A bacterial agent for inhibiting chloride ion absorption, a preparation method and application thereof

Abstract

The application provides a bacterium agent for inhibiting absorption of chloride ions, a preparation method and application thereof, and relates to the technical field of microbial engineering. The application first adopts marshy Rhodopseudomonas LM6-purple and Baccillus velezensis LGT-1 to obtain a composite bacterium agent which can effectively inhibit absorption of chloride ions in tobacco in soil, and provides a new effective component and functional strain for a chloride ion inhibitor; in the application, the salt tolerance of Baccillus velezensis LGT-1 is domesticated, so that it is more suitable for soil with high chloride ion content, the application scene is expanded, and a new fertilizer component is provided for tobacco planting in saline-alkali soil and improvement; the application provides the preparation method of the composite bacterium agent, the production cost is low, the production method is simple, and the application is suitable for wide promotion.

Description

Technical Field

[0001] This application relates to the field of microbial engineering technology, and in particular to a bacterial agent that inhibits chloride ion absorption, its preparation method, and its application. Background Technology

[0002] As the world's largest tobacco-producing country, my country's tobacco plays a vital role in the national economy. With the increasing area under tobacco cultivation, the quality requirements for tobacco leaves have also risen. Combustion performance, a crucial indicator of tobacco quality, has always been highly valued. The combustibility of tobacco leaves is primarily related to the potassium-chlorine ratio; therefore, improving combustibility can begin by adjusting the chlorine content. Chlorine is one of the essential nutrients for tobacco growth. Appropriate chlorine levels help increase cell turgor pressure, enhance drought resistance, and reduce the occurrence of leaf blight. It can also lower protein, nicotine, and total nitrogen content while increasing total sugar and reducing sugar content, thus improving the flavor of the tobacco. However, excessively high chloride ion content can severely impact tobacco quality; therefore, proper control of chloride ion content is crucial for tobacco growth.

[0003] Currently, chloride ions in flue-cured tobacco mainly originate from soil, irrigation water, rainfall, and various fertilizers, with soil chloride being the primary source of chloride absorption by tobacco plants. Although chloride levels can be reduced to some extent through agronomical management practices, breeding, and chloride ion inhibitors, the problem of excessively high chloride ion concentrations in tobacco leaves persists. Furthermore, while using inhibitors under salt stress can reduce the chloride content in plants and thus mitigate the harmful effects of chloride, this also places demands on the salt tolerance of the inhibitors themselves.

[0004] Therefore, it is necessary to continue exploring a practical and feasible solution to improve the chloride ion content in tobacco leaves. Summary of the Invention

[0005] The purpose of this invention is to provide a salt-resistant, green, highly efficient and feasible effective component for chloride ion inhibition.

[0006] On the one hand, this application provides a bacterial agent for inhibiting chloride ion absorption, the bacterial agent comprising Bacillus velezensis and / or Rhodopseudomonas palustris.

[0007] Preferably, the microbial agent includes Bacillus velezensis and Rhodopseudomonas palustris.

[0008] Further, the *Bacillus belyssus* is *Bacillus belyssus* LGT-1, with accession number CGMCC No. 24342; preferably, the viable count of the *Bacillus belyssus* is 9.21 × 10⁻⁶. 8 cfu·mL -1 .

[0009] Further, the *Rhodopseudomonas palustris* is *Rhodopseudomonas palustris* LM6-purple, with accession number CGMCC No. 1.8929; preferably, the viable count of the *Rhodopseudomonas palustris* is 3 × 10⁻⁶. 9 cfu / mL.

[0010] This application is the first to use Bacillus belye and Rhodopseudomonas palustris to obtain a bacterial agent for inhibiting chloride ion absorption in tobacco. It has the effect of inhibiting the absorption of chloride ions by tobacco, and therefore can be used as a chloride ion absorption inhibitor, providing a new effective ingredient for reducing the chloride ion content in tobacco and producing low-chlorine tobacco.

[0011] Those skilled in the art will understand that *Bacillus bellis* in this application may be *Bacillus bellis*, *Bacillus bellis* bacterial agent, *Bacillus bellis* dead bacterial suspension, *Bacillus bellis* metabolites, or *Bacillus bellis* extract; and that *Rhodopseudomonas palustris* in this application may be *Rhodopseudomonas palustris*, *Rhodopseudomonas palustris* bacterial agent, *Rhodopseudomonas palustris* dead bacterial suspension, *Rhodopseudomonas palustris* metabolites, or *Rhodopseudomonas palustris* extract.

[0012] On the other hand, this application also provides a method for preparing the aforementioned microbial agent, the method comprising the following steps:

[0013] Step 1: Ferment Bacillus belye to obtain Bacillus belye fermentation broth for later use; preferably, the viable count of the Bacillus belye is 9.21 × 10⁻⁶. 8 cfu·mL -1 ;

[0014] Step 2: Fermenting *Rhodopseudomonas palustris* to obtain *Rhodopseudomonas palustris* fermentation broth for later use; preferably, the viable count of the *Rhodopseudomonas palustris* is 3 × 10⁻⁶. 9 cfu / mL;

[0015] Step 3: Mix the Bacillus vesicularis fermentation broth and Rhodopseudomonas palustris fermentation broth to obtain the inoculum.

[0016] Preferably, the Bacillus berberis fermentation medium in step one comprises: tryptone, yeast extract, and sodium chloride; more preferably, the tryptone content is 1-10 g / L. -1 1-5g / L of yeast powder -1 NaCl 1-10 g·L-1 More preferably, tryptone 10 g / L -1 5g / L of yeast powder -1 NaCl 10 g·L -1 ;

[0017] Preferably, the pH of the Bacillus belye fermentation medium is 7-7.5; more preferably, 7.2.

[0018] Preferably, the volume ratio of the Bacillus vesicularis fermentation broth to the Rhodopseudomonas palustris fermentation broth is 1:(1-5); more preferably, 1:1.

[0019] The volume ratio of the Bacillus vesicularis fermentation broth to the Rhodopseudomonas palustris fermentation broth can be any value among 1:1, 1:2, 1:3, 1:4, and 1:5.

[0020] Preferably, the Bacillus belesii is Bacillus belesii LGT-1, with accession number CGMCC No. 24342.

[0021] Preferably, step one further includes the step of activating Bacillus belye, which can be activated by those skilled in the art using common methods.

[0022] The method for activating Bacillus belyss includes: inoculating Bacillus belyss LGT-1 strain into Bacillus belyss activation medium, incubating at 25℃-40℃ for 12-24 hours in a constant temperature incubator, picking single colonies and inoculating them into Bacillus belyss LGT-1 seed medium, incubating at 20℃-40℃ and 100-300 rpm. -1 Under the specified conditions, shake culture on a shaker for 12-24 hours yields Bacillus belysin LGT-1 seed culture.

[0023] Preferably, the *Bacillus vesiculus* LGT-1 strain is inoculated into *Bacillus vesiculus* activation medium and incubated at 30°C for 24 hours. Single colonies are then picked and inoculated into *Bacillus vesiculus* LGT-1 seed culture medium at 30°C and 200 rpm. -1 The seed culture of Bacillus belye LGT-1 was obtained by shaking and culturing in a shaker for 24 hours under the specified conditions.

[0024] Preferably, the OD of the Bacillus belyss LGT-1 seed liquid... 600 The value is 0.6-1.

[0025] Preferably, the Bacillus berreatus activation medium is Bacillus berreatus LGT-1 plate solid medium.

[0026] Preferably, the Bacillus belye LGT-1 plate solid culture medium comprises: 10 g / L peptone-1 5g / L yeast powder -1 10 g·L of sodium chloride -1 10 g / L agar -1 The remainder is water, with a pH of 7.2-7.4.

[0027] Preferably, the Bacillus belye LGT-1 seed culture medium comprises: 10 g / L peptone -1 5g / L yeast powder -1 10 g·L of sodium chloride -1 The remainder is water, with a pH of 7.2-7.4.

[0028] Preferably, the preparation of the Bacillus vesiculus LGT-1 fermentation broth includes: inoculating Bacillus vesiculus LGT-1 seed culture into Bacillus vesiculus fermentation medium at an inoculation rate of 1%-5%, and culturing at 20℃-40℃, 100-200r / min for 24-48h to prepare Bacillus vesiculus LGT-1 fermentation broth.

[0029] Preferably, the Bacillus berleis LGT-1 seed culture is inoculated into the Bacillus berleis fermentation medium at an inoculation rate of 5%, and cultured at 30°C and 200 r / min for 24 h to prepare the Bacillus berleis LGT-1 fermentation broth.

[0030] Preferably, the Rhodopseudomonas palustris is Rhodopseudomonas palustris LM6-purple, with accession number CGMCCNo.1.8929.

[0031] Preferably, step two further includes activating *Rhodopseudomonas palustris*, which can be activated using common methods by those skilled in the art.

[0032] The activation method of Rhodopseudomonas palustris includes: inoculating Rhodopseudomonas palustris into a culture medium, adding 1-5 g / L of carbon source, culturing at 25℃-40℃ for 24-48 h, picking a single colony and inoculating it into a culture medium, adding 1-5 g / L of carbon source, and anaerobic culturing at 20℃-40℃ for 24-48 h to obtain Rhodopseudomonas palustris LM6-purple seed culture.

[0033] Preferably, the activation method of Rhodopseudomonas palustris includes: inoculating Rhodopseudomonas palustris into a Rhodopseudomonas palustris culture medium, adding 5 g / L carbon source, culturing at 30°C for 48 h, picking a single colony and inoculating it into a Rhodopseudomonas palustris culture medium, adding 5 g / L carbon source, and culturing at 30°C under light and anaerobic conditions for 48 h to obtain Rhodopseudomonas palustris LM6-purple seed culture.

[0034] Preferably, the OD of the *Rhodopseudomonas palustris* LM6-purple seed liquid...600 The value is 0.6-1.

[0035] Preferably, the preparation of the Rhodopseudomonas palustris LM6-purple fermentation broth includes: inoculating the Rhodopseudomonas palustris LM6-purple seed culture into the Rhodopseudomonas palustris culture medium at an inoculation rate of 1%-5%, adding 1-5 g / L carbon source, and anaerobic culture at 20℃-40℃ for 24-48 h to obtain the Rhodopseudomonas palustris LM6-purple fermentation broth.

[0036] Preferably, the seed culture of Rhodopseudomonas palustris LM6-purple is inoculated into the Rhodopseudomonas palustris culture medium at a ratio of 1%, and 5 g / L carbon source is added. The culture is carried out under light and anaerobic conditions at 30°C for 48 h to obtain the Rhodopseudomonas palustris LM6-purple fermentation broth.

[0037] The culture medium for Rhodopseudomonas swampus is PM medium.

[0038] Preferably, the activated medium for Rhodopseudomonas palustris is PM medium, and the carbon source is selected from one or more of sodium acetate, sodium malate, glycerol, sodium succinate, sodium bicarbonate, and carbon dioxide.

[0039] Preferably, step three further includes centrifugation followed by resuspending of the fermentation broth.

[0040] The resuspended material-to-liquid ratio is 1:1.

[0041] In a preferred embodiment, the method includes the following steps:

[0042] Step 1: Inoculate the *Bacillus belyssus* LGT-1 seed culture at an inoculum rate of 1%-5% into the *Bacillus belyssus* fermentation medium, and incubate at 20℃-40℃ and 100-200 rpm for 24-48 hours to prepare the *Bacillus belyssus* LGT-1 fermentation broth for later use. The *Bacillus belyssus* fermentation medium comprises: 1-10 g / L tryptone. -1 1-5g / L of yeast powder -1 NaCl 1-10 g·L -1 ;

[0043] Step 2: Inoculate the Rhodopseudomonas palustris LM6-purple seed culture into the Rhodopseudomonas palustris culture medium at an inoculation rate of 1%-5%, add 1-5 g / L carbon source, and culture anaerobically under light at 20℃-40℃ for 24-48 h to obtain the Rhodopseudomonas palustris LM6-purple fermentation broth for later use.

[0044] Step 3: Centrifuge and resuspend the Bacillus vesicularis fermentation broth and Rhodopseudomonas palustris fermentation broth respectively, and mix them at a volume ratio of 1:(1-5) to obtain the inoculum.

[0045] On the other hand, this application also provides a chloride ion absorption inhibitor, which includes the bacterial agent.

[0046] Preferably, the chloride ion absorption inhibitor is a chloride ion absorption inhibitor for tobacco, which has the effect of inhibiting the absorption of chloride ions from the soil by tobacco.

[0047] On the other hand, this application also provides a fertilizer, the microbial agent or the chloride ion absorption inhibitor.

[0048] Those skilled in the art will understand that the fertilizer of this application may also contain excipients, which may be appropriate solvents, solubilizers, co-solvents, emulsifiers, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, preservatives, suspending agents, coating materials, anti-adhesion agents, integrators, penetration promoters, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, filter aids, release inhibitors, etc.

[0049] On the other hand, this application also provides the application of the microbial agent, the chloride ion absorption inhibitor, or the fertilizer in inhibiting the absorption of chloride ions by tobacco, reducing the chloride ion content in tobacco, improving the combustion performance of tobacco, and / or increasing the chloride ion content in soil.

[0050] Preferably, the tobacco includes tobacco leaves.

[0051] In a preferred embodiment, Yan'an No. 6 tobacco was used as the test plant in this application. Those skilled in the art will understand that the method of this application can be extended to the planting of different varieties of tobacco and achieve the same technical effect. Considering the experimental cost and experimental time, it will not be listed one by one in this application.

[0052] Preferably, the soil is soil used for growing tobacco.

[0053] More preferably, the chloride ion content of the soil is 80-90 mg / kg.

[0054] On the other hand, this application also provides a method for inhibiting the absorption of chloride ions by tobacco, reducing the chloride ion content of tobacco and / or improving the combustion performance of tobacco, the method comprising: cultivating tobacco using the microbial agent or the chloride ion absorption inhibitor or the fertilizer.

[0055] Those skilled in the art can use common methods to cultivate tobacco using the microbial agent, the chloride ion absorption inhibitor, or the fertilizer described in this application.

[0056] The microbial agent can be applied directly or diluted by watering, dipping, or drenching the roots. It can also be added to fertilizers to make fertilizer products that inhibit the absorption of chloride ions by tobacco plants, and applied by methods such as pond application, hole application, strip application, or ring application.

[0057] In a preferred embodiment, the amount of the microbial agent used is 1-100 mL / strain; preferably, 10 mL / strain.

[0058] This application utilizes a compound microbial agent obtained from Bacillus belyssus and Rhodopseudomonas palustris to cultivate tobacco. This agent can inhibit the absorption of chloride ions by tobacco and reduce the chloride ion content in tobacco. Furthermore, this method is environmentally friendly and conducive to the sustainable use of soil, providing a new active ingredient for tobacco cultivation.

[0059] On the other hand, this application also provides tobacco cultivated by the method described above.

[0060] Preferably, the tobacco has a low chloride ion content; specifically, the chloride ion content in the tobacco is <0.8%.

[0061] Preferably, the tobacco comprises tobacco leaves; more preferably, the tobacco leaves comprise upper leaves, middle leaves, and lower leaves; even more preferably, lower leaves.

[0062] This application provides a tobacco with low chloride ion content. Compared with ordinary tobacco, the tobacco leaves of this tobacco have a lower chloride ion content, better combustibility, and are more suitable for making cigarettes, thus providing an excellent material for cigarette production.

[0063] On the other hand, this application also provides the use of the tobacco in the preparation of cigarettes.

[0064] Preferably, the cigarette has good combustibility.

[0065] The present invention has the following beneficial effects:

[0066] 1. This invention is the first to use Rhodopseudomonas palustris LM6-purple and Bacillus belye LGT-1 to obtain a compound bacterial agent that can effectively inhibit the absorption of chloride ions in soil by tobacco, providing a new effective ingredient and functional strain for chloride ion inhibitors;

[0067] 2. In this invention, Bacillus belye LGT-1 was fermented and cultured, which expanded its application scenarios and provided a new fertilizer component for tobacco planting and improvement in saline-alkali land;

[0068] 3. This invention provides a method for preparing the above-mentioned compound microbial agent, which has low production cost, simple production method and is suitable for promotion;

[0069] 4. The present invention uses microbial technology to control chloride ions in tobacco and its soil, providing a new approach to chloride ion control in this field. Compared with traditional chloride ion control technologies, it is more convenient, lower in cost, has less side effects on soil and tobacco, and has a significant effect on improving the combustibility of tobacco, thus improving the quality of tobacco leaves. Attached Figure Description

[0070] The accompanying drawings, which are included to provide a further understanding of this application and form part of this application, illustrate exemplary embodiments and are used to explain this application, but do not constitute an undue limitation of this application. In the drawings:

[0071] Figure 1 The effect of different culture media on the viable cell count of Bacillus vesiculosus LGT-1 fermentation broth;

[0072] Figure 2 Chloride ion content in potted soil of different treatment groups at different time periods;

[0073] Figure 3 Chloride ion content of tobacco leaves in each treatment group after control measures. Detailed Implementation

[0074] To more clearly illustrate the overall concept of this application, a detailed description is provided below with reference to the accompanying drawings and embodiments. Numerous specific details are set forth in the following description to provide a more thorough understanding of the invention. However, it will be apparent to those skilled in the art that the invention can be practiced without one or more of these details. In other instances, certain technical features well-known in the art have not been described to avoid confusion with the invention.

[0075] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0076] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to this application. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0077] Unless otherwise specified, in the following embodiments, reagents or instruments whose manufacturers are not indicated are all conventional products that can be purchased commercially.

[0078] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention. Test methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the respective manufacturers.

[0079] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0080] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques in the fields of microbiology, biochemistry, analytical chemistry, cell culture, and related areas.

[0081] Among them, Rhodopseudomonas palustris LM6-purple, with accession number CGMCC No:1.8929, was purchased from the China General Microbiological Culture Collection Center (CGMCC); Bacillus velezensis LGT-1 was deposited at the China General Microbiological Culture Collection Center on January 19, 2022, with accession number CGMCC No.24342, and has been published in Chinese patent document with patent number ZL202210302471.2.

[0082] Bacillus belye LGT-1 plate solid medium: peptone 10 g·L -1 5g / L yeast powder -1 10 g·L of sodium chloride -1 10 g / L agar -1 The remainder is water, with a pH of 7.2-7.4.

[0083] Bacillus belyssus LGT-1 seed culture medium: peptone 10 g·L -1 5g / L yeast powder -1 10 g·L of sodium chloride -1 The remainder is water, with a pH of 7.2-7.4.

[0084] PM liquid culture medium (1000mL): 1.701125g potassium dihydrogen phosphate, 1g ammonium sulfate, 1.7745g disodium hydrogen phosphate, 1mL concentrate, 100μL 0.1mol / L sodium thiosulfate pentahydrate, 1000mL distilled water. Sterilize at 0.15MPa and 121℃ for 30min. The concentrate (100mL) is formulated as follows: 2g aminotriacetic acid, 2.89g anhydrous magnesium sulfate, 5.03g calcium chloride, and 0.0198g ferrous sulfate heptahydrate. Sterilize at 121℃ for 20min.

[0085] PM solid culture medium (1000mL): 1.701125g potassium dihydrogen phosphate, 1g ammonium sulfate, 1.7745g disodium hydrogen phosphate, 1mL concentrate, 100μL 0.1mol / L sodium thiosulfate pentahydrate, 18g agar powder, 1000mL distilled water. Sterilize at 0.15MPa and 121℃ for 30min. The concentrate (100mL) is formulated as follows: 2g aminotriacetic acid, 2.89g anhydrous magnesium sulfate, 5.03g calcium chloride, and 0.0198g ferrous sulfate heptahydrate. Sterilize at 121℃ for 20min.

[0086] The soil used in this experiment was potted soil from Jimo District, Qingdao City, Shandong Province, with a chloride ion content of 80-90 mg / kg.

[0087] In addition, the "water" mentioned in this invention includes any feasible water that can be used in the art, such as deionized water, distilled water, ion-exchanged water, double-distilled water, high-purity water, and purified water.

[0088] In the following examples, unless otherwise specified, % means wt%, i.e., weight percentage.

[0089] Example 1: Screening of Bacillus belyss LGT-1 fermentation medium

[0090] This embodiment provides a Bacillus belyss LGT-1 fermentation broth and screens the components of its fermentation culture medium, specifically including:

[0091] Activation of Bacillus vesiculus LGT-1: Under aseptic conditions, Bacillus vesiculus LGT-1 strain stored at -80℃ was transferred to Bacillus vesiculus LGT-1 plate solid culture medium and incubated at 30℃ for 24 hours before use.

[0092] Preparation of Bacillus belyss LGT-1 seed culture: One loop of activated Bacillus belyss LGT-1 was inoculated into a 100 mL Erlenmeyer flask containing 40 mL of Bacillus belyss LGT-1 seed culture medium, and incubated at 30 °C and 200 rpm. -1The seed culture of Bacillus belye LGT-1 was obtained by shaking and culturing in a shaker for 24 hours under the specified conditions.

[0093] Bacillus belye LGT-1 fermentation broth: Bacillus belye LGT-1 seed culture was inoculated at a rate of 5% into 100mL Erlenmeyer flasks containing 40mL of different fermentation media. The inoculum was then incubated at 30℃ and 200 rpm. -1 The *Bacillus vesiculosus* LGT-1 fermentation broth was obtained by shaking and culturing on a shaker for 24 hours under the specified conditions. The composition of the fermentation medium is shown in Table 1. After fermentation, the viable cell count of the *Bacillus vesiculosus* LGT-1 fermentation broth was measured using the dilution plate count method.

[0094] Table 1. Culture medium composition

[0095]

[0096] Experimental results are as follows Figure 1 As shown, Bacillus belye LGT-1 exhibited the highest viable cell count per unit volume (9.21 × 10⁻⁶) during shake-flask fermentation in medium 2. 8 cfu·mL -1 The viable cell count per unit volume of fermentation broth in culture medium No. 1 was 8.13 × 10⁻⁶. 8 cfu·mL -1 The viable cell count per unit volume of fermentation broth in culture medium No. 3 was 6.52 × 10⁻⁶. 8 cfu·mL -1 The viable cell count per unit volume of fermentation broth in culture medium No. 4 was 8.55 × 10⁻⁶. 8 cfu·mL -1 Therefore, medium No. 2 was selected as the basal medium for shake-flask fermentation of Bacillus belyss LGT-1.

[0097] Example 2: Preparation of Compound Microbial Agent

[0098] Based on the preferred results of Example 1, this example provides a method for preparing Bacillus belyss LGT-1 fermentation broth:

[0099] (1) Under aseptic conditions, the Bacillus vesiculus LGT-1 strain stored at -80℃ was transferred to Bacillus vesiculus LGT-1 plate solid culture medium and incubated at 30℃ for 24 hours in a constant temperature incubator before use.

[0100] (2) Select a single colony of activated Bacillus belye LGT-1 and inoculate it into a 100 mL Erlenmeyer flask containing 40 mL of Bacillus belye LGT-1 seed culture medium. Incubate at 30℃ with shaking at 200 r / min for about 24 h, until the OD reaches 0.5. 600If the value is greater than 0.6, prepare Bacillus belyss LGT-1 seed culture;

[0101] (3) Transfer the seed culture to a fermenter at an inoculation rate of 5% (wherein, the fermentation medium formula is: 10 g / L tryptone). -1 5g / L of yeast powder -1 NaCl 10 g·L -1 The Bacillus berberis LGT-1 fermentation broth was prepared by incubating at 30℃ and 200r / min for about 24 hours (pH 7.2).

[0102] This embodiment provides a method for preparing fermentation broth of Rhodopseudomonas palustris LM6-purple strain:

[0103] (1) Under aseptic conditions, Rhodopseudomonas palustris LM6-purple, which was stored at -80℃, was inoculated into PM plate (solid) medium, 5 g / L carbon source was added, and the culture was carried out at 30℃ for 48 h, and activated 3 times.

[0104] (2) Select a single colony and transfer it to PM liquid medium, add 5 g / L carbon source, and culture under light and anaerobic conditions at 30℃ for 48 h to obtain Rhodopseudomonas palustris LM6-purple seed culture.

[0105] (3) Inoculate the seed culture at a ratio of 1% into PM liquid culture medium, add 5g / L carbon source, and culture under light and anaerobic conditions at 30℃ for 48h to obtain the fermentation broth of Rhodopseudomonas palustris LM6-purple.

[0106] The carbon source includes one of sodium acetate, sodium malate, glycerol, sodium succinate, sodium bicarbonate, and carbon dioxide.

[0107] This embodiment also provides a method for preparing a compound microbial agent: the above-mentioned Bacillus vesicle LGT-1 fermentation broth and Rhodopseudomonas palustris LM6-purple fermentation broth are centrifuged at 6000 r / min and then resuspended to eliminate the influence of salt in the fermentation broth on the subsequent soil and tobacco. The resuspended liquids are mixed in a volume ratio of 1:1 to prepare a compound microbial agent.

[0108] Example 3: Pot experiment with compound microbial agent

[0109] In this embodiment, the compound bacterial agent prepared by the method of Example 2 was used to study its chloride ion absorption inhibition function.

[0110] The experimental site was located at the Qingdao Experimental Base of the Tobacco Research Institute of the Chinese Academy of Agricultural Sciences in Jimo District, Qingdao City, Shandong Province, China. The experimental tobacco seedling variety was Yan'an No. 1.

[0111] Plastic flowerpots with an inner diameter of 40 cm and a height of 35 cm were used as experimental containers, each filled with 20.0 kg of soil. The potted plants were arranged at a row spacing of 120 cm × 50 cm. This study included four control groups (CK, T1, T2, and T3) for comparison. The CK treatment served as the control group, receiving conventional fertilization according to local standards. T1, T2, and T3 were the treatment groups. In addition to conventional fertilization, the T1 treatment group received 10 mL of the aforementioned compound microbial agent per plant at the crowning stage, vigorous growth stage, and maturity stage. T2 used an equal amount of Bacillus belyssus LGT-1 fermentation broth instead of the compound microbial agent, and T3 used an equal amount of Rhodopseudomonas palustris LM6-purple instead of the compound microbial agent. All other treatments were identical.

[0112] Furthermore, the chloride ion content in the potting soil was tested before transplanting, and transplanting was carried out according to standard procedures. The chloride ion content in the soil was measured during the rosette stage, vigorous growth stage, and maturity stage, using the silver nitrate titration method (NY / T1378-2007). Simultaneously, samples of tobacco leaves at different growth stages were taken, and the chloride ion content was measured directly after blanching, without curing. The chloride ion content in the tobacco leaves was determined using Fourier transform near-infrared spectroscopy.

[0113] 1. Chloride ion content in soil and tobacco leaves at different periods

[0114] Soil chloride ion content was determined using the silver nitrate titration method (NY / T1378-2007), covering four stages from pre-transplanting to maturity (pre-transplanting, seedling stage, vigorous growth stage, and maturity stage). Rhizosphere soil samples were collected by digging 5 cm of soil from the rhizosphere with a shovel. Five pots were randomly selected for each treatment, and a five-point sampling method was used, collecting a total of 200 g of soil from each treatment. The test results are shown in Table 2. Figure 2 .

[0115] Table 2

[0116]

[0117] From Table 2 and Figure 2 The results showed that the soil chloride ion content in the CK group was slightly lower than that in the T1 group during the three stages of the seedling stage, vigorous growth stage, and maturity stage. There were no significant changes in either treatment group before transplanting. Therefore, the data from the two groups at the four stages indicate that the compound microbial agent inhibited the absorption of chloride ions from the soil by tobacco, leaving some chloride ions in the soil.

[0118] 2. Chloride ion content in mature tobacco leaves

[0119] Chloride ion detection in tobacco leaves was performed using Fourier transform near-infrared spectroscopy. Five tobacco plants were randomly selected from each treatment group after three rounds of control measures. Three leaves each from the upper, middle, and lower parts of the leaves were taken, dried, and then ground into powder. 5g of sample was taken from each treatment group for testing. The results are shown in Table 3. (Table 3 is used to prepare the sample.) Figure 3 .

[0120] Table 3

[0121]

[0122] From Table 3 and Figure 3 The results showed that the chloride ion content in the tobacco leaves of the CK group was significantly higher than that in the tobacco leaves of the T1 group, indicating that the above-mentioned compound microbial agent has a certain effect on inhibiting chloride ions in tobacco leaves, and provides new ideas and methods for the control of chloride ions in tobacco leaves.

[0123] The above description is merely an embodiment of this application and is not intended to limit the scope of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the scope of the claims of this application.

Claims

1. A microbial agent for inhibiting chloride ion absorption in tobacco, characterized in that, The bacterial agent is composed of Bacillus vesiculosus (B. vesiculosus) Bacillus velezensis ) and Rhodopseudomonas palustris ( Rhodopseudomonas palustris The composition includes: Bacillus belyssus LGT-1, with accession number CGMCC No. 24342; and Rhodopseudomonas palustris LM6-purple, with accession number CGMCC No. 1.8929.

2. The method for preparing the microbial agent for inhibiting chloride ion absorption in tobacco as described in claim 1, characterized in that, The method includes the following steps: Step 1: Ferment Bacillus belyssus to obtain Bacillus belyssus fermentation broth for later use; the Bacillus belyssus is Bacillus belyssus LGT-1, with preservation number CGMCC No. 24342; Step 2: Ferment Rhodopseudomonas palustris to obtain Rhodopseudomonas palustris fermentation broth for later use; the Rhodopseudomonas palustris is Rhodopseudomonas palustris LM6-purple, with the preservation number CGMCC No.1.8929; Step 3: Mix the Bacillus vesicularis fermentation broth and Rhodopseudomonas palustris fermentation broth to obtain the inoculum.

3. The preparation method according to claim 2, characterized in that, The viable count of the *Bacillus belyssus* was 9.21 × 10⁻⁶. 8 cfu / mL.

4. The preparation method according to claim 2, characterized in that, The viable count of the *Rhodopseudomonas palustris* was 3 × 10⁻⁶. 9 cfu / mL.

5. The preparation method according to claim 2, characterized in that, The volume ratio of the Bacillus vesicularis fermentation broth to the Rhodopseudomonas palustris fermentation broth is 1:(1-5).

6. The preparation method according to claim 5, characterized in that, The volume ratio of the Bacillus vesicularis fermentation broth to the Rhodopseudomonas palustris fermentation broth is 1:

1.

7. The method according to claim 2, characterized in that, The Bacillus berberis fermentation medium in step one includes: tryptone, yeast extract, and sodium chloride.

8. The method according to claim 7, characterized in that, The Bacillus berberis fermentation medium in step one includes: 1-10 g·L⁻¹ tryptone. -1 1-5 g / L of yeast powder -1 NaCl 1-10 g·L -1 .

9. The method according to claim 8, characterized in that, The Bacillus berberis fermentation medium in step one includes: 10 g·L⁻¹ tryptone. -1 5 g·L yeast powder -1 NaCl 10 g·L -1 .

10. The method according to claim 7, characterized in that, The pH of the Bacillus berberis fermentation medium is 7-7.

5.

11. The method according to claim 10, characterized in that, The pH of the Bacillus berberis fermentation medium was 7.

2.

12. The application of the microbial agent for inhibiting chloride ion absorption in tobacco as described in claim 1, or the preparation method as described in any one of claims 2-11, in inhibiting chloride ion absorption in tobacco, reducing chloride ion content in tobacco, improving tobacco combustion performance, and / or increasing chloride ion content in soil.

13. A method for inhibiting the absorption of chloride ions in tobacco, reducing the chloride ion content in tobacco, and / or improving the combustion performance of tobacco, characterized in that, The method includes: cultivating tobacco using the microbial agent for inhibiting chloride ion absorption in tobacco as described in claim 1.

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