WO2024047025A1

WO2024047025A1 - Derivative bacillus strains for high abiotic stress

Info

Publication number: WO2024047025A1
Application number: PCT/EP2023/073637
Authority: WO
Inventors: Ronnie MACHIELSEN; Raquel AZEVEDO; Sailesh MALLA; Jan GERWIG; Cesar FONSECA; Lorenzo FIMOGNARI; Lars MOELBAK
Original assignee: Chr. Hansen A/S
Priority date: 2022-08-30
Filing date: 2023-08-29
Publication date: 2024-03-07
Also published as: AU2023331894A1

Abstract

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

Description

DERIVATIVE BACILLUS STRAINS FOR HIGH ABIOTIC STRESS

Technical field of the invention

Background of the invention

The rapid population growth combined with climate change create a big challenge for crop production and yield globally. In one hand, there is an increasing demand of agricultural yield while on the other hand various biotic and abiotic issues significantly reduce crop production.

Soil salinity is an abiotic stress that poses a great threat to agriculture. Major crop losses annually occur due to toxic amounts of salts in the soil, particularly sodium chloride (NaCI). About 20% of the world cultivated area and around 50% of the world irrigated lands are affected by increased soil salinity, causing significant abiotic stress to plants. The main cause of increased salinity in irrigated areas are due to soluble salts carried in the irrigation water, which remain in the soil after evaporation and transpiration of the water. Unless these salts are leached from the soil, after prolonged time they accumulate to levels that are inhibitory to plant growth. This will also result in degradation of the soil structure affecting water and root penetration. Typically, plants stressed with NaCI are characterized by slower growth, premature leaf senescence, reduced tillering and lower yield. Sodium ions (Na⁺) are particularly damaging in high cytosolic concentration in leaf cells since they intervene with metabolic processes, and in particular, photosynthesis. Hence, among abiotic stresses, soil salinity and drought are considered a major issue.

Plant and soil microbes interact to help each other for their growth and development as well as to maintain the terrestrial eco-system. Plants can also use these growth promoting microbes as allies to withstand abiotic stress. Notably, plant growth promoting bacteria (PGPR) may help plants to grow in a high salinity soil by various mechanisms such as i) providing compatible solutes/osmolytes to the plants ii) by improving biofilm formation and root colonization to trap water molecules inside the biofilms, iii) modulating plant growth through secretion of growth regulators (e.g. auxins, gibberellins) and/or iv) by balancing K⁺ and Na⁺ ion ratio in the plants. Growth promoting microbes include Bacillus which are Gram-positive bacteria characterized by having thick cell walls and the absence of outer membranes. Much of the cell wall of Gram-positive bacteria is composed of peptidoglycan. Gram-positive species are divided into groups according to their morphological and biochemical characteristics. The genus Bacillus is belonging to the group of sporulating bacteria. Bacterial spores are one of the most resilient cell types; they resist many environmental changes, withstand dry heat and certain chemical disinfectants and may persist for years on dry land.

Accordingly, plant growth promoting biostimulant Bacillus strains have been applied, both naturally and commercially, as biofertilizers and as plant protectants. However, creating Bacillus strains with phenotypes adapted for stimulating plant growth at high soil salinity is very challenging without thorough knowledge of the target gene(s) involved. Typically, a combination of strain mutagenesis, smart screening, and careful selection of candidate strains is necessary to identify and isolate candidate derivative strains with desired phenotypes. Candidate derivative strains may then be evaluated diligently at different levels, such as in vitro, in vivo/in planta and/or in field trials, for determining their performance. Accordingly, it is a complex task to obtain derivative Bacillus strains capable of promoting plant growth under high salinity conditions. Further development of such Bacillus strains would come with significant economic savings and improve the ability to meet the increasing global demands for crop production as the world population grow and climate changes enhance abiotic stress on farmland.

Thus, there is an unmet need for supplying improved Bacillus strains to aid plants challenged by abiotic stress and improve their yield under sub-optimal growth conditions.

Hence, it would be advantageous to provide Bacillus strains capable of proliferating under abiotic stress conditions, such as high salinity or drought. Specifically, it would be advantageous to provide improved Bacillus strains which efficiently promote plant health and growth under conditions of high salinity.

Summary of the invention

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 parental Bacillus strains which may be applied to plants or the habitat of the plant thereby forming a colonization of beneficial bacteria on and around the plant, to provide the plant with better conditions for retrieval of necessary nutrients from the soil, thereby mitigating the stress exerted on the plant and freeing energy for plant growth. The derivative Bacillus strains are prepared through natural methods of classical strain improvement by exposure of a parental Bacillus strain to an adapted evolution campaign followed by identification and selection of improved strains. Accordingly, the present invention makes available improved Bacillus strains to improve yield of plants or crops under conditions of high salinity.

Thus, an object of the present invention relates to the provision of improved Bacillus strains capable of promoting plant growth and resilience under abiotic stress.

In particular, it is an object of the present invention to provide derivative strains of Bacillus that increases yield of plants or crops under conditions of high salinity or drought.

Thus, an aspect of the present invention relates to a derivative Bacillus strain, or variant thereof, with increased salt tolerance compared to a parental strain of Bacillus, wherein the derivative Bacillus strain, or variant thereof, is a derivative strain of said parental strain of Bacillus.

Another aspect of the present invention relates to a derivative Bacillus strain, or variant thereof, with increased salt tolerance compared to a deposited parental strain of Bacillus selected from the group consisting of;

- DSM34004 (deposited on 24 August 2021),

- DSM34003 (deposited on 24 August 2021),

- DSM33240 (deposited on 14 August 2019),

- DSM17231 (deposited on 7 April 2005),

- DSM33110 (deposited on 8 May 2019), and

- DSM32324 (deposited on 8 June 2016), wherein the deposited strain is deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Inhoffenstr. 7B, D-38124 Braunschweig, Germany, by Chr. Hansen A/S, Horsholm, Denmark, and wherein the derivative Bacillus strain, or variant thereof, is a derivative strain of said parental strain of Bacillus.

A further aspect of the present invention relates to a method for preparing a derivative Bacillus strain as described herein, said method comprising the steps of:

(i) providing a parental strain of Bacillus

(ii) growing said parental strain of Bacillus under conditions of high salinity, and

(iii) selecting a derivative Bacillus strain with increased salt tolerance that proliferates under the growth conditions of step (ii). Yet another aspect of the present invention relates to a derivative Bacillus strain with increased salt tolerance compared to a parental strain of Bacillus obtainable by the method as described herein.

Still another aspect of the present invention relates to a composition comprising the derivative Bacillus strain with increased salt tolerance compared to a parental strain of Bacillus as described herein.

A still further aspect of the present invention relates to a plant or seed coated with a composition as described herein.

An even further aspect of the present invention relates to a method of increasing resistance of a plant against a condition of abiotic stress, said method comprising applying a derivative Bacillus strain with increased salt tolerance compared to a parental strain of Bacillus or a composition as described herein to the plant, to a part of the plant and/or to the habitat of the plant.

Yet another aspect of the present invention relates to use of a derivative Bacillus strain with increased salt tolerance compared to a parental strain of Bacillus or a composition as described herein as a biostimulant, a growth promoter and/or to increase resistance of a plant against a condition of abiotic stress.

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

(i) a derivative Bacillus strain with increased salt tolerance compared to a parental strain of Bacillus, a composition, or coated plant seeds as described herein;

(ii) a container; and

(iii) optionally, instructions for use.

Brief description of the figures

Figure 1 shows growth of various Bacillus strains in CSE media and CSE media supplemented with NaCI. (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.

Figure 2 shows inhibition curves of several Bacillus strains upon supplementation of various concentrations of NaCI 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. Figure 3 shows the inhibitory concentrations of NaCI to inhibit 50% (IC50) and 90% (IC90) of Bacillus strain growth in CSE media at 47 h of incubation at 250 RPM and 30°C. EC: Effective concentration. For each Bacillus strain are given from left to right: IC50, EC50, IC90 and EC90.

Figure 4 shows growth phenotypes under salt stress condition of the some of the selected evolved strains compared to their parental strains. (A) SA70 vs. SA-P5, (B) SA63 vs. SA-P1, (C) SA60 vs. SA-P4, (D) SA59 vs. SA-), and (E) SA40 vs. SA-P7. Light grey (open circles): evolved strains, CSE (or low salt). Dark grey (open circles): parent strain, CSE (or low salt). Dark grey (closed circles): evolved strain, high salt. Black (closed circles): parent strain, high salt.

Figure 5 shows production of osmolytes (A) proline and (B) citrulline, respectively, by parental Bacillus strain and SALTY-ALE derivative strains in CSE media (dark grey bar) and CSE supplemented with 0.35 M NaCI media (light grey bar). (C) Comparison of the extracellular amino acid levels of SALTY-ALE derivative SA60 and its parental strain SA- P4. Cultivation was conducted either in CSE or CSE-0.35 M NaCI medium for 28 hrs.

Figure 6 shows (A) germination of Arabidopsis thaliana seedlings under saline conditions (90 mM NaCI) with and without supplementation of compatible solutes. (B) Fresh weight of 15-days old seedlings with and without supplementation of proline grown under conditions of no salt or 90 mM NaCI. Five-days old seedlings were transferred to V2MS media without and with 1 mM proline.

Figure 7 shows influence of derivative Bacillus strains on plant growth under salt stress conditions. Corn plants were treated with 150 mM NaCI with or without a derivative Bacillus strain. (A) 8 biological replicates were employed for each treatment (B) 10 biological replicated were employed and an additional control was employed where plants were well watered without NaCI.

The present invention will in the following be described in more detail.

Detailed description of the invention

Definitions

Prior to outlining the present invention in more details, a set of terms and conventions is first defined:

Plant biostimulant In the present context, the term "plant biostimulant" refers to any substance or microorganism applied to plants with the ability to enhance nutrition efficiency, abiotic stress tolerance and/or crop quality traits, regardless of its nutrients content. By extension, plant biostimulants also designate commercial products containing mixtures of such substances and/or microorganisms.

Plant growth promoting agent

In the present context, the term "plant growth promoting agent" or "plant growth promoting microorganism" refers to a microorganism with the ability to colonize roots and/or inner plant tissues and promote plant growth and health by either acting as a biofertilizer, biostimulant or via biological control of plant disease.

Parental strain

In the present context, the term "parental strain" refers to a microorganism that is the origin to one or more derived strains, i.e. it is designating the first generation giving rise to one or more succeeding generation.

Derivative strain

In the present context, the term "derivative strain" refers to a microorganism that is a second generation derived from a parental strain. The derivative strain may be developed by mutagenesis, wherein one or more mutations are introduced into the genome of the parental strain. The mutations may be introduced via adaptive laboratory evolution by exposing the parental strain to increasing concentrations of salt, such as NaCI.

Identifying characteristics

In the present context, the term "identifying characteristics" refers to the phenotype of a microorganism, i.e. the set of observable characteristics or traits of the microorganism. Particularly, the identifying characteristic can be increased salt tolerance.

Microorganisms sharing all identifying characteristics can have different non-identical genomic sequences. This may be the case if mutations are silent or conservative, i.e. the new codon gives rise to the same amino acid or the new amino acid have similar biochemical properties (e.g. charge or hydrophobicity), respectively.

Salt tolerance In the present context, the term "salt tolerance" refers to the concentration of salt, such as NaCI, needed to inhibit the proliferation of a cell population, such as a Bacillus strain, by 50%. This is also called the half maximal inhibitory concentration (IC50).

"Salt tolerance" may be measured with respect to typical salts found in irrigation water including sodium chloride (NaCI), sodium sulphate (Na2SC>4), sodium bicarbonate (NaHCCh), magnesium sulphate (MgSC ), calcium sulphate (CaSC ), calcium chloride (CaCh), potassium chloride (KCI), and potassium sulphate (K2SO4).

Preferably, "salt tolerance" is measured with respect to NaCI.

Osmolyte

In the present context, the term "osmolyte" refers to highly water soluble and low molecular weight organic compounds that assist a plant in withstanding osmotic imbalances. They may protect the integrity of cells by influencing viscosity, melting point and/or ionic strength of biological fluids. In particular, osmolytes may relieve swelling of cells caused by osmotic pressure by efflux through membrane channels of osmolytes carrying water with them.

Osmolytes can be categorized in three major groups: amino acids, quaternary and tertiary onium compounds, and polyol/small sugars. Examples of osmolytes include, but are not limited to, proline, citrulline, spermidine, glycine-betain, glycerol, mannitol, sorbitol, trimethylamine N-oxide (TMAO), dimethylsulfoniopropionate, sarcosine, betaine, glycerophosphorylcholine, myo-inositol, taurine, and glycine.

In some context, osmolytes may also be referred to as "compatible solutes". Accordingly, the terms "osmolyte" and "compatible solute" are used interchangeably herein.

Variant or variant strain

In the present context, the term "variant" or "variant strain" refers to a strain which is functionally equivalent to a strain of the invention, e.g. having substantially the same, or improved, properties (e.g. regarding salt tolerance). Such variants, which may be identified using appropriate screening techniques, are a part of the present invention.

The term "variant” or "variant strain" is a strain derived, or a strain which can be derived, from a strain of the invention by means of mutagenesis e.g. genetic engineering, adaptive laboratory evolution, physical and/or chemical treatment. Especially, the term "variant" or "variant strain" refers to a strain obtained by subjecting a strain of the invention to any conventionally used mutagenesis treatment including classical strain improvement, treatment with a chemical mutagen such as ethane methane sulphonate (EMS) or N-methyl-N'-nitro-N-nitroguanidine (NTG), UV light, or to a spontaneously occurring mutant. A variant or variant strain may have been subjected to several mutagenesis treatments (a single treatment should be understood as one mutagenesis step followed by a screening/selection step), but it is presently preferred that no more than 20, or no more than 10, or no more than 5, treatments (or screening/selection steps) are carried out.

In a presently preferred variant or variant strain, less than 5%, or less than 1% or even less than 0.1% of the nucleotides in the bacterial genome have been modified with another nucleotide, or deleted, compared to the parental strain.

The terms "variant" and "variant strain" are used interchangeably herein.

Nomenclature of mutation

In the present context, the conventional one-letter and three-letter codes for amino acid residues are used. For ease of reference, amino acid changes in variants of the invention are described by use of the following nomenclature: amino acid residue in the parent enzyme; position; substituted amino acid residue(s).

According to this nomenclature, the substitution of, for instance, an alanine residue for a glycine residue at position 20 is indicated as Ala20Gly or A20G. The deletion of alanine in the same position is shown as Ala20* or A20*. The insertion of an additional amino acid residue (e.g. a glycine) is indicated as Ala20AlaGly or A20AG. The deletion of a consecutive stretch of amino acid residues (e.g. between alanine at position 20 and glycine at position 21) is indicated as DELTA(Ala20-Gly21) or DELTA(A20-G21). When a parent enzyme sequence contains a deletion in comparison to the enzyme sequence used for numbering an insertion in such a position (e.g. an alanine in the deleted position 20) is indicated as *20Ala or *20A. Multiple mutations are separated by a plus sign or a slash. For example, two mutations in positions 20 and 21 substituting alanine and glutamic acid for glycine and serine, respectively, are indicated 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 a comma or a slash. For example, substitution of alanine at position 30 with either glycine or glutamic acid is indicated as A20G,E or A20G/E, or A20G, A20E. When a position suitable for modification is identified herein without any specific modification being suggested, it is to be understood that any amino acid residue may be substituted for the amino acid residue present in the position. Thus, for instance, when a modification of an alanine in position 20 is mentioned but not specified, it is to be understood that the alanine may be deleted or substituted for any other amino acid residue (/.e. any one of R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, V).

Mutation

In the present context, the term "mutation" refers to an alteration in the nucleotide sequence of the genome of an organism resulting in changes in the phenotype of said organism, wherein the alteration may be a deletion of a nucleotide, a substitution of a nucleotide by another nucleotide, an insertion of a nucleotide, or a frameshift.

A deletion is to be understood as a genetic mutation resulting in the removal of one or more nucleotides of a nucleotide sequence of the genome of an organism; an insertion is to be understood as the addition of one or more nucleotides to the nucleotide sequence; a substitution (or point mutation) is to be understood as a genetic mutation where a nucleotide of a nucleotide sequence is substituted by another nucleotide; a frameshift is to be understood as a genetic mutation caused by a insertion or deletion of a number of nucleotides in a nucleotide sequence that is not divisible by three, therefore changing the reading frame and resulting in a completely different translation from the original reading frame; an introduction of a stop codon is to be understood as a point mutation in the DNA sequence resulting in a premature stop codon; an inhibition of substrate binding of the encoded protein is to be understood as any mutation in the nucleotide sequence that leads to a change in the protein sequence responsible for preventing binding of a substrate to its catalytic site of the protein.

Furthermore, a knockout mutant is to be understood as genetic mutation resulting 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.

In the present description and claims the conventional one-letter code for nucleotides is used following the analogous principles as described for amino acids nomenclature supra.

About

Wherever the term "about" is employed herein in the context of amounts, for example absolute amounts, such as numbers, purities, concentrations, weights, sizes, etc., or relative amounts (e.g. percentages, equivalents or ratios), timeframes, and parameters such as temperatures, pressure, etc., it will be appreciated that such variables are approximate and as such may vary by ±10%, for example ± 5% and preferably ± 2% (e.g. ± 1%) from the actual numbers specified. This is the case even if such numbers are presented as percentages in the first place (for example 'about 10%' may mean ±10% about the number 10, which is anything between 9% and 11%).

Sequence identity

In the present context, the term "sequence identity" is here defined as the sequence identity between proteins at the amino acid level. The protein sequence identity may be determined by comparing the amino acid sequence in a given position in each sequence when the sequences are aligned.

To determine the percent identity of two amino acid sequences, the sequences are aligned for optimal comparison purposes (e.g. gaps may be introduced in the sequence of a first amino acid sequence for optimal alignment with a second amino acid sequence). The amino acid residues at corresponding amino acid positions are then compared. When a position in the first sequence is occupied by the same amino acid residue as the corresponding position in the second sequence, then 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 = # of identical positions/total # of positions (e.g., overlapping positions) x 100).

In one embodiment, the two sequences are the same length. In another embodiment, the two sequences are of different length and gaps are seen as different positions.

One may manually align the sequences and count the number of identical amino acids. Alternatively, alignment of two sequences for the determination of percent identity may be accomplished using a mathematical algorithm. Such an algorithm is incorporated into the XBLAST program of (Altschul et al. 1990). BLAST protein searches may be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to a protein molecule of the invention.

To obtain gapped alignments for comparison purposes, Gapped BLAST may be utilized. Alternatively, PSI-Blast may be used to perform an iterated search, which detects distant relationships between molecules. When utilizing the XBLAST and Gapped BLAST programs, the default parameters of the respective programs may be used. See http://www.ncbi.nlm.nih.gov. Alternatively, sequence identity may be calculated after the sequences have been aligned e.g. by the BLAST program in the EMBL database (www.ncbi.nlm.gov/cgi-bin/BLAST). Generally, the default settings with respect to e.g. "scoring matrix" and "gap penalty" may be used for alignment.

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

Derivative Bacillus strains

Agriculture is globally faced with the challenge of maximizing agricultural yield to meet the steep global demands of large quantities of plants or crops produced under conditions that are becoming gradually more challenging. Specifically, abiotic stress, such as high salinity and drought, are increasingly more frequent events caused by e.g. climate changes and/or intensive irrigated farming.

A source of salinity issues is irrigation water, wherein salts provided with the irrigation water accumulate in the soil over time to a point at which more salt is provided than the plants can uptake. Salt may leach out of the soil with irrigation water or rainwater moving materials, including salt and organic material, downward through the soil. However, leaching may be inhibited by high clay content or compaction of the soil, wherein water remains at the surface and evaporates, depositing the salt content but not leaching undissolved salts below the root zone. Problems with high salinity is therefore compounded in arid or semi-arid regions or under conditions of drought. If the salt concentration in the soil becomes greater than the salt concentration in the plant, there will be a net movement of water from the plant into the soil, which will inhibit plant growth or ultimately lead to plant death.

There are strategies to mitigate the negative effect of high salinity soils in farmlands. These include improved drainage, leaching (e.g. by increased watering with saltdeficient water), and reducing surface water evaporation. However, none of these strategies eliminate the challenges with high salinity in the soil. Notably, tillage is a nonpermanent treatment of the soil that may temporarily increase drainage, but with the soil likely re-sealing. Leaching is only effective if the soil drains well and even then, it is a costly and non-environmentally friendly procedure that necessitates application of huge amount of water. Evaporation can be reduced by covering the soil with residue or mulch but comes with economic drawbacks and the risk of creating an anaerobic environment that facilitates fungal diseases to develop at plant stems and/or roots. Therefore, solutions to enable cultivation under abiotic stress, such as high salinity or drought, are in demand. Bacillus strains can be used in biological agriculture with different benefits depending on the bacterial strain used. Biological agriculture or food industries can rely on natural methods of classical strain improvement (CSI) techniques to create improved strains and products. This approach is guided by introduction of random mutations to a parental strain followed by screening and selection of improved variants. Mutations of classical strain techniques are random by nature and can be either natural or induced. Accordingly, the entire genome of the parental strain is probed in contrast to modern era site-directed genome engineering, such as CRISPR, affecting exclusively specific target genes. A benefit hereof is that improved complex phenotypes which may be governed by the interaction between multiple genes can be identified. In absence of thorough understanding of the parental strain genome, such types of improved complex phenotypes are unlikely to be identified by specific genomic substitutions. Moreover, strains developed by the classical strain improvement approach are considered non- genetically modified organisms (GMO) which negates the commercial barriers caused by the strict GMO regulations of, for example, the EU.

The derivative strains of Bacillus disclosed herein are obtained by mutagenesis of a parental strain with desired traits through an adaptive laboratory evolution (ALE) campaign. ALE is used to improve strain performance and stability through (accumulation of) beneficial mutations. With this approach, microbial strains are cultivated under specified growth conditions for prolonged periods of time, in the range from weeks to months or years, with regular passages of the cells in fresh growth media. Directed by the set of growth conditions, the microbial strain will adapt and accumulate beneficial mutations as part of a natural evolution scheme. Notably, the ALE campaign spurs also genome wide mutations that aid the fitness and growth of the microbial strain.

Accordingly, mutagenesis may be accomplished by an ALE campaign wherein a parental Bacillus strain is exposed to a stress condition, such as elevated saline levels, to purposefully introduce mutations in the genome. Therefore, the evolution campaign is referred to as Salt Tolerance Adaptive Laboratory Evolution (SALTY-ALE).

Herein are disclosed derivatives obtained from parental Bacillus strains which through mutagenesis have acquired increased salt tolerance and ability to promote plant health under conditions of abiotic stress, such as high salinity or drought.

It is to be understood that derivative Bacillus strains with identical or similar phenotypes also forms part of the invention. These may be obtained by the method described herein or by further evolution of derivative strains disclosed herein producing new variants with identical or similar phenotypes. Such strains may be said to have all of the identifying characteristics of the derivative strains disclosed herein. Accordingly, strains sharing all identifying characteristics can have different non-identical genomic sequences. The identifying characteristics may include, but is not limited to, the increased salt tolerance and/or increased production of one or more osmolytes.

Thus, an embodiment of the present invention relates to a derivative Bacillus strain, or variant thereof, with all the identifying characteristics thereof, with increased salt tolerance compared to a parental strain of Bacillus.

The main cause of increased soil salinity is the application of irrigation water which carries along soluble salt which stay in the soil after water has evaporated and transpired. The state of high salinity is compounded in dry periods or even drought. Typical salts found in irrigation water include sodium chloride (NaCI), sodium sulphate (Na2SC>4), sodium bicarbonate (NaHCCh), magnesium sulphate (MgSC ), calcium sulphate (CaSC ), calcium chloride (CaCh), potassium chloride (KCI), and potassium sulphate (K2SO4). In particular, high concentration of sodium chloride can be detrimental to plant health.

An embodiment of the present invention relates to the derivative Bacillus strain as described herein, wherein said derivative Bacillus strain has increased salt tolerance compared to said parental strain of Bacillus, when cultured under the same conditions.

Parental strains of Bacillus may have varying baseline salt tolerance, i.e. the resistance to elevated salt concentrations before they are adapted through an evolution campaign. However, parental Bacillus strains with a lower baseline salt tolerance may be a desirable starting point because they have other desired traits, such as enhanced biostimulant properties. Accordingly, for some applications it is worth assessing the improved salt tolerance as a fold-change compared to baseline salt tolerance.

Thus, an embodiment of the present invention relates to the derivative Bacillus strain as described herein, wherein said derivative Bacillus strain has a salt tolerance which is at least 2.5 fold increased compared to a parental strain of Bacillus, such as at least 3 fold increased, such as 3.5 fold increased, such as 4 fold increased compared to a parental strain of Bacillus.

For other applications it may be anticipated that the derivative Bacillus strain is exposed to a given concentration of salt over extended periods of time or frequent enough to warrant a target absolute salt tolerance.

Accordingly, an embodiment of the present invention relates to the derivative Bacillus strain as described herein, wherein said derivative Bacillus strain has a salt tolerance of at least 0.8 M NaCI, such as at least 0.9 M NaCI, such as at least 1.0 M NaCI, such as at least 1.1 M NaCI, such as at least 1.2 M NaCI, such as at least 1.3 M NaCI, such as at least 1.4 M NaCI, such as at least 1.5 M NaCI.

It is to be understood that the salt tolerance of a derivative Bacillus strains is the concentration at which proliferation of the cell population is inhibited by 50% (IC50). The IC50 may be determined as described in Example 1.

Another embodiment of the present invention relates to the derivative Bacillus strain as described herein, wherein said salt tolerance is defined by the half-maximal inhibitory concentration (IC50).

The IC50 value can be determined by inoculating a Bacillus culture in liquid growth medium supplemented with a pre-determined concentration of NaCI followed by incubation. Optical density measurement of cultures incubated at different NaCI concentrations can be collated and plotted as a growth curve, which may subsequently be converted to inhibition curves from which the IC50 value can be read from the fit.

A further embodiment of the present invention relates to the derivative Bacillus strain as described herein, wherein the IC50 is measured as described in Example 1.

For some Bacillus strains small additions of NaCI enhances the growth which will cause an offset of the inhibition curve to negative absolute inhibitory values at low NaCI concentration. In these cases, an effective concentration (EC) may be utilized if desired. The EC50 is the concentration of NaCI that gives half-maximal response.

Therefore, an embodiment of the present invention relates to the derivative Bacillus strain as described herein, wherein said salt tolerance is defined by the EC50. The salt tolerance of the derivative Bacillus strain is to be distinguished from the actual salt levels in the habitat of the plant, e.g. the soil. The salt levels in soil are less than the salt levels under which salt tolerance of the derivative Bacillus strain is measured since plants cannot survive at these elevated salt levels. However, supplying derivative Bacillus strains with increased salt tolerance as described herein benefit the health of plants growing in habitats with increased salt levels.

The inventors have identified several derivative Bacillus species and strains thereof that are favorable for use as parental strains to undergo the salt evolution campaign. These are not only naturally gifted with an adequate baseline salt tolerance but also possess properties that are useful for their role as biostimulants and/or biopesticides to promote plant health.

Therefore, an embodiment of the present invention relates to the derivative Bacillus strain as described herein, wherein said parental strain of Bacillus is of a species selected from the group consisting of Bacillus velezensis, Bacillus paralicheniformis, Bacillus amyloliquefaciens, and Bacillus subtilis.

Another embodiment of the present invention relates to the derivative Bacillus strain as described herein, wherein said parental strain of Bacillus is a deposited strain selected from the group consisting of;

- DSM34004 (deposited on 24 August 2021),

- DSM34003 (deposited on 24 August 2021),

- DSM33240 (deposited on 14 August 2019),

- DSM17231 (deposited on 7 April 2005),

- DSM33110 (deposited on 8 May 2019), and

- DSM32324 (deposited on 8 June 2016), wherein the deposited strain is deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Inhoffenstr. 7B, D-38124 Braunschweig, Germany, by Chr. Hansen A/S, Horsholm, Denmark.

A preferred embodiment of the present invention relates to the derivative Bacillus strain as described herein, wherein said parental strain of Bacillus is deposited as DSM34004 at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Inhoffenstr. 7B, D-38124 Braunschweig, Germany, by Chr. Hansen A/S, Horsholm, Denmark on 24 August 2021.

The SALTY-ALE campaign promotes random mutations to a parental strain followed by screening and selection of improved variants. Thus, the derivative Bacillus strains comprise genomes that are distinct from their parental strain and in turn result in improved phenotypes, such as increased salt tolerance.

Thus, an embodiment of the present invention relates to the derivative Bacillus strain as described herein, wherein said derivative Bacillus strain is genetically distinct from said parental strain of Bacillus.

Another embodiment of the present invention relates to the derivative Bacillus strain as described herein, wherein said derivative Bacillus strain comprises one or more mutations compared to said parental strain of Bacillus.

A mutation is an alteration in the nucleotide sequence of the genome of an organism resulting in changes in the phenotype of said organism, wherein the alteration may 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 the subsequent screening and evaluation of the evolved Bacillus strains several target genes were revealed as particularly promising for producing derivative Bacillus strains with improved properties.

An embodiment of the present invention relates to the derivative Bacillus strain as described herein, wherein said one or more mutations are deletion(s), substitution(s), insertion(s) and/or frame shifts.

Another embodiment of the present invention relates to the derivative Bacillus strain as described herein, wherein the one or more mutations are located in one or more genes of said parental strain of Bacillus selected from the group consisting of:

- yjcG encoding putative phosphoesterase YjcG; comP encoding sensor histidine kinase ComP; dnaA-1 encoding chromosomal replication initiator protein;

- sigB encoding RIMA polymerase sigma-B factor;

- flhB-2 encoding flagellar biosynthetic protein FlhB;

- rny encoding ribonuclease Y;

- ilvE encoding branched-chain-amino-acid aminotransferase; dcuS-1 encoding sensor histidine kinase DcuS;

- rsbT encoding serine/threonine-protein kinase RsbT;

- fliM encoding flagellar motor switch protein FliM;

- mgsR-1 encoding regulatory protein MgsR;

- opuE encoding osmoregulated proline transporter OpuE;

- bshA-1 encoding N-acetyl-alpha-D-glucosaminyl L-malate synthase; - metAA encoding homoserine O-acetyltransferase;

- motB encoding mobility protein B;

- kinA-1 encoding sporulation kinase A; comA encoding transcriptional regulatory protein comA;

- rpoC encoding DNA-directed RIMA polymerase subunit beta'; eamA-1 encoding putative amnio-acid metabolite efflux pump; and

- rpoB encoding DNA-directed RNA polymerase subunit beta.

A further embodiment of the present invention relates to the derivative Bacillus strain as described herein, wherein said one or more mutations are located in one or more genes of said parental strain of Bacillus 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 (s/gB), SEQ ID NO: 5 (flhB-2), SEQ ID NO: 6 (rny), SEQ ID NO: 7 (/7v5), SEQ ID NO:8 (cfcuS-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-l ), 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 (eamA-1), SEQ ID NO:20 (rpoB), and combinations thereof.

A still further embodiment of the present invention relates to the derivative Bacillus strain as described herein, wherein said nucleic acid sequence of the derivative Bacillus strain has at least 90% sequence identity to the nucleic acid sequence of the parental strain of Bacillus, such as at least 95% sequence identity, such as least 98% sequence identity, such as at least 99% sequence identity to the nucleic acid sequence of the parental strain of Bacillus.

Without being bound by theory, it is contemplated herein that mutations resulting in inactivating or downregulating of the energy-consuming general stress response under high salt concentrations can be favorable for improving salt tolerance.

A common way in bacteria to mediate differential gene expression is by applying alternative sigma factors that guide the RNA polymerase to other gene targets than the house keeping sigma factor A. One of the most powerful and striking re-modulations of gene expression in Bacillus, such as B. subtilis, is guided by the alternative sigma factor B (SigB) in response to different stresses and starvation. The SigB protein is highly conserved in Bacillus and controls the expression of about 150 genes, which are involved in adapting the cells to a variety of different stresses, such as high salt concentration, ethanol, nitric oxide, acids, nutrient starvation and high/low temperatures. Due to the broad response and the rather unspecific adaption to a variety of stresses, it is usually referred to as a "general stress response" (GSR). The induction of SigB-dependent GSR constitutes a big burden for the cells, because e.g. big parts of the translation machinery are busy with producing stress connected proteins. Thus, Bacillus ensures a tight repression of GSR during the exponential growth phase and a strong and immediate induction if required. This tight regulation is achieved by a complex signal transduction cascade involving a 25S multiprotein complex, called the stressosome. The activity of this multiprotein complex involves the serine kinase RsbT and the protein RsbS which is activated by RsbT trough phosphorylation.

A secondary stress response of the bacteria is induction of sporulation to produce mature and highly resistant spores. Involved in this process is the phosphorelay which initiates a cascade of events to promote sporulation. The phosphorelay includes a series of sporulation kinases A-E.

The inventors have found that strains with mutations leading to impairment of the GSR and/or sporulation response have improved salt tolerance. It is contemplated that these strains develop other traits to allow long-term growth under high salinity conditions, such as (i) SigB-independent up-regulation of osmolyte uptake, (ii) Re-routing of amino acid synthesis towards osmolyte production (e.g. proline), and/or (iii) increased synthesis of osmolytes.

Thus, an embodiment of the present invention relates to the derivative Bacillus strain as described herein, wherein said one or more mutations compared to said parental strain of Bacillus at least comprises one or more mutations in sigB, rsbT, and/or kinA.

Another embodiment of the invention relates to the derivative Bacillus strain as described herein, wherein said one or more mutations compared to said parental strain of Bacillus at least comprises one or more mutations in sigB.

Without being bound by theory, it is also contemplated herein that mutations affecting amino acid synthesis and transport can also lead to improved salt tolerance.

Bacillus, such as B. subtilis, comprise transporters for osmoprotective compatible solutes. One of these transporters is OpuE which facilitates uptake of proline. Defects in this protein may eventually lead to less uptake of protein. Mutations in the ilvE gene encoding the IlvE protein involved in branched chain amino acid synthesis and in the rny gene encoding the endonuclease RNAse Y, which is involved in the T-box mediated control of anabolic proline production also seem relevant to salt tolerance. Indeed, the inventors have found that derivative Bacillus strains containing combinations of mutations in these genes lead to strains that are capable of increasing extracellular proline levels without showing growth defects under high salinity conditions (see Example 4 (Figure 5A).

Moreover, mutations related to eamA-1, encoding a putative amino acid metabolite efflux pump, have been identified to play roles for extracellular amino acid levels. Notably, it appears that inactivation of the putative amino acid transporter EamA-1 leads to increased extracellular amino acid levels, hereunder proline levels, e.g. by a rerouting of amino acid export in the absence of a functional EamA-1 protein (see Example 4 (Figure 5C). These findings are of great interest because a higher efflux of osmoprotective amino acids, such as proline, can cause a growth promotion effect on plants.

Thus, an embodiment of the present invention relates to the derivative Bacillus strain as described herein, wherein said one or more mutations compared to said parental strain of Bacillus at least comprises one or more mutations in opuE, ilvE, rny and/or eamA-1.

Another embodiment of the present invention relates to the derivative Bacillus strain as described herein, wherein said one or more mutations compared to said parental strain of Bacillus at least comprises one or more mutations in opuE.

Yet another embodiment of the present invention relates to the derivative Bacillus strain as described herein, wherein said one or more mutations compared to said parental strain of Bacillus at least comprises one or more mutations in ilvE.

A still further embodiment of the present invention relates to the derivative Bacillus strain as described herein, wherein said one or more mutations compared to said parental strain of Bacillus at least comprises one or more mutations in eamA-1.

Without being bound by theory, it is further contemplated herein that mutations affecting the C4 organic acid/sodium symporter can favor improved salt tolerance.

As a sensory histidine kinase, the DcuS-1 (MalK) protein is involved in the regulation of gene expression in response to an external trigger. The DcuS-1 protein senses C4- dicarboxylic acids (herein succinate in the media in the evolution campaign) and upregulates (through the kinase cascade) the C4-dicarboxylic acid/sodium symporter (MaeN). The increased organic acid uptake is, therefore, coupled to an increased uptake of the sodium cation. The increased intracellular sodium concentration is toxic to the cell and may impact cell viability if accumulated at a high rate.

Evolved strains with mutations in the dcuS-1 gene were identified during the SALTY- ALE campaign. It is contemplated that these mutations are a survival mechanism that prevent fast accumulation of harmful sodium cations under high salinity by decreasing or downregulating the sodium symport uptake systems.

Thus, an embodiment of the present invention relates to the derivative Bacillus strain as described herein, wherein said one or more mutations compared to said parental strain of Bacillus at least comprises one or more mutations in dcuS-1.

Other favorable mutations that resulted in increased salt tolerance were identified during the SALTY-ALE campaign, hereunder mutations affecting another sensory histidine kinase, ComP, as well as in a phosphoesterase, YjcG, and a chromosomal replication initiator protein, DnaA-1.

Therefore, an embodiment of the present invention relates to the derivative Bacillus strain as described herein, wherein said one or more mutations are located in one or more genes of said parental strain of Bacillus selected from the group consisting of yjcG, comP, dnaA-1, and combinations thereof.

It is to be understood that the identified genes in which mutations lead to increased salt tolerance in derivative Bacillus strains may be combined to achieve a concerted advantage from several mechanism affecting salt tolerance. This may be illustrated e.g. by a derivative Bacillus strain benefitting from mutations inactivating or downregulating the GSR and from mutations affecting the sodium symport uptake systems.

Therefore, an embodiment of the present invention relates to the derivative Bacillus strain as described herein, wherein said one or more mutations are located in one or more genes of said parental strain of Bacillus selected from the group consisting of sigB, rsbT, kinA, opuE, ilvE, rny, dcuS-1, yjcG, comP, dnaA-1, and combinations thereof.

Another embodiment of the present invention relates to the derivative Bacillus strain as described herein, wherein said one or more mutations compared to said parental strain of Bacillus at least comprises 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. A further embodiment of the present invention relates to the derivative Bacillus strain as described herein, wherein said one or more mutations compared to said parental strain of Bacillus at least comprises one or more mutations in one or more genes selected from the group consisting of sig B, opuE, ilvE, rny, dcuS-1, and comP.

The mutations promoted by the SALTY-ALE campaign produce derivative Bacillus strains with new phenotypes. Favorable phenotypes have resulted in increased salt tolerance and/or osmolyte production or transport. These phenotypes are a result of changes to the proteome of the evolved Bacillus strains.

Thus, an embodiment of the present invention relates to the derivative Bacillus strain as described herein, wherein said one or more mutations lead to one or more modifications in one or more proteins of said parental strain of Bacillus 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 (ComA), SEQ ID NO:38 (RpoC), SEQ ID NO:39 (EamA-1), SEQ ID NO:40 (RpoB), and combinations thereof.

Another embodiment of the present invention relates to the derivative Bacillus strain as described herein, wherein said one or more mutations lead to one or more modifications compared to said parental strain of Bacillus selected from the group consisting of YjcG- (458-514/516nt), ComP-Y372C, DnaA-l-R262Q, and combinations thereof.

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

Particular advantageous derivative Bacillus strains include those with high salt tolerance and good biostimulant properties.

A preferred embodiment of the present invention relates to the derivative Bacillus strain as described herein, wherein said derivative Bacillus strain is deposited as DSM34005 at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Inhoffenstr. 7B, D-38124 Braunschweig, Germany, by Chr. Hansen A/S, Horsholm, Denmark on 24 August 2021.

In plants grown at high salinity or drought conditions, various protective mechanisms have been found to adapt against unfavourable environments for their survival and growth. One of the major mechanisms found in plant is accumulation of highly soluble organic compounds known as compatible solutes. The compatible solutes are osmoprotectants which acts as osmolytes to help organisms to survive in extreme osmotic stress. Osmolytes are highly water soluble and low molecular weight organic compounds which may be categorized into three major groups: amino acids (e.g. proline), quaternary and tertiary onium compounds (e.g. glycine betaine, dimethylsulfoniopropionate) and polyol/small sugars (e.g. mannitol, trehalose).

Compatible solutes or osmolytes may be beneficial to plant health under conditions of high salinity since the availability of osmolytes in the external environment reduce the energy necessary for the plant to spend on synthesis of these osmolytes and hence the available carbon source can be directed for their growth and development. It is therefore advantageous if the evolved Bacillus strains make available elevated levels of osmolytes.

Therefore, an embodiment of the present invention relates to the derivative Bacillus strain as described herein, wherein the derivative Bacillus strain has high production of one or more osmolytes under conditions of high salinity.

Another embodiment of the present invention relates to the derivative Bacillus strain as described herein, wherein said one or more osmolytes are selected from the group consisting of proline, citrulline, spermidine, glycine-betain, glycerol, amino acids, sugars, polyols, mannitol and sorbitol.

Yet another embodiment of the present invention relates to the derivative Bacillus strain as described herein, wherein said one or more osmolytes are proline and/or citrulline.

A further embodiment of the present invention relates to the derivative Bacillus strain as described herein, wherein high production of said one or more osmolytes is defined as equal or increased production of said osmolytes compared to said parental strain of Bacillus, preferably increased production compared to said parental strain of Bacillus.

A still further embodiment of the present invention relates to the derivative Bacillus strain as described herein, wherein high production of said one or more osmolytes is defined as at least 2-fold increase, such as at least 3-fold increase, such as at least 4- fold increase, such as at least 5-fold increase, such as at least 10-fold increase, in production of said osmolytes compared to said parental strain of Bacillus.

An even further embodiment of the present invention relates to the derivative Bacillus strain as described herein, wherein said high production of said one or more osmolytes is determined in presence of at least 0.2 M NaCI, such as at least 0.3 M NaCI, such as 0.35 M NaCI in the culture medium.

Another embodiment of the present invention relates to the derivative Bacillus strain as described herein, wherein 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.

Still another embodiment of the present invention relates to the derivative Bacillus strain according to any one of items Y21-Y23, wherein 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.

Gram-positive bacteria, such as Bacillus, are capable of forming spores, typically in the form of intracellular spores called endospores, as a surviving mechanism. These endospores are very retractile 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 nutrition depletion. As the surrounding environment favors bacterial proliferation, the bacterial spores will germinate back into vegetative cells, i.e. an active bacterial cell undergoing metabolism.

Accordingly, spore-forming bacteria are preferred in the present context as they possess the ability to lay dormant if conditions in the field does not favor survival. Thus, this risk of losing the derivative Bacillus strain after application to the plant or the soil is reduced for spore-forming bacteria.

Accordingly, an embodiment of the present invention relates to the derivative Bacillus strain as described herein, wherein the derivative Bacillus strain is in the form of spores or vegetative cells, preferably spores. Development of derivative Bacillus strains with increased salt tolerance may be promoted by the SALTY-ALE campaign described herein. The methodology comprises at least three steps, starting with identification and provision of a parental Bacillus strain that has desired traits. The parental strain may be selected based on criteria such as its baseline salt tolerance or biostimulant properties. The selected parental strain is then cultivated under conditions of high salinity to promote mutations that will allow the Bacillus to survive under the increased abiotic stress. When a population of evolved Bacillus has been prepared, the population is cultured under conditions that allows selection of colonies with increased salt tolerance.

Accordingly, an aspect of the present invention relates to a method for preparing a derivative Bacillus strain as described herein, said method comprising the steps of:

(i) providing a parental strain of Bacillus

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

An embodiment of the present invention relates to the method as described herein, wherein step (ii) comprises growing said parental strain of Bacillus under conditions of high salinity to obtain an evolved population.

The SALTY-ALE campaign is by nature random, and it is not possible a priori to know the properties of the evolved population. An advantage of this type of random mutagenesis evolution is that a large spectrum of genes and the interrelations are probed as opposed to site-directed approaches wherein only few, but specific genes are mutated. In this manner it is possible to identify even complex phenotypes. At the selection step, the derivative Bacillus strains are selected only based on the evolutionary parameter of high salinity, preferably in comparison to the parental Bacillus strain.

Thus, an embodiment of the present invention relates to the method as described herein, wherein said conditions of high salinity is defined as a growth medium with a NaCI concentration of at least 0.8 M NaCI, such as at least 0.9 M NaCI, such as at least 1.0 M NaCI, such as at least 1.1 M NaCI, such as at least 1.2 M NaCI, such as at least 1.3 M NaCI, such as at least 1.4 M NaCI, such as at least 1.5 M NaCI.

Another embodiment of the present invention relates to the method as described herein, wherein said conditions of high salinity is obtained by incremental increase of the salinity level. A further embodiment of the present invention relates to the method as described herein, wherein said incremental increase of the salinity level starts from a growth medium with no salt.

Herein are used parental Bacillus strains selected based on their baseline salt tolerance, i.e. prior to any salt tolerance evolution. The initial pool of parental Bacillus candidates included species with biostimulant properties to optimize the SALTY-ALE campaign towards obtaining derivative Bacillus strains with increased salt tolerance and properties beneficial for plant health.

Therefore, an embodiment of the present invention relates to the method as described herein, wherein said parental strain of Bacillus is of a species selected from the group consisting of Bacillus velezensis, Bacillus paralicheniformis, Bacillus amyloliquefaciens, and Bacillus subtilis.

Another embodiment of the present invention relates to the method as described herein, wherein said parental strain of Bacillus is a deposited strain selected from the group consisting of;

- DSM34004 (deposited on 24 August 2021),

- DSM34003 (deposited on 24 August 2021),

- DSM33240 (deposited on 14 August 2019),

- DSM17231 (deposited on 7 April 2005),

- DSM33110 (deposited on 8 May 2019), and

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

Thus, an embodiment of the present invention relates to the method as described herein, wherein the selection step (iii) comprises providing the evolved population of step (ii) to a solid growth medium. Several strategies may be employed for selecting derivative Bacillus strains with beneficial phenotypes. The salt concentration may be adjusted to make the selection pressure more or less harsh. Typically, very high salt concentration will decrease the number of colonies making screening faster but also less explorative/diverse. Alternatively, the evolved population may be provided to solid growth media comprising different concentrations of salt to increase the diversity of the colonies from which candidate derivative Bacillus strains are selected. Thus, colonies of derivative Bacillus strains may be selected either from solid growth media of a single salt concentration or of multiple salt concentrations.

An embodiment of the present invention relates to the method as described herein, wherein the selection step (iii) comprises providing the evolved population of step (ii) to solid growth media of different salt concentration.

The evolved population obtained from step (ii) may be applied directly to the solid growth medium in the selection step (iii) or undergo treatment prior to the selection step (iii).

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

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

Without being bound by theory, it is contemplated that it advantageous to pass the evolved population through a rich medium to eliminate the salt stress prior to plating and selecting the evolved population on the solid growth medium. Relaxation of the evolved population can eliminate strains that appear to be improved based solely on an induced salt stress response and can ultimately result in more stable strains.

Therefore, an embodiment of the present invention relates to the method as described herein, wherein the evolved population is passed through a rich medium before plating on the solid growth medium in the selection step (iii).

Another embodiment of the present invention relates to the method as described herein, wherein the rich medium is fresh liquid medium, such as fresh LB medium. Derivative Bacillus strain candidates should be able to proliferate during the selection step (iii). Proliferation may be determined by visual confirmation or more quantitatively by optical density (OD) measurements.

Thus, an embodiment of the present invention relates to the method as described herein, wherein proliferation is defined as visual formation of colonies and/or by optical density measurement.

Selected and isolated derivative Bacillus strains are not guaranteed to provide beneficial impact 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 derivative Bacillus strains. These steps may include characterization of osmolyte production and/or genetic analysis of the derivative Bacillus strain.

Therefore, an embodiment of the present invention relates to the method as described herein, wherein step (iii) is followed by:

(i) a step of determining the production of one or more osmolytes of said derivative Bacillus strain with increased salt tolerance; and/or

(ii) a step of sequencing and identifying SNPs of said derivative Bacillus strain with increased salt tolerance.

Another embodiment of the present invention relates to the method as described herein, wherein said one or more osmolytes are selected from the group consisting of proline, citrulline, spermidine, glycine-betain, glycerol, amino acids, sugars, polyols, mannitol and sorbitol.

A further embodiment of the present invention relates to the method as described herein, wherein said method further comprises a step of selecting a derivative Bacillus strain with high production of said one or more osmolytes.

A still further embodiment of the present invention relates to the method as described herein, wherein high production of said one or more osmolytes is defined as equal or increased production of said osmolytes compared to said parental strain of Bacillus, preferably increased production compared to said parental strain of Bacillus.

The methods described herein may be carried out to produce and select derivative Bacillus strains with increased salt tolerance in a systematic and optimized manner. The derivative Bacillus strains may advantageously be used as part of compositions with beneficial properties for plant health under conditions of abiotic stress, such as high salinity or drought.

Accordingly, an aspect of the present invention relates to a derivative Bacillus strain with increased salt tolerance compared to a parental strain of Bacillus obtainable by the method as described herein.

Another aspect of the present invention relates to a composition comprising the derivative Bacillus strain with increased salt tolerance compared to a parental strain of Bacillus as described herein.

The derivative Bacillus strains are preferably spore-forming since endospores have significantly enhanced ability to withstand any stress condition. Spores of the derivative Bacillus strains will therefore increase the robustness and longevity of the composition, especially when applied under harsh conditions.

Thus, an embodiment of the present invention relates to the composition as described herein, wherein said composition comprises spores of said derivative Bacillus strain.

The compositions may comprise additional ingredients that improve the physical or functional properties of the composition. Thus, additional ingredients may benefit e.g. stability, deliverability, wetting, penetration or retention. Additional active ingredient beyond the derivative Bacillus strain can afford the composition with dual mode of action and include standard ingredients that are typically used in formulations of plant growth promoting agents, plant biostimulants, or biopesticides.

Therefore, an embodiment of the present invention relates to the composition as described herein, wherein said composition further comprises one or more agrochemically acceptable excipients, carriers, surfactants, dispersants and yeast extracts.

Another embodiment of the present invention relates to the composition as described herein, wherein the agrochemically acceptable excipients or carriers are selected from the group consisting of maltodextrine, silicon dioxide, modified zeolite, kaolinite, lignin, starch, chitosan, and calcium carbonate.

A still further embodiment of the present invention relates to the composition as described herein, wherein said composition further comprises one or more active ingredients. An even further embodiment of the present invention relates to the composition as described herein, wherein said one or more active ingredients are selected from the group consisting of an insecticide, fungicide, nematicide, bactericide, herbicide, plant extract, plant growth regulator, a plant growth stimulator, and a fertilizer.

Yet another embodiment of the present invention relates to the composition as described herein, wherein said one or more active ingredients are of microbial, biological or chemical origin.

Another embodiment of the present invention relates to the composition as described herein, wherein said insecticide is selected from the group consisting of pyrethroids, bifenthrin, tefluthrin, zeta-cypermethrin, organophosphates, chlorethoxyphos, chlorpyrifos, tebupirimfos, cyfluthrin, fiproles, fipronil, nicotinoids, and clothianidin, and combinations thereof.

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

The composition may comprise more than one strain of bacteria. Such consortia of bacteria can work in synergy to increase the beneficial effects of the recipient plant. The bacterial consortium may comprise bacteria that e.g. promote plant health by making osmolytes available to the plant, bacteria that functions as biopesticide, and bacteria that acts as biofertilizers.

Therefore, an embodiment of the present invention relates to the composition as described herein, wherein said one or more active ingredients are selected from one or more second strains of bacteria different from said derivative Bacillus strain with increased salt tolerance compared to a parental strain of Bacillus.

Another embodiment of the present invention relates to the composition as described herein, wherein said second strain of bacteria is a biostimulant strain, preferably a biostimulant Bacillus strain.

The composition can be provided in a variety of different forms including, but not limited to, a liquid, an oil dispersion, a dust, a dry wettable powder, a spreadable granule, or a dry wettable granule. More specifically the composition may for example be an emulsion concentrate (EC), a suspension concentrate (SC), a water dispersible granule (WG), an emulsifiable granule (EG), a water-in-oil emulsion (EO), an oil-in-water emulsion (EW), a micro-emulsion (ME), an oil dispersion (OD), an oil miscible liquid (OL), a soluble concentrate (SL), a dispersible concentrate (DC), or a wettable powder (WP).

Thus, an embodiment of the present invention relates to the composition as described herein, wherein said composition is a form selected from the group consisting of a liquid, a wettable powder, a granule, a spreadable granule, a wettable granule, a microencapsulation, and a planting matrix.

Coating polymers are useful for providing solid entities with an outer shell that can be protective or add extra properties to the entity it is coating. Coating polymers may be part of liquid formulations which are then applied to another entity. For compositions in solid form, such as powders or granules, a coating polymer may therefore form an outer shell on the particles. For compositions in liquid form, a coating polymer may make the composition suitable for coating of other entities, such as seeds or plants.

Therefore, an embodiment of the present invention relates to the composition as described herein, wherein said composition further comprises a coating polymer.

A further embodiment of the present invention relates to the composition as described herein, wherein said composition is a liquid formulation.

An aspect of the present invention relates to a plant or seed coated with a composition as described herein.

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, in particular seeds or plants that will be exposed to abiotic stress, such as high salinity or drought. For example, by coating seeds with the composition germination can be improved for seeds sown in soil which are or will become subject abiotic stress.

The plant seed can include, but is not limited to, the seed of monocots, dicots, cereals, corn, sweet corn, popcorn, seed corn, silage corn, field corn, rice, wheat, barley, sorghum, asparagus, berry, blueberry, blackberry, raspberry, loganberry, huckleberry, cranberry, gooseberry, elderberry, currant, caneberry, bush berry brassica vegetables, broccoli, cabbage, cauliflower, brussels sprouts, collards, kale, mustard greens, kohlrabi, bulb vegetables, onion, garlic, shallots, citrus, orange, grapefruit, lemon, tangerine, tangelo, pomelo, fruiting vegetables, pepper, tomato, eggplant, ground cherry, tomatillo, okra, grape, herbs/spices, cucurbit vegetables, cucumber, cantaloupe, melon, muskmelon, squash, watermelon, pumpkin, leafy vegetables, lettuce, celery, spinach, parsley, radicchio, leg umes/veg etab les (succulent and dried beans and peas), beans, green beans, snap beans, shell beans, soybeans, dry beans, garbanzo beans, lima beans, peas, chick peas, split peas, lentils, oil seed crops, canola, castor, coconut, cotton, flax, oil palm, olive, peanut, rapeseed, safflower, sesame, sunflower, soybean, pome fruit, apple, crabapple, pear, quince, mayhaw, root/tuber and corm vegetables, carrot, potato, sweet potato, beets, ginger, horseradish, radish, ginseng, turnip, stone fruit, apricot, cherry, nectarine, peach, plum, prune, strawberry, tree nuts, almond, pistachio, pecan, walnut, filberts, chestnut, cashew, beechnut, butternut, macadamia, kiwi, banana, agave, ornamental plants, poinsettia, hardwood cuttings, oak, maple, sugarcane, sugarbeet, grass, or turf grass.

An embodiment of the present invention relates to the plant or seed as described herein, wherein said composition is present in an amount suitable to benefit plant growth.

Another embodiment of the present invention relates to the seed as described herein, wherein said composition comprises a number of vegetative cells or spores of the Bacillus strain from about 1.0x l0² CFU/seed to about l.OxlO⁹ CFU/seed, such as about l.OxlO³ CFU/seed to about l.OxlO⁹ CFU/seed, such as about l.OxlO⁴ CFU/seed to about l.Ox lO⁹ CFU/seed.

Yet another embodiment of the present invention relates to the seed as described herein, wherein said composition comprises a number of vegetative cells or spores of the Bacillus strain from about 1.0 x 10⁶ CFU/g of seed to about 1.0 x 10¹¹ CFU/g of seed.

A further embodiment of the present invention relates to the plant as described herein, wherein said composition comprises a number of vegetative cells or spores of the Bacillus strain from about l.Ox lO⁴ CFU/g of roots to about l.OxlO¹⁰ CFU/g of roots, such as about l.OxlO⁵ CFU/g of roots to about l.OxlO⁹ CFU/g of roots.

The Bacillus strains and compositions comprising them can advantageously be utilized for increasing the resistance of plants against abiotic stress, such as high salinity or drought. Application may be directly to the plant, i.e. foliar application, or into the habitat of the plant. Thus, an aspect of the present invention relates to a method of increasing resistance of a plant against a condition of abiotic stress, said method comprising applying a derivative Bacillus strain with increased salt tolerance compared to a parental strain of Bacillus or a composition as described herein to the plant, to a part of the plant and/or to the habitat of the plant.

Another embodiment of the present invention relates to the method as described herein, wherein said condition of abiotic stress is high salinity, drought and/or nutrient deficiency.

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

It is to be understood that high salinity refers to the salt levels in the habitat of the plant, such as the soil. The salinity level in the habitat of the plant will affect its ability to grow. High salinity levels will typically cause a significant decrease in plant mass and growth speed and under severe circumstances lead to plant death. Normal NaCI levels in soil are in the range of approx. 10-40 mM.

Thus, an embodiment of the present invention relates to the method as described herein, wherein high salinity is defined by a NaCI concentration in the habitat of the plant of more than about 50 mM, such as more than about 60 mM, such as more than about 70 mM, such as more than about 80 mM, such as more than about 90 mM, such as more than about 100 mM, such as more than about 110 mM, such as more than about 120 mM, such as more than about 130 mM, such as more than about 140 mM, such as more than about 150 mM.

Another embodiment of the present invention relates to the method as described herein, wherein high salinity is defined by a NaCI concentration in the habitat of the plant in the range of about 50 mM to about 150 mM, such as about 100 mM to about 150 mM.

Soil salinity may be measured by the electrical conductivity (EC) of the of the soil given in the unit dS/m. 10 mM NaCI has an EC close to 1 dS/m. 100 mM NAcI has an EC close to 9.8 dS/m.

The method of increasing resistance of a plant against a condition of abiotic stress is not limited to any particular plant since most plants will be affected by abiotic stress, such as high salinity or drought. Typically, the method is mainly relevant for agriculture, because relatively small improvements in yield can make a great difference in an industrial setting. Moreover, the prospect of being able to improve yield in a climatefriendly manner is attractive and preferred over traditional agrochemicals that cause widespread ecological damage.

An embodiment of the present invention relates to the method as described herein, wherein the plant is selected from the group consisting of a crop, a monocotyledonous plant, a dicotyledonous plant, a tree, a herb, a bush, a grass, a vine, a fern, and a moss.

Other plants that may benefit from the method include, but are not limited to, main crops, fruticulture, horticulture and floriculture. Main crops may be, but is not limited to, sugar cane, coffee, soybeans, cotton, corn, potatoes, tomatoes, tobacco, banana, rice, wheat, avocado, pineapple, squash, cacao, coconut, oats, onion, lettuce, beet, carrot, cassava, beans, sunflower, pepper, turnip, apple, strawberry, okra, radish and onion. Fruticulture includes, but are not limited to, citrus, grape, guava, papaya, fig, peach, plum and loquat. Floriculture may be rose, chrysanthemum, lisianthus, gerbera, amaryllis, begonia and celosia.

An embodiment of the present invention relates to the method as described herein, wherein the plant is selected from the group consisting of wheat, barley, oats, small cereal grains, corn, rice, sugar cane, soybean, potato, carrot, coffee and banana.

An embodiment of the present invention relates to the method as described herein, wherein the part of the plant is selected from the group consisting of a seed, fruit, root, stem, leaf, corm, tuber, bulb and rhizome.

The derivative Bacillus strain or composition benefit plant health under field conditions if they are capable of colonizing the plant, more specifically the roots. Therefore, it is preferred to apply the derivative Bacillus strain or composition to the habitat of the plant.

Thus, an embodiment of the present invention relates to the method as described herein, wherein said derivative Bacillus strain or said composition is applied to the habitat of the plant.

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

The Bacillus strain may also be used for combatting abiotic stress of green algae. Accordingly, an aspect of the present invention relates to a method of increasing resistance of a green algae against a condition of abiotic stress, said method comprising applying a derivative Bacillus strain with increased salt tolerance compared to a parental strain of Bacillus or a composition as described herein to the green algae and/or to the habitat of the green algae.

For reduction of abiotic stress in green algae, the habitat may be a liquid, such as water.

The derivative Bacillus strain or composition may be provided to the plant, to a part of the plant and/or to the habitat of the plant. Depending on the form of the derivative Bacillus strain or composition, application can be performed by dusting or spraying. Dusting, as used herein, refers to distribution of dry, finely powdered or granular compositions, typically after mixing with an inert carrier. Such application can typically be implemented in most agricultural settings without the need for investment in additional equipment.

A further embodiment of the present invention relates to the method as described herein, wherein said derivative Bacillus strain or said composition is applied to the habitat of the plant by dusting or spraying.

Application of the derivative Bacillus strain or composition may be performed as a preparatory measure prior to sowing or planting, or when the plant is already growing in the habitat, e.g. on the field.

Therefore, an embodiment of the present invention relates to the method as described herein, wherein the derivative Bacillus strain or said composition is applied before, during or after the plant or part of the plant comes into contact with the habitat.

The derivative Bacillus strain or composition may be provided to the soil or growth medium surrounding the plant; soil or growth medium before sowing seed of the plant in the soil or growth medium; or soil or growth medium before planting the plant, the plant cutting, the plant graft, or the plant callus tissue in the soil or growth medium.

An embodiment of the present invention relates to the method as described herein, wherein said derivative Bacillus strain or said 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 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 part of the plant comes into contact with the habitat.

Another embodiment of the present invention relates to the method as described herein, wherein said derivative Bacillus strain or said composition is applied to said seed at a rate of about 1 x 10⁴ to about 1 x 10⁸ cfu per seed, such as 1 x 10⁵ to about 5 x 10⁷ cfu per seed.

A further embodiment of the present invention relates to the method as described herein, wherein said derivative Bacillus strain or said composition is applied at a rate of about 1 x 10⁷ to about 1 x 10¹⁴ cfu per acre, such as about 1 x 10⁸ to about 1 x 10¹³ cfu per acre, such as about 1 x 10⁹ to about 1 x 10² cfu per acre.

Prior to application of the derivative Bacillus strain or composition, the habitat may be analyzed to identify any condition of abiotic stress. This order of action is preferred to avoid wasting material if no action is needed. Traditional means for measuring e.g. salinity levels in soil can be applied.

Thus, an embodiment of the present invention relates to the method as described herein, wherein said condition of abiotic stress is identified prior to application of said derivative Bacillus strain or said composition.

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

A further embodiment of the present invention relates to the method as described herein, wherein TSS or EC is measured of a 1:5 distilled water:soil dilution or a saturated paste extract.

The derivative Bacillus strain or composition is capable of colonizing the root system of plants and provide a biostimulatory effect. The effect hereof may vary between plants.

Thus, an embodiment of the present invention relates to the method as described herein, wherein increasing said resistance of a plant against a condition of abiotic stress facilitates improved seedling vigor, improved root development, improved plant growth, improved plant health, increased yield, improved appearance, or a combination thereof. Another aspect of the present invention relates to use of a derivative Bacillus strain with increased salt tolerance compared to a parental strain of Bacillus or a composition as described herein as a biostimulant, a growth promoter and/or to increase resistance of a plant against a condition of abiotic stress.

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

Another embodiment of the present invention relates to the use as described herein, wherein increasing said resistance of a plant against a condition of abiotic stress facilitates improved seedling vigor, improved root development, improved plant growth, improved plant health, increased yield, improved appearance, reduced pathogenic infection, or a combination thereof.

The derivative Bacillus strain, composition or coated seeds may conveniently be provided as a kit for easy application. The kit may comprise other active ingredients for mixing prior to distribution to the plants or their habitat.

Therefore, an aspect of the present invention relates to a kit comprising:

(ii) a container; and

(iii) optionally, instructions for use.

An embodiment of the present invention relates to the kit as described herein, wherein the kit further comprises one or more active ingredients selected from the group consisting of an insecticide, fungicide, nematicide, bactericide, herbicide, plant extract, plant growth regulator, a plant growth stimulator, and fertilizer.

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

The listing or discussion of an apparently prior published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge. Preferences, options and embodiments for a given aspect, feature or parameter of the invention should, unless the context indicates otherwise, be regarded as having been disclosed in combination 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 the derivative strains and all its features, which may readily be part of the part of the method or use for promoting health and growth of a plant under conditions of abiotic stress, such as high salinity. Embodiments and features of the present invention are also outlined in the following items.

Items

Yl. A derivative Bacillus strain, or variant thereof, with increased salt tolerance compared to a parental strain of Bacillus, wherein the derivative Bacillus strain, or variant thereof, is a derivative strain of said parental strain of Bacillus.

Y2. The derivative Bacillus strain according to item Yl, wherein said derivative Bacillus strain has increased salt tolerance compared to said parental strain of Bacillus, when cultured under the same conditions.

Y3. The derivative Bacillus strain according to any one of items Yl or Y2, wherein said derivative Bacillus strain has a salt tolerance which is at least 2.5 fold increased compared to a parental strain of Bacillus, such as at least 3 fold increased, such as 3.5 fold increased, such as 4 fold increased compared to a parental strain of Bacillus.

Y4. The derivative Bacillus strain according to any one of the preceding items, wherein said derivative Bacillus strain has a salt tolerance of at least 0.8 M NaCI, such as at least 0.9 M NaCI, such as at least 1.0 M NaCI, such as at least 1.1 M NaCI, such as at least 1.2 M NaCI, such as at least 1.3 M NaCI, such as at least 1.4 M NaCI, such as at least 1.5 M NaCI.

Y5. The derivative Bacillus strain according to any one of the preceding items, wherein said salt tolerance is defined by the half-maximal inhibitory concentration (IC50).

Y6. The derivative Bacillus strain according to any one of the preceding items, wherein said parental strain of Bacillus is of a species selected from the group consisting of Bacillus velezensis, Bacillus paralicheniformis, Bacillus amyloliquefaciens, and Bacillus subtil is. Y7. The derivative Bacillus strain according to any one of the preceding items, wherein said parental strain of Bacillus is a deposited strain selected from the group consisting of;

- DSM34004 (deposited on 24 August 2021),

- DSM34003 (deposited on 24 August 2021),

- DSM33240 (deposited on 14 August 2019),

- DSM17231 (deposited on 7 April 2005),

- DSM33110 (deposited on 8 May 2019), and

Y8. The derivative Bacillus strain according to any one of the preceding items, wherein said parental strain of Bacillus is deposited as DSM34004 at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Inhoffenstr. 7B, D-38124 Braunschweig, Germany, by Chr. Hansen A/S, Horsholm, Denmark on 24 August 2021.

Y9. The derivative Bacillus strain according to any one of the preceding items, wherein said derivative Bacillus strain is genetically distinct from said parental strain of Bacillus.

Y10. The derivative Bacillus strain according to any one of the preceding items, wherein said derivative Bacillus strain comprises one or more mutations compared to said parental strain of Bacillus.

Yll. The derivative Bacillus strain according to item Y10, wherein said one or more mutations are deletion(s), substitution(s), insertion(s) and/or frame shifts.

Y12. The derivative Bacillus strain according to any one of items Y10 or Yll, wherein the one or more mutations are located in one or more genes of said parental strain of Bacillus selected from the group consisting of:

- sigB encoding RIMA polymerase sigma-B factor;

- flhB-2 encoding flagellar biosynthetic protein FlhB;

- rny encoding ribonuclease Y;

- ilvE encoding branched-chain-amino-acid aminotransferase; dcuS-1 encoding sensor histidine kinase DcuS; - rsbT encoding serine/threonine-protein kinase RsbT;

- fliM encoding flagellar motor switch protein FliM;

- mgsR-1 encoding regulatory protein MgsR;

- opuE encoding osmoregulated proline transporter OpuE;

- bshA-1 encoding N-acetyl-alpha-D-glucosaminyl L-malate synthase;

- metAA encoding homoserine O-acetyltransferase;

- motB encoding mobility protein B;

- rpoC encoding DNA-directed RNA polymerase subunit beta'; eamA-1 encoding putative amnio-acid metabolite efflux pump; and

- rpoB encoding DNA-directed RNA polymerase subunit beta.

Y13. The derivative Bacillus strain according to any one of items Y10-Y12, wherein said one or more mutations are located in one or more genes of said parental strain of Bacillus 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 (s/gB), SEQ ID NO:5 {flhB-2), SEQ ID NO:6 (rny), SEQ ID NO:7 (/7v5), SEQ ID NO:8 (cfcuS-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-l), 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 {eamA-1), SEQ ID NO:20 {rpoB), and combinations thereof.

Y14. The derivative Bacillus strain according to any one of items Y10-Y13, wherein said one or more mutations are located in one or more genes of said parental strain of Bacillus selected from the group consisting of yjcG, comP, dnaA-1, and combinations thereof.

Y15. The derivative Bacillus strain according to any one of items Y10-Y14, wherein said one or more mutations lead to one or more modifications in one or more proteins of said parental strain of Bacillus 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 (ComA), SEQ ID NO:38 (RpoC), SEQ ID NO:39 (EamA-1), SEQ ID NO:40 (RpoB), and combinations thereof. Y16. The derivative Bacillus strain according to any one of items Y10-Y15, wherein said one or more mutations lead to one or more modifications compared to said parental strain of Bacillus selected from the group consisting of YjcG-delta(458-514), ComP- Y372C, DnaA-l-R262Q, and combinations thereof.

Y17. The derivative Bacillus strain according to any one of the preceding items, wherein said derivative Bacillus strain is deposited as DSM34005 at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Inhoffenstr. 7B, D-38124 Braunschweig, Germany, by Chr. Hansen A/S, Horsholm, Denmark on 24 August 2021.

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

Y19. The derivative Bacillus strain according to any one of the preceding items, wherein the derivative Bacillus strain has high production of one or more osmolytes under conditions of high salinity.

Y20. The derivative Bacillus strain according to item Y19, wherein said one or more osmolytes are selected from the group consisting of proline, citrulline, spermidine, glycine-betain, glycerol, amino acids, sugars, polyols, mannitol and sorbitol.

Y21. The derivative Bacillus strain according to any one of items Y19 or Y20, wherein said one or more osmolytes are proline and/or citrulline.

Y22. The derivative Bacillus strain according to any one of items Y19-Y21, wherein high production of said one or more osmolytes is defined as equal or increased production of said osmolytes compared to said parental strain of Bacillus, preferably increased production compared to said parental strain of Bacillus.

Y23. The derivative Bacillus strain according to any one of items Y21 or Y22, wherein 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.

Y24. The derivative Bacillus strain according to any one of items Y21-Y23, wherein 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.

XI. A method for preparing a derivative Bacillus strain according to any one of the preceding items, said method comprising the steps of:

(i) providing a parental strain of Bacillus

X2. The method according to item XI, wherein said conditions of high salinity is defined as a growth medium with a NaCI concentration of at least 0.8 M NaCI, such as at least

0.9 M NaCI, such as at least 1.0 M NaCI, such as at least 1.1 M NaCI, such as at least

1.2 M NaCI, such as at least 1.3 M NaCI, such as at least 1.4 M NaCI, such as at least

1.5 M NaCI.

X3. The method according to any one of items XI or X2, wherein said conditions of high salinity is obtained by incremental increase of the salinity level.

X4. The method according to any one of items X1-X3, wherein said parental strain of Bacillus is of a species selected from the group consisting of Bacillus velezensis, Bacillus paralicheniformis, Bacillus amyloliquefaciens, and Bacillus subtilis.

X5. The method according to any one of items X1-X4, wherein said parental strain of Bacillus is a deposited strain selected from the group consisting of;

- DSM34004 (deposited on 24 August 2021),

- DSM34003 (deposited on 24 August 2021),

- DSM33240) (deposited on 14 August 2019),

- DSM17231 (deposited on 7 April 2005),

- DSM33110 (deposited on 8 May 2019), and

X6. The method according to any one of items X1-X5, wherein proliferation is defined as visual formation of colonies and/or by optical density measurement. X7. The method according to any one of the items X1-X6, wherein step (iii) is followed by:

X8. The method according to item X7, wherein said one or more osmolytes are selected from the group consisting of proline, citrulline, spermidine, glycine-betain, glycerol, amino acids, sugars, polyols, mannitol and sorbitol.

X9. The method according to any one of items X7 or X8, wherein said method further comprises a step of selecting a derivative Bacillus strain with high production of said one or more osmolytes.

X10. The method according to item X9, wherein high production of said one or more osmolytes is defined as equal or increased production of said osmolytes compared to said parental strain of Bacillus, preferably increased production compared to said parental strain of Bacillus.

Pl. A derivative Bacillus strain with increased salt tolerance compared to a parental strain of Bacillus obtainable by the method according to any one of items X1-X10.

QI. A composition comprising the derivative Bacillus strain with increased salt tolerance compared to a parental strain of Bacillus according to any one of items Y1-Y24 or Pl.

Q2. The composition according to item QI, wherein said composition comprises spores of said derivative Bacillus strain.

Q3. The composition according to any one of items QI or Q2, wherein said composition further comprises one or more agrochemically acceptable excipients, carriers, surfactants, dispersants and yeast extracts.

Q4. The composition according to any one of items Q1-Q3, wherein said composition further comprises one or more active ingredients.

Q5. The composition according to item Q4, wherein said one or more active ingredients are selected from the group consisting of an insecticide, fungicide, nematicide, bactericide, herbicide, plant extract, plant growth regulator, a plant growth stimulator, and a fertilizer.

Q6. The composition according to any one of items Q4 or Q5, wherein said one or more active ingredients are of microbial, biological or chemical origin.

Q7. The composition according to any one of items Q5 or Q6, wherein said insecticide is selected from the group consisting of pyrethroids, bifenthrin, tefluthrin, zeta- cypermethrin, organophosphates, chlorethoxyphos, chlorpyrifos, tebupirimphos, cyfluthrin, fiproles, fipronil, nicotinoids, and clothianidin, and combinations thereof.

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

Q9. The composition according to item Q4, wherein said one or more active ingredients are selected from one or more second strains of bacteria different from said derivative Bacillus strain with increased salt tolerance compared to a parental strain of Bacillus.

Q10. The composition according to item Q4, wherein said second strain of bacteria is a biostimulant strain, preferably a biostimulant Bacillus strain.

Qll. The composition according to any one of items Q1-Q10, wherein said composition is a form selected from the group consisting of a liquid, a wettable powder, a granule, a spreadable granule, a wettable granule, a microencapsulation, and a planting matrix.

Q12. The composition according to any one of items Ql-Qll, wherein said composition further comprises a coating polymer.

Q13. The composition according to any one of items Q1-Q12, wherein said composition is a liquid formulation.

Rl. A plant or seed coated with a composition according to any one of items Q1-Q13.

R2. The plant or seed according to item Rl, wherein said composition is present in an amount suitable to benefit plant growth.

R3. The seed according to any one of items Rl or R2, wherein said composition comprises a number of vegetative cells or spores of the derivative Bacillus strain from about l.OxlO² CFU/seed to about l.OxlO¹¹ CFU/seed, such as about 1.0xl0³ CFU/seed to about l.OxlO¹⁰ CFU/seed, such as about l.OxlO⁴ CFU/seed to about l.OxlO⁹ CFU/seed.

Zl. A method of increasing resistance of a plant against a condition of abiotic stress, said method comprising applying a derivative Bacillus strain with increased salt tolerance compared to a parental strain of Bacillus according to any one of items Yl- Y24 or Pl or a composition according to any one of items Q1-Q13 to the plant, to a part of the plant and/or to the habitat of the plant.

Z2. The method according to item Zl, wherein said condition of abiotic stress is high salinity, drought and/or nutrient deficiency.

Z3. The method according to any one of items Zl or Z2, wherein said condition of abiotic stress is high salinity.

Z4. The method according to any one of items Z2 or Z3, wherein high salinity is defined by a NaCI concentration in the habitat of the plant of more than about 50 mM, such as more than about 60 mM, such as more than about 70 mM, such as more than about 80 mM, such as more than about 90 mM, such as more than about 100 mM, such as more than about 110 mM, such as more than about 120 mM, such as more than about 130 mM, such as more than about 140 mM, such as more than about 150 mM.

Z5. The method according to any one of items Z2-Z4, wherein high salinity is defined by a NaCI concentration in the habitat of the plant in the range of about 50 mM to about 150 mM, such as about 100 mM to about 150 mM.

Z6. The method according to any one of items Z1-Z5, wherein the plant is selected from the group consisting of a crop, a monocotyledonous plant, a dicotyledonous plant, a tree, a herb, a bush, a grass, a vine, a fern, and a moss.

Z7. The method according to any one of items Z1-Z6, wherein the plant is selected from the group consisting of wheat, barley, oats, small cereal grains, corn, rice, sugar cane, soybean, potato, carrot, coffee and banana.

Z8. The method according to any one of items Z1-Z7, wherein the part of the plant is selected from the group consisting of a seed, fruit, root, stem, leaf, corm, tuber, bulb and rhizome. Z9. The method according to any one of items Z1-Z8, wherein said derivative Bacillus strain or said composition is applied to the habitat of the plant.

Z10. The method according to any one of items Z1-Z9, wherein the habitat of the plant is a liquid or soil, preferably soil.

Zll. The method according to any one of items Z1-Z10, wherein the derivative Bacillus strain or said composition is applied before, during or after the plant or part of the plant comes into contact with the habitat.

Z12. The method according to any one of items Zl-Zl 1, wherein said condition of abiotic stress is identified prior to application of said derivative Bacillus strain or said composition.

Z13. The method according to item Z12, wherein said condition of abiotic stress is identified by measuring the total soluble salts by evaporation of a soil water extract (TSS) or the electrical conductivity.

Z14. The method according to item Z13, wherein TSS or EC is measured of a 1 :5 distilled water:soil dilution or a saturated paste extract.

Z15. The method according to any one of items Z1-Z14, wherein increasing said resistance of a plant against a condition of abiotic stress facilitates improved seedling vigor, improved root development, improved plant growth, improved plant health, increased yield, improved appearance, or a combination thereof.

VI. Use of a derivative Bacillus strain with increased salt tolerance compared to a parental strain of Bacillus according to any one of items Y1-Y24 or Pl or a composition according to any one of items Q1-Q13 as a biostimulant, a growth promoter and/or to increase resistance of a plant against a condition of abiotic stress.

V2. The use according to item VI, wherein said condition of abiotic stress is high salinity, drought and/or nutrient deficiency, preferably high salinity.

V3. The use according to any one of items VI or V2, wherein increasing said resistance of a plant against a condition of abiotic stress facilitates improved seedling vigor, improved root development, improved plant growth, improved plant health, increased yield, improved appearance, reduced pathogenic infection, or a combination thereof. Tl. A kit comprising:

(i) a derivative Bacillus strain with increased salt tolerance compared to a parental strain of Bacillus according to any one of items Y1-Y24 or Pl, a composition according to any one of items Q1-Q13, or coated plant seeds according to any one of items RR-RR;

(ii) a container; and

(iii) optionally, instructions for use.

T2. The kit according to item Tl, wherein the kit further comprises one or more active ingredients selected from the group consisting of an insecticide, fungicide, nematicide, bactericide, herbicide, plant extract, plant growth regulator, a plant growth stimulator, and fertilizer.

T3. The kit according to item T2, wherein said derivative Bacillus strain and said one or more active ingredients are provided in separate compartments in the container.

The invention will now be described in further details in the following non-limiting examples.

Examples

Example 1: Selection of parental Bacillus strains for adaptive laboratory evolution (ALE) campaign for increasing salt tolerance

Growth conditions were established that were suitable for determining the effect on the growth of a range of parental Bacillus strains when exposed to increased salt levels. Minimum inhibitory concentration (MIC) was determined to select parental Bacillus strains for the ALE campaign.

Method:

Strains, media and culture conditions

Bacillus strains were cultured at 30 °C (or 37 °C) in Luria-Bertani (LB) broth or on LB agar. The salt tolerance campaign was carried out in modified CSE media (10.46 g/L of MOPS, 3.3 g/L of (NH₄)₂SO₄, 0.13 g/L of KH2PO4, 0.267 g/L of K₂HPO₄, 3.23 mg/L of MnSO₄-4H₂O, 120 mg/L of MgSO₄-7H₂O, 22 pg/L of ammonium ferric citrate, 0.8% of potassium glutamate, 0.6% of sodium succinate, pH 7).

To make NaCI supplemented CSE media, various concentration of NaCI was added in the media by adjusting the volume of MQ-water. For sporulating the strains, SP media (8 g/L of Difco nutrient broth, 1 g/L of KCI, 0.25 g/L of MgSO₄-7H₂O, 10 pM of MnCI₂, 0.5 mM of CaCI₂, and 4.4 pg/L of Ammonium ferric citrate) and LB adapted for sporulation (LBSpore) media (10 g/L tryptone, 5 g/L Yeast extract, 10 g/L NaCI, 10 mM of MgCh, 10 mM of MgSO4-7H2O, 50 pM of MnCh and 0.5 mM of CaCh) were used to grow the Bacillus strains and the salt evolved isolates.

To investigate the effect of salt (sodium chloride) in the growth of Bacillus strains, 19 different Bacillus strains belonging to various species, including the lab model strain Bacillus subtilis 168, were tested. The strains were cultivated in CSE media supplemented with 0 to 2 M of NaCI at 30 °C and 225 RPM in a Growth Profiler and a picture of the culture plates was taken every 30 min to assess the bacterial growth over time.

Analysis of Bacillus growth using a Growth Profiler setup

Single colonies of Bacillus strains from LB-agar plates were inoculated into 400 pl of CSE media in 96 deep well-plate. Then, the plate was incubated at 30 °C and 250 RPM for 20-24 hours. After 10- to 20-fold dilutions, ODeoonm was determined. The saturated overnight pre-cultures were diluted 100-fold into 250 pl of CSE media or CSE media supplemented with a pre-determined concentration of NaCI. Subsequently, the culture plates were incubated in a Growth Profiler (Enzyscreen, Heemstede, The Netherlands) at 30 °C and 250 RPM for 2-3 days with constant monitoring of the growth by scanning the plate every 30 minutes. Using the GP software (GP960Viewer version 1.0.0.4, Enzyscreen, Heemstede, The Netherlands), the pixel data of the pictures was converted into digital ODeoonm values and the growth curves over time were generated.

Minimum inhibitory concentration (MIC) and IC90 determination

For MIC determination, a fresh single colony of Bacillus strain was inoculated in 400 pl of CSE media in a 96-deep well plate and then the plate was incubated at 37 °C and 200 RPM overnight. The overnight cultures were inoculated in 250 pl of CSE media supplemented with a pre-determined concentration of NaCI (ranging from 0 to 2 M) at the ratio of 1: 100 fold dilution (/.e. 2.5 pl of overnight culture in 250 pl of medium). The culture plate was subsequently incubated at 225 RPM and 30°C. MICs were determined using a logarithmic NaCI concentration gradient. At 24 h and 47 h of incubation, optical density of the culture at 600nm wavelength (ODeoonm) was measured (background- subtracted). Growth inhibition of the Bacillus strains was plotted against NaCI concentration with a polynomial interpolation between neighboring data points using R software (Free Software provided by the R Foundation). The percentage of inhibition was calculated using the formula :

[1 ^— (ODeoonm NaCl/ODeOOnm control) ] X 100% The inhibitory concentrations termed IC50 and IC90 were defined as the lowest concentration of the NaCI that inhibited 50% and 90% of the growth of the strain tested, respectively.

Results:

Most of the 19 tested Bacillus strains grew well in CSE media. Upon increasing NaCI concentration, the growth rate gradually decreased until a concentration was reached that no longer supported growth (Figure 1). Figure 1 displays a selection of growth profiles of the tested Bacillus strains and illustrates the fact that different Bacillus strains have different NaCI tolerance levels.

The inhibition curves were plotted for the Bacillus strains using the log(NaCI) concentration as X-axis and inhibition percentage as Y-axis (Figure 2). From these inhibition curves, the IC50 and IC90 values of NaCI were determined for the Bacillus strains (Figure 3). Practically, the addition of a small amount (e.g. 200 mM or 350 mM) of salt enhances the growth of some Bacillus strains resulting in growth promotion rather than inhibition. In such cases, the absolute inhibitory value becomes negative at low salt concentration (Figure 2). To account for this complexity, an effective concentration (EC) was used. The EC50 is the concentration of NaCI that gives half-maximal response. EC50 = IC50, for the ideal inhibition curves whereas IC50>EC50 if there is negative inhibition value to consider (Figure 3).

Comparison of the growth behavior of the Bacillus strains in CSE media supplemented with NaCI showed that in the natural state the most salt tolerant strains are of the species B. paralicheniformis (IC50 of 1.0 -1.2 M at 47 h) followed by B. velezensis (IC50 of 1.0 - 1.1 mM at 47 h) and B. subtilis (IC50 of 0.7 - 0.9 M at 47 h). The least salt tolerant strain is a B. amyloliquefaciens strain (IC50 of 0.5 M at 47 h) (Figure 3).

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

Table 1.

Conclusion:

This example demonstrates that conditions suitable for evaluating salt tolerance was established and candidate parental strains were purposefully selected for the ALE campaign based on their baseline salt tolerance.

Example 2: Salt evolution campaign and selection of derivative Bacillus strains with improved salt tolerance from the evolved populations

Method:

Each selected parental strain of Example 1 was subjected to a Salt Tolerance Adaptive Laboratory Evolution (SALTY-ALE) campaign carried out by linearly increasing NaCI concentration in the growth medium in triplicate tubes (see Table 1). Three different methods were utilized for selecting derivative strains with increased salt tolerance from the evolved populations of the SALTY-ALE campaign.

Method 1 : Relaxation step in rich media and plating

The evolved populations in the salt supplemented CSE media were first transferred in LB media to eliminate the salt stress before plating in LB agar supplemented with various concentrations of NaCI. Briefly, 500 pl of the evolved strains from the SALTY-ALE campaign were centrifuged at 5000 x g for 5 min and the supernatant was discarded (done for all three lineages from each parental strain). The cell pellet was resuspended into 500 pl of fresh LB media. Then the resuspended cells were transferred into 4.5 ml of LB media and the culture tubes were incubated overnight at 30°C and 250 RPM. The parental strain was also inoculated in 5 ml of LB media (as control) and also incubated overnight at 30°C and 250 RPM. Next day, the overnight cultures were diluted in LB media to reach a final ODeoonm of 1. These OD-adjusted cultures were streaked or spread on LB agar and LB agar supplemented with various concentrations of NaCI. For instance, in LB agar, LB+ 1M NaCI agar plates the cells were streaked out, whereas 10-25 ul of the cells were spread on LB+ 1.4 M NaCI, LB+ 1.6 M NaCI, and 25-100 ul of the cultures were plated in LB+ 1.89 M NaCI, LB+ 2 M NaCI and LB+2.2 M NaCI agar plates. The plates were incubated at 37°C until pickable colonies were observed, i.e. after about 24-48 h.

Method 2: Direct streaking of the population from the evolved culture tubes Few microliters of the growing cells from the SALTY-ALE culture tubes were directly streaked out on LB agar supplemented with various concentrations of NaCI. Briefly, 2-5 pl of the culture broth (ODeoonm between 1-1.5) from the SALTY-ALE campaign culture tubes were directly streaked on LB agar plates supplemented with various concentrations of NaCI. Then the plates were incubated at 37°C for 24 to 48 h.

Method 3: Direct streaking of the cultures from the evolved culture tubes after washing

Before streaking the cells from the SALTY-ALE culture tubes, the metabolite produced in the culture broths were removed and the cells were streaked out in LB agar supplemented with various concentrations of NaCI. Briefly, 500 pl of the culture broth from the SALTY-ALE tubes was centrifuged at 5000 x g for 5 min and the supernatant was discarded. The cell pellet was resuspended in 1 ml of CSE media with NaCI (same concentration as in the SALTY-ALE tubes) and centrifuged at 5000 x g for 5 min and the supernatant was discarded. Then the washed cells were resuspended in 500 pl of LB media and 2-5 pl of the resuspended cells were streaked out in LB agar plate supplemented with various concentrations of NaCI. Finally, the plates were incubated at 37°C for 24 to 48 h.

Comparison of growth parameters of the selected isolates from the ALE populations

To select improved salt tolerance isolates after the SALTY-ALE experiment, the growth parameters of the colonies (isolates) obtained from methods 1-3 described above were analyzed in CSE media and CSE supplemented with NaCI media using the Growth Profiler setup.

At first, we checked the growth parameters in singlet isolates, and then the growth improved isolates (with relatively higher growth rate and/or short lag time with respect to the parental strain in NaCI supplemented media) were further analyzed to confirm their improvement by testing in triplicates. In all Growth Profiler experiments, the most improved strains were selected based on (i) improved growth rate and/or (ii) reduced lag phase in the NaCI supplemented media. These two criteria were compared to the performance of the parental strain under identical conditions as a benchmark.

For the Growth Profiler experiments, the starting ODeoonm of all the strains, including the parental strain, was kept within the range 0.02-0.05 to achieve comparable results.

Results:

The results are exemplified by the SALTY-ALE campaign conducted based on the parental Bacillus paralicheniformis strain termed SA-P7. Each of the three methods were evaluated for the suitability to select derivative strains with increased salt tolerance from the evolved populations.

For method 1, the number of colonies growing on plates of increasing salt concentration were higher for the salt evolved culture tubes compared to the parental strain. Growth of the salt evolved cultures were especially favored at high salt load.

Methods 2 and 3 confirmed that the salt evolved culture tubes contained evolved populations that formed colonies at high salt concentrations. Especially, method 2 produced colonies at high salt concentration. Without being bound by theory, the high salt tolerance might be due to the induced salt stress and/or presence of metabolites from the ALE cultures during direct streaking.

Comparison of growth curves of the isolates from all three methods in CSE supplemented with 1 M NaCI showed that most of the isolates had improved growth rates.

Utilizing the procedure described above, a selection of lead candidate derivative Bacillus strains was isolated from the colonies growing at high salt concentration. The lead candidate derivative Bacillus strains are listed in Table 2 below.

Table 2.

Growth phenotypes for a selection of these lead candidates depicted in figure 4 and show that the derivative Bacillus strains that have been subjected to the SALTY-ALE campaign outperforms their corresponding parental strain.

Conclusion: This example demonstrates that parental Bacillus strains subjected to the SALTY-ALE campaign improved their salt tolerance significantly. Several methods were used and found useful for selecting and isolating improved derivative strains from colonies.

Example 3: Determination of sporulation capacity of selected isolated derivative Bacillus strains

Method:

To test the spore forming capacity of the derivative Bacillus strains, the strains were cultured in Sporulation (SP) medium, or LB adapted for sporulation medium (LBSpore) and the culture tubes were incubated at 37°C for 24-26 h. Then 40 pl of the fully grown cultures was heated at 80°C for 20 min. A non-sporulating strain was taken as a negative control.

The heat-treated and heat-untreated cultures were 10-fold serially diluted from 10° to IO^-7 in LB media and 5 pl 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. Next day, the plates were analyzed for growth of the improved derivatives at the different dilutions.

In this assay, strains capable of producing spores will be able to grow in both heat- treated and heat-untreated samples whereas the sporulation negative strain can grow only in heat-untreated condition.

Results:

Lead candidates (table 2) were all shown to be spore-positives in the assay as described above, i.e. they were able to survive heat treatment and subsequently grow under normal conditions. Absence of growth in the heated samples from the negative control strain validated the sporulation test.

Conclusion:

This example demonstrates that the SALTY-ALE campaign was successful in producing candidates that retained their sporulation capacity throughout the evolution.

Example 4: Determination of osmolyte production under salt stress conditions of selected isolated derivative Bacillus strains

Osmolyte production under salt stress conditions of a selected set of improved derivatives was investigated by biochemical analysis. Methods

Single colonies of Bacillus strains were inoculated in 400 pl of LB media in 96-deep well plate. The plates were incubated overnight at 37°C and 250 RPM. Next morning, 25 pl of the saturated overnight cultures were inoculated in 500 pl of CSE or 350 mM (final concentration) NaCI supplemented CSE media in 96 deep well plate in triplicates and the plate was incubated at 30°C and 250 RPM for 28 h. The optical density (ODeoonm) of the cultures were measured after 20-fold dilution. The culture plate was centrifuged at 3700 RPM for 30 min and 450 pl of the supernatant were taken in to 96 deep well plate. The plates were submitted for quantification of various amino acids and compatible solutes (for LC Mass analysis).

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 Ultra RP Column (130A, 1.7 pm, 2.1 mm x 100 mm, Waters Corporation, Milford. MA, USA).

A 50 pL aliquot was transferred from the plate to a new plate and 950 pL of MilliQ water was added to the aliquot. The amino acid derivatization was performed using the AccQ- Tag™ Ultra Derivatization Kit (Waters Corporation, Milford. MA, USA). Forty microliters of sample were mixed with 40 pL of an internal standard solution containing ca. 1 pM of all the targets labelled with 13C. The mix was buffered using 50 pL of borate buffer and 20 pL of the derivatization reagent was added. The solution was mixed and heated at 55°C at 2000 rpm for 10 min before being analyzed on the LC-MS.

Results

Herein was tested and quantified the extracellular concentrations of various compatible solutes by the salt tolerant improved lead Bacillus strains.

In many of the improved salt tolerant Bacillus strains, the amount of proline was markedly increased as compared to their corresponding parental strains when cultivated in high salt condition, such as SA60 which had more than 7-fold increased extracellular proline concentration compared to its parental strain SA-P4 (Figure 5A).

Upon analysis of extracellular concentration of another compatible solute, citrulline, it can be concluded that several improved salt tolerant Bacillus strains have increased extracellular concentration of citrulline as compared to their parental strains under high salinity condition (Figure 5B). Further analysis of the amino acid levels of one of the improved salt tolerant Bacillus strains, SA60, showed elevated amino acid levels for a broad selection of amino acid (Figure 5C). For several amino acids the levels were in particular increased under conditions of high salinity. Besides proline and citrulline, this was the case for e.g. glycine, caline, leucine, isoleucine and arginine.

Conclusion

Many of the improved salt tolerant derivative Bacillus strains adapted during the SALTY- ALE campaign by evolving their ability to produce osmolytes and amino acids.

Example 5: Effect of osmolytes in protection of Arabidopsis thaliana germination and growth under saline stress conditions

To investigate the effect of osmolytes on plant growth under elevated salt concentration, determination of the germination and growth of Arabdopsis thaliana supplemented with osmolytes, and in particular proline, was performed.

Methods

Germination of Arabidopsis thaliana seedlings

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

Growth of Arabidopsis thaliana seedlings

Five-days old Arabidopsis thaliana seedlings were transferred to ¹/2 Murashige and Skoog supplemented with 1 mM proline with or without NaCI. The whole plant biomass was measured on 15 days old plants.

Results

Germination of Arabidopsis thaliana seedlings

The four osmolytes L-proline, L-citrulline, glycine-betaine and spermidine were all able to improve seed germination frequency for Arabidopsis thaliana at 90 mM NaCI (Figure 6A). Different concentrations of the osmolytes resulted in optimal response to the salt stress conditions. Thus, optimal response for mitigating the salt stress conditions was observed for 1 mM proline, 0.5 mM citrulline, 30 mM glycine-betaine or 0.15 mM spermidine.

Growth ofArabidopsis tha liana seed linos

Increased fresh weight biomass of Arabidopsis thaliana was detected under conditions of high salt concentration (90 mM NaCI) when the growth medium was supplemented with proline as compared to the control without proline (Figure 6B). The data support that proline can alleviate salt stress in plants.

Conclusion

This example demonstrates that osmolytes have a beneficial effect on plant growth for plants subject to salt stress. This would indicate that a derivative Bacillus strain having evolved both salt tolerance and the ability to produce osmolytes could improve plant growth if supplemented to plants growing in a habitat with high salinity levels.

Example 6: Evaluation of plant growth when supplemented with derivative Bacillus strains

To investigate the beneficial effect of derivative Bacillus strains on plant growth, greenhouse trials with corn were performed.

Methods

Plant trials in the greenhouse were started by embedding 50 corn seeds (C4E8 maize) with a solution of each Bacillus strain at a concentration of OD600=1 for 1 hour. Afterwards, the seeds were moved to a petri dish containing 12 mL of 100 mM NaCI and left to germinate for 12 days in a growth chamber at 16 hours light - 8 hours night cycle. After germination, seeds were transplanted to 250 mL pots containing clay field soil and watered with 150 mM NaCI. Plants were grown for 3 weeks in the greenhouse and at each watering, pots were fully soaked with 150 mM NaCI and the excess water was removed, to assure that all pots were equilibrating to 150 mM NaCI at each watering. Plants were harvested at the end of the experiment and the dry weight was recorded.

Results

Salt evolved derivative Bacillus strains are presented as write columns immediately to the right of their parental strain (Figure 7A-B). The data show that supplement of the salt evolved derivative Bacillus strains (SA3, SA9, SA21, SA40, SA59, SA60, SA63, SA64, SA70) increase plant dry matter compared to the respective parental Bacillus strains (SA-P1, SA-P3, SA-P4, SA-P5, SA-P7, SA-P9) and the controls (control salt, control salty water) without any bacteria. Thus,

Conclusion

This example demonstrates that supplementation with the salt adapted derivative Bacillus strains promote plant tolerance to salt stress.

Example 7: Genetic analysis of selected isolated derivative Bacillus strains

The improved derivative Bacillus strains of Table 2 were subjected to genetic analysis to detect mutations, hereunder single nucleotide polymorphisms (SNPs), and thereby identify key target genes.

Methods

A single colony of the improved derivative Bacillus strains were streaked in a LB plate for three rounds of purification steps. Then 5 colonies of the purified strain were inoculated in 5 ml of LB media and the tube was incubated overnight at 30 °C. Next morning, 500 pl of the overnight culture was transferred in 30 ml of fresh LB media. The flask was incubated at 37 °C until the ODeoonm reached about 1.0-1.5. Then, whole genome sequencing samples were prepared.

Cell pellets from 1.5 ml of 1.5 OD equivalent cultures were collected by centrifuging the cultures at 5000 x g for 10 min. Then the cell pellet was submitted for gDNA isolation and whole genome sequencing.

Results

Whole genome sequencing of the derivative Bacillus strains detected genetic changes and identified key target genes considered to play a role for improved salt tolerance. These target genes include exporter genes involved in metabolite transport in and out the cells, genes regulating symporters and genes related to quorum sensing.

Conclusion

Key target genes were identified based on genetic analysis of derivative Bacillus strains with improved salt tolerance.

References

• Altschul et a/. (1990), J. Mol. Biol., 215, 403-410

Deposits and Expert Solution The applicant requests that a sample of the deposited microorganisms stated in table 3 below may only be made available to an expert, until the date on which the patent is granted.

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The deposits were made according to the Budapest treaty on the international recognition of the deposit of microorganisms for the purposes of patent procedure at at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Inhoffenstr. 7B, D-38124 Braunschweig, Germany.

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

Table 3. Deposited strains made at a depositary institution.

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Claims

58 Claims

1. A derivative Bacillus strain, or variant thereof, with increased salt tolerance compared to a deposited parental strain of Bacillus selected from the group consisting of;

- DSM34004 (deposited on 24 August 2021),

- DSM34003 (deposited on 24 August 2021),

- DSM33240 (deposited on 14 August 2019),

- DSM17231 (deposited on 7 April 2005),

- DSM33110 (deposited on 8 May 2019), and

2. The derivative Bacillus strain according to claim 1, wherein said derivative Bacillus strain has a salt tolerance which is at least 2.5 fold increased compared to a parental strain of Bacillus, such as at least 3 fold increased, such as 3.5 fold increased, such as 4 fold increased compared to a parental strain of Bacillus.

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

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

5. The derivative Bacillus strain according to claim 4, wherein the one or more mutations are located in one or more genes of said parental strain of Bacillus selected from the group consisting of:

- sigB encoding RNA polymerase sigma-B factor;

- flhB-2 encoding flagellar biosynthetic protein FlhB;

- rny encoding ribonuclease Y; 59

- rsbT encoding serine/threonine-protein kinase RsbT;

- fliM encoding flagellar motor switch protein FliM;

- mgsR-1 encoding regulatory protein MgsR;

- opuE encoding osmoregulated proline transporter OpuE;

- bshA-1 encoding N-acetyl-alpha-D-glucosaminyl L-malate synthase;

- metAA encoding homoserine O-acetyltransferase;

- motB encoding mobility protein B;

- rpoB encoding DNA-directed RNA polymerase subunit beta.

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

7. The derivative Bacillus strain according to any one of the preceding claims, wherein the derivative Bacillus strain has high production of one or more osmolytes under conditions of high salinity, wherein high production of said one or more osmolytes is defined as equal or increased production of said osmolytes compared to said parental strain of Bacillus, preferably increased production compared to said parental strain of Bacillus.

8. The derivative Bacillus strain according to any one of the preceding claims, wherein said derivative Bacillus strain is deposited as DSM34005 at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Inhoffenstr. 7B, D-38124 Braunschweig, Germany, by Chr. Hansen A/S, Horsholm, Denmark on 24 August 2021.

9. A method for preparing a derivative Bacillus strain according to any one of the preceding claims, said method comprising the steps of:

(i) providing a parental strain of Bacillus

(iii) selecting a derivative Bacillus strain with increased salt tolerance that proliferates under the growth conditions of step (ii). 60

10. A derivative Bacillus strain with increased salt tolerance compared to a parental strain of Bacillus obtainable by the method according to claim 9.

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

12. The composition according to claim 11, wherein said composition comprises spores of said derivative Bacillus strain.

13. The composition according to any one of claims 11 or 12, wherein said composition is a form selected from the group consisting of a liquid, a wettable powder, a granule, a spreadable granule, a wettable granule, a microencapsulation, and a planting matrix.

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

15. Use of a derivative Bacillus strain with increased salt tolerance compared to a parental strain of Bacillus according to any one of claims 1-8 or 10 or a composition according to any one of claims 11-13 as a biostimulant, a growth promoter and/or to increase resistance of a plant against a condition of abiotic stress.