Bacillus firmus BCK018 and application thereof

Abstract

The invention discloses a bacillus firmus BCK018 strain and an application of the bacillus firmus BCK018 strain. The bacillus firmus BCK018 is preserved in the China General Microbiological Culture Collection Center on July 18, 2024, and the preservation number of the bacillus firmus BCK018 is CGMCC No. 31342. The bacillus firmus BCK018 has the advantages that the fatality rate of the bacillus firmus BCK018 on meloidogyne incognita J2 reaches 86.3%, the growth inhibition rate of fusarium oxysporum is 74.4%, the inhibition rate of the bacillus firmus BCK018 on ralstonia solanacearum is 88.1%, and the microbial agent prepared from the bacillus firmus BCK018 can be used for preventing and treating meloidogyne and soil-borne diseases of various crops and has the advantages of being efficient, safe, environmentally friendly and the like.

Description

Technical Field

[0001] The present application belongs to the field of microbial technology, and in particular relates to a strain of Bacillus firmus BCK018 and its application. Background Art

[0002] Root-knot nematode complex diseases of crops refer to diseases caused by root-knot nematodes and soil-borne pathogens that inhabit the same ecological niche and infect the same crop together. Root-knot nematodes and various plant pathogenic fungi and bacteria cause complex diseases caused by the combined infection. For example, complex diseases caused by the combined infection of pathogenic bacteria Pseudomonas, Rolbachia, Chryseobacterium and pathogenic fungi Fusarium oxysporum, Phytophthora scaber, and Sphaerocephala are extremely common and have a great impact on modern agricultural production. The wounds caused by the second-instar larvae of root-knot nematodes infecting the root system of crops are conducive to the infection of various soil-borne pathogenic fungi and bacteria. Root-knot nematodes have been widely reported in my country's economic crops such as melons, Chinese medicinal materials, tobacco, fruits, cotton, bananas, and beans, causing serious economic losses.

[0003] At present, it has become urgent to prevent crop diseases. The main ways to prevent and control diseases are chemical control, physical control, biological control, etc. There are few studies on bacterial microbial diseases for the prevention and control of root-knot nematodes, and the prevention and control effect needs to be improved. Biological control refers to the use of one organism to fight against another organism, so as to achieve the purpose of preventing and controlling crop diseases. Microbial pesticides can effectively reduce the occurrence of nematodes, inhibit the pathogens carried by nematodes, improve the root microecological environment, and stimulate crop growth; microbial control has gradually become a hot topic in the research of plant disease prevention and control. This patent proposes that Bacillus firmus BCK018 can prevent and control root-knot nematode diseases, which can inhibit eggs, infect larvae and female insects, and significantly reduce the harm of plant nematode diseases such as root-knot nematodes, cyst nematodes, and stem nematodes in various crops. Bacillus firmus BCK018 has the characteristics of high efficiency, safety, and environmental friendliness. It has achieved remarkable results in the prevention and control of crop diseases and has a wide range of uses for biological control bacteria to prevent and control crop diseases. Summary of the invention

[0004] In view of the above problems, the purpose of the present invention is to provide a strain of Bacillus firmus BCK018 and the application of the strain in preventing and controlling soil-borne diseases of root-knot nematodes in crops.

[0005] The invention of the present application is achieved as follows: a strain of Bacillus firmus (Bacillus firmus strain) BCK018, wherein the Bacillus firmus BCK018 was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on July 18, 2024, with a deposit number of CGMCCNO.31342.

[0006] Another object of the present invention is a microbial agent comprising the above-mentioned Bacillus firmus BCK018, with a live bacteria content of 0.2-100 billion CFU / gram.

[0007] Preferably, the Bacillus firmus BCK018 and the microbial agent containing Bacillus firmus BCK018 are used for the treatment of root-knot nematode diseases, root rot and canker diseases of crops.

[0008] Preferably, the root-knot nematode disease is root-knot nematodes incognita, the pathogenic fungus of root rot is Fusarium oxysporum, and the pathogen of bacterial wilt is Ralstonia solanacearum.

[0009] Preferably, the microbial agent is applied by spraying, seed mixing, root dipping, furrow application, or hole application, and the application amount is 2-5 kg / mu.

[0010] The present invention has the following advantages:

[0011] The Bacillus firmus BCK018 of the present invention is separated from the roots of crops seriously affected by soil-borne diseases, can resist root-knot nematodes, and inhibit pathogenic bacteria and pathogenic fungi. The microbial agent prepared by the Bacillus firmus BCK018 strain can effectively prevent and control root-knot nematodes and complex diseases of crops, and has the characteristics of low use cost, no residue, and safety for humans and animals. It is a very safe biological control product, indicating that the bacteria has great potential to be developed into a commercialized biological pesticide. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 This is the colony morphology and bacterial cell characteristics of Bacillus firmus BCK018.

[0013] Figure 2 Schematic diagram of Bacillus firmus BCK018 inhibiting root-knot nematodes.

[0014] Figure 3 Schematic diagram of the inhibition of Fusarium by Bacillus firmus BCK018.

[0015] Figure 4 Schematic diagram of the inhibition of Ralstonia by Bacillus firmus BCK018. DETAILED DESCRIPTION

[0016] In order to make the purpose, technical solution and advantages of the present application more clear, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0017] Example 1

[0018] Isolation, Screening and Identification of Bacillus firmus BCK018

[0019] 1. Strain isolation and screening

[0020] The roots of melons infected with root-knot nematodes were collected from vegetable planting areas in Jingyang County, Xianyang City, Shaanxi Province. After the surface of the nodules was cleaned and disinfected, the egg capsules and female worms of the root-knot nematodes were separated and picked, mixed with physiological saline and inoculated on nutrient agar plates. After culturing at 25°C for 7 days, a total of 48 strains of bacteria were obtained by the traditional streak separation method. These 48 strains of bacteria were cultured in LB liquid culture medium at 32°C and 150rpm for 3 days to obtain bacterial fermentation liquid. The lethality of the fermentation liquid of each strain to southern root-knot nematode J2 was determined. The results showed that the lethality of 25 strains to J2 was above 70%. Then, the pathogenic fungus Fusarium oxysporum and the pathogenic bacteria Rothschildi of the root-knot nematode complex disease were used as targets to determine the antagonism of the fermentation liquid of these 25 fungi to the two pathogens. The results showed that among the 25 strains, 4 strains (BCK006, BCK011, BCK018 and BCK025) could resist three target pathogens at the same time, among which BCK018 strain had the best activity, with a lethality rate of 86.3% against southern root-knot nematode J2, a growth inhibition rate of 74.4% against Fusarium oxysporum, and an inhibition rate of 88.1% against Ralstonia solanacearum (Table 1). This shows that Bacillus firmus BCK018 strain has good potential for biological control applications.

[0021] Table 1 Determination of the inhibitory effect of fungi on the pathogens of root-knot nematode complex disease

[0022]

[0023] 2. Identification of strain Bacillus firmus BCK018

[0024] The screened strain Bacillus firmus BCK018 was inoculated into LB medium and cultured at 35°C. Observation showed that the colonies of the strain were earthy white, with concave and convex edges, flat colonies, and short rod-shaped bacteria.

[0025] The strain was inoculated in liquid LB medium for fermentation. After culturing at 28°C for 5 days, the bacteria were collected and ground with liquid nitrogen. The fungal strain DNA was extracted using a bacterial genomic DNA extraction kit (Beijing Tiangen Biotechnology Co., Ltd.). The extraction steps were referred to the kit instructions. The strain DNA was PCR amplified using bacterial 16S rDNA universal primers. The amplified product was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequence was Blast compared using NCBI data, and phylogenetic analysis was performed using MEGA7.0 software. After phylogenetic analysis and combined with bacterial morphological observation, the strain BCK018 was determined to be a bacillus firmus strain. The strain was deposited in the General Microbiology Center (CGMCC) of the China Microbiological Culture Collection Administration Committee with a deposit number of CGMCC NO.31342 and a deposit date of July 18, 2024.

[0026] Example 2

[0027] Cultivation of Bacillus firmus BCK018 and preparation of microbial inoculants

[0028] 1. Test tube slant seed culture of strain Bacillus firmus BCK018: inoculate the strain onto a conventional LB medium slant and culture at 35°C for 2 days to obtain slant seeds.

[0029] 2. Liquid seed culture of strain Bacillus firmus BCK018: inoculate the slant seeds into a Erlenmeyer flask filled with LB liquid culture medium, and culture in a shaking incubator at 35° C. and 160 rpm for 2 days to obtain liquid seeds.

[0030] 3. Large-scale culture of strain Bacillus firmus BCK018 in a fermenter: In a 1000-liter fermenter, the liquid seeds were inoculated into a sterilized composite culture medium containing soybean meal and bran at a ratio of 5% (V / V). The fermentation conditions were a stirring speed of 180 rpm, a temperature of 35°C, an aeration volume of 340 L / min, a fermentation time of 2 days, and the fermentation was terminated when the bacterial content of the fermentation liquid was above 50 billion CFU / mL.

[0031] 4. Preparation of the bacterial agent of Bacillus firmus BCK018: The bacterial bodies and their metabolites obtained by fermentation tank culture are evenly mixed with appropriate amounts of diatomaceous earth, Tween 80 and starch, and then vacuum dried at 45°C to a moisture content of 5%. The fineness of the obtained powder is 100 mesh using a cyclone mill to obtain a Bacillus firmus BCK018 bacterial agent with a content of 50 billion CFU / g.

[0032] Application Example 3

[0033] Experimental study on the efficacy of Bacillus firmus BCK018 against melon root-knot nematodes and their combined diseases

[0034] Test location: melon planting area in Yanliang District, Xi'an City, Shaanxi Province. The test site is a greenhouse where southern root-knot nematode disease occurs severely.

[0035] Experimental crop: Ximi No. 3 melon.

[0036] Test agents: Bacillus firmus BCK018 microbial agent, with a content of 10 billion CFU / g, prepared according to the method of the present invention; the control pesticide for controlling root-knot nematodes is 10% thiathiazolyl granules (Syngenta Crop Protection Co., Ltd., Switzerland), the control pesticide for controlling soil-borne fungal diseases is 30% carbendazim aqueous solution (Weifang Huanuo Biotechnology Co., Ltd.), and the control pesticide for controlling soil-borne bacterial diseases is 20% thiophanate-copper suspension (Yancheng Limin Agrochemical Co., Ltd., Jiangsu Province). Melon seedlings were raised according to conventional methods and the drugs were used when transplanting.

[0037] Experimental method: There are three treatments as follows, each with 3 replications and each replication covering 0.3 mu.

[0038] Treatment 1: Bacillus firmus BCK018 microbial agent, 2.0 kg / mu. Take 1.0 kg of the agent when transplanting, dilute it with 200 kg of water and apply it to the roots; take 1.0 kg of the agent 30 days after transplanting, dilute it with 800 kg of water and then irrigate the roots.

[0039] Treatment 2: During land preparation, spread 10% thiamethoxam granules (1500 g / mu) on the soil and mix it evenly into the ridges. Seven days after transplanting, dilute 30% carbendazim aqueous solution (500 ml / mu) and 20% thiophanate-methyl suspension (500 ml / mu) with water according to the instructions and then irrigate the roots.

[0040] Treatment 3: blank control without any pesticide application.

[0041] Investigation and statistical methods: Six months after transplanting, 100 melon plants were randomly selected from each treatment, and the plant disease levels for root-knot nematode disease and combined diseases were divided, and the disease index and prevention effect were calculated.

[0042] Calculation of root-knot nematode index and control effectiveness survey

[0043] The root knot grading standards are as follows: Level 0, no root knots are found on the roots; Level 1, a small number of root knots can be found through careful identification; Level 2, no root knots on the main root, and a small number of root knots on the fibrous roots can be clearly identified; Level 3, no root knots on the main root, and slightly larger root knots can be found on the fibrous roots; Level 4, no root knots on the main root, and mainly larger root knots on the fibrous roots; Level 5, 50% of the roots are infected, and the main root is very slightly infected; Level 6, root knots are found on the main root; Level 7, most of the main roots are infected by root knots; Level 8, all main roots are infected or very few roots are not infected; Level 9, all roots can be severely infected or the plant usually dies; Level 10, all roots are severely infected and there are no roots.

[0044] The root knot index and relative control effect are calculated as follows:

[0045] Root knot index = [∑(number of diseased plants at each level × representative value of each level) / (total number of plants surveyed × highest representative value)] × 100.

[0046] Relative control effect = (root knot index of control group - root knot index of treatment group) / root knot index of control group × 100%.

[0047] Calculation of soil-borne disease classification and control effectiveness survey method

[0048] Grading standard: Level 0: no disease symptoms on the whole plant; Level 1: mild vascular discoloration, no external symptoms; Level 2: moderate vascular discoloration, no external symptoms; Level 3: the lower leaves turn yellow and gradually wilt; Level 4: most of the leaves wilt; Level 5: the plant dies.

[0049] Disease index = [∑(number of diseased plants at each level × representative value at each level) / (total number of plants surveyed × highest representative value)] × 100.

[0050] Control effect = (control disease index - treatment disease index) / control disease index × 100%

[0051] Table 2 Determination of the efficacy of BCK018 microbial agent against the pathogens of melon root-knot nematode complex disease

[0052]

[0053]

[0054] Test results: Table 2 shows that in treatment 1, 3.0 kg of Bacillus firmus BCK018 microbial agent was applied per mu, with a live bacteria content of 2 billion CFU / g, and the control effects on melon root-knot nematode disease and combined diseases were 80.89% and 69.47%, respectively; in treatment 2, three pesticides for combined diseases of root-knot nematodes, fungi and bacteria were applied, and the control effects on melon root-knot nematode disease and combined diseases of root-knot nematodes were 81.12% and 72.03%, respectively. The two treatments of microbial agent and chemical drug had equivalent control effects on root-knot nematode disease and combined diseases of greenhouse melons.

[0055] Application Example 4

[0056] Experiment on the efficacy of Bacillus firmus BCK018 against kiwifruit knot nematodes and their combined diseases

[0057] Test location: kiwifruit base in Zhouzhi County, Shaanxi Province, the test site is a field where root-knot nematode disease occurs severely.

[0058] Experimental crop: Actinidia chinensis.

[0059] Test agent: Bacillus firmus BCK018 microbial agent, with a content of 50 billion CFU / g, prepared according to the method of the present invention. The control pesticide for controlling root-knot nematodes is 10% thiazolyl granules (Syngenta Crop Protection Co., Ltd., Switzerland), the control pesticide for controlling soil-borne fungal diseases is 30% carbendazim aqueous solution (Weifang Huanuo Biotechnology Co., Ltd.), and the control pesticide for controlling soil-borne bacterial diseases is 20% thiophanate-copper suspension (Yancheng Limin Agrochemical Co., Ltd., Jiangsu Province). Kiwifruit was planted and managed according to conventional methods, and the root was irrigated with drugs.

[0060] Experimental method: There are three treatments as follows, each with 3 replications and each replication covering 0.5 mu.

[0061] Treatment 1: 6.0kg / mu of Bacillus firmus BCK018 microbial agent. In March, take 2.0kg of the agent, dilute it with 500kg of water and irrigate the roots; after May, take 2.0kg of the agent, dilute it with 500 times of water and irrigate the roots once; in August, take 2.0kg of the agent, dilute it with 500 times of water and irrigate the roots once, and manage water and fertilizer normally.

[0062] Treatment 2: In March, 10% thiamethoxam granules (2000 g / mu) were spread on the ground and evenly mixed into the tillage layer. In May, 30% carbendazim aqueous solution (800 ml / mu) and 20% thiophanate-methyl suspension (800 ml / mu) were diluted with water according to the instructions and then irrigated the roots.

[0063] Treatment 3: blank control with no treatment.

[0064] Investigation and statistical methods: 9 months after transplanting, statistics were conducted after the disease was fully developed. A five-point sampling method was used for each plot, with 5-10 plants checked at each point. The root-knot nematode disease and combined disease were classified into plant disease levels, and the disease index and prevention effect were calculated according to the method in Example 3.

[0065] Table 3 Determination of the efficacy of BCK018 microbial agent against the pathogens of kiwifruit root-knot nematode complex disease

[0066]

[0067] Test results: Table 3 shows that in treatment 1, 6.0 kg of Bacillus firmus BCK018 with a content of 50 billion CFU / g per mu was applied, and the control effects on kiwifruit root knot nematode disease and its composite disease were 81.83% and 74.10%, respectively. In treatment 2, chemical pesticides targeting root knot nematodes, pathogenic fungi and pathogenic bacteria were applied simultaneously, and the control effects on kiwifruit root knot nematode disease and composite diseases were 77.57% and 54.32%, respectively. The control effect of Bacillus firmus BCK018 microbial agent on root knot nematode disease was higher than that of chemical pesticides, and the control effect on composite diseases was significantly higher than that of chemical pesticides.

[0068] In summary, the microbial agent prepared by using Bacillus firmus BCK018 in the present invention has a prevention effect of more than 80% on the root-knot nematode disease of melon and kiwi fruit, and a prevention effect of more than 69% on the root-knot nematode complex disease. It can be seen that the microbial agent can effectively prevent the root-knot nematode disease of various crops, and also has a certain prevention effect on soil-borne fungal diseases and bacterial diseases. It has the characteristics of low use cost, no residue, and safety for humans and animals.

[0069] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A Bacillus firmus strain BCK018 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on July 18, 2024, with the deposit number CGMCC NO.31342.

2. A solid microbial agent, characterized in that: The invention comprises the Bacillus firmus strain BCK018 as claimed in claim 1, and has a live bacteria content of 0.2-100 billion CFU / g.

3. The use of the Bacillus firmus BCK018 according to claim 1 or the microbial agent according to claim 2, characterized in that: Used for the prevention and control of root-knot nematode disease, root rot and bacterial wilt of crops.

4. The use of the microorganism as claimed in claim 3, characterized in that The root-knot nematode disease to be controlled is the southern root-knot nematode, the pathogenic fungus of root rot is Fusarium oxysporum, and the pathogen of bacterial wilt is Ralstonia solanacearum.

5. The use of the microbial agent according to claim 2, characterized in that: The application methods are spraying, seed mixing, root dipping, furrow application, and hole application. The application amount is 2-5 kg / mu.

Images