODCC.1, which enhances the plant's capacity to mitigate oxidative stress through reactive oxygen species (ROS) detoxification, concurrently, with the upregulation of Pr-1A provides a robust mechanism for maintaining cellular integrity and physiological functions in plants exposed to salt stress, by activating Systemic Acquired Resistance (SAR) responses. To access gene expression quantification samples were grinded in liquid nitrogen in a sterilized mortar and turned into a fine powder using a pestle. RNA was extracted using the Spectrum Plant Total RNA kit (STRN250-1KT, Sigma), according to the manufacturer’s instructions. cDNA synthesis was performed using the SuperScript IV first-strand synthesis system, according to the manufacturer’s instructions. Gene expression quantification was accessed using the Supreme NZY taq II 2x Green master mix. Samples with the mix were pooled in a 96- well qPCR plate and measured in CFX Connect TM Real-Time System. Specific primer pairs were used for SODCC.1 (5’CTATTACCGACAAGCAGATTCC3’, SEQ ID NO 1 and 3’AATACCACAAGCAATCCTTCC5’, SEQ ID NO 2) (Della et al., 2022) and Pr-1A (5’TCTTGTGAGGCCCAAAATTC3’, SEQ ID NO 3 and 3’TAGTCTGGCCTCTCGGACA5’, SEQ ID NO 4) (Samaras et al., 2021) transcripts amplification. For a reliable quantitative PCR’s, a reference gene expression, ELONGATION FACTOR (EF) (Acession number: X14449; GenBank) was chosen to normalize the data, using the specific primer pair 5’GGAACTTGAGAAGGAGCCTAAG3’, SEQ ID NO 5 and 3’CAACACCAACAGCAACAGTCT5’, SEQ ID NO 6(Løvdal T. and Lillo C., 2009). The method used to analyze the qPCR data was the relative quantification
method, or 2- ΔΔCT method, where the ΔΔCT value = (Cq Target – Cq Reference) (Livak and Schmittgen, 2001). The normalized data was statistically analysed using t-test at p ≤ 0.05. [00113] an embodiment, the primer sequences can be selected from a list consisting of: Qualifier Seq. Seq. Organism Molecule Molecule Residues ID name name type Type Seq. SODCC.1 Forward synthetic ID. DNA Other DNA CTATTACCGACAAGCAGATTCC Primer construct NO 1 Seq. SODCC.1 Reverse synthetic ID. DNA Other DNA CCTTCCTAACGAACACCATAA Primer construct NO 2 Seq. Pr-1A Forward synthetic ID. DNA Other DNA TCTTGTGAGGCCCAAAATTC Primer construct NO 3 Seq. Pr-1A Reverse synthetic ID. DNA Other DNA ACAGGCTCTCCGGTCTGAT Primer construct NO 4 Seq. Elongation Factor synthetic ID. DNA Other DNA CAACACCAACAGCAACAGTCT (EF) Forward Primer construct NO 5
Qualifier Seq. Seq. Organism Molecule Mo Residues ID lecule name name type Type Seq. Elongation Factor synthetic ID. DNA Other DNA TCTGACAACGACAACCACAAC (EF) Reverse Primer construct NO 6 13. Solid formulation of I21 B. velezensis [00114] A bacterial suspension was prepared by growing I21 B. velezensis for 72 h at 28 °C with agitation at 200 rpm in 2 L baffled Erlenmeyer flasks containing 1 L of liquid LB, then the growth medium was discarded after centrifugation and the pellet resuspended in sterile water. The aqueous bacterial suspension was used (1 mL/g of solids) to prepare a paste by blending bentonite clay (1:3), soy flour (1:3), and tapioca flour (1:3) to be used as carriers and nutrients to sustain bacterial growth and incorporate bacteria into the soil. Small pellets (Ø = 1-5 mm; average length = 3-10 mm) were prepared by extrusion and dried by using a fluid bed dryer at 40 °C for 20 min, followed by drying at room temperature until reaching a final moisture level that is equal or lower than 10 %. The final bacterial concentration of the solid formulation ranged between 2 × 108 and 1 × 109 CFU/g of formulation. 14. Liquid formulation concentrate of I21 B. velezensis [00115] An I21 bacterial suspension was prepared by growing a 10 mL pre-inoculum of I21 B. velezensis in LB media for 24 h at 28 °C and 200 rpm in a 100 mL Erlenmeyer. Then, the pre-inoculum was used to set the OD of a new LB culture media (100 mL) to 0.1, which was incubated for 72 h at 28 °C and 200 rpm in a 250 mL Erlenmeyer. A 3 % (w/v) potato starch solution was prepared. To make the liquid formulation, 60 mL of potato starch solution was added to 20 mL of agave syrup, 40 mL of deionized water and 60 mL of I21 B. velezensis inoculum and stirred at 400 rpm for 5 min at room temperature. The final concentration of bacteria in the concentrate was 1 x 1011 CFU/mL. Before applying, the concentrate was diluted either 100 or 1,000 times. [00116] In an embodiment, I21 B. velezensis displayed a higher capacity to produce the hormone auxin, known to enhance plant development (Fig.1). [00117] In an embodiment, the disclosed I21 B. velezensis displayed ACC deaminase activity, known to indirectly increase plant tolerance to abiotic stresses (Fig.2).
[00118] In an embodiment, I21 B. velezensis showed catalase activity as observed by bubble production resulting from release of oxygen (Fig.3). This suggests that I21 B. velezensis should survive in harsh environments because of its inherent capacity for auto-protection against oxidative stress. [00119] In an embodiment, plants treated with I21 B. velezensis showed greater seedling establishment (Fig.4) and plant growth at 5 weeks (Fig.5), as well as higher fruit production (Fig.6). [00120] In an embodiment, plants treated with I21 B. velezensis are colonised by the bacteria (Fig.7). [00121] In an embodiment, plants treated with I21 B. velezensis demonstrated superior growth as determined by a significantly higher shoot height and shoot and root dry weights, of tomato plants (Fig. 4 and Fig.5), and a significant shoot fresh weight of lettuce (Fig.11), compared to the control. [00122] In an embodiment, tomato plants treated with I21 B. velezensis show significantly higher levels of Ca, Cb, Ca+b and carotenoids during the vegetative stage compared to the control (Fig.8). [00123] In an embodiment, tomato plants treated with I21 B. velezensis show significantly higher CCI and lower AntM and FlavM content until fruit production compared to control (Fig.8). [00124] In an embodiment, as shown in Fig. 9, the photosynthetic quantum yield of cherry tomato plants treated with I21 B. velezensis was greater in the vegetative and fruit production stages than in the control. [00125] In an embodiment, cherry tomato plants treated with I21 B. velezensis show a higher number of fruits and total fruit mass per plant compared to the control (Fig.6). [00126] In an embodiment, plants treated with I21 B. velezensis after salt stress show significant recovery of biomass growth, determined by a significantly higher shoot height and shoot and root dry weights, compared to the non-treated stressed controls (Fig.10). [00127] In an embodiment, plants treated with I21 B. velezensis after salt stress show significant upregulation of SODCC.1 (superoxide dismutase copper chaperone 1) and Pr-1A (pathogenesis-related protein 1A) genes, compared to the non-treated stressed controls (Fig.17). [00128] In an embodiment, the formulated I21 B. velezensis strain shows equivalent effects on tomato plants’ early vegetative growth (Fig. 13 and Fig. 15), and significantly higher values of fruit production (Fig.14). [00129] The term 'comprising’ whenever used in this document is intended to indicate the presence of stated features, integers, steps, or components, but not to preclude the presence or addition of one or more other features, integers, steps, components, or groups thereof. [00130] All references recited in this document are incorporated herein in their entirety by reference, as if each and every reference had been incorporated by reference individually.
[00131] Those skilled in the art will recognise or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above description, but rather is as set forth in the appended claims. [00132] Where singular forms of elements or features are used in the specification of the claims, the plural form is also included, and vice-versa, if not specifically excluded. For example, the term ‘a bacterium’ or ‘the bacterium’ also includes the plural forms ‘bacteria’ or ‘the bacteria’, and vice versa. In the claims, articles such as ‘a’, ‘an’, and ‘the’ may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include ‘or’ between one or more members of a group are considered satisfied if one, more than one, or all the group members are present in, employed in or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in or otherwise relevant to a given product or process. The invention also included embodiments in which more than one or all of the group members are present in, employed in or otherwise relevant to a given product or process. [00133] Furthermore, it is to be understood that the invention encompasses all variations, combinations and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the claims or from relevant portions of the description is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Furthermore, when the claims recite a composition, it is to be understood that methods of using the composition for any of the purposes disclosed herein are included, and methods of making the composition according to any of the methods of making disclosed herein or other methods known in the art are included, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise. [00134] Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. It is also to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any subrange within the given range, wherein the endpoints of the subrange are expressed to the same degree of accuracy as the tenth of the unit of the lower limit of the range.
[00135] In addition, it is to be understood that any particular embodiment of the present invention may be explicitly excluded from any one or all of the claims. Where ranges are given, any value within the range may be explicitly excluded from any one or all of the claims. Any embodiment, element, feature, application or aspect of the compositions and/or uses and methods of the invention can be excluded from any one or more of the claims. For purposes of brevity, all embodiments in which one or more elements, features, purposes or aspects are excluded are not set out forth explicitly herein. [00136] The above-described embodiments are combinable. [00137] The following claims further set out particular embodiments of the disclosure. References Alenezi, F.N., Slama, H.B., Bouket, A.C., Cherif-Silini, H., Silini, A., Luptakova, L., Nowakowska, J.A., Oszako, T. & Belbahri, L. (2021) Bacillus velezensis: A Treasure House of Bioactive Compounds of Medicinal, Biocontrol and Environmental Importance. Forests,12, 1714. Della Lucia M.C., Baghdadi A., Mangione F., Borella M., Zegada-Lizarazu W., Ravi S., Deb S., Broccanello C., Concheri G., Monti A., Stevanato P. & Nardi S. (2022) Transcriptional and Physiological Analyses to Assess the Effects of a Novel Biostimulant in Tomato. Frontiers in Plant Science.12, 781993. Fan, B., Wang, C., Song, X., Ding, X., Wu, L., Wu, H., Gao, X. & Borriss, R. (2018) Bacillus velezensis FZB42 in 2018: The Gram-Positive Model Strain for Plant Growth Promotion and Biocontrol. Frontiers in Microbiology, 9, 2491. Gharsa, H.B., Bouri, M., Mougou, H.A., Schuster, C., Leclerque, A. & Rhouma, A. (2021) Bacillus velezensis strain MBY2, a potential agent for the management of crown gall disease. PLoS ONE 16(6): e0252823. Gilbert, S., Xu, J., Acosta, K., Poulev, A., Lebeis, S. & Lam, E. (2018) Bacterial Production of Indole Related Compounds Reveals Their Role in Association Between Duckweeds and Endophytes. Frontiers in Chemistry, 6, 265. Hansen, S. L., & Stewart, B. J. (1978) Slide Catalase: A Reliable Test for Differentiation and Presumptive Identification of Certain Clinically Significant Anaerobes. American Journal of Clinical Pathology, 69(1), 36-40. Joly, P., Calteau, A., Wauquier, A., Dumas, R., Beuvin, M., Vallenet, D., Crovadore, J., Cochard, B., Lefort, F. & Berthon, J.-Y. (2021) From Strain Characterization to Field Authorization: Highlights on Bacillus velezensis Strain B25 Beneficial Properties for Plants and Its Activities on Phytopathogenic Fungi. Microorganisms, 9, 1924. Kim, Y.S., Lee, Y., Cheon, W. et al. (2021) Characterization of Bacillus velezensis AK-0 as a biocontrol agent against apple bitter rot caused by Colletotrichum gloeosporioides. Sci. Rep.11, 626. Lichtenthaler, H.K. & Buschmann, C. (2001) Chlorophylls and Carotenoids: Measurement and Characterization by UV-VIS Spectroscopy. Current Protocols in Food Analytical Chemistry, 1: F4.3.1-F4.3.8. Løvdal T. & Lillo C. (2009) Reference gene selection for quantitative real-time PCR normalization in tomato subjected to nitrogen, cold, and light stress. Analytical Biochemistry, 387:2, 238-242. Myo, E.M., Than, W.M., Win, T. & Khai, A.A. (2021) Evaluation of the symbiotic performance of tomato plants inoculated with Bacillus velezensis NKG-2 and challenged with Fusarium wilt pathogen. Archives of Phytopathology and Plant Protection, 54(17-18), 1391.
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