Halalkalibacter sp. strain HAAS-P013 and application thereof

Abstract

The application relates to the technical field of agricultural microorganisms, in particular to a Halalkalibacter new species strain HAAS-P013 and application thereof. The strain HAAS-P013 is preserved in the China General Microbiological Culture Collection Center on June 9, 2025, and the preservation number is CGMCC No. 34803. The application also provides a culture, a fermentation liquor, a bacterial suspension, a bacterial agent and a bacterial powder containing the above-mentioned strain. In addition, the application also provides application of the above-mentioned strain and the culture, the fermentation liquor, the bacterial suspension, the bacterial agent and the bacterial powder thereof in plant growth promotion. The strain HAAS-P013 provided by the application can solve the problems of weak stress resistance, poor colonization ability, lack of active alkali reduction capacity and the like of the existing saline-alkali soil improvement microbial fertilizer.

Description

Technical Field

[0001] This application relates to the technical field of agricultural microorganisms, and more particularly to a strain of... Halalkalibacter A new species of strain HAAS-P013 and its applications. Background Technology

[0002] my country has a vast area of ​​saline-alkali land, and the salinization of arable land in some areas is intensifying, making comprehensive improvement and utilization of saline-alkali land of significant practical importance. Traditional physical and chemical methods for improving saline-alkali land suffer from drawbacks such as high application costs, limited improvement effects, and a tendency to cause secondary soil pollution. Against this backdrop, utilizing salt-tolerant microorganisms for the biological improvement of saline-alkali land has become a research hotspot in this field.

[0003] Halophilic microorganisms, as a special type of extremophile microorganisms, can grow and reproduce normally in high-salt environments (NaCl concentration up to 25%) and a wide pH range (pH 4.5-10.5), showing great application potential in the fields of saline-alkali land improvement and bioremediation. In recent years, bioremediation technology based on functional microorganisms has gradually become the mainstream research direction in the field of saline-alkali land management due to its environmentally friendly and sustainable characteristics.

[0004] Previous studies have shown that plant rhizosphere growth-promoting bacteria (PGPRs) can promote plant growth through nitrogen fixation, phosphorus solubilization, and the secretion of plant hormones. However, in practical applications, most known PGPRs, such as Pseudomonas (…), are not readily available. Pseudomonas ), Bacillus ( Bacillus In strongly alkaline environments with pH > 9.0, the activity of microorganisms decreases significantly or even becomes inactive, making it difficult for them to stably colonize and continuously function in saline-alkali soils. Furthermore, existing microbial agents generally lack the ability to actively reduce alkali content, failing to fundamentally alleviate soil alkalization and severely limiting their application in in-situ remediation of severely saline-alkali lands.

[0005] Although some halophilic bacteria ( Halophiles While some strains have been reported to possess certain salt tolerance, strains with highly efficient plant growth-promoting functions remain extremely scarce. For example, some strains reported in existing literature... Halomonas genus or Alkalibacillus Although the strains can tolerate high-salt environments, their metabolic activity is insufficient under alkaline conditions, and there is no literature to record their ability to systematically regulate rhizosphere pH and actively improve the alkaline microenvironment of the rhizosphere.

[0006] In summary, existing microbial strains for improving saline-alkali land generally suffer from technical shortcomings such as poor adaptability to extreme saline-alkali environments, weak rhizosphere colonization ability, lack of active alkali reduction function, and insufficient comprehensive growth-promoting effect. The industry urgently needs to develop a new type of functional microbial resource that can not only survive stably in extreme saline-alkali environments, but also actively improve the rhizosphere microenvironment and effectively promote plant growth. Summary of the Invention

[0007] This application provides one plant Halalkalibacter This application relates to a novel strain, HAAS-P013, and its applications. Halalkalibacter This is a novel bacterium, strain number HAAS-P013, capable of stable growth in extreme saline-alkali environments with pH 9.0-11 and NaCl 5-10% (w / v). The application of this strain in plant growth promotion can address the problems of weak stress resistance, poor colonization ability, and lack of active alkali reduction capacity found in existing microbial fertilizers for saline-alkali land improvement.

[0008] Firstly, this application provides a plant Halalkalibacter The novel strain HAAS-P013 was developed using the following technical approach:

[0009] This application provides Halalkalibacter New species strain HAAS-P013 ( Halalkalibacter sp. HAAS-P013 was deposited on June 9, 2025 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 34803.

[0010] This application successfully isolated a novel halophilic bacterium, HAAS-P013, from typical soda-saline soil in the Dongfeng Grassland of Daqing City, Heilongjiang Province. Multiphase taxonomic analysis confirmed it to be... Halalkalibacter This is a new species in the genus. In particular, strain HAAS-P013 exhibits a unique dual function of "stress resistance-growth promotion": on the one hand, it carries an ectoine synthesis gene cluster, endowing it with excellent osmotic protection capabilities; on the other hand, genome prediction shows that it contains a type III polyketide synthase (PKS III) gene cluster, which may participate in the biosynthesis of flavonoids, thereby inducing plant defense responses and growth and development. Experimental verification shows that this strain can significantly reduce the pH of the culture medium during growth, possessing the ability to actively regulate the alkaline environment. These characteristics make it an ideal candidate strain for solving the three major bottlenecks in current bioremediation of soda-saline-alkali land: "difficult colonization, weak function, and lack of alkalinity reduction."

[0011] Secondly, this application provides a culture. The culture includes the above-described... Halalkalibacter It belongs to a new species strain HAAS-P013.

[0012] Thirdly, this application provides a fermentation broth. The fermentation broth includes the above-mentioned... Halalkalibacter It belongs to a new species strain HAAS-P013.

[0013] Fourthly, this application provides a bacterial suspension. The bacterial suspension includes the above-mentioned components. Halalkalibacter It belongs to a new species strain HAAS-P013.

[0014] Fifthly, this application provides a microbial agent. The microbial agent comprises the above-mentioned... Halalkalibacter It belongs to a new species strain HAAS-P013.

[0015] Optionally, the above-mentioned microbial agents can be any one of liquid preparations, powders, or granules.

[0016] Sixthly, this application provides a bacterial powder. The bacterial powder includes the above-mentioned... Halalkalibacter It belongs to a new species strain HAAS-P013.

[0017] Seventhly, this application provides a aforementioned Halalkalibacter Application of the new species strain HAAS-P013, its culture, fermentation broth, bacterial suspension, bacterial agent or bacterial powder in plant growth promotion.

[0018] In summary, this application includes at least one of the following beneficial technical effects:

[0019] This application provides a plant Halalkalibacter New species strain HAAS-P013 ( Halalkalibacter sp. HAAS-P013 was deposited on June 9, 2025 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 34803.

[0020] The strain HAAS-P013 exhibits a unique combination of "stress resistance and growth promotion": on the one hand, it carries a tetrahydropyrimidine (ectoine) synthesis gene cluster, which endows it with excellent osmotic protection ability; on the other hand, genome prediction shows that it contains a type III polyketide synthase (PKS III) gene cluster, which may be involved in the biosynthesis of flavonoids, thereby inducing plant defense response and growth and development.

[0021] The application of this strain in plant growth promotion can solve the problems of weak stress resistance, poor colonization ability, and lack of active alkali reduction ability in existing saline-alkali land improvement microbial fertilizers. Attached Figure Description

[0022] Figure 1 This is an electron micrograph of strain HAAS-P013.

[0023] Figure 2 This is a photograph of the colony morphology of strain HAAS-P013.

[0024] Figure 3 Phylogenetic tree of strain HAAS-P013 constructed based on 16S rRNA gene sequence.

[0025] Figure 4The results show the alkali-reducing ability of strain HAAS-P013.

[0026] Figure 5 The results of measuring the growth-promoting ability (root length) of strain HAAS-P013 on rice seeds under different salt stress conditions.

[0027] Figure 6 The results of the determination of the growth-promoting ability (sprout length) of strain HAAS-P013 on rice seeds under different salt stress conditions.

[0028] Figure 7 The results of measuring the growth-promoting ability (root length) of strain HAAS-P013 on rice seeds under different alkaline stress conditions.

[0029] Figure 8 The results of the determination of the growth-promoting ability (sprout length) of strain HAAS-P013 on rice seeds under different alkaline stress conditions.

[0030] Figure 9 The results of measuring the growth-promoting ability (root length) of strain HAAS-P013 on rice under salt-alkali stress are shown.

[0031] Figure 10 The results show the effect of strain HAAS-P013 on promoting rice growth (sprout length) under salt-alkali stress. Detailed Implementation

[0032] Before describing the embodiments of this application in detail, it should be understood that the terminology used herein is for the purpose of describing a particular embodiment only. Unless otherwise defined, 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 term pertains.

[0033] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this application, unless otherwise stated, "multiple" means two or more.

[0034] The endpoints and any values ​​of the ranges disclosed in this application are not limited to the precise ranges or values, and such ranges or values ​​should be understood to include values ​​close to such ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0035] In this application, the terms "comprising" or "including" are open-ended expressions, meaning they include the content specified in this application but do not exclude other aspects.

[0036] This application provides a Halalkalibacter New species strain HAAS-P013 ( Halalkalibacter sp.HAAS-P013), the Halalkalibacter The novel strain HAAS-P013 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 34803. This application also provides methods for the isolation, purification, and cultivation of this strain. Furthermore, it provides cultures, fermentation broths, bacterial suspensions, bacterial powders, and inoculum preparations containing this strain. Halalkalibacter The study focuses on the novel strain HAAS-P013 and its applications in promoting plant growth, including its cultures, fermentation broths, suspensions, powders, and agents.

[0037] To make the objectives, technical solutions, and advantages of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. All other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort are within the scope of protection of this application. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application.

[0038] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0039] The reagents, solvents, and other experimental materials used in the following examples are all commercially available.

[0040] The present application will be further described in detail below with reference to the embodiments, accompanying drawings and test results.

[0041] Example 1 – Screening of Strains

[0042] This example provides the screening process for strain HAAS-P013.

[0043] Fresh soil samples were collected from severely soda-saline-alkali land in Dongfeng Grassland, Daqing City, Heilongjiang Province. Microorganisms were isolated and purified using the dilution-spread plate method. The culture medium was an alkaline, salt-containing R2A solid medium with the following formula (1 L): yeast extract 0.5 g, acid-hydrolyzed casein 0.5 g, glucose 0.5 g, soluble starch 0.5 g, K2HPO4 0.3 g, MgSO4·7H2O 0.1 g, sodium pyruvate 0.3 g, and agar 20 g. The pH of the culture medium was adjusted to 10.0 using separately sterilized anhydrous Na2CO3 solution, and the NaCl concentrations were set to 5%, 10%, and 20% (w / v).

[0044] After spreading the soil suspension onto plates, incubate at 37°C for 3-5 days. Select well-grown single colonies and repeatedly streak them on R2A plates for purification until a pure culture is obtained, yielding strain number HAAS-P013. Store the purified strain in cryovials containing 20% ​​glycerol at -80°C for later use.

[0045] Example 2 – Identification of the strain

[0046] In this example, the strain HAAS-P013 was biologically identified.

[0047] (a) Morphological characteristics

[0048] Figure 1 This is an electron micrograph of strain HAAS-P013.

[0049] Depend on Figure 1 It can be seen that this strain is Gram-positive, rod-shaped, with a cell size of (0.2-1.0) × (1.5-4.7) μm, and can produce spores.

[0050] (II) Cultivation Characteristics

[0051] Strain strain HAAS-P013 was inoculated onto R2A plates at pH 10 with 7% NaCl (w / v) and incubated at 37°C until the logarithmic growth phase. Colony characteristics were then observed. Results are as follows: Figure 2 As shown.

[0052] Depend on Figure 2 It can be seen that after culturing the strain on R2A plates for 2-3 days, the colonies are white, round, flat, and have irregular edges; after culturing for more than 7 days, the colonies are cream-colored.

[0053] (III) Physiological and Biochemical Characteristics

[0054] Referring to the instructions for the API 20NE, API ZYM, and API 50CHB kits, strain HAAS-P013 and its closely related species were analyzed. H. hemicellulosilyticus JCM 9152 TPhysiological and biochemical characteristics were tested.

[0055] The main measurement results are shown in Table 1.

[0056] Table 1. Comparison of strain HAAS-P013 and model strain JCM 9152 T Comparison of physiological and biochemical characteristics

[0057]

[0058] As shown in Table 1, API 20NE analysis revealed that strain HAAS-P013 was positive for urease, β-galactosidase, and arabinose assimilation reactions; while it was negative for glucosidase, nitrate reduction, indole formation, glucose, mannose, mannitol, N-acetylglucosamine, maltose, potassium gluconate, adipic acid, trisodium citrate, and phenylacetic acid assimilation reactions. API ZYM enzyme profile analysis showed that it was positive for alkaline phosphatase, cystine aromatic aminoaminase, and naphthol-AS-BI-phosphohydrolase, while it was negative for esterase (C4), lipolipase (C8), lipase (C14), leucine aromatic aminoaminase, valine aromatic aminoaminase, β-glucuronidase, trypsin, chymotrypsin, acid phosphatase, α-galactosidase, N-acetyl-β-D-glucosidase, α-mannosidase, and α-fucosidase. API 50CHB results showed that strain HAAS-P013 could only utilize esculin (ferric citrate) as a carbon source.

[0059] Under the same experimental conditions, closely related species H. hemicellulosilyticus JCM 9152 T It can utilize aescin and produces glucosidase and β-galactosidase. It is positive for alkaline phosphatase, esterase (C4), lipoesterase (C8), valine aromatic amino acid enzyme, and naphthol-AS-BI-phosphohydrolase, but does not produce urease and cystine aromatic amino acid enzyme. The remaining physiological and biochemical characteristics are consistent with those of the strains mentioned above.

[0060] In summary, the above-mentioned strains are similar to JCM 9152. T Their physiological and biochemical characteristics are generally similar, with differences only in a few traits.

[0061] (iv) Molecular biological characteristics

[0062] (1) 16S rDNA sequencing

[0063] Genomic DNA was extracted from strain HAAS-P013 using the TIANamp Bacteria DNA Kit. Using the genomic DNA as a template, PCR amplification of the 16S rRNA gene was performed using universal bacterial primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3').

[0064] The PCR amplification system consisted of 40 μl of PCR amplification material, including 1 μl each of upstream and downstream primers, 2 μl of template DNA, 21 μl of Taq PCR Mix, and 15 μl of ddH2O. The PCR reaction program was as follows: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 1 min, for a total of 30 cycles; and a final extension at 72℃ for 5 min. The PCR amplification products were detected by electrophoresis, purified, and then sequenced.

[0065] The 16S rRNA gene sequence of strain HAAS-P013 is 1478 bp in length, as shown in SEQ ID NO.1. The obtained sequence was aligned to the EzBioCloud database (https: / / www.ezbiocloud.net / ), and a phylogenetic tree was constructed using MEGA X software with a neighbor-joining method (e.g., ...). Figure 3 (As shown).

[0066] The results showed that strain HAAS-P013 and Halalkalibacter Other species in the genus are clustered together with other species. H. hemicellulosilyticus JCM 9152 T The 16S rRNA gene sequence showed the highest homology, at 97.35% (less than the 98.7% species threshold), preliminarily indicating that this strain is... Halalkalibacter A potential new species of the genus.

[0067] (2) Whole genome sequencing and analysis

[0068] Whole-genome sequencing of the bacteria was performed using the Illumina high-throughput sequencing platform. The genome size of strain HAAS-P013 was 4,681,515 bp, with a DNA G+C content of 37.8%. Genome-to-genome distance calculator (GGDC) and average nucleotide identity (ANI) analysis were used to compare the genome of this strain with that of its closest relatives. H. hemicellulosilyticus JCM 9152 T The genomes were compared. The detection results are shown in Table 2.

[0069] Table 2 HAAS-P013 andHalalkalibacter hemicellulosilyticus JCM 9152 T Genome alignment results

[0070]

[0071] The results showed that strain HAAS-P013 was related to species Halalkalibacter hemicellulosilyticus JCM9152 T The DNA-DNA hybridization (DDH) predicted value was 14.0%, and the ANI value was 72.31%, both values ​​being far below the thresholds for prokaryotic species classification (DDH < 70%, ANI < 95-96%). This further confirms at the genomic level that strain HAAS-P013 is a prokaryotic species. Halalkalibacter A new species of the genus.

[0072] Will Halalkalibacter The novel strain HAAS-P013 was deposited on June 9, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 34803. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0073] Example 3

[0074] This embodiment provides a Halalkalibacter Fermentation broth of a new strain HAAS-P013.

[0075] The specific method for preparing the above fermentation broth is as follows:

[0076] The strain HAAS-P013 was activated and transferred to R2A liquid medium with pH 10 and 7% NaCl (w / v). The culture was carried out in a shaker at 37℃ and 180 r / min for 48 h to obtain the fermentation broth of strain HAAS-P013.

[0077] Example 4

[0078] This embodiment provides a Halalkalibacter Fermentation supernatant of the new species strain HAAS-P013.

[0079] The specific method for preparing the above-mentioned fermentation supernatant is as follows:

[0080] The strain HAAS-P013 was activated and transferred to R2A liquid medium with pH 10 and 7% NaCl (w / v). The medium was cultured in a shaker at 37℃ and 180 r / min for 48 h to obtain the fermentation broth of strain HAAS-P013. The fermentation broth was centrifuged and the supernatant was collected as the fermentation supernatant of strain HAAS-P013.

[0081] Example 5

[0082] This embodiment provides a Halalkalibacter A suspension (bacterial suspension) of the new species strain HAAS-P013.

[0083] The specific preparation method of the above-mentioned HAAS-P013 suspension is as follows:

[0084] The activated strain HAAS-P013 was inoculated into R2A liquid medium at pH 10 and 7% NaCl (w / v), and cultured at 37℃ and 180 rpm for 24 h with shaking to obtain the seed culture. The OD was then adjusted. 600 The inoculum concentration was 0.5. Seed culture was inoculated at a rate of 5% (v / v) into 100 mL (250 mL Erlenmeyer flask) of LB liquid medium and cultured at 28℃ and 180 rpm for 48 h with constant temperature shaking to obtain bacterial culture. The bacterial culture was centrifuged at 4℃ (8000 rpm, 10 min) to remove the supernatant. After washing twice with sterile water and centrifuging again, the suspension was resuspended in sterile water. The optical density of the bacterial suspension was adjusted to 0.5 ± 0.02 at 600 nm to obtain HAAS-P013 suspension, which was stored at 4℃ for later use.

[0085] Example 6

[0086] This example demonstrates the determination of the salt and alkali tolerance of strain HAAS-P013.

[0087] (a) Alkali resistance test

[0088] The activated strain HAAS-P013 was inoculated into R2A liquid medium at pH 10 and 7% NaCl (w / v) and cultured with shaking at 37℃ and 180 rpm for 24 h to obtain the seed culture. The seed culture was then inoculated at 1% (v / v) into R2A liquid medium at pH values ​​of 5.0–12.0 (intervals of approximately 1.0), with three replicates for each pH. After culturing at 37℃ and 180 rpm for 72 h, the OD of the culture medium was measured. 600 value.

[0089] The test results are shown in Table 3.

[0090] Table 3. Results of alkali resistance test for strain HAAS-P013

[0091]

[0092] As shown in Table 3, strain HAAS-P013 can grow in the pH range of 6.0-11.0, with the optimal growth pH being 10.0.

[0093] (II) Salt tolerance test

[0094] The above seed culture was inoculated at a 1% (v / v) inoculation rate into R2A liquid medium (pH adjusted to 10.0) containing 0%, 2%, 5%, 7%, 10%, 15%, 20%, and 25% (w / v) NaCl, respectively. Three replicates were set for each salt concentration. After incubation at 37°C and 180 rpm for 72 h with shaking, the OD of the culture medium was measured. 600 value.

[0095] The test results are shown in Table 4.

[0096] Table 4. Results of salt tolerance test for strain HAAS-P013

[0097]

[0098] As shown in Table 4, strain HAAS-P013 can grow in the range of NaCl concentration from 0 to 15% (w / v), with the optimal growth salt concentration being 7% (w / v).

[0099] Example 7

[0100] This example demonstrates the prediction of functional gene clusters in strain HAAS-P013.

[0101] Using antiSMASH ( https: / / antismash.secondarymetabolites.org Online tools were used to predict and analyze the secondary metabolite biosynthesis gene clusters of the whole genome sequence of strain HAAS-P013. Specific information is shown in Table 5.

[0102] Table 5. Prediction of gene clusters for the biosynthesis of secondary metabolites in the genome (HAAS-P013)

[0103]

[0104] Table 5 shows that the genome of strain HAAS-P013 contains multiple gene clusters related to the synthesis of bioactive substances. Among them, tetrahydropyrimidine (ectoine) is closely related to the strain's salt tolerance and osmotic pressure tolerance. In addition, type III polyketide synthase (Type III PKS) can catalyze the synthesis of polyketide backbones and dominates the synthesis of flavonoids, stilbenes, and aromatic polyketides in plants and microorganisms. It is a key enzyme in plant defense, pigment formation, and the synthesis of medicinal components.

[0105] Example 8

[0106] This example demonstrates the ability of strain HAAS-P013 to reduce alkali.

[0107] The activated strain HAAS-P013 was inoculated into R2A liquid medium at pH 10 and 7% (w / v) NaCl, and cultured with shaking at 37°C and 180 rpm for 24 h to obtain the seed culture. The seed culture was then inoculated into R2A liquid medium at pH 10 and 7% (w / v) NaCl at a 1% (v / v) inoculation rate. Each treatment was performed in triplicate, with the uninoculated medium serving as a control. The culture was then cultured with shaking at 37°C and 180 rpm, and the pH of the culture medium was measured every 12 h.

[0108] The measurement results are as follows Figure 4 As shown.

[0109] Depend on Figure 4 It was found that after 48 hours of cultivation, strain HAAS-P013 could lower the pH of the culture medium to 7.84. This result indicates that strain HAAS-P013 has a significant alkalinity-reducing ability and can effectively lower the pH value of highly alkaline environments.

[0110] Example 9

[0111] This example investigates the effects of strain HAAS-P013 on rice seed growth under salt-alkali stress.

[0112] (a) Determination of the growth-promoting ability of the strain on agar plates

[0113] (1) Preparation of HAAS-P013 suspension: namely, the HAAS-P013 suspension provided in Example 5.

[0114] (2) Rice seed treatment: Select uniform and plump rice seeds, disinfect them with 75% ethanol at 180 r / min for 30 min, and then wash them with sterile water until no ethanol residue remains on the seed surface. Soak the treated seeds in HAAS-P013 suspension at 28℃ for 3 h.

[0115] (3) Preparation of rice germination bed: Sterile petri dishes were selected for the experiment. Agar was added to solutions with different NaCl concentrations (0, 50, 100, 150 mmol / L) and different pH values ​​(8.0, 9.0, 10.0) to simulate different salt and alkali stress environments. Rice seeds treated with HAAS-P013 suspension were placed under salt and alkali stress environments as the experimental group (HAAS-P013). At the same time, a group of rice seeds soaked in sterile water was set up as the control group (CK). Each group contained 16 rice seeds, with 3 replicates. After the rice seeds were cultured in the dark at 28℃ for 7 days, the root length and shoot length of the rice seeds were measured.

[0116] The results of the determination of the growth-promoting ability of strain HAAS-P013 on rice seeds under different salt stress conditions are as follows:Figure 5 (Root length) and Figure 6 (Sprout length) is shown. The results of the determination of the growth-promoting ability of strain HAAS-P013 on rice seeds under different alkaline stresses are shown below. Figure 7 (Root length) and Figure 8 As shown in (bud length).

[0117] Depend on Figure 5 and Figure 6 It can be seen that with the increase of salt concentration, the root length and shoot length of rice seeds in both the control group and the treatment group gradually decreased. However, compared with the control group, the root length and shoot length of rice seeds treated with strain HAAS-P013 were significantly increased under salt stress of 50, 100 and 150 mmol / L NaCl.

[0118] Depend on Figure 7 and Figure 8 It can be seen that as the pH value increases, the root length and shoot length of rice seeds in both the control group and the treatment group gradually decrease. However, compared with the control group, under different alkaline stress conditions of pH 8.0-10.0, the root length and shoot length of rice seeds treated with strain HAAS-P013 are significantly increased.

[0119] (II) Determination of the hydroponic growth-promoting ability of the strain

[0120] (1) Preparation of HAAS-P013 suspension: namely, the HAAS-P013 suspension provided in Example 5.

[0121] (2) Hydroponic environment: In order to simulate the environment of soda saline-alkali soil, distilled water was adjusted to pH 8.5 with 42.8 g / L Na2CO3 as the hydroponic environment for rice.

[0122] (3) Rice seed treatment: Select rice seeds of uniform size and plumpness, disinfect them with 75% ethanol at 180 r / min for 30 min, and then wash them with sterile water until there is no ethanol residue on the seed surface. Soak the treated seeds in sterile water for 24 h, and then germinate them at a temperature of about 30℃ for 48 h. Sow the seeds when they "show white".

[0123] (4) Hydroponic treatment: After germination, rice seeds with uniform growth were selected and planted in hydroponic boxes, which were then placed in an artificial climate chamber at 28℃ for 14 hours of light and 10 hours of darkness. When the rice reached the two-leaf-one-heart stage, 10 mL of HAAS-P013 suspension was inoculated as the experimental group (HAAS-P013). At the same time, a group inoculated with 10 mL of sterile water was set up as the control group (CK), with 3 replicates in each group. The length of rice seedlings and roots were measured 4 weeks after planting.

[0124] The results of the determination of the growth-promoting ability of strain HAAS-P013 on rice under salt-alkali stress are as follows: Figure 9(Root length) and Figure 10 As shown in (bud length).

[0125] Depend on Figure 9 and Figure 10 It can be seen that, compared with the control group, the root length and seedling length of rice seeds treated with strain HAAS-P013 under saline-alkali stress conditions were significantly increased.

[0126] Example 10

[0127] This embodiment provides a Halalkalibacter Solid bacterial powder of the new species strain HAAS-P013.

[0128] The specific method for preparing the above-mentioned solid bacterial powder is as follows:

[0129] The strain HAAS-P013 was activated and transferred to R2A liquid medium with pH 10 and NaCl 7% (w / v). The culture was carried out in a shaker at 37℃ and 180 r / min for 72 h to obtain the fermentation broth of strain HAAS-P013.

[0130] Centrifuge the fermentation broth at 4℃ and 4000rpm for 10-15min, discard the supernatant, and collect the bacterial precipitate; wash the bacterial cells twice with sterile physiological saline or phosphate buffer (PBS, pH 7.0-7.4).

[0131] Subsequently, the washed bacterial cells were suspended in a solution containing a preservative (formulation: 10% skim milk powder, 8% sucrose, 3% trehalose) to achieve a final concentration of 10. 9 -10 11 CFU / mL, to obtain bacterial suspension.

[0132] The bacterial suspension was dispensed into freeze-drying trays and pre-frozen at -80℃ for 8-12 hours. Then, it was placed in a freeze dryer and freeze-dried at -50℃ or below, under a vacuum of 10 Pa or less, for 24-48 hours to obtain loose, blocky, or powdery solid bacterial powder. The obtained solid bacterial powder was further pulverized, sealed, and dried at 4℃ for storage.

[0133] Example 11

[0134] This example investigates the effect of strain HAAS-P013 on improving moderately saline-alkali soil in dryland areas.

[0135] (a) Test materials

[0136] The soybean variety used in this example is "Heinong 531", and the sowing rate is 4 kg / mu. The tested inoculum powder is HAAS-P013 solid inoculum powder (the solid inoculum powder prepared in Example 10), with an effective viable count ≥1.0×10⁻⁶. 9The concentration of CFU / g was 5 kg / mu. The tested soil was soda saline-alkali soil with the following basic physicochemical properties: pH 8.83, electrical conductivity (EC) 335.16 uS / cm, and alkalinity (ESP) 19.88%.

[0137] (II) Experimental Design

[0138] This experiment employed a field plot design. Each plot was 75 m², and the experiment included two treatments, with three replicate plots per treatment, arranged in a randomized block design. The replicate plots were as follows:

[0139] Treatment group (microbial powder treatment): The microbial powder is applied by side application, evenly applied 3-5cm below and to the side of the soybean seeds.

[0140] Control group (blank control): No bacterial agent was applied.

[0141] (III) Field Management

[0142] Before sowing, routine land preparation is carried out. After sowing, field management (including irrigation, weeding, and pest and disease control) is kept consistent in each plot, and no other microbial fertilizers are applied.

[0143] Soil samples were collected from the root zone in each plot during the soybean seedling stage (25-30 days after emergence) using the "S"-shaped five-point sampling method. After mixing, the samples were air-dried and sieved. The following soil salinity and alkalinity indicators were determined: pH value: determined by potentiometric method (soil-water ratio 1:2.5); Alkalinity (ESP): the exchangeable sodium and cation exchange capacity of the soil were determined and their ratio was calculated.

[0144] The salinity and alkalinity of the rhizosphere soil were measured during the soybean seedling stage. The results are shown in Table 6.

[0145] Table 6. Changes in soil salinity and alkali index in the root zone of soybean seedlings under different treatments.

[0146]

[0147] As shown in Table 6, compared with the control group, the soil pH value of the treatment group treated with HAAS-P013 solid bacterial powder decreased from 8.83 to 8.09, a decrease of 8.38% (p < 0.05); the soil alkalinity (ESP) decreased from 19.88% to 15.41%, a decrease of 22.5% (p > 0.05); and the electrical conductivity (EC) decreased from 335.16 μS / cm to 297.62 μS / cm, a decrease of 11.20% (p > 0.05).

[0148] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0149] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of this application.

Claims

1. A plant Halalkalibacter The new species strain HAAS-P013 is characterized by, The Halalkalibacter The novel strain HAAS-P013 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 34803.

2. A culture, characterized in that, The culture comprises that described in claim 1. Halalkalibacter It belongs to a new species strain HAAS-P013.

3. A fermentation broth, characterized in that, The fermentation broth comprises the product of claim 1. Halalkalibacter It belongs to a new species strain HAAS-P013.

4. A bacterial suspension, characterized in that, The bacterial suspension comprises the one described in claim 1. Halalkalibacter It belongs to a new species strain HAAS-P013.

5. A microbial agent, characterized in that, The microbial agent includes the one described in claim 1. Halalkalibacter It belongs to a new species strain HAAS-P013.

6. The microbial agent according to claim 5, characterized in that, The bacterial agent can be any one of liquid preparation, powder, or granule.

7. A bacterial powder, characterized in that, The bacterial powder includes the components described in claim 1. Halalkalibacter It belongs to a new species strain HAAS-P013.

8. A device as claimed in claim 1 Halalkalibacter The application of the new species strain HAAS-P013, the culture according to claim 2, the fermentation broth according to claim 3, the bacterial suspension according to claim 4, the bacterial agent according to claim 5, and the bacterial powder according to claim 7 in promoting plant growth.

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