Halotolerant Bacillus, microbial agent and their applications

Abstract

The present invention relates to the field of microbial technology, and discloses a salt-tolerant Bacillus sp., a microbial agent and their applications. The salt-tolerant Bacillus sp. provided by the present invention has excellent ability to colonize in the rhizosphere of plants and soil, can secrete auxin, produce siderophores, and also has good abilities of phosphorus solubilization, potassium solubilization, and degradation of autotoxic substances that may be generated during crop planting such as benzoic acid, and can improve the soil and promote crop growth. In addition, through tests, it is also found that this strain also has good antagonistic effects on the pathogens of various plant soil-borne diseases, and can be used for the prevention and control of plant diseases and achieve good prevention and control effects.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and particularly relates to a halotolerant Bacillus, a microbial agent and their applications. Background Art

[0002] With the development of modern agriculture towards large-scale, intensive and monoculture, continuous cropping patterns are very common, resulting in frequent occurrence of continuous cropping obstacles such as inhibited crop growth, high incidence of soil-borne diseases and sharp reduction in yield. The secretion of autotoxic substances by plant roots is one of the main factors leading to this phenomenon. With the development of society and economy, there is an urgent need for a more green, environmentally friendly and sustainable way of agricultural production.

[0003] Beneficial rhizosphere microorganisms can provide more available nutrient elements for plants and effectively inhibit or even kill plant pathogens by dissolving phosphorus, secreting phytohormones, and producing antibacterial active substances during their growth and metabolism. Beneficial rhizosphere microorganisms can also utilize autotoxic substances in the soil to reduce or eliminate the adverse effects caused by the accumulation of autotoxic substances on the soil or plants. Therefore, it has great application value and potential in the biological control of plant diseases. In addition, the application of beneficial rhizosphere microorganisms can reduce the use of pesticides and chemical fertilizers, which can reduce production costs on the one hand and meet the requirements of modern agriculture for sustainable development on the other hand, and is of great significance for the development and transformation of modern agricultural production.

[0004] However, at present, the beneficial rhizosphere strains that can be used in actual production are still very limited, and the effects of existing strains need to be improved. Moreover, the development of most current beneficial plant rhizosphere strains relies on artificial mutagenesis and screening, and the obtained strains are prone to problems such as difficult colonization or far from reaching the laboratory detection level in actual production. To meet the needs of agricultural production, there is an urgent need to develop new beneficial rhizosphere strains with better and more stable growth-promoting effects and easier colonization, so as to be applicable to actual production. Summary of the Invention

[0005] The purpose of the present invention is to overcome the above problems existing in the prior art, and provide a halotolerant Bacillus, a microbial agent and their applications. The halotolerant Bacillus provided by the present invention is a forest rhizosphere bacterium collected from a primeval forest. After application, it has good colonization ability. Moreover, through detection, this strain can efficiently degrade benzoic acid, and has good abilities of secreting auxin, producing siderophores, dissolving phosphorus and potassium, has broad-spectrum antibacterial activity against a variety of pathogenic bacteria and has a significant promoting effect on plant growth.

[0006] To achieve the above purpose, in the first aspect of the present invention, a halotolerant Bacillus (Bacillus halotolerans) is provided, and the preservation number of this strain is CCTCC NO: M 20232276.

[0007] In the second aspect of the present invention, a bacterial agent is provided, which comprises the halotolerant Bacillus sp. described in the first aspect.

[0008] In the third aspect of the present invention, there is provided the use of the halotolerant Bacillus sp. described in the first aspect, or the bacterial agent described in the second aspect, in degrading benzoic acid, dissolving phosphorus, dissolving potassium, producing auxin, producing siderophores, antagonizing pathogenic bacteria or alleviating the inhibitory effect of benzoic acid on seed germination.

[0009] In the fourth aspect of the present invention, a method for preparing auxin and / or siderophores is provided, which comprises culturing the halotolerant Bacillus sp. described in the first aspect and collecting the culture product.

[0010] In the fifth aspect of the present invention, there is provided the use of the halotolerant Bacillus sp. described in the first aspect, or the bacterial agent described in the second aspect, in improving soil, and / or promoting plant growth, and / or promoting plant seed germination, and / or preventing and controlling plant soil-borne diseases.

[0011] In the sixth aspect of the present invention, a method for preventing and controlling plant soil-borne diseases and / or promoting plant growth is provided, which comprises applying the halotolerant Bacillus sp. described in the first aspect and / or its metabolites, or the bacterial agent described in the second aspect, to the rhizosphere soil of plants.

[0012] Through the above technical solutions, the present invention can at least achieve the following beneficial effects:

[0013] (1) The halotolerant Bacillus sp. provided by the present invention has good effects in degrading benzoic acid, dissolving phosphorus and dissolving potassium, and can also produce auxin and siderophores. In addition, this bacterium also has a broad-spectrum inhibitory effect on a variety of pathogenic bacteria, thus having the effect of improving soil quality and increasing the content of nutrients that plants can utilize in the soil, so as to play a role in promoting plant growth and preventing and controlling plant diseases.

[0014] (2) The halotolerant Bacillus sp. provided by the present invention is a natural forest rhizosphere bacterium isolated from the unique habitat of the primary forest. Compared with the strains screened by laboratory mutagenesis or artificially constructed by genetic engineering, it has better adaptability to the planting environment, can colonize in the soil better, and thus can more stably play the role of promoting growth and preventing diseases. Description of the Drawings

[0015] Figure 1 It is a colony morphology diagram of the strain YNK-FB0022 in Example 1.

[0016] Figure 2 It is a phylogenetic tree of the strain YNK-FB0022 in Example 1.

[0017] Figure 3It is the broad-spectrum antibacterial effect diagram of strain YNK-FB0022 in Example 2.

[0018] Figure 4 It is the effect diagram of strain YNK-FB0022 in Example 6 to relieve the inhibition of seed germination by benzoic acid.

[0019] Biological deposit

[0020] The halotolerant Bacillus provided by the present invention, classified and named as Bacillus halotolerans YNK-FB0022, was deposited at the China Center for Type Culture Collection on November 20, 2023. The address is Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the deposit number is CCTCC NO: M 20232276.

[0021] The Streptomyces albidoflavus provided by the present invention, classified and named as Streptomyces alboflavus YNK-FS0019, was deposited at the China Center for Type Culture Collection on September 7, 2023. The address is Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the deposit number is CCTCC NO: M 20231644.

[0022] The Streptomyces blastmyceticus provided by the present invention, classified and named as Streptomyces blastmyceticus YNK-FS0018, was deposited at the China Center for Type Culture Collection on September 7, 2023. The address is Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the deposit number is CCTCC NO: M 20231643. Detailed implementation manners

[0023] In the ranges disclosed herein, the endpoints and any values are not limited to the exact ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, between the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values, they can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0024] In the present invention, without special instructions, "halotolerant Bacillus / strain CCTCC NO:M20232276", "halotolerant Bacillus / strain YNK-FB0022", "halotolerant Bacillus provided by the present invention" and the like all refer to the same strain, that is, the halotolerant Bacillus strain with the deposit number of CCTCC NO:M 20232276. The above expressions have the same meaning and can be used interchangeably.

[0025] In the present invention, "phosphorus solubilization" means that the content of P available for plants in the soil is increased through the action of the strains provided by the present invention. For example, it promotes the conversion of organic phosphorus components (such as lecithin, inositol hexaphosphate, etc.) in the soil into forms that can be utilized by plants, thereby promoting the absorption and utilization of phosphorus elements in the soil by plants. For example, after continuous planting of crops such as tomatoes, tobacco, and cucumbers, phenolic acid autotoxic substances such as benzoic acid will be secreted. The accumulation of these phenolic acid autotoxic substances in the soil is likely to cause problems such as growth inhibition of crops, which is not conducive to the continuous planting of crops. "Benzoic acid degradation" means that the benzoic acid autotoxic substances secreted by plant roots can be degraded through the action of the strains provided by the present invention, thereby reducing or eliminating the adverse effects on the soil or plants caused by the accumulation of autotoxic substances; microorganisms secrete siderophores to chelate Fe in the environment 3+ is considered to be one of the main ways of the effective iron source for plants, and it has the effect of promoting plant growth. Secondly, by competing with plant pathogens for iron ions by strains capable of secreting siderophores, it can also inhibit the growth of pathogens and play the effect of preventing and controlling crop diseases. "Siderophore secretion" means that the strains provided by the present invention can secrete siderophores under iron-deficient conditions to help microorganisms absorb Fe from the environment 3+ , increasing Fe in the environment 3+ mobility, and promoting the absorption of Fe by crops 3+ .

[0026] During the research process, the inventors of the present invention accidentally isolated a halotolerant Bacillus from the rhizosphere soil of forest trees in Wuliangshan National Nature Reserve. After detection, it was found that this strain has functions such as highly efficient degradation of benzoic acid, phosphorus solubilization, potassium solubilization, auxin production, siderophore secretion, and antagonism against various pathogens. This strain can not only degrade phenolic acid autotoxic substances secreted by plant roots, but also convert organic phosphorus and insoluble potassium elements into available phosphorus and available potassium that can be absorbed by plants. Through further research, the inventors also found that applying this strain to the rhizosphere soil of plants can effectively promote plant growth, prevent and control the occurrence of tomato wilt disease, improve the soil environment, increase agricultural productivity, and promote the green and sustainable development of ecological agriculture

[0027] Based on the above findings, on the one hand, the present invention provides a halotolerant Bacillus (Bacillus halotolerans), and the preservation number of this strain is CCTCC NO: M 20232276

[0028] On the second aspect, the present invention provides a microbial agent, and the microbial agent contains the halotolerant Bacillus described in the first aspect (as an active ingredient).

[0029] In the microbial agent provided by the present invention, only the halotolerant Bacillus CCTCC NO: M 20232276 can be used as the sole active ingredient, or other strains can be added to form a common active ingredient

[0030] The microbial inoculant provided by the present invention can be used for degrading benzoic acid, alleviating the inhibition of benzoic acid on the germination of crop seeds (preferably at least one of tomato, pepper and cucumber), decomposing phosphorus (especially organic phosphorus), decomposing potassium, preventing plant diseases caused by microbial infection, preparing auxin, preparing siderophores, etc.

[0031] According to some preferred embodiments of the present invention, the inoculant may further contain optional excipients. "Optional" means that in this inoculant, the excipients can be selected whether to be added according to the actual situation and needs, and are not essential components of the inoculant. The excipients can be any excipients that can be used in the preparation of microbial inoculants in the art, and can play roles such as protecting halotolerant Bacillus spores, improving the activity and survival rate of viable bacteria in the inoculant during long-term storage or transportation, etc. For example, it may include various culture media that can be used for the culture, storage and transportation of halotolerant Bacillus spores; microbial protectants (such as lyophilization protectants, etc.); buffering agents, etc.

[0032] According to some preferred embodiments of the present invention, the inoculant can be a liquid inoculant.

[0033] In the present invention, there is no particular limitation on the content of halotolerant Bacillus spores CCTCC NO: M 20232276 in the inoculant, as long as the above-mentioned purposes can be achieved.

[0034] According to some preferred embodiments of the present invention, the content of the halotolerant Bacillus spores in the inoculant is not less than 1×10 7 CFU / mL, preferably 1×10 7 -1×10 10 CFU / mL. For example, it can be 1×10 7 CFU / mL, 5×10 7 CFU / mL, 1×10 8 CFU / mL, 5×10 8 CFU / mL, 1×10 9 CFU / mL, 5×10 9 CFU / mL, 1×10 10 CFU / mL, or it can also be a range composed of any two of the above values, or any intermediate value within this range.

[0035] The third aspect of the present invention provides the application of the halotolerant Bacillus spores described in the first aspect, or the inoculant described in the second aspect, in degrading benzoic acid, decomposing phosphorus, decomposing potassium, producing auxin, producing siderophores, antagonizing pathogenic bacteria or alleviating the inhibitory effect of benzoic acid on seed germination.

[0036] The present invention further provides a method for degrading benzoic acid, which includes contacting benzoic acid with the halotolerant Bacillus sp. CCTCC NO: M 20232276.

[0037] According to some preferred embodiments of the present invention, the method may include evenly spreading the halotolerant Bacillus sp. CCTCC NO: M 20232276 (such as its bacterial agent or culture solution) into the soil to alleviate the accumulation of benzoic acid caused by continuous cropping of crops or soil pollution, thereby solving problems such as adverse effects on crop planting.

[0038] The present invention further provides a method for dissolving phosphorus and / or potassium, which includes contacting the above-mentioned halotolerant Bacillus sp. with a phosphorus-containing compound and / or a potassium-containing compound.

[0039] Preferably, the phosphorus-containing compound includes lecithin.

[0040] Preferably, the potassium-containing compound includes potassium feldspar.

[0041] The present invention further provides a method for alleviating the inhibition of benzoic acid on the germination of crop seeds, which includes contacting the seeds with the halotolerant Bacillus sp. CCTCC NO: M 20232276 during the germination process.

[0042] According to a preferred embodiment of the present invention, the method may include:

[0043] i. Soaking the seeds in water for 2 - 4 h to obtain soaked seeds;

[0044] ii. Contacting the halotolerant Bacillus sp. CCTCC NO: M 20232276 with the soaked seeds obtained in step i to obtain soaked seeds with alleviated germination inhibition.

[0045] Preferably, in step ii, the method of contact includes immersing the soaked seeds in the culture solution of the halotolerant Bacillus sp. CCTCC NO: M 20232276. Preferably, the concentration of the halotolerant Bacillus sp. CCTCC NO: M 20232276 in the culture solution is 10 7 -10 8 CFU / mL (that is, the content of the halotolerant Bacillus sp. in the culture solution reaches 10 7 -10 8 CFU / mL level, that is, the content of the halotolerant Bacillus sp. in the culture solution can be in the range of greater than or equal to 1×10 7 CFU / mL to less than 1×10 9 CFU / mL).

[0046] Preferably, the method may further include:

[0047] iii. Place the soaked seeds with alleviated germination inhibition obtained in step ii under seed germination conditions to allow them to germinate.

[0048] More preferably, step iii can be carried out by soaking the soaked seeds with alleviated germination inhibition in water to allow them to germinate. Step iii can also be carried out by burying the soaked seeds with alleviated germination inhibition in soil to allow them to germinate.

[0049] In the above method provided by the present invention, "soaking" refers to the process of contacting the seeds with water or the culture solution of Bacillus halotolerans CCTCC NO: M20232276, so that the seeds are in a wet state to promote seed germination. To prevent the seeds from being unable to germinate due to lack of oxygen, usually the soaking liquid can be used in a way that does not completely submerge the seeds, or the seeds can be placed on a paper material such as absorbent paper, and then the paper material is wetted with the soaking liquid for "soaking" treatment.

[0050] According to a preferred embodiment of the present invention, the crop is at least one of tomato, pepper and cucumber.

[0051] The fourth aspect of the present invention provides a method for preparing auxin and / or siderophore, the method comprising culturing the above-mentioned Bacillus halotolerans and collecting the culture product.

[0052] Any method and conditions capable of fermenting and culturing Bacillus halotolerans to produce auxin and / or siderophore can be applied to the present invention.

[0053] According to a preferred embodiment of the present invention, the conditions of the (fermentation) culture include: a culture temperature of 28 - 32 °C and a culture time of 24 - 72 h.

[0054] According to a preferred embodiment of the present invention, when the fermentation culture is carried out by shake flask fermentation, the culture can be carried out by oscillating at a rotation speed of 100 - 200 rpm during the fermentation process.

[0055] The fifth aspect of the present invention provides the application of the Bacillus halotolerans described in the first aspect, or the microbial agent described in the second aspect, in improving soil, and / or promoting plant growth, and / or promoting plant seed germination, and / or preventing the spread of plant soil-borne diseases.

[0056] In the present invention, "improving soil" means increasing the content of nutrients in the soil that can be absorbed and utilized by plants, thereby improving the soil quality and making the soil more suitable for plant growth. The soil quality can be determined by measuring the physical and chemical properties of the soil, such as the pH value of the soil, the nutrient content, etc. It can also be determined by measuring the enzyme content such as soil invertase, urease, acid phosphatase, catalase, etc. in the soil to determine the transformation of available nutrients (i.e., nutrients that can be absorbed and utilized by plants, such as available phosphorus, available nitrogen, available potassium, etc.) in the soil, so as to determine the change of soil quality. Generally, the higher the content of nutrients, especially available nutrients, in the soil, the higher the content of the above enzymes in the soil, indicating better soil quality.

[0057] In the present invention, "promoting plant growth" means promoting the absorption of nutrient elements in the soil by plants, increasing the growth rate of plants, such as increasing the plant height, stem length, root length, plant weight, fruit weight and quality of plants, or shortening the growth cycle of plants, promoting the growth and development of plant roots, etc.

[0058] In the present invention, "preventing and controlling plant soil-borne diseases" can include aspects such as "preventing and controlling the occurrence of plant soil-borne diseases" and "preventing and controlling the spread of plant soil-borne diseases", specifically including having a prevention and control effect on plant soil-borne diseases (such as diseases caused by microbial infections such as tomato wilt), significantly reducing the incidence of plant soil-borne diseases in fields where plant soil-borne diseases have occurred, preventing the occurrence of soil-borne diseases in fields where no soil-borne diseases have occurred, and ensuring the healthy growth of plants. Generally, the prevention and control effect of plant soil-borne diseases can be detected by means of crop cultivation experiments.

[0059] According to some preferred embodiments of the present invention, wherein the plant soil-borne diseases include diseases caused by at least one of Fusarium oxysporum, Exserohilum turcicum, Colletotrichum micotianae, Phoma matteuciicola, Phytophthora parasitica, and Fusarium oxysporum fsp.Cubenserace4 (Foc4).

[0060] Preferably, the plant soil-borne disease includes tomato wilt.

[0061] The present invention further provides a method for inhibiting plant pathogens, which includes contacting the salt-tolerant Bacillus sp. described in the first aspect or the microbial agent described in the second aspect with plant pathogens.

[0062] Preferably, the plant pathogenic bacteria are selected from at least one of Fusarium oxysporum f. sp. lycopersici, Exserohilum turcicum, Colletotrichum tabacum, Phyllosticta zingiberi, Phytophthora parasitica var. nicotianae, and Fusarium oxysporum f. sp. cubense.

[0063] The sixth aspect of the present invention provides a method for controlling plant soil-borne diseases and / or promoting plant growth, which includes applying the halotolerant Bacillus sp. and / or its metabolites described in the first aspect, or the microbial agent described in the second aspect, to the rhizosphere soil of plants.

[0064] According to a preferred embodiment of the present invention, the application amount of the halotolerant Bacillus sp. is not less than 1×10 8 CFU / strain / time, preferably 1×10 9 -1×10 12 CFU / strain / time.

[0065] According to a preferred embodiment of the present invention, the dosage of the microbial agent is such that the application amount of the halotolerant Bacillus sp. is not less than 1×10 8 CFU / strain / time, preferably 1×10 9 -1×10 12 CFU / strain / time. Preferably, the application frequency of the halotolerant Bacillus sp. or the microbial agent is 1-3 times per crop.

[0066] According to a preferred embodiment of the present invention, the application amount of the metabolite is not less than 100 mL / strain / time, preferably 100-200 mL / strain / time.

[0067] Preferably, the application frequency of the metabolite is 1-3 times per crop.

[0068] More preferably, the metabolite includes auxin and / or siderophore.

[0069] According to a preferred embodiment of the present invention, the metabolite is provided by the fermentation broth of the halotolerant Bacillus sp. The fermentation broth can be obtained by the fermentation culture method as described above, and the specific method and conditions are not elaborated here.

[0070] Preferably, in the fermentation broth, the content of auxin can be 20-40% (w / v). Preferably 30-37% (w / v).

[0071] More preferably, in the fermentation broth, the content of auxin (indoleacetic acid) can be 10 - 40 μg / mL, preferably 25 - 35 μg / mL. For example, it can be 25 μg / mL, 26 μg / mL, 27 μg / mL, 28 μg / mL, 29 μg / mL, 30 μg / mL, 31 μg / mL, 32 μg / mL, 33 μg / mL, 34 μg / mL, 35 μg / mL, or it can also be the range formed by any two of the above values, or any intermediate value within this range.

[0072] Preferably, in the fermentation broth, the content of siderophore can be 15 - 40%.

[0073] In the present invention, the fermentation broth can be directly applied (after dilution / concentration to make the content of auxin / siderophore reach the above levels), or the auxin / siderophore in the fermentation broth can be purified and then made into a preparation for separate application. The purification method can be any method that can be used in the art to separate and purify auxin / siderophore.

[0074] Preferably, the seeds are selected from Solanaceae plants, preferably Solanum plants, most preferably tomatoes; and / or, selected from Solanaceae plants, preferably Capsicum plants, most preferably peppers; and / or, selected from Cucurbitaceae plants, preferably Cucumis plants, most preferably cucumbers.

[0075] According to some preferred embodiments of the present invention, wherein the method further includes applying Streptomyces albidoflavus CCTCC NO: M 20231644 and / or Streptomyces blastmycinus CCTCC NO: M 20231643 to the rhizosphere soil of plants.

[0076] Preferably, the application amount of Streptomyces albidoflavus CCTCC NO: M 20231644 is not less than 1×10 8 CFU / plant / time, preferably 1×10 9 - 1×10 12 CFU / plant / time.

[0077] More preferably, the application frequency of Streptomyces albidoflavus CCTCC NO: M 20231644 is 1 - 3 times per crop.

[0078] Preferably, the application amount of Streptomyces blastmycinus CCTCC NO: M 20231643 is not less than 1×10 8 CFU / plant / time, preferably 1×10 9 - 1×10 12 CFU / plant / time.

[0079] More preferably, the application frequency of Streptomyces blastmycinus CCTCC NO: M 20231643 is 1-3 times per crop.

[0080] In the method provided by the present invention, the salt-tolerant Bacillus sp. and Streptomyces albidoflavus CCTCC NO: M 20231644 and / or Streptomyces blastmycinus CCTCC NO: M 20231643 provided by the present invention can be applied to the rhizosphere soil of plants together, or can be applied in batches in sequence.

[0081] The present invention will be described in detail below through examples. It should be understood that the following examples are only used to further explain and illustrate the content of the present invention by way of example, and are not used to limit the present invention.

[0082] In the following examples, unless otherwise specified, the reagents and materials used are all commercially available products purchased from regular chemical / biological reagent or material suppliers, and the reagents are all of analytical grade.

[0083] In the following examples, unless otherwise specified, the operating temperature is room temperature (25±5°C).

[0084] Example 1

[0085] This example is used to illustrate the acquisition, identification and preservation of salt-tolerant Bacillus sp. CCTCC NO: M 20232276.

[0086] (I) Strain isolation and purification

[0087] NA medium was used during the strain isolation and purification process, and the preparation method is as follows: Weigh 10.00 g of peptone, 3.00 g of beef powder, and 5.00 g of sodium chloride, dissolve them in 1000 mL of deionized water, adjust the pH value to 7.30±0.12, add 15.00 g of agar, and sterilize with steam at 121°C for 20 min for later use.

[0088] A strain of bacteria was isolated and purified from the rhizosphere soil sample of Pinus armandii collected from the Wuliangshan National Nature Reserve by the dilution plating method and the streak plate method, and named YNK-FB0022.

[0089] (II) Strain identification

[0090] 1. Identification of bacterial morphological characteristics and physiological and biochemical characteristics

[0091] The physiological and biochemical properties of strain YNK-FB0022 were determined with reference to Bergey's Manual of Determinative Bacteriology and Manual of Systematic Identification of Common Bacteria, and the colony morphological characteristics were described.

[0092] Colony & cell morphology: Figure 1The colony morphology of strain YNK-FB0022 on NA medium is shown. It can be seen from the figure that the colonies of this strain are nearly circular, opaque, with a smooth surface and milky white in color.

[0093] Physiological and biochemical characteristics: The surface of the colonies of YNK-FB0022 is moist and smooth, opaque, Gram-positive, can utilize maltose and glucose, can hydrolyze amylase, the methyl red reaction is positive, and cannot hydrolyze fat.

[0094] 2. Molecular identification

[0095] Table 1 PCR system and conditions

[0096]

[0097] The total DNA of strain YNK-FB0022 was extracted by the Chelex extraction method as a template. 27F (5’-AGAGTTTGATCCTGGCTCAG-3’) was used as the upstream primer and 1492R (5’-TAC GGCTACCTTGTTACGACTT-3’) was used as the downstream primer. 16S rRNA amplification was carried out using the reaction system and conditions in Table 1.

[0098] The amplified products were electrophoresed on 1% agarose gel, purified and recovered using the gel recovery and purification kit produced by Guangzhou Meiji Biotechnology Co., Ltd., and then sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The sequencing results were compared and analyzed in the NCBI database (https: / / www.ncbi.nlm.nih.gov / ) and the EZBiocloud database (https: / / www.ezbiocloud.net / ). Using the phylogenetic analysis method, the 16S rRNA sequences of the type strains with higher homology were selected as the reference objects, and multiple sequence alignment was carried out using Clustal X 1.8 software to calculate the similarity between the sequences of the tested strain and the reference strains. When performing phylogenetic analysis, base deletion sites were excluded, and the neighbor-joining method was used to construct the phylogenetic tree between the tested strain and the reference strains using MEGA 7.0. Among them, the Bootstrap value was set to 1000, and the rest were default values.

[0099] Figure 2 The phylogenetic tree of strain YNK-FB0022 is shown. It can be seen from it that YNK-FB0022 has the highest homology with Bacillus halotolerans LPVF01000003.

[0100] 3. Identification results

[0101] Combined with the molecular detection results of strain YNK-FB0022, as well as the detection results of bacterial morphological characteristics and physiological and biochemical characteristics, this strain was identified as Bacillus halotolerans.

[0102] (III) Strain preservation

[0103] The obtained Bacillus halotolerans YNK-FB0022 was preserved in the China Center for Type Culture Collection on November 20, 2023. The address is Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province. The preservation number is CCTCC NO: M 20232276.

[0104] Example 2

[0105] This example is used to illustrate the effects of Bacillus halotolerans CCTCC NO: M 20232276 in decomposing organic phosphorus, potassium, and antagonizing various pathogenic bacteria.

[0106] (I) Decomposing organic phosphorus

[0107] Preparation method of organic phosphorus solid medium: Weigh 10 g of glucose, 0.5 g of ammonium sulfate, 0.5 g of yeast extract powder, 0.3 g of sodium chloride, 0.3 g of potassium chloride, 0.3 g of magnesium sulfate, 0.03 g of ferrous sulfate, 0.03 g of manganese sulfate, 0.2 g of lecithin, and 1 g of calcium carbonate, add them to 1000 mL of water and dissolve. Adjust the pH to 7.3 ± 0.2, and add 18 ± 2 g of agar. Autoclave at 121 °C for 20 min.

[0108] Using the plate four-point inoculation method, inoculate the strain YNK-FB0022 obtained in Example 1 on the organic phosphorus culture plate, and repeat the inoculation on 3 plates. After inoculation, place the plates in an incubator at 30 °C for 5 days, and observe the bacterial growth status and the formation of clear zones around the colonies every day during the cultivation period.

[0109] It can be seen from the results that the strain YNK-FB0022 can produce a clear zone when cultured on the organic phosphorus culture plate. Using the cross method to measure the size of the clear zone, the ratio of the clear zone diameter (D) to the colony diameter (d) was calculated as D / d = 3.021 ± 0.64. This indicates that the strain has good ability to decompose organic phosphorus (II) Decomposing potassium

[0110] Preparation method of potassium-decomposing medium: Weigh 10 g of sucrose, 1 g of Na 2 HPO 4 、0.5 g of (NH 4 )2SO 4 、1 g of MgSO 4 ·7H 20, 0.2 g of yeast powder, 0.1 g of NaCl, 0.1 g of CaCO 3 , 0.005 g of FeCl 3 , 5 g of potassium feldspar powder (washed 5 times with distilled water before use and then dried), added to 1000 mL of water and dissolved, with the pH being natural. Subsequently, 15 g of agar was added, and it was autoclaved at 121 °C for 20 min.

[0111] Using the plate four-point inoculation method, the strain YNK-FB0022 obtained in Example 1 was inoculated on the potassium-dissolving culture plate, and 3 plates were inoculated repeatedly. After inoculation, the plates were placed in an incubator at 30 °C for 5 days, and the growth status of the bacteria and the formation of transparent zones around the colonies were observed every day during the cultivation. The size of the transparent zone was measured by the cross-cross method, and the ratio of the diameter of the transparent zone (D) to the diameter of the colony (d), D / d = 2.025 ± 0.17, was calculated. This indicates that the strain has good potassium-dissolving ability.

[0112] (III) Broad-spectrum antibacterial property of YNK-FB0022

[0113] Use an inoculation needle to pick up a mycelium block of the pathogen and place it in a PDA culture dish for 5 days. Use a borer to punch a 0.50 cm pathogen cake at the edge of the pathogen, place it in the center of a new PDA culture dish, and use an inoculation needle to pick up the activated YNK-FB0022 from the culture dish and inoculate the strain crosswise 2.50 cm away from the pathogen. Use the culture dish inoculated only with the pathogen as a control, and place it in an incubator at 28 °C for 3 - 5 days (the specific cultivation time is determined according to the growth of the pathogen and YNK-FB0022. When the plate inoculated only with the pathogen is covered with mycelium, the cultivation can be stopped). The inhibitory effect on the pathogen is expressed by the inhibition rate, and the calculation method is as follows. Table 2 and Figure 3 shows the inhibitory effects of the YNK-FB0022 strain on different pathogens.

[0114] Inhibition rate (%) = [(control colony diameter - treated colony diameter) / control colony diameter] × 100%.

[0115] Table 2 Inhibitory rates of YNK-FB0022 on different pathogens

[0116] Pathogenic bacteria Inhibitory rate of YNK-FB0022 (%) Fusarium oxysporum causing tomato wilt 83.65 Exserohilum turcicum causing northern leaf blight of maize 83.91 Colletotrichum micotianae causing anthracnose of tobacco 74.36 Phoma matteuciicola causing leaf spot of tsaoko 88.23 Phytophthora parasitica causing black shank of tobacco 78.91 Fusarium oxysporum fsp. Cubense race4 (Foc4) causing banana wilt 65.48

[0117] It can be seen from the data in the table that the inhibitory rates of YNK-FB0022 on the above-mentioned pathogens are all over 65%, among which the inhibitory rates on Fusarium oxysporum f. sp. lycopersici, Exserohilum turcicum, and Phyllosticta sp. are over 80%, and the highest inhibitory rate on Phyllosticta sp. is 88.23%. This indicates that the halotolerant Bacillus sp. has good application prospects in biological control.

[0118] Example 3

[0119] This example is used to illustrate the effect of the salt-tolerant Bacillus sp. CCTCC NO: M 20232276 in secreting auxin.

[0120] The activated salt-tolerant Bacillus sp. YNK-FB0022 was inoculated into KB medium and cultured with shaking at 30 °C and 180 rpm for 24 h. Under sterile conditions, 1 mL of the fermentation broth was aspirated into a centrifuge tube and quickly mixed with 4 mL of Sackowcki's chromogenic reagent. It was left to develop color in the dark at room temperature for 40 min, and the color change was observed and recorded. If it turned pink, it was positive, indicating that the strain could secrete IAA.

[0121] After culturing the strain in NB medium for 1 day, a seed solution was prepared and inoculated into KB liquid medium containing L-tryptophan at an inoculation amount of 1% (the initial content of YNK-FB0022 in the culture system after inoculation was about 2.5×10 6 CFU / mL). It was cultured with shaking at 30 °C in a constant temperature shaker at 180 rpm. Every day, 4 mL of the supernatant was aspirated and mixed with 4 mL of Sackowcki's chromogenic reagent. After standing in the dark for 40 min, the OD value was measured at a wavelength of 535 nm. The obtained absorbance value was substituted into the standard curve for calculation to obtain the indoleacetic acid content in the fermentation broth. It was determined that the indoleacetic acid content in the fermentation broth of the strain YNK-FB0022 was the highest on the 3rd day, which was 28.87±0.12 μg / mL.

[0122] Example 4

[0123] This example is used to illustrate the effect of the salt-tolerant Bacillus sp. CCTCC NO: M 20232276 in producing siderophores.

[0124] Preparation method of CAS chromogenic reagent:

[0125] Solution A: 60.5 mg of Chrome Azurol S (CAS) was added to 50 mL of deionized water, and then mixed with 10 mL of Fe 3+ solution (1 mM FeCl 3 ·6H 2 O, 10 mM HCl). All of the above Solution A and Solution B were mixed and filtered through a 0.2 μm filter membrane to obtain the CAS chromogenic reagent.

[0126] Preparation method of CAS chromogenic double-layer medium:

[0127] Add 1.5 g of agar to 100 mL of distilled water, and autoclave at 121 °C for 20 min to obtain water agar. When the water agar cools to 50 - 60 °C, uniformly add 10% of the CAS detection solution to the medium, and then pour a relatively large amount of the medium as the lower-layer medium. After the water agar medium cools and solidifies, pour the iron-free Czapek medium to form a double-layer plate.

[0128] Qualitative test:

[0129] Inoculate the test strain on the CAS chromogenic double-layer medium by the method of 4-point inoculation, with 3 replicates for each treatment. Place it in an incubator at 30 °C and continuously observe whether a chromogenic ring is produced around the colony. Also, measure the ratio between the diameter (D) of the chromogenic ring and the diameter (d) of the colony produced. The D / d value of strain YNK-FB0022 is 3.4 ± 0.17.

[0130] Quantitative test:

[0131] Strain YNK-FB0022 is cultured in an iron-free Czapek liquid medium on a constant-temperature shaker at 30 °C and 150 rpm for 48 h. After the culture is completed, pipette 2 - 5 mL of the culture solution, filter it through a 0.22-μm sterile filter membrane, and then add an equal volume of the CAS detection solution. After standing for 1 h, use a full-wavelength microplate reader to measure the OD of the inoculated bacterial solution 630 (denoted as “As”), and measure the OD of the uninoculated liquid medium in the same way 630 as the reference value (denoted as “Ar”). The concentration of the siderophore is expressed in siderophore units (SU), SU = [(Ar - As) / Ar] × 100%. The measurement is repeated 3 times, and the concentration of the siderophore of strain YNK-FB0022 is 31%.

[0132] Example 5

[0133] This example is used to illustrate the effect of Bacillus halodurans CCTCC NO: M 20232276 in degrading benzoic acid.

[0134] Benzoic acid inorganic salt medium: MgSO 4 ·7H 2 O 0.20 g; (NH4) 2 SO 4 1 g; KH 2 PO 4 0.50 g; NaCl 0.50 g; K 2 HPO 4 1.50 g, benzoic acid 0.1 g, distilled water 1000 mL, pH natural; autoclave at 121 °C for 20 min.

[0135] The YNK-FB0022 screened in Example 1 was activated and inoculated into 20 mL of NB liquid medium, and cultured in a shaker at 30 °C and 180 rpm for 24 h as the seed solution. 1 mL of the seed solution was taken and inoculated into 50 mL of benzoic acid inorganic salt liquid medium. Zero-time samples and final-time samples after shaking culture at 37 °C and 180 rpm for 4 d were taken respectively, filtered, and the substrate amount was detected by HPLC, so as to calculate the substrate degradation efficiency.

[0136] Conditions for high performance liquid chromatography: Take the fermentation broth to be tested, add it to a 50 mL centrifuge tube, centrifuge at 4 °C and 8000 rpm for 10 min, take 1.00 mL of the supernatant and filter it through a 0.22 μm microporous membrane as the sample for injection. The chromatographic column used is an Agillent C18 column (250 mm × 4.0 mm × 5 μm); the column temperature is 35 °C; the ultraviolet detection wavelength is 230 nm; the mobile phase is methanol: 0.02 mol / L ammonium acetate = 5:95; the flow rate is 1.00 mL / min; the injection volume is 10.00 μL, and isocratic elution is used.

[0137] Degradation rate = [(substrate amount at zero time - substrate amount at final time) / substrate amount at zero time] × 100%

[0138] The results showed that after culturing YNK-FB0022 in benzoic acid inorganic salt medium for four days, the content of benzoic acid was detected to decrease by high performance liquid chromatography, and at the same time, the cell weight increased, indicating that the strain utilized benzoic acid. After calculation, the degradation rate of YNK-FB0022 to benzoic acid reached 64.91%.

[0139] Example 6

[0140] This example is used to illustrate the effect of Bacillus halodurans CCTCC NO: M 20232276 on alleviating the inhibition of seed germination by benzoic acid.

[0141] Three treatments were set up in the experiment, namely: 1) water control (CK1); 2) 1.00 g / L benzoic acid treatment (CK2); 3) YNK-FB0022 bacterial suspension (experimental group).

[0142] Preparation method of the bacterial suspension in the experimental group: Inoculate the YNK-FB0022 strain into Gause's No. 1 liquid medium at an inoculation amount of 1%, and culture it in a constant temperature shaker at 30 °C and 180 rpm for 48 h to make the strain concentration reach about 2.5×10 8 CFU / mL, centrifuge at 4 °C and 8000 rpm for 10 min, pour off the supernatant, resuspend with sterile water and dilute to the above concentration for use.

[0143] Tomato, pepper, and cucumber seeds were selected as experimental subjects. The seeds were soaked in 70% alcohol for 10 min and then washed 3 times with sterile water to complete disinfection. The seeds that sank to the bottom of the water were taken. After soaking in water at room temperature for 12 h, the seeds of each group were placed in a 9.00 cm transparent petri dish lined with 2 - 3 layers of sterilized filter paper. There were 10 seeds per petri dish for each treatment, and the experiment was repeated 3 times. For treatment 3), first, 2.00 mL of the bacterial suspension was injected into the petri dish to moisten the sterilized paper and culture the seeds on the moist sterilized paper for 24 h. Then, 2.00 mL of benzoic acid was continued to be injected into the petri dish to culture the seeds on the sterilized paper containing the benzoic acid solution for 24 h. Subsequently, 2.00 mL of sterile water was injected into the petri dish every 24 h to keep the sterilized paper always moist. For other treatments, 2.00 mL of the required liquid was injected into the petri dish, and 2.00 mL of sterile water was added every 24 h. The seeds germinated in an artificial climate chamber at 26°C, with the light and dark alternating for 16 h and 8 h. The seed germination rate and the whole plant length were measured 7 days after the start of the culture with the bacterial suspension treatment. Table 3 and Figure 4 shows the effect of strain YNK - FB0022 in alleviating the inhibition of benzoic acid on plant germination.

[0144] Determination of germination index: Whether the seeds germinated after treatment was determined by the standard that the radicle emergence > 1 / 2 of the seed length. The seed germination rate (%) = (number of germinated seeds / number of tested seeds) × 100%.

[0145] Table 3 Results of YNK - FB0022 in alleviating the inhibition of benzoic acid on seed germination

[0146]

[0147] From the results, it can be seen that benzoic acid inhibited the germination rates of tomato, pepper, and cucumber seeds. After treatment with strain YNK - FB0022, the seed germination rates increased. Moreover, the germination rate of cucumber in the experimental group also increased compared with the water control, indicating that YNK - FB0022 can degrade benzoic acid, alleviate the inhibition of benzoic acid on seeds, and promote seed germination. In addition, the whole plant lengths of tomato, cucumber, and pepper were all increased compared with the control, indicating that the strain has the effect of promoting seed growth.

[0148] Example 7

[0149] This example is used to illustrate the control effect of halotolerant Bacillus sp. CCTCC NO: M 20232276 on tomato wilt.

[0150] Preparation of the bacterial agent: The strain YNK - FB0022 obtained in Example 1 was inoculated into NA liquid medium and cultured with shaking at 180 rpm at 30°C for 72 h. The resulting fermentation broth was the bacterial agent of strain YNK - FB0022 (the viable cell count was about 7×10 8 CFU / mL).

[0151] Plant planting: Add the same weight of soil to each flower pot, randomly group them into 10 pots for each treatment, and plant 1 tomato seedling with similar growth in each pot.

[0152] Verification of the effect of controlling Fusarium wilt: On the 3rd day after transplanting tomato seedlings into flower pots, irrigate the roots of tomato seedlings with Fusarium oxysporum. Make a small hole at the root and stem of the seedlings during irrigation. After three days of irrigation with Fusarium oxysporum to allow the pathogen to colonize, irrigate the roots of tomato seedlings with the above-mentioned bacterial agent (100 mL / plant), and at the same time, use the same amount of sterile water for the same treatment as the control group (CK).

[0153] The tomato seedlings after root irrigation treatment are placed in the greenhouse to grow naturally. 45 days after root irrigation treatment, measure the incidence and disease index of tomato plants. The results are shown in Table 4.

[0154] The disease conditions of each treatment are in accordance with the disease grading standard of tomato Fusarium wilt: 0: asymptomatic; 1: one or two leaves turn yellow; 2: three or four true leaves turn yellow and the leaves wilt and droop; 3: five or six true leaves turn yellow or the true leaves wilt and droop; 4: the whole plant severely wilts and dies.

[0155] Disease index = (number of diseased plants at each level × the value of this disease level) / (total number of plants × the highest disease level value) × 100.

[0156] Control effect = (control group disease index - treatment group disease index) / control group disease index × 100.

[0157] Table 4 Control effect of YNK-FB0022 on tomato Fusarium wilt

[0158] Determination index CK Experimental group Disease index 25.83±0.21a 5.00±0.013b Control effect (%) - 80.65±0.29

[0159] "-" indicates no control effect

[0160] From the above data, it can be seen that after applying the bacterial agent YNK-FB0022, the disease index of tomato Fusarium wilt decreased significantly, and the incidence was significantly controlled, indicating that this strain can prevent and control tomato Fusarium wilt.

[0161] Example 8

[0162] This example is used to illustrate the growth promotion effect and soil improvement effect of salt-tolerant Bacillus sp. CCTCC NO: M 20232276.

[0163] (I) Verification of growth promotion effect

[0164] Add the same weight of soil to each flower pot, randomly group them into 10 pots for each treatment, and plant 1 tomato seedling with similar growth in each pot. On the 3rd day after transplanting the tomato seedlings into the flower pots, drench the roots of the tomato seedlings with the microbial agent in Example 7 at a dosage of 100 mL / plant, and at the same time, perform the same treatment with an equal amount of sterile water as Control Group 1 (CK1), and perform the same treatment with an equal amount of NB medium as Control Group 2 (CK2).

[0165] Place the tomato seedlings after root drenching treatment in the greenhouse to grow naturally. 45 days after the root drenching treatment, measure the above-ground fresh weight and dry weight, underground (root) fresh weight and dry weight, stem diameter, root length, and plant height of the tomato plants. The results are shown in Table 5 for details.

[0166] The specific measurement methods are as follows:

[0167] Above-ground fresh weight & dry weight: Cut off the part above the base of the tomato plant and weigh the above-ground fresh weight on an analytical balance, retaining 1 decimal place. Put the above-ground part of the plant that has been weighed for fresh weight into a paper bag and place it in an oven. Blanch at 100 ± 5 °C for 10 min, then reduce the oven temperature to 75 ± 5 °C and dry to a constant weight, and weigh on an analytical balance to obtain the above-ground dry weight, retaining 1 decimal place.

[0168] Underground fresh weight & dry weight: Cut off the part below the base of the tomato plant and weigh the underground fresh weight on an analytical balance, retaining 1 decimal place. Put the underground part of the plant that has been weighed for fresh weight into a paper bag and place it in an oven. Blanch at 100 ± 5 °C for 10 min, then reduce the oven temperature to 75 ± 5 °C and dry to a constant weight, and weigh on an analytical balance to obtain the underground dry weight, retaining 1 decimal place.

[0169] Stem diameter: Measure the diameter of the thickest part of the plant using a vernier caliper.

[0170] Root length: Straighten the root and measure the root length using a ruler.

[0171] Plant height: Straighten the plant and measure the above-ground length from the highest point of the leaves using a ruler.

[0172] Table 5 Growth status of tomato seedling plants

[0173] Growth index CK1 CK2 Experimental group Fresh weight of aboveground part (g) 31.93±1.78b 30.15±1.12b 46.21±0.60a Dry weight of aboveground part (g) 5.71±0.33b 5.15±0.11b 7.22±0.15a Root weight (g) 7.07±0.12c 8.12±0.14b 12.39±0.41a Stem diameter (mm) 5.24±0.07b 5.11±0.04b 5.71±0.03a Root length (cm) 12.03±0.60b 13.18±0.66b 22.83±1.46a Plant height (cm) 84.23±3.43b 86.23±3.27b 104.90±1.61a

[0174] * Different letters in the data in the table represent significant differences

[0175] As can be seen from the data in the above table, after the tomato plants were treated with the YNK-FB0022 microbial agent by root irrigation, the plant height, stem diameter, fresh weight of the above-ground part, root length, and fresh and dry root weights of the tomato plants were all significantly increased compared with the control group (CK1) treated with clear water and the control group (CK2) treated with NB medium. After calculation, the net increase in plant height of the tomatoes in the experimental group reached 24.50% compared with CK1, the stem diameter increased by 9%, the fresh weight of the above-ground part increased by 44.7%, the dry weight of the above-ground part increased by 26.40%, the root length increased by 26.30%, and the root weight increased by 75.10%, indicating that the strain YNK-FB0022 can effectively promote plant growth. There were significant differences between the control groups (P<0.05) except for the stem diameter. It shows that inoculating the rhizosphere strain YNK-FB0022 has a significant promoting effect on the growth of tomatoes.

[0176] (II) Detection of soil physical and chemical properties

[0177] After the cultivation of tomatoes (45 days) ended, the rhizosphere soil of tomatoes in each group was collected, and the obtained soil samples were detected for soil physical and chemical property parameters such as pH, organic matter, total nitrogen, total phosphorus, total potassium, available nitrogen, available phosphorus, and available potassium to verify the improvement effect of the strain YNK-FB0022 on the soil. The detection results are shown in Table 6.

[0178] The specific detection methods are as follows:

[0179] The determination of organic matter was carried out by the chromic acid redox titration method, the pH value was determined by the potentiometric method, the total nitrogen was determined by the Kjeldahl method, the total phosphorus was determined by the sodium hydroxide alkali fusion-molybdenum antimony anti-colorimetric method, the total potassium and available potassium were determined by the flame photometry method, the available nitrogen was determined by the diffusion absorption method, and the available phosphorus was determined by the 0.5mol / L NaHCO 3 -molybdenum antimony anti-colorimetric method.

[0180] Table 6 Detection results of soil physical and chemical properties of soil samples

[0181] Detection index CK1 CK2 Experimental group pH 7.1 6.9 6.9 Organic matter (g / kg) 163.8 182.3 86 Total nitrogen (g / kg) 3.75 3.54 3.97 Total phosphorus (g / kg) 1.09 1.03 1.21 Total potassium (g / kg) 6.35 6.85 6.98 Hydrolyzable nitrogen (mg / kg) 289 302 261 Available phosphorus (mg / kg) 115.6 127.5 79.4 Available potassium (mg / kg) 216 245 176

[0182] From the data in the table, there were no obvious differences in the pH value, total phosphorus, total nitrogen, and total potassium among the three treatments. However, the contents of available potassium, organic matter, total nitrogen, available phosphorus, and hydrolyzable nitrogen were all lower than those of the control group. By jointly analyzing with the tomato plant growth data in Table 5, it can be inferred that because the strain YNK-FB0022 has the functions of decomposing organic phosphorus and potassium, it transforms the nutrients in the soil into available phosphorus and available potassium that can be absorbed by plants and is fully absorbed by the tomato roots, resulting in an increase in the organic matter content and nutrient accumulation of the tomato plants, and thus the contents of available phosphorus and potassium in the soil environment after planting decrease. It proves that the strain YNK-FB0022 has a good improvement effect on the soil and can thus have an obvious promoting effect on the growth of tomatoes.

[0183] (3) Verification of the improvement effect of soil enzyme activity

[0184] After the cultivation of tomatoes (45 days) ended, the rhizosphere soil of tomatoes in each group was collected, and the obtained soil samples were tested for soil physical and chemical property parameters such as urease, sucrase, acid phosphatase, and catalase to verify the improvement effect of strain YNK-FB0022 on the soil. The test results are shown in Table 7.

[0185] The specific detection methods are as follows:

[0186] The urease activity was determined by the indophenol colorimetric method; the sucrase activity was determined by the 3,5-dinitrosalicylic acid colorimetric method; the acid phosphatase activity was determined by the phenyl disodium phosphate colorimetric method; the catalase activity was determined by the potassium permanganate titration method.

[0187] Table 7 Detection results of soil enzyme activities

[0188] Detection index CK1 CK2 Experimental group <![CDATA[Invertase (mg·d -1 ·g -1 )]]> 4.39 4.65 6.45 <![CDATA[Urease (μg·d -1 ·g -1 )]]> 286.31 295.3 326.54 <![CDATA[Acid phosphatase (μmol·d -1 ·g -1 )]]> 8.56 9.02 11.98 Catalase (mg / g·d) 2.13 2.41 4.75

[0189] From the data in the table, it can be seen that the soil sucrase, urease, acid phosphatase, and catalase were all increased compared with the two controls. Combining with the changes in the soil physical and chemical index data in Table 6, it shows that strain YNK-FB0022 increased the content of these enzymes in the soil, thereby promoting the transformation of available phosphorus, available nitrogen, and available potassium in the soil, providing more nutrient elements for plants, and thus promoting the healthy growth of plants.

[0190] Example 9

[0191] This example is used to illustrate the growth-promoting effects of the composite bacterium agents of Streptomyces alboflavus CCTCC NO: M 20231644 and Bacillus halodurans CCTCC NO: M20232276, and the composite bacterium agents of Streptomyces blastmycinus CCTCC NO: M20231643 and Bacillus halodurans CCTCC NO: M20232276.

[0192] (1) Verification of growth-promoting effect

[0193] The same weight of soil was added to each flower pot, and the flower pots were randomly grouped according to 10 pots for each treatment, and 1 tomato seedling with similar growth vigor was planted in each pot. The treatment groups were set as follows:

[0194] 1) T1: On the 3rd day after transplanting tomato seedlings into the flower pots, the tomato seedlings were irrigated with a liquid bacterium agent of Streptomyces blastmycinus CCTCC NO: M20231643 with a bacterial content of 6×10 8 CFU / mL at 100 mL / plant. Three days later, a liquid bacterium agent with a bacterial content of 7×10 8The tomato seedlings were treated by root irrigation with a liquid bacterial agent of salt-tolerant Bacillus sp. YNK-FB0022 at a concentration of 8 CFU / mL; 2) T2: On the 3rd day after transplanting tomato seedlings into flower pots, the seedlings were treated by root irrigation with a liquid bacterial agent of Streptomyces albidoflavus CCTCC NO: M20231644 at a concentration of 1.2×10 8 CFU / mL, and three days later, they were treated by root irrigation with a liquid bacterial agent of salt-tolerant Bacillus sp. YNK-FB0022 at a concentration of 7×10 8 CFU / mL; 3) T3: On the 3rd day after transplanting tomato seedlings into flower pots, the seedlings were treated by root irrigation with salt-tolerant Bacillus sp. YNK-FB0022 at a concentration of 7×10

[0195] CFU / mL; 4) CK: The same treatment was carried out with an equal amount of sterile water as the control group. The tomato seedlings after root irrigation treatment were placed in the greenhouse for natural growth.

[0196] Above-ground fresh weight & dry weight: Cut off the part above the root base of the tomato plant and weigh the above-ground fresh weight on an analytical balance, retaining 1 decimal place. The above-ground part of the plant with the fresh weight measured was put into a paper bag and placed in an oven. It was blanched at 100±5°C for 10 min, then the oven temperature was reduced to 75±5°C and dried to a constant weight, and the above-ground dry weight was weighed on an analytical balance, retaining 1 decimal place.

[0197] Below-ground fresh weight & dry weight: Cut off the part below the root base of the tomato plant and weigh the below-ground fresh weight on an analytical balance, retaining 1 decimal place. The below-ground part of the plant with the fresh weight measured was put into a paper bag and placed in an oven. It was blanched at 100±5°C for 10 min, then the oven temperature was reduced to 75±5°C and dried to a constant weight, and the below-ground dry weight was weighed on an analytical balance, retaining 1 decimal place.

[0198] Stem diameter: Measure the diameter of the thickest part of the plant using a vernier caliper.

[0199] Root length: Measure the root length using a ruler after straightening the root.

[0200] Plant height: Measure the above-ground length using a ruler from the highest point of the leaves after straightening the plant.

[0201] Table 8 Growth status of tomato seedling plants

[0202] Growth index CK T1 T2 T3 Fresh weight of aboveground part (g) 31.93±1.78c 56.48±1.58a 51.26±1.07a 46.21±0.60b Dry weight of aboveground part (g) 5.71±0.33c 9.57±0.24a 8.31±0.25a 7.22±0.15b Root weight (g) 7.07±0.12b 13.47±0.11a 13.67±0.13a 12.39±0.41a Stem diameter (mm) 5.24±0.07b 6.13±0.09a 5.78±0.10a 5.71±0.03a Root length (cm) 12.03±0.60bc 23.26±0.37a 19.16±0.52a 22.83±1.46a Plant height (cm) 84.23±3.43b 116.89±0.59b 108.85±0.45a 104.90±1.61b

[0203] * Different letters in the data in the table represent significant differences

[0204] From the data in the above table, it can be seen that the growth status of tomato seedlings of T1 and T2 is improved compared with that of T3 and CK, indicating that after root irrigation with the combined application of Streptomyces albidoflavus CCTCC NO: M 20231644 and YNK-FB0022, or the combined application of Streptomyces blastmycinus CCTCC NO: M 20231643 and YNK-FB0022, the growth-promoting effect on tomato seedlings is better. After calculation, the fresh weight of the above-ground part of tomatoes in T1 increased by 22.22% and 60.54% respectively compared with T3 and CK; the dry weight of the above-ground part increased by 32.55% and 67.6% respectively; the root weight increased by 8.72% and 90.52% respectively; the root length increased by 1.88% and 93.35% respectively; the fresh weight of the above-ground part of tomatoes in T2 increased by 10.93% and 60.54% respectively compared with T3 and CK; the dry weight of the above-ground part increased by 15.1% and 45.53% respectively; the root weight increased by 10.33% and 93.35% respectively.

[0205] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited thereto. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, including any other suitable combination of each technical feature. These simple modifications and combinations should also be regarded as the content disclosed by the present invention and fall within the protection scope of the present invention.

Claims

1. A strain of halotolerant Bacillus ( Bacillus halotolerans ), characterized in that The deposit number of this strain is CCTCC NO:M 20232276.

2. A bacterial agent, characterized in that The bacterial agent comprises the halotolerant Bacillus according to claim 1.

3. The bacterial agent according to claim 2, wherein The bacterial agent is a liquid bacterial agent.

4. The bacterial agent according to claim 3, wherein The content of the salt-tolerant Bacillus in the bacterial agent is not less than 1×10 7 CFU / mL.

5. The bacterial agent according to claim 4, wherein The content of the salt-tolerant Bacillus in the bacterial agent is 1×10 7 -1×10 10 CFU / mL.

6. The use of the halotolerant Bacillus according to claim 1, or the bacterial agent according to any one of claims 2 to 5 in degrading benzoic acid, degrading organic phosphorus, degrading potassium, producing indoleacetic acid, producing siderophores, antagonizing pathogens or alleviating the inhibitory effect of benzoic acid on seed germination; in, The pathogens are tomato wilt pathogen, corn leaf spot pathogen, tobacco anthracnose pathogen, tsaoko leaf spot pathogen, tobacco black shank pathogen and banana wilt pathogen.

7. A method for preparing indoleacetic acid and / or siderophore, characterized in that: The method comprises culturing the halotolerant Bacillus according to claim 1 and collecting the culture product.

8. The method according to claim 7, wherein: The culture conditions include: culture temperature 28-32° C., and culture time 24-72 h.

9. Use of the salt-tolerant Bacillus according to claim 1 or the bacterial agent according to any one of claims 2 to 5 in improving soil, and / or promoting plant growth, and / or promoting plant seed germination, and / or preventing and controlling soil-borne plant diseases, wherein: The soil-borne diseases are diseases caused by at least one of tomato wilt pathogen, corn leaf blight pathogen, tobacco anthracnose pathogen, tsaoko leaf spot pathogen, tobacco black shank pathogen and banana wilt pathogen.

10. A method for preventing and controlling soil-borne plant diseases and / or promoting plant growth, characterized in that: The method comprises applying the halotolerant Bacillus of claim 1 or the bacterial agent of any one of claims 2 to 5 to the rhizosphere soil of the plant; Wherein, the plant is selected from Solanaceae and / or Cucurbitaceae plants; The soil-borne diseases are diseases caused by at least one of tomato wilt pathogen, corn leaf blight pathogen, tobacco anthracnose pathogen, tsaoko leaf spot pathogen, tobacco black shank pathogen and banana wilt pathogen.

11. The method according to claim 10, wherein: The application amount of the salt-tolerant Bacillus is not less than 1×10 8 CFU / strain / time; or The dosage of the bacterial agent is such that the dosage of the salt-tolerant Bacillus is not less than 1×10 8 CFU / strain / time; or And / or, the application frequency of the halotolerant Bacillus or bacterial agent is 1-3 times per crop.

12. The method according to claim 11, wherein: The application amount of the salt-tolerant Bacillus is 1×10 9 -1×10 12 CFU / strain / time; or The dosage of the bacterial agent is such that the dosage of the halodurogenic Bacillus is 1×10 9 -1×10 12 CFU / strain / time.

13. The method according to claim 11, wherein: The plant is selected from at least one of tomato, pepper and cucumber.

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