Chloride ion absorption inhibitor for tobacco and preparation method and application thereof

Abstract

The invention provides a chloride ion absorption inhibitor for tobacco and a preparation method and application thereof, and relates to the technical field of microbial engineering. According to the invention, vibrio natriticus and rhodopseudomonas palustris are adopted for the first time to construct the complex microbial inoculant as the tobacco chloride ion absorption inhibitor, tobacco pot experiment verification proves that the inhibition effect of the tobacco leaves on chloride ion absorption can reach 37.6% at most, the inhibition effect is obvious, two new functional strains are provided for functional components for inhibiting tobacco leaf chloride ion absorption, and the method is suitable for industrial production. In addition, the vibrio natrieus is a salt-tolerant microorganism, the rhodopseudomonas palustris is a photosynthetic bacterium, and both the vibrio natrieus and the rhodopseudomonas palustris are easy to ferment and produce, low in production cost and suitable for The microbial community structure of the soil can be improved, the synergistic absorption of elements such as potassium and nitrogen of tobacco plants is enhanced, the soil fertility is improved to a certain degree, and the method has the characteristic of being green and environment-friendly.

Description

Technical Field

[0001] The present application relates to the field of microbial engineering technology, and in particular to a chloride ion absorption inhibitor for tobacco, and a preparation method and application thereof. Background Art

[0002] Tobacco is an important economic crop in my country, and the improvement of its quality and yield is of great significance. Chlorine is an essential trace element for tobacco, which has an important impact on tobacco growth and development, tobacco leaf yield and quality. Combustibility is the most important physical property of tobacco leaves and tobacco products. It is also an important indicator for measuring tobacco safety and is closely related to the quality of tobacco leaves. Chlorine is one of the main chemical components that affect the combustibility of tobacco leaves. The higher its content, the worse the combustibility of tobacco leaves. Therefore, it is necessary to reasonably regulate the chlorine content in tobacco to eliminate the adverse effects of excessive chlorine on tobacco leaf quality.

[0003] The existing solutions to reduce the chloride ion content in tobacco leaves include reasonable irrigation, scientific fertilization, and reasonable layout of farming systems, which are conventional methods that have been used for a long time. In addition, there is also a chemical method of reducing chlorine by adding zinc sulfate heptahydrate. However, the above methods are difficult to apply, cannot be effectively promoted, and have adverse effects on the environment.

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

[0005] The purpose of the present invention is to provide a chloride ion absorption inhibitor for tobacco and a preparation method and application thereof.

[0006] In one aspect, the present application provides a chloride ion absorption inhibitor, which includes: Vibrio neptunius and / or Rhodopseudomonas palustris.

[0007] Preferably, Vibrio neptunius and Rhodopseudomonas palustris.

[0008] Furthermore, the Vibrio natriureticum is Vibrio natriureticum NH87-81, with a deposit number of CGMCC No. 1.8755; preferably, the viable count of the Vibrio natriureticum is 5×10 9 cfu / mL-2.0×10 10 cfu / mL; more preferably, 2.0×10 10 cfu / mL.

[0009] Furthermore, the palustris Rhodopseudomonas is palustris LM6-purple, with a preservation number of CGMCC No. 1.8929; preferably, the viable count of the palustris Rhodopseudomonas is 5×10 9 cfu / mL-2.0×10 10 cfu / mL; more preferably, 2.0×10 10 cfu / mL.

[0010] In the present application, Vibrio natrii and Rhodopseudomonas palustris are compounded for the first time to obtain a composite bacterial agent, which has the effect of inhibiting tobacco from absorbing chloride ions, and can therefore 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 can understand that the Vibrio natrii in the present application can be Vibrio natrii, a Vibrio natrii bacterial agent, a Vibrio natrii dead bacterial suspension, a Vibrio natrii metabolite, or a Vibrio natrii extract; the Rhodopseudomonas palustris in the present application can be Rhodopseudomonas palustris, a Vibrio natrii bacterial agent, a Vibrio natrii dead bacterial suspension, a Vibrio natrii metabolite, or a Vibrio natrii extract.

[0012] Preferably, the Vibrio natriuresis is a fermentation broth of Vibrio natriuresis, and the Rhodopseudomonas palustris is a fermentation broth of Rhodopseudomonas palustris.

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

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

[0015] Step 1: fermenting Vibrio natrii to obtain Vibrio natrii fermentation broth for standby use; preferably, the number of viable bacteria of Vibrio natrii in the Vibrio natrii fermentation broth is 5×10 9 cfu / mL-2.0×10 10 cfu / mL; more preferably, 2.0×10 10 cfu / mL;

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

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

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

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

[0020] Preferably, step one also includes an activation step, and those skilled in the art can activate it in a general manner, wherein:

[0021] The activation method of Vibrio natrii comprises: inoculating Vibrio natrii into a culture medium, culturing at 25-40°C for 24-48 hours, picking a single colony and inoculating it into the culture medium, culturing at 20-40°C and 100-300r / min for 24-48 hours, and obtaining a Vibrio natrii seed solution.

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

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

[0024] The activation method of Rhodopseudomonas palustris comprises: inoculating Rhodopseudomonas palustris into a culture medium, adding 10-50 mL of a 0.1-0.5 mMol / L carbon source, culturing at 25°C-40°C for 24h-48h, picking a single colony and inoculating it into the culture medium, adding 10-50 mL of a 0.1-0.5 mMol / L carbon source, culturing at 20°C-40°C for 24h-48h anaerobically, and obtaining a seed solution of Rhodopseudomonas palustris.

[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 comprises: inoculating Rhodopseudomonas palustris into a culture medium, adding 30 mL of a 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 a 0.5 mMol / L carbon source, 30°C, and culturing anaerobically for 24 h to obtain a seed solution of Rhodopseudomonas palustris.

[0027] Preferably, the Rhodopseudomonas palustris seed solution is fermented according to a conventional method to obtain a Rhodopseudomonas palustris fermentation solution with a viable cell count of 2.0×1010 cfu / mL is sufficient.

[0028] Preferably, the Vibrio natrii seed solution is fermented according to a conventional method to obtain a Vibrio natrii fermentation solution with a viable cell count of 2.0×10 10 cfu / mL is sufficient.

[0029] In a preferred embodiment, the fermentation method is a multi-stage fermentation, the inoculation amount of the seed liquid is 2%, the inoculation amount from the first stage to the next stage of fermentation is 10%, and the fermentation temperature is controlled at 30°C.

[0030] Specifically, the fermentation method of Vibrio natrii includes: inoculating Vibrio natrii with an inoculation amount of 2% into LB medium with 3 times sodium chloride, controlling the fermentation temperature at 30°C, and performing secondary fermentation. The inoculation amount from the first stage to the next stage is 10%, and the number of viable bacteria in the final fermentation liquid is 2.0×10 10 cfu / mL.

[0031] The fermentation method of Rhodopseudomonas palustris comprises: inoculating Rhodopseudomonas palustris into PM culture medium at an inoculation amount of 2%, controlling the fermentation temperature at 30°C, and performing secondary fermentation. The inoculation amount from the first stage to the next stage of fermentation is 10%, and the number of live bacteria in the final fermentation liquid is 2.0×10 10 cfu / mL.

[0032] The remaining fermentation conditions can be adjusted by technicians in this field according to the number of viable bacteria to ensure that the number of viable bacteria in the fermentation liquid is 2.0×10 10 cfu / mL is sufficient and is not specifically limited here.

[0033] On the other hand, the present application also provides a fertilizer, which contains the chloride ion absorption inhibitor.

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

[0035] A1) inhibiting tobacco from absorbing chloride ions;

[0036] A2) reducing the chloride ion content of tobacco;

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

[0038] A4) Improve soil microbial community structure;

[0039] A5) Enrich soil microbial flora.

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

[0041] In a preferred embodiment, Yan'an No. 6 tobacco is used as a test plant for experiments in this application. Those skilled in the art can understand that the method of this application can be extended to the cultivation of different varieties of tobacco to achieve the same technical effect. Considering the test cost and experimental time issues, they are not exemplified 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 physical and chemical properties includes increasing soil pH, increasing soil AK, AP, NO 3- -N, NH 4+ -N content, that is, improving soil acidity and alkalinity, and improving soil fertility by increasing the effective ingredients in the soil.

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

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

[0046] On the other hand, the present application also provides a method for inhibiting tobacco from absorbing chloride ions, reducing the chloride ion content of tobacco, improving soil physical and chemical properties, improving soil microbial community structure and / or enriching soil microbial flora, the method comprising: cultivating tobacco using the chloride ion absorption inhibitor or the fertilizer.

[0047] Those skilled in the art can adopt a general method to cultivate tobacco using the chloride ion absorption inhibitor or the fertilizer described in this application.

[0048] In a preferred embodiment, the method comprises: directly diluting the tobacco leaf chloride ion absorption inhibitor for use, for example, diluting the composite bacterial agent by 50-100 times, and applying it by pouring, dipping the roots, or irrigating the roots.

[0049] In another preferred embodiment, the method comprises: adding a tobacco leaf chloride ion absorption inhibitor to a fertilizer to prepare a fertilizer product that inhibits tobacco plants from absorbing chloride ions for use, for example, diluting the composite bacterial agent 50-100 times and adding it to the fertilizer, and applying it by pond application, hole application, strip application, ring application, etc.

[0050] Preferably, the fertilizer may be an organic fertilizer, more preferably, a tobacco compound fertilizer.

[0051] The present application adopts a composite bacterial agent obtained by compounding Vibrio natrii and Rhodopseudomonas palustris to cultivate tobacco, which can inhibit tobacco from absorbing chloride ions and reduce the chloride ion content of tobacco. In the planting process, it can improve the physical and chemical properties of the soil, improve the structure of the soil microbial community and enrich the soil microbial flora, which is beneficial to the sustainable use of the soil and green environmental protection, and provides a new effective ingredient for tobacco planting.

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

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

[0054] Preferably, the tobacco includes tobacco leaves; more preferably, the tobacco leaves include upper leaves, middle leaves, and lower leaves; more preferably, lower leaves.

[0055] The present application provides tobacco with a low chloride ion content. Compared with ordinary tobacco, the tobacco leaves of the tobacco have a low chloride ion content and good combustibility, are more suitable for making cigarettes, and provide an excellent material for cigarette making.

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

[0057] Preferably, the cigarette has good combustibility.

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

[0059] 1. The present invention adopts Vibrio natrii and Rhodopseudomonas palustris to construct a composite bacterial agent for the first time as a tobacco chloride ion absorption inhibitor. The tobacco pot test shows that the inhibition effect of tobacco leaf absorption of chloride ions can reach up to 37.6%, and the inhibition effect is obvious. Two new functional strains are provided for the functional components inhibiting the absorption of chloride ions by tobacco leaves. Moreover, Vibrio natrii is a salt-tolerant microorganism, and Rhodopseudomonas palustris is a photosynthetic bacterium. Both are easy to ferment and produce, with low production cost, and are suitable for wide promotion.

[0060] 2. The tobacco chloride ion absorption inhibitor (composite bacterial agent) provided by the present invention contains two salt-tolerant bacteria, has the advantages of easy operation, no pollution, low cost, high activity, etc., can also improve the structure of soil microbial communities, enhance the synergistic absorption of elements such as potassium and nitrogen in tobacco plants, and has a certain effect on improving soil fertility, and has the characteristics of being green and environmentally friendly. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] The drawings described herein are used to provide a further understanding of the present application and constitute a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings:

[0062] Figure 1 This is a graph of chloride ion content in different parts of tobacco leaves;

[0063] Figure 2 This is a graph of chloride ion content in tobacco-growing soil at different periods;

[0064] Figure 3 This is a bar graph of soil microbial communities in tobacco-growing soils;

[0065] Figure 4 This is the Venn diagram of species composition of tobacco-growing soil. DETAILED DESCRIPTION

[0066] In order to more clearly explain the overall concept of the present application, the following is described in detail in conjunction with the accompanying drawings of the specification by way of embodiment. In the following description, a large number of specific details are provided to provide a more thorough understanding of the present invention. However, it is obvious to those skilled in the art that the present invention can be implemented without one or more of these details. In other examples, in order to avoid confusion with the present invention, some technical features well known in the art are not described.

[0067] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0068] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0069] Unless otherwise specified, in the following embodiments, the reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased commercially.

[0070] Before further describing the 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 terms used in the examples of the present invention are intended to describe specific embodiments, rather than to limit the scope of protection of the present invention. The test methods in the following examples without specifying specific conditions are generally carried out under conventional conditions or under conditions recommended by the manufacturers.

[0071] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to realize the present invention.

[0072] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional techniques in microbiology, biochemistry, analytical chemistry, cell culture, and related fields conventional in the technical field.

[0073] Among them, the preservation number of Vibrio neptunius NH87-81 is CGMCC No.1.8755, which was purchased from the General Microbiology Center of China Culture Collection Administration (CGMCC); the preservation number of Rhodopseudomonas palustris LM6-purple is CGMCC No.1.8929, which was purchased from the General Microbiology Center of China Culture Collection Administration (CGMCC).

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

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

[0076] LB solid medium (1000 mL) with 3 times sodium chloride: 10.0 g of peptone, 15.0 g of sodium chloride, 10.0 g of yeast extract, 18 g of agar powder, the balance is water, pH 7.2. Sterilize at 0.15 MPa, 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, and the balance is water. Sterilize at 0.15MPa, 121℃ for 30min and set aside. The formula of the concentrate (100mL) is: 2g aminotriacetic acid, 2.89g anhydrous magnesium sulfate, 5.03g calcium chloride, 0.0198g ferrous sulfate heptahydrate. Sterilize at 121℃ for 20min and set aside.

[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, and the balance is water. Sterilize at 0.15MPa, 121℃ for 30min and set aside. The formula of the concentrate (100mL) is: 2g aminotriacetic acid, 2.89g anhydrous magnesium sulfate, 5.03g calcium chloride, 0.0198g ferrous sulfate heptahydrate. Sterilize at 121℃ for 20min and set aside.

[0079] In the following embodiments, the composite bacterial agent of chloride ion absorption inhibitor for tobacco leaves can be directly diluted and used as a microbial agent, for example, the composite bacterial agent is diluted 50-100 times and applied by pouring, dipping roots, or irrigating roots; it can also be added to fertilizers to make fertilizer products that inhibit tobacco plants from absorbing chloride ions, for example, the composite bacterial agent is diluted 50-100 times by weight and added to organic fertilizers to make biological organic fertilizers, which are applied by pond application, hole application, strip application, ring application, etc.

[0080] In addition, the "water" mentioned in the present invention includes any feasible water that can be used in the art, such as deionized water, distilled water, ion exchange 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 Composite bacterial agent for tobacco leaf chloride ion absorption inhibitor

[0083] The present embodiment provides a composite bacterial agent of tobacco leaf chloride absorption inhibitor having the function of inhibiting tobacco leaf from absorbing chloride ions, comprising Vibrio natrii and Rhodopseudomonas palustris, wherein Vibrio natrii is numbered NH87-81 and Rhodopseudomonas palustris is numbered LM6-purple.

[0084] The preparation method comprises the following steps:

[0085] Step 1: Under sterile conditions, inoculate the Vibrio natrii NH87-81 stored at -80°C in a LB plate (solid) medium containing 3 times sodium chloride, culture at 37°C for 48 hours, then pick a single colony and inoculate it into a LB liquid medium containing 3 times sodium chloride, and culture it at 30°C and 220r / min for 24 hours to obtain a Vibrio natrii seed solution (OD 600 The value is about 2), for backup;

[0086] Under sterile conditions, the palustris Rhodopseudomonas LM6-purple stored at -80°C was inoculated into a PM plate (solid) medium, 30 mL of 0.5 mMol / L carbon source was added, and cultured at 30°C for 48 h. A single colony was picked and transferred to a PM liquid medium, 30 mL of 0.5 mMol / L carbon source was added, and cultured at 30°C for 24 h under light anaerobically to obtain a palustris Rhodopseudomonas seed solution (OD 600 The carbon source comprises one of sodium acetate, sodium malate, glycerol, sodium succinate, sodium bicarbonate and carbon dioxide.

[0087] Step 2: The Vibrio natrii seed solution and Rhodopseudomonas palustris seed solution obtained in step 1 are fermented in corresponding culture medium (culture medium and seed solution culture stage are the same), and fermentation is performed according to conventional methods to obtain Vibrio natrii fermentation solution (viable cell count is 2.0×10 10 cfu / mL), Rhodopseudomonas palustris fermentation broth (viable count 2.0×10 10 cfu / mL). The fermentation method can be multi-stage fermentation, the inoculation amount of the seed liquid is 2%, the inoculation amount from the first stage to the next stage of fermentation is 10%, and the fermentation temperature is controlled at 30°C. The two fermentation broths obtained are mixed in a volume ratio of Vibrio natrii: Rhodopseudomonas palustris = 1.0:2.0 to obtain a composite bacterial agent of chloride ion absorption inhibitor for tobacco leaves.

[0088] Example 2 Tobacco strain chloride ion absorption inhibition experiment

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

[0090] The test container is a plastic flower pot with an inner diameter of 40 cm and a height of 35 cm, and each pot is filled with 20.0 kg of soil. Potted plants are placed according to a row spacing of 120 cm × 50 cm. The experimental group (T1) is set as follows: the compound bacterial agent of the tobacco leaf chloride ion absorption inhibitor in Example 1 is added twice during the clustering period and the vigorous growth period, and the soil is taken 20 days after the control to determine the soil chloride ion content; at the same time, the tobacco leaf samples in the mature period are sampled by different parts, and the chloride ion content is directly measured after the greening; the effect of adding the compound bacterial agent of the tobacco leaf chloride ion absorption inhibitor on soil microorganisms is analyzed. The compound bacterial agent is diluted 50 times when added, and a total of 1L of water is added for root irrigation. An equal amount of Rhodomonas palustris is used to replace the compound bacterial agent of the tobacco leaf chloride ion absorption inhibitor as the experimental group (T2). An equal amount of Vibrio natrii is used to replace the compound bacterial agent of the tobacco leaf chloride ion absorption inhibitor as the experimental group (T3). The control group (CK) also adds 1L of clean water for root irrigation. The rest of the farming operations are exactly the same.

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

[0092] In this embodiment, the chloride ion content of tobacco leaves is extracted by hot distilled water method, potassium chromate is used as an indicator, and silver nitrate standard solution is used for titration to calculate the chloride ion content of different parts of tobacco leaves. Figure 1 And as shown in Table 1.

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

[0094]

[0095] From Table 1 and Figure 1 It can be seen that the chloride ion content of tobacco leaves at different parts of maturity after adding the compound bacterial agent of tobacco leaf chloride ion absorption inhibitor was lower than that of the control group, and the inhibition effect was most significant in the upper leaves, with an inhibition rate of 37.6%.

[0096] The chloride ion content of tobacco leaves in the treatment group with single R. palustris (R.pal) was basically the same as that in the control group, and the chloride ion content of tobacco leaves in the treatment group with single Vibrio natrii (Vn) was lower than that in the control group and the treatment group with single R. palustris, but higher than that in the treatment group with both R. palustris and R. palustris. This proves that the main function of R. palustris is to promote photosynthetic carbon fixation in plant roots, and that the combination of R. palustris and Vibrio natrii can play a synergistic role.

[0097] 2. Determination of Chloride Ion Content in Tobacco Planting Soil at Different Stages

[0098] In this embodiment, the determination of chloride ion content in tobacco-growing soil is carried out using the determination method of soil chloride ion content in the agricultural industry standard of the People's Republic of China NY / T1378-2007. Figure 2 And as shown in Table 2.

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

[0100]

[0101] From Table 2 and Figure 2 It can be concluded that the chloride ion content in the tobacco-planting soil of the group treated with the composite bacterial agent which is a chloride ion absorption inhibitor for tobacco leaves was higher than that in the control group during the clumping and maturity stages, that is, the residual chloride ion content in the soil was high and the tobacco leaves absorbed less chloride ions, which proves that the composite bacterial agent was effective in inhibiting the absorption of chloride ions by tobacco.

[0102] 3. Determination of soil physical and chemical properties

[0103] The physical and chemical properties of tobacco-growing soil were determined by mixing 10 g of air-dried soil with 25 mL of distilled water using a glass electrode digital pH meter to determine the pH value in the sample. Soil available phosphorus (AP) was extracted with 0.5 M sodium bicarbonate and measured using the molybdenum blue colorimetric method. Available potassium in the soil was extracted with ammonium acetate, and the available potassium (AK) content was measured using flame photometry. According to the Agricultural Industry Standards of the People's Republic of China, the chloride ion content of the soil extract was titrated with silver nitrate to measure the chloride ion content of the soil. Nitrate nitrogen (NO 3- -N); soil ammonium nitrogen (NH 4+ -N). The soil physical and chemical properties data are shown in Table 3.

[0104] Table 3 Physical and chemical data of tobacco-growing soil

[0105]

[0106] It can be concluded from Table 3 that adding the tobacco leaf chloride ion absorption inhibitor and composite bacterial agent to the tobacco-growing soil will change the physical and chemical properties of the soil, enhance the synergistic absorption of potassium, nitrogen and other elements by tobacco plants, and also have a certain effect on improving soil fertility.

[0107] 4. Effects of compound bacterial agents of tobacco leaf chloride ion absorption inhibitor on soil microorganisms

[0108] In this example, 16S RNA high-throughput sequencing was performed on mature tobacco-growing soil to analyze the soil diversity composition.

[0109] Soil microbial community bar chart Figure 3 As shown, it can be seen that compared with the control group, the group treated with the compound bacterial agent of tobacco leaf chloride ion inhibitor can improve the structure of microbial communities and enhance the proportion of dominant bacteria, especially Actinobacteriota, Proteobacteria and Chloroflexi.

[0110] The species composition Venn diagram is shown in Figure 1. Figure 4 As shown, it can be seen that the treatment group with the addition of the tobacco leaf chloride ion absorption inhibitor and the composite bacterial agent can significantly enhance the number of soil microbial flora and enrich the soil flora.

[0111] In summary, adding the compound bacterial agent of chloride ion absorption inhibitor to tobacco leaves can inhibit the absorption of chloride ions in the soil by tobacco plants, causing chloride ions to be deposited in the soil. At the same time, it can improve the physical and chemical indicators of tobacco-growing soil, significantly increase the number of soil microbial flora, and enrich the soil flora. Therefore, the compound bacterial agent of chloride ion absorption inhibitor to tobacco leaves has played a role in inhibiting the absorption of chloride ions by tobacco plants, improving the flora of tobacco-growing soil, and achieving the purpose of "reducing chlorine" in tobacco leaves.

[0112] The above is only an embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the scope of the claims of the present application.

Claims

1. A chloride ion absorption inhibitor, characterized in that The chloride ion absorption inhibitor includes: Vibrio neptunius and / or Rhodopseudomonas palustris.

2. The chloride ion absorption inhibitor according to claim 1, characterized in that The Vibrio natrii is Vibrio natrii NH87-81, with a deposit number of CGMCC No.1.8755; preferably, the viable cell count of the Vibrio natrii is 5×10 9 cfu / mL-2.0×10 10 cfu / mL; more preferably, 2.0×10 10 cfu / mL.

3. The chloride ion absorption inhibitor according to claim 1, characterized in that The palustris Rhodopseudomonas is Rhodopseudomonas palustris LM6-purple, with a preservation number of CGMCC No.1.8929; preferably, the viable count of the palustris Rhodopseudomonas is 5×10 9 cfu / mL-2.0×10 10 cfu / mL; more preferably, 2.0×10 10 cfu / mL.

4. The chloride ion absorption inhibitor according to claim 1, characterized in that The volume ratio of Vibrio natriuresis to Rhodopseudomonas palustris is 1:(1-5); preferably, 1:

2.

5. The method for preparing the chloride ion absorption inhibitor according to any one of claims 1 to 4, characterized in that: The steps include: Step 1: fermenting Vibrio natrii to obtain Vibrio natrii fermentation broth for standby use; preferably, the number of viable bacteria of Vibrio natrii in the Vibrio natrii fermentation broth is 5×10 9 cfu / mL-2.0×10 10 cfu / mL; more preferably, 2.0×10 10 cfu / mL; Step 2: ferment Rhodopseudomonas palustris to obtain Rhodopseudomonas palustris fermentation broth for later use; preferably, the number of viable Rhodopseudomonas palustris in the Rhodopseudomonas palustris fermentation broth is 5×10 9 cfu / mL-2.0×10 10 cfu / mL; more preferably, 2.0×10 10 cfu / mL; Step 3, mixing the fermentation broth of Vibrio natriuresis and the fermentation broth of Rhodopseudomonas palustris to obtain a chloride ion absorption inhibitor; Preferably, the volume ratio of the Vibrio natriuresis fermentation broth to the Rhodopseudomonas palustris fermentation broth is 1:(1-5); preferably, 1:

2.

6. A fertilizer, characterized in that: The fertilizer comprises the chloride ion absorption inhibitor according to any one of claims 1 to 4.

7. Use of the chloride ion absorption inhibitor according to any one of claims 1 to 4 or the fertilizer according to claim 6 in any one of the following A1) to A5): A1) inhibiting tobacco from absorbing chloride ions; A2) reducing the chloride ion content of tobacco; A3) Improve soil physical and chemical properties; A4) Improve soil microbial community structure; A5) Enrich soil microbial flora.

8. A method for inhibiting tobacco from absorbing chloride ions, reducing tobacco chloride ion content, improving soil physical and chemical properties, improving soil microbial community structure and / or enriching soil microbial flora, characterized in that: The method comprises: cultivating tobacco using the chloride ion absorption inhibitor according to any one of claims 1 to 4 or the fertilizer according to claim 6.

9. Tobacco cultivated by the method of claim 8.

10. Use of the tobacco according to claim 9 in the preparation of cigarettes.

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