Lysobacter enzymogenes LJ6-4 and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and discloses a lysobacter enzymogenes strain and application thereof, lysobacter enzymogenes LJ6-4 has been preserved in China General Microbiological Culture Collection Center on October 18, 2023, and has a preservation number of CGMCC No.28658. The lysobacter enzymogenes LJ6-4 has been preserved in China General Microbiological Culture Collection Center on October 18, 2023; the lysobacter enzymogenes LJ6-4 aqueous solution can be applied to preparation of drugs for preventing and treating tobacco bacterial wilt and panax notoginseng root rot; the preparation method of the lysobacter enzymogenes LJ6-4 water aqua comprises the following steps: strain culture, including test tube strain culture and strain shaking table amplification culture; the fermentation comprises the steps of seed suspension preparation, inoculation, liquid fermentation and aqueous solution preparation. The lysobacter enzymogenes LJ6-4 disclosed by the invention has good comprehensive characters, can be used for preventing and treating tobacco bacterial wilt and panax notoginseng root rot, and is safe to use and free of residues.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to an enzyme-producing Lysozyme Bacillus LJ6-4 and an application thereof. Background Art

[0002] Tobacco bacterial wilt, a soil-borne bacterial disease caused by Ralstonia solanacearum, has a serious impact on tobacco production. The most typical symptom is the wilting of green leaves, hence the name "bacterial wilt." The wilting often occurs on one side of the plant, while leaves on the other side grow normally. As the disease progresses, chlorotic spots appear on one side of the plant stem, gradually developing into black streaks. The roots of the branch on this side often turn black and rot. In later stages, the pith at the base of the tobacco stem completely rots, forming a cavity, and eventually the entire plant turns yellow and dies. The disease can occur throughout the tobacco plant's growth period.

[0003] The prevention and control of tobacco bacterial wilt has always been a global challenge, attracting the attention of scientists both domestically and internationally. Despite recent efforts and attempts by domestic and international scientists to prevent and control tobacco bacterial wilt, and significant progress, there is still no single, effective prevention and control measure. Prevention and control measures for tobacco bacterial wilt mainly include chemical control, breeding of resistant varieties, soil improvement, crop rotation, and biological control. While chemical control is convenient and effective, it is costly and environmentally destructive. Long-term application can easily lead to bacterial wilt resistance. Breeding disease-resistant varieties takes a long time, and the physiological races of pathogens are diverse and variable. Prolonged cultivation of resistant varieties can lead to degradation or even loss of resistance. Long-term soil improvement measures not only alter and damage soil structure but also have limited effectiveness in disease prevention and control. Crop rotation cannot be integrated with local economic development, and its long rotation cycle makes it unsuitable for tobacco cultivation in areas where tobacco is grown. Microbial control, due to its abundant resources, safety, and environmental advantages, plays a key role in promoting sustainable agricultural development and has gradually become the mainstream trend in global agricultural production for plant disease prevention and control.

[0004] Chemical control is an important means of controlling soil-borne diseases, but the extensive use of chemical agents can lead to pesticide residues, environmental pollution, and disruption of ecological balance. Biological control, with its characteristics of no residual pesticides, environmental friendliness, and resistance to pesticides, has gradually replaced chemical control methods and become a new research hotspot. Microbial control refers to the use of specific beneficial microorganisms to inhibit the growth or reproduction of pathogens. Its disease prevention and control mechanisms include: regulating the soil microecology to promote plant growth and enhance plant disease resistance; inducing systemic resistance in plants to pathogens; suppressing pathogen populations through competition for nutrients and ecological niches by beneficial microorganisms; and killing pathogens through the secretion of metabolites or parasitism by beneficial microorganisms.

[0005] Based on preliminary research using Maldi-Tof-Ms and metabolomics, the strain LJ6-4 has been shown to produce antimicrobial substances such as le-pyrrolopyrazines, HSFAs, and lipopeptides. The types and amounts of these active substances play a crucial role in the biocontrol of tobacco bacterial wilt. Enzyme activity assays have shown that the strain LJ6-4 secretes cellulases, proteases, and chitinases, suggesting that its antimicrobial activity is related to the secretion of extracellular hydrolases. Red fluorescent labeling of the strain LJ6-4 has confirmed its excellent colonization of tobacco root tissues, its strong adaptability to the ecological environment, and its potential for the control of tobacco bacterial wilt. Therefore, the strain LJ6-4 possesses significant potential for development.

[0006] Through the above analysis, the problems and defects of the existing technology are as follows:

[0007] (1) With the rapid diversity and mutation of the physiological subspecies of bacterial wilt pathogen, the damage caused by tobacco bacterial wilt has shown a significant trend of increasing severity. The prevention and control of crop bacterial wilt is not ideal and has certain limitations.

[0008] (2) Existing soil improvement is a long-term measure that not only destroys the soil structure but also has little effect on disease prevention and control in most cases.

[0009] (3) Although the current method of using chemical agents to control soil-borne crop diseases is effective, the accumulation of pesticide residues, environmental pollution, and destruction of ecological balance caused by excessive use are contrary to the green and sustainable development principles pursued by modern agriculture. Summary of the Invention

[0010] In view of the problems existing in the prior art, the present invention provides an enzyme-producing Lysozyme Bacillus LJ6-4 and applications thereof.

[0011] The present invention is achieved as follows: a strain of enzyme-producing lytic Bacillus LJ6-4, which was deposited in the General Microbiology Center of the China Culture Collection Administration on October 18, 2023, with the deposit number: CGMCC No. 28658.

[0012] The present invention aims to provide an application of an aqueous solution prepared by the enzyme-producing Lysozyme Bacillus LJ6-4 in preventing and treating diseases such as tobacco bacterial wilt and Panax notoginseng root rot.

[0013] Furthermore, the preparation process of the enzyme-producing Lysobacterium LJ6-4 aqueous solution is as follows:

[0014] Step 1: bacterial culture, i.e. bacterial expansion culture;

[0015] Step 2, fermentation, includes seed liquid preparation, inoculation, liquid fermentation and aqueous solution preparation.

[0016] Furthermore, the bacterial culture in step 1 includes:

[0017] The enzyme-producing Lysozyme Bacillus LJ6-4 was inoculated into KB solid medium and cultured at 28°C for 36 hours.

[0018] Furthermore, the formula of the KB culture medium is: 1.5 g of dipotassium hydrogen phosphate, 1.5 g of magnesium sulfate heptahydrate, 10 mL of glycerol, 17.0-20.0 g of agar powder, 1000 mL of distilled water, and pH 7.0.

[0019] Furthermore, the seed solution preparation in step 2 includes:

[0020] (1) Inoculate a single colony from a plate into liquid KB medium and culture at 28°C for 24 h to obtain the primary seed solution.

[0021] (2) Add the fermentation medium into the fermentation tank, and inoculate the first-level seed liquid into the seed tank at an inoculation rate of 1% to 4% by volume, and culture for 24 hours to obtain the second-level seed liquid.

[0022] Furthermore, the inoculation in step 2 includes:

[0023] The secondary seed liquid is injected into the culture medium in the fermentation tank through the inoculation port with the help of pressure difference at an inoculation amount of 1% to 4% of the volume of the fermentation culture medium.

[0024] The fermentation tank culture medium has the following formula: 0.25% tryptone, 0.5% glucose, 0.025% dipotassium hydrogen phosphate, 1000 mL distilled water, and a pH of 7.0 before sterilization.

[0025] Furthermore, the liquid fermentation in step 2 includes:

[0026] After the fermentation tank culture medium is inoculated, the culture cycle is set at 48-60 hours; during this period, the temperature of the fermentation tank is maintained at 27-30℃ and the pressure in the tank is maintained at 0.5kg / cm 2 At the same time, the stirring speed in the tank was set to 160 rpm, the ventilation volume was maintained at 1:0.5-1 (V / V·min), and the defoaming agent was 1% vegetable oil; during the inoculation and pressure increase stages, the tank pressure was ensured not to exceed 1.5 kg / cm 2 .

[0027] Furthermore, the preparation of the aqueous solution in step 2 includes:

[0028] The fermentation liquid is mixed evenly with 3% Tween 80 (surfactant), 3% carboxymethyl cellulose (thickener), and 1% ascorbic acid (ultraviolet protection agent) to obtain an enzyme-producing Lysozyme Bacillus LJ6-4 aqueous solution.

[0029] Furthermore, the method of using the enzyme-producing Lysobacterium LJ6-4 aqueous solution is root irrigation.

[0030] In combination with the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solutions to be protected by the present invention are analyzed as follows:

[0031] First, the enzyme-producing Lysozyme Bacillus LJ6-4 provided by the present invention is isolated from the rhizosphere soil of Panax notoginseng in Xiaonuohei Village, Guishan Town, Shilin County, Yunnan Province. The enzyme-producing Lysozyme Bacillus LJ6-4 can be made into an aqueous solution, which can act on economic crops such as tobacco, Panax notoginseng, small berries and vegetables, and is used to prevent and control tobacco bacterial wilt and Panax notoginseng root rot.

[0032] The enzyme-producing Lysozyme Bacillus LJ6-4 provided by the present invention can be prepared into an aqueous solution. It inhibits pathogens by producing secondary metabolites, including le-pyrrolopyrazines, HSFAs, lipopeptides, and other active substances, and is used to control tobacco bacterial wilt and Panax notoginseng root rot. Furthermore, the aqueous solution is environmentally safe, leaves no residue, and exhibits strong and stable control effects against various diseases, making it suitable for industrial production and use.

[0033] The Lysobacter enzymogenes LJ6-4 provided by the invention has good comprehensive properties and can be used for preventing and controlling tobacco bacterial wilt and Panax notoginseng root rot.

[0034] Second, the enzymogenes Lysobacterium LJ6-4 provided by the present invention was isolated from the rhizosphere soil of Panax notoginseng in Xiaonuohei Village, Guishan Town, Shilin County, Yunnan Province, and was deposited in the General Microbiology Center of China Culture Collection of Microorganisms on October 18, 2023, with the deposit number: CGMCC No. 28658. The enzymogenes Lysobacterium LJ6-4 aqueous solution can be used to prepare a solution for preventing and treating tobacco bacterial wilt and Panax notoginseng root rot; the preparation method of the enzymogenes Lysobacter LJ6-4 aqueous solution includes: strain culture, including test tube strain culture and strain shaker expansion culture; fermentation, including preparation of seed suspension, inoculation, liquid fermentation and aqueous solution preparation. The enzymogenes Lysobacter (Lysobacter enzymogenes) LJ6-4 of the present invention has good comprehensive properties, can be used to prevent and treat tobacco bacterial wilt and Panax notoginseng root rot, and is safe to use and has no residue.

[0035] Based on preliminary research using Maldi-Tof-Ms and metabolomics, the strain LJ6-4 of Lysozyme-producing Bacillus lysozyme has been shown to produce antimicrobial substances such as le-pyrrolopyrazines, HSFAs, and lipopeptides. The types and amounts of these active substances play an important role in the biocontrol of tobacco bacterial wilt. Enzyme activity assays have shown that the strain LJ6-4 secretes cellulases, proteases, and chitinases, and its antimicrobial activity is related to the secretion of extracellular hydrolases.

[0036] The expected benefits and commercial value of the technical solution after transformation are that Lysobacter enzymogenes LJ6-4 is easy to culture and isolate, has good colonization characteristics in tobacco root tissue, has strong adaptability to the ecological environment, and is more conducive to the prevention and treatment of tobacco bacterial wilt. Therefore, Lysobacter enzymogenes LJ6-4 has great potential for development and prospects.

[0037] Third, the present invention ensures the high activity of enzyme-producing Lysozyme Bacillus LJ6-4 in liquid culture medium by optimizing the culture and fermentation conditions. 2 , the pressure rise does not exceed 1.5kg / cm 2 By adjusting key parameters such as stirring speed (160 rpm), and seed incubation time (24 hours), the number of viable bacteria in the culture solution increased by over 30%, while also reducing bacterial attenuation during the fermentation process, ensuring the stability and long-term effectiveness of the culture solution during application. Compared to existing technologies, this method effectively reduces bacterial inactivation rates and improves the efficiency of the aqueous solution.

[0038] The fermentation process of traditional microbial preparations is easily affected by the culture medium formula, resulting in uneven bacterial growth or metabolic abnormalities. The present invention adopts a specific ratio of KB culture medium (1.5g dipotassium hydrogen phosphate, 1.5g magnesium sulfate heptahydrate, 10mL glycerol, 17-20g agar powder, pH 7.0) and fermentor medium (0.25% tryptone, 0.5% glucose, 0.025% dipotassium hydrogen phosphate, pH 7.0), which optimizes the bacterial growth environment, increases the bacterial reproduction rate, and increases the bacterial concentration per unit time by 25%. Compared with the existing culture method, it can shorten the fermentation time by 6-8 hours, improve the fermentation yield, and reduce the culture medium cost by 10-15%.

[0039] When existing microbial preparations are applied in the field, they have low bacterial survival rates and are easily affected by the external environment, resulting in unstable disease control effects. The present invention improves the survival rate of the LJ6-4 strain in the aqueous solution by 35% by optimizing the bacterial culture and fermentation environment. In addition, the aqueous solution is applied by root irrigation, which avoids the inactivation of the bacteria due to ultraviolet rays or dry environments during the spraying process, improves the rhizosphere colonization ability, and improves the control effect by more than 40%. Compared with traditional biological pesticides, the aqueous solution of the present invention has a longer field residual time, can continuously inhibit the reproduction of pathogens, and enhances the control effect against tobacco wilt and Panax notoginseng root rot.

[0040] Currently, the primary control method for tobacco bacterial wilt and Panax notoginseng root rot in the market still relies on chemical pesticides, but long-term use can lead to soil microecological imbalance, pesticide resistance, and environmental pollution. The microbial aqueous solution of the present invention effectively reduces the frequency of chemical use through a highly effective biological antagonism mechanism, lowering the risk of chemical residues and reducing field pesticide usage by 30%, achieving the development goal of green agriculture. Compared to traditional biological agents, the strain of the present invention, due to its efficient enzyme production and rhizosphere competition advantages, can function over the long term while reducing environmental pollution, improving crop health and achieving sustainable disease management. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to intuitively illustrate the specific implementation scheme of the present invention, the accompanying drawings used will be briefly introduced below. Obviously, the accompanying drawings shown here are only presentations of several examples of the present invention. For ordinary technicians in this technical field, more accompanying drawings can be derived based on these accompanying drawings without performing innovative work.

[0042] Figure 1 The embodiment of the present invention provides Figure 1 A. Gram staining of Lysobacter enzymogenes LJ6-4; Figure 1 B. Line drawing of Lysobacterenzymogenes LJ6-4 after pure culture.

[0043] Figure 2 This is a test on the protective effect of the Lysobacterium enzymogenes LJ6-4 greenhouse potted plants on tobacco bacterial wilt provided by the embodiments of the present invention. Figure 2 A is the LJ6-4 fermentation broth treatment; Figure 2 B was treated with 3% Zhongshengmycin wettable powder; Figure 2 C is the clear water control. DETAILED DESCRIPTION

[0044] In order to more clearly illustrate the objectives, technical implementations and advantages of the present invention, the present invention will be further analyzed through specific examples. It is important to recognize that the specific examples described herein are intended to illustrate rather than limit the scope of the present invention.

[0045] In view of the limitations of the prior art, the present invention innovatively proposes Lysobacter enzymogenes LJ6-4 and its application, and describes the present invention in detail through the accompanying drawings.

[0046] In order to ensure that professionals in this field fully understand the specific implementation methods of the present invention, this section elaborates on the explanation and embodiments of the technical solutions in the claims.

[0047] The LJ6-4 strain provided in the embodiment of the present invention is classified and named Lysobacter enzymogenes. The enzyme-producing Lysobacter enzymogenes strain LJ6-4 was deposited in the General Microbiology Center of the China Culture Collection Administration on October 18, 2023, and its preservation number is CGMCC No. 28658.

[0048] The application of the enzyme-producing Lysozyme Bacillus LJ6-4 provided in an embodiment of the present invention is: the enzyme-producing Lysozyme Bacillus LJ6-4 is prepared into an aqueous solution for the prevention and treatment of diseases such as tobacco bacterial wilt and Panax notoginseng root rot.

[0049] The specific steps of the preparation method of Lysobacter enzymogenes LJ6-4 aqueous solution provided in the embodiment of the present invention are as follows:

[0050] The first step is to culture the strain and expand the strain culture; the second step is fermentation, including the preparation of seed suspension, inoculation, liquid fermentation and aqueous preparation.

[0051] The Lysobacter enzymogenes LJ6-4 provided in an embodiment of the present invention is prepared as an aqueous solution, and the preparation method thereof comprises the following steps:

[0052] (1) Bacterial culture

[0053] Bacterial culture: Lysobacter enzymogenes LJ6-4 was inoculated into KB solid medium and cultured at 28°C for 36 h.

[0054] The formula of the KB medium is: 1.5 g of dipotassium hydrogen phosphate, 1.5 g of magnesium sulfate heptahydrate, 10 mL of glycerol, 17.0-20.0 g of agar powder, 1000 mL of distilled water, pH 7.0;

[0055] (2) Fermentation

[0056] a. Preparation of seed solution: Inoculate a single colony from a plate into liquid KB medium and incubate at 28°C for 24 hours to obtain a primary seed solution;

[0057] The fermentation medium is added to the fermentation tank, and the first-level seed liquid is inoculated into the seed tank at an inoculum amount of 1% to 4% by volume, and cultured for 24 hours to obtain the second-level seed liquid;

[0058] b. Inoculation: Through the inoculation port, the secondary seed liquid is injected into the fermentation tank medium at an inoculum volume of 1% to 4% by pressure differential according to the fermentation medium;

[0059] The formula and processing method of the fermentation tank culture medium are as follows:

[0060] Tryptone 0.25%, glucose 0.5%, dipotassium hydrogen phosphate 0.025%, distilled water 1000mL, pH 7.0 before sterilization.

[0061] c. Liquid fermentation: After the fermentation tank culture medium is inoculated, the culture cycle is set at 48-60 hours; during this period, the temperature of the fermentation tank is maintained at 27-30°C and the pressure in the tank is maintained at 0.5kg / cm 2 At the same time, the stirring speed in the tank was set to 160 rpm, the ventilation volume was maintained at 1:0.5-1 (V / V·min), and the defoaming agent was 1% vegetable oil; during the inoculation and pressure increase stages, the tank pressure was ensured not to exceed 1.5 kg / cm 2 ;

[0062] d. Aqueous preparation: The fermentation broth was mixed with 3% Tween 80 (surfactant), 3% carboxymethyl cellulose (thickener), and 1% ascorbic acid (UV protectant) to obtain an aqueous solution of Bacillus lysozyme LJ6-4.

[0063] Furthermore, the liquid fermentation in step 2 includes:

[0064] The method for using the Lysobacter enzymogenes LJ6-4 aqueous solution provided in the embodiment of the present invention is root irrigation.

[0065] The Lysobacter enzymogenes LJ6-4 strain described in the present invention has the following morphological characteristics:

[0066] Lysobacter enzymogenes LJ6-4 colony Figure 1 As shown, the colonies are yellow-green, with a flat surface and moist, mucus-like appearance. After 48 hours of incubation, the bacteria exhibit a sliding phenomenon. Gram staining is negative, and the optimal growth temperature is 28°C.

[0067] Example 1 Preparation of the aqueous solution of Lysobacterium enzyme-producing LJ6-4.

[0068] (1) Bacterial culture

[0069] Bacteria culture (the following are all weight percentages)

[0070] Prepare a plate with KB culture medium, inoculate Lysobacter enzymogenes LJ6-4, and culture at 28°C for 36 hours.

[0071] (2) Fermentation

[0072] The specific fermentation production process is as follows:

[0073] a. Preparation of seed solution: Inoculate a single colony from a plate into liquid KB medium and incubate at 28°C for 24 hours to obtain a primary seed solution;

[0074] The fermentation medium is added to the fermentation tank, and the first-level seed liquid is inoculated into the seed tank at an inoculum amount of 1% to 4% by volume, and cultured for 24 hours to obtain the second-level seed liquid;

[0075] b. Inoculation: The seed suspension is passed through the inoculation port, and the secondary seed liquid is injected into the fermentation medium in the fermenter by pressure difference at an inoculum amount of 1% to 4% of the fermentation medium volume (the fermenter medium formula is: 0.25% tryptone, 0.5% glucose, 0.025% dipotassium hydrogen phosphate, 1000mL distilled water, pH 7.0 before sterilization. The fermenter tank temperature is 27-30 ° C, and the tank pressure is 0.5kg / cm 2 The stirring speed in the tank is 160 rpm, the ventilation volume is maintained at 1:0.5-1 (V / V·min), and the defoaming agent is 1% vegetable oil; pay attention to avoid contamination, and during the inoculation and pressure-increasing process, the tank pressure does not exceed 1.5 kg / cm 2 .

[0076] c. Liquid fermentation:

[0077] After the seed liquid is inoculated into the fermentation tank, it is cultured for 48 to 60 hours until the number of bacteria no longer increases, and then the tank is opened and the plate count method is used to determine whether the number of viable bacteria reaches 1.5 billion CFU / mL.

[0078] d. Aqueous preparation: The fermentation broth was mixed with 3% Tween 80 (surfactant), 3% carboxymethyl cellulose (thickener), and 1% ascorbic acid (UV protectant) to obtain an aqueous solution of Bacillus lysozyme LJ6-4.

[0079] This preparation can be applied by root irrigation at the early stage of disease after transplanting, which can improve the agronomic traits of the plants and disease prevention and control. It can be applied three times in areas where the disease is seriously occurred.

[0080] Example 2: In vitro antibacterial test of Lysobacter enzymogenes LJ6-4 against different pathogens.

[0081] The culture medium used is as follows:

[0082] KB medium, NA medium, PDA medium.

[0083] In vitro antibacterial test of the bacterial agent of Lysobacter enzymogenes LJ6-4 against Ralstonia solanacearum and other pathogens.

[0084] Test pathogens:

[0085] These include Ralstonia solanacearum, Fusarium oxysporum, Fusarium solani, Phytophthora capsici, Botrytis cinerea, Xanthomonas fragariae, Agrobacterium tumefactions, Colletotrichum siamense, etc. All of the above pathogens were provided by the Bacteria Research Laboratory of Yunnan Agricultural University.

[0086] Test method:

[0087] Activation of pathogenic fungi: Pick up pathogenic fungal hyphae and place them in PDA culture medium, and culture them at a constant temperature of 28°C for later use.

[0088] Activation of bacteria: Pipette pathogenic bacteria stored in cryopreservation tubes and activate them in NA medium, culture at 28℃ for 48h, pick out single colonies and streak for later use.

[0089] Determination method:

[0090] Measurement of the fungal inhibition rate: The inhibitory ability of strain LJ6-4 against pathogenic fungi was tested using the plate standoff method. A 5 mm diameter cake was obtained from a plate containing mature pathogenic fungi using a hole punch and inoculated onto the center of a PDA solid medium. Strain LJ6-4 was then inoculated onto the outer edge of the cake at a distance of 2.5 cm. A control plate inoculated only with pathogenic fungi was also set up. Three replicates were performed for each pathogen. Plates were incubated at a constant temperature of 28°C. Once the control fungi had completely grown on the plate, the size of the inhibition zone was measured and recorded.

[0091] Measurement of the inhibition rate of pathogenic bacteria: Pick a single colony of the purified pathogen in NA liquid culture medium, and culture it with shaking at 28°C and 160 rpm for 36 hours to prepare a bacterial suspension. Take 100 μL of the bacterial suspension and spread it on the NA medium. Then, inoculate the strain LJ6-4 at the center of the plate after spreading. After inverted culture at 28°C for 48 hours, measure the size of the inhibition zone.

[0092] Test results and analysis:

[0093] The test results are shown in Table 1.

[0094] Table 1 Determination of the inhibitory effect of Lysozyme Bacillus LJ6-4 on the growth of different pathogens

[0095]

[0096] Table 1 Determination of the inhibitory effect of Lysozyme-producing Bacillus LJ6-4 on the growth of different pathogens

[0097]

[0098] As shown in Table 1, Lysobacter enzymogenes LJ6-4 has a strong antagonistic effect on the eight pathogenic bacteria and fungi tested. Among them, Lysobacter enzymogenes LJ6-4 has the strongest inhibitory effect on Ralstonia solanacearum, followed by the inhibitory effect on Colletotrichum siamense.

[0099] Example 3: Greenhouse Control Effect of Lysobacter enzymogenes LJ6-4 on Tobacco Bacterial Wilt

[0100] Test crop: Tobacco variety Yunyan 87, moderately resistant to tobacco bacterial wilt.

[0101] Test culture medium: KB liquid culture medium

[0102] Experimental location: Greenhouse of Yunnan Agricultural University, Kunming, Yunnan Province

[0103] Preparation of biocontrol agents:

[0104] A single colony of Lysobacterium zymogenes LJ6-4 was inoculated into KB liquid medium and cultured at 28°C with a shaking rate of 160 rpm for 2 days until the OD value of the fermentation liquid reached 0. 600nm value = 0.5, at this time the fermentation liquid concentration is adjusted to 1×10 8 CFU / mL for subsequent use.

[0105] Experimental methods:

[0106] Tobacco seedlings at the 6-7 leaf stage were selected and transplanted into pots for a tobacco bacterial wilt prevention test. Three treatments were set up in the test: LJ6-4 fermentation liquid treatment, water control, and a control of the antibiotic. The pesticide application method was root irrigation. The experimental treatment was carried out 5 days after the tobacco plants were transplanted. Each tobacco plant was watered with 100 mL of the fermentation liquid of the enzyme-producing strain LJ6-4 (approximately 1×10 8 CFU / mL, and 24 h later, the roots were inoculated with 30 mL of tobacco bacterial wilt pathogen fermentation liquid (about 1×10 8 CFU / mL). After inoculation with tobacco bacterial wilt pathogen, two applications of biocontrol bacteria fermentation broth were applied, 7 days apart. The pesticide used was a 3% Zhongshengmycin wettable powder diluted 800-fold. Each treatment involved 20 tobacco plants. The seedlings were maintained in a constant-temperature, light-treated greenhouse at the Yunnan Agricultural University Engineering Center, with 16 hours of light per day, a temperature of 30°C, and 80% humidity. Disease investigations were conducted 14 days after treatment. Tobacco disease classification and investigation methods were based on the national standard GB / T23222-2008 (Table 2).

[0107] Table 2 Grading standards for tobacco bacterial wilt

[0108]

[0109] Incidence rate = (number of diseased plants / number of surveyed plants) × 100%

[0110] Disease index = Σ(number of diseased plants × corresponding disease level) / (total number of plants × highest disease level) × 100 Relative control efficacy = (control disease index - treatment disease index) / control disease index × 100%

[0111] Table 3 Greenhouse efficacy of biocontrol agent LJ6-4 against tobacco bacterial wilt

[0112]

[0113] Under greenhouse conditions, the control effect of strain LJ6-4 on tobacco bacterial wilt in potted plants was determined. The results showed that the disease index of strain LJ6-4 was 18.67 (Table 3), which was significantly lower than the disease index of 30.05 treated with Zhongshengmycin and the disease index of 69.78 in the control group ( Figure 2 C). The relative protection efficiency of strain LJ6-4 against tobacco bacterial wilt was 73.25% ( Figure 2 A), which is also significantly higher than the relative efficacy of 56.93% ( Figure 2 B).

[0114] Example 4: Field test of the efficacy of Lysobacter enzymogenes LJ6-4 against tobacco bacterial wilt

[0115] Test crop: Tobacco variety Yunyan 87, moderately resistant to tobacco bacterial wilt.

[0116] Test culture medium: KB liquid culture medium

[0117] Location: Kangnong Village Group, Xi'e Village Committee, Yongping Town, Jinggu County, Pu'er City

[0118] Preparation of biocontrol agents:

[0119] A single colony of Lysobacterium zymogenes LJ6-4 was inoculated into KB liquid medium and cultured at 28°C with a shaking rate of 160 rpm for 2 days until the OD value of the fermentation liquid reached 0. 600nm value = 0.5, at this time the fermentation liquid concentration is adjusted to 1×10 8 CFU / mL for subsequent use.

[0120] Experimental methods:

[0121] The experiment set up three treatments: biocontrol agent treatment, water control, and pesticide control. The tobacco plantations in the experimental field were planted at a spacing of 0.5×1.2 (m). The experimental area was divided into three rectangular plots with an aspect ratio of 2:1. Each row in the plot was a treatment, and each treatment was repeated three times. Each treatment had 50 tobacco seedlings, and two protective rows were set around the plot. 7 days after the tobacco was transplanted, 3% Zhongshengmycin wettable powder was diluted 800 times, and 300 mL / plant was used for root irrigation. At the same time, each tobacco plant was irrigated with 300 mL of fermentation liquid of the enzyme-producing Lysozyme Bacillus LJ6-4 strain (about 1×10 8 CFU / ml) as a biocontrol agent, with 3-4 applications at 7-day intervals. Field management was carried out according to conventional tobacco planting procedures. Fifty days after transplanting, a disease survey was conducted, recording the incidence and disease index of tobacco bacterial wilt. Relative control efficacy was then calculated and analyzed.

[0122] The tobacco disease classification and investigation methods were carried out in accordance with Example 3.

[0123] Table 4 Field efficacy of biocontrol agent LJ6-4 against tobacco bacterial wilt

[0124]

[0125] Field plot tests showed that treatment with LJ6-4 fermentation broth significantly reduced the disease index of tobacco bacterial wilt in the field compared to the control, achieving a relative control efficacy of 69.71% (Table 4), significantly higher than that achieved with the chemical agent, zhongshengyin, showing the most significant control effect. This suggests that the biocontrol agent, Lysobacter enzymogenes LJ6-4, has a strong control effect against tobacco bacterial wilt, and the test is reproducible. Greenhouse and field trials demonstrated that the biocontrol agent, Lysobacter enzymogenes LJ6-4, exhibits significant and stable control efficacy against tobacco bacterial wilt, demonstrating the foundation for its widespread application in agricultural production.

[0126] Example 5: Disease prevention test of Lysobacter enzymogenes LJ6-4 against root rot of biennial Panax notoginseng grown in greenhouses.

[0127] Test crops: Biennial Panax notoginseng

[0128] Test culture medium: KB liquid culture medium

[0129] Experimental location: Panax notoginseng farmer's greenhouse in Pingyuan Street, Yanshan County, Wenshan Prefecture

[0130] Preparation of biocontrol agents:

[0131] A single colony of Lysobacterium zymogenes LJ6-4 was inoculated into KB liquid medium and cultured at 28°C with a shaking rate of 160 rpm for 2 days until the OD value of the fermentation liquid reached 0. 600nm value = 0.5, at this time the fermentation liquid concentration is adjusted to 1×10 8 CFU / mL for subsequent use.

[0132] Test method:

[0133] The experiment set up two treatments: biocontrol agent treatment and clear water control, with 3 replicates for each treatment. All treatments were applied by root irrigation. 500 mL of biocontrol agent solution was irrigated to the roots of each two-year-old Panax notoginseng tree. The biocontrol agent was applied once every 7 days for 3 consecutive times. The disease was investigated 90 days after application. Ten two-year-old Panax notoginseng trees were randomly selected from the replicate plots of each treatment for investigation. The root rot disease was graded and the incidence rate, disease index, and disease prevention effect were calculated. The grading standards are as follows:

[0134] Table 5 Field grading standards for Panax notoginseng root rot:

[0135]

[0136] Table 5: Field grading standards for Panax notoginseng root rot:

[0137]

[0138] Incidence rate = (number of diseased plants / number of surveyed plants) × 100%

[0139] Disease index = Σ(number of diseased plants × corresponding disease level) / (total number of plants × highest disease level) × 100 Relative control efficacy = (control disease index - treatment disease index) / control disease index × 100%

[0140] Test results and analysis

[0141] The test results are shown in Table 6.

[0142] Table 6 The disease prevention effect of Lysozyme-producing Bacillus LJ6-4 on Panax notoginseng root rot

[0143]

[0144] As shown in Table 6, the application of Lysobacter enzymogenes LJ6-4 had a certain preventive effect on the root rot of biennial Panax notoginseng, and significantly reduced the incidence rate and disease index of the root rot of biennial Panax notoginseng.

[0145] Example 6: Field control trial of Lysobacter enzymogenes LJ6-4 against clubroot disease of Chinese cabbage.

[0146] Test crop: Chinese cabbage (Luchunbai No. 1), susceptible to clubroot.

[0147] Test culture medium: KB liquid culture medium

[0148] Experimental location: Chinese cabbage experimental field in Dabai Village, Songhua Street, Panlong District, Kunming City, Yunnan Province

[0149] Preparation of biocontrol agents:

[0150] A single colony of Lysobacterium zymogenes LJ6-4 was inoculated into KB liquid medium and cultured at 28°C with a shaking rate of 160 rpm for 2 days until the OD value of the fermentation liquid reached 0. 600nm value = 0.5, at this time the fermentation liquid concentration is adjusted to 1×10 8 CFU / mL for subsequent use.

[0151] Experimental methods:

[0152] The experiment included three treatments: a biocontrol agent treatment, a water control, and a pesticide control, with three replicates per treatment. All treatments were applied via root irrigation, with 300 mL of the biocontrol solution irrigated onto the roots of each Chinese cabbage plant. The first application of the biocontrol agent occurred at the early onset of clubroot, followed by topdressing every seven days for a total of three applications. After 50 days of treatment, a disease survey was conducted, and the incidence and disease index of clubroot in Chinese cabbage were recorded. The control efficacy was calculated and analyzed. Thirty Chinese cabbage plants were randomly selected from each treatment for the survey, with three replicates. The treatments were graded based on clubroot disease incidence (see Table 7). The incidence, disease index, and control efficacy of each treatment were calculated using the following method.

[0153] Table 7 Clubroot grading standards

[0154]

[0155] Incidence rate = (number of diseased plants / number of surveyed plants) × 100%

[0156] Disease index = Σ(number of diseased plants × corresponding disease level) / (total number of plants × highest disease level) × 100 Relative control efficacy = (control disease index - treatment disease index) / control disease index × 100%

[0157] Table 8 Field efficacy of biocontrol agent LJ6-4 against Chinese cabbage clubroot

[0158]

[0159] As shown in Table 8, field application of the biocontrol agent Lysobacter enzymogenes LJ6-4 demonstrated significant control effects against Chinese cabbage clubroot, achieving a 64.68% efficacy. Compared to the control and the pesticide cyazofamid, application of the biocontrol agent LJ6-4 significantly reduced both the incidence and disease index, demonstrating the most significant control effect. This demonstrates that the biocontrol agent LJ6-4 exhibits significant and consistent control effects against Chinese cabbage clubroot, and the reproducibility of its experimental results further confirms the agent's high efficacy and reliability. This discovery not only provides a powerful biological control method for Chinese cabbage clubroot but also lays a solid foundation for its large-scale adoption in agricultural production.

[0160] The above is only a specific embodiment of the present invention, and its protection scope is not limited thereto. Any person skilled in the art who is familiar with this technical field can make modifications, equivalent replacements or optimizations within the technical scope disclosed in the present invention, which should all be considered as the protection scope of the present invention.

Claims

1. A Lysobacter enzyme-producing strain LJ6-4, characterized in that The enzyme-producing Lysozyme Bacillus LJ6-4 was deposited in the General Microbiology Center of the China Culture Collection Administration on October 18, 2023, with the preservation number: CGMCC No. 28658.

2. Use of the enzyme-producing Lysozyme Bacillus strain LJ6-4 as claimed in claim 1 in preparing an aqueous solution for preventing and treating tobacco bacterial wilt and Panax notoginseng root rot.

3. Use of an enzyme-producing Lysozyme Bacillus strain LJ6-4 in the preparation of an aqueous solution for preventing and treating tobacco bacterial wilt and Panax notoginseng root rot, characterized in that: The preparation method of the aqueous solution comprises the following steps: S1. Bacterial culture: Inoculate Lysobacterium enzymogenes LJ6-4 onto KB solid medium and incubate at 28°C for 36 h. S2. Fermentation: S2.

1. Seed solution preparation: ① Inoculate a single colony from a streak plate into liquid KB medium and incubate at 28°C for 24 h to obtain a primary seed solution; ② Add the fermentation medium to the fermentation tank, and inoculate the first-level seed solution at a volume of 1% to 4% of the first-level seed solution into the seed tank, and culture for 24 hours to obtain the second-level seed solution; S2.2 inoculation: Through the inoculation port, the secondary seed liquid is injected into the fermentation tank medium at an inoculum volume of 1% to 4% by pressure differential; S2.

3. Liquid fermentation: After inoculation, the fermentation tank culture medium is cultured for 48 to 60 hours, with the tank temperature at 27 to 30°C and the tank pressure at 0.5 kg / cm 2 The stirring speed in the tank is 160 rpm, the ventilation volume is maintained at 1:0.5-1 (V / V·min), the defoaming agent is 1% vegetable oil, and the tank pressure does not exceed 1.5 kg / cm during the inoculation and pressure increase process. 2 ; S2.

4. Preparation of aqueous solution: The fermentation broth is mixed evenly with 3% Tween 80 (surfactant), 3% carboxymethyl cellulose (thickener), and 1% ascorbic acid (UV protectant) to obtain the enzyme-producing Lysozyme Bacillus LJ6-4 aqueous solution. S3. Root irrigation: apply the aqueous solution to the roots of tobacco or Panax notoginseng by root irrigation.

4. The use according to claim 3, characterized in that The formula of the KB culture medium is: 1.5 g of dipotassium hydrogen phosphate, 1.5 g of magnesium sulfate heptahydrate, 10 mL of glycerol, 17.0-20.0 g of agar powder, 1000 mL of distilled water, and pH 7.

0.

5. The use according to claim 3, characterized in that The formula of the fermentation tank culture medium is: 0.25% tryptone, 0.5% glucose, 0.025% dipotassium hydrogen phosphate, 1000 mL distilled water, and pH 7.0 before sterilization.

6. The use according to claim 3, characterized in that During the bacterial culture process, solid culture medium is used for preliminary culture to improve the activity of the subsequent fermentation bacterial liquid.

7. The use according to claim 3, characterized in that During the preparation of the seed solution, the liquid KB medium was cultured at 28°C for 24 h to obtain a bacterial suspension of appropriate concentration.

8. The use according to claim 3, characterized in that During the liquid fermentation process, the initial tank pressure of the fermentation tank was controlled at 0.5 kg / cm 2 , gradually increase the pressure to 1.5kg / cm 2 To ensure cell growth and metabolic balance.

9. The use according to claim 3, characterized in that During the fermentation process, the stirring speed in the tank was set at 160 rpm to maintain uniform distribution of bacteria and improve fermentation efficiency.

10. The use according to claim 3, characterized in that The use of the aqueous solution includes at least one root irrigation application to ensure that the microorganisms effectively act on the root environment of tobacco and Panax notoginseng.

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