A chloride ion absorption inhibitor for tobacco, its preparation method and application

Abstract

This invention provides a chloride ion absorption inhibitor for tobacco, its preparation method, and its application, relating to the field of microbial engineering technology. This invention is the first to use a compound bacterial agent constructed from *Vibrio natans* and *Rhodopseudomonas palustris* as a chloride ion absorption inhibitor for tobacco. Verification through tobacco pot experiments demonstrated that its inhibitory effect on chloride ion absorption in tobacco leaves can reach up to 37.6%, showing a significant inhibitory effect. It provides two new functional strains for inhibiting chloride ion absorption in tobacco leaves. Furthermore, *Vibrio natans* is a salt-tolerant microorganism, and *Rhodopseudomonas palustris* is a photosynthetic bacterium; both are easy to ferment and produce, with low production costs, making them suitable for widespread application. It can also improve the soil microbial community structure, enhance the synergistic absorption of potassium, nitrogen, and other elements by tobacco plants, and has a certain effect on improving soil fertility, exhibiting green and environmentally friendly characteristics.

Description

Technical Field

[0001] This application relates to the field of microbial engineering technology, and in particular to a chloride ion absorption inhibitor for tobacco, its preparation method, and its application. Background Technology

[0002] Tobacco is an important economic crop in my country, and improving its quality and yield is of great significance. Chlorine is an essential trace element for tobacco, significantly impacting its growth, development, leaf yield, and quality. Combustibility is the most important physical characteristic of tobacco leaves and tobacco products, and also a crucial indicator of tobacco safety, closely related to leaf quality. Chlorine is one of the main chemical components affecting tobacco leaf combustibility; higher chlorine content results in poorer combustibility. Therefore, it is necessary to rationally regulate the chlorine content in tobacco to eliminate the adverse effects of excessive chlorine on leaf quality.

[0003] Current methods for reducing chloride ion content in tobacco leaves include rational irrigation, scientific fertilization, and reasonable cropping systems. These are all conventional methods that have been used for a long time. In addition, there is the chemical method of adding zinc sulfate heptahydrate to reduce chloride content. However, all of the above methods have problems such as being difficult to apply, not being able to be effectively promoted, and having adverse environmental impacts.

[0004] Therefore, there is an urgent need for a green, environmentally friendly, efficient, and feasible method to reduce chloride ions in tobacco leaves. Summary of the Invention

[0005] The purpose of this invention is to provide a chloride ion absorption inhibitor for tobacco, its preparation method, and its application.

[0006] On one hand, this application provides a chloride ion absorption inhibitor, which includes: Vibrio neptunius and / or Rhodopseudomonas palustris.

[0007] Preferred species are Vibrio neptunius and Rhodopseudomonas palustris.

[0008] Further, the sodium-dependent Vibrio is sodium-dependent Vibrio NH87-81, with accession number CGMCC No. 1.8755; preferably, the viable count of the sodium-dependent Vibrio is 5 × 10⁻⁶. 9 cfu / mL -2.0×10 10 cfu / mL; more preferably, 2.0 × 10⁻⁶. 10 cfu / mL.

[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 5 × 10⁻⁶. 9 cfu / mL -2.0×10 10 cfu / mL; more preferably, 2.0 × 10⁻⁶. 10 cfu / mL.

[0010] This application is the first to use a compound microbial agent made from a combination of sodium-dependent Vibrio and Rhodopseudomonas palustris. This compound microbial agent 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 the sodium-dependent Vibrio in this application can be sodium-dependent Vibrio, sodium-dependent Vibrio inoculum, sodium-dependent Vibrio dead bacterial suspension, sodium-dependent Vibrio metabolites, or sodium-dependent Vibrio extract; and the Rhodopseudomonas palustris in this application can be Rhodopseudomonas palustris, Rhodopseudomonas palustris inoculum, Rhodopseudomonas palustris dead bacterial suspension, Rhodopseudomonas palustris metabolites, or Rhodopseudomonas palustris extract.

[0012] Preferably, the sodium-dependent Vibrio is a sodium-dependent Vibrio fermentation broth, and the Rhodopseudomonas palustris is a Rhodopseudomonas palustris fermentation broth.

[0013] Furthermore, the volume ratio of the sodium-dependent Vibrio fermentation broth to the Rhodopseudomonas palustris fermentation broth is 1:(1-5); preferably, 1:2.

[0014] On the other hand, this application also provides a method for preparing the aforementioned chloride ion absorption inhibitor, comprising the following steps:

[0015] Step 1: Fermentation of sodium-dependent Vibrio bacteria to obtain sodium-dependent Vibrio fermentation broth for later use; preferably, the viable count of sodium-dependent Vibrio bacteria in the sodium-dependent Vibrio fermentation broth is 5 × 10⁻⁶. 9 cfu / mL -2.0×10 10 cfu / mL; more preferably, 2.0 × 10⁻⁶. 10 cfu / mL;

[0016] Step 2: Fermenting *Rhodopseudomonas palustris* to obtain *Rhodopseudomonas palustris* fermentation broth for later use; preferably, the viable count of *Rhodopseudomonas palustris* in the fermentation broth is 5 × 10⁻⁶. 9 cfu / mL -2.0×10 10 cfu / mL; more preferably, 2.0 × 10⁻⁶. 10 cfu / mL;

[0017] Step 3: Mix the fermentation broth of *Vibrio natans* and the fermentation broth of *Rhodopseudomonas palustris* to obtain a chloride ion absorption inhibitor.

[0018] Preferably, the volume ratio of the sodium-dependent Vibrio fermentation broth to the Rhodopseudomonas palustris fermentation broth is 1:(1-5); more preferably, 1:2.

[0019] The volume ratio of the sodium-dependent Vibrio broth to the Rhodopseudomonas palustris broth can be any value among 1:1, 1:2, 1:3, 1:4, and 1:5.

[0020] Preferably, step one further includes an activation step, which can be activated using general methods by those skilled in the art.

[0021] The activation method of sodium-dependent Vibrio includes: inoculating sodium-dependent Vibrio into a culture medium and culturing at 25℃-40℃ for 24h-48h; picking a single colony and inoculating it into a culture medium and culturing at 20℃-40℃ and shaking at 100-300r / min for 24h-48h to obtain sodium-dependent Vibrio seed culture.

[0022] Preferably, the culture medium is LB medium with 3 times the amount of sodium chloride.

[0023] Preferably, the activation method of sodium-dependent Vibrio includes: inoculating sodium-dependent Vibrio into a culture medium, culturing at 37°C for 48 hours, picking a single colony and inoculating it into a culture medium, and culturing at 30°C and 220 r / min with shaking for 24 hours to obtain sodium-dependent Vibrio seed culture.

[0024] The activation method of Rhodopseudomonas palustris includes: inoculating Rhodopseudomonas palustris into a culture medium, adding 10-50 mL of 0.1-0.5 mmol / L carbon source, culturing at 25℃-40℃ for 24-48 h, picking a single colony and inoculating it into a culture medium, adding 10-50 mL of 0.1-0.5 mmol / L carbon source, and anaerobic culturing at 20℃-40℃ for 24-48 h to obtain Rhodopseudomonas palustris seed culture.

[0025] Preferably, the culture medium is PM culture medium, and the carbon source is selected from one or more of sodium acetate, sodium malate, glycerol, sodium succinate, sodium bicarbonate and carbon dioxide.

[0026] Preferably, the activation method of Rhodopseudomonas palustris includes: inoculating Rhodopseudomonas palustris into a culture medium, adding 30 mL of 0.5 mmol / L carbon source, culturing at 30°C for 48 h, picking a single colony and inoculating it into a culture medium, adding 30 mL of 0.5 mmol / L carbon source, and anaerobic culturing at 30°C for 24 h to obtain Rhodopseudomonas palustris seed culture.

[0027] Preferably, the *Rhodopseudomonas palustris* seed culture is fermented using conventional methods to obtain *Rhodopseudomonas palustris* fermentation broth, with a viable cell count of 2.0 × 10⁻⁶.10 cfu / mL is sufficient.

[0028] Preferably, the sodium-dependent Vibrio seed culture is fermented using conventional methods to obtain a sodium-dependent Vibrio fermentation broth with a viable cell count of 2.0 × 10⁻⁶. 10 cfu / mL is sufficient.

[0029] In a preferred embodiment, the fermentation method is a multi-stage fermentation, with an inoculum size of 2% for the seed liquid, an inoculum size of 10% for each stage of fermentation, and the fermentation temperature controlled at 30°C.

[0030] Specifically, the fermentation method for the sodium-dependent Vibrio includes: inoculating the sodium-dependent Vibrio at an inoculum size of 2% into LB medium containing 3 times the amount of sodium chloride; controlling the fermentation temperature at 30°C; conducting a two-stage fermentation; with the inoculum size for transitioning from the first stage to the next stage being 10%; and finally achieving a viable cell count of 2.0 × 10⁻⁶ in the fermentation broth. 10 cfu / mL.

[0031] The fermentation method of *Rhodopseudomonas palustris* includes: inoculating *Rhodopseudomonas palustris* at an inoculum size of 2% into PM medium, controlling the fermentation temperature at 30℃, performing two-stage fermentation, with the inoculum size for each stage being 10%, and the final viable cell count in the fermentation broth being 2.0 × 10⁻⁶. 10 cfu / mL.

[0032] Other fermentation conditions can be adjusted by those skilled in the art based on the number of viable cells to ensure that the number of viable cells in the fermentation broth is 2.0 × 10⁻⁶. 10 The concentration of cfu / mL is sufficient; no specific limit is specified here.

[0033] On the other hand, this application also provides a fertilizer containing the aforementioned chloride ion absorption inhibitor.

[0034] On the other hand, this application also provides the application of the chloride ion absorption inhibitor or the fertilizer in any of the following A1)-A5):

[0035] A1) Inhibits the absorption of chloride ions by tobacco;

[0036] A2) Reduce the chloride ion content in tobacco;

[0037] A3) Improves soil physical and chemical properties;

[0038] A4) Improve the structure of the soil microbial community;

[0039] A5) Enrich the soil microbial community.

[0040] Preferably, the tobacco described in A1) and A2) includes tobacco leaves.

[0041] 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.

[0042] Among them, the soil described in A3)-A5) is soil for growing tobacco.

[0043] The improvement of soil physicochemical properties includes increasing soil pH and increasing soil AK, AP, and NO. 3- -N, NH 4+ -N content improves soil pH and enhances soil fertility by increasing the effective components in the soil.

[0044] Preferably, improving the soil microbial community structure includes increasing the proportion of dominant phyla in the soil, including Actinobacteriota, Proteobacteria, and Chloroflexi.

[0045] Preferably, enriching the soil microbial community includes increasing the number of soil microbial communities.

[0046] 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, improving the physical and chemical properties of soil, improving the structure of soil microbial communities and / or enriching soil microbial communities, the method comprising: cultivating tobacco using the chloride ion absorption inhibitor or the fertilizer.

[0047] Those skilled in the art can use common methods to cultivate tobacco using the chloride ion absorption inhibitor or fertilizer described in this application.

[0048] In a preferred embodiment, the method includes: directly diluting and using a tobacco chloride ion absorption inhibitor, for example, diluting a compound microbial agent 50-100 times, and applying it by watering, dipping the roots, or drenching the roots.

[0049] In another preferred embodiment, the method includes: adding a tobacco chloride ion absorption inhibitor to fertilizer to make a fertilizer product that inhibits the absorption of chloride ions by tobacco plants for use, for example, diluting a compound microbial agent 50-100 times and adding it to fertilizer, and applying it by means of pond application, hole application, strip application, ring application, etc.

[0050] Preferably, the fertilizer is an organic fertilizer, and more preferably, a tobacco compound fertilizer.

[0051] This application uses a compound microbial agent obtained by combining sodium-dependent Vibrio and Rhodopseudomonas palustris to cultivate tobacco. It can inhibit the absorption of chloride ions by tobacco, reduce the chloride ion content of tobacco, and improve the physical and chemical properties of soil, improve the structure of soil microbial community and enrich soil microbial flora during the planting process. This is conducive to the sustainable use of soil, green and environmentally friendly, and provides a new functional component for tobacco planting.

[0052] On the other hand, this application also provides tobacco cultivated by the method described above.

[0053] Preferably, the tobacco has a low chloride ion content; specifically, the chloride ion content in the tobacco is <0.8%; more preferably, <0.46%.

[0054] Preferably, the tobacco comprises tobacco leaves; more preferably, the tobacco leaves comprise upper leaves, middle leaves, and lower leaves; even more preferably, lower leaves.

[0055] 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.

[0056] On the other hand, this application also provides the use of the tobacco in the preparation of cigarettes.

[0057] Preferably, the cigarette has good combustibility.

[0058] The present invention has the following beneficial effects:

[0059] 1. This invention is the first to use a compound bacterial agent constructed from sodium-dependent Vibrio and Rhodopseudomonas palustris as an inhibitor of chloride ion absorption in tobacco. Verification through tobacco pot experiments showed that its inhibitory effect on chloride ion absorption in tobacco leaves can reach up to 37.6%, demonstrating a significant inhibitory effect. This invention provides two new functional strains for the functional component of inhibiting chloride ion absorption in tobacco leaves. Furthermore, sodium-dependent Vibrio is a salt-tolerant microorganism, and Rhodopseudomonas palustris is a photosynthetic bacterium; both are easy to ferment and produce, have low production costs, and are suitable for widespread application.

[0060] 2. The tobacco chloride ion absorption inhibitor (compound microbial agent) provided by this invention contains two salt-tolerant bacteria. It has the advantages of convenient operation, no pollution, low cost, and high activity. It can also improve the soil microbial community structure, enhance the synergistic absorption of elements such as potassium and nitrogen in tobacco plants, and has a certain effect on improving soil fertility. It has green and environmentally friendly characteristics. Attached Figure Description

[0061] 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:

[0062] Figure 1 A graph showing the chloride ion content of tobacco leaves in different parts;

[0063] Figure 2 A graph showing the chloride ion content in soil during different tobacco-growing periods;

[0064] Figure 3 A bar chart of soil microbial community in tobacco-growing soil;

[0065] Figure 4 Venn diagram showing the soil species composition for tobacco cultivation. Detailed Implementation

[0066] 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.

[0067] 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.

[0068] 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.

[0069] Unless otherwise specified, in the following embodiments, reagents or instruments whose manufacturers are not indicated are all conventional products that can be purchased commercially.

[0070] 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.

[0071] 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.

[0072] 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.

[0073] Among them, Vibrio neptunius NH87-81, with accession number CGMCC No. 1.8755, was purchased from the China General Microbiological Culture Collection Center (CGMCC); Rhodopseudomonas palustris LM6-purple, with accession number CGMCC No. 1.8929, was also purchased from the China General Microbiological Culture Collection Center (CGMCC).

[0074] The culture medium used in this invention is as follows:

[0075] LB liquid medium (1000 mL) with 3 times sodium chloride: 10.0 g peptone, 15.0 g sodium chloride, 10.0 g yeast extract, balance water, pH 7.2. Sterilize at 0.15 MPa and 121 °C for 30 min and set aside.

[0076] LB solid medium (1000 mL) with 3 times sodium chloride: 10.0 g peptone, 15.0 g sodium chloride, 10.0 g yeast extract, 18 g agar powder, balance water, pH 7.2. Sterilize at 0.15 MPa and 121 °C for 30 min and set aside.

[0077] 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, balance 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.

[0078] 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, balance water. Sterilize at 0.15MPa, 121℃ for 30min. The concentrate (100mL) is formulated as follows: 2g aminotriacetic acid, 2.89g anhydrous magnesium sulfate, 5.03g calcium chloride, 0.0198g ferrous sulfate heptahydrate. Sterilize at 121℃ for 20min.

[0079] In the following examples, the compound microbial agent for inhibiting chloride ion absorption in tobacco leaves can be used directly by diluting it as a microbial agent. For example, the compound microbial agent can be diluted 50-100 times and applied by watering, dipping roots, or drenching roots. Alternatively, it can be added to fertilizers to make fertilizer products that inhibit the absorption of chloride ions by tobacco plants. For example, the compound microbial agent can be diluted 50-100 times by weight and added to organic fertilizers to make bio-organic fertilizers, which can be applied by pond application, hole application, strip application, or ring application.

[0080] 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.

[0081] In the following examples, unless otherwise specified, % means wt%, i.e., weight percentage.

[0082] Example 1: Compound microbial agent for inhibiting chloride ion absorption in tobacco leaves

[0083] This embodiment provides a compound microbial agent that inhibits the absorption of chloride ions by tobacco leaves, comprising *Vibrio natophilus* and *Rhodopseudomonas palustris*, wherein *Vibrio natophilus* is designated NH87-81 and *Rhodopseudomonas palustris* is designated LM6-purple.

[0084] The preparation method includes the following steps:

[0085] Step 1: Under aseptic conditions, inoculate sodium-dependent Vibrio NH87-81, stored at -80℃, onto LB agar plates (solid) containing 3 times the amount of sodium chloride. Incubate at 37℃ for 48 hours. Then, pick a single colony and inoculate it onto LB liquid medium containing 3 times the amount of sodium chloride. Incubate at 30℃ with shaking at 220 rpm for 24 hours to obtain the sodium-dependent Vibrio seed culture (OD). 600 (Value around 2), for backup;

[0086] Under aseptic conditions, *Rhodopseudomonas palustris* LM6-purple, preserved at -80℃, was inoculated onto PM agar plates (solid) with 30 mL of 0.5 mmol / L carbon source. The plates were incubated at 30℃ for 48 h. Single colonies were then transferred to PM liquid medium, and 30 mL of 0.5 mmol / L carbon source was added. The plates were then incubated under anaerobic light at 30℃ for 24 h to obtain the *Rhodopseudomonas palustris* seed culture (OD). 600 (Value around 2), for later use. The carbon source includes one of sodium acetate, sodium malate, glycerol, sodium succinate, sodium bicarbonate, and carbon dioxide.

[0087] Step 2: Ferment the sodium-dependent Vibrio spp. seed culture and Rhodopseudomonas palustris seed culture obtained in Step 1 in their respective culture media (the culture media and seed culture stages are the same). Ferment according to conventional methods to obtain sodium-dependent Vibrio spp. fermentation broth (viable cell count 2.0 × 10⁻⁶) respectively. 10 CFU / mL), Rhodopseudomonas palustris fermentation broth (viable count 2.0 × 10⁻⁶). 10 (cfu / mL). The fermentation method can be multi-stage fermentation, with an inoculum size of 2% for the seed culture and 10% for each subsequent fermentation stage. The fermentation temperature is controlled at 30℃. The two fermentation broths are mixed at a volume ratio of sodium-dependent Vibrio: Rhodopseudomonas palustris = 1.0:2.0 to obtain a compound microbial agent for inhibiting chloride ion absorption in tobacco leaves.

[0088] Example 2: Chloride Ion Absorption Inhibition Experiment in Tobacco Plants

[0089] In this embodiment, a pot experiment was conducted using Yan'an No. 6 tobacco as the test plant, specifically including:

[0090] Plastic flowerpots with an inner diameter of 40cm and a height of 35cm were used as experimental containers, each containing 20.0kg of soil. The potted plants were arranged with a row spacing of 120cm × 50cm. The experimental group (T1) was set up as follows: the tobacco chloride ion absorption inhibitor compound microbial agent from Example 1 was added twice during the rosette stage and vigorous growth stage. Soil chloride ion content was measured 20 days after the control period. Simultaneously, samples of mature tobacco leaves were taken from different parts, and chloride ion content was measured directly after blanching. The effects of adding the tobacco chloride ion absorption inhibitor compound microbial agent on soil microorganisms were analyzed. The compound microbial agent was diluted 50 times and added to the roots by irrigating with 1L of water. An equal amount of *Rhodotorula glutinis* was used to replace the tobacco chloride ion absorption inhibitor compound microbial agent as experimental group (T2). An equal amount of *Vibrio natans* was used to replace the tobacco chloride ion absorption inhibitor compound microbial agent as experimental group (T3). The control group (CK) was also irrigated with 1L of water. All other agricultural operations were completely identical.

[0091] 1. Determination of chloride ion content in different parts of tobacco leaves at maturity

[0092] In this embodiment, the chloride ion content of tobacco leaves was extracted from different parts of the leaves using hot distillation water. Potassium chromate was used as an indicator, and the chloride ion content in different parts of the tobacco leaves was calculated by titration with silver nitrate standard solution. The chloride ion content results are as follows: Figure 1 As shown in Table 1.

[0093] Table 1. Chloride ion content of tobacco leaves at different parts during maturity.

[0094]

[0095] From Table 1 and Figure 1 It can be seen that the chloride ion content of mature tobacco leaves in different parts of the plant with the compound microbial agent containing chloride ion absorption inhibitor was lower than that of the control group, and the upper leaves showed the most significant inhibitory effect, with an inhibition rate of 37.6%.

[0096] The chloride ion content of tobacco leaves treated with *Rhodopseudomonas palustris* (R. pal) was basically the same as that of the control group. The chloride ion content of tobacco leaves treated with *Vibrio natans* (Vn) was lower than that of the control group and the group treated with *Rhodopseudomonas palustris* alone, but higher than that of the group treated with both *Vibrio natans* and *Rhodopseudomonas palustris*. This demonstrates that the main function of *Rhodopseudomonas palustris* is to promote photosynthetic carbon fixation in plant roots, and that the combination of *Rhodopseudomonas palustris* and *Vibrio natans* can have a synergistic effect.

[0097] 2. Determination of chloride ion content in tobacco-growing soil at different stages

[0098] In this embodiment, the chloride ion content in the tobacco-growing soil was determined according to the People's Republic of China Agricultural Industry Standard NY / T1378-2007, "Determination Method for Soil Chloride Ion Content". The results of the chloride ion content in the tobacco-growing soil are as follows: Figure 2 As shown in Table 2.

[0099] Table 2 Chloride ion content in tobacco-growing soil at different stages

[0100]

[0101] From Table 2 and Figure 2 It can be concluded that the soil in the tobacco-growing group treated with the compound microbial agent that adds chloride ion absorption inhibitors has a higher chloride ion content than the control group during the rosette and maturity stages. This means that the soil has a higher residual chloride ion content and the tobacco leaves absorb less chloride ion, proving that the compound microbial agent has a significant effect on inhibiting the absorption of chloride ions by tobacco.

[0102] 3. Determination of soil physicochemical properties

[0103] The physicochemical properties of tobacco-grown soil were determined using the following methods: 10g of air-dried soil was mixed with 25mL of distilled water using a glass electrode digital pH meter to determine the pH value of the sample. Available phosphorus (AP) in the soil was extracted with 0.5M sodium bicarbonate and measured using the molybdenum blue colorimetric method. Available potassium (AK) in the soil was extracted with ammonium acetate and its content was measured using flame photometry. The chloride ion content in the soil was measured by titrating the soil extract with silver nitrate according to the Agricultural Industry Standard of the People's Republic of China. Nitrate nitrogen (NOx) was measured using the phenol disulfonic acid colorimetric method. 3- -N); Soil ammonium nitrogen (NH4+) was measured using the KCl extraction-indophenol blue colorimetric method. 4+ -N). Soil physicochemical properties data are shown in Table 3.

[0104] Table 3 Physicochemical data of tobacco-growing soil

[0105]

[0106] Table 3 shows that the addition of compound microbial agents to the tobacco plantation soil treated with chloride ion inhibitors alters the soil's physical and chemical properties, enhances the synergistic absorption of potassium, nitrogen, and other elements by tobacco plants, and also has a certain effect on improving soil fertility.

[0107] 4. The effects of compound microbial agents containing chloride ion inhibitors from tobacco leaves on soil microorganisms

[0108] In this embodiment, 16S RNA high-throughput sequencing was performed on tobacco-grown soil at maturity to analyze soil diversity composition.

[0109] Soil microbial community bar chart as follows Figure 3 As shown in the figure, compared with the control group, the compound microbial agent added to the tobacco chloride ion absorption inhibitor treatment group can improve the microbial community structure and enhance the proportion of dominant phyla, especially Actinobacteriota, Proteobacteria and Chloroflexi.

[0110] Venn diagram of species composition Figure 4 As shown in the figure, the treatment group with added tobacco chloride ion absorption inhibitor compound microbial agent can significantly increase the number of soil microbial communities and enrich the soil flora.

[0111] In summary, the addition of a compound microbial agent to inhibit chloride ion absorption from tobacco leaves can suppress the absorption of chloride ions from the soil by tobacco plants, causing chloride ions to deposit in the soil. Simultaneously, it can improve the physicochemical properties of tobacco-growing soil, significantly increase the number of soil microorganisms, and enrich the soil flora. Therefore, the compound microbial agent to inhibit chloride ion absorption from tobacco plants and improve the soil flora achieves the goal of "chlorine reduction" in tobacco leaves.

[0112] 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 chloride ion absorption inhibitor for tobacco, characterized in that, The chloride ion absorption inhibitor is composed of sodium-dependent Vibrio ( Vibrio neptunius ) and Rhodopseudomonas palustris ( Rhodopseudomonas palustris The composition includes: the sodium-dependent Vibrio species is sodium-dependent Vibrio NH87-81, with accession number CGMCC No. 1.8755; the Rhodopseudomonas palustris species is Rhodopseudomonas palustris LM6-purple, with accession number CGMCC No. 1.8929.

2. The chloride ion absorption inhibitor for tobacco according to claim 1, characterized in that, The volume ratio of the sodium-dependent Vibrio and Rhodopseudomonas palustris is 1:(1-5).

3. The chloride ion absorption inhibitor for tobacco according to claim 2, characterized in that, The volume ratio of the sodium-dependent Vibrio and Rhodopseudomonas palustris is 1:

2.

4. The method for preparing a chloride ion absorption inhibitor for tobacco according to any one of claims 1-3, characterized in that, Includes the following steps: Step 1: Fermentation of sodium-dependent Vibrio to obtain sodium-dependent Vibrio fermentation broth for later use; the sodium-dependent Vibrio is sodium-dependent Vibrio NH87-81, with accession number CGMCC No.1.8755; 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 fermentation broth of *Vibrio natans* and the fermentation broth of *Rhodopseudomonas palustris* to obtain a chloride ion absorption inhibitor.

5. The preparation method according to claim 4, characterized in that, The viable count of natriuretic Vibrio in the fermentation broth was 5 × 10⁻⁶. 9 cfu / mL -2.0×10 10 cfu / mL.

6. The preparation method according to claim 5, characterized in that, The viable count of sodium-dependent Vibrio in the fermentation broth was 2.0 × 10⁻⁶. 10 cfu / mL.

7. The preparation method according to claim 4, characterized in that, The viable count of *Rhodopseudomonas palustris* in the fermentation broth was 5 × 10⁻⁶. 9 cfu / mL -2.0×10 10 cfu / mL.

8. The preparation method according to claim 7, characterized in that, The viable count of *Rhodopseudomonas palustris* in the fermentation broth was 2.0 × 10⁻⁶. 10 cfu / mL.

9. The preparation method according to claim 4, characterized in that, The volume ratio of the sodium-dependent Vibrio broth to the Rhodopseudomonas palustris broth is 1:(1-5).

10. The preparation method according to claim 9, characterized in that, The volume ratio of the sodium-dependent Vibrio broth to the Rhodopseudomonas palustris broth is 1:

2.

11. A fertilizer, characterized in that, The fertilizer contains a chloride ion absorption inhibitor for tobacco as described in any one of claims 1-3.

12. The use of the chloride ion absorption inhibitor for tobacco as described in any one of claims 1-3 or the fertilizer as described in claim 11 in any one of the following A1)-A5): A1) Inhibits the absorption of chloride ions by tobacco; A2) Reduce the chloride ion content in tobacco; A3) Improves soil physical and chemical properties; A4) Improve the structure of the soil microbial community; A5) Enrich the soil microbial community.

13. A method for inhibiting the absorption of chloride ions by tobacco, reducing the chloride ion content in tobacco, improving soil physicochemical properties, improving soil microbial community structure, and / or enriching soil microbial flora, characterized in that, The method includes cultivating tobacco using a chloride ion absorption inhibitor for tobacco as described in any one of claims 1-3 or a fertilizer as described in claim 11.

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