Derivative bacillus strains for high abiotic stress

Abstract

The present invention relates to the provision of bacteria for promoting plant health under abiotic stress conditions. In particular, the present invention relates to the provision of derived Bacillus strains for increasing plant growth at high salinity.

Description

Technical Field

[0001] The present invention relates to providing bacteria for promoting plant health under abiotic stress conditions. In particular, the present invention relates to providing derivative Bacillus strains for increasing plant growth under high salinity. Background Art

[0002] Rapid population growth coupled with climate change poses a huge challenge to global crop production and yields. On the one hand, the demand for agricultural output is increasing, while on the other hand, various biotic and abiotic problems significantly reduce crop yields.

[0003] Soil salinity is an abiotic stress that poses a serious threat to agriculture. Every year, toxic levels of salt in the soil, especially sodium chloride (NaCl), cause a loss in the yields of major crops. About 20% of the world's cultivated area and nearly 50% of irrigated land are affected by increased soil salinity, which poses a serious abiotic stress to plants. The main reason for the increase in salinity in irrigated areas is that soluble salts are carried in irrigation water, which remain in the soil after evaporation and transpiration. Unless these salts are leached from the soil, they will accumulate to levels that inhibit plant growth over time. This will also lead to degradation of soil structure and affect water and root penetration. Typically, plants stressed by NaCl are characterized by slow growth, premature leaf senescence, reduced tillering and reduced yield. Sodium ions (Na + ) are particularly damaging at high cytosolic concentrations in leaf cells, as they interfere with metabolic processes, especially photosynthesis. Therefore, among abiotic stresses, soil salinity and drought are considered major problems.

[0004] Plants and soil microorganisms interact to promote each other's growth and development and to maintain terrestrial ecosystems. Plants can also use these growth-promoting microorganisms as allies to resist abiotic stresses. Notably, plant growth-promoting bacteria (PGPR) can help plants grow in high-salinity soils through multiple mechanisms, such as i) providing compatible solutes / osmoregulatory substances to plants, ii) trapping water molecules within biofilms by improving biofilm formation and root colonization, iii) regulating plant growth by secreting growth regulators (e.g., auxins, gibberellins), and / or iv) balancing K in plants. + and Na + Ion ratio.

[0005] Growth-promoting microorganisms include the genus Bacillus, which are Gram-positive bacteria characterized by a thick cell wall and the absence of an outer membrane. The cell wall of Gram-positive bacteria is mostly composed of peptidoglycan. Gram-positive species are divided into several groups based on their morphological characteristics and biochemical features. The genus Bacillus belongs to the class of spore-forming bacteria. Bacterial spores are one of the most resistant cell types; they can resist many environmental changes, tolerate dry heat and certain chemical disinfectants, and can survive in dry land for years.

[0006] Therefore, biostimulant Bacillus strains that promote plant growth have been used both naturally and commercially as biofertilizers and plant protection agents. However, it is extremely challenging to create Bacillus strains with a phenotype suitable for stimulating plant growth under high soil salinity without a deep understanding of the target genes involved. Typically, a combination of strain mutagenesis, intelligent screening and careful selection of candidate strains are required to identify and isolate candidate derivative strains with the desired phenotype. The candidate derivative strains can then be carefully evaluated at different levels (e.g., in vitro, in vivo / in planta and / or field trials) to determine their performance. Therefore, obtaining derivative Bacillus strains that can promote plant growth under high salinity conditions is a complex task. As the world's population grows and climate change intensifies abiotic stresses on agricultural fields, further development of such Bacillus strains will bring significant economic savings and improve the ability to meet the growing global crop production needs.

[0007] Therefore, there is an unmet need for the supply of improved Bacillus strains to help plants cope with the challenges of abiotic stress and improve their yield under suboptimal growth conditions.

[0008] Therefore, it would be advantageous to provide Bacillus strains that are able to proliferate under abiotic stress conditions, such as high salinity or drought. In particular, it would be advantageous to provide improved Bacillus strains that effectively promote plant health and growth under high salinity conditions. Summary of the invention

[0009] The present invention relates to Bacillus strains that are capable of promoting plant health and growth under conditions of abiotic stress, such as high salinity. In particular, the present invention discloses derivatives of parent Bacillus strains that can be applied to plants or plant habitats to form colonization of beneficial bacteria on and around the plants, providing better conditions for the plants to obtain necessary nutrients from the soil, thereby reducing the stress on the plants and releasing energy for plant growth. The derivative Bacillus strains are prepared by natural methods of classical strain improvement, which exposes the parent Bacillus strain to adaptive evolutionary activities, followed by identification and selection of improved strains. Therefore, the present invention provides improved Bacillus strains to increase the yield of plants or crops under conditions of high salinity.

[0010] Therefore, one object of the present invention relates to providing improved Bacillus strains capable of promoting plant growth and enhancing its resistance under abiotic stress.

[0011] In particular, it is an object of the present invention to provide derived Bacillus strains that increase plant or crop yield under high salinity or drought conditions.

[0012] Therefore, one aspect of the present invention relates to a derivative Bacillus strain or a variant thereof having increased salt tolerance compared to a parent Bacillus strain, wherein the derivative Bacillus strain or the variant thereof is a derivative strain of the parent Bacillus strain.

[0013] Another aspect of the present invention relates to a derivative Bacillus strain or a variant thereof having increased salt tolerance compared to a deposited parent Bacillus strain, the parent Bacillus strain being selected from the group consisting of:

[0014] -DSM34004 (deposited on August 24, 2021),

[0015] -DSM34003 (deposited on August 24, 2021),

[0016] -DSM33240 (deposited on August 14, 2019),

[0017] - DSM 17231 (deposited on April 7, 2005),

[0018] -DSM33110 (deposited on 8 May 2019) and

[0019] - DSM 32324 (deposited on 8 June 2016),

[0020] The deposited strains are deposited by Chr. Hansen GmbH, Hirsholm, Denmark, at the German Collection of Microorganisms and Cell Cultures (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, DSMZ), D-38124 Inhofenstrasse 7B, Brunswick, Germany, and

[0021] The derived Bacillus strain or its variant is a derivative strain of the parent Bacillus strain.

[0022] A further aspect of the invention relates to a method of preparing the derivative Bacillus strain described herein, the method comprising the steps of:

[0023] (i) Providing a parent Bacillus strain

[0024] (ii) growing the parent Bacillus strain under high salinity conditions, and

[0025] (iii) selecting a derivative Bacillus strain having increased salt tolerance that proliferates under the growth conditions of step (ii).

[0026] Yet another aspect of the present invention relates to a derivative Bacillus strain having increased salt tolerance compared to a parent Bacillus strain, which strain is obtainable by the method described herein.

[0027] Still another aspect of the present invention relates to a composition comprising a derivative Bacillus strain having increased salt tolerance compared to the parent Bacillus strain described herein.

[0028] Still further aspects of the invention relate to a plant or seed coated with a composition as described herein.

[0029] Even further aspects of the invention relate to a method of increasing resistance of a plant to abiotic stress conditions, the method comprising applying to the plant, a part of the plant and / or the habitat of the plant a derivative Bacillus strain or a composition as described herein having increased salt tolerance compared to a parent Bacillus strain.

[0030] Yet another aspect of the present invention relates to the use of a derivative Bacillus strain or a composition as described herein having increased salt tolerance compared to a parent Bacillus strain as a biostimulant, growth promoter and / or for increasing resistance of a plant to abiotic stress conditions.

[0031] Another aspect of the present invention relates to a kit comprising:

[0032] (i) a derivative Bacillus strain, composition or coated plant seed as described herein having increased salt tolerance compared to a parent Bacillus strain;

[0033] (ii) containers; and

[0034] (iii) optionally, instructions for use. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The growth of various Bacillus strains in CSE medium and CSE medium supplemented with NaCl is shown. (A) SA-P4, (B) SA-P3, (C) SA-P5, (D) SA-P7, (E) SA-P8, (F) SA-P9, (G) SA-P1, and (H) SA-P2.

[0036] Figure 2Shown are inhibition curves of several Bacillus strains after supplementation of various concentrations of NaCl in CSE growth medium. The inhibition curves were determined at 47 h of incubation time. (A) SA-P4, (B) SA-P3, (C) SA-P5, (D) SA-P7, (E) SA-P8, (F) SA-P9, (G) SA-P1, and (H) SA-P2.

[0037] Figure 3 The NaCl inhibition concentrations that inhibited the growth of Bacillus strains by 50% (IC50) and 90% (IC90) at 47h of incubation in CSE medium at 250RPM and 30°C are shown. EC: effective concentration. For each Bacillus strain, from left to right: IC50, EC50, IC90 and EC90 are given.

[0038] Figure 4 The growth phenotypes of some selected evolved strains compared to their parent strains under salt stress conditions are shown. (A) SA70 relative to SA-P5, (B) SA63 relative to SA-P1, (C) SA60 relative to SA-P4, (D) SA59 relative to SA-) and (E) SA40 relative to SA-P7. Light grey (open circles): evolved strains, CSE (or low salt). Dark grey (open circles): parent strain, CSE (or low salt). Dark grey (filled circles): evolved strains, high salt. Black (filled circles): parent strain, high salt.

[0039] Figure 5 Osmoregulatory substances produced by the parent Bacillus strain and the SALTY-ALE derivative strain in CSE medium (dark grey bars) and CSE medium supplemented with 0.35 M NaCl (light grey bars), respectively: (A) proline and (B) citrulline. (C) Comparison of extracellular amino acid levels of the SALTY-ALE derivative strain SA60 and its parent strain SA-P4. Cultivation in CSE or CSE-0.35 M NaCl medium was continued for 28 h.

[0040] Figure 6 Shown are: (A) Arabidopsis thaliana seedling germination under salt conditions (90 mM NaCl) with and without supplementation of compatible solutes. (B) Fresh weight of 15-day-old seedlings grown under salt-free or 90 mM NaCl conditions with and without proline supplementation. Five-day-old seedlings were transferred to 1 / 2 MS medium without and with 1 mM proline supplementation.

[0041] Figure 7The effect of derived Bacillus strains on plant growth under salt stress conditions is shown. Corn plants were treated with 150 mM NaCl, supplemented or not with derived Bacillus strains. (A) 8 biological replicates were used for each treatment (B) 10 biological replicates were used, and an additional control was used, where the plants were well watered and not supplemented with NaCl.

[0042] The present invention will be described in more detail below. DETAILED DESCRIPTION

[0043] definition

[0044] Before outlining the present invention in more detail, a set of terms and conventions are first defined:

[0045] Plant biostimulants

[0046] In the context of the present invention, the term "plant biostimulant" refers to any substance or microorganism applied to plants that has the ability to enhance nutrient efficiency, abiotic stress tolerance and / or crop quality traits, regardless of its nutritional content. Further, plant biostimulants also refer to commercial products containing mixtures of such substances and / or microorganisms.

[0047] Plant growth promoter

[0048] In the context of the present invention, the term "plant growth promoter" or "plant growth promoting microorganism" refers to a microorganism that has the ability to colonize the roots and / or internal tissues of the plant and promotes plant growth and health by acting as a biofertilizer, biostimulant or through biological control of plant diseases.

[0049] Parent strain

[0050] In the context of the present invention, the term "parent strain" refers to the microorganism that is the origin of one or more derived strains, ie it refers to the first generation that produces one or more generations of offspring.

[0051] Derivative strains

[0052] In the context of the present invention, the term "derivative strain" refers to a second generation microorganism derived from a parent strain. Derivative strains can be produced by mutagenesis, wherein one or more mutations are introduced into the genome of the parent strain. These mutations can be introduced via laboratory adaptive evolution, which is exposing the parent strain to gradually increasing concentrations of salt, such as NaCl.

[0053] Identification features

[0054] In the context of the present invention, the term "identifying characteristic" refers to the phenotype of a microorganism, i.e. a set of observable characteristics or traits of the microorganism. In particular, an identifying characteristic may be increased salt tolerance.

[0055] Microorganisms that share all identifying characteristics may also have different, non-identical genomic sequences. This may be the case if the mutation is a silent mutation or a conservative mutation, i.e., the new codon produces the same amino acid or the new amino acid has similar biochemical properties (e.g., charge or hydrophobicity), respectively.

[0056] Salt tolerance

[0057] In the context of the present invention, the term "salt tolerance" refers to the concentration of salt (eg NaCl) required to inhibit the proliferation of a cell population (eg Bacillus strain) by 50%. This is also known as the half maximal inhibitory concentration (IC50).

[0058] “Salt tolerance” can be measured against typical salts found in irrigation water, including sodium chloride (NaCl), sodium sulfate (Na2SO4), sodium bicarbonate (NaHCO3), magnesium sulfate (MgSO4), calcium sulfate (CaSO4), calcium chloride (CaCl2), potassium chloride (KCl), and potassium sulfate (K2SO4).

[0059] Preferably, "salt tolerance" is measured against NaCl.

[0060] Osmotic regulating substances

[0061] In the context of the present invention, the term "osmotic regulating substances" refers to highly water-soluble and low molecular weight organic compounds that help plants resist osmotic imbalance. They can protect the integrity of cells by affecting the viscosity, melting point and / or ionic strength of biological fluids. In particular, osmotic regulating substances can relieve cell swelling caused by osmotic pressure by carrying water through membrane channels.

[0062] Osmotic regulating substances can be classified into three major groups: amino acids, quaternary and tertiary onium compounds, and polyols / small molecule sugars. Examples of osmotic regulating substances include, but are not limited to, proline, citrulline, spermidine, glycine betaine, glycerol, mannitol, sorbitol, trimethylamine N-oxide (TMAO), dimethylmercaptopropionic acid, sarcosine, betaine, glycerophosphorylcholine, inositol, taurine, and glycine.

[0063] In certain contexts, an osmotic regulating substance may also be referred to as a “compatible solute.” Thus, the terms “osmotic regulating substance” and “compatible solute” may be used interchangeably herein.

[0064] Variant or variant strain

[0065] In the context of the present invention, the term "variant" or "variant strain" refers to a bacterial strain that is functionally equivalent to the bacterial strain of the present invention, for example, having substantially the same or improved properties (for example in terms of salt tolerance). Such variants can be identified using appropriate screening techniques and are a part of the present invention.

[0066] The term "variant" or "variant strain" refers to a strain derived or derivable from a strain of the present invention by mutagenesis (eg genetic engineering, laboratory adaptive evolution, physical and / or chemical treatment).

[0067] In particular, the term "variant" or "variant strain" refers to a strain obtained by subjecting the strain of the invention to any conventionally used mutagenic treatment, including classical strain improvement, treatment with chemical mutagens (e.g. ethane methane sulfonate (EMS) or N-methyl-N'-nitro-N-nitroguanidine (NTG)), UV light treatment, or to a spontaneously generated mutant. A variant or variant strain may have been subjected to several mutagenic treatments (a single treatment is understood to be a mutagenic step followed by a screening / selection step), but it is currently preferred to perform no more than 20, or no more than 10, or no more than 5 treatments (or screening / selection steps).

[0068] In currently preferred variants or variant strains, less than 5% or less than 1% or even less than 0.1% of the nucleotides in the bacterial genome have been modified by another nucleotide or have been deleted compared to the parent strain.

[0069] The terms "variant" and "variant strain" are used interchangeably herein.

[0070] Nomenclature of mutations

[0071] In the context of the present invention, the conventional single-letter and three-letter codes for amino acid residues are used.For ease of reference, the amino acid changes in the variants of the present invention are described by using the following nomenclature: amino acid residue in the parent enzyme; position; substituted amino acid residue.

[0072] According to this nomenclature, for example, the substitution of an alanine residue with a glycine residue at position 20 is represented as Ala20Gly or A20G. At the same position, the deletion of alanine is represented as Ala20* or A20*. The insertion of an additional amino acid residue (e.g., glycine) is represented as Ala20AlaGly or A20AG. The deletion of a continuous stretch of amino acid residues (e.g., between an alanine at position 20 and a glycine at position 21) is represented as δ(Ala20-Gly21) or δ(A20-G21). When the parent enzyme sequence contains a deletion compared to the enzyme sequence used for numbering, an insertion at such a position (e.g., an alanine is inserted at position 20 of the deletion) is represented as *20Ala or *20A. Multiple mutations are separated by a plus sign or a slash. For example, two mutations at positions 20 and 21, replacing alanine and glutamic acid with glycine and serine, respectively, are represented as A20G+E21S or A20G / E21S. When an amino acid residue at a given position is substituted with two or more alternative amino acid residues, these residues are separated by commas or slashes. For example, the alanine at position 30 is substituted with glycine or glutamic acid, which is represented as "A20G, E", or "A20G / E", or "A20G, A20E".

[0073] When a position suitable for modification is identified herein, but no specific modification is mentioned, it is understood that the amino acid residue present at that position can be substituted with any amino acid residue. Thus, for example, when the alanine at position 20 is mentioned for modification but not specified, it is understood that the alanine can be deleted or substituted with any other amino acid residue (i.e., any of R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, V).

[0074] mutation

[0075] In the context of the present invention, the term "mutation" refers to a change in the nucleotide sequence of the genome of an organism, resulting in a change in the phenotype of the organism, wherein the change may be a deletion of a nucleotide, a substitution of a nucleotide by another nucleotide, an insertion of a nucleotide or a frameshift.

[0076] Deletion is understood as a genetic mutation resulting in the removal of one or more nucleotides in the nucleotide sequence of the genome of an organism; insertion is understood as the addition of one or more nucleotides to a nucleotide sequence; substitution (or point mutation) is understood as a genetic mutation in which a nucleotide in a nucleotide sequence is replaced by another nucleotide; frameshift is understood as a genetic mutation caused by the insertion or deletion of a certain number of nucleotides in the nucleotide sequence that is not divisible by three, thereby changing the reading frame and resulting in a translation that is completely different from the original reading frame; introduction of a stop codon is understood as a point mutation in the DNA sequence resulting in a premature stop codon; inhibition of substrate binding of the encoded protein is understood as any mutation in the nucleotide sequence that results in a change in the protein sequence, thereby preventing the binding of the substrate to its protein catalytic site.

[0077] Furthermore, a knockout mutant is to be understood as a genetic mutation which results in the removal or deletion of a gene (such as an entire gene or an entire open reading frame) from the genome of an organism.

[0078] In the present specification and claims, the conventional single-letter code for nucleotides is used, following similar principles to the amino acid nomenclature described above.

[0079] about

[0080] Wherever the term "about" is used herein in the context of an amount (e.g., an absolute amount, such as number, purity, concentration, weight, size, etc.), or a relative amount (e.g., a percentage, equivalent, or ratio), a time frame, and a parameter (e.g., temperature, pressure, etc.), it is understood that such variables are approximate and therefore may vary by ±10%, such as ±5%, preferably ±2% (e.g., ±1%) from the actual number specified. This is true even if such a number is initially presented as a percentage (e.g., 'about 10%' may mean ±10% of the number 10, i.e., anywhere between 9% and 11%).

[0081] Sequence identity

[0082] In the context of the present invention, the term "sequence identity" is defined herein as sequence identity between proteins at the amino acid level. When sequences are aligned, protein sequence identity can be determined by comparing the amino acid sequence at a given position in each sequence.

[0083] To determine the percent identity of two amino acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of the first amino acid for optimal alignment with the second amino acid sequence). Then, the amino acid residues at corresponding amino acid positions are compared. When a position in the first sequence is occupied by the same amino acid residue as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions (e.g., overlapping positions) x 100).

[0084] In one embodiment, the two sequences are of the same length. In another embodiment, the two sequences are of different lengths, and gaps are considered as different positions.

[0085] The sequences can be aligned manually and the number of identical amino acids calculated. Alternatively, a mathematical algorithm can be used to align two sequences to determine the percent identity. This algorithm is incorporated into the XBLAST program (Altschul et al. 1990). BLAST protein searches can be performed using the XBLAST program (score = 50, word length = 3) to obtain amino acid sequences homologous to protein molecules of the invention.

[0086] In order to obtain gap comparisons for comparison purposes, gap BLAST can be utilized. Alternatively, PSI-Blast can be used for iterative searches, which detect distant relationships between molecules. When using XBLAST and gap BLAST programs, the default parameters of the corresponding programs can be used. See http: / / www.ncbi.nlm.nih.gov. Alternatively, after the sequences are compared, for example, by the BLAST program (www.ncbi.nlm.gov / cgi-bin / BLAST) in the EMBL database, sequence identity can be calculated. Typically, comparisons can be performed using the default settings of aspects such as "scoring matrix" and "gap penalty".

[0087] The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. When calculating percent identity, only exact matches are counted.

[0088] Derived Bacillus strains

[0089] Global agriculture faces the challenge of maximizing agricultural production to meet the urgent global demand for large quantities of plants or crops produced under increasingly harsh conditions. In particular, abiotic stresses, such as high salinity and drought, are increasingly frequent events caused by climate change and / or intensive irrigated agriculture, among others.

[0090] The source of salinity problems is irrigation water, where the salts provided by irrigation water accumulate in the soil over time to the point where more salt is provided than the plants can take up. Salts may be leached from the soil as irrigation water or rainwater percolates downward through the soil, including salts and organic matter. However, leaching may be inhibited by high clay content or soil compaction, where water remains at the surface and evaporates, depositing salts but not leaching undissolved salts below the root zone. Thus, high salinity problems are more severe in arid or semi-arid regions or under drought conditions. If the salt concentration in the soil is higher than that in the plant, there will be a net flow of water from the plant to the soil, which will inhibit plant growth or eventually kill the plant.

[0091] Strategies exist that can mitigate the negative effects of high-salinity soils in agricultural fields. These include improving drainage, leaching (e.g. by increasing irrigation with water lacking salt), and reducing surface water evaporation. However, none of these strategies can eliminate the challenges posed by high soil salinity. It is important to note that tillage is a non-permanent treatment to the soil and may temporarily increase drainage, but the soil is likely to reseal. Leaching is only effective if the soil is well-drained, and even then it is an expensive and environmentally unfriendly procedure that requires large amounts of water. Evaporation can be reduced by covering the soil with residues or mulch, but this has economic disadvantages and risks creating anaerobic environments that favor the development of fungal diseases of plant stems and / or roots. Therefore, solutions are needed that enable cultivation under abiotic stresses, such as high salinity or drought.

[0092] Bacillus strains can be used in biological agriculture, with different benefits depending on the bacterial strain used. The biological agriculture or food industry can rely on the natural method of classical strain improvement (CSI) technology to create improved strains and products. This method is based on the introduction of random mutations into the parent strain, followed by screening and selection of improved variants. The mutations in classical strain technology are random in nature and can be natural or induced. Therefore, compared with modern site-directed genome engineering (such as CRISPR, which only affects specific target genes), the entire genome of the parent strain is probed. The benefit is that improved complex phenotypes that may be controlled by interactions between multiple genes can be identified. In the absence of a deep understanding of the genome of the parent strain, this type of improved complex phenotype is unlikely to be identified by specific genomic replacement. In addition, strains developed by classical strain improvement methods are considered non-genetically modified organisms (GMOs), which eliminates commercial barriers such as strict GMO regulations in the EU.

[0093] The derived Bacillus strains disclosed herein are obtained by mutagenizing parent strains with desired traits through laboratory adaptive evolution (ALE) activities. ALE is used to improve strain performance and stability by (accumulating) beneficial mutations. In this method, microbial strains are cultured under specific growth conditions for a prolonged period of time, ranging from weeks to months or years, and cells are regularly passaged in fresh growth medium. Under the guidance of a range of growth conditions, microbial strains will adapt and accumulate beneficial mutations as part of a natural evolutionary program. It is worth noting that ALE activities can also induce whole genome mutations, which contribute to the adaptability and growth of microbial strains.

[0094] Therefore, mutagenesis can be achieved through an ALE campaign, in which a parental Bacillus strain is exposed to stress conditions (e.g., elevated salt levels) to purposefully introduce mutations into the genome. Hence, this evolution campaign is referred to as salt tolerance laboratory adaptive evolution (SALTY-ALE).

[0095] Derivatives obtained from a parent Bacillus strain that have been induced to have increased salt tolerance and the ability to promote plant health under abiotic stress conditions (eg, high salinity or drought) are disclosed herein.

[0096] Therefore, one aspect of the present invention relates to a derivative Bacillus strain or a variant thereof having increased salt tolerance compared to a parent Bacillus strain, wherein the derivative Bacillus strain or the variant thereof is a derivative strain of the parent Bacillus strain.

[0097] It should be understood that derivative Bacillus strains with identical or similar phenotypes also form a part of the present invention. These strains can be obtained by the methods described herein, or obtained by further evolution of the derivative strains disclosed herein, producing new variants with identical or similar phenotypes. Such strains can be said to have all the identification characteristics of the derivative strains disclosed herein. Therefore, microorganisms with all identification characteristics can also have different, inconsistent genome sequences. Identification characteristics can include but are not limited to increased salt tolerance and / or an increase in the output of one or more osmotic regulating substances.

[0098] Thus, one embodiment of the present invention relates to a derivative Bacillus strain or variant thereof having all of the identifying characteristics of the strain, with increased salt tolerance compared to the parent Bacillus strain.

[0099] The main reason for increased soil salinity is the use of irrigation water, which carries soluble salts that remain in the soil after evaporation and transpiration. High salinity conditions are exacerbated during dry periods or even droughts. Typical salts found in irrigation water include sodium chloride (NaCl), sodium sulfate (Na2SO4), sodium bicarbonate (NaHCO3), magnesium sulfate (MgSO4), calcium sulfate (CaSO4), calcium chloride (CaCl2), potassium chloride (KCl) and potassium sulfate (K2SO4). In particular, high concentrations of sodium chloride can be harmful to plant health.

[0100] One embodiment of the present invention relates to the derivative Bacillus strain described herein, wherein the derivative Bacillus strain has increased salt tolerance compared to the parent Bacillus strain when cultured under the same conditions.

[0101] Parent Bacillus strains may have different baseline salt tolerance, i.e., resistance to elevated salt concentrations before they adapt through evolutionary activity. However, parent Bacillus strains with lower baseline salt tolerance may be a desirable starting point because they have other desirable traits, such as enhanced biostimulant properties. Therefore, for some applications, it is valuable to assess improved salt tolerance as a fold change compared to baseline salt tolerance.

[0102] Therefore, one embodiment of the present invention relates to the derivative Bacillus strain described herein, wherein the salt tolerance of the derivative Bacillus strain is at least increased by 2.5 times compared to the parent Bacillus strain, such as at least increased by 3 times, such as increased by 3.5 times, such as increased by 4 times compared to the parent Bacillus strain.

[0103] For other applications, it may be contemplated that the derived Bacillus strain will be exposed to a given concentration of salt for an extended period of time, or with sufficient frequency to ensure a target absolute salt tolerance.

[0104] Therefore, one embodiment of the present invention relates to the derived Bacillus strain described herein, wherein the derived Bacillus strain has a salt tolerance of at least 0.8 M NaCl, such as at least 0.9 M NaCl, such as at least 1.0 M NaCl, such as at least 1.1 M NaCl, such as at least 1.2 M NaCl, such as at least 1.3 M NaCl, such as at least 1.4 M NaCl, such as at least 1.5 M NaCl.

[0105] It is understood that the salt tolerance of a derived Bacillus strain is the concentration at which proliferation of the cell population is inhibited by 50% (IC50). The IC50 can be determined as described in Example 1.

[0106] Another embodiment of the present invention relates to the derived Bacillus strain described herein, wherein the salt tolerance is defined by a half maximal inhibitory concentration (IC50).

[0107] IC50 values ​​can be determined by inoculating a Bacillus culture in a liquid growth medium supplemented with a predetermined concentration of NaCl followed by incubation. Optical density measurements of cultures incubated at different NaCl concentrations are collated and plotted as a growth curve, which can then be converted into an inhibition curve from which the IC50 value can be read based on the fit results.

[0108] Additional embodiments of the present invention relate to the derived Bacillus strains described herein, wherein the IC50 is measured as described in Example 1.

[0109] For some Bacillus strains, a small amount of NaCl is added to enhance growth, which will result in an inhibition curve shifting to a negative absolute inhibition value at low NaCl concentrations. In these cases, an effective concentration (EC) can be used if necessary. EC50 is the NaCl concentration that produces a half-maximal response.

[0110] Therefore, one embodiment of the present invention relates to the derived Bacillus strains described herein, wherein the salt tolerance is defined by EC50.

[0111] The salt tolerance of the derived Bacillus strain should be distinguished from the actual salt level in the plant's habitat (e.g., soil). The salt level in the soil is lower than the salt level at which the derived Bacillus strain salt tolerance is measured because the plant cannot survive at these elevated salt levels. However, providing the derived Bacillus strains described herein with increased salt tolerance is beneficial to the health of plants grown in habitats with increased salt levels.

[0112] The inventors have identified several derivative Bacillus species and strains thereof that are suitable as parent strains for salt evolution activities. These strains not only naturally possess sufficient baseline salt tolerance, but also possess properties that are useful for the role of these strains as biostimulants and / or biopesticides to promote plant health.

[0113] Thus, one embodiment of the present invention relates to the derived Bacillus strain described herein, wherein the parent Bacillus strain belongs to a species selected from the group consisting of Bacillus velezensis, Bacillus paralicheniformis, Bacillus amyloliquefaciens and Bacillus subtilis.

[0114] Another embodiment of the present invention relates to the derived Bacillus strain described herein, wherein the parent Bacillus strain is a deposited strain selected from the group consisting of:

[0115] -DSM34004 (deposited on August 24, 2021),

[0116] -DSM34003 (deposited on August 24, 2021),

[0117] -DSM33240 (deposited on August 14, 2019),

[0118] - DSM 17231 (deposited on April 7, 2005),

[0119] -DSM33110 (deposited on 8 May 2019) and

[0120] - DSM 32324 (deposited on 8 June 2016),

[0121] The deposited strains are deposited by Chr. Hansen AG, Hirsholm, Denmark at the German Collection of Microorganisms and Cell Cultures (DSMZ), Inhofenstrasse 7B, D-38124 Braunschweig, Germany.

[0122] A preferred embodiment of the present invention relates to the derived Bacillus strain described herein, wherein the parent Bacillus strain was deposited as DSM34004 by Chr. Hansen AG, Hirsholm, Denmark on August 24, 2021 at the German Collection of Microorganisms and Cell Cultures (DSMZ), Inhofenstrasse 7B, D-38124 Braunschweig, Germany.

[0123] SALTY-ALE activity promotes random mutagenesis of the parent strain, followed by screening and selection of improved variants. Thus, the derivative Bacillus strain contains a genome that is different from its parent strain, resulting in an improved phenotype, such as increased salt tolerance.

[0124] Thus, one embodiment of the present invention relates to a derivative Bacillus strain as described herein, wherein the derivative Bacillus strain is genetically different from the parent Bacillus strain.

[0125] Another embodiment of the present invention relates to a derivative Bacillus strain described herein, wherein the derivative Bacillus strain comprises one or more mutations compared to the parent Bacillus strain.

[0126] A mutation is a change in the nucleotide sequence of the genome of an organism that results in a change in the phenotype of the organism, wherein the change can be a deletion of a nucleotide, a substitution of a nucleotide by another nucleotide, an insertion of a nucleotide, or a frameshift. During the SALTY-ALE campaign and subsequent screening and evaluation of evolved Bacillus strains, several target genes were found to be particularly promising for generating derivative Bacillus strains with improved properties.

[0127] One embodiment of the present invention relates to the derived Bacillus strain described herein, wherein the one or more mutations are deletions, substitutions, insertions and / or frameshifts.

[0128] Another embodiment of the present invention relates to the derivative Bacillus strain described herein, wherein one or more mutations are in one or more genes of the parent Bacillus strain selected from the group consisting of:

[0129] -yjcG encoding the putative phosphatase YjcG;

[0130] -comP encoding the sensor histidine kinase ComP;

[0131] -dnaA-1, encoding the chromosome replication initiation protein;

[0132] -sigB, encoding RNA polymerase sigma-B factor;

[0133] - flhB-2 encoding the flagellar biosynthesis protein FlhB;

[0134] -rny encoding ribonuclease Y;

[0135] -ilvE encoding branched-chain amino acid aminotransferase;

[0136] -dcuS-1, encoding the sensor histidine kinase DcuS;

[0137] -rsbT encoding the serine / threonine protein kinase RsbT;

[0138] - fliM encoding the flagellar motor switch protein FliM;

[0139] - mgsR-1, encoding the regulatory protein MgsR;

[0140] - opuE encoding the osmoregulatory proline transporter OpuE;

[0141] - bshA-1 encoding N-acetyl-α-D-glucosaminyl L-malate synthase;

[0142] - metAA encoding homoserine O-acetyltransferase;

[0143] -motB encoding movement protein B;

[0144] -kinA-1 encoding sporulation kinase A;

[0145] -comA, encoding the transcriptional regulator protein comA;

[0146] - rpoC, encoding the β' subunit of the DNA-directed RNA polymerase;

[0147] - eamA-1 encoding a putative amino acid metabolite efflux pump; and

[0148] -rpoB, encoding the β subunit of DNA-directed RNA polymerase.

[0149]

[00136] Additional embodiments of the invention relate to the derived Bacillus strains described herein, wherein the one or more mutations are in one or more genes of the parent Bacillus strain comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1 (yjcG), SEQ ID NO: 2 (comP), SEQ ID NO: 3 (dnaA-1), SEQ ID NO: 4 (sigB), SEQ ID NO: 5 (flhB-2), SEQ ID NO: 6 (rny), SEQ ID NO: 7 (ilvE), SEQ ID NO: 8 (dcuS-1), SEQ ID NO: 9 (rsbT), SEQ ID NO: 10 (fliM), SEQ ID NO: 11 (mgsR-1), SEQ ID NO: 12 (opuE), SEQ ID NO: 13 (bshA-1), SEQ ID NO: 14 (metAA), SEQ ID NO: 15 (motB), SEQ ID NO: 16 (kinA-1), SEQ ID NO: 17 (comA), SEQ ID NO: 18 (rpoC), SEQ ID NO: 19 (rny), SEQ ID NO: 20 (ilvE), SEQ ID NO: 21 (dcuS-1), SEQ ID NO: 22 (rsbT), SEQ ID NO: 23 (fliM), SEQ ID NO: 24 (mgsR-1), SEQ ID NO: 25 NO:19 (eamA-1), SEQ ID NO:20 (rpoB) and combinations thereof.

[0150] Still further embodiments of the invention relate to the derivative Bacillus strains described herein, wherein the nucleic acid sequence of the derivative Bacillus strain has at least 90% sequence identity with the nucleic acid sequence of the parent Bacillus strain, e.g., at least 95% sequence identity, e.g., at least 98% sequence identity, e.g., at least 99% sequence identity with the nucleic acid sequence of the parent Bacillus strain.

[0151] Without being bound by theory, it is contemplated herein that mutations that inactivate or downregulate general stress responses that result in energy expenditure under high salinity concentrations may be beneficial for improving salt tolerance.

[0152] In bacteria, a common way to mediate differential gene expression is the use of alternative sigma factors, which direct RNA polymerase to other gene targets than the housekeeping sigma A factor. In Bacillus (e.g., B. subtilis), one of the most powerful and significant re-regulations of gene expression is directed by the alternative sigma B factor (SigB) in response to different stresses and starvation. The SigB protein is highly conserved in Bacillus and controls the expression of about 150 genes that are involved in adapting cells to a variety of different stresses, such as high salt concentrations, ethanol, nitric oxide, acid, nutrient starvation, and high / low temperature. Due to the broad response to a variety of stresses and the rather non-specific adaptation, it is often referred to as the "general stress response" (GSR).

[0153] The induction of SigB-dependent GSR is a great burden on the cell, since, for example, a large part of the translation machinery is busy producing stress-related proteins. Therefore, Bacillus ensures a strict repression of GSR during the exponential growth phase and a strong and immediate induction if necessary. This tight regulation is achieved via a complex signal transduction cascade involving a multiprotein complex of 25S, called the stresssome. The activity of this multiprotein complex involves the serine kinase RsbT and the protein RsbS, which is activated by RsbT via phosphorylation.

[0154] The secondary stress response of bacteria is to induce sporulation to produce mature and highly resistant spores. Involved in this process is a phosphorylation pathway that initiates a series of events to promote sporulation. The phosphorylation pathway includes a series of sporulation kinases, AEs.

[0155] The inventors have found that strains with mutations that result in impaired GSR and / or sporulation responses have improved salt tolerance. It is contemplated that these strains will develop other traits to enable long-term growth under high salinity conditions, such as (i) SigB-independent upregulation of osmotic regulator uptake, (ii) redirection of amino acid synthesis to osmotic regulator production (e.g., proline), and / or (iii) increased osmotic regulator synthesis.

[0156] Therefore, one embodiment of the present invention relates to the derived Bacillus strain described herein, wherein the one or more mutations comprise at least one or more mutations in sigB, rsbT and / or kinA compared to the parent Bacillus strain.

[0157] Another embodiment of the present invention relates to the derivative Bacillus strain described herein, wherein the one or more mutations comprise at least one or more mutations in sigB compared to the parent Bacillus strain.

[0158] Without being bound by theory, it is also contemplated herein that mutations affecting amino acid synthesis and transport may also result in increased salt tolerance.

[0159] Bacillus species (e.g., B. subtilis) contain transporters for osmoprotective compatible solutes. One of these transporters is OpuE, which facilitates the uptake of proline. Defects in this protein may ultimately lead to reduced protein uptake. Mutations in the ilvE gene, encoding the IlvE protein involved in the synthesis of branched-chain amino acids, and in the rny gene, encoding the endonuclease RNase Y involved in the control of T-box-mediated anabolic proline production, also appear to be associated with salt tolerance. In fact, the inventors found that derived Bacillus strains containing combined mutations in these genes enabled the strains to increase extracellular proline levels under high salinity conditions without showing growth defects (see Example 4 ( Figure 5 A)).

[0160] Furthermore, mutations associated with eamA-1, encoding a putative amino acid metabolite efflux pump, have been identified that play a role in extracellular amino acid levels. Notably, inactivation of the putative amino acid transporter EamA-1 appears to lead to increased extracellular amino acid levels, including proline levels, for example by redirecting amino acid export in the absence of a functional EamA-1 protein (see Example 4 ( Figure 5 C)). These findings are very interesting because a higher efflux of osmoprotective amino acids such as proline could have a growth-promoting effect on plants.

[0161] Therefore, one embodiment of the present invention relates to the derived Bacillus strain described herein, wherein the one or more mutations comprise at least one or more mutations in opuE, ilvE, rny and / or eamA-1 compared to the parent Bacillus strain.

[0162] Another embodiment of the present invention relates to the derivative Bacillus strain described herein, wherein the one or more mutations comprise at least one or more mutations in opuE compared to the parent Bacillus strain.

[0163] Yet another embodiment of the present invention relates to the derivative Bacillus strain described herein, wherein the one or more mutations comprise at least one or more mutations in ilvE compared to the parent Bacillus strain.

[0164] Still further embodiments of the present invention relate to the derivative Bacillus strains described herein, wherein the one or more mutations comprise at least one or more mutations in eamA-1 compared to the parent Bacillus strain.

[0165] Without wishing to be bound by theory, it is further contemplated herein that mutations affecting the C4 organic acid / sodium symporter may be beneficial in improving salt tolerance.

[0166] As a sensing histidine kinase, the DcuS-1 (MalK) protein is involved in regulating gene expression in response to external triggers. The DcuS-1 protein senses C4-dicarboxylic acids (succinate in the culture medium in this context) and upregulates (via a kinase cascade) the C4-dicarboxylic acid / sodium symporter (MaeN). Thus, the increase in organic acid uptake is coupled to an increase in sodium cation uptake. Increased intracellular sodium concentrations are toxic to cells and, if accumulated at high rates, may affect cell viability.

[0167] During the SALTY-ALE campaign, strains evolving mutations in the dcuS-1 gene were identified. These mutations are considered a survival mechanism that prevents the rapid accumulation of harmful sodium cations under high salinity by reducing or downregulating the sodium symporter uptake system.

[0168] Therefore, one embodiment of the present invention relates to the derived Bacillus strain described herein, wherein the one or more mutations comprise at least one or more mutations in dcuS-1 compared to the parent Bacillus strain.

[0169] During the SALTY-ALE campaign, other favorable mutations leading to increased salt tolerance were identified, including mutations affecting another sensing histidine kinase, ComP, as well as the phosphatase YjcG and the chromosome replication initiation protein DnaA-1.

[0170] Therefore, one embodiment of the present invention relates to the derived Bacillus strain described herein, wherein the one or more mutations are located in one or more genes of the parent Bacillus strain selected from the group consisting of yjcG, comP, dnaA-1 and combinations thereof.

[0171] It will be appreciated that the identified genes, mutations of which result in increased salt tolerance in the derived Bacillus strains, can be combined to achieve synergistic advantages from several mechanisms affecting salt tolerance. This can be illustrated, for example, by the following, where the derived Bacillus strains benefit from mutations that inactivate or downregulate the GSR and mutations that affect the sodium symporter uptake system.

[0172] Thus, one embodiment of the present invention relates to a derived Bacillus strain as described herein, wherein the one or more mutations are located in one or more genes of the parent Bacillus strain selected from the group consisting of: sigB, rsbT, kinA, opuE, ilvE, rny, dcuS-1, yjcG, comp, dnaA-1, and combinations thereof.

[0173] Another embodiment of the present invention relates to the derived Bacillus strain described herein, wherein the one or more mutations comprise at least one or more mutations in one or more genes selected from the group consisting of: sigB, rsbT, kinA, opuE, ilvE, rny, dcuS-1, yjcG, comp, and dnaA-1, compared to the parent Bacillus strain.

[0174] Additional embodiments of the present invention relate to the derived Bacillus strains described herein, wherein the one or more mutations comprise at least one or more mutations in one or more genes selected from the group consisting of: sigB, opuE, ilvE, rny, dcuS-1 and compP as compared to the parent Bacillus strain.

[0175] Mutations promoted by SALTY-ALE activity have resulted in derived Bacillus strains with new phenotypes. The favorable phenotypes have resulted in increased salt tolerance and / or production or transport of osmotic regulating substances. These phenotypes are the result of changes in the proteome of the evolved Bacillus strains.

[0176] : SEQ ID NO: 29 (FlhB-2), SEQ ID NO: 30 (FliM), SEQ ID NO: 31 (MgsR-1), SEQ ID NO: 32 (OpuE), SEQ ID NO: 33 (BshA-1), SEQ ID NO: 34 (MetAA), SEQ ID NO: 35 (MotB), SEQ ID NO: 36 (KinA-1), SEQ ID NO: 37 (ComA), SEQ ID NO: 38 (SigB), SEQ ID NO: 39 (FlhB-2), SEQ ID NO: 40 (Rny), SEQ ID NO: 41 (IlvE), SEQ ID NO: 42 (DcuS-1), SEQ ID NO: 43 (RsbT), SEQ ID NO: 44 (FliM), SEQ ID NO: 45 (MgsR-1), SEQ ID NO: 46 (OpuE), SEQ ID NO: 47 (BshA-1), SEQ ID NO: 48 (MetAA), SEQ ID NO: 49 (MotB), SEQ ID NO: 50 (KinA-1), SEQ ID NO: 51 (ComA), SEQ ID NO: 52 (SigB), SEQ ID NO: 53 (FlhB-2), SEQ ID NO: NO:38 (RpoC), SEQ ID NO:39 (EamA-1), SEQ ID NO:40 (RpoB) and combinations thereof.

[0177] Another embodiment of the present invention relates to the derived Bacillus strain described herein, wherein the one or more mutations result in one or more modifications selected from the group consisting of: YjcG-(458-514 / 516nt), ComP-Y372C, DnaA-1-R262Q and combinations thereof, compared to the parent Bacillus strain.

[0178] Yet another embodiment of the present invention relates to the derivative Bacillus strain described herein, wherein the genome and / or proteome of the derivative Bacillus strain is at least 95% identical, such as at least 98% identical, such as at least 99% identical, such as at least 99.5% identical, such as at least 99.8% identical, such as at least 99.9% identical to the genome and / or proteome of the parent Bacillus strain.

[0179] Particularly advantageous derived Bacillus strains include those having high salt tolerance and good biostimulant properties.

[0180] A preferred embodiment of the present invention relates to the derived Bacillus strain described herein, wherein the derived Bacillus strain was deposited as DSM34005 by Chr. Hansen AG, Hirsholm, Denmark at the German Collection of Microorganisms and Cell Cultures (DSMZ), Inhofenstrasse 7B, D-38124, Brunswick, Germany on August 24, 2021.

[0181] Various protective mechanisms have been found in plants grown under conditions of high salinity or drought that allow them to adapt to environments that are unfavorable for their survival and growth. One of the main mechanisms found in plants is the accumulation of highly soluble organic compounds called compatible solutes. Compatible solutes are osmoprotectants that act as osmotic regulators to help organisms survive extreme osmotic pressures. Osmoregulators are highly water-soluble and low molecular weight organic compounds that can be classified into three major groups: amino acids (e.g., proline), quaternary and tertiary onium compounds (e.g., glycine betaine, dimethylmercaptopropionic acid), and polyols / small molecule sugars (e.g., mannitol, trehalose).

[0182] Under conditions of high salinity, compatible solutes or osmolytes may be beneficial to plant health because the availability of osmolytes in the external environment reduces the energy that the plant needs to expend to synthesize these osmolytes, so the available carbon source can be directed to plant growth and development. Therefore, it would be advantageous if evolved Bacillus strains could provide elevated levels of osmolytes.

[0183] Therefore, one embodiment of the present invention relates to a derived Bacillus strain as described herein, wherein the derived Bacillus strain produces one or more osmotic regulating substances in large amounts under high salinity conditions.

[0184] Another embodiment of the present invention relates to the derived Bacillus strain described herein, wherein the one or more osmotic regulating substances are selected from the group consisting of proline, citrulline, spermidine, glycine betaine, glycerol, amino acids, sugars, polyols, mannitol and sorbitol.

[0185] Yet another embodiment of the present invention relates to the derived Bacillus strain described herein, wherein the one or more osmotic regulating substances are proline and / or citrulline.

[0186] Further embodiments of the present invention relate to the derivative Bacillus strains described herein, wherein the high production of the one or more osmotic regulating substances is defined as equal or increased production of the osmotic regulating substances compared to the parent Bacillus strain, preferably increased production compared to the parent Bacillus strain.

[0187] Still further embodiments of the present invention relate to the derivative Bacillus strains described herein, wherein the high production of the one or more osmotic regulating substances is defined as at least a 2-fold increase in the production of the osmotic regulating substances, such as at least a 3-fold increase, such as at least a 4-fold increase, such as at least a 5-fold increase, such as at least a 10-fold increase, compared to the parent Bacillus strain.

[0188] Even further embodiments of the invention relate to the derived Bacillus strains described herein, wherein said high production of said one or more osmotic regulating substances is determined in the presence of at least 0.2 M NaCl, such as at least 0.3 M NaCl, such as 0.35 M NaCl in the culture medium.

[0189] Another embodiment of the present invention relates to the derived Bacillus strain described herein, wherein the high production of proline corresponds to a proline concentration of at least about 0.01 mM, such as at least about 0.02 mM, such as at least about 0.05 mM, such as at least about 0.1 mM, such as at least about 0.2 mM, such as at least about 0.3 mM, such as at least about 0.4 mM, such as at least about 0.5 mM.

[0190] Still another embodiment of the present invention relates to a derived Bacillus strain according to any one of items Y21-Y23, wherein the high production of citrulline corresponds to a citrulline concentration of at least about 0.01 mM, such as at least about 0.02 mM, such as at least about 0.03 mM, such as at least about 0.04 mM.

[0191] Gram-positive bacteria, such as Bacillus, are able to form spores, typically in the form of intracellular spores called endospores, as a survival mechanism. These endospores are extremely contractile and thick-walled structures that constitute the most dormant form of bacteria, as they exhibit minimal metabolism, respiration, and enzyme production. Such bacterial spores are highly resistant to temperature fluctuations, chemical agents, UV radiation, pH gradients, drought, and nutrient depletion. When the surrounding environment is favorable for bacterial proliferation, bacterial spores will germinate back into vegetative cells, i.e., metabolically active bacterial cells.

[0192] Therefore, in the context of the present invention, spore-forming bacteria are preferred because they have the ability to remain dormant if field conditions are not favorable for survival. Thus, for spore-forming bacteria, the risk of losing the derived Bacillus strain after application to plants or soil is reduced.

[0193] Therefore, one embodiment of the present invention relates to the derived Bacillus strain described herein, wherein the derived Bacillus strain is in the form of spores or vegetative cells, preferably spores.

[0194] The SALTY-ALE activity described herein can facilitate the development of derivative Bacillus strains with increased salt tolerance. The method includes at least three steps, first identifying and providing a parent Bacillus strain with a desired trait. The parent strain can be selected based on criteria such as its baseline salt tolerance or biostimulant properties. The selected parent strain is then cultured under high salinity conditions to promote mutations that allow the Bacillus to survive increased abiotic stress. Once an evolved Bacillus population has been prepared, the population is cultured under conditions that allow selection of colonies with increased salt tolerance.

[0195] Therefore, one aspect of the present invention relates to a method for preparing the derivative Bacillus strain described herein, the method comprising the following steps:

[0196] (i) Providing a parent Bacillus strain

[0197] (ii) growing the parent Bacillus strain under high salinity conditions, and

[0198] (iii) selecting a derivative Bacillus strain having increased salt tolerance that proliferates under the growth conditions of step (ii).

[0199] One embodiment of the present invention relates to the method described herein, wherein step (ii) comprises growing the parent Bacillus strain under high salinity conditions to obtain an evolved population.

[0200] The SALTY-ALE activity is essentially random and it is not possible to know in advance the properties of the evolving population. An advantage of this type of random mutagenesis evolution is that, in contrast to fixed-site approaches, where only a few specific genes are mutated, a wide range of genes and their interrelationships are explored. In this way, even complex phenotypes may be identified. In the selection step, the derivative Bacillus strains are selected based solely on the evolutionary parameter of high salinity, preferably in comparison with the parent Bacillus strain.

[0201] Thus, one embodiment of the invention relates to the methods described herein, wherein the high salinity conditions are defined as a growth medium having a NaCl concentration of at least 0.8 M NaCl, such as at least 0.9 M NaCl, such as at least 1.0 M NaCl, such as at least 1.1 M NaCl, such as at least 1.2 M NaCl, such as at least 1.3 M NaCl, such as at least 1.4 M NaCl, such as at least 1.5 M NaCl.

[0202] Another embodiment of the present invention relates to the method described herein, wherein the high salinity condition is obtained by increasing the salinity level.

[0203] Additional embodiments of the invention relate to the methods described herein, wherein the increasing salinity level begins with a growth medium without salt.

[0204] The parent Bacillus strains used herein were selected based on their baseline salt tolerance (i.e., before any salt tolerance evolution). The initial pool of parent Bacillus candidate strains included species with biostimulant properties to optimize SALTY-ALE activity toward obtaining derived Bacillus strains with increased salt tolerance and beneficial properties for plant health.

[0205] Thus, one embodiment of the present invention relates to the methods described herein, wherein the parent Bacillus strain belongs to a species selected from the group consisting of Bacillus velez, Bacillus paralicheniformis, Bacillus amyloliquefaciens, and Bacillus subtilis.

[0206] Another embodiment of the present invention relates to the methods described herein, wherein the parent Bacillus strain is a deposited strain selected from the group consisting of:

[0207] -DSM34004 (deposited on August 24, 2021),

[0208] -DSM34003 (deposited on August 24, 2021),

[0209] -DSM33240 (deposited on August 14, 2019),

[0210] - DSM 17231 (deposited on April 7, 2005),

[0211] -DSM33110 (deposited on 8 May 2019) and

[0212] - DSM 32324 (deposited on 8 June 2016),

[0213] The deposited strains are deposited by Chr. Hansen AG, Hirsholm, Denmark at the German Collection of Microorganisms and Cell Cultures (DSMZ), Inhofenstrasse 7B, D-38124 Braunschweig, Germany.

[0214] At the end of step (ii), i.e. after growing the parent Bacillus strain under high salinity conditions, an evolved Bacillus population has been prepared. This population will contain a pool of different bacterial cells (strains) that can be selected and isolated in the subsequent step (iii). The selection step can be performed in different ways. Preferably, the evolved population is streaked or spread on a solid growth medium, such as an agar plate, and colonies are manually selected therefrom.

[0215] Thus, one embodiment of the present invention relates to the method described herein, wherein the selecting step (iii) comprises providing the evolved population of step (ii) to a solid growth medium.

[0216] Several strategies can be adopted to select the derived Bacillus strain with a beneficial phenotype. The salt concentration can be adjusted to make the severity of the selection pressure greater or less. Typically, very high salt concentrations reduce the number of colonies, making the screening faster, but the exploratory / diversity will also be reduced. Alternatively, the evolved population can be provided to a solid growth medium containing different salt concentrations to increase the diversity of the bacterium colonies from which the candidate derived Bacillus strains are selected. Therefore, the bacterium colonies of the derived Bacillus strains can be selected from the solid growth medium of a single salt concentration or the solid growth medium of multiple salt concentrations.

[0217] One embodiment of the present invention relates to the method described herein, wherein the selecting step (iii) comprises providing the evolved population of step (ii) to a solid growth medium of varying salt concentrations.

[0218] The evolved population obtained from step (ii) can be applied directly to the solid growth medium in the selection step (iii) or can be treated prior to the selection step (iii).

[0219] Thus, one embodiment of the present invention relates to the method described herein, wherein the evolved population of step (ii) is plated directly on a solid growth medium in the selection step (iii).

[0220] Another embodiment of the present invention relates to the method described herein, wherein the evolved population of step (ii) is subjected to a washing step prior to plating on solid growth medium in the selection step (iii).

[0221] Without being bound by theory, it is contemplated that it is advantageous to subject the evolved population to a rich medium to eliminate salt stress before plating onto solid growth medium and selecting the evolved population thereon. Relaxing the evolved population can eliminate strains that appear to have been improved solely based on an induced salt stress response, and can ultimately result in more stable strains.

[0222] Thus, one embodiment of the present invention relates to the method described herein, wherein the evolved population is passed through a rich medium prior to plating it onto a solid growth medium in the selection step (iii).

[0223] Another embodiment of the present invention relates to the method described herein, wherein the rich culture medium is fresh liquid culture medium, such as fresh LB medium.

[0224] The candidate derivative Bacillus strain should be able to proliferate in the selection step (iii). Proliferation can be determined by visual confirmation or more quantitatively by optical density (OD) measurement.

[0225] Thus, one embodiment of the present invention relates to the methods described herein, wherein proliferation is defined as visual formation of colonies and / or as measured by optical density.

[0226] The selected and isolated derived Bacillus strains are not guaranteed to have a beneficial effect on plant growth in high soil salinity. Therefore, it may be advantageous to add one or more additional steps to further screen the selected and isolated derived Bacillus strains. These steps may include characterizing the osmotic regulating substance production of the derived Bacillus strains and / or performing genetic analysis on the derived Bacillus strains.

[0227] Therefore, one embodiment of the present invention relates to the method described herein, wherein after step (iii) is performed:

[0228] (i) determining the production of one or more osmotic regulating substances of the derived Bacillus strain having increased salt tolerance; and / or

[0229] (ii) a step of sequencing the derived Bacillus strain having increased salt tolerance and identifying SNPs.

[0230] Another embodiment of the present invention relates to the methods described herein, wherein the one or more osmotic regulating substances are selected from the group consisting of proline, citrulline, spermidine, glycine betaine, glycerol, amino acids, sugars, polyols, mannitol and sorbitol.

[0231] Additional embodiments of the present invention relate to the methods described herein, wherein the methods further comprise the step of selecting a derived Bacillus strain having a high production of the one or more osmotic regulating substances.

[0232] Still further embodiments of the present invention relate to the methods described herein, wherein the high production of the one or more osmotic regulating substances is defined as equal or increased production of the osmotic regulating substances compared to the parent Bacillus strain, preferably increased production compared to the parent Bacillus strain.

[0233] The methods described herein can be performed in a systematic and optimized manner to generate and select derivative Bacillus strains with increased salt tolerance. These derivative Bacillus strains can be advantageously used as part of compositions that have beneficial properties for plant health under abiotic stress conditions such as high salinity or drought.

[0234] Thus, one aspect of the present invention relates to a derivative Bacillus strain having increased salt tolerance compared to a parent Bacillus strain, which strain is obtainable by the method described herein.

[0235] Another aspect of the present invention relates to a composition comprising a derivative Bacillus strain having increased salt tolerance compared to the parent Bacillus strain described herein.

[0236] The derived Bacillus strain is preferably spore-forming, since endospores have a significantly enhanced ability to withstand any stress conditions. Thus, spores of the derived Bacillus strain will increase the robustness and durability of the composition, especially when applied under harsh conditions.

[0237] Thus, one embodiment of the present invention relates to the composition described herein, wherein the composition comprises spores of the derived Bacillus strain.

[0238] The composition may contain additional ingredients that improve the physical or functional properties of the composition. Thus, the additional ingredients may benefit, for example, stability, deliverability, wettability, penetration or retention. The additional active ingredients in addition to the derived Bacillus strain may provide a dual mechanism of action for the composition and include standard ingredients typically used in the formulation of plant growth promoters, plant biostimulants or biopesticides.

[0239] Thus, one embodiment of the present invention relates to the composition described herein, wherein the composition further comprises one or more agrochemically acceptable excipients, carriers, surfactants, dispersants and yeast extracts.

[0240] Another embodiment of the present invention relates to the composition described herein, wherein the agrochemically acceptable excipient or carrier is selected from the group consisting of maltodextrin, silicon dioxide, modified zeolite, kaolinite, lignin, starch, chitosan and calcium carbonate.

[0241] Still further embodiments of the present invention are directed to the compositions described herein, wherein the compositions further comprise one or more active ingredients.

[0242] Even further embodiments of the present invention relate to the compositions described herein, wherein the one or more active ingredients are selected from the group consisting of insecticides, fungicides, nematicides, bactericides, herbicides, plant extracts, plant growth regulators, plant growth stimulants and fertilizers.

[0243] Yet another embodiment of the present invention relates to the compositions described herein, wherein the one or more active ingredients are derived from microorganisms, organisms or chemicals.

[0244] Another embodiment of the present invention relates to the composition described herein, wherein the insecticide is selected from the group consisting of pyrethroids, bifenthrin, tefluthrin, zeta-cypermethrin, organophosphates, chlorethoxyfos, chlorpyrifos, butylpirimiphos, cyfluthrin, fipronil, fipronil, nicotinoids and clothianidin, and combinations thereof.

[0245] Still another embodiment of the present invention relates to the composition described herein, wherein the fungicide is selected from the group consisting of fluopyram+tebuconazole, thiophanate-methyl, thiophanate-methyl, prothioconazole and copper hydroxide, and combinations thereof.

[0246] The composition may contain more than one bacterial strain. Such bacterial consortia may act synergistically to enhance the beneficial effects on the recipient plant. The bacterial consortia may contain, for example, bacteria that promote plant health by providing osmotic regulating substances to the plant, bacteria that function as biopesticides, and bacteria that act as biofertilizers.

[0247] Thus, one embodiment of the present invention relates to a composition as described herein, wherein the one or more active ingredients are selected from one or more second bacterial strains different from the derived Bacillus strain, the derived Bacillus strain having increased salt tolerance compared to the parent Bacillus strain.

[0248] Another embodiment of the present invention relates to the composition described herein, wherein the second bacterial strain is a biostimulant strain, preferably a biostimulant Bacillus strain.

[0249] The composition can be provided in a variety of different forms, including but not limited to liquids, oil dispersants, dusts, dry wettable powders, spreadable granules or dry wettable granules. More particularly, the composition can be, for example, an emulsion concentrate (EC), a suspension concentrate (SC), a water dispersible granule (WG), an emulsifiable granule (EG), an oil-in-water emulsion (EO), an oil-in-water emulsion (EW), a microemulsion (ME), an oil dispersant (OD), an oil miscible liquid (OL), a soluble concentrate (SL), a dispersible concentrate (DC) or a wettable powder (WP).

[0250] Therefore, one embodiment of the present invention relates to the composition described herein, wherein the composition is in a form selected from the group consisting of: a liquid, a wettable powder, a granule, a spreadable granule, a wettable granule, a microcapsule, and a planting matrix.

[0251] Coating polymers can be used to provide a shell for a solid entity, which can provide protection or add additional properties to the entity it is coated with. The coating polymer can be part of a liquid formulation that is subsequently applied to another entity. For compositions in solid form, such as powders or granules, the coating polymer can thus form a shell on the particles. For compositions in liquid form, the coating polymer can make the composition suitable for coating other entities, such as seeds or plants.

[0252] Thus, one embodiment of the present invention relates to the composition described herein, wherein the composition further comprises a coating polymer.

[0253] Additional embodiments of the present invention relate to the compositions described herein, wherein the composition is a liquid formulation.

[0254] One aspect of the present invention relates to a plant or seed coated with a composition as described herein.

[0255] The composition is not limited to coating a particular type of seed or plant. It is contemplated that any seed or plant may benefit from coating with the composition, particularly seeds or plants that will be exposed to abiotic stresses (e.g., high salinity or drought). For example, by coating seeds with the composition, the germination rate of seeds sown in soil that is or will be subjected to abiotic stresses may be increased.

[0256] Plant seeds may include, but are not limited to, monocots, dicots, grains, corn, sweet corn, popcorn corn, seed corn, silage corn, field corn, rice, wheat, barley, sorghum, asparagus, berries, blueberries, blackberries, raspberries, loganberries, huckleberries, cranberries, gooseberries, elderberries, currants, cranberries, bush berries, brassica vegetables, broccoli, cabbage, cauliflower, Brussels sprouts, collard greens, kale, mustard greens, kohlrabi, bulb vegetables, onions, garlic, shallots, citrus, oranges, grapefruit, lemons, tangerines, tangelos, pomelo, fruiting vegetables, peppers, tomatoes, eggplants, tomatillos, tomatillos, okra, grapes, herbs / spices, cucurbit vegetables, cucumbers, cantaloupe, melons, cantaloupe, winter squash, watermelon, pumpkins, leafy greens, lettuce, celery, spinach, parsley, endive, beans / vegetables ( succulents and dry beans and peas), beans, mung beans, snap beans, beans with pods, soybeans, dry beans, chickpeas, lima beans, peas, garbanzo beans, split peas, lentils, oilseed crops, canola, castor, coconut, cotton, flax, oil palm, olives, peanuts, rapeseed, safflower, sesame, sunflower, soybeans, pome fruits, apples, crab apples, pears, quinces, summer flowering hawthorn, root / tuber and bulb vegetables, carrots carrots, potatoes, sweet potatoes, beets, ginger, horseradish, radish, ginseng, turnips, stone fruits, apricots, cherries, nectarines, peaches, plums, prunes, strawberries, tree nuts, almonds, pistachios, pecans, walnuts, hazelnuts, chestnuts, cashews, beech nuts, butternuts, macadamia, kiwi, bananas, agave, ornamentals, poinsettias, hardwood cuttings, oak, maple, sugar cane, sugar beets, grass or turf grass.

[0257] One embodiment of the present invention relates to the plant or seed described herein, wherein the composition is present in an amount suitable to benefit the growth of the plant.

[0258] Another embodiment of the present invention relates to the seeds described herein, wherein the composition comprises about 1.0×10 2 CFU / seed is about 1.0×10 9 CFU / seed, e.g. about 1.0×10 3 CFU / seed is about 1.0×10 9 CFU / seed, for example, about 1.0×10 4 CFU / seed is about 1.0×10 9 CFU / seed.

[0259] Yet another embodiment of the present invention relates to the seeds described herein, wherein the composition comprises about 1.0×10 6 CFU / g seeds is about 1.0×10 11 CFU / g seeds.

[0260] Another embodiment of the present invention relates to the plant described herein, wherein the composition comprises about 1.0×10 vegetative cells or spores of the Bacillus strain. 4 CFU / g root is about 1.0×10 10 CFU / g root, for example, about 1.0×10 5 CFU / g root is about 1.0×10 9 CFU / g root.

[0261] Bacillus strains and compositions comprising them can be advantageously used to increase the resistance of plants to abiotic stresses, such as high salinity or drought.Application can be either directly to the plant, ie foliar application, or in the plant's habitat.

[0262] Thus, one aspect of the present invention relates to a method of increasing resistance of a plant to abiotic stress conditions, the method comprising applying to the plant, a part of the plant and / or the habitat of the plant a derivative Bacillus strain or a composition as described herein having increased salt tolerance compared to a parent Bacillus strain.

[0263] Another embodiment of the present invention relates to the methods described herein, wherein the abiotic stress condition is high salinity, drought and / or nutrient deficiency.

[0264] A preferred embodiment of the present invention relates to the method described herein, wherein said abiotic stress condition is high salinity.

[0265] It should be understood that high salinity refers to the salt level in the plant's habitat (e.g., soil). The salinity level in the plant's habitat will affect its ability to grow. High salinity levels will typically result in a significant decrease in plant quality and growth rate, and in severe cases, plant death. Normal NaCl levels in soil range from about 10-40 mM.

[0266] Therefore, one embodiment of the present invention relates to the methods described herein, wherein high salinity is defined as a NaCl concentration in the plant's habitat greater than about 50 mM, such as greater than about 60 mM, such as greater than about 70 mM, such as greater than about 80 mM, such as greater than about 90 mM, such as greater than about 100 mM, such as greater than about 110 mM, such as greater than about 120 mM, such as greater than about 130 mM, such as greater than about 140 mM, such as greater than about 150 mM.

[0267] Another embodiment of the present invention relates to the methods described herein, wherein high salinity is defined as NaCl concentration in the habitat of the plant ranging from about 50 mM to about 150 mM, such as from about 100 mM to about 150 mM.

[0268] Soil salinity can be measured by the electrical conductivity (EC) of the soil, which is measured in dS / m. The EC of 10mM NaCl is close to 1dS / m. The EC of 100mM NAcl is close to 9.8dS / m.

[0269] Methods for increasing plant resistance to abiotic stress conditions are not limited to any particular plant, as most plants are susceptible to abiotic stresses (e.g., high salinity or drought). Typically, the method is primarily relevant to agriculture, as relatively small increases in yield can also make a big difference in an industrial setting. In addition, the prospect of being able to increase yield in an environmentally friendly manner is very attractive, and is preferred over conventional agrochemicals that cause extensive ecological damage.

[0270] One embodiment of the invention relates to the methods described herein, wherein the plant is selected from the group consisting of crops, monocots, dicots, trees, herbs, shrubs, grasses, vines, ferns and mosses.

[0271] Other plants that may benefit from the method include but are not limited to staple crops, fruit tree cultivation plants, horticultural plants and flower cultivation plants. Staple crops can be but are not limited to sugar cane, coffee, soybean, cotton, corn, potato, tomato, tobacco, banana, rice, wheat, avocado, pineapple, winter squash, cocoa, coconut, oat, onion, lettuce, beet, carrot, cassava, kidney bean, sunflower, pepper, turnip, apple, strawberry, okra, radish and onion. Fruit tree cultivation plants include but are not limited to citrus, grape, guava, papaya, fig, peach, plum and loquat. Flower cultivation plants can be rose, chrysanthemum, lisianthus, gerbera, amaryllis, begonia and gentiana.

[0272] One embodiment of the present invention relates to the methods described herein, wherein the plant is selected from the group consisting of wheat, barley, oats, small grain cereals, corn, rice, sugar cane, soybean, potato, carrot, coffee and banana.

[0273] One embodiment of the present invention relates to the methods described herein, wherein the part of the plant is selected from the group consisting of seeds, fruits, roots, stems, leaves, corms, tubers, bulbs and rhizomes.

[0274] If the derived Bacillus strain or composition is able to colonize the plant, more particularly the root, then they are beneficial to the plant health under field conditions. Therefore, it is preferred that the derived Bacillus strain or composition is applied to the plant's habitat.

[0275] Thus, one embodiment of the present invention relates to the methods described herein, wherein the derivative Bacillus strain or the composition is applied to a habitat of a plant.

[0276] Another embodiment of the present invention relates to the method described herein, wherein the habitat of the plant is liquid or soil, preferably soil.

[0277] Bacillus strains can also be used to combat abiotic stresses against green algae.

[0278] Thus, one aspect of the present invention relates to a method of increasing resistance of green algae to abiotic stress conditions, the method comprising applying a derivative Bacillus strain or composition described herein having increased salt tolerance compared to a parent Bacillus strain to green algae and / or a habitat of green algae.

[0279] To alleviate abiotic stresses on the green algae, the habitat may be a liquid, such as water.

[0280] The derived Bacillus strain or composition can be provided to a plant, a part of a plant and / or a habitat of a plant. Depending on the form of the derived Bacillus strain or composition, application can be carried out by dusting or spraying. Dusting as used herein refers to spreading a dry, finely powdered or granular composition, typically after mixing with an inert carrier. Such application can typically be implemented in most agricultural environments without the need to invest in additional equipment.

[0281] Additional embodiments of the present invention relate to the methods described herein, wherein the derivative Bacillus strain or the composition is applied to the habitat of the plant by dusting or spraying.

[0282] Application of the derived Bacillus strain or composition can be performed as a preparatory measure before sowing or planting, or when the plants are already growing in a habitat, such as a field.

[0283] Thus, one embodiment of the present invention relates to the methods described herein, wherein the derived Bacillus strain or the composition is applied before, during or after contacting the plant or a part of a plant with a habitat.

[0284] The derived Bacillus strain or composition can be provided to: the soil or growth medium surrounding a plant; the soil or growth medium before sowing plant seeds in the soil or growth medium; or the soil or growth medium before planting a plant, plant cutting, plant graft or plant callus in the soil or growth medium.

[0285] One embodiment of the invention relates to the methods described herein, wherein the derivative Bacillus strain or the composition is applied at least about 365 days, such as at least about 200 days, such as at least about 100 days, such as at least about 30 days, such as at least about 10 days, such as at least about 5 days, such as at least 1 day, such as at least about 12 hours, such as at least about 5 hours, such as at least about 1 hour, before the plant or a part of the plant is contacted with the habitat.

[0286] Another embodiment of the present invention relates to the method described herein, wherein the derived Bacillus strain or the composition is present at about 1×10 4 About 1×10 8 cfu are applied to the seeds at a rate of, for example, 1×10 5 About 5×10 7 cfu.

[0287] Additional embodiments of the present invention relate to the methods described herein, wherein the derived Bacillus strain or the composition is present at a concentration of about 1×10 7 About 1×10 14 cfu per acre, for example, about 1×10 8 About 1×10 13 cfu, for example, about 1×10 per acre 9 About 1x 10 2 cfu.

[0288] Prior to applying the derived Bacillus strain or composition, the habitat may be analyzed to identify any abiotic stress conditions. This sequence of actions is preferred in order to avoid wasting materials when no action is needed. Traditional methods of measuring, for example, soil salinity levels may be employed.

[0289] Therefore, one embodiment of the present invention relates to the methods described herein, wherein said abiotic stress condition is identified prior to administering said derivative Bacillus strain or said composition.

[0290] Another embodiment of the present invention relates to the method described herein, wherein said abiotic stress condition is identified by measuring total soluble salts by evaporating soil water extract (TSS) or by measuring electrical conductivity.

[0291] Further embodiments of the invention relate to the methods described herein, wherein TSS or EC is measured on a 1 :5 distilled water:soil dilution or a saturated paste extract.

[0292] The derived Bacillus strain or composition is able to colonize the plant root system and provide a biostimulatory effect. The effect may vary from plant to plant.

[0293] Thus, one embodiment of the present invention relates to the methods described herein, wherein increasing a plant's resistance to abiotic stress conditions contributes to increased seedling vigor, improved root development, increased plant growth, improved plant health, increased yield, improved appearance, or a combination thereof.

[0294] Another aspect of the present invention relates to the use of a derivative Bacillus strain or a composition as described herein having increased salt tolerance compared to a parent Bacillus strain as a biostimulant, growth promoter and / or for increasing resistance of a plant to abiotic stress conditions.

[0295] One embodiment of the present invention relates to the use as described herein, wherein the abiotic stress condition is high salinity, drought and / or nutrient deficiency, preferably high salinity.

[0296] Another embodiment of the present invention relates to the uses described herein, wherein increasing the resistance of a plant to abiotic stress conditions contributes to improving seedling vigor, improving root development, promoting plant growth, improving plant health, increasing yield, improving appearance, reducing pathogen infection, or a combination thereof.

[0297] The derived Bacillus strain, composition or coated seed may conveniently be provided as a kit for easy application. The kit may contain other active ingredients for mixing prior to spreading on plants or their habitats.

[0298] Therefore, one aspect of the present invention relates to a kit comprising:

[0299] (i) a derivative Bacillus strain, composition or coated plant seed as described herein having increased salt tolerance compared to a parent Bacillus strain;

[0300] (ii) containers; and

[0301] (iii) optionally, instructions for use.

[0302] One embodiment of the present invention relates to the kit described herein, wherein the kit further comprises one or more active ingredients selected from the group consisting of insecticides, fungicides, nematicides, bactericides, herbicides, plant extracts, plant growth regulators, plant growth stimulants and fertilizers.

[0303] Another embodiment of the present invention relates to the kit described herein, wherein said derivative Bacillus strain and said one or more active ingredients are provided in separate compartments of a container.

[0304] The listing or discussion of an apparently prior-published document in this specification should not be taken as an admission that the document is part of the state of the art or is common general knowledge.

[0305] Unless the context indicates otherwise, preferences, options and embodiments for a given aspect, feature or parameter of the invention should be considered to have been disclosed in conjunction with any and all preferences, options and embodiments for all other aspects, features and parameters of the invention. This is especially true for the description of derivative strains and all their features, which can easily become part of a method or use portion for promoting plant health and growth under abiotic stress conditions (e.g., high salinity). Embodiments and features of the invention are also summarized in the following items.

[0306] project

[0307] Y1. A derivative Bacillus strain or a variant thereof having increased salt tolerance compared to a parent Bacillus strain,

[0308] The derived Bacillus strain or its variant is a derivative strain of the parent Bacillus strain.

[0309] Y2. The derivative Bacillus strain according to item Y1, wherein the derivative Bacillus strain has increased salt tolerance compared to the parent Bacillus strain when cultured under the same conditions.

[0310] Y3. A derived Bacillus strain according to any one of items Y1 or Y2, wherein the salt tolerance of the derived Bacillus strain is at least increased by 2.5 times compared to the parent Bacillus strain, for example at least increased by 3 times, for example increased by 3.5 times, for example increased by 4 times compared to the parent Bacillus strain.

[0311] Y4. The derived Bacillus strain according to any of the preceding items, wherein the salt tolerance of the derived Bacillus strain is at least 0.8 M NaCl, such as at least 0.9 M NaCl, such as at least 1.0 M NaCl, such as at least 1.1 M NaCl, such as at least 1.2 M NaCl, such as at least 1.3 M NaCl, such as at least 1.4 M NaCl, such as at least 1.5 M NaCl.

[0312] Y5. A derived Bacillus strain according to any of the preceding items, wherein the salt tolerance is defined by half maximal inhibitory concentration (IC50).

[0313] Y6. The derived Bacillus strain according to any of the preceding items, wherein the parent Bacillus strain belongs to a species selected from the group consisting of Bacillus Velezii, Bacillus paralicheniformis, Bacillus amyloliquefaciens and Bacillus subtilis.

[0314] Y7. The derived Bacillus strain according to any one of the preceding items, wherein the parent Bacillus strain is a deposited strain selected from the group consisting of:

[0315] -DSM34004 (deposited on August 24, 2021),

[0316] -DSM34003 (deposited on August 24, 2021),

[0317] -DSM33240 (deposited on August 14, 2019),

[0318] - DSM 17231 (deposited on April 7, 2005),

[0319] -DSM33110 (deposited on 8 May 2019) and

[0320] - DSM 32324 (deposited on 8 June 2016),

[0321] The deposited strains are deposited by Chr. Hansen AG, Hirsholm, Denmark at the German Collection of Microorganisms and Cell Cultures (DSMZ), Inhofenstrasse 7B, D-38124 Braunschweig, Germany.

[0322] Y8. A derived Bacillus strain according to any of the preceding items, wherein the parent Bacillus strain is deposited as DSM34004 by Chr. Hansen AG, Hirsholm, Denmark on August 24, 2021 at the German Collection of Microorganisms and Cell Cultures (DSMZ), Inhofenstrasse 7B, D-38124, Brunswick, Germany.

[0323] Y9. A derived Bacillus strain according to any of the preceding items, wherein the derived Bacillus strain is genetically different from the parent Bacillus strain.

[0324] Y10. A derived Bacillus strain according to any of the preceding items, wherein the derived Bacillus strain comprises one or more mutations compared to the parent Bacillus strain.

[0325] Y11. A derived Bacillus strain according to item Y10, wherein the one or more mutations are deletions, substitutions, insertions and / or frameshifts.

[0326] Y12. The derived Bacillus strain according to any one of items Y10 or Y11, wherein one or more mutations are in one or more genes of the parent Bacillus strain selected from the group consisting of:

[0327] -yjcG encoding the putative phosphatase YjcG;

[0328] -comP encoding the sensor histidine kinase ComP;

[0329] -dnaA-1, encoding the chromosome replication initiation protein;

[0330] -sigB, encoding RNA polymerase sigma-B factor;

[0331] - flhB-2 encoding the flagellar biosynthesis protein FlhB;

[0332] -rny encoding ribonuclease Y;

[0333] -ilvE encoding branched-chain amino acid aminotransferase;

[0334] -dcuS-1, encoding the sensor histidine kinase DcuS;

[0335] -rsbT encoding the serine / threonine protein kinase RsbT;

[0336] - fliM encoding the flagellar motor switch protein FliM;

[0337] - mgsR-1, encoding the regulatory protein MgsR;

[0338] - opuE encoding the osmoregulatory proline transporter OpuE;

[0339] - bshA-1 encoding N-acetyl-α-D-glucosaminyl L-malate synthase;

[0340] - metAA encoding homoserine O-acetyltransferase;

[0341] -motB encoding movement protein B;

[0342] -kinA-1 encoding sporulation kinase A;

[0343] -comA, encoding the transcriptional regulator protein comA;

[0344] - rpoC, encoding the β' subunit of the DNA-directed RNA polymerase;

[0345] - eamA-1 encoding a putative amino acid metabolite efflux pump; and

[0346] -rpoB, encoding the β subunit of DNA-directed RNA polymerase.

[0347] Y13. A derived Bacillus strain according to any one of items Y10 to Y12, wherein the one or more mutations are in one or more genes of the parent Bacillus strain, which genes comprise a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1 (yjcG), SEQ ID NO: 2 (comP), SEQ ID NO: 3 (dnaA-1), SEQ ID NO: 4 (sigB), SEQ ID NO: 5 (flhB-2), SEQ ID NO: 6 (rny), SEQ ID NO: 7 (ilvE), SEQ ID NO: 8 (dcuS-1), SEQ ID NO: 9 (rsbT), SEQ ID NO: 10 (fliM), SEQ ID NO: 11 (mgsR-1), SEQ ID NO: 12 (opuE), SEQ ID NO: 13 (bshA-1), SEQ ID NO: 14 (metAA), SEQ ID NO: 15 (motB), SEQ ID NO: 16 (kinA-1), SEQ ID NO: 17 (comA), SEQ ID NO: 18 (motB), SEQ ID NO: 19 (kinA-2), SEQ ID NO: 20 (comA), SEQ ID NO: 21 ( NO:18 (rpoC), SEQ ID NO:19 (eamA-1), SEQ ID NO:20 (rpoB) and combinations thereof.

[0348] Y14. A derived Bacillus strain according to any one of items Y10-Y13, wherein the one or more mutations are located in one or more genes of the parent Bacillus strain, which genes are selected from the group consisting of: yjcG, comP, dnaA-1 and combinations thereof.

[0349] Y15. A derived Bacillus strain according to any one of items Y10-Y14, wherein the one or more mutations result in one or more modifications in one or more proteins of the parent Bacillus strain, the proteins comprising an amino acid sequence selected from the group consisting of SEQ ID NO:21 (YjcG), SEQ ID NO:22 (ComP), SEQ ID NO:23 (DnaA-1), SEQ ID NO:24 (SigB), SEQ ID NO:25 (FlhB-2), SEQ ID NO:26 (Rny), SEQ ID NO:27 (IlvE), SEQ ID NO:28 (DcuS-1), SEQ ID NO:29 (RsbT), SEQ ID NO:30 (FliM), SEQ ID NO:31 (MgsR-1), SEQ ID NO:32 (OpuE), SEQ ID NO:33 (BshA-1), SEQ ID NO:34 (MetAA), SEQ ID NO:35 (MotB), SEQ ID NO:36 (KinA-1), SEQ ID NO:37 (MetAA), SEQ ID NO:38 (MotB), SEQ ID NO:39 (KinA-2), SEQ ID NO:40 ( NO:37 (ComA), SEQ ID NO:38 (RpoC), SEQ ID NO:39 (EamA-1), SEQ ID NO:40 (RpoB) and combinations thereof.

[0350] Y16. A derived Bacillus strain according to any one of items Y10-Y15, wherein the one or more mutations result in one or more modifications selected from the group consisting of: YjcG-δ(458-514), ComP-Y372C, DnaA-1-R262Q and combinations thereof, compared to the parent Bacillus strain.

[0351] Y17. A derived Bacillus strain according to any of the preceding items, wherein the derived Bacillus strain is deposited as DSM34005 by Chr. Hansen AG, Hersholm, Denmark on August 24, 2021 at the German Collection of Microorganisms and Cell Cultures (DSMZ), Inhofenstrasse 7B, D-38124, Brunswick, Germany.

[0352] Y18. The derived Bacillus strain according to any one of the preceding items, wherein the derived Bacillus strain is in the form of spores or vegetative cells, preferably spores.

[0353] Y19. The derived Bacillus strain according to any one of the preceding items, wherein the derived Bacillus strain produces a large amount of one or more osmotic regulating substances under high salinity conditions.

[0354] Y20. A derived Bacillus strain according to item Y19, wherein the one or more osmotic regulating substances are selected from the group consisting of: proline, citrulline, spermidine, glycine betaine, glycerol, amino acids, sugars, polyols, mannitol and sorbitol.

[0355] Y21. A derived Bacillus strain according to any one of items Y19 or Y20, wherein the one or more osmotic regulating substances are proline and / or citrulline.

[0356] Y22. A derived Bacillus strain according to any one of items Y19-Y21, wherein the high production of one or more osmotic regulating substances is defined as the same or increased production of the osmotic regulating substances compared to the parent Bacillus strain, preferably an increased production compared to the parent Bacillus strain.

[0357] Y23. A derived Bacillus strain according to any one of items Y21 or Y22, wherein the high production of proline corresponds to a proline concentration of at least about 0.01 mM, such as at least about 0.02 mM, such as at least about 0.05 mM, such as at least about 0.1 mM, such as at least about 0.2 mM, such as at least about 0.3 mM, such as at least about 0.4 mM, such as at least about 0.5 mM.

[0358] Y24. A derived Bacillus strain according to any one of items Y21-Y23, wherein the high production of citrulline corresponds to a citrulline concentration of at least about 0.01 mM, such as at least about 0.02 mM, such as at least about 0.03 mM, such as at least about 0.04 mM.

[0359] X1. A method for preparing a derivative Bacillus strain according to any one of the preceding items, the method comprising the following steps:

[0360] (i) Providing a parent Bacillus strain

[0361] (ii) growing the parent Bacillus strain under high salinity conditions, and

[0362] (iii) selecting a derivative Bacillus strain having increased salt tolerance that proliferates under the growth conditions of step (ii).

[0363] X2. A method according to item X1, wherein the high salinity condition is defined as a growth medium having a NaCl concentration of at least 0.8 M NaCl, for example, at least 0.9 M NaCl, for example, at least 1.0 M NaCl, for example, at least 1.1 M NaCl, for example, at least 1.2 M NaCl, for example, at least 1.3 M NaCl, for example, at least 1.4 M NaCl, for example, at least 1.5 M NaCl.

[0364] X3. A method according to any one of items X1 or X2, wherein the high salinity condition is obtained by increasing the salinity level.

[0365] X4. The method according to any one of items X1-X3, wherein the parent Bacillus strain belongs to a species selected from the group consisting of Bacillus Velezii, Bacillus paralicheniformis, Bacillus amyloliquefaciens and Bacillus subtilis.

[0366] X5. A method according to any one of items X1 to X4, wherein the parent Bacillus strain is a deposited strain selected from the group consisting of:

[0367] -DSM34004 (deposited on August 24, 2021),

[0368] -DSM34003 (deposited on August 24, 2021),

[0369] -DSM33240 (deposited on August 14, 2019),

[0370] - DSM 17231 (deposited on April 7, 2005),

[0371] -DSM33110 (deposited on 8 May 2019) and

[0372] - DSM 32324 (deposited on 8 June 2016),

[0373] The deposited strains are deposited by Chr. Hansen AG, Hirsholm, Denmark at the German Collection of Microorganisms and Cell Cultures (DSMZ), Inhofenstrasse 7B, D-38124 Braunschweig, Germany.

[0374] X6. A method according to any one of items X1-X5, wherein proliferation is defined as visual formation of colonies and / or measured by optical density.

[0375] X7. A method according to any one of items X1 to X6, wherein after step (iii):

[0376] (i) determining the production of one or more osmotic regulating substances of the derived Bacillus strain having increased salt tolerance; and / or

[0377] (ii) a step of sequencing the derived Bacillus strain having increased salt tolerance and identifying SNPs.

[0378] X8. A method according to item X7, wherein the one or more osmotic regulating substances are selected from the group consisting of: proline, citrulline, spermidine, glycine betaine, glycerol, amino acids, sugars, polyols, mannitol and sorbitol.

[0379] X9. A method according to any one of items X7 or X8, wherein the method further comprises the step of selecting a derived Bacillus strain having a high yield of the one or more osmotic regulating substances.

[0380] X10. A method according to item X9, wherein the high production of the one or more osmotic regulating substances is defined as the production of the osmotic regulating substances being equal to or increased compared to the parent Bacillus strain, preferably the production being increased compared to the parent Bacillus strain.

[0381] P1. A derivative Bacillus strain having increased salt tolerance compared to a parent Bacillus strain, the derivative Bacillus strain being obtainable by a method according to any one of items X1 to X10.

[0382] Q1. A composition comprising a derivative Bacillus strain according to any one of items Y1-Y24 or P1 having increased salt tolerance compared to a parent Bacillus strain.

[0383] Q2. A composition according to item Q1, wherein the composition comprises spores of the derived Bacillus strain.

[0384] Q3. A composition according to any one of items Q1 or Q2, wherein the composition further comprises one or more agrochemically acceptable excipients, carriers, surfactants, dispersants and yeast extracts.

[0385] Q4. A composition according to any one of items Q1-Q3, wherein the composition further comprises one or more active ingredients.

[0386] Q5. A composition according to item Q4, wherein the one or more active ingredients are selected from the group consisting of insecticides, fungicides, nematicides, bactericides, herbicides, plant extracts, plant growth regulators, plant growth stimulants and fertilizers.

[0387] Q6. A composition according to any one of items Q4 or Q5, wherein the one or more active ingredients are derived from microorganisms, organisms or chemicals.

[0388] Q7. A composition according to any one of items Q5 or Q6, wherein the insecticide is selected from the group consisting of pyrethroids, cypermethrin, fluthrin, ζ-cypermethrin, organophosphates, chlorethoxyfos, chlorpyrifos, butylpyrimidophos, flucythrin, fipronil, fipronil, nicotinoids and clothianidin and combinations thereof.

[0389] Q8. The composition according to any one of items Q5-Q7, wherein the fungicide is selected from the group consisting of fluopyram+tebuconazole, thiophanate-methyl, methyl thiophanate, prothioconazole and copper hydroxide, and combinations thereof.

[0390] Q9. A composition according to item Q4, wherein the one or more active ingredients are selected from one or more second bacterial strains different from the derived Bacillus strain, and the derived Bacillus strain has increased salt tolerance compared to the parent Bacillus strain.

[0391] Q10. The composition according to item Q4, wherein the second bacterial strain is a biostimulant strain, preferably a biostimulant Bacillus strain.

[0392] Q11. A composition according to any one of items Q1-Q10, wherein the composition is in a form selected from the group consisting of: liquid, wettable powder, granules, spreadable granules, wettable granules, microcapsules and planting matrix.

[0393] Q12. A composition according to any one of items Q1-Q11, wherein the composition further comprises a coating polymer.

[0394] Q13. A composition according to any one of items Q1-Q12, wherein the composition is a liquid formulation.

[0395] R1. A plant or seed coated with the composition according to any one of items Q1 to Q13.

[0396] R2. The plant or seed according to item R1, wherein the composition is present in an amount suitable for benefiting the growth of the plant.

[0397] R3. The seed according to any one of items R1 or R2, wherein the composition comprises about 1.0×10 vegetative cells or spores of the derived Bacillus strain. 2 CFU / seed is about 1.0×10 11 CFU / seed, e.g. about 1.0×10 3 CFU / seed is about 1.0×10 10 CFU / seed, for example, about 1.0×10 4CFU / seed is about 1.0×10 9 CFU / seed.

[0398] Z1. A method for increasing resistance of a plant to abiotic stress conditions, the method comprising applying a derivative Bacillus strain having increased salt tolerance compared to a parent Bacillus strain according to any one of items Y1-Y24 or P1, or a composition according to any one of items Q1-Q13 to the plant, a part of the plant and / or the habitat of the plant.

[0399] Z2. A method according to item Z1, wherein the abiotic stress condition is high salinity, drought and / or nutrient deficiency.

[0400] Z3. A method according to any one of items Z1 or Z2, wherein the abiotic stress condition is high salinity.

[0401] Z4. A method according to any one of items Z2 or Z3, wherein high salinity is defined as a NaCl concentration in the plant's habitat greater than about 50 mM, for example greater than about 60 mM, for example greater than about 70 mM, for example greater than about 80 mM, for example greater than about 90 mM, for example greater than about 100 mM, for example greater than about 110 mM, for example greater than about 120 mM, for example greater than about 130 mM, for example greater than about 140 mM, for example greater than about 150 mM.

[0402] Z5. A method according to any one of items Z2-Z4, wherein high salinity is defined as a NaCl concentration in the plant's habitat in the range of about 50 mM to about 150 mM, for example about 100 mM to about 150 mM.

[0403] Z6. A method according to any one of items Z1-Z5, wherein the plant is selected from the group consisting of crops, monocots, dicots, trees, herbs, shrubs, grasses, vines, ferns and mosses.

[0404] Z7. A method according to any one of items Z1-Z6, wherein the plant is selected from the group consisting of wheat, barley, oats, small grain cereals, corn, rice, sugarcane, soybeans, potatoes, carrots, coffee and bananas.

[0405] Z8. A method according to any one of items Z1 to Z7, wherein the part of the plant is selected from the group consisting of seeds, fruits, roots, stems, leaves, corms, tubers, bulbs and rhizomes.

[0406] Z9. The method according to any one of items Z1 to Z8, wherein the derived Bacillus strain or the composition is applied to a habitat of a plant.

[0407] Z10. A method according to any one of items Z1-Z9, wherein the habitat of the plant is liquid or soil, preferably soil.

[0408] Z11. A method according to any one of items Z1 to Z10, wherein the derived Bacillus strain or the composition is applied before, during or after the plant or a part of the plant is contacted with a habitat.

[0409] Z12. The method according to any one of items Z1-Z11, wherein the abiotic stress condition is identified prior to applying the derived Bacillus strain or the composition.

[0410] Z13. A method according to item Z12, wherein the abiotic stress condition is identified by measuring total soluble salts by evaporating soil water extract (TSS) or by measuring electrical conductivity.

[0411] Z14. A method according to item Z13, wherein TSS or EC is measured on a 1:5 distilled water:soil dilution or saturated paste extract.

[0412] Z15. A method according to any one of items Z1-Z14, wherein increasing the plant's resistance to abiotic stress conditions contributes to improving seedling vigor, improving root development, promoting plant growth, improving plant health, increasing yield, improving appearance, or a combination thereof.

[0413] V1. Use of a derivative Bacillus strain according to any one of items Y1-Y24 or P1 having increased salt tolerance compared to the parent Bacillus strain, or a composition according to any one of items Q1-Q13 as a biostimulant, growth promoter and / or for increasing the resistance of a plant to abiotic stress conditions.

[0414] V2. The use according to item V1, wherein the abiotic stress condition is high salinity, drought and / or nutrient deficiency, preferably high salinity.

[0415] V3. The use according to any one of items V1 or V2, wherein increasing the resistance of the plant to abiotic stress conditions contributes to improving seedling vigor, improving root development, promoting plant growth, improving plant health, increasing yield, improving appearance, reducing pathogen infection or a combination thereof.

[0416] T1. A kit comprising:

[0417] (i) a derivative Bacillus strain according to any one of items Y1-Y24 or P1 having increased salt tolerance compared to the parent Bacillus strain, a composition according to any one of items Q1-Q13, or a coated plant seed according to any one of items RR-RR;

[0418] (ii) containers; and

[0419] (iii) optionally, instructions for use.

[0420] T2. A kit according to item T1, wherein the kit further comprises one or more active ingredients selected from the group consisting of insecticides, fungicides, nematicides, bactericides, herbicides, plant extracts, plant growth regulators, plant growth stimulants and fertilizers.

[0421] T3. A kit according to item T2, wherein the derived Bacillus strain and the one or more active ingredients are provided in separate compartments of a container.

[0422] The present invention will now be described in further detail in the following non-limiting examples.

[0423] Examples

[0424] Example 1: Selection of parent Bacillus strains for laboratory adaptive evolution (ALE) campaigns to increase salt tolerance

[0425] Growth conditions were established that were suitable for determining the effect on the growth of a range of parental Bacillus strains when exposed to increasing levels of salt. Minimum inhibitory concentrations (MICs) were determined to select parental Bacillus strains for ALE activities.

[0426] method:

[0427] Strains, media, and culture conditions

[0428] Bacillus strains were cultured in LB (Luria-Bertani) broth or on LB agar at 30° C. (or 37° C.). Salt tolerance activity was performed in modified CSE medium (10.46 g / L MOPS, 3.3 g / L (NH4)2SO4, 0.13 g / L KH2PO4, 0.267 g / L K2HPO4, 3.23 mg / L MnSO4·4H2O, 120 mg / L MgSO4·7H2O, 22 μg / L ammonium ferric citrate, 0.8% potassium glutamate, 0.6% sodium succinate, pH 7).

[0429] To prepare CSE medium supplemented with NaCl, various concentrations of NaCl were added to the medium by adjusting the volume of MQ water. To sporulate the strains, SP medium (8 g / L Difco nutrient broth, 1 g / L KCl, 0.25 g / L MgSO4·7H2O, 10 μM MnCl2, 0.5 mM CaCl2, and 4.4 μg / L ferric ammonium citrate) and LB medium suitable for sporulation (LB Spore) (10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, 10 mM MgCl2, 10 mM MgSO4·7H2O, 50 μM MnCl2, and 0.5 mM CaCl2) were used to grow Bacillus strains and salt-evolved isolates.

[0430] To investigate the effect of salt (sodium chloride) on the growth of Bacillus strains, 19 different Bacillus strains belonging to different species were tested, including the laboratory model strain Bacillus subtilis 168. These strains were cultured in CSE medium supplemented with 0 to 2 M NaCl at 30°C and 225 RPM in a growth profiler, and pictures of the plates were taken every 30 min to assess bacterial growth over time.

[0431] Analysis of the growth of Bacillus spp. using the growth profiler setup

[0432] A single colony of the Bacillus strain from the LB-agar plate was inoculated into 400 μl of CSE medium in a 96-well deep-well plate. The plate was then incubated at 30°C and 250 RPM for 20-24 hours. After 10-fold to 20-fold dilution, the OD 600nm . The saturated overnight preculture was diluted 100-fold in 250 μl of CSE medium or CSE medium supplemented with a predetermined concentration of NaCl. The culture plates were then incubated at 30°C and 250 RPM for 2-3 days in a growth profiler (Enzyscreen; Heemstede, The Netherlands), where growth was continuously monitored by scanning the plates every 30 minutes. The pixel data of the photographs were converted into digital OD values ​​using GP software (GP960Viewer version 1.0.0.4, Enzyscreen, Heemstede, The Netherlands). 600nm values ​​and generate a growth curve over time.

[0433] Determination of Minimum Inhibitory Concentration (MIC) and IC90

[0434] For the determination of MIC, a fresh single colony of the Bacillus strain was inoculated into 400 μl CSE medium in a 96-well deep well plate, and the plate was then incubated overnight at 37°C and 200 RPM. The overnight culture was inoculated into 250 μl CSE medium supplemented with a predetermined concentration of NaCl (ranging from 0 to 2 M) at a 1:100 dilution ratio (i.e., 2.5 μl overnight culture in 250 μl medium). Subsequently, the culture plate was incubated at 225 RPM and 30°C. The MIC was determined using a logarithmic NaCl concentration gradient. At the 24th and 47th hours of incubation, the optical density (OD) of the culture at a wavelength of 600 nm was measured. 600nm , background subtracted). Growth inhibition of Bacillus strains was plotted against NaCl concentration using R software (free software provided by the R Foundation) by polynomial interpolation between adjacent data points. Percent inhibition was calculated using the following formula:

[0435] [1–(OD 600nm NaCl / OD 600nm(对照) )]x100%

[0436] The inhibitory concentrations, called IC50 and IC90, were defined as the lowest NaCl concentrations that inhibited the growth of the tested strain by 50% and 90%, respectively.

[0437] result:

[0438] Most of the 19 tested Bacillus strains grew well in CSE medium. As the NaCl concentration increased, the growth rate gradually decreased until it reached a concentration that no longer supported growth ( Figure 1 ). Figure 1 A selected series of growth curves of the tested Bacillus strains are shown, illustrating the fact that different Bacillus strains have different levels of NaCl tolerance.

[0439] The inhibition curve of Bacillus strains was plotted with log(NaCl) concentration as X-axis and inhibition percentage as Y-axis ( Figure 2 From these inhibition curves, the NaCl IC50 and IC90 values ​​of the Bacillus strains were determined ( Figure 3 In fact, the addition of small amounts (e.g., 200 mM or 350 mM) of salt enhances the growth of some Bacillus strains, resulting in growth promotion rather than inhibition. In this case, the absolute inhibition value becomes negative at low salt concentrations ( Figure 2 ). To account for this complexity, the effective concentration (EC) is used. EC50 is the concentration of NaCl that produces a half-maximal response. For an ideal inhibition curve, EC50 = IC50, while if negative inhibition values ​​are to be considered, IC50>EC50 ( Figure 3 ).

[0440] Comparison of the growth behavior of Bacillus strains in CSE medium supplemented with NaCl showed that, under natural conditions, the most salt-tolerant strains belonged to the following species: Bacillus paralicheniformis (IC50 1.0-1.2 M at 47 h), followed by Bacillus velezensis (IC50 1.0-1.1 mM at 47 h) and Bacillus subtilis (IC50 0.7-0.9 M at 47 h). The least salt-tolerant strain was the Bacillus amyloliquefaciens strain (IC50 0.5 M at 47 h) ( Figure 3 ).

[0441] Based on the MIC values, eight Bacillus strains were selected for ALE activities to further improve their salt tolerance. The selected parent strains (SA-P1 to SA-P5 and SA-P7 to SA-P9) are listed in Table 1 below.

[0442]

[0443] Table 1.

[0444] in conclusion:

[0445] This example demonstrates that suitable conditions for assessing salt tolerance were established and candidate parent strains were purposefully selected for ALE activities based on their baseline salt tolerance.

[0446] Example 2: Salt evolution campaign and selection of derived Bacillus strains with improved salt tolerance from the evolved population

[0447] method:

[0448] Salt tolerance laboratory adaptive evolution (SALTY-ALE) campaigns were performed in triplicate tubes by linearly increasing the NaCl concentration in the growth medium for each parent strain selected in Example 1 (see Table 1). Three different methods were used to select derivative strains with increased salt tolerance from the evolved populations of the SALTY-ALE campaign.

[0449] Method 1: Relaxation step and plating in rich medium

[0450] The evolved population in the salt-supplemented CSE medium was first transferred to LB medium to eliminate salt stress before plating on LB agar supplemented with various concentrations of NaCl. Briefly, 500 μl of the evolved strain from the SALTY-ALE activity was centrifuged at 5000 x g for 5 min, and the supernatant was discarded (this was done for all three lineages of each parent strain). The cell pellet was resuspended in 500 μl of fresh LB medium. The resuspended cells were then transferred to 4.5 ml of LB medium, and the culture tubes were incubated overnight at 30°C and 250 RPM. The parent strain was also inoculated into 5 ml of LB medium (as a control) and incubated overnight at 30°C and 250 RPM. The next day, the overnight culture was diluted in LB medium to a final OD 600nm The OD adjusted cultures were streaked or plated on LB agar and LB agar supplemented with various concentrations of NaCl. For example, the cells were streaked on LB agar, LB + 1M NaCl agar plates, 10-25 μl of the cells were plated on LB + 1.4M NaCl, LB + 1.6M NaCl, and 25-100 μl of the cultures were plated on LB + 1.89M NaCl, LB + 2M NaCl, and LB + 2.2M NaCl agar plates. The plates were incubated at 37°C until pickable colonies were observed, i.e., after about 24-48 hours.

[0451] Method 2: Direct streaking of the population in the evolving culture tube

[0452] Several microliters of growing cells from the SALTY-ALE tubes were streaked directly onto LB agar supplemented with various concentrations of NaCl. Briefly, 2-5 μl of culture medium (OD 600nm The 100 μg / ml PBS (10 μg / ml) was directly streaked onto LB agar plates supplemented with various concentrations of NaCl. The plates were then incubated at 37°C for 24 to 48 h.

[0453] Method 3: After the washing step, streak the culture in the evolved culture tube directly

[0454] Before streaking the cells in the SALTY-ALE culture tubes, the metabolites produced in the culture solution were removed and the cells were streaked onto LB agar supplemented with various concentrations of NaCl. In brief, 500 μl of the culture solution from the SALTY-ALE tube was centrifuged at 5000 x g for 5 min, and the supernatant was discarded. The cell pellet was resuspended in 1 ml of CSE medium containing NaCl (the NaCl concentration was the same as in the SALTY-ALE tube) and centrifuged at 5000 x g for 5 min, and the supernatant was discarded. Then, the washed cells were resuspended in 500 μl LB medium, and 2-5 μl of the resuspended cells were streaked onto LB agar plates supplemented with various concentrations of NaCl. Finally, the plates were incubated at 37 ° C for 24 to 48 h.

[0455] Comparison of growth parameters of isolates selected from the ALE population

[0456] To select isolates with improved salt tolerance after SALTY-ALE experiments, growth parameters of colonies (isolates) obtained from methods 1-3 above were analyzed in CSE medium and CSE medium supplemented with NaCl using a growth profiler setup.

[0457] First, we examined growth parameters for individual isolates, and then further analyzed isolates with improved growth (relatively higher growth rate and / or shorter lag time relative to the parent strain in NaCl-supplemented medium) by confirming their improvement by testing in triplicate. In all growth profiler experiments, the strains with the greatest improvement were selected based on: (i) improved growth rate and / or (ii) shortened lag period in NaCl-supplemented medium. Both criteria were compared to the performance of the parent strain under the same conditions as a benchmark.

[0458] For growth profiler experiments, the starting OD of all strains including the parental strain was 600nm Keep it within the 0.02-0.05 range to achieve comparable results.

[0459] result:

[0460] The results are exemplified by SALTY-ALE activity based on the parental B. paralicheniformis strain designated SA-P7. The suitability of each of the three approaches to select derivative strains with increased salt tolerance from the evolved population was evaluated.

[0461] For method 1, the salt-evolved culture tubes had a greater number of colonies growing on plates with increasing salt concentrations than the parent strain. Growth of the salt-evolved cultures was particularly favored under high salt loads.

[0462] Methods 2 and 3 demonstrated that the salt-evolved culture tubes contained evolved populations that formed colonies at high salinity concentrations. In particular, method 2 produced colonies at high salinity concentrations. Without being bound by theory, the high salt tolerance may be attributed to the presence of metabolites in the ALE culture during the induced salt stress and / or direct streaking.

[0463] Comparison of the growth curves of isolates from all three methods in CSE supplemented with 1 M NaCl showed that the growth rate of most isolates was enhanced.

[0464] Using the above procedure, a selected series of lead candidate derivative Bacillus strains were isolated from colonies grown under high salt concentrations. The lead candidate derivative Bacillus strains are listed below.

[0465] Table 2.

[0466]

[0467]

[0468] Table 2.

[0469] The growth phenotypes of a selected series of these lead candidate strains are depicted in Figure 4 It was shown that the derived Bacillus strains that had been subjected to SALTY-ALE activity outperformed their corresponding parent strains.

[0470] in conclusion:

[0471] This example shows that parental Bacillus strains subjected to SALTY-ALE activity significantly improved their salt tolerance. Several methods were used and found to be useful for selecting and isolating improved derivative strains from colonies.

[0472] Example 3: Method for determining the spore-forming ability of selected isolated derivative Bacillus strains:

[0473] To test the ability of the derived Bacillus strains to form spores, these strains were cultured in sporulation (SP) medium or LB medium suitable for sporulation (LB Spore) and the culture tubes were incubated at 37°C for 24-26h. Then 40 μl of the fully grown culture was heated at 80°C for 20 min. Strains that did not form spores were taken as negative controls.

[0474] Heat-treated and non-heat-treated cultures were grown in LB medium from 10 0 to 10 -710-fold serial dilutions were performed and 5 μl of the serially diluted cultures were spotted on LB agar plates. The spotted cultures were allowed to dry and the plates were incubated overnight at 30° C. The next day, the plates were analyzed for growth of the modified derivatives at different dilutions.

[0475] In this assay, strains capable of spore formation will be able to grow in both heat-treated and non-heat-treated samples, whereas spore-negative strains will only grow under non-heat-treated conditions.

[0476] result:

[0477] As described above, the lead candidate strains (Table 2) were all positive for sporulation in the assay, i.e. they were able to survive the heat treatment and subsequently grow under normal conditions. The absence of growth in the heated samples from the negative control strain validated the sporulation test.

[0478] in conclusion:

[0479] This example demonstrates that SALTY-ALE activity successfully generated candidate strains that retained their spore-forming ability throughout evolution.

[0480] Example 4: Determination of the production of osmotic regulating substances by selected isolated derivative Bacillus strains under salt stress conditions

[0481] A selected group of improved derivatives were investigated for the production of osmotic regulating substances under salt stress conditions by biochemical analysis.

[0482] method

[0483] A single colony of a Bacillus strain was inoculated into 400 μl LB medium in a 96-well deep-well plate. The plates were incubated overnight at 37°C and 250 RPM. The next morning, 25 μl of the saturated overnight culture was inoculated in triplicate into 500 μl CSE or CSE medium supplemented with 350 mM (final concentration) NaCl in a 96-well deep-well plate, and the plate was incubated at 30°C and 250 RPM for 28 h. The optical density (OD) of the culture was measured after 20-fold dilution. 600nm ). The plates were centrifuged at 3700 RPM for 30 min and 450 μl of the supernatant was transferred to a 96-well deep well plate. These plates were submitted for quantification of various amino acids and compatible solutes (for LC-mass spectrometry analysis).

[0484] The concentrations of various amino acids were determined on a liquid chromatography-mass spectrometer (LC-MS) (Acquity UPLC and Xevo TQ-XS, Waters Corporation, Milford, MA, USA) equipped with an AccQ-Tag UltraRP column ( 1.7 μm, 2.1 mm × 100 mm, Waters Corporation, Milford, MA, USA).

[0485] A 50 μL aliquot was transferred from this plate to a new plate and 950 μL of MilliQ water was added to the aliquot. TM Amino acid derivatization was performed using the Ultra Derivatization Kit (Waters Corporation, Milford, MA, USA). 40 μL of sample was mixed with 40 μL of internal standard solution containing approximately 1 μM of all targets labeled with 13C. The mixture was buffered with 50 μL of borate buffer and 20 μL of derivatization reagent was added. The solution was mixed and heated at 55°C, 2000 rpm for 10 min and then analyzed on LC-MS.

[0486] result

[0487] Here, the extracellular concentrations of various compatible solutes in a salt-tolerant improved lead Bacillus strain were tested and quantified.

[0488] In many improved halotolerant Bacillus strains, the amount of proline increased significantly when cultured under high salt conditions compared to their corresponding parent strains. For example, the extracellular proline concentration of SA60 increased more than 7-fold compared to its parent strain SA-P4 ( Figure 5 A).

[0489] Analysis of the extracellular concentration of another compatible solute, citrulline, concluded that under high salinity conditions, several modified halotolerant Bacillus strains had increased extracellular citrulline concentrations compared to their parent strains ( Figure 5 B).

[0490] Further analysis of the amino acid levels of one of the modified halotolerant Bacillus strains, SA60, showed that for a broad range of selected amino acids, the amino acid levels were elevated ( Figure 5 C) Under high salinity conditions, the levels of several amino acids are particularly increased. In addition to proline and citrulline, this is also true for, for example, glycine, valine, leucine, isoleucine and arginine.

[0491] in conclusion

[0492] Many of the modified halotolerant derivative Bacillus strains adapted during the SALTY-ALE campaign by evolving their ability to produce osmoregulatory substances and amino acids.

[0493] Example 5: Protective effects of osmotic regulating substances on Arabidopsis germination and growth under salt stress

[0494] To investigate the effects of osmotic regulators on plant growth under conditions of elevated salt concentration, the germination and growth of Arabidopsis thaliana supplemented with osmotic regulators, particularly proline, were measured.

[0495] method

[0496] Germination of Arabidopsis seedlings

[0497] Arabidopsis seeds were surface sterilized by incubation in 96% ethanol for 2 min, in 10% (v / v) commercial bleach for 5 min, and washed thoroughly four times in sterile water. The water was removed and the seeds were stratified at 4°C for 2 days. Sterile stock solutions of individual compounds (L-proline, L-citrulline, glycine betaine and spermidine, Sigma) were mixed directly with warm 1 / 2 Murashige and Skoog (MS) basal medium (M5519, Merck) pH 5.8 containing 0.8% plant agar (P1003, Duchefa Biochemie) supplemented with sodium chloride to a final concentration of 90 mM NaCl. Seeds were sown on plant culture plates and grown in a Percival plant growth chamber under long day conditions (16 h light / 8 h dark) with a light intensity of 115 μmol m2 s -1 and incubate at 22°C / 20°C.

[0498] Growth of Arabidopsis seedlings

[0499] Five-day-old Arabidopsis seedlings were transferred to 1 / 2 Murashige and Skoog medium supplemented with 1 mM proline (with or without NaCl). Whole plant biomass was measured on 15-day-old plants.

[0500] result

[0501] Germination of Arabidopsis seedlings

[0502] Four osmotic regulating substances, L-proline, L-citrulline, glycine betaine and spermidine, all increased the seed germination frequency of Arabidopsis thaliana at 90 mM NaCl ( Figure 6 A). Different concentrations of osmotic regulating substances produce the best response to salt stress conditions. Thus, it was observed that 1 mM proline, 0.5 mM citrulline, 30 mM glycine betaine or 0.15 mM spermidine had the best response to alleviate salt stress conditions.

[0503] Growth of Arabidopsis seedlings

[0504] When the growth medium was supplemented with proline, an increase in the fresh biomass of Arabidopsis thaliana was detected under high salinity concentration (90 mM NaCl) compared to the control without proline ( Figure 6 B) The data support that proline can alleviate salt stress in plants.

[0505] in conclusion

[0506] This example demonstrates that osmotic regulating substances have a beneficial effect on plant growth in plants subjected to salt stress. This suggests that if derived Bacillus strains that have evolved both salt tolerance and the ability to produce osmotic regulating substances are supplemented to plants growing in habitats with high salinity levels, plant growth can be improved.

[0507] Example 6: Evaluation of plant growth when supplemented with derived Bacillus strains

[0508] To investigate the beneficial effects of derived Bacillus strains on plant growth, greenhouse trials with maize were conducted.

[0509] method

[0510] Plant test in greenhouse begins with 50 corn seeds (C4E8 maize) being embedded 1 hour with every kind of bacillus strain solution, and this solution concentration is OD600=1.Then, seed is moved in the culture dish containing 12mL 100mM NaCl, and in growth chamber, it is sprouted 12 days with the cycle of 16 hours illumination-8 hours darkness.After sprouting, seed is transplanted in the 250mL flowerpot containing clay field soil, and is watered with 150mM NaCl.Plant is grown 3 weeks in greenhouse, and when watering at every turn, with 150mM NaCl, flowerpot is fully soaked, and removes unnecessary water, to guarantee that all flowerpots are balanced to 150mM NaCl when watering at every turn.Gather plant at the end of experiment, and record dry weight.

[0511] result

[0512] The salt-evolved derivative Bacillus strains are presented as white bars immediately to the right of their parent strains ( Figure 7 AB). The data showed that supplementation with salt-evolved derivative Bacillus strains (SA3, SA9, SA21, SA40, SA59, SA60, SA63, SA64, SA70) increased plant dry matter compared to the corresponding parental Bacillus strains (SA-P1, SA-P3, SA-P4, SA-P5, SA-P7, SA-P9) and controls without any bacteria (control salt, control brine). Therefore,

[0513] in conclusion

[0514] This example demonstrates that supplementation with salt-adapted derivative Bacillus strains promotes plant tolerance to salt stress.

[0515] Example 7: Genetic Analysis of Selected Isolated Derivative Bacillus Strains

[0516] The modified derivative Bacillus strains in Table 2 were subjected to genetic analysis to detect mutations, including single nucleotide polymorphisms (SNPs), to identify key target genes.

[0517] method

[0518] Streak a single colony of the modified derivative Bacillus strain onto an LB plate and perform three rounds of purification steps. Then, inoculate 5 colonies of the purified strain into 5 ml of LB medium and incubate the tube at 30°C overnight. The next morning, transfer 500 μl of the overnight culture into 30 ml of fresh LB medium. Incubate the flask at 37°C until the OD 600nm Then, prepare the whole genome sequencing sample.

[0519] Cell pellets were collected from 1.5 OD equivalent of 1.5 ml culture by centrifugation of the culture at 5000 x g for 10 min. The cell pellets were then submitted for gDNA isolation and whole genome sequencing.

[0520] result

[0521] Whole genome sequencing of the derived Bacillus strains detected genetic changes and identified key target genes believed to play a role in improving salt tolerance. These target genes include exporter genes involved in the transport of metabolites into and out of the cell, genes regulating symporters, and genes related to quorum sensing.

[0522] in conclusion

[0523] Based on genetic analysis of derived Bacillus strains with improved salt tolerance, key target genes were identified.

[0524] References

[0525] Altschul et al. (1990), J. Mol. Biol., 215, 403-410

[0526] Preservation and Expert Solutions

[0527] The applicant requests that, prior to the date of grant of the patent, samples of the deposited microorganisms described in Table 3 below be provided only to experts.

[0528] The applicant requested that the availability of the deposited microorganisms referred to in Article 33 EPC should only be made by issuing samples to an independent expert appointed by the applicant (Rule 32(1) EPC). If an expert solution has been requested, restrictions on the provision of samples apply.

[0529] Deposited under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure at the German Collection of Microorganisms and Cell Cultures (DSMZ), Inhofenstrasse 7B, D-38124 Braunschweig, Germany.

[0530] The Budapest Treaty provides that any restrictions on public access to samples of deposited biological material must be irrevocably lifted from the date of grant of the relevant patent.

[0531] strain name Accession number Date of deposit Bacillus amyloliquefaciens SA-P1 DSM34003 August 24, 2021 Bacillus subtilis SA-P3 DSM17231 April 7, 2005 Bacillus subtilis SA-P4 DSM32324 June 8, 2016 Bacillus Velez SA-P5 DSM34004 August 24, 2021 Bacillus paralicheniformis SA-P7 DSM33110 May 8, 2019 Bacillus paralicheniformis SA-P9 DSM33240 August 14, 2019 Bacillus Velez SA70 DSM34005 August 24, 2021

[0532] Table 3. Strains deposited at depository institutions.

Claims

1. A derivative Bacillus strain or variant thereof having increased salt tolerance compared to a deposited parent Bacillus strain, the parent Bacillus strain being selected from the group consisting of: -DSM34004 (deposited on August 24, 2021), -DSM34003 (deposited on August 24, 2021), -DSM33240 (deposited on August 14, 2019), -DSM17231 (deposited on April 7, 2005), -DSM33110 (deposited on May 8, 2019) and - DSM 32324 (deposited on 8 June 2016), The deposited strains are deposited by Chr. Hansen AG, Hirsholm, Denmark, at the German Collection of Microorganisms and Cell Cultures (DSMZ), Inhofenstrasse 7B, D-38124, Brunswick, Germany, and The derived Bacillus strain or its variant is a derivative strain of the parent Bacillus strain.

2. The derived Bacillus strain according to claim 1, wherein the salt tolerance of the derived Bacillus strain is at least increased by 2.5 times compared to the parent Bacillus strain, such as at least increased by 3 times, such as increased by 3.5 times, such as increased by 4 times compared to the parent Bacillus strain.

3. The derived Bacillus strain according to any one of claims 1 or 2, wherein the derived Bacillus strain has a salt tolerance of at least 0.8 M NaCl, such as at least 0.9 M NaCl, such as at least 1.0 M NaCl, such as at least 1.1 M NaCl, such as at least 1.2 M NaCl, such as at least 1.3 M NaCl, such as at least 1.4 M NaCl, such as at least 1.5 M NaCl.

4. The derived Bacillus strain according to any one of the preceding claims, wherein the derived Bacillus strain comprises one or more mutations compared to the parent Bacillus strain.

5. The derived Bacillus strain of claim 4, wherein the one or more mutations are located in one or more genes of the parent Bacillus strain selected from the group consisting of: -yjcG encoding the putative phosphatase YjcG; -comP encoding the sensor histidine kinase ComP; -dnaA-1, encoding the chromosome replication initiation protein; -sigB, encoding RNA polymerase sigma-B factor; - flhB-2 encoding the flagellar biosynthesis protein FlhB; -rny encoding ribonuclease Y; -ilvE encoding branched-chain amino acid aminotransferase; -dcuS-1, encoding the sensor histidine kinase DcuS; -rsbT encoding the serine / threonine protein kinase RsbT; - fliM encoding the flagellar motor switch protein FliM; - mgsR-1, encoding the regulatory protein MgsR; - opuE encoding the osmoregulatory proline transporter OpuE; - bshA-1 encoding N-acetyl-α-D-glucosaminyl L-malate synthase; - metAA encoding homoserine O-acetyltransferase; -motB encoding movement protein B; -kinA-1 encoding sporulation kinase A; -comA, encoding the transcriptional regulator protein comA; - rpoC, encoding the β' subunit of the DNA-directed RNA polymerase; - eamA-1 encoding a putative amino acid metabolite efflux pump; and -rpoB, encoding the β subunit of DNA-directed RNA polymerase.

6. The derived Bacillus strain according to any one of claims 4 or 5, wherein the one or more mutations are located in one or more genes of the parent Bacillus strain selected from the group consisting of yjcG, comP, dnaA-1 and combinations thereof.

7. The derived Bacillus strain according to any one of the preceding claims, wherein the derived Bacillus strain produces one or more osmotic regulating substances in large amounts under high salinity conditions, The high production of the one or more osmotic regulating substances is defined as the production of the osmotic regulating substances being equal to or increased compared to the parent Bacillus strain, preferably increased compared to the parent Bacillus strain.

8. The derived Bacillus strain according to any of the preceding claims, wherein the derived Bacillus strain was deposited as DSM34005 by Chr. Hansen AG, Hirsholm, Denmark on August 24, 2021 at the German Collection of Microorganisms and Cell Cultures (DSMZ), Inhofenstrasse 7B, D-38124 Braunschweig, Germany.

9. A method for preparing a derived Bacillus strain according to any one of the preceding claims, the method comprising the following steps: (i) Providing a parent Bacillus strain (ii) growing the parent Bacillus strain under high salinity conditions, and (iii) selecting a derivative Bacillus strain having increased salt tolerance that proliferates under the growth conditions of step (ii).

10. A derivative Bacillus strain having increased salt tolerance compared to a parent Bacillus strain, the derivative Bacillus strain being obtainable by the method according to claim 9.

11. A composition comprising the derivative Bacillus strain according to any one of claims 1-8 or 10 having increased salt tolerance compared to a parent Bacillus strain.

12. The composition of claim 11, wherein the composition comprises spores of the derived Bacillus strain.

13. The composition according to any one of claims 11 or 12, wherein the composition is in a form selected from the group consisting of: liquid, wettable powder, granules, spreadable granules, wettable granules, microcapsules and planting substrates.

14. A method of increasing resistance of a plant to abiotic stress conditions, the method comprising applying to the plant, a part of the plant and / or the habitat of the plant a derivative Bacillus strain having increased salt tolerance compared to a parent Bacillus strain according to any one of claims 1 to 8 or 10, or a composition according to any one of claims 11 to 13.

15. Use of a derivative Bacillus strain according to any one of claims 1 to 8 or 10 having increased salt tolerance compared to a parent Bacillus strain, or a composition according to any one of claims 1 1 to 13 as a biostimulant, growth promoter and / or for increasing resistance of plants to abiotic stress conditions.

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