Bacillus siamensis and application of bacillus siamensis in aspects of plant disease control, growth promotion and yield increase

Abstract

The invention belongs to the field of biological agents, and particularly relates to bacillus siamensis and application thereof in the aspects of plant disease control, growth promoting and yield increasing. The bacillus siamensis XNC-3 provided by the invention is wide in antibacterial spectrum, can effectively inhibit various plant pathogenic fungi including phytophthora parasitica var nicotianae, has broad-spectrum antibacterial activity, and can be used for preventing and treating plant diseases, especially tobacco diseases. The bacillus siamensis has a promoting effect on tobacco seedling growth and seed germination, has growth-promoting and yield-increasing effects on tobacco, can remarkably increase the tobacco yield and improve the tobacco quality, is safe to the environment and has a very good application prospect.

Description

Technical Field

[0001] This application belongs to the field of biological agents, and particularly relates to a Bacillus siamensis and its application in the prevention and control of plant diseases and the promotion of growth and yield increase. Background Art

[0002] Tobacco (scientific name: Nicotiana tabacum L.) is an annual herbaceous plant of the genus Nicotiana in the Solanaceae family. It is native to South America and is widely cultivated in various provinces and regions in the north and south of China. It is one of the important source crops for fiscal revenue. It likes warm and sunny environments, is not cold-tolerant, is relatively heat-tolerant, likes fertile, deep, and well-drained soil, and mainly reproduces by tillering and sowing. Tobacco black shank is one of the most devastating diseases in tobacco production. It is caused by the infection of Phytophthora parasitica var. nicotianae (Breda de hean) Tuker. Phytophthora parasitica belongs to the class Oomycetes, the order Peronosporales, and the genus Phytophthora. High humidity and high temperature are conducive to its reproduction and infection. In recent years, due to the rapid expansion of the national tobacco-growing area, the continuous cropping tobacco fields and the continuous cropping years have been increasing year by year, and the flue-cured tobacco varieties widely planted in production are mostly susceptible to black shank. As a result, the prevalence of this disease has caused heavy economic losses to the flue-cured tobacco industry.

[0003] The pathogen of tobacco black shank has the characteristics of wide distribution, strong adaptability, easy mutation and drug resistance, etc. It is extremely difficult to control and the control effect is not ideal. At present, production mainly focuses on prevention. Before ridging, organic fertilizers containing Bacillus subtilis and tobacco special fertilizers are applied together as base fertilizers by furrow application. When transplanting, chemical agents such as metalaxyl-manganese zinc are used for root irrigation, and comprehensive control methods such as agricultural control are combined. There is still no effective prevention and control method for tobacco black shank in production. Chemical control is still the main means, but the effect is not good. The control effect is significantly reduced or even lost after continuous application for 1-2 years, and the large amount of chemical agents used will cause soil pollution in tobacco fields. The research on the ecological control measures of harmful organisms mainly based on green and safe biological control has become the focus of preventing and controlling tobacco black shank.

[0004] There are many reported biocontrol bacteria. The biocontrol bacteria most used in tobacco field production mainly include Trichoderma harzianum and Bacillus subtilis, but the control effect on tobacco black shank is not good, which may be related to the strain type, adaptability, etc. Bacillus is one of the earliest studied and applied microorganisms in biological control. It is widely distributed in soil, rivers, plants and animals, etc. It has significant effects on promoting growth and preventing diseases, rich secondary metabolites, and has high development and utilization value. However, there are still some problems in using Bacillus for disease control at present. Some Bacillus have inhibitory effects on one pathogen but no inhibitory effects on other pathogens; they have growth-promoting effects but no disease-preventing effects; they have disease-preventing and growth-promoting effects but the control object is single and cannot compete with agents with broad-spectrum antibacterial activity, so their application is limited.

[0005] Therefore, there is an urgent need in the art for a biological agent with a broad antibacterial spectrum that can effectively prevent and control plant diseases, especially tobacco diseases, and has the effect of promoting growth and increasing yield. Summary of the Invention

[0006] To solve the problems existing in the prior art, the present invention provides a strain of Bacillus siamensis XNC-3, which has a broad antibacterial spectrum, can effectively inhibit a variety of plant pathogenic fungi including tobacco black shank disease, can be used for the prevention and control of plant diseases, especially the prevention and control of tobacco diseases, and has the effect of promoting growth and increasing yield. The Bacillus siamensis has a good control effect on tobacco black shank disease, has a good control effect in field application, significantly increases the tobacco yield, improves the tobacco quality, and is safe for the environment.

[0007] In a first aspect, the present application provides a Bacillus siamensis XNC-3, which was deposited at the China Center for Type Culture Collection on March 27, 2025, with the deposit number CCTCC NO: M2025600 and the deposit address being Wuhan University, Wuchang District, Wuhan City, Hubei Province.

[0008] The 16S rDNA of the Bacillus siamensis XNC-3 is as shown in SEQ ID NO.1.

[0009] In a second aspect, the present application provides a microbial agent, which contains the Bacillus siamensis XNC-3 described in the first aspect, and / or the fermentation product of the Bacillus siamensis XNC-3 described in the first aspect. The fermentation product herein refers to the fermentation broth, fermentation filtrate; or the concentrate, dried product or dilution of the fermentation broth, fermentation filtrate; or the metabolite in the fermentation broth.

[0010] In some preferred technical solutions, the fermentation broth contains at least 1×10 7 cfu / mL of the Bacillus siamensis XNC-3. Specifically, the fermentation broth may contain 1×10 7 -1×10 9 cfu / mL, such as 1×10 7 , 2×10 7 , 3×10 7 , 4×10 7 , 5×10 7 , 6×10 7 , 7×10 7 , 8×10 7 , 9×10 7 , 1×10 8 , 2×10 8 , 3×10 8 , 4×10 8 、5×108 , 6×10 8 , 7×10 8 , 8×10 8 , 9×10 8 , 1×10 9 cfu / mL, preferably 2×10 7 -1×10 8 cfu / mL of the Bacillus siamensis XNC-3. The fermentation filtrate is the fermentation filtrate after the fermentation broth is centrifuged and filtered (such as microfiltration membrane filtration). The dilution is a water dilution of the fermentation broth or the fermentation filtrate (such as dilution with clear water or sterile water).

[0011] In some preferred technical solutions, the preparation of the fermentation broth includes the following steps: culturing the Bacillus siamensis XNC-3 in an LB liquid medium; the culture conditions are 25-30°C, 150-200 rpm, and culturing (such as dark culturing) for 24-72 h. Specifically, the culture temperature is 25-30°C, such as 25, 26, 27, 28, 29, 30°C, the rotation speed can be 150-200 rpm, such as 150, 160, 170, 180, 190, 200 rpm, and the culture time can be 24-72 h, such as 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72 h.

[0012] In some preferred technical solutions, 0.5-2% (volume ratio) of the seed culture solution of Bacillus siamensis XNC-3 is added to the LB liquid medium for culture. The inoculation amount is 0.5-2%, such as 0.5%, 1%, 1.5%, 2%. The preparation of the seed culture solution of Bacillus siamensis XNC-3 includes the following steps: culturing the Bacillus siamensis XNC-3 in an LB liquid medium to obtain a seed culture solution; the culture conditions are 25-30°C, 150-200 rpm, and culturing (such as dark culturing) for 8-30 h. Specifically, the culture temperature can be 25-30°C, such as 25, 26, 27, 28, 29, 30°C, the rotation speed can be 150-200 rpm, such as 150, 160, 170, 180, 190, 200 rpm, and the culture time can be 8-24 h, such as 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 h. The seed solution contains 1×10 5 -1×10 7 cfu / mL of the Bacillus siamensis XNC-3. Specifically, the seed solution can contain, for example, 1×10 5 , 2×10 5 , 3×10 5 , 4×10 5 , 5×10 5 , 6×105 , 7×10 5 , 8×10 5 , 9×10 5 , 1×10 6 , 2×10 6 , 3×10 6 , 4×10 6 , 5×10 6 , 6×10 6 , 7×10 6 , 8×10 6 , 9×10 6 , 1×10 7 cfu / mL, preferably 5×10 5 -5×10 6 cfu / mL of the Bacillus siamensis XNC-3.

[0013] When cultured confrontationally in a petri dish, the Bacillus siamensis XNC-3 has an inhibitory effect on a variety of phytopathogenic fungi, such as Sclerotinia sclerotiorum, Phytophthora nicotianae, Boeremia exigua, Botrytis cinerea, Dothiorella gregaria, Alternaria alternata, Stagonosporopsis ligulicola, Thielaviopsis basicola, Colletotrichum capsici, Rhizoctonia solani, Fusarium oxysporum and other 11 tobacco pathogens, and has good antibacterial activity. The antibacterial rate in the petri dish ranges from 46.53% to 65.30%. The fermentation filtrate of Bacillus siamensis XNC-3 also has a good inhibitory effect on 11 tobacco pathogens. It shows that the Bacillus siamensis has broad-spectrum antibacterial activity and can effectively inhibit a variety of phytopathogens, especially phytopathogenic fungi; such as tobacco pathogens (such as Phytophthora nicotianae).

[0014] In a third aspect, the present application provides the use of the Bacillus siamensis described in the first aspect or the microbial preparation described in the second aspect, which is used for plant disease control, inhibiting plant pathogens, promoting the growth and yield increase of plants or promoting the germination of plant seeds.

[0015] The plant diseases described in the present application are preferably plant diseases caused by plant pathogens.

[0016] In some preferred technical solutions, the plant is preferably tobacco, such as Yunyan 99.

[0017] In some preferred technical solutions, the plant pathogen is a plant pathogenic fungus, preferably Fusarium, Rhizoctonia, Botrytis, Colletotrichum; more preferably a tobacco plant pathogenic fungus, including Sclerotinia sclerotiorum, Phytophthora nicotianae, Boeremia exigua, Botrytis cinerea, Dothiorella gregaria, Alternaria alternata, Stagonosporopsis ligulicola, Thielaviopsis basicola, Colletotrichum capsici, Rhizoctonia solani, Fusarium oxysporum. Preferably, the plant pathogen is Phytophthora nicotianae.

[0018] In some preferred technical solutions, the Bacillus siamensis described in the first aspect or the microbial preparation described in the second aspect is applied by irrigation or spraying. The application method can be root irrigation or foliar spraying at the roots of the plant.

[0019] In specific applications, those skilled in the art can select and adjust the application method, application amount, etc. according to the specific conditions of plant diseases, plant pathogens, and the specific conditions of the Bacillus siamensis or the microbial preparation.

[0020] In a fourth aspect, the present application provides a method for preventing and controlling diseases and promoting growth and increasing yield of tobacco, which includes the following steps:

[0021] (1) When transplanting tobacco in the tobacco field, apply the fermentation broth of the Bacillus siamensis XNC-3 containing 1×10 7 -1×10 8 cfu / mL at a rate of 1-10 kg per mu, and apply a 50-500-fold dilution of the fermentation broth per plant by hole application, 2×10 7 -5×10 8 cfu / plant;

[0022] (2) After transplanting, apply the fermentation broth of the Bacillus siamensis XNC-3 containing 1×10 7 -1×10 8The fermentation broth of the Bacillus siamensis XNC-3 at cfu / mL, foliar spray the 5-20 times diluted solution of the fermentation broth, and continuously spray 2-6 times every 10-20 days after transplanting.

[0023] In the method, in step (1), apply (such as by irrigation) the fermentation broth of the Bacillus siamensis XNC-3 in an amount of 1-10 kg per mu, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 kg. The fermentation broth contains 1×10 7 -1×10 8 cfu / mL, such as 1×10 7 、2×10 7 、3×10 7 、4×10 7 、5×10 7 、6×10 7 、7×10 7 、8×10 7 、9×10 7 、1×10 8 cfu / mL of Bacillus siamensis XNC-3. Apply the 50-500 times, such as 50, 100, 150, 200, 250, 300, 350, 400, 450, 500 times diluted solution of the fermentation broth per plant by hole application. The diluted solution applied per plant contains 2×10 7 -5×10 8 cfu / plant, such as 2×10 7 、3×10 7 、4×10 7 、5×10 7 、6×10 7 、7×10 7 、8×10 7 、9×10 7 、1×10 8 、2×10 8 -5×10 8 、4×10 8 、5×10 8 cfu / plant of Bacillus siamensis XNC-3. The volume of the diluted solution applied per plant by hole application in step (1) is 200 mL - 1000 mL, such as 200, 300, 400, 500, 600, 700, 800, 900, 1000 mL.

[0024] In the method, in step (2), apply the fermentation broth of the Bacillus siamensis XNC-3 in an amount of 1-10 kg per mu, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 kg. The fermentation broth contains 1×10 7 -1×10 8 cfu / mL, such as 1×107 、2×10 7 、3×10 7 、4×10 7 、5×10 7 、6×10 7 、7×10 7 、8×10 7 、9×10 7 、1×10 8 cfu / mL of the Bacillus siamensis XNC-3. Foliar spray with 5-20 times the fermentation broth, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 times dilution. Every 10-20 days after transplanting, such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 days, continuously spray 2-6 times, such as 2, 3, 4, 5, 6 times.

[0025] In some preferred technical solutions, in step (1), apply the fermentation broth containing 2×10 7 -5×10 7 cfu / mL of the Bacillus siamensis XNC-3 at a rate of 2-5 kg per mu. Apply a 100-200 times dilution of the fermentation broth to each plant hole, 5×10 7 -2×10 8 cfu / plant; preferably, apply 500 mL of the dilution to each plant hole.

[0026] In some preferred technical solutions, in step (1), apply the fermentation broth containing 2×10 7 cfu / mL of the Bacillus siamensis XNC-3 at a rate of 2.5-5 kg per mu. Apply a 100-200 times dilution of the fermentation broth to each plant hole, 5×10 7 -1×10 8 cfu / plant; preferably, apply 500 mL of the dilution to each plant hole. More preferably, in step (1), apply the fermentation broth containing 2×10 7 cfu / mL of the Bacillus siamensis XNC-3 at a rate of 5 kg per mu. Apply a 100 times dilution of the fermentation broth to each plant hole, and the volume of the dilution applied to each plant hole is 500 mL, containing 1×10 8 cfu / plant.

[0027] In some preferred technical solutions, in step (2), apply the fermentation broth containing 2×10 7 -5×10 7 cfu / mL of the Bacillus siamensis XNC-3 at a rate of 2-5 kg per mu. Foliar spray with a 10-20 times dilution of the fermentation broth, and continuously spray 2-6 times every 10-20 days after transplanting.

[0028] In some preferred technical solutions, in step (2), the fermentation broth containing Bacillus siamensis XNC-3 at 2×10 7 cfu / mL is applied at a rate of 2.5 - 5 kg per mu, and a 10 - 20-fold dilution of the fermentation broth is sprayed on the leaves, and it is continuously sprayed 2 - 6 times every 10 - 20 days after transplanting. Preferably, in step (2), the fermentation broth containing Bacillus siamensis XNC-3 at 2×10 7 cfu / mL is applied at a rate of 2.5 kg per mu, and a 10-fold dilution of the fermentation broth is sprayed on the leaves, and it is continuously sprayed 4 times every 15 days after transplanting.

[0029] In some preferred technical solutions, the fermentation broth and its preparation are as described in the second aspect. The dilution of the fermentation broth refers to the water dilution of the fermentation broth (such as dilution with clear water or sterile water).

[0030] In some preferred technical solutions, the tobacco is Yunyan 99.

[0031] In the fifth aspect, the present application provides a method for germinating tobacco seeds, which includes the following steps: soaking tobacco seeds in a dilution containing 5 - 50% of the fermentation broth of Bacillus siamensis XNC-3, and then germinating them. That is, the volume fraction of the fermentation broth in the dilution is 5 - 50%.

[0032] In some preferred technical solutions, the tobacco seeds are soaked in a dilution containing 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50% of the fermentation broth of Bacillus siamensis XNC-3. Preferably, the tobacco seeds are soaked in a dilution containing 10 - 30% of the fermentation broth of Bacillus siamensis XNC-3.

[0033] In some preferred technical solutions, the soaking time is 8 - 24 h, such as 8, 10, 12, 14, 16, 18, 20, 22, 24 h. After soaking, rinse with water and then germinate. It is preferably rinsed with sterile water 2 - 5 times, such as 3 - 4 times.

[0034] In some preferred technical solutions, after disinfecting the tobacco seeds, rinse them with water, and then soak them in a dilution containing 10 - 30% of the fermentation broth of Bacillus siamensis XNC-3 for 8 - 24 h.

[0035] In some preferred technical solutions, the fermentation broth contains Bacillus siamensis XNC-3 at 1×10 7 -1×10 8 cfu / mL.

[0036] In some preferred technical solutions, tobacco seeds are disinfected with ethanol and sodium hypochlorite solution and then rinsed with water (such as sterile water). Specifically, they are disinfected successively with 75% ethanol, 1% sodium hypochlorite solution, and rinsed with water. Specifically, they are disinfected with 75% ethanol for 10 s, 1% sodium hypochlorite solution for 30 s, and rinsed with sterile water.

[0037] In some preferred technical solutions, the fermentation broth and its preparation are as described in the second aspect. The dilution of the fermentation broth refers to the water dilution of the fermentation broth (such as dilution with clear water or sterile water).

[0038] In some preferred technical solutions, the tobacco is Yunyan 99.

[0039] Those skilled in the art can combine common knowledge to combine the above-mentioned technical solutions to obtain more technical solutions related to this application. For example, the features described or illustrated as part of some technical solutions can be used in other technical solutions to produce further technical solutions.

[0040] Through research, it is found that compared with the prior art, this application has at least the following beneficial effects:

[0041] (1) In this application, a strain of Bacillus siamensis XNC-3 is isolated from the rhizosphere soil of healthy plants in tobacco black shank fields. The confrontation in the petri dish and the fermentation filtrate of the Bacillus siamensis can effectively inhibit the growth of 11 tobacco pathogens such as Fusarium, Rhizoctonia, Botrytis cinerea, and Colletotrichum. The inhibition rates of the fermentation filtrate with different volume fractions on Phytophthora parasitica var. nicotianae can reach 89.88% and 100%. The pot control effect of the Bacillus siamensis on tobacco black shank in the laboratory is higher than 90%. It shows that the Bacillus siamensis XNC-3 has broad-spectrum antibacterial activity and can be used for the prevention and control of plant diseases, especially tobacco diseases.

[0042] (2) The fermentation broth of the Bacillus siamensis has a promoting effect on the growth of tobacco seedlings and seed germination. When the Bacillus siamensis is applied in tobacco fields, such as applying the fermentation broth of the Bacillus siamensis by root irrigation during tobacco transplanting and spraying the fermentation broth on the leaves after transplanting, the agronomic traits such as plant height, number of leaves, stem girth, maximum leaf length, and maximum leaf width of tobacco plants during the vigorous growth period and the dome period are significantly improved, and the opening degree of the top 4 leaves is good 100 days after planting; the yield per mu increases by 25.68 kg after harvesting, a 12.74% increase; the output value per mu increases by 998.87 yuan, a 17.04% increase; the proportion of upper and middle-grade tobacco increases by 4.76 percentage points, and the new benefit per mu is 936.37 yuan. The field control effect on tobacco black shank is 88.64%. It shows that the Bacillus siamensis has a significant control effect on tobacco black shank, has both growth promotion and yield increase effects, significantly improves the quality of tobacco, and has good development and application potential in the prevention and control of tobacco diseases and growth promotion and yield increase. Description of the Drawings

[0043] To more clearly illustrate the technical solutions of the specific embodiments of the present application, the following will briefly introduce the drawings required for the description of the specific embodiments. Obviously, the drawings in the following description are some embodiments of the present application. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0044] Figure 1 Colony morphology and spore morphology of strain XNC-3.

[0045] Figure 2 Phylogenetic tree constructed based on the 16S rDNA sequence of strain XNC-3.

[0046] Figure 3 Inhibitory effect of strain XNC-3 on 11 tobacco pathogenic fungi.

[0047] Figure 4 Inhibitory effect of fermentation filtrates of strain XNC-3 with different volume fractions on 11 tobacco pathogenic fungi.

[0048] Figure 5 Pot control efficacy of the fermentation broth of strain XNC-3 against tobacco black shank.

[0049] Figure 6 Effect of the fermentation broth of strain XNC-3 on the growth of tobacco seedlings.

[0050] Figure 7 Effect of the fermentation broth of strain XNC-3 applied in the field at the vigorous growth stage on the agronomic traits of Yunyan 99.

[0051] Figure 8 Effect of the fermentation broth of strain XNC-3 applied in the field at the domed stage on the agronomic traits of Yunyan 99.

[0052] Figure 9 Effect of the fermentation broth of strain XNC-3 applied in the field on the leaf expansion of the top 4 leaves of Yunyan 99.

[0053] Bacillus siamensis strain XNC-3 was deposited at the China Center for Type Culture Collection on March 27, 2025, with the deposit number CCTCC NO: M2025600 and the deposit address being Wuhan University, Wuchang District, Wuhan City, Hubei Province. Specific Embodiments

[0054] The following will elaborate on the specific embodiments of the present application. It should be understood that the specific embodiments described herein are only for the purpose of illustrating and explaining the present application, and are not used to limit the present application.

[0055] Unless otherwise specified, the meanings of all terms (including technical and scientific terms) used to disclose this application are the same as those commonly understood by those of ordinary skill in the art to which this application pertains. Through further guidance, the following definitions are used to better understand the teachings of this application. The terms used in the specification of this application are for the purpose of describing specific embodiments only and are not intended to limit this application.

[0056] As used herein, the alternative scopes of the terms "and / or", "or / and", and "and / or" include any one of two or more related listed items, as well as any and all combinations of the related listed items. The said any and all combinations include combinations of any two related listed items, any more related listed items, or all related listed items. It should be noted that when at least three items are connected by at least two conjunctive combinations selected from "and / or", "or / and", and "and / or", it should be understood that in this application, this technical solution undoubtedly includes the technical solution connected by "logical AND", and also undoubtedly includes the technical solution connected by "logical OR". For example, "A and / or B" includes three parallel solutions: A, B, and A + B. Another example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, it includes combinations of any two or any three of A, B, C, and D, and also includes the combination of the four items A, B, C, and D (that is, the technical solution connected by "logical AND").

[0057] The terms "comprising", "containing", and "including" used in this application are synonyms, which are inclusive or open-ended and do not exclude additional, unrecited members, elements, or method steps.

[0058] In this application, the numerical ranges indicated by endpoints include all the numerical values and fractions contained within that range, as well as the recited endpoints.

[0059] In this application, regarding concentration values, their meanings include fluctuations within a certain range. For example, it can fluctuate within the corresponding precision range. For example, for 2%, a fluctuation within the range of ±0.1% is allowed. For larger numerical values or those that do not require overly precise control, larger fluctuations are also allowed for their meanings. For example, for 100 mM, fluctuations within the ranges of ±1%, ±2%, ±5%, etc. are allowed. Regarding molecular weight, a fluctuation of ±10% is allowed for its meaning.

[0060] In this application, regarding descriptions such as "a plurality of", "a variety of", etc., unless otherwise specified, it means greater than or equal to 2 in quantity.

[0061] In this application, among the technical features described in an open-ended manner, there are included closed technical solutions composed of the listed features, as well as open technical solutions containing the listed features.

[0062] In this application, "preferably", "better", "more preferably", and "it is advisable to" are only used to describe embodiments or examples with better effects. It should be understood that they do not constitute a limitation on the protection scope of this application.

[0063] In this application, "optionally", "optional", "option", "alternatively", "alternative", "alternate" mean that it may or may not be present, that is, it refers to any one of the two alternative options of "present" or "absent". If there are multiple "optionally" or "alternatively" in a technical solution, without special instructions and without contradictions or mutual constraints, each "optionally" or "alternatively" is independent.

[0064] In this application, the composition of the LB solid medium is: 1% peptone, 0.5% yeast extract, 1% sodium chloride, and the remaining 1.5% is water (preferably distilled water).

[0065] The composition of the LB liquid medium is: 1% peptone, 0.5% yeast extract, 1% sodium chloride, and the rest is water (preferably distilled water).

[0066] Next, the implementation solutions of this application will be described in detail in conjunction with the examples. It should be understood that these examples are only used to illustrate this application and not to limit the scope of this application. For the experimental methods without specific conditions in the following examples, the guidance given in this application is preferably referred to, and it can also be carried out according to the experimental manuals or conventional conditions in the art, and other experimental methods known in the art can also be referred to, or according to the conditions recommended by the manufacturer.

[0067] In the following specific examples, for the measurement parameters of the raw material components, without special instructions, there may be slight deviations within the weighing accuracy range. For the temperature and time parameters, acceptable deviations caused by the instrument test accuracy or operation accuracy are allowed.

[0068] The experimental methods in the following examples are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0069] Example 1 Identification of Strain XNC-3

[0070] 1.1 Isolation of the Strain

[0071] This strain was isolated by the inventor from the root soil of healthy plants in the Yunyan 99 plot with tobacco black shank disease in Fancun, Luonan County, Shangluo City, Shaanxi Province in June 2024 using the dilution isolation method, and was numbered XNC-3 in the example.

[0072] 1.2 Ecological identification of strain XNC-3

[0073] Observation of single colony morphology: Strain XNC-3 was cultured in LB liquid medium at a culture temperature of 28 °C, 180 rpm, for 24 h. Prepare bacterial suspensions of 10 -8 、10 -7 、10 -6 、10 -5 、10 -4 , and streak-inoculate them on the air-dried LB solid plates in sequence. After culturing in a 25 °C incubator for 48 h, take pictures and observe.

[0074] Gram staining: Drop 2 μL of bacterial liquid on a clean glass slide, spread it evenly, and fix it; drop and add ammonium oxalate crystal violet for staining for 1 min, and wash with water; drop and add Gram iodine solution (iodine-potassium iodide solution) for staining for 1 min, and wash with water; drop and add ethanol with a volume fraction of 95%, wash with water after about 45 s; drop and add safranin staining solution for staining for 3 min, wash with water and dry it; drop and add cedarwood oil and observe under a 100-fold microscope. Gram-positive bacteria are purple, and Gram-negative bacteria are red.

[0075] Electron microscopy observation: The bacterial cells were obtained by centrifuging the fermentation broth at 4000 r / min and rinsed 3 times with PBS; impregnated and fixed with 2.5% glutaraldehyde at room temperature for more than 1 hour and overnight at 4 °C; washed 2-3 times with PBS buffer solution with a pH value of 7.2 and a concentration of 0.1 M, 10 minutes each time; washed once with 30%, 50%, 70%, 80%, 90% ethanol, 10 minutes each time; washed twice with 100% ethanol, 10 minutes each time; dried overnight; glued to the stage, sputter-coated with gold, and then photographed and observed.

[0076] As Figure 1 shown, strain XNC-3 is a Gram-positive bacterium, with spores 0.75 - 1.5 microns, and the bacterial cells are rod-shaped. The colonies on the LB medium are milky white, with regular edges, no pigment production, rough and opaque surfaces, and folds.

[0077] 1.3 Molecular biological identification of strain XNC-3

[0078] Using the genomic DNA of strain XNC-3 as a template, amplify its 16S rDNA sequence using the primers and amplification system described in Table 1-2. The amplification program is: pre-denaturation at 94 °C for 5 min; denaturation at 94 °C for 1 min, annealing at 56 °C for 1 min, extension at 72 °C for 90 s, for 35 cycles; extension at 72 °C for 10 min.

[0079] Table 1 shows the primer sequences and annealing temperatures:

[0080]

[0081] Table 2 shows the 16S rDNA PCR amplification system

[0082]

[0083] The primers used were ordered from Shaanxi Zhongke Yutong Biotechnology Co., Ltd. The amplified products were sequenced by Shaanxi Qingke Biotechnology Co., Ltd. The 16S rDNA gene sequence of the above strain XNC-3 is shown in SEQ ID NO.1 (1420bp):

[0084]

[0085] The obtained 16S rDNA sequences were analyzed for sequence similarity in the NCBI database, and a phylogenetic tree was constructed using MEGA 7.0 software, as shown in Figure 2 The results showed that the 16S rDNA gene sequence of strain XNC-3 was 1420 bp, and the phylogenetic tree showed that strain XNC-3 and Bacillus siamensis (NR 117274) were clustered in one branch.

[0086] Based on phylogenetic analysis and ecological identification, strain XNC-3 was identified as Bacillus siamensis.

[0087] Example 2 Activation and fermentation culture of strain XNC-3

[0088] The activated strain XNC-3 was cultured on a plate using LB solid medium at a temperature of 28° C. for 72 h.

[0089] The activated strain XNC-3 (bacteria cake diameter d = 9 mm) was inoculated into LB liquid medium at a temperature of 28 ° C, 180 r / min, and a culture time of 24 h to obtain a seed culture solution with a bacterial content of 3 × 10 6 cfu / mL.

[0090] Inoculate 1 mL of seed culture medium into 100 mL of LB liquid medium (packed in 250 mL conical flasks) and culture in the dark at 28 °C, 160 rpm, and shake for 48 h to obtain a fermentation broth with a bacterial count of 2 × 10 7 cfu / mL.

[0091] Example 3 Determination of the antibacterial spectrum of strain XNC-3

[0092] 3.1 Test pathogens

[0093] The tested tobacco pathogenic fungi include: Sclerotinia sclerotiorum, Phytophthora nicotianae, Boeremia exigua, Botrytis cinerea, Dothiorella gregaria, Alternaria alternata, Stagonosporopsis ligulicola, Thielaviopsis basicola, Colletotrichum capsici, Rhizoctonia solani, and Fusarium oxysporum, all of which were isolated, identified, and preserved by the Vegetable Disease and Biological Control Laboratory of the College of Plant Protection, Northwest A&F University.

[0094] The above-mentioned tested plant pathogenic bacteria can be provided by the applicant and are accessible to the public. They are only used for repeating the test process and results of the present invention and not for commercial purposes.

[0095] 3.2 Determination of the antibacterial spectrum of strain XNC-3

[0096] Using the confrontation culture method, a sterilized borer was used to take an activated test pathogenic bacteria cake (d = 9 mm) and inoculate it in the center of a PDA plate. 2 μL of the fermentation broth of strain XNC-3 was added dropwise at a distance of 1 cm from the left and right edges of the plate. A PDA plate inoculated only with the pathogenic bacteria cake was used as a control. Incubate in the dark at 25 °C for 7 d, with 3 replicates for each treatment. Measure the colony radius and calculate the inhibition rate. The results are as Figure 3 shown in Table 3.

[0097] Inhibition rate (%) = (colony radius of the control group - colony radius of the treatment group) / colony radius of the control group × 100.

[0098] Table 3 shows the inhibitory effects of strain XNC-3 on 11 kinds of tobacco pathogenic fungi

[0099]

[0100] Note: The data in the table are mean ± standard deviation. Different lowercase letters indicate significant differences (P < 0.05) tested by Duncan's new multiple range method. The same below.

[0101] The results show that the in-dish inhibition rate of strain XNC-3 against the 11 tested tobacco pathogenic fungi ranges from 46.53% to 65.30%.

[0102] 3.3 Inhibitory effects of fermentation filtrates of strain XNC-3 with different volume fractions on pathogenic fungi

[0103] The fermentation broth of strain XNC-3 was aliquoted into centrifuge tubes and centrifuged at 4000 r / min for 10 min. The supernatant was aspirated and filtered through a microporous membrane (pore size 0.22 μm) to collect the fermentation filtrate. The fermentation filtrate was added to the PDA medium and mixed evenly. The volume fractions of the fermentation filtrate in the PDA medium were 10% and 20% respectively. At the same time, a PDA plate medium added with an equal amount of PDA liquid medium was set as a control. Eleven tobacco pathogenic fungi were inoculated respectively. A sterilized borer was used to pick the activated pathogen cake (d = 9 mm) and place it in the center of the PDA plate containing the fermentation filtrate of XNC-3 with different volume fractions. Each group had 3 replicates. After culturing in the dark at 25 °C for 7 d, the cross method was used to measure the colony diameters of the 11 pathogens and calculate the inhibition rate. The results are as Figure 4 shown in Table 4.

[0104] Inhibition rate (%) = (colony diameter of the control group - colony diameter of the treatment group) / (colony diameter of the control group - diameter of the pathogen cake) × 100.

[0105] Table 4 shows the inhibitory effects of the fermentation filtrate of strain XNC-3 on 11 tobacco pathogenic fungi

[0106]

[0107] Note: The data in the table are mean ± standard deviation. Different lowercase letters indicate significant differences (P < 0.05) tested by Duncan's new multiple range method. The same below.

[0108] The results showed that the fermentation filtrate of strain XNC-3 with a volume fraction of 20% had good inhibitory activity against 11 tobacco pathogenic fungi. Especially, it had prominent inhibitory effects on Sclerotinia sclerotiorum, Phytophthora nicotianae, and Botrytis cinerea, and the inhibition rates in the petri dish were all 100%. Except for Fusarium oxysporum, the inhibition rates of this strain against other pathogens were all higher than 60%.

[0109] Example 4 Pot control efficacy of the fermentation broth of strain XNC-3 against tobacco black shank

[0110] Two treatments were tested, namely adding 20 mL of a 10-fold dilution of the fermentation broth of strain XNC-3 (diluted with sterile water) and a sterile water control. Tobacco seedlings at the 4-6 leaf stage and 8-10 leaf stage were selected for the experiment respectively.

[0111] Protection test: Diluted solutions of 20 mL of the strain fermentation broth were applied around the rhizosphere of tobacco seedlings in each treatment. After 24 h, 10 mL of a zoospore suspension of Phytophthora parasitica var. nicotianae at a concentration of 1×10 4 cfu / mL was inoculated; Treatment test: First, 10 mL of a zoospore suspension of Phytophthora parasitica var. nicotianae at a concentration of 1×10 4 cfu / mL was inoculated per plant. After 24 h, 20 mL each of the diluted solution of the strain fermentation broth and sterile water were applied around the rhizosphere of each tobacco seedling. There were 15 plants in each treatment, with 3 replicates. The plants were managed at 25°C in a greenhouse. After 14 d, the disease incidence of tobacco plants was investigated, the incidence rate and disease index were statistically analyzed, and the control effect was calculated. The calculation formula is as follows:

[0112]

[0113] The results are shown in Figure 5 Table 5.

[0114] The grading standard for tobacco black shank disease refers to "Grading and Investigation Methods for Tobacco Diseases and Insect Pests" (GB / T 23222—2008): Grade 0, the whole plant is disease-free; Grade 1, the lesion on the stem does not exceed 1 / 3 of the stem circumference, or the leaves below 1 / 3 wither; Grade 3, the lesion on the stem encircles 1 / 3 - 1 / 2 of the stem circumference, or 1 / 3 - 1 / 2 of the leaves are slightly withered, or a few lower leaves show lesions; Grade 5, the lesion on the stem exceeds 1 / 2 of the stem circumference but does not completely encircle the stem circumference, or 1 / 2 - 2 / 3 of the leaves wither; Grade 7, the lesion on the stem completely encircles the stem circumference, or more than 2 / 3 of the leaves wither; Grade 9, the diseased plant is basically dead.

[0115] Table 5 shows the pot control effect of the fermentation broth of strain XNC-3 against tobacco black shank

[0116]

[0117] The results showed that the control effects of the fermentation broth of strain XNC-3 in the pot protection test for the 4 - 6 leaf stage and 8 - 10 leaf stage were 94.74% and 90.58% respectively, and the control effects in the pot treatment test were 62.11% and 50.22% respectively, and the protective effect was better than the treatment effect.

[0118] Example 5 Effect of the fermentation broth of strain XNC-3 on the growth of tobacco seedlings

[0119] Cultivate Yunyan 99 until it reaches the 4-5 true leaf stage, select tobacco seedlings with consistent growth vigor for transplanting, fill 180 g ± 2.5 g of substrate soil into each seedling-raising pot, with 6 plants per treatment and repeated 3 times. The experiment was divided into 5 treatments. After transplanting, add 10 mL of clear water, LB liquid medium, the original fermentation broth of strain XNC-3, 10-fold diluted fermentation broth (diluted with clear water), and 50-fold diluted fermentation broth (diluted with clear water) into the seedling-raising pots respectively. Irrigate the roots again after 7 days. During this period, the temperature, water, and fertilizer management were the same, with the temperature at (25 ± 0.5) °C, relative humidity at 50% - 60%, and light-dark cycle at 14L / 10D. After 30 days, measure plant height, root length, stem girth, fresh and dry weights of the above-ground and underground parts, maximum leaf width, maximum leaf length, etc., and calculate the root-shoot ratio. The results are as Figure 6 shown in Tables 6-7.

[0120] Table 6 Effects of the fermentation broth of strain XNC-3 on the agronomic traits of tobacco seedlings

[0121]

[0122] Table 7 Effects of the fermentation broth of strain XNC-3 on the biomass of tobacco seedlings

[0123]

[0124] The results showed that the fermentation broth of strain XNC-3 could significantly promote the growth of tobacco seedlings. Root irrigation with the 50-fold diluted fermentation broth could significantly increase plant height, root length, internode distance between the 5th and 6th nodes, stem girth, number of leaves, maximum leaf length, maximum leaf width, fresh and dry weights of the above-ground and underground parts.

[0125] Example 6 Effects of the fermentation broth of strain XNC-3 on the germination of tobacco seeds

[0126] The naked seeds of Yunyan 99 were successively disinfected with 75% ethanol for 10 s, 1% sodium hypochlorite solution for 30 s, rinsed thoroughly with sterile water, and then soaked in the fermentation broth of strain XNC-3 with different volume fractions (sterile water, 10%, 20%, 30%, diluted with sterile water) for 16 h. After rinsing 3-4 times with sterile water and air-drying, the seeds were neatly placed in a petri dish moisturized with filter paper. Using the disinfected seeds soaked in sterile water for the same time as the control, with 50 seeds per petri dish, 2 petri dishes per treatment, and repeated 3 times. The germination index was based on the emergence of the radicle. The number of germinated seeds was counted on the 4th day, and the germination rate was calculated. The results are shown in Table 8.

[0127] Table 8 Effects of the fermentation broth of strain XNC-3 on the germination of tobacco seeds

[0128]

[0129] The results showed that the fermentation broth of strain XNC-3 could significantly promote the germination of tobacco seeds. When the content of the fermentation broth was 30%, the effect of promoting germination was the most obvious.

[0130] Field Application of Fermentation Broth of Strain XNC-3 in Example 7

[0131] 7.1 Method for Field Application of Fermentation Broth of Strain XNC-3

[0132] Treatment 1 (F1): When transplanting tobacco seedlings of Yunyan 99 in the field, drench the roots with 2.5 kg / mu of the fermentation broth of strain XNC-3, apply 500 mL / plant (bacterial amount 5×10 7 cfu / plant) by hole application after diluting the fermentation broth 200-fold (diluted with clear water); after transplanting, spray the leaves with 2.5 kg / mu of the fermentation broth diluted 10-fold, and spray once each at 30 days, 45 days, 60 days, and 75 days, for a total of 4 sprays. Treatment 2 (F2): When transplanting tobacco seedlings of Yunyan 99 in the field, drench the roots with 5.0 kg / mu of the fermentation broth of strain XNC-3, apply 500 mL / plant (bacterial amount 1×10 8 cfu / plant) by hole application after diluting the fermentation broth 100-fold (diluted with clear water); after transplanting, spray the leaves with 2.5 kg / mu of the fermentation broth diluted 10-fold, and spray once each at 30 days, 45 days, 60 days, and 75 days, for a total of 4 sprays. Treatment 3 (CK): Treated with clear water.

[0133] 7.2 Effects of Field Application of Fermentation Broth of Strain XNC-3 on Agronomic Traits of Yunyan 99 at the Fast-Growing Stage and the Round-Top Stage

[0134] According to the tobacco industry standard "YC / T 142-2010 Investigation Method for Tobacco Agronomic Traits", investigate the agronomic traits at the fast-growing stage and the round-top stage, including plant height, stem girth, internode distance, number of effective leaves, maximum length of the middle leaf, and maximum width of the middle leaf. Randomly investigate 15 plants for each treatment, take the average value, and repeat each treatment 3 times. The results are shown in Figures 7-8 Tables 9-10.

[0135] Table 9 Effects of Field Application of Fermentation Broth of Strain XNC-3 on Agronomic Traits of Yunyan 99 at the Fast-Growing Stage

[0136]

[0137]

[0138] Table 10 Effects of Field Application of Fermentation Broth of Strain XNC-3 on Agronomic Traits of Yunyan 99 at the Round-Top Stage

[0139]

[0140] After the field application of the fermentation broth of strain XNC-3, the agronomic traits such as plant height, number of leaves, stem girth, maximum leaf length, and maximum leaf width of Yunyan 99 at the fast-growing stage and the round-top stage were significantly improved.

[0141] 7.3 Effect of field application of XNC-3 fermentation liquid on the opening of the fourth leaf of Yunyan 99

[0142] After the fermentation liquid of strain XNC-3 was applied in the field, the length and width of the first, second, third and fourth leaves from the top were measured 100 days after planting. Fifteen plants were randomly surveyed and the average value was taken. The results were repeated three times. Figure 9 , as shown in Table 11-12.

[0143] Table 11 Effects of field application of fermentation liquid of strain XNC-3 on the length of the four leaves of Yunyan 99

[0144]

[0145] Table 12 Effects of field application of XNC-3 fermentation liquid on the width of four leaves of Yunyan 99

[0146]

[0147] After the fermentation liquid of strain XNC-3 was applied in the field, 100 days after planting, the top four leaves opened well, and the leaf length and width were significantly better than those of the control.

[0148] 7.4 Effects of field application of XNC-3 fermentation broth on the economic traits of Yunyan 99

[0149] After field application of the fermentation liquid of strain XNC-3, the flue-cured tobacco was graded according to the national standard "GB 2635-1992 Flue-cured Tobacco," and the weight of each grade of tobacco leaf was measured. The yield, output value, average price, and proportion of medium and high-quality tobacco leaves for each treatment were calculated, with three replicates. The results are shown in Table 13.

[0150] Table 13 Effects of field application of fermentation liquid of strain XNC-3 on the economic traits of Yunyan 99

[0151]

[0152] Results showed that after field application of the fermentation broth of strain XNC-3 as described above, per-acre yield, per-acre output value, proportion of high-quality tobacco, proportion of medium tobacco, and proportion of high-medium tobacco all significantly increased. Excluding costs, the additional benefits per-acre for Treatment 1 (F1) and Treatment 2 (F2) were 406.45 yuan and 936.37 yuan, respectively. Treatment 2 (F2) significantly increased yield and income, with per-acre yield increasing by 25.68 kg, a 12.74% increase; per-acre output value increasing by 998.87 yuan, a 17.04% increase; and the proportion of high-quality tobacco increased by 4.76 percentage points. Excluding costs, the additional benefits per-acre were 936.37 yuan.

[0153] 7.5 Field control efficacy of strain XNC-3 fermentation liquid against tobacco black shank.

[0154] After the fermentation broth of strain XNC-3 was applied in the field, according to the tobacco industry standard "GB / T 23222-2008 Tobacco Disease Grading and Investigation Methods", the occurrence of black shank was investigated 60 days after transplanting. All plants in each treatment were investigated, with 3 replicates, and the total number of plants and the number of diseased plants at each level in each treatment were recorded; at the same time, the incidence rate, disease index and control effect were statistically calculated, and attention was paid to observing whether there was any phytotoxicity. The calculation formulas are as follows:

[0155]

[0156] The results are shown in Table 14.

[0157] Table 14 Field control effect of the fermentation broth of strain XNC-3 on tobacco black shank

[0158]

[0159] The results showed that after the fermentation broth of strain XNC-3 was applied in the field, the field control effect on tobacco black shank was 88.64%.

[0160] According to the description of the specific implementation manners of the present application, a strain of Bacillus siamensis XNC-3 was isolated in the present application, which has antibacterial activity against 11 kinds of tobacco pathogenic bacteria, has a broad antibacterial spectrum, can be used for plant diseases, especially the prevention and control of tobacco diseases, and has the effect of promoting growth and increasing yield. The Bacillus siamensis XNC-3 shows a good control effect on tobacco black shank, and the field control effect is up to more than 88% when applied in the field, and it can significantly increase the tobacco yield, improve the tobacco quality, and improve the economic benefits, and has good development and application potential in the biological control of tobacco diseases.

[0161] The above embodiments only represent one implementation manner of the present application, and the description thereof is relatively specific and detailed, but it should not be construed as a limitation on the patent scope of the present application. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present application, several modifications and improvements can still be made, and these all belong to the protection scope of the present application.

Claims

1. A Bacillus siamensis XNC-3, which was deposited at the China Center for Type Culture Collection on March 27, 2025, with the deposit number CCTCC NO: M2025600, and the deposit address is Wuhan University, Wuchang District, Wuhan City, Hubei Province.

2. A microbial preparation, characterized in that, It contains the Bacillus siamensis XNC-3 described in claim 1, and / or the fermentation product of the Bacillus siamensis XNC-3 described in claim 1; preferably, the fermentation product refers to the fermentation broth, fermentation filtrate; or the concentrate, dried product or diluent of the fermentation broth, fermentation filtrate; or the metabolites in the fermentation broth.

3. Use of Bacillus siamensis XNC-3 according to claim 1 or the microbial preparation according to claim 2, characterized in that, It is used for plant disease control, inhibiting plant pathogens, promoting plant growth and increasing yield, or promoting the germination of plant seeds.

4. The application according to claim 3, wherein The plant is tobacco.

5. The application according to claim 3 or 4, characterized in that, The plant disease is a disease caused by plant pathogens; The plant pathogens are plant pathogenic fungi, preferably one or more of Sclerotinia sclerotiorum, Phytophthora nicotianae, Boeremia exigua, Botrytis cinerea, Dothiorella gregaria, Alteraria alternata, Stagonosporopsis ligulicola, Thielaviopsis basicola, Colletotrichum capsici, Rhizoctonia solani, Fusarium oxysporum.

6. The application according to claim 3 or 4, characterized in that, The Bacillus siamensis XNC-3 or the microbial preparation is applied by irrigation or spraying, preferably by root irrigation or foliar spraying at the roots of the plant.

7. A method for preventing and controlling tobacco diseases and promoting growth and increasing yield, characterized in that: It comprises the following steps, (1) When transplanting tobacco in tobacco fields, apply the fermentation broth of Bacillus siamensis XNC-3 described in claim 1 containing 1×10 7 -1×10 8 cfu / mL at a rate of 1-10 kg per mu. Apply the 50-500-fold dilution of the fermentation broth to each hole, with 2×10 7 -5×10 8 cfu / plant; (2) After transplanting, apply the fermentation broth of Bacillus siamensis XNC-3 described in claim 1 containing 1×10 7 -1×10 8 cfu / mL at a rate of 1-10 kg per mu, and foliar spray a 5-20-fold dilution of the fermentation broth, continuously spraying 2-6 times every 10-20 days after transplanting.

8. The method according to claim 7, wherein Step (1) Apply 2×10 7 -5×10 7 cfu / mL of the fermentation liquid of Bacillus siamensis XNC-3, 100-200 times dilution of the fermentation liquid was applied to each hole, 5×10 7 -2×10 8 cfu / strain; and / or Step (2) Apply the fermentation broth of the Bacillus siamensis XNC-3 containing 2×10 7 -5×10 7 cfu / mL at a rate of 2-5 kg per mu, and foliar spray a 10-20-fold dilution of the fermentation broth. Continuously spray 2-6 times every 10-20 days after transplanting.

9. A method for seed germination of tobacco, characterized in that, It comprises the following steps: soaking tobacco seeds in a diluent containing 5-50% of the fermentation broth of Bacillus siamensis XNC-3 described in claim 1, and then germinating them; preferably, the fermentation broth contains 1×10 7 -1×10 8 cfu / mL of Bacillus siamensis XNC-3.

10. The method according to claim 9, characterized in that, After disinfecting the tobacco seeds, rinsing them with water, and then soaking them in a diluent containing 10-30% of the fermentation broth for 8-24 hours.

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