Halotolerant kushneria isolates and related products, systems, and methods for stimulating plant growth in saline conditions

Abstract

Disclosed are halotolerant Kushneria isolates and related products, systems, and methods for stimulating plant growth in saline conditions. Illustrative halotolerant Kushneria isolates include Kushneria sp. A3 and Kushneria sp. B5. Systems for adapting a plant for improved growth in salinated soil may include at least two strains of halotolerant rhizobacteria, such as Kushneria sp. A3 and Kushneria sp. B5. Illustrative products include the at least two strains of halotolerant rhizobacteria, such as Kushneria sp. A3 and Kushneria sp. B, and a diluent or spreading agent. Illustrative methods include applying at least two strains of halotolerant rhizobacteria, such as Kushneria sp. A3 and Kushneria sp. B, to soil having a salt concentration of about 1% w / v to about 10% w / v, and cultivating a plant in the soil.

Description

[0001]23903.1a Halotolerant Kushneria isolates and related products, systems, and methods for stimulating plant growth in saline conditions Background Soil quality in many parts of the U.S. and worldwide is susceptible to a variety of stresses including drought, erosion, and increasing salinity due to evaporation and / or poor irrigation practices leading to accumulating presence of salts. At the same time, the human population is growing, and in many regions, high-quality agricultural land is decreasing due to the expansion of urban areas. According to the USDA Salinity Laboratory about 15% of cultivated land globally is irrigated, but this acreage yields up to 40% of the total food harvest. A key barrier to crop production is soil salinity, which is a serious and growing problem world-wide due to inadequate water drainage, saline ground water, or inadequate rainfall to wash away soil salts. Salinity increases (builds up) in irrigated areas due to soluble salts that are carried and transported in the irrigation water and remain in the soil after evaporation and transpiration, imposing ionic stress and disrupting cellular processes in plants. In the absence of adequate leaching mechanisms, these salts amass to levels inhibitory to plant growth and may lead to the transformation of soils into sodic conditions. This can extend to the degradation of soil structure, impinging upon both water retention and root penetration. The growing challenge of salinization presents a serious menace to global food security, leading to the abandonment of an estimated 700,000 hectares of arable land annually. As of 2022, salinity issues affect approximately 1 billion hectares of arable land globally, with the annual economic toll surpassing $27 billion. In the United States, soil and water salinity affects about 30% of all irrigated land, while worldwide this number is about 50%. In the United States, soil salinity translates into an annual crop production loss of $3.1 billion. The reduction in crop yield induced by salinity is a universal phenomenon, with nations such as Australia experiencing losses of up to 50% in wheat yields due to the impact of salinity. With few exceptions, crops and other vegetation fail to thrive in high saline soils. Crop failure and underproduction can have widespread and lasting effects on food supply and the sustenance of life and health. The severity of increasing soil salinity is likely to intensify with growing food demand and degradation of prime agricultural land. These factors will require new approaches to maintain adequate food supplies globally. Existing strategies for alleviating the effects of soil salinity include specialized irrigation, reconditioning, and resting (fallowing) of soil plots for one or more vegetative cycles to allow the soil to recover. These strategies can be expensive, wasteful, and pollutive and may result in under- or non-production of food crops in high-need areas. Alternative strategies may 23903.1a include specialized plant breeding to select for plants with enhanced salinity tolerance, which can be a time-consuming process and leads to differing results depending on the crop species. Thus, soil salinity represents a worldwide issue that requires immediate attention. Summary The present disclosure relates to halotolerant Kushneria isolates and related products, systems, and methods for stimulating plant growth in saline conditions. Illustrative halotolerant Kushneria isolates include Kushneria sp. A3 and Kushneria sp. B5. Systems for adapting a plant for improved growth in salinated soil may include at least one or at least two strains of halotolerant rhizobacteria, such as Kushneria sp. A3 and Kushneria sp. B5. Illustrative products include at least one or at least two strains of halotolerant rhizobacteria, such as Kushneria sp. A3 and Kushneria sp. B, and a diluent or spreading agent. Illustrative methods include applying at least one or at least two strains of halotolerant rhizobacteria, such as Kushneria sp. A3 and Kushneria sp. B, to soil having a salt concentration of about 1% w / v to about 10% w / v, and cultivating a plant in the soil. At least one aspect or embodiment of the present disclosure includes a method. The method can successfully adapt plants for growth in saline conditions. Indeed, the method can stimulate plant growth in high salt soil. In some embodiments, the method can include the step of applying at least one strain or at least two strains of halotolerant rhizobacteria to soil having a salt concentration of about 1% w / v to about 10% w / v. The at least one or at least two strains of halotolerant rhizobacteria can illustratively comprise Kushneria sp. A3 and / or Kushneria sp. B5. In some embodiments, the method can further include cultivating a plant in the soil. Illustratively, the at least one or at least two strains of halotolerant rhizobacteria can colonize root tissue of the plant during cultivation. Colonization of the root tissue by the at least one or at least two strains of halotolerant rhizobacteria can adapt the plant for growth in the soil having a salt concentration of about 1% w / v to about 10% w / v. In some embodiments, the at least one or at least two strains of halotolerant rhizobacteria can improve growth of the plant in the soil having a salt concentration of about 1% w / v to about 10% w / v, as compared to a corresponding plant cultivated in soil having a salt concentration of about 1% w / v to about 10% w / v without the at least two strains of halotolerant rhizobacteria. Illustratively, the at least two strains of halotolerant rhizobacteria are applied to the soil: as a mixture of the at least two strains of halotolerant rhizobacteria; or in separate applications applied sequentially or simultaneously. In some embodiments, cultivating the plant in the soil can comprise planting a seed in the soil, the seed sprouting to produce the plant. In other embodiments, cultivating the plant in the soil can comprise transplanting the plant into the soil. 23903.1a In at least one embodiment, the plant comprises a legume crop. Illustratively, the legume crop can be or comprise a Fabaceae sp. plant, such as beans (e.g., kidney beans, pinto beans, lima beans), peas, lentils, chickpeas, peanuts, soybeans, lupins, and / or alfalfa. Another aspect or embodiment of the present disclosure includes a system for adapting a plant for improved growth in salinated soil. The system can include at least one or at least two strains of halotolerant rhizobacteria. The at least one or at least two strains of halotolerant rhizobacteria can illustratively comprise Kushneria sp. A3 and / or Kushneria sp. Another aspect or embodiment of the present disclosure includes a product. The product can include at least one or at least two strains of halotolerant rhizobacteria and a diluent or spreading agent. The at least one or at least two strains of halotolerant rhizobacteria can illustratively comprise Kushneria sp. A3 and / or Kushneria sp. Illustratively, the at least one or at least two strains of halotolerant rhizobacteria can be in a substantially dry form and / or solid state. Alternatively, the at least one or at least two strains of halotolerant rhizobacteria can be in liquid form. The diluent or spreading agent can include any material or component suitable for application to crop soil and that improves the spreadability of the strain(s) of halotolerant rhizobacteria. Illustratively, the diluent or spreading agent can be or comprise one or more naturally-occurring earth component, such as a phyllosilicate, illustratively of the 1:1 or 2:1 clay mineral variety. In at least one embodiment, the phyllosilicate can be selected from the group consisting of aliettite, attapulgite, bentonite, chlorite, dickite, halloysite, hectorite, illite, kaolinite, montmorillonite, nacrite, nontronite, palygorskite, saponite, sauconite, sepiolite, serpentine, talc, and vermiculite. In at least one embodiment, the diluent or spreading agent can be or comprise diatomaceous earth or a plant-based extract or component. Illustratively, the diluent or spreading agent can be in a substantially dry form and / or solid state. The earth component preferably has an average particle size between about 20 – 297 µm. The earth component preferably has a moisture content between about 0.25% and about 20% by weight. Some embodiments may include any of the features, options, and / or possibilities set out elsewhere in the present disclosure, including in other aspects or embodiments of the present disclosure. It is also noted that each of the foregoing, following, and / or other features described herein represent a distinct embodiment of the present disclosure. Moreover, combinations of any two or more of such features represent distinct embodiments of the present disclosure. Such features or embodiments can also be combined in any suitable combination and / or order without departing from the scope of this disclosure. Thus, each of the features described herein can be combinable with any one or more other features described herein in 23903.1a any suitable combination and / or order. Accordingly, the present disclosure is not limited to the specific combinations of exemplary embodiments described in detail herein. Additional features and advantages of exemplary embodiments of the present disclosure will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by the practice of such exemplary embodiments. The features and advantages of such embodiments may be realized and obtained by means of the instruments and combinations particularly pointed out in the appended claims. These and other features will become more fully apparent from the following description and appended claims, or may be learned by the practice of such exemplary embodiments as set forth hereinafter. Brief Description of the Drawings In order to describe the manner in which the above-recited and other advantages and features of the present disclosure can be obtained, a more particular description of the embodiments briefly described above will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings. For better understanding, the like elements have been designated by like reference numbers throughout the figure(s). Understanding that these drawings depict only typical embodiments of the invention and are not therefore to be considered to be limiting of its scope, the invention will be described and explained with additional specificity and detail through the use of the accompanying drawing(s) in which: Figure 1 depicts a graphical representation of isolation, screening of halotolerant bacteria and its effect on alfalfa plant growth under salt stress. Figure 2 depicts ten strains, inoculated on NBRIP and Pikovskaya's agar medium to determine their phosphate solubilizing ability. A) Pikovskaya's medium and B) NBRIP medium for phosphate solubilization, C) mineral salts medium for zinc solubilization, and D) IAA production assay in a 96-well plate. Figure 3 depicts screening of selected strains under different NaCl stress conditions, testing halotolerant strains on LB agar with 0M (0% w / v), 1M (2.7% w / v), 2M (5.4% w / v), 3M (8.1% w / v), or 4M (10.8% w / v) NaCl. Figure 4 illustrates the effect of Kushneria strains on alfalfa plant growth in open pot trials. A) photographs of plants after harvesting from each treatment (No salt control without bacteria, Salt control without bacteria, A3 inoculation, B5 inoculation). B) the total plant fresh weight is shown. Data points indicate means ± SEM of at least 10 biological replicates. The statistically significant difference is marked with asterisks (Tukey’s HSD, P < 0.05). Significance: P<0.001 ‘***’. C) Plants in open pots before harvesting. 23903.1a Figure 5 illustrates Alfalfa plant growth trial with Kushneria strains under salt stress conditions. Harvested alfalfa plants are shown at the bottom. Each pot was inoculated with the indicated bacterial isolate. Figure 6 illustrates the effect of Kushneria strain inoculation on alfalfa in growth chamber trials. A) plant height, B) root length, C) plant fresh weight, and D) plant dry weight. Data points show the means ± SEM of at least 15 biological replicates. Statistically significant differences are marked with asterisks (Tukey’s HSD, P < 0.05). Significance codes: 0.001 ‘***’; 0.01 ‘**’; 0.05 ‘*’. Figure 7 illustrates Alfalfa plant growth trial with Kushneria strains under salt stress conditions (top panel) and harvested alfalfa plants (bottom panel). All plants were grown in closed pots with a single watering upon planting and inoculated with the indicated bacterial isolate except for the controls (uninoculated No salt & salt). Figure 8 illustrates the effect of Kushneria strain on alfalfa plants under field conditions A) shows shoot length, B) root length, C) shoot fresh weight, and D) root fresh weight. Data points show the means ± SEM of at least 13 biological replicates except for E4 which has 10 replicates and the unstressed control which has 4 replicates. Statistically significant differences are marked with asterisks (Tukey’s HSD, P < 0.05). Significance codes: 0.001 ‘***’; 0.01 ‘**’; 0.05 ‘*’. Figure 9 illustrates survival determination of inoculated bacteria in alfalfa pots on 1M NaCl + LB media with 10-6dilution after harvesting A) No inoculation B) A3 inoculation C) B5 inoculation. Figure 10 depicts a visualization of B5 strain expressing GFP in alfalfa root tissue. A & B) No inoculation, C & D) B5+GFP inoculation. Root sample from alfalfa inoculated with B5 strain, constitutively expressing the GFP gene as compared to control, visualized using the Echo Revolve microscope. Fluorescent GFP signal highlights bacterial colonization within the root tissue. Figure 11 illustrates a proposed mechanism for salt-tolerant plant growth promoting rhizobacterial mitigation of salt stress in plants. Figure 12 depicts harvested alfalfa plants. All plants were grown in closed pots with a single watering upon planting. Watered with 0.5X Hoagland’s solution containing 1% NaCl (except for the no salt control). Each pot was inoculated with the indicated bacterial isolate. When harvested the plants were washed and length and weight of shoot and root tissues were measured (both fresh and dry weights). 23903.1a Figure 13 depicts open pot alfalfa growth trial. Experiment performed as before but in open pots (soil was not autoclaved) with regular watering with 0.5X Hoagland’s solution containing 1% NaCl, except for the no salt control. Inoculation was with the bacterial isolates indicated Figure 14 illustrates the effect of Kushneria strains on alfalfa plants in closed pots. A) shows plant height, B) root length, C) Plant Fresh weight, and D) plant dry weight. Data points show means ± Std of at least 15 biological replicates except A3 + B5 which have four replicates. Statistically significant differences are marked with asterisks (Tukey’s HSD, P < 0.05). Significance codes: 0.001 ‘***’; 0.01 ‘**’; 0.05 ‘*’. Figures 15A-15D illustrate the effect of Kushneria strains on alfalfa plant growth in closed pot trials. Figure 15A shows shoot length, Figure 15B shows root length, Figure 15C shows Shoot Fresh weight, and Figure 15D shows Root fresh weight. Data points show means ± Std of at least 13 biological replicates except for E4 which have 10 replicates. Statistically significant differences are marked with asterisks (Tukey’s HSD, P < 0.05). Significance codes: 0.001 ‘***’; 0.01 ‘**’; 0.05 ‘*’. Figures 16A and 16B illustrate the effect of Kushneria strains on alfalfa plant growth in open pot trials. Figure 16A depicts photographs of two plants from each treatment (-salt control, + salt control, B5 inoculation, A3 inoculation). Figure 16A illustrates total plant fresh weight. Data points indicate means ± Std of at least 1o biological replicates. Statistically significant difference is marked with an asterisk (Tukey’s HSD, P < 0.05). Significance code: 0.001 ‘***’. Detailed Description Before describing various aspects and embodiments of the present disclosure in detail, it is to be understood that this disclosure is not limited to the specific features of the particularly exemplified systems, methods, and / or products described herein that may vary from one embodiment to the next. Thus, while certain embodiments of the present disclosure will be described in detail, with reference to specific features (e.g., components, ingredients, parts, portions, elements, members, combinations, configurations, parameters, properties, steps, etc.), the descriptions are illustrative and are not to be construed as limiting the scope of the present disclosure and / or the claimed invention. It is also to be understood that much, if not all of the terminology used herein is only for the purpose of describing particular embodiments of the present disclosure, and is not necessarily intended to limit the scope of the disclosure in any particular manner. 23903.1a Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure pertains. Various aspects of the present disclosure, including systems, methods, and / or products may be illustrated with reference to one or more embodiments or implementations, which are exemplary in nature. As used herein, the terms “embodiment” and “implementation” mean “serving as an example, instance, or illustration,” and should not necessarily be construed as preferred or advantageous over other aspects disclosed herein. In addition, reference to an “implementation” of the present disclosure or invention includes a specific reference to one or more embodiments thereof, and vice versa, and is intended to provide illustrative examples without limiting the scope of the invention, which is indicated by the appended claims rather than by the description thereof. As used throughout this disclosure, the words “can” and “may” are used in a permissive sense (i.e., meaning having the potential to), rather than the mandatory sense (i.e., meaning must). Additionally, the terms “including,” “having,” “involving,” “containing,” “characterized by,” variants thereof (e.g., “includes,” “has,” and “involves,” “contains,” etc.), and similar terms as used herein, including the claims, shall be inclusive and / or open-ended, shall have the same meaning as the word “comprising” and variants thereof (e.g., “comprise” and “comprises”), and do not exclude additional, un-recited elements or method steps, illustratively. As used in this specification and the appended claims, the singular forms “a,” “an” and “the” each contemplate, include, and specifically disclose both the singular and plural referents, unless the context clearly dictates otherwise. For example, reference to a “carrier” contemplates and specifically discloses one, as well as two or more nucleic acids. Similarly, use of a plural referent does not necessarily require a plurality of such referents, but contemplates, includes, and specifically discloses one, as well as two or more of such referents, unless the context clearly dictates otherwise. It will also be appreciated that where two or more values, or a range of values (e.g., less than, greater than, at least, and / or up to a certain value, and / or between two recited values) is disclosed or recited, any specific value or range of values falling within the disclosed values or range of values is likewise specifically disclosed and contemplated herein. Thus, disclosure of an illustrative measurement (e.g., length, width, thickness, etc.) that is less than or equal to about 10 units or between 0 and 10 units includes, illustratively, a specific disclosure of: (i) a measurement of 9 units, 5 units, 1 units, or any other value between 0 and 10 units, including 23903.1a 0 units and / or 10 units; and / or (ii) a measurement between 9 units and 1 units, between 8 units and 2 units, between 6 units and 4 units, and / or any other range of values between 0 and 10 units. Various aspects or embodiments of the present disclosure can be illustrated by describing components that are bound, coupled, attached, connected, and / or joined together. As used herein, the terms “bound,” “coupled”, “attached”, “connected,” and / or “joined” are used to indicate either a direct association between two components or, where appropriate, an indirect association with one another through intervening or intermediate components. In contrast, when a component is referred to as being “directly bound,” “directly coupled”, “directly attached”, “directly connected,” and / or “directly joined” to another component, no intervening elements are present or contemplated. Furthermore, binding, coupling, attaching, connecting, and / or joining can comprise mechanical, physical, and / or chemical association. In addition, various aspects or embodiments of the present disclosure can be illustrated by describing components that are mixed together. As used herein, “mixed,” “mixing,” and similar terms indicate a physical combining or combination of two or more components. In some embodiments, the physical combining or combination results in a (chemical and / or physical) reaction. Such chemical reactions can be evidenced by a change in the chemical composition, pH, or other indicator relative to the components prior to being mixed (or as expected after being mixed absent the reaction). Thus, mixing and / or mixed components can include reacting and / or reacted components in certain embodiments. Accordingly, reference to mixing or mixed components includes a reference to reacting or reacted components. To facilitate understanding, like reference numerals have been used, where possible, to designate like elements common to the figures. Specifically, in the exemplary embodiments illustrated in the figures, like structures will be provided with similar reference designations, where possible. Specific language will be used herein to describe the exemplary embodiments. Nevertheless, it will be understood that no limitation of the scope of the disclosure is thereby intended. Rather, it is to be understood that the language used to describe the exemplary embodiments is illustrative only and is not to be construed as limiting the scope of the disclosure (unless such language is expressly described herein as essential). While the detailed description is separated into sections, the section headers and contents within each section are for organizational purposes only and are not intended to be self-contained descriptions and embodiments or to limit the scope of the description or the claims. Rather, the contents of each section within the detailed description are intended to be read and understood as a collective whole, where elements of one section may pertain to and / or 23903.1a inform other sections. Accordingly, embodiments specifically disclosed within one section may also relate to and / or serve as additional and / or alternative embodiments in another section having the same and / or similar products, methods, and / or terminology. Embodiments of the present disclosure include halotolerant Kushneria isolates and related products, systems, and methods for stimulating plant growth in saline conditions. Illustrative halotolerant Kushneria isolates include Kushneria sp. A3 and Kushneria sp. B5. Systems for adapting a plant for improved growth in salinated soil may include at least one or at least two strains of halotolerant rhizobacteria, such as Kushneria sp. A3 and Kushneria sp. B5. Illustrative products include at least one or at least two strains of halotolerant rhizobacteria, such as Kushneria sp. A3 and Kushneria sp. B, and a diluent or spreading agent. Illustrative methods include applying at least one or at least two strains of halotolerant rhizobacteria, such as Kushneria sp. A3 and Kushneria sp. B, to soil having a salt concentration of about 1% w / v to about 10% w / v, and cultivating a plant in the soil. At least one aspect or embodiment of the present disclosure includes a method. The method can successfully adapt plants for growth in saline conditions. Indeed, the method can stimulate plant growth in high salt soil. In some embodiments, the method can include the step of applying at least one strain or at least two strains of halotolerant rhizobacteria to soil having a salt concentration of about 1% w / v to about 10% w / v. The at least one or at least two strains of halotolerant rhizobacteria can illustratively comprise Kushneria sp. A3 and / or Kushneria sp. B5. In some embodiments, the method can further include cultivating a plant in the soil. Illustratively, the at least one or at least two strains of halotolerant rhizobacteria can colonize root tissue of the plant during cultivation. Colonization of the root tissue by the at least one or at least two strains of halotolerant rhizobacteria can adapt the plant for growth in the soil having a salt concentration of about 1% w / v to about 10% w / v. In some embodiments, the at least one or at least two strains of halotolerant rhizobacteria can improve growth of the plant in the soil having a salt concentration of about 1% w / v to about 10% w / v, as compared to a corresponding plant cultivated in soil having a salt concentration of about 1% w / v to about 10% w / v without the at least two strains of halotolerant rhizobacteria. Illustrate, the at least two strains of halotolerant rhizobacteria are applied to the soil: as a mixture of the at least two strains of halotolerant rhizobacteria; or in separate applications applied sequentially or simultaneously. In some embodiments, cultivating the plant in the soil can comprise planting a seed in the soil, the seed sprouting to produce the plant. In other embodiments, cultivating the plant in the soil can comprise transplanting the plant into the soil. 23903.1a In at least one embodiment, the plant comprises a legume crop. Illustratively, the legume crop can be or comprise a Fabaceae sp. plant, such as beans (e.g., kidney beans, pinto beans, lima beans), peas, lentils, chickpeas, peanuts, soybeans, lupins, and / or alfalfa. Another aspect or embodiment of the present disclosure includes a system for adapting a plant for improved growth in salinated soil. The system can include at least one or at least two strains of halotolerant rhizobacteria. The at least one or at least two strains of halotolerant rhizobacteria can illustratively comprise Kushneria sp. A3 and / or Kushneria sp. Another aspect or embodiment of the present disclosure includes a product. The product can include at least one or at least two strains of halotolerant rhizobacteria and a diluent or spreading agent. The at least one or at least two strains of halotolerant rhizobacteria can illustratively comprise Kushneria sp. A3 and / or Kushneria sp. Illustratively, the at least one or at least two strains of halotolerant rhizobacteria can be in a substantially dry form and / or solid state. Alternatively, the at least one or at least two strains of halotolerant rhizobacteria can be in liquid form. The diluent or spreading agent can include any material or component suitable for application to crop soil and that improves the spreadability of the strain(s) of halotolerant rhizobacteria. Illustratively, the diluent or spreading agent can be or comprise one or more naturally-occurring earth component, such as a phyllosilicate, illustratively of the 1:1 or 2:1 clay mineral variety. In at least one embodiment, the phyllosilicate can be selected from the group consisting of aliettite, attapulgite, bentonite, chlorite, dickite, halloysite, hectorite, illite, kaolinite, montmorillonite, nacrite, nontronite, palygorskite, saponite, sauconite, sepiolite, serpentine, talc, and vermiculite. In at least one embodiment, the diluent or spreading agent can be or comprise diatomaceous earth or a plant-based extract or component. Illustratively, the diluent or spreading agent can be in a substantially dry form and / or solid state. The earth component preferably has an average particle size between about 20 – 297 µm. The earth component preferably has a moisture content between about 0.25% and about 20% by weight. Some plant-associated microbes have been shown to form a relationship with plants to stimulate growth under salty soil conditions. In the present disclosure, a variety of bacterial samples from native salt-tolerant plants in Utah were isolated and screened for the ability to stimulate plant growth in salty soil. Identified genera included: Kushneria, Halomonas, Bacillus (Bacillus aryabhattai, Bacillus zanthoxyli, Bacillus atrophaeus and other species), Microbacterium, Virgibacillus, Oceanobacillus, Zhihengliuella, Vibrio, Bacillaceae, Priestia, and Planococcus. Different species were identified for nearly all genera, but based on partial sequence analysis, it is most reliable to identify these strains by the genus name. 23903.1a In all, a total of 180 bacterial strains were isolated and about 110 strains were screened. See Genbank IDs: MK873873-MK873913. Selected strains showed promise when used to inoculate illustrative plant varieties (e.g., legume crops, such as alfalfa) that were grown in salty soil conditions. Several strains show some stimulation effect. The most consistent results were observed with two strains of Kushneria; designated “A3” and “B5”, and the combination of A3 and B5, which exhibited synergistic effects with greater growth stimulation than either strain alone. In addition to testing A3 and B5, in combination, we tested the Halomonas A9 strain with either A3 or B5 Kushneria, and also the Bacillus B1 strain with A9 and with B5. A3 plus B5 gives the most surprising, unexpected, additive, and / or synergistic effect. Inoculation of crop plants with these Kushneria strains could allow farmers to increase yields from land affected by salinity. Thus, there is substantial promise for plant-associated microbes isolated from halophytes (salt-tolerant plants) to enhance growth of salt-sensitive crop plants in salty soils. Several of the isolated and screened bacteria were characterized by 16S ribosomal RNA (Sanger dideoxy) sequencing. Table A, below, presents six (6) illustrative isolated and screened bacteria. Corresponding sequences are presented in the Sequence Listing document submitted herewith, the entirety of which is incorporated herein by reference. Table A SEQ ID NO: Bacteria Designator Accession # Taxon 1 K h i A A PP4 12 8 9 6 is identifiable by its 16S ribosomal RNA sequence presented in the Sequence Listing document submitted herewith. For example, Kushneria sp. A3 is identifiable by and, therefore, corresponds to / with SEQ ID NO:1 and Kushneria sp. B5 is identifiable by and, therefore, corresponds to / with SEQ ID NO:1=2, etc. Thus, Kushneria sp. A3 may be termed a (first) Kushneria species according to SEQ ID NO:1 (or having a 16S ribosomal RNA sequence according to SEQ ID NO:1) and Kushneria sp. B5 may be termed a (second) Kushneria species according to SEQ ID NO:2 (or having a 16S ribosomal RNA sequence according to SEQ ID NO:2), etc. 23903.1a The use of microbes that associate with salt-tolerant plants is a natural means to stimulate plant growth under adverse saline conditions and can be effective immediately upon inoculation of young plants with the bacteria. Work by others has shown the general potential of some bacteria to stimulate plant growth (as biofertilizers), but the present disclosure addresses the ability of two illustrative Kushneria strains of bacteria to stimulate plant growth under salty conditions. Also, the combination of two strains showed a synergistic effects when both were used as the inoculum. During initial investigations, several salt-tolerant (i.e. halophilic) bacterial strains were previously isolated from native halophytes (salt-tolerant plants) growing near Goshen, Utah. Soil and root samples were collected as previously described and bacteria isolated on LB agar plates containing 1 M NaCl. Pure colonies were isolated and frozen stock cultures were stored at -80 C. DNA was recovered from each isolate and used as template for PCR amplification of a portion of the 16S rRNA gene. PCR products were subjected to Sanger sequencing, and the results used in NCBI BLAST searches to identify the bacteria. Individual isolates were used to inoculate alfalfa seedlings to test for the ability to stimulate plant growth in the presence of salt. Seedlings were transplanted into sterile soil in magenta boxes with 80 ml of 0.5 X Hoagland’s solution containing 0 (control) or 1% NaCl. We transplanted three seedlings into each pot and used three pots for each inoculum and salt concentration treatment. The watering solution contained 1 ml of LB or 1 ml of liquid culture for each strain being tested. Boxes were covered with an inverted magenta box allowing a small passage for air, and plants were placed in a controlled light / temperature room for growth. After 6 weeks, plants were harvested and analyzed for fresh weight, height, length of roots. Individual harvested plants are shown in Figure 12. Plants growing in open pots in a plant growth chamber are shown in Figure 13. Analyzed data is summarized in Figure 14 and Figure 15. Figures 16A and 16B show the results of plants grown in open pots in the greenhouse. Open pots have unsterilized soil and are watered regularly with the same watering solution. The most consistent results were observed with Kushneria strains A3 and B5, and the combination of A3 and B5 showed synergistic effects with greater growth stimulation than either strain alone. Additional Kushneria strains also showed promise for effectiveness, but additional studies were needed. The objective of the present disclosure was to identify salt-tolerant bacteria that grow in close association with native halophytes and characterize their ability to stimulate the growth of alfalfa in salty soil conditions. Several halotolerant bacteria, including Kushneria, Halomonas, and Bacillus, were identified from the rhizosphere or roots of three halophyte species in a saline area south of Utah 23903.1a Lake, Utah, USA. The isolates were characterized for various properties commonly associated with salt-tolerant, plant growth-promoting bacteria (ST-PGPB). The isolated strains were screened for the ability to stimulate growth of alfalfa in controlled laboratory experiments. A GFP-expressing strain of Kushneria was used to detect the presence of various bacteria in inoculated plants. Among the species identified, two independent isolates of the genus Kushneria were found to have significant growth-promoting activity for inoculated alfalfa plants grown under saline conditions (1.2 % NaCl). The biochemical properties, including indole acetic acid production, biofilm formation, phosphate solubilization and siderophore production activities, which have been associated with PGP activity, were characterized for several isolates. The presence of GFP-expressing bacteria was visualized in roots of inoculated plants. Plants inoculated with a combination of two Kushneria strains that have PGPB properties exhibited a 20-30% increase in fresh and dry weight over uninoculated plants. A GFP marker confirmed bacterial presence in the roots of inoculated plants. Bacteria with ST- PGP activity will be a key resource to facilitate increased crop yield from land affected by salinity. Several species of plant growth-promoting rhizobacteria (PGPR) have been found to stimulate plant growth under a variety of conditions. One well-studied example is Bacillus subtilis (GB03), a rhizosphere bacterium that stimulates the growth of white clover (a legume) under saline conditions. This bacterial strain produces volatile compounds that enhance plant photosynthetic capacity and chlorophyll content, induces increased endogenous sugar content, and suppresses abscisic acid (ABA)-induced RNA transcripts. Other effects include alterations in plant gene expression such as upregulating expression of the HKT1 sodium transporter gene in shoots and downregulating its expression in roots, resulting in lower sodium accumulation throughout the plant. We previously reported the identification of several halotolerant bacterial strains from the rhizosphere of three halophyte species in a saline area south of Utah Lake. In that study, two strains (Halomonas sp. and Bacillus sp.) were identified that when introduced to young seedlings significantly stimulated the growth of alfalfa in the presence or absence of 1% NaCl. We have collected additional soil and root samples from the halophytes at the same site. We report here the identification of additional strains, some of which have growth-promoting activity when used to inoculate alfalfa plants grown under saline conditions. Two Kushneria strains show promise in growth stimulation studies with alfalfa in greenhouse and growth 23903.1a chamber trials in saline soil conditions, and their potential PGPB properties have been characterized. Methods Bacterial strains used in this Disclosure We collected 100 bacterial isolates from halophyte plants (root and soil rhizosphere samples, in a highly saline area near Goshen, Utah as previously described. Initial isolation was performed on Luria Bertani (LB) agar plates containing 1M NaCl using serial dilution. Pure colonies were isolated and stock cultures were stored at -80°C. DNA was recovered from each isolate as described and used as a template for PCR amplification of a portion of the 16S rRNA gene. PCR products were subjected to Sanger sequencing, and the results were used in NCBI BLAST searches to identify the bacteria. The flowchart in Figure 1 illustrates the steps involved. Specifically, Figure 1 depicts a graphical representation of isolation, screening of halotolerant bacteria and its effect on alfalfa plant growth under salt stress. In-vitro screening of halotolerant bacteria for the detection of plant growth promoting traits PO4 - solubilizing activity assay Pure bacterial colonies were screened for phosphate solubilizing activity in NBRIP with 1% NaCl (10 g / L) (20 g Glucose, 5.0 g Ca3(PO4)2, 0.1 g (NH4)2SO4, 0.25 g MgSO4.7H2O, 10 g MgCl2.6H2O, 0.2 g KCl, 0.1g yeast extract) and Pikovskaya's agar (per L) (10.0 g Glucose, 5.0 g Ca3(PO4)2, 0.5 g (NH4)2SO4, 0.2 g NaCl, 0.1 g MgSO4.7H2O, 0.2 g KCl, 0.5 g yeast extract, 0.002 g MnSO4.H2O, 0.002 g FeSO4.7H2O), pH 7.0, 15.0 g Agar. A loop of fresh pure colonies of bacteria was spotted on both media. Plates were incubated at 30±2°C for seven days and observed for halo zone formation around bacterial growth. The presence of a halo zone indicated positive phosphate solubilization activity. Zn- solubilizing activity assay All halotolerant isolates were inoculated into mineral salts medium (MSM) with 1% NaCl (g / L) as described by Saravanan et al. (10.0g containing dextrose, 1.0g (NH4)2SO4, 0.2g KCl, 0.1g K2HPO4, 0.2g MgSO4, 1g ZnO, pH: 7.0; 15.0 g Agar), autoclaved at 121 °C for 20 min. Plates were incubated at 30±2 °C for seven days and observed for halo zone formation around bacterial growth. The presence of a halo zone indicated positive zinc solubilization activity. IAA production assay Indole acetic acid (IAA) production of halotolerant bacteria was determined by inoculating a bacterial colony into LB broth containing 1% NaCl with L-tryptophan 0.1% (w / v) 23903.1a and incubated at 30°C at 125 rpm for four days. The broth was then centrifuged at 10,000 rpm for 10 min and 1 mL of supernatant was mixed (1:1 v / v) with Salkowski reagent (98 mL of 35% HClO4 and 2 mL of 0.5 M FeCl3) and incubated at room temperature. IAA was indicated by pink color development and the optical density was recorded at 530^nm after 2^h. Siderophore production assay For siderophore production, selected halotolerant strains were examined on the Chrome Azurol’s (CAS) agar medium with 1% NaCl The plates were incubated at 30°C for 7 days. Pink color development around the colony was indicative of siderophore production. Biofilm production assay To test whether the halotolerant strains can form biofilms, which may contribute to the plant growth stimulation activity, a 96-well plate crystal violet binding assay was conducted. Biofilm formation was characterized and values measured for each isolate as described previously. Biolog assay We tested halotolerant strains for the BIOLOG assay following the instructions outlined in the BIOLOG GEN III MicroPlateTMmanual (Biolog, Inc., Hayward, CA). Pure fresh cultures of the selected bacterial isolates were grown on LB agar plates with 1% NaCl at 30°C for 48 hours, as freshly grown cells are needed due to the potential loss of viability and metabolic vigor in the stationary phase, which could impact the assay. To ensure proper calibration, we first tested the blank without bacterial inoculation to calibrate the turbidimeter to 100% transmittance. Bacterial colonies were collected using a sterilized wooden stick and mixed with the liquid provided by the BIOLOG GEN III MicroPlateTMkit. The target cell density was adjusted to between 90-98%T for this protocol. The cell suspension was poured into the multichannel pipette reservoir, and each well in the MicroPlate was filled with 100 µl of the suspension, being careful to not splash from one well into another. The MicroPlate was covered with its lid and incubated at 30°C. Readings were taken after 8 hours and 22 hours. Minimum inhibitory concentration (MIC) of NaCl for selected isolates To evaluate the salt tolerance potential of the halotolerant strains, we determined the minimum inhibitory concentration (MIC) of NaCl for each. Selected strains were inoculated into LB broth and agar plates with 0M (0% w / v), 1M (2.7% w / v), 2M (5.4% w / v), 3M (8.1% w / v), or 4M (10.8% w / v) NaCl. After incubation at 30°C for 48 hours, the NaCl inhibitory concentration was recorded. Greenhouse trial of inoculated alfalfa growth promotion under salt stress 23903.1a All isolates were initially screened on alfalfa plants for the ability to stimulate growth in saline conditions and the ten most promising isolates were selected for further experiments. Kushneria strains A3 and B5 were inoculated into alfalfa (Vernal variety, Granite Seeds, Lehi, Utah) to examine growth stimulation in open pots in the presence and absence of salt. Seeds were planted directly into pots containing potting soil and watered with 100 ml of 0.5X Hoagland’s solution containing 0 or 1.2 % NaCl. The initial watering solution contained 1 ml of LB or 1 ml of liquid culture (1 x 109CFU / ml) for each strain tested. Plants were grown at the BYU Greenhouse complex with watering every third day. After 4 weeks in the open environment, plants were harvested and analyzed for length of roots, height, and weight. Growth chamber trials for alfalfa plant growth stimulation with selected halotolerant bacterial isolates Alfalfa seeds were surface sterilized with dilute bleach (1% sodium hypochlorite) followed by extensive washing with sterile water and germination in a sterile petri dish. After 24-36 hours the seedlings were transplanted into autoclaved soil (1:1:1 potting soil:clay:sand) in a clear magenta box. A specific volume (100 ml) of 0.5X Hoagland’s basic nutrient solution containing 0 or 1.2% NaCl along with 1 ml of the bacterial culture (containing 1 x 109CFU / ml) was added as inoculum or sterile LB broth as control was added to each box. Out of the ten isolates that were initially screened for properties associated with PGP activity, five strains were selected for further trials based on those that had the most plant growth promotion properties and performed the best in preliminary growth trials (A3, B5, B2, D8, E4). Five seedlings were transplanted into each of six boxes. A second magenta box was inverted and taped in place to allow air exchange, and the plants were placed in the growth room at 25°C with a 16 hr light / 8 hr dark cycle. After four weeks of growth, total plant height and weight were measured and analyzed for statistical significance. Determination of bacterial survival in alfalfa pots under salt stress conditions To determine whether the bacterial inoculum could colonize the soil and / or become endophytic in alfalfa roots, soil and root samples were collected when the alfalfa plants were harvested. Soil was diluted in sterile PBS and spread on LB agar plates containing 1 M NaCl. Roots were surface sterilized, ground in sterile PBS, and similarly spread on plates. The identification was based on colony characteristics of the bacterial colonies and 16S rRNA gene sequencing as above. Preparation and in-plant analysis of GFP-expressing B5 strain A constitutively expressing GFP gene was introduced into the B5 Kushneria strain. The plasmids pJMP1039 and pJMP2834, developed by Peters et al., were ordered from Addgene. 23903.1a Cultures of single colony isolated from these plasmid-bearing strains were grown overnight in average LB with 50 ug / ml carbenicillin in a 37C incubator at 220 rpm. Each plasmid was extracted from the culture using the Qiagen Midi Prep Kit (Qiagen). Kushneria (250 ml) was grown to exponential phase and made electrocompetent via multiple centrifugations and resuspensions in 10% glycerol, then stored on ice in 50 ul aliquots for immediate use. To generate a green fluorescent protein (GFP) encoding strain of Kushneria, electrocompetent Kushneria cells were electroporated in a 1 mm electroporation cuvette with 100 ng of pJMP1039 and 100 ng of pJMP2834. After electroporation, the cells were incubated at 30^C and shaken at 220 rpm for 2 hours for recovery. The culture was grown overnight in 5% NaCl growth media with 30 ug / ml kanamycin for selection. This strain was inoculated into alfalfa as above. Upon harvest, root samples were examined using an ECHO Revolve Fluorescence Microscope (Model RVL2-K3) at the BYU Microscopy Center. Statistical analyses All statistical analyses were conducted using R environment 4.1.3 for Windows. The data for shoot length, root length, total plant length, and plant biomass were analyzed through one-way analysis of variance (ANOVA) to identify the significant effects of bacterial treatments on these traits, keeping P = 0.05. The R package, Agricolae, was used to carry out Tukey’s Honest Significant Difference (HSD) Post Hoc test to identify which group means differ from each other after finding a significant difference in the overall ANOVA. Experimental Results One hundred isolates were initially isolated and screened for the ability to stimulate alfalfa growth in salty conditions. Many of these were identified as similar or duplicate isolates based on 16S rDNA sequence analysis. Ten distinct isolates were analyzed for potential plant growth promotion properties as follows (summarized in Table 1). Table 1: Potential halophilic bacterial isolate identification and plant growth promoting traits Accession numbers of 16S rDNA sequences are given for the five strains tested for plant growth promotion activity. (+++) exhibited the highest PGP properties among those evaluated, (++) moderate activity, (+) slight / minimal activity, and (-) no activity. See Methods for explanation of numerical values. 23903.1a Table 1 Halophilic Host Biofilm PO4Zinc Siderophore IAA bacteria Halophyte solubil. solubil. (%) production Accession number A1 Halomonas sp. A. 0.089 - - 0 ++ occidentalis soil A3 Kushneria A. 0.125 +++ ++ 44.9 +++ marisflavi occidentalis PP463953 roots A5 Bacillus S. utahensis 0.06 ++ - 14.2 ++ aryabhattai roots A9 Halomonas A. 0.005 ++ +++ 13.4 + elongata occidentalis MK873884 roots B1 Bacillus S. utahensis 0.058 + - 0 ++ zanthoxyli roots B2 Kushneria sp. S. rubrum 0.201 ++ ++ 36.4 +++ MK873883 soil B3 S. rubrum 0.066 + - 0 - Microbacterium roots sp. B5 Kushneria sp. S. utahensis 0.059 +++ +++ 25.1 + PP463954 roots D8 Bacillus sp. S. utahensis 0.010 ++ +++ 15.8 + PP446494 soil E4 Halomonas sp. A. 0.087 - + 12.5 +++ PP446495 occidentalis soil PO4solubilization The ten strains were inoculated on NBRIP and Pikovskaya's agar medium to determine their phosphate solubilizing ability (Figure 2). Figure 2 depicts ten strains, inoculated on NBRIP and Pikovskaya's agar medium to determine their phosphate solubilizing ability. 23903.1a A) Pikovskaya's medium and B) NBRIP medium for phosphate solubilization, C) mineral salts medium for zinc solubilization, and D) IAA production assay in a 96-well plate. Results are summarized in Table 1. Of these, eight strains showed phosphate solubilizing activity, with A3 and B5 Kushneria isolates showing the greatest P solubilization activity among the evaluated strains. Zn- solubilizing activity assay Zinc is an important micronutrient for plants and is involved in many key cellular functions such as metabolic and physiological processes, enzyme activation, and ion homeostasis. Bacteria with this property are capable of solubilizing inorganic zinc and making it bioavailable, which helps in the growth promotion of plants. For further characterization of PGP-traits for these halotolerant bacterial isolates, we screened them on mineral salts medium (MSM) for zinc solubilization activity (Figure 2). Out of ten strains, only six showed zinc solubilizing activity. Maximum Zn-solubilization activity was observed for the A9 Halomonas sp, B5 Kushneria and D8 Bacillus strain. Minimum Zn-solubilizing activity was found in E4. However, no zone formation was recorded for A1, A5, B1 and B3 strains. Siderophore production assay Siderophores are chelating molecules secreted by microorganisms under low iron stress. They scavenge Fe from the environment and form Fe (III)-siderophore complexes, making the otherwise insoluble iron available for bacterial uptake. They play a crucial role in facilitating iron uptake by plants, ultimately promoting their growth and development. To test siderophore production activity strains were spotted on Chrome Azurol’s (CAS) agar medium. Strains A3, B2 and B5 showed the maximum siderophore production, while minimum siderophore production was observed in A5 (Bacillus sp.) and A9 (Halomonas sp.). Biofilm production The ability to form biofilms has been identified as a potential contributing factor to plant growth promotion, as biofilms can help control nutrient and ion access from the soil to the roots. To test whether the bacterial strains can form biofilms, which may contribute to the plant growth stimulation activity, a plate assay was conducted. The A3 and B2 Kushneria isolates showed strong biofilm formation, while other isolates, including the Bacillus and Halomonas isolates, showed lower biofilm activity (Table 1). A3 and B2 have the highest biofilm activity, while that of B5 is lower. Table 1 provides a summary of these analyses, and show that the A3, B2 and B5 Kushneria strains have high phosphate and zinc solubilization activity, produce IAA and have 23903.1a the highest siderophore activity as compared to the Bacillus (A5 and B1) and Halomonas (A9) isolates. Kushneria strains A3 and B2 exhibit the highest biofilm activity. Biolog assays The Biolog plates include different tests in a 96 well plate, allowing screening of strains for the ability to metabolize different sugars and substrates, resistance to antibiotics, and the ability to grow at different salt concentrations or in mildly acidic conditions (pH 6). The results of Biolog analysis of ten strains are shown in Supplementary Table 1. The strains exhibit different properties; all can grow in glucose and many of the other sugars. As expected, all ten strains showed growth at 8% NaCl, and most can grow at pH6 (except the A9 and D8 strains). All except the D8 strain were positive for growth in lithium chloride. Testing of halotolerant strains for NaCl tolerance Selected strains were screened with different NaCl stress conditions (0M (0% w / v), 1M (2.7% w / v), 2M (5.4% w / v), 3M (8.1% w / v), or 4M (10.8% w / v) of NaCl) on plates and in liquid media. Out of ten strains only six strains tolerate 1 M NaCl (A3, A9, B2, B5, D8 & E4). Five of these six strains grew with up to 4M NaCl, except for E4 (as shown in Figure 3). Figure 3 depicts screening of selected strains under different NaCl stress conditions, testing halotolerant strains on LB agar with 0M (0% w / v), 1M (2.7% w / v), 2M (5.4% w / v), 3M (8.1% w / v), or 4M (10.8% w / v) NaCl. No growth was observed on plates with 0M salt, which indicates that all the strains are halophilic and require salt for growth. Greenhouse trials of inoculated alfalfa To examine the growth stimulation potential of the strains under different conditions, alfalfa plants were grown in open pots in the BYU greenhouse with and without inoculation with the Kushneria strains (A3 and B5) grown in the presence 1.2 % NaCl. Results are shown in Figure 4. Figure 4 illustrates the effect of Kushneria strains on alfalfa plant growth in open pot trials. A) photographs of plants after harvesting from each treatment (No salt control without bacteria, Salt control without bacteria, A3 inoculation, B5 inoculation). B) the total plant fresh weight is shown. Data points indicate means ± SEM of at least 10 biological replicates. The statistically significant difference is marked with asterisks (Tukey’s HSD, P < 0.05). Significance: P<0.001 ‘***’. C) Plants in open pots before harvesting. Clear differences in growth of roots and shoot tissue were observed between the inoculated plants and the control uninoculated plants grown in the presence of salt. The B5 Kushneria strain yielded the greatest increase in plant fresh weight. At the same time, A3, while appearing visually to result in modest enhanced growth, did not show a statistically significant 23903.1a difference (Figure. 4B). These two strains were used for more detailed analysis in the growth chamber as individual inoculants and in combination. Alfalfa plant growth trial in the growth chamber under salt stress Alfalfa plants were grown with and without inoculation with the Kushneria sp. A3, B5 or co-inoculation of A3 and B5 isolates, in the presence and absence of 1.2 % NaCl in the watering solution. We selected isolates A3 and B5 for co-inoculation as these two strains produced the best results when tested individually among other strains from preliminary experiments (Figures 5 and 6). Figure 5 illustrates Alfalfa plant growth trial with Kushneria strains under salt stress conditions. Harvested alfalfa plants are shown at the bottom. Each pot was inoculated with the indicated bacterial isolate. See Figure 6 for the data analysis. Figure 6 illustrates the effect of Kushneria strain inoculation on alfalfa in growth chamber trials. A) plant height, B) root length, C) plant fresh weight, and D) plant dry weight. Data points show the means ± SEM of at least 15 biological replicates. Statistically significant differences are marked with asterisks (Tukey’s HSD, P < 0.05). Significance codes: 0.001 ‘***’; 0.01 ‘**’; 0.05 ‘*’ Individual isolates A3 and B5 have significant effects on plant height (M = 35.8, P = 0.0003 and M = 38.07, P = 0.0001), respectively) and root length (M = 13.3, P = 0.01 and M = 16.17, P = 0.002, respectively) in alleviating salt stress. These isolates also increased total plant biomass compared to the plus salt control (Figures.6C, D). Without being bound to any particular theory, it was proposed that the two strains might contribute to plant growth when inoculated together. Indeed, the combination of A3+B5 resulted in the largest increase in plant height (Figure 6) (M = 40.7); it also shows the most reproducible positive results in both plant fresh (M = 1.76) and dry weight (M = 0.36), and the B5 strain showed the most promising result for root length as compared with others. These findings indicate that the combination of A3+B5 is the best for promoting alfalfa growth in the presence of 1.2 % NaCl as compared to individual isolates. Other strains appeared to be inhibitory or had little effect on plant growth (not shown). Additional trials Three other strains were tested for plant growth promotion activity. Alfalfa plants were grown with and without inoculation with the Kushneria strain B2, Bacillus strain D8, and Halomonas strain E4. in the presence and absence of 1.2 % NaCl in the watering solution. Results are shown in Figures 7 and 8 (harvested plants and data analysis of shoot length, root length, shoot and root fresh fresh weight). Figure 7 illustrates Alfalfa plant growth trial with Kushneria strains under salt stress conditions (top panel) and harvested alfalfa plants (bottom 23903.1a panel). All plants were grown in closed pots with a single watering upon planting and inoculated with the indicated bacterial isolate except for the controls (uninoculated No salt & salt). Figure 8 illustrates the effect of Kushneria strain on alfalfa plants under field conditions A) shows shoot length, B) root length, C) shoot fresh weight, and D) root fresh weight. Data points show the means ± SEM of at least 13 biological replicates except for E4 which has 10 replicates and the unstressed control which has 4 replicates. Statistically significant differences are marked with asterisks (Tukey’s HSD, P < 0.05). Significance codes: 0.001 ‘***’; 0.01 ‘**’; 0.05 ‘*’. Strains B2 and D8 stimulated growth (shoot length) in the presence of 1.2 % salt (M = 10.33, and M = 10.33, respectively). However, Halomonas strain E4 inhibited plant growth when compared to other isolates (M = 4.82). The Kushneria B2 strain showed the highest growth stimulation for root length compared to other isolates (M = 16.63), and D8 and E4 did not show any significant effect on root length (M= 15.15; P = 0.2897 and M = 11.10; P = 0.999, respectively). Both strains supported the greatest promotion of shoot fresh weight. These strains also show the reproducible effects but statistically not significant increases in root fresh weight (P = 0.4570 and P = 0.9999, respectively (Figure. 7). Strain E4 appeared to have an inhibitory effect or no effect on plant shoot (M = 0.01) and root fresh weight (M = 0.02). Confirmation of re-isolated colonies from alfalfa pots Root tissue and soil from around the roots of plants inoculated with Kushneria were harvested, ground in buffer, and plated out with different dilutions to recover bacteria. Uninoculated soil and roots were used as controls. Colonies recovered included the same bacteria used to inoculate the plants. However, contaminating bacteria were also obtained, including mostly Bacillus species. Even though autoclaved soil was used in the growth chamber trials, it was difficult to sterilize the soil completely, potentially leading to contamination (Figure 9). Figure 9 illustrates survival determination of inoculated bacteria in alfalfa Pots on 1M NaCl + LB media with 10-6dilution after harvesting A) No inoculation B) A3 inoculation C) B5 inoculation. The Kushneria strains have a distinctive orange pigment that aids in identification, which was confirmed by partial 16S rRNA gene sequencing. Visualization of B5 expressing GFP in inoculated plants Inoculation with the B5 strain carrying a constitutively expressing GFP gene allowed visualization of bacterial colonization within the root tissue of alfalfa plants (Fig. 10). Figure 10 depicts a visualization of B5 strain expressing GFP in alfalfa root tissue. A & B) No inoculation, C & D) B5+GFP inoculation. Root sample from alfalfa inoculated with B5 strain, constitutively expressing the GFP gene as compared to control, visualized using the Echo 23903.1a Revolve microscope. Fluorescent GFP signal highlights bacterial colonization within the root tissue. The root samples were examined using an ECHO Revolve Fluorescence Microscope (Model RVL2-K3), which provided high-resolution images of GFP-expressing bacteria in the plant root tissue. Figure 10 compares root tissues inoculated with the B5 strain and control root tissues that were not inoculated. In the GFP-inoculated samples, the B5 strain is visible as bright green fluorescent punctae (Fig. 10 C,D), indicating successful colonization and persistence of the bacteria within the plant roots. Only background fluorescence was observed in the control non-inoculated root samples (Fig.10 A,B). The challenge of producing adequate food for the growing world population is complicated by many biotic and abiotic factors. One serious problem is soil salinity, which affects many agricultural areas. The accumulation of surplus sodium and chloride ions within plant tissues plays a critical role in plant growth. The inherent toxicity of these ions to plants impedes essential metabolic pathways and compromises the plant's homeostatic equilibrium. Soil salinity is spreading in many places due to climate change and poor irrigation practices. We initiated this research to identify salt-tolerant bacterial strains with the potential for inoculation of salt-sensitive crops to increase yield in salt-affected soils. Identifying salt- tolerant (halotolerant) bacteria with PGP properties (ST-PGPB) is one approach to help address the problem of soil salinity affecting crop productivity. In the present disclosure, we identified two strains of Kushneria that each stimulate alfalfa growth in salty soil conditions. When inoculated together in young alfalfa seedlings, the A3 and B5 strains (Figures. 4-5) showed a synergistic effect on plant growth. The analysis of biochemical activities of these strains and eight others, including additional Kushneria isolates and Bacillus and Halomonas strains, led to the identification of potentially important traits of the strains that most effectively stimulate alfalfa growth in the presence of 1.2 % NaCl in the watering solution. In particular, the A3 and B5 strains showed relatively high activity for phosphate and zinc solubilization and siderophore production. Both produce IAA, with A3 showing higher activity. Both exhibited biofilm formation, while A3 again was higher. The B2 Kushneria strain was also high for these activities and appears to be closely related to the A3 strain based on properties and rDNA sequence analysis (Table 1). In another study, Kushneria marisflavi, along with Pseudomonas stutzeria, were found to have the ability to reduce salinity stress-induced damage in barley, sunflower and lettuce (Szymańska et al., 2022). When alfalfa plants were exposed to NaCl stress without added bacteria, a notable reduction in their growth was observed (Figures. 4-7, salt-stressed plants), with more pronounced effects on roots. The higher inhibition by NaCl stress on roots is plausible due to 23903.1a their direct contact with a higher salt concentration in the soil. In contrast, promoting root growth is a potential solution for enhancing water absorption under salt stress. There is a growing number of publications on plant growth promotion by salt-tolerant (halotolerant) rhizobacteria associated with halophyte species in saline soils. Some of these bacteria are closely associated with the roots, while others are established within plant tissues (endophytes). Mechanisms by which these bacteria enhance plant growth may include enhanced nutrient acquisition and changes in bacterial and host plant gene expression. For example, ACC (1-aminocyclopropane-1-carboxylase) deaminase is a bacterial enzyme found in many endophytes that stimulates nutrient acquisition and plant growth by reducing the amount of ACC converted to ethylene, a known inhibitor of plant growth in response to salt, drought and other environmental stresses. Burkholderia phytofirmans is an endophyte that alters the expression of plant transcription factors known to regulate the expression of plant stress genes. Some bacterial endophytes (Sphingomonas, Pantoea, Bacillus and Enterobacter) have been reported to enhance the salt tolerance of hybrid elephant grass. The genus Kushneria has been characterized as a plant growth-promoting rhizobacteria (PGPR) known to produce IAA, biofilm, and siderophores, as well as solubilize zinc and phosphate. In our work, we observed a differential growth response to NaCl stress when plants were inoculated with different bacterial strains. Out of 10 strains, only a few (A3, B5, B2 and D8) exhibited significant effects. The varying performance of these strains may be linked to the differential production of IAA, with the A3 Kushneria strain exhibiting the highest IAA levels. Further investigation is needed to elucidate the correlation between plant growth promotion and Indole-3-acetic acid (IAA), especially considering that certain strains in the present disclosure, such as E4, produced IAA but did not support growth stimulation in plant trials. As the main auxin in plants, IAA plays a crucial role in modulating plant growth by influencing cell division, stimulating stem elongation, and enhancing root branching through increased cell wall synthesis. The observed variations in plant growth responses to IAA- producing strains might also be influenced by factors such as the concentration of IAA produced, the timing of its release, and the specific mechanisms through which these strains interact with plant tissues. The IAA producing bacterial strains P. aureantiaca and P. extremorientalis alleviated the reductive effect of salt stress on percentage of germination (up to 79%), probably through their ability to produce IAA. Solubilizing inorganic phosphate is a key mechanism through which plant-associated bacteria promote plant growth. This process involves the release of organic acids by bacteria into the soil, leading to the solubilization of phosphate complexes. This transformation converts 23903.1a them into ortho-phosphate, a form readily available for plant uptake and utilization. Hence, this activity plays an essential role in phosphorus cycling and promoting plant growth by increasing phosphorus uptake in rhizosphere soils. We assessed the phosphorus-solubilizing capability of the ten selected isolates at high salt concentrations. Our findings align with previous research indicating that phosphorus-solubilizing bacteria exhibit enhanced solubilization of inorganic phosphate in the presence of NaCl. However, it is also reported that there is a decrease in phosphate solubilization activity in stress-tolerant phosphate-solubilizing strains at salt concentrations exceeding 0.4 M. In the present disclosure, all tested strains demonstrated a high phosphorus-solubilizing ability even in the presence of 1 M NaCl, except for A1 and E4. This suggests that halophile and halotolerant microorganisms with phosphate-solubilizing capabilities have the potential to enhance crop yield under salt stress. Notably, strains A3, B5, and D8 displayed efficient phosphate solubilization, highlighting their potential as candidates for promoting crop growth under saline conditions. Zinc, a pivotal component for plant development, was investigated for its solubilization by the selected bacterial strains. All tested strains exhibited zinc solubilization activity associated with plant growth promotion except A1, A5, B1 and B3. This observation is consistent with findings from previous studies that demonstrated that microbes can influence the uptake of toxic ions and nutrients by modulating host physiology or directly reducing the accumulation of Na+and Cl−, while concurrently increasing the uptake and translocation of other cations, such as zinc. Despite this, the exact mechanisms underlying these processes remain unknown. In the present disclosure, strains A3, B5, and D8 demonstrated significant zinc solubilization activity, indicating their potential as promising candidates for fostering crop growth in saline conditions. This suggests that inoculation with these strains enhances the bioavailability of zinc to alfalfa plant roots compared to un-inoculated controls, ultimately promoting robust plant growth. Earlier research has shown that applying PGPR can enhance zinc translocation to rice and wheat grains. This capability of rhizobacteria or endophytic bacteria (PGPB) is associated with their proficiency in facilitating successful plant-microbe interactions, including the induction of physiological processes, mineralization, and solubilization. The present findings align with previous studies highlighting that biofilm-producing bacteria enhance soil structure, increasing soil porosity and facilitating water and nutrient movement from soil to plant. The biofilm structures, characterized by extracellular polymeric substances (EPS), adhere to soil particles, serving as an effective matrix for soil moisture retention. Biofilms not only protect roots from desiccation but also mitigate the impact of salt 23903.1a stress by sequestering toxic Na+and Cl−ions, as previously reported. Nevertheless, based on our observations, it appears that not all bacteria exhibiting high biofilm production necessarily promote plant growth under salt stress. A summary of potential bacterial growth promotion properties and their effect on plant growth is shown in Fig.11. Salinity affects various growth parameters of Lactuca sativa, including the number of leaves and plant fresh weight. In the present disclosure, a salinity effect on alfalfa growth parameters, as plant height and plant dry weight were compromised without inoculation of bacteria compared to plants with bacterial inoculation under salt stress, is demonstrated. This suggests that different types of plants may exhibit considerable differences in salinity tolerance in response to inoculation with halotolerant bacteria. The intricate interplay between inoculation, salinity levels, and plant species-specific responses underscores the complexity of plant-microbe interactions in the context of salt stress. One notable observation of the present disclosure is that, in the context of salt stress, the inoculation of plants with both A3 and B5 strains resulted in a notable synergistic increase in growth parameters, including shoot length, root length, and both fresh and dry weights, as shown in Figures 4 and 6. Conversely, the E4 strain significantly reduced these growth parameters, suggesting a potential competition for nutrients with other bacterial isolates present in the environment. This halotolerant strain appears unable to ameliorate the stress effectively. This work provides promising support for using halotolerant bacterial strains, initially isolated from halophytes, as inoculants of crop plants to stimulate growth in salty soil conditions. The A3 and B5 Kushneria strains have shown encouraging results with alfalfa, and we have initiated testing of these strains with other plant species including rice, corn, and wheat grass. Further studies are needed to identify the optimal isolates and combinations for each crop plant. In summary, several halotolerant strains of bacteria isolated from halophytes were found to have potential plant growth promotion properties, including phosphate and zinc solubilization, as well as IAA, siderophore and biofilm production. Some of these strains have shown promising results for the stimulation of growth of alfalfa in the presence of salt in the greenhouse and growth chamber. We have observed the most consistent results with Kushneria strains A3 and B5, and the combination of A3 and B5 shows synergistic effects with greater growth stimulation than either strain alone. In other preliminary studies we have observed growth stimulation of other crop plants, including rice, wheat grass and rye grass. These results are an important step in developing new approaches to help farmers deal with decreased crop 23903.1a yields caused by salinity. Further research to examine the potential of these strains under field conditions and with different crop species is warranted. Genbank accession numbers for partial 16S rRNA sequences of the strains used in the present disclosure are provided in Table 1, and include PP446494, PP446495, PP463953, PP463954, MK873883, MK873884. All other data generated and analyzed in connection with the present disclosure are included in Table 2. Table 2 Biolog test for selected salt-tolerant and halophilic bacterial isolates BIOLOG Test A1 A3 A5 A9 B1 B2 B5 D8 E4 23903.1a D-Gluconic acid ++ ++ + ++ + ++ ++ + + 23903.1a L-Malic acid ++ - - ++ + ++ ++ ++ + reaction; +, slight reaction with light color; -, no reaction or color. A3 and B5, being closely related, share many properties but there are some important differences for some properties. Conclusion While the foregoing detailed description makes reference to specific exemplary embodiments, the present disclosure may be embodied in other specific forms without departing from its spirit or essential characteristics. Accordingly, the described embodiments are to be considered in all respects only as illustrative and not restrictive. For instance, various substitutions, alterations, and / or modifications of the inventive features described and / or illustrated herein, and additional applications of the principles described and / or illustrated herein, which would occur to one skilled in the relevant art and having possession of this disclosure, can be made to the described and / or illustrated embodiments without departing from 23903.1a the spirit and scope of the invention as defined by the appended claims. Such substitutions, alterations, and / or modifications are to be considered within the scope of this disclosure. The scope of the invention is, therefore, indicated by the appended claims rather than by the foregoing description. The limitations recited in the claims are to be interpreted broadly based on the language employed in the claims and not limited to specific examples described in the foregoing detailed description, which examples are to be construed as non-exclusive and non-exhaustive. All changes which come within the meaning and range of equivalency of the claims are to be embraced within their scope. It will also be appreciated that various features of certain embodiments can be compatible with, combined with, included in, and / or incorporated into other embodiments of the present disclosure. For instance, systems, methods, and / or products according to certain embodiments of the present disclosure may include, incorporate, or otherwise comprise features described in other embodiments disclosed and / or described herein. Thus, disclosure of certain features relative to a specific embodiment of the present disclosure should not be construed as limiting application or inclusion of said features to the specific embodiment. In particular, ingredients and components disclosed in the present application are intended to be independently added or removed from embodiments of the present disclosure, without regard to any example, listing, series, or combination in which they are disclosed. Similarly, method steps disclosed in the present application are intended to be independently added or removed from embodiments of the present disclosure, without regard to any example, listing, series, or combination in which they are disclosed. Likewise, any steps recited in any method or process described herein and / or recited in the claims can be executed in any suitable order and are not necessarily limited to the order described and / or recited, unless otherwise stated (explicitly or implicitly). Such steps can, however, also be required to be performed in a specific order or any suitable order in certain embodiments of the present disclosure. In addition, unless a feature is described as being required in a particular embodiment, features described in the various embodiments can be optional and may not be included in other embodiments of the present disclosure. Moreover, unless a feature is described as requiring another feature in combination therewith, any feature herein may be combined with any other feature of a same or different embodiment disclosed herein. It will be appreciated that while features may be optional in certain embodiments, when features are included in such embodiments, they can be required to have a specific configuration as described in the present disclosure.

Claims

23903.1a CLAIMS I claim:

1. A method, comprising: applying at least two strains of halotolerant rhizobacteria to soil having a salt concentration of about 1% w / v to about 10% w / v, the at least two strains of halotolerant rhizobacteria comprising Kushneria sp. A3 and Kushneria sp. B5; and cultivating a plant in the soil.

2. The method of claim 1, wherein the at least two strains of halotolerant rhizobacteria colonize root tissue of the plant during cultivation.

3. The method of claim 2, wherein colonization of the root tissue by the at least two strains of halotolerant rhizobacteria adapts the plant for growth in the soil having a salt concentration of about 1% w / v to about 10% w / v.

4. The method of claim 1, wherein the at least two strains of halotolerant rhizobacteria improve growth of the plant in the soil having a salt concentration of about 1% w / v to about 10% w / v, as compared to a corresponding plant cultivated in soil having a salt concentration of about 1% w / v to about 10% w / v without the at least two strains of halotolerant rhizobacteria.

5. The method of claim 1, wherein the at least two strains of halotolerant rhizobacteria are applied to the soil: as a mixture of the at least two strains of halotolerant rhizobacteria; or in separate applications applied sequentially or simultaneously.

6. The method of claim 1, wherein cultivating the plant in the soil comprises planting a seed in the soil, the seed sprouting to produce the plant.

7. The method of claim 1, wherein cultivating the plant in the soil comprises transplanting the plant into the soil.

8. The method of claim 1, wherein the plant comprises a legume crop.

9. A system for adapting a plant for improved growth in salinated soil, the system comprising at least two strains of halotolerant rhizobacteria comprising Kushneria sp. A3 and Kushneria sp. B5.

10. A product, comprising at least two strains of halotolerant rhizobacteria comprising Kushneria sp. A3 and Kushneria sp. B; and a diluent or spreading agent.

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