Bacillus subtilis MJ-4 as well as fungicide, preparation method and application thereof

Abstract

The invention provides bacillus subtilis MJ-4 as well as a microbial agent, a preparation method and application thereof, and relates to the technical field of microbial fertilizers, the bacillus subtilis MJ-4 is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No. 30850. The bacillus subtilis MJ-4 strain provided by the invention is a strain MJ-4 which is separated from sick bark beetle bodies and has a relatively strong lethal effect on pine bark beetles, and is high in propagation speed and stable in performance. The bacillus subtilis strain is identified as gram positive bacillus subtilis. The microbial agent prepared by fermentation of the bacillus subtilis MJ-4 has the advantages that the corrected death rate of bark beetles in contact with pine trees can reach 90.04%, the corrected death rate of stomach toxicity can reach 57.84%, and the microbial agent has a good prevention and control effect on the bark beetles in the pine trees.

Description

Technical Field

[0001] This invention relates to the field of microbial fertilizer technology, and in particular to a Bacillus subtilis MJ-4, its inoculum, preparation method and application. Background Technology

[0002] There are approximately 500 recorded species of bark beetles in my country, among which the most serious forest pests include the red turpentine beetle (Gnaphalium affineum). Dendroctonus valens ), larch bark beetle ( Ips subelongatus ), spruce bark beetle ( Ips typograpHus ), spruce borer (D. micans Huashan pine borer ( D. amandi ), six-toothed bark beetle ( I. duplicatus Yunnan pine shoot beetle ( Tomicus yunnanensis ), Horizontal pit tip beetle ( Tomicus minor Pine bark beetles, one of the most serious borer pests in forestry, are widely distributed and numerous, causing severe damage to various forest ecosystems. Due to their strong concealment and wide adaptability, and the spread of their population caused by global warming, traditional chemical agents are ineffective, making control increasingly difficult. Biological control is considered one of the most promising methods for pest control and has broad application prospects.

[0003] The control strategy of bark beetle pests is mainly to pay attention to the self-control ability of forest ecosystem, based on quarantine measures and forest management measures (carrying out preventive health measures, cutting down infested trees, and strengthening forest tending), and to comprehensively use physical control (trapping), biological control (protecting and utilizing natural enemies), and chemical control as a supplement (or emergency measure). (1) Strengthening plant quarantine is a fundamental measure to prevent the spread of forestry pests. It is strictly prohibited to transport infested trees or to import seedlings (scions), logs, and their products from bark beetle pest areas. Infested trees should be treated with chemicals or bark removal in a timely manner to prevent the spread of bark beetles through seedlings, wood, and their products, and to protect local forest resources. (2) During afforestation, mixed forests (needle-broadleaf mixed forests, etc.) and complex layer forests (arbor, shrub, and grass complex layer forests, etc.) should be created as much as possible. The forest stand in the insect source area should be transformed to increase biodiversity and enhance the natural control of the forest ecosystem on bark beetles, improve the resistance of trees, and the stability of the forest ecosystem. Attention should be paid to water and fertilizer management, and trees should be harvested at the appropriate age or under suitable conditions (stump height below 5 cm). Infested trees, wind-fallen trees, dead standing trees, wind-broken trees, and crushed trees should be removed in a timely manner. Fruit trees should be pruned in winter to completely remove diseased and weak branches, improve ventilation and light conditions, and enhance tree vigor and resistance. Removing infested trees is the preferred method for eliminating bark beetles. For heavily infested trees with high insect density, they should be removed and debarked in a timely manner. Infested trees, weak trees, dead standing trees, and other factors that can induce bark beetles, such as dead branches, insect branches, and stumps, should be removed and destroyed to reduce the insect source and prevent residual bark beetles from laying eggs on stumps, insect branches, and dead branches. Winter plowing of tree pits can destroy overwintering sites for bark beetles. During the adult emergence period, low branches with entry holes should be manually removed. Before the end of March, debarking should be done on harvested logs that have not been transported to prevent overwintering bark beetles from spreading. Infested logs should be soaked in water for 15 days to kill live insects. (3) Physical control methods include the following: During the adult emergence period, appropriate sex pheromone traps can be used to trap adults according to the specific species. For forest stands with a bark beetle infestation rate of less than 2%, bait wood can be used for trapping. Before the middle of April, bait wood should be placed in the forest edge or open area (forest gap) at a height of about 1 m. The number of bait wood should be 1-2 pieces per mu. The bait wood can be wind-fallen trees, original logs, or small-diameter logs with fresh bark. During the period when the eggs of dominant species or pioneer species hatch into larvae and the larvae have not yet pupated (or before the new adults emerge), debarking or chemical treatment should be performed. During the overwintering adult emergence period in spring and the adult emergence period in summer, coarse extracts of aggregation pheromones and synthetic attractants (such as Xiongkeng cut branch bark beetle attractant and Larix gmelinii eight-toothed bark beetle attractant) can be used to trap adult bark beetles by hanging traps in the forest.(4) The methods of biological control mainly include the following methods: using natural enemies of insects is a kind of biological control, and is an important means to realize sustainable management. Factory breeding parasitic bees and other natural enemies, releasing them in time and proper amount according to the development stage of the bark beetle, controlling the population number of the bark beetle, and achieving the purpose of control. Common natural enemies include nematodes (Steinernema spp.), parasitic bees (Ceratocystes spp., Sclerodermus spp., Tenthredinidae spp., etc.), predatory insects (Dendrolimus punctatus, Carabus, snakefly, etc.), mites, and parasitic flies, etc. The first batch of 100,000 ceratocystes is released during the oviposition period of the bark beetle. 2 The second batch is released during the larval period, and the third batch is released during the pupal period. The effect of Sclerodermus parasitizing the larvae and pupae of the bark beetle is also good. The predation rate of Dendrolimus punctatus on the larvae and pupae of the bark beetle is more than 10%. The use of natural enemy microorganisms, such as Paecilomyces militaris, Beauveria bassiana, and Metarhizium spp., can effectively parasitize the bark beetle. Paecilomyces militaris (spore content ≥10 billion / g) is commonly used for control in the light damage area and the medium and heavy damage area of the bark beetle. During the adult flying period and the beginning of the branch turning dry period, 1 kg of Paecilomyces militaris powder is sprayed for control of the adult twice or three times per mu.

[0004] As can be seen from the above, the biological control method has a wide application prospect in forest pest control, is friendly to the environment, does not have negative effects on the ecological system, and can effectively control the occurrence and spread of target pests. However, there are still some challenges and difficulties in practical application, such as the selection of suitable biological control agents and the search for effective application methods. SUMMARY

[0005] The present application provides a kind of Bacillus subtilis MJ-4 and its bacterial agent, preparation method and application, to solve the technical problems of poor effect of preventing bark beetle in prior art measures. The preferred technical solutions in many technical effects of the technical solutions provided in the present application are described in detail below.

[0006] To achieve the above-mentioned purpose, the present application provides the following technical solutions: In one or more embodiments of the present application, the 16S rDNA sequence of the Bacillus subtilis MJ-4 is shown in SEQ ID NO. 1. Bacillus subtilis The Bacillus subtilis MJ-4 is deposited with the China General Microbiological Culture Collection Center, and the deposit number is CGMCC No. 30850.

[0007] In one or more embodiments of the present application, the 16S rDNA sequence of the Bacillus subtilis MJ-4 is shown in SEQ ID NO. 1.

[0008] In a second aspect, the present application provides a microbial inoculant containing the Bacillus subtilis MJ-4 of the first aspect, wherein the microbial inoculant is a fermentation broth prepared by fermentation of the Bacillus subtilis MJ-4.

[0009] In a third aspect, the present application provides a preparation method of the microbial inoculant containing the Bacillus subtilis MJ-4 of the second aspect, comprising the following steps: (1) preparing a liquid LB medium, and dispensing the prepared liquid LB medium into containers, sterilizing at high temperature, and obtaining a standby medium after sterilization and cooling; (2) inoculating a colony of the Bacillus subtilis MJ-4 into the standby medium prepared in step (1); (3) placing the inoculated culture bottle obtained in step (2) in a shaking bed for culture to obtain a seed liquid of the Bacillus subtilis MJ-4, wherein the bacterial amount in the obtained seed liquid of the Bacillus subtilis MJ-4 is 5×10 8 ~1×10 9 CFU / mL; (4) inoculating the seed liquid of the Bacillus subtilis MJ-4 of the above concentration obtained in step (3) into a 1-10 ton volume fermenter at a bacterial amount of 1%-2% for industrial aerobic fermentation, and when the bacterial amount reaches 2-3×10 10 CFU / mL, the fermenter is discharged to obtain a microbial inoculant.

[0010] In one or more embodiments of the present application, in step (1), the liquid LB medium dispensed into the containers is sterilized at 121°C for 30 min, and then cooled to 37°C to obtain the standby medium.

[0011] In one or more embodiments of the present application, in step (3), the inoculated culture bottle obtained in step (1) is placed in a shaking bed with a rotation speed of 160 r / min and a temperature of 37°C for culture for 24 h, and the pH is 7 to obtain the seed liquid of the Bacillus subtilis MJ-4.

[0012] In one or more embodiments of the present application, in step (4), the culture conditions of the bacteria in the fermenter are as follows: rotation speed is 160 r / min, temperature is 37°C, pH is 7, and aerobic fermentation time is 24-30 hours.

[0013] In a fourth aspect, the present application provides an application of the Bacillus subtilis MJ-4 of any one of the first aspect in a product for preventing and controlling Ips pines.

[0014] In a fifth aspect, the present application provides an application of the microbial inoculant containing the Bacillus subtilis MJ-4 prepared by the preparation method of any one of the third aspect in a product for preventing and controlling Ips pines.

[0015] Based on the above technical scheme, the Bacillus subtilis MJ-4 and the microbial agent, the preparation method and the application thereof have at least the following beneficial technical effects: The Bacillus subtilis MJ-4 strain of the application is a pathogenic strain MJ-4 with strong lethal effect on Ips typographus, which is isolated from diseased Ips typographus and identified as a gram-positive bacterium. The Bacillus subtilis strain has fast reproduction speed, high concentration, and is easy to produce. It has good stability in indoor and field effect determination, and has broad-spectrum contact and stomach toxicity effect on Coleoptera and Lepidoptera. It is harmless to human body, plants and livestock, and has biological safety. It can stably colonize in plants, establish a green barrier to avoid pests, and decompose trace elements in soil, which has certain growth-promoting effect on plants. The microbial agent prepared by fermentation of the Bacillus subtilis MJ-4 has a corrected mortality of 90.04% on Ips typographus, and a corrected mortality of 57.84% on Ips typographus, which has good prevention and control effect on Ips typographus. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 is the morphological structure of the Bacillus subtilis MJ-4 strain, wherein Figure 1 A is a streaked colony morphology, Figure 1 B is a plated colony morphology; Figure 2 is a 16s rDNA primer phylogenetic tree analysis diagram of the Bacillus subtilis MJ-4; Figure 3 is an analysis of the insecticidal mortality of the fermentation liquid of the Bacillus subtilis MJ-4 in the field test; Figure 4 is the growth condition of the strain MJ-4 in different element culture media (A and B are phosphorus-dissolving, C is potassium-dissolving, D is nitrogen-fixing, and E is ferriphilin-producing). DETAILED DESCRIPTION

[0017] In order to make the purpose, technical scheme and advantages of the present application clearer, the technical scheme of the present application will be described in detail below. Obviously, the described embodiments are only part of the embodiments of the present application, not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0018] Example 1 Isolation of Bacillus subtilis MJ-4

[0019] The diseased bark beetles are picked up with tweezers in the tunnels invaded by the bark beetles, and the bark beetles are placed in a sterile culture dish. The bark beetles are first washed on the surface with sterile water, then surface-disinfected with 75% (v / v) alcohol for 1 min, then washed with sterile water for 5 times, and then the water on the surface of the bark beetles is absorbed with sterile filter paper. The bark beetles are placed in a sterile mortar, sterile water is added, and the grinding liquid is absorbed 100 μL to LB solid medium plates, and the plates are evenly coated with a triangular applicator. The LB solid medium is prepared on the basis of LB liquid, and 15 g of agar powder is added per liter. The composition of the LB liquid medium includes 10 g / mL of sodium chloride, 5 g / mL of yeast extract, 10 g / mL of peptone, and 1000 mL of water. At the same time, 100 μL of sterile water rinsing liquid is absorbed three times to coat the plates, so as to detect whether the surface disinfection of the bark beetles is complete. Finally, the plates are inverted and cultured at 37°C in the dark for 1-2 days until colonies grow, the colony morphology is observed, and colonies with different morphologies are picked up and streaked on LB solid medium plates for purification culture. A total of 56 strains are isolated, and the strains are stored in a -80°C refrigerator with 40% glycerol.

[0020] Example 2 Screening of bark beetle killing strains and obtaining of Bacillus subtilis MJ-4.

[0021] The 56 strains isolated from the diseased bark beetles are inoculated in LB liquid medium with pH = 7, and are fermented at 37°C and 160 rpm for 24 h. The obtained fermentation liquid is sprayed on the body surface of live bark beetles, 30 bark beetles are placed in each dish, and a blank control (LB liquid medium) is set. Each treatment is repeated 3 times, the growth of the bark beetles in the experiment is observed, and the survival condition is recorded. A strain with a bark beetle killing effect is obtained, and is named MJ-4. The strain is stored in a -80°C refrigerator with 40% glycerol.

[0022] The strain MJ-4 is inoculated in LB liquid medium and shaken to obtain fermentation liquid with a bacterial concentration of 10 7 cfu / ml, 10 8 cfu / ml, and 10 9 cfu / ml, respectively. The fermentation liquid is uniformly sprayed on the back and side of the bark beetles distributed on the pine trees, and a blank control (LB liquid medium) is set. Each treatment is repeated 3 times, the survival condition of the bark beetles is recorded, and the corrected mortality rate is calculated. The corrected mortality rate is calculated by using the Abbott's correction formula: mortality rate = (number of dead insects ÷ total number of treated insects) × 100%, and corrected mortality rate = [(number of treated dead insects - number of control dead insects) ÷ (1 - control mortality rate)] × 100%.

[0023] The results in Tables 1 and 2 prove that the concentration of 1 × 10 9The fermentation broth at CFU / mL showed the best contact killing effect on bark beetles, with a contact-corrected mortality rate of 90.04% and a stomach poison-corrected mortality rate of 57.84%.

[0024] Table 1. Analysis of the effect of fermentation broth with different bacterial cell concentrations on the mortality rate of bark beetles.

[0025] Table 2. Analysis of the effect of fermentation broth with different cell concentrations on the corrected mortality rate of bark beetles.

[0026] Example 3 Identification of Bacillus subtilis strain MJ-4.

[0027] 3.1 Morphological characteristics and physiological and biochemical measurements: According to the "Handbook of Systematic Identification of Common Bacteria" published by Science Press in February 2001, strain MJ-4 was morphologically and physiologically and biochemically identified to determine its taxonomic position. For example... Figure 1 As shown, the biological characteristics of this strain are as follows: the optimal growth temperature is 37℃. On LB agar, the colonies are milky white, translucent, with a rough, dull surface, round or oval edges, and small pentagonal or triangular protrusions. The surface is dry and wrinkled. Physiological and biochemical assays were performed using the laboratory-sequentially sequenced Bacillus subtilis B9601-Y2 strain as a positive control. It was Gram-positive, non-motile, aerobic, and showed positive results for citrate utilization, malonate utilization, hydrogen sulfide production, gelatin liquefaction, VP reaction, glucose utilization, starch hydrolysis, catalase, indole production, methyl red test, and KOH reaction (see Table 3).

[0028] Table 3. Physiological and biochemical identification results of Bacillus subtilis strain MJ-4

[0029] 3.2 16S rDNA gene sequencing: The *Bacillus subtilis* MJ-4 provided by this invention was isolated from *Bacillus thunbergii*, a diseased species found in the tunnels of *Pinus yunnanensis*. The 16S rDNA gene sequence of the isolated strain MJ-4 was determined, and the sequence is shown in SEQ ID NO. 1. The results showed that the 16S rDNA sequence of strain MJ-4 provided by this invention had the highest homology with *Bacillus subtilis* XYBJ13, with a similarity of 94%. Combined with physiological and biochemical assays, the isolated strain was confirmed to be *Bacillus subtilis*. Bacillus subtilis MJ-4.

[0030] The Bacillus subtilis (Bacillus subtilis) Bacillus subtilis ) MJ-4 was deposited in the China General Microbiological Culture Collection Center (CGMCC) on June 3, 2024, and the deposit number is CGMCC No. 30850. The address of the CGMCC is No. 1, Beichen West Road, Yard 3, Chaoyang District, Beijing, China, and the postal code is 100101. The strain was checked by the CGMCC on June 3, 2024, and was found to be alive.

[0031] Results The extracted Bacillus subtilis MJ-4 strain genomic DNA was directly subjected to PCR amplification, and a bright band of about 1400 bp was obtained, which is consistent with the report that 16S rDNA is highly conserved and constant in length (about 1.5 kb) in all bacteria. The 16s rDNA sequence of strain MJ-4 was subjected to homology comparison with the existing sequences in the NCBI nucleic acid database BLAST, and it was determined that strain MJ-4 belongs to the same genus as Bacillus subtilis sp., and has 100% homology with Bacillus subtilis. Bacillus Bacillus subtilis In combination with the morphological characteristics, physiological and biochemical properties, and 16s rDNA phylogenetic analysis of the above strain MJ-4, as shown in Figure 2 , strain MJ-4 was identified as Bacillus subtilis (Bacillus subtilis) Bacillus subtilis ) MJ-4.

[0032] Example 4 The present embodiment provides a preparation method of a microbial agent containing Bacillus subtilis MJ-4 strain, comprising the following steps: (1) First, prepare liquid LB medium with the following components: sodium chloride 10 g / mL, yeast extract 5 g / mL, and protein peptone 10 g / mL, and water 1000 mL. Then, the prepared medium is divided into containers and sterilized at 121°C for 30 min, and then cooled to 37°C to obtain a standby medium; (2) Take one single colony of Bacillus subtilis (Bacillus subtilis) Bacillus subtilis ) MJ-4, i.e. 1 microliter of bacterial body, and inoculate it into the obtained standby medium; (3) Place the obtained inoculated culture bottle in a shaker at a speed of 160 r / min and a temperature of 37°C for 24 h, pH=7, to obtain Bacillus subtilis MJ-4 seed liquid. The bacterial amount in the obtained Bacillus subtilis (Bacillus subtilis) Bacillus subtilis ) MJ-4 seed liquid is 5×10 8 ~1×10 9 CFU / mL; ​(4) The MJ-4 seed culture of the above concentration was inoculated into a fermenter with a volume of 1-10 tons at a bacterial count of 1%~2% for industrial aerobic fermentation. The culture conditions were: rotation speed of 160 r / min, temperature of 37℃, culture medium pH=7, aerobic fermentation for 24-30 hours, and bacterial count of 2~3×10⁻⁶. 10 When the concentration reaches CFU / mL, transfer the product to a container to obtain the fermentation broth product.

[0033] Example After conducting field insecticidal tests on 20 common insects using the Bacillus subtilis MJ-4 fermentation broth obtained above, the following field test insecticidal mortality data were obtained, as detailed in the appendix. Figure 3 The field test results show the insecticide mortality rate analysis.

[0034] From the appendix Figure 3 The mortality rates of the insecticides were as follows: inchworm 73.3%, leaf beetle 53.3%, golden apple snail 13.3%, red imported fire ant 66.7%, tobacco aphid 93%, whitefly 90%, fall armyworm 26.67%, ground beetle 53.3%, scarab beetle 60%, snail 26.7%, two-spotted leaf beetle 66.7%, cabbage bug 20%, Dubia roach 73.3%, citrus mealybug 80%, spider mite 60%, corn aphid 93%, corn weevil 93%, pear sword-shaped moth 86.7%, fungus gnat 80%, and slug 66.7%.

[0035] The MJ-4 bacterial suspension obtained above was dropped onto five special nutrient media and cultured. After 7 days of culture, colonies grew on each treatment medium and showed obvious clear zones, indicating that strain MJ-4 has the ability to solubilize phosphorus, solubilize potassium, fix nitrogen, and produce ferrophosphate. Figure 4 As shown.

[0036] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A Bacillus subtilis MJ-4, characterized in that, The Bacillus subtilis ( Bacillus subtilis MJ-4 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.30850.

2. The Bacillus subtilis MJ-4 according to claim 1, characterized in that, The 16S rDNA sequence of Bacillus subtilis MJ-4 is shown in SEQ ID NO.

1.

3. A microbial inoculant of Bacillus subtilis MJ-4 as described in claim 1 or 2, characterized in that, The microbial agent is a fermentation broth made from Bacillus subtilis MJ-4 through fermentation.

4. A method for preparing a microbial inoculant of Bacillus subtilis MJ-4 as described in claim 3, characterized in that, Includes the following steps: (1) Prepare liquid LB medium with the following components: sodium chloride 10 g / mL, yeast extract 5 g / mL, peptone 10 g / mL, and water 1000 mL. Dispense the prepared liquid LB medium into containers and sterilize them at high temperature. After sterilization, cool the medium to obtain the ready-to-use medium. (2) Take the colonies of Bacillus subtilis MJ-4 and inoculate them into the prepared culture medium in step (1); (3) The inoculation culture bottle obtained in step (2) is placed in a shaker for culture to obtain Bacillus subtilis MJ-4 seed culture, wherein the bacterial count of the obtained Bacillus subtilis MJ-4 seed culture is 5×10⁻⁶. 8 ~1×10 9 CFU / mL; (4) The Bacillus subtilis MJ-4 seed culture obtained in step (3) is inoculated into a fermenter with a volume of 1 to 10 tons at a bacterial count of 1% to 2% for industrial aerobic fermentation until the bacterial count reaches 2 to 3 × 10⁻⁶. 10 When the concentration of CFU / mL is reached, the mixture is placed in a container to obtain the microbial inoculant.

5. The method for preparing the Bacillus subtilis MJ-4 microbial inoculant according to claim 4, characterized in that, In step (1), the liquid LB medium dispensed into the container is sterilized at 121°C for 30 min, and then cooled to 37°C to obtain a spare medium.

6. The method for preparing the microbial inoculant of Bacillus subtilis MJ-4 according to claim 4, characterized in that, In step (3), the inoculation culture bottle obtained in step (1) is placed in a shaker with a rotation speed of 160 r / min and a temperature of 37°C for 24 h and a pH of 7 to obtain Bacillus subtilis MJ-4 seed liquid.

7. The method for preparing the Bacillus subtilis MJ-4 microbial inoculant according to claim 4, characterized in that, The culture conditions for bacteria in the fermenter in step (4) are: rotation speed of 160 r / min, temperature of 37℃, pH=7, and aerobic fermentation time of 24-30 hours.

8. The application of Bacillus subtilis MJ-4 as described in claim 1 or 2 in products for controlling bark beetles of pine trees.

9. The application of the microbial agent containing Bacillus subtilis MJ-4 prepared by the preparation method according to any one of claims 4 to 7 in products for controlling pine bark beetles.

Images