Fungicide for inhibiting chloride ion absorption as well as preparation method and application thereof

Abstract

The invention provides a fungicide for inhibiting chloride ion absorption as well as a preparation method and application thereof, and relates to the technical field of microbial engineering. According to the invention, the rhodopseudomonas palustris LM6-purkle and the bacillus velezensis LGT-1 are compounded for the first time to obtain the complex microbial inoculant capable of effectively inhibiting the tobacco from absorbing the chloride ions in the soil, and a new effective component and a new functional strain are provided for the chloride ion inhibitor; salt tolerance domestication is carried out on the bacillus velezensis LGT-1, so that the bacillus velezensis LGT-1 is more suitable for soil with high chloride ion content, the application scene of the bacillus velezensis LGT-1 is expanded, and a new fertilizer component is provided for tobacco planting and improvement in saline-alkali soil; the invention provides a preparation method of the complex microbial inoculant, and the preparation method is low in manufacturing cost, simple in production method and suitable for wide popularization.

Description

Technical Field

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

[0002] As the world's largest tobacco-growing country, my country plays an important role in my country's economy. As the area of ​​tobacco cultivation in my country increases, the quality requirements for tobacco leaves have also increased. Among them, combustibility has always been highly valued as an important indicator of tobacco quality. The combustibility of tobacco leaves is mainly related to the potassium-chlorine ratio. Therefore, the combustibility of tobacco leaves can be improved by adjusting the chlorine content. Chlorine is one of the important nutrients required for tobacco growth. An appropriate amount of chlorine content helps to increase the turgor pressure of tobacco leaf cells, enhance drought resistance, and reduce the occurrence of tobacco leaf necrotic spots. It can also reduce the protein content, nicotine and total nitrogen content in tobacco leaves, while increasing the content of total sugar and reducing sugar, thereby improving the taste of tobacco leaves. However, excessive chloride ion content will seriously affect the quality of tobacco leaves, so reasonable control of chloride ion content is crucial to the growth of tobacco.

[0003] At present, the chloride ions in flue-cured tobacco mainly come from soil, irrigation water, rainfall and various fertilizers. Soil chloride is the main source of chloride absorption by tobacco plants. Although the effect of reducing chloride can be achieved to a certain extent through agronomic trait management measures, breeding, chloride ion inhibitors, etc., the problem of excessive chloride ion concentration in tobacco leaves still exists. In addition, the use of inhibitors under salt stress conditions can reduce the chloride content of plants, thereby reducing the harm of chlorine to plants, but it also puts forward salt tolerance requirements for the inhibitors themselves.

[0004] Therefore, we need to continue to explore a feasible way to improve the chloride ions in tobacco leaves. Summary of the invention

[0005] The purpose of the present invention is to provide a salt-tolerant, green, efficient and feasible chloride ion inhibitor effective ingredient.

[0006] In one aspect, the present application provides a bacterial agent for inhibiting chloride ion absorption, wherein the bacterial agent comprises Bacillus velezensis and / or Rhodopseudomonas palustris.

[0007] Preferably, the bacterial agent comprises Bacillus velezensis and Rhodopseudomonas palustris.

[0008] Furthermore, the Velez subtilis is Velez subtilis LGT-1, with a deposit number of CGMCC No. 24342; preferably, the viable cell count of the Velez subtilis is 9.21×10 8 cfu·mL -1 .

[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 3×10 9 cfu / mL.

[0010] In the present application, for the first time, Bacillus Velez and Rhodopseudomonas palustris are compounded to obtain a bacterial agent for inhibiting chloride ion absorption for tobacco. The bacterial agent has the effect of inhibiting the absorption of chloride ions by tobacco, and can therefore be used as a chloride ion absorption inhibitor, providing a new active ingredient for reducing the chloride ion content in tobacco and producing low-chloride tobacco.

[0011] Those skilled in the art can understand that the Bacillus Velez in the present application can be Bacillus Velez, Bacillus Velez inoculant, dead bacteria suspension of Bacillus Velez, metabolites of Bacillus Velez, and Bacillus Velez extract; the Rhodopseudomonas palustris in the present application can be Rhodopseudomonas palustris, Rhodopseudomonas palustris inoculant, dead bacteria suspension of Rhodopseudomonas palustris, metabolites of Rhodopseudomonas palustris, and extracts of Rhodopseudomonas palustris.

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

[0013] Step 1: fermenting Bacillus Velez subtilis to obtain Bacillus Velez subtilis fermentation liquid for standby use; preferably, the viable cell count of the Bacillus Velez subtilis is 9.21×10 8 cfu·mL -1 ;

[0014] Step 2: Fermentation of Rhodopseudomonas palustris to obtain Rhodopseudomonas palustris fermentation liquid for later use; preferably, the viable count of Rhodopseudomonas palustris is 3×10 9 cfu / mL;

[0015] Step 3: Mix the Bacillus Velez fermentation broth and the Rhodopseudomonas palustris fermentation broth to obtain a bacterial agent.

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

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

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

[0019] The volume ratio of the Bacillus Velez 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, the Bacillus Velez subtilis is Bacillus Velez subtilis LGT-1, and its deposit number is CGMCC No.24342.

[0021] Preferably, the step 1 further comprises a step of activating Bacillus velez. Those skilled in the art can activate it in a general manner, wherein:

[0022] The method for activating Bacillus Velez subtilis comprises: inoculating Bacillus Velez subtilis LGT-1 strain into Bacillus Velez subtilis activation medium, culturing in a constant temperature incubator at 25°C-40°C for 12-24 hours, picking a single colony and inoculating it into Bacillus Velez subtilis LGT-1 seed medium, 20°C-40°C, 100-300r·min -1 The culture was carried out under shaking conditions for 12-24 hours to obtain the seed solution of Bacillus velezensis LGT-1.

[0023] Preferably, the Velez Bacillus LGT-1 strain is inoculated into the Velez Bacillus activation medium, cultured in a constant temperature incubator at 30°C for 24 hours, and a single colony is picked and inoculated into the Velez Bacillus LGT-1 seed medium at 30°C and 200 r·min. -1 The culture was carried out under shaking conditions for 24 hours to obtain the seed solution of Bacillus velezensis LGT-1.

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

[0025] Preferably, the Bacillus Velez subtilis activation culture medium is a Bacillus Velez subtilis LGT-1 flat solid culture medium.

[0026] Preferably, the solid medium for Bacillus Velezii LGT-1 includes: peptone 10 g·L-1 , yeast powder 5g·L -1 , sodium chloride 10g·L -1 , agar 10 g·L -1 , the balance is water, pH value 7.2-7.4.

[0027] Preferably, the seed culture medium of Bacillus Velezii LGT-1 comprises: peptone 10 g·L -1 , yeast powder 5g·L -1 , sodium chloride 10g·L -1 , the balance is water, pH value 7.2-7.4.

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

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

[0030] Preferably, Rhodopseudomonas palustris is Rhodopseudomonas palustris LM6-purple, and its deposit number is CGMCC No.1.8929.

[0031] Preferably, the step 2 further comprises the step of activating Rhodopseudomonas palustris, and those skilled in the art can activate it in a general manner, wherein:

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

[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 hours, picking a single colony and inoculating it into a Rhodopseudomonas palustris culture medium, adding 5 g / L carbon source, 30°C, and anaerobic culturing under light for 48 hours to obtain Rhodopseudomonas palustris LM6-purple seed solution.

[0034] Preferably, the OD of the Rhodopseudomonas palustris LM6-purple seed solution is600 The value is 0.6-1.

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

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

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

[0038] Preferably, the Rhodopseudomonas palustris activation medium 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, the step three further comprises resuspending the fermentation broth after centrifugation.

[0040] The ratio of resuspension material to liquid is 1:1.

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

[0042] Step 1: Inoculate the seed liquid of Bacillus Velez LGT-1 into the fermentation medium of Bacillus Velez at an inoculation rate of 1%-5%, culture at 20°C-40°C, 100-200r / min, and culture for 24-48h to prepare the fermentation liquid of Bacillus Velez LGT-1 for standby use; the fermentation medium of Bacillus Velez includes: 1-10g·L tryptone -1 , yeast powder 1-5g·L -1 ,NaCl1-10g·L -1 ;

[0043] Step 2: Inoculate the Rhodopseudomonas palustris LM6-purple seed solution in a Rhodopseudomonas palustris culture medium at an inoculum amount of 1%-5%, add 1-5 g / L carbon source, culture at 20°C-40°C, and perform light anaerobic culture for 24-48 hours to obtain the Rhodopseudomonas palustris LM6-purple fermentation liquid for later use;

[0044] Step 3: Centrifuge the Bacillus Velez fermentation broth and the Rhodopseudomonas palustris fermentation broth, resuspend them, and mix them in a volume ratio of 1:(1-5) to obtain a bacterial agent.

[0045] On the other hand, the present 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 function of inhibiting tobacco from absorbing chloride ions in the soil.

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

[0048] Those skilled in the art will appreciate that the fertilizer of the present application may further include auxiliary materials, which may be appropriate solvents, solubilizers, cosolvents, emulsifiers, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, preservatives, suspending agents, coating materials, anti-adhesives, integrities, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, filter aids, release retardants, and the like.

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

[0050] Preferably, the tobacco comprises tobacco leaves.

[0051] 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.

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

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

[0054] 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 and / or improving tobacco combustion performance, the method comprising: cultivating tobacco using the bacterial agent or the chloride ion absorption inhibitor or the fertilizer.

[0055] Those skilled in the art can adopt a general method to cultivate tobacco using the bacterial agent, the chloride ion absorption inhibitor or the fertilizer described in this application.

[0056] The fungal agent can be applied directly or after dilution by watering, dipping roots, or irrigating roots. It can also be added to fertilizers to make fertilizer products that inhibit tobacco plants from absorbing chloride ions, and applied by pond application, hole application, strip application, ring application, etc.

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

[0058] The present invention adopts a composite bacterial agent obtained by compounding Bacillus Velez and Rhodopseudomonas palustris to cultivate tobacco, which can inhibit tobacco from absorbing chloride ions and reduce the chloride ion content of tobacco. The method is green and environmentally friendly, is conducive to the sustainable use of soil, and provides a new effective ingredient for tobacco planting.

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

[0060] Preferably, the chloride ion content in the tobacco is low, and specifically, the chloride ion content in the tobacco is less than 0.8%.

[0061] Preferably, the tobacco includes tobacco leaves; more preferably, the tobacco leaves include upper leaves, middle leaves, and lower leaves; more preferably, lower leaves.

[0062] 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.

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

[0064] Preferably, the cigarette has good combustibility.

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

[0066] 1. The present invention uses Rhodopseudomonas palustris LM6-purple and Bacillus Velezii LGT-1 for the first time to obtain a composite bacterial agent that can effectively inhibit tobacco from absorbing chloride ions in the soil, providing a new effective ingredient and functional strain for chloride ion inhibitors;

[0067] 2. In the present invention, fermentation culture is carried out on Bacillus Velezii LGT-1, which expands its application scenarios and provides a new fertilizer component for tobacco planting and improvement in saline-alkali land;

[0068] 3. The present invention provides a method for preparing the composite bacterial agent, which has low production cost and simple production method and is suitable for promotion;

[0069] 4. The present invention adopts microbial technology to control chloride ions in tobacco and its soil, which provides a new idea for chloride ion control in this field. Compared with traditional chloride ion control technology, it is more convenient, has lower cost, has less side effects on soil and tobacco, and has a significant improvement on the combustibility of tobacco, thereby improving the quality of tobacco leaves. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] 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:

[0071] Figure 1 The effect of different culture media on the number of live bacteria in the fermentation broth of Bacillus Velez LGT-1;

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

[0073] Figure 3 Chloride ion content in tobacco leaves of each treatment group after control. DETAILED DESCRIPTION

[0074] 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.

[0075] 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.

[0076] 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.

[0077] 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.

[0078] 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.

[0079] 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.

[0080] 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.

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

[0082] Bacillus Velez LGT-1 solid medium: peptone 10 g L -1 , yeast powder 5g·L -1 , sodium chloride 10g·L -1 , agar 10 g·L -1 , the balance is water, pH value 7.2-7.4.

[0083] Bacillus Velez LGT-1 seed culture medium: peptone 10 g L -1 , yeast powder 5g·L -1 , sodium chloride 10g·L -1 , the balance is water, pH value 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, 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.

[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, 121℃ for 30min and set aside. The concentrate (100mL) formula 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.

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

[0087] 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.

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

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

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

[0091] Activation of Bacillus Velez LGT-1: Under sterile conditions, transfer the Bacillus Velez LGT-1 strain stored at -80°C to the Bacillus Velez LGT-1 flat solid culture medium, and culture it in a constant temperature incubator at 30°C for 24 hours before use.

[0092] Preparation of Velez Bacillus LGT-1 seed solution: Take a loop of activated Velez Bacillus LGT-1 and inoculate it into a 100 mL Erlenmeyer flask containing 40 mL Velez Bacillus LGT-1 seed medium, and incubate at 30 °C and 200 r / min. -1The culture was carried out under shaking conditions for 24 hours to obtain the seed solution of Bacillus velezensis LGT-1.

[0093] Velez Bacillus LGT-1 fermentation broth: 5% of the Velez Bacillus LGT-1 seed liquid was inoculated into 100 mL Erlenmeyer flasks containing 40 mL of different fermentation mediums and incubated at 30 °C and 200 r / min. -1 The culture was shaken on a shaker for 24 hours under the condition of , and the fermentation liquid of Bacillus Velez LGT-1 was obtained. The components of the fermentation medium are shown in Table 1. After the fermentation, the number of viable bacteria in the fermentation liquid of Bacillus Velez LGT-1 was measured by the dilution plate count method.

[0094] Table 1 Culture medium components

[0095]

[0096] The experimental results are as follows Figure 1 As shown in the figure, the number of viable bacteria per unit volume of Bacillus Velezii LGT-1 was the highest when cultured in shake flasks in medium No. 2, which was 9.21×10 8 cfu·mL -1 The number of viable bacteria per unit volume of fermentation liquid in medium No. 1 is 8.13×10 8 cfu·mL -1 The number of viable bacteria per unit volume of fermentation liquid in medium No. 3 was 6.52×10 8 cfu·mL -1 The number of viable bacteria per unit volume of the fermentation liquid of medium No. 4 is 8.55×10 8 cfu·mL -1 Therefore, medium No. 2 was selected as the basic medium for shake flask fermentation of Bacillus velez LGT-1.

[0097] Example 2 Preparation of composite bacterial agent

[0098] According to the preferred results of Example 1, a method for preparing a fermentation broth of Bacillus Velezii LGT-1 is provided in this example:

[0099] (1) Under sterile conditions, the Bacillus Velez LGT-1 strain stored at -80°C was transferred to a solid medium of Bacillus Velez LGT-1 and cultured in a constant temperature incubator at 30°C for 24 hours for later use;

[0100] (2) A single colony of the activated Bacillus Velez subtilis LGT-1 was picked and inoculated into a 100 mL Erlenmeyer flask containing 40 mL of the seed culture medium of Bacillus Velez subtilis LGT-1. The culture was shaken at 200 rpm and 30 °C for about 24 h until the OD 600The value was greater than 0.6, and the seed solution of Bacillus velez LGT-1 was prepared;

[0101] (3) The seed liquid was transferred into the fermentation tank at an inoculum rate of 5% (wherein the fermentation medium formula: tryptone 10g·L -1 , yeast powder 5g·L -1 ,NaCl 10g·L -1 , pH 7.2), 30°C, 200r / min, and culture for about 24h to prepare the fermentation liquid of Bacillus Velez LGT-1.

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

[0103] (1) Under sterile conditions, Rhodopseudomonas palustris LM6-purple stored at -80°C was inoculated into PM plate (solid) culture medium, 5 g / L carbon source was added, cultured at 30°C for 48 h, and activated three times;

[0104] (2) Pick a single colony and transfer it to PM liquid culture medium, add 5 g / L carbon source, and culture it anaerobically at 30°C for 48 h under light to obtain the seed liquid of Rhodopseudomonas palustris LM6-purple;

[0105] (3) The seed liquid was inoculated into PM liquid culture medium at a ratio of 1%, 5 g / L carbon source was added, and the culture was carried out under light anaerobically at 30° C. for 48 h to obtain the fermentation liquid 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 composite bacterial agent: the fermentation liquid of the Bacillus Velez LGT-1 and the fermentation liquid of Rhodopseudomonas palustris LM6-purple are centrifuged at 6000 r / min and then resuspended to eliminate the influence of salt in the fermentation liquid on subsequent soil and tobacco, and the resuspended liquids are mixed in a volume ratio of 1:1 to prepare a composite bacterial agent.

[0108] Example 3 Potted Plant Experiment of Composite Bacterial Agent

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

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

[0111] The test container was a plastic flower pot with an inner diameter of 40 cm and a height of 35 cm, and each pot was filled with 20.0 kg of soil. The potted plants were placed according to the row spacing of 120 cm × 50 cm. This study set up CK and T1, T2, and T3 for comparison. The CK treatment was the control group, and fertilization was carried out according to the local conventional fertilization treatment. T1, T2, and T3 were the treatment groups. In addition to conventional fertilization, the T1 treatment group also applied the above-mentioned composite bacterial agent in the clustering period, the vigorous growth period, and the mature period, respectively, with 10 mL of the bacterial agent applied to each plant. T2 used an equal amount of Bacillus Velez LGT-1 fermentation liquid instead of the composite bacterial agent, and T3 used an equal amount of Rhodopseudomonas palustris LM6-purple instead of the composite bacterial agent. The rest of the treatments were exactly the same.

[0112] In addition, the chloride ion content in the potting soil was tested before transplanting, and transplanting was carried out according to standard operations. The chloride ion content in the soil was tested during the clustering period, the vigorous growth period, and the maturity period according to the growth and development process. The soil chloride ion content was tested using the silver nitrate titration method (NY / T1378-2007). At the same time, tobacco leaf samples were taken at different growth periods, and the chloride ion content was directly measured after withering without baking. The tobacco leaf chloride ion detection was determined using Fourier transform near-infrared spectroscopy.

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

[0114] The soil chloride content was tested by silver nitrate titration (NY / T1378-2007), and four periods were tested from before transplanting to maturity (before transplanting, clustering period, vigorous growth period, and maturity period). The sample soil was rhizosphere soil, and 5 cm of rhizosphere soil was dug with a shovel. Five pots were randomly selected for each treatment, and a five-point sampling method was used. A total of 200 g of soil was taken under the same treatment. The test results are shown in Table 2, which is obtained from 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 clustering period, vigorous growth period, and mature period, and there was no significant change in the two groups before transplanting. Therefore, the two groups of data in the four periods showed that the composite bacterial agent inhibited tobacco from absorbing chloride ions in the soil and left some chloride ions in the soil.

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

[0119] Tobacco leaf chloride ion detection was carried out by Fourier transform near infrared spectroscopy. Tobacco plants after three controls were selected, and 5 plants were randomly selected for each treatment group. Three leaves of the upper, middle and lower leaves were taken, and the leaves were ground into powder after drying. 5g of samples were taken from each treatment group for testing. The test results are shown in Table 3. Figure 3 .

[0120] Table 3

[0121]

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

[0123] 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 bacterial agent for inhibiting chloride ion absorption, characterized in that: The bacterial agent includes Bacillus velezensis and / or Rhodopseudomonas palustris.

2. The bacterial agent according to claim 1, characterized in that The Velez Bacillus is Velez Bacillus LGT-1, with a deposit number of CGMCC No. 24342; preferably, the viable cell count of the Velez Bacillus is 9.21×10 8 cfu·mL -1 .

3. The bacterial agent 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 3×10 9 cfu / mL.

4. The method for preparing the bacterial agent according to any one of claims 1 to 3, characterized in that: The method comprises the following steps: Step 1: fermenting Bacillus Velez subtilis to obtain Bacillus Velez subtilis fermentation liquid for standby use; preferably, the viable cell count of the Bacillus Velez subtilis is 9.21×10 8 cfu·mL -1 ; Step 2: Fermentation of Rhodopseudomonas palustris to obtain Rhodopseudomonas palustris fermentation liquid for later use; preferably, the viable count of Rhodopseudomonas palustris is 3×10 9 cfu / mL; Step 3: Mix the Bacillus Velez fermentation broth and the Rhodopseudomonas palustris fermentation broth to obtain a bacterial agent; preferably, the volume ratio of the Bacillus Velez fermentation broth to the Rhodopseudomonas palustris fermentation broth is 1:(1-5); preferably, 1:

1.

5. The method according to claim 4, characterized in that In the step 1, the fermentation medium of Bacillus velezinii comprises: tryptone, yeast powder, and sodium chloride; more preferably, 1-10 g·L tryptone -1 , yeast powder 1-5g·L -1 ,NaCl1-10g·L -1 More preferably, tryptone 10 g L -1 , yeast powder 5g·L -1 ,NaCl 10g·L -1； Preferably, the pH of the Bacillus Velez fermentation medium is 7-7.5; more preferably, 7.

2.

6. A chloride ion absorption inhibitor, characterized in that The chloride ion absorption inhibitor comprises the bacterial agent according to any one of claims 1-3.

7. A fertilizer, characterized in that The fertilizer comprises the bacterial agent according to any one of claims 1 to 3 or the chloride ion absorption inhibitor according to claim 6.

8. Use of the bacterial agent according to any one of claims 1 to 4, the chloride ion absorption inhibitor according to claim 6, or the fertilizer according to claim 7 for inhibiting tobacco from absorbing chloride ions, reducing the chloride ion content of tobacco, improving tobacco combustion performance and / or increasing the chloride ion content in soil.

9. A method for inhibiting tobacco from absorbing chloride ions, reducing the chloride ion content of tobacco and / or improving the combustion performance of tobacco, characterized in that: The method comprises: cultivating tobacco using the bacterial agent according to any one of claims 1 to 4 or the chloride ion absorption inhibitor according to claim 6 or the fertilizer according to claim 7.

10. Tobacco grown according to the method of claim 9.

Images