Lysinic Bacillus and its biological preparation and application

Abstract

This application discloses a lysinibacillus, its biological preparation, and its application, belonging to the field of microbial technology. The lysinibacillus of this application, deposited with CCTCC NO: M 20251099, exhibits high control efficacy against major rice pests and diseases, rice planthoppers and rice blast. Furthermore, since rice planthoppers and rice blast can occur at both the seedling and panicle stages, with overlapping occurrence phases, lysinibacillus possessing both rice planthopper and rice blast bioinhibitory activity can effectively reduce the frequency of pest and disease control efforts and the investment in control products.

Description

Technical Field

[0001] The present application relates to the field of microbial technology, and in particular to a lysinic Bacillus and its biological preparation and application. Background Art

[0002] Bacteria are important resources for green pest control. Bacillus thuringiensis and Bacillus subtilis are widely used in rice pest and disease control. However, due to the physiological characteristics of Hemipteran pests such as rice planthoppers, such as their piercing-sucking mouthparts, digestive tract filtration chambers, and the lack of a peritrophic membrane, the vast majority of discovered insecticidal bacteria have low bioactivity against them. Currently, isolated bacteria with insecticidal activity against Hemipteran pests include Bacillus thuringiensis, Bacillus subtilis, Xenorhabditis nematophila, Photorhabdus luminescens, Serratia marcescens, Pseudomonas putida, and Dictyosphaeria davidianensis. Fewer bacteria have insecticidal activity against rice planthoppers, including Bacillus thuringiensis, Serratia marcescens, Pseudomonas aeruginosa, and Elizabethia spp. Among these, Serratia marcescens, Pseudomonas aeruginosa, and Elizabethia spp. are opportunistic pathogens and pose certain safety risks. Most Bacillus species are non-pathogenic, harmless to animals, plants, and humans, and are therefore relatively safe. Furthermore, Bacillus species are widely found in nature and come from a variety of sources, including seawater, soil, plant rhizospheres or tissues, and animal digestive tracts and excreta. Bacillus species from different sources may exhibit varying properties. Currently, Bacillus species isolated from rice with biocontrol activity include Bacillus velezensis, Bacillus mojavei, Bacillus eudicots, Bacillus amyloliquefaciens, Bacillus megaterium, Paenibacillus polymyxa, Bacillus subtilis, and Bacillus vannesii. These species exhibit limited biocontrol activity against sheath blight, rice blast, bacterial base rot, false smut, and seedling rot. However, reports on endophytic lysinibacillus species for biocontrol are limited. Lysinibacillus species used in rice applications are all derived from soil and have been shown to improve soil quality or exhibit fungicidal activity against false smut and sheath blight. There are no reports on endophytic lysinibacillus species for controlling rice planthoppers or blast. Summary of the Invention

[0003] In view of this, the present application provides a novel lysinophilic Bacillus HA-B2, the classification name and Latin name of which is Lysinibacillus sp. The preservation information is: Preservation unit: China Center for Type Culture Collection (CCTCC), preservation address: Wuhan University, Wuhan, China, preservation number: CCTCC NO: M 20251099, preservation time: May 19, 2025, Bacillus lysinicola HA-B2 has biological activity against rice planthoppers and rice blast, and can be effectively used in the control of rice diseases and pests.

[0004] The embodiment of the present application is implemented as follows:

[0005] In a first aspect, the present application provides a lysinic Bacillus, the deposit number of which is CCTCC NO: M 20251099.

[0006] In some embodiments, the lysinibacillus is an endophytic lysinibacillus of rice, derived from rice stems; and / or the sequence of the 16s rDNA base sequence fragment of the lysinibacillus is shown in SEQ ID NO: 1.

[0007] In some embodiments, the lysinophilic Bacillus is cultured on a plate at 28-37° C. for 24-48 hours, and the colonies are: neatly edged, creamy yellow or white, flat, round, slightly raised, opaque, and smooth and moist on the surface.

[0008] In a second aspect, the present application provides a biological preparation, which includes the above-mentioned lysinic Bacillus, or includes a fermentation product obtained by fermenting the above-mentioned lysinic Bacillus.

[0009] In some embodiments, the fermentation conditions of the lysinic Bacillus are: inoculation into liquid culture medium, culturing for 16 to 20 hours at a pH of 6.5 to 7.5, a temperature of 28 to 37° C., and a shaking speed of 150 to 250 rpm.

[0010] In some embodiments, the fermentation products include active proteins and secondary metabolites separated after fermentation, proteins and secondary metabolites screened and developed using the lysinic Bacillus as a source, and protein gene products with insecticidal or antibacterial activity screened or modified based on the lysinic Bacillus.

[0011] In a third aspect, the present application provides the use of the lysinic Bacillus or the biological agent in controlling rice diseases and pests.

[0012] In some embodiments, the lysinic Bacillus or the biological agent takes into account the prevention and control of both rice planthoppers and rice blast.

[0013] In some embodiments, the rice planthopper is one or more of the group consisting of the brown planthopper, the brown planthopper, and the white-backed planthopper; and / or the causative agent of rice blast is the blast fungus Magnaporthe oryzae ; and / or, the lysinic Bacillus or the biological preparation is also used for the prevention and treatment of sheath blight.

[0014] The lysinic acid Bacillus of the present application has high insecticidal or antibacterial activity against the major rice pests and diseases, rice planthoppers and rice blast, and thus has a dual control effect on rice planthoppers and rice blast. The occurrence phases of rice pests and diseases overlap; during the same growth period, multiple pests and diseases may occur simultaneously or around the same time. For example, rice planthoppers and rice blast may occur at both the seedling and panicle stages. Therefore, the use of a lysinic acid Bacillus that controls both rice planthoppers and rice blast can effectively reduce the number of pest and disease control procedures or the investment in control products. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application. For those skilled in the art, other drawings can be obtained based on these drawings without creative work.

[0016] Figure 1 The colony morphology is that of Bacillus lysinicus HA-B2;

[0017] Figure 2 is the Gram staining image of Bacillus lysinicola HA-B2;

[0018] Figure 3 This is a spore staining image of Bacillus lysinicus HA-B2;

[0019] Figure 4 is the growth curve of Bacillus lysinicus HA-B2;

[0020] Figure 5 The toxicity of Bacillus lysinicola HA-B2 to the second-instar nymphs of Laodelphax striatellus is shown;

[0021] Figure 6 The toxicity of Bacillus lysinicola HA-B2 to the second-instar nymphs of brown planthopper is shown;

[0022] Figure 7 This is a diagram showing the inhibitory effect of Bacillus lysinicola HA-B2 on rice blast fungus;

[0023] Figure 8 This is a diagram showing the inhibitory effect of Bacillus lysinicola HA-B2 on sheath blight. DETAILED DESCRIPTION

[0024] The technical solutions in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without making creative work are within the scope of protection of this application. In addition, it should be understood that the specific embodiments described herein are only used to illustrate and explain the present application and are not used to limit the present application. In addition, in the description of the present application, the term "including" means "including but not limited to".

[0025] Various embodiments of the present application may be presented in the form of a range; it should be understood that the description in the form of a range is only for convenience and brevity and should not be understood as a hard limitation on the scope of the present application; therefore, the range description should be considered to have specifically disclosed all possible sub-ranges and single numbers within the range. For example, the description of a range from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the range, such as 1, 2, 3, 4, 5 and 6, which applies regardless of the range. In addition, whenever a numerical range is indicated herein, it is meant to include any cited number (fractional or integer) within the indicated range.

[0026] In this application, "and / or" describes the association relationship of associated objects, indicating that three relationships may exist. For example, A and / or B can represent: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural.

[0027] In this application, "at least one" means one or more, and "more than one" means two or more. "At least one", "at least one of the following" or similar expressions refer to any combination of these items, including any combination of single or plural items. For example, "at least one of a, b, or c", or "at least one of a, b, and c" can all mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, and c can be single or plural, respectively.

[0028] The present application provides a lysinophilic Bacillus HA-B2, which is classified and named in Latin Lysinibacillus sp.The deposit information is as follows: Depository: China Center for Type Culture Collection (CCTCC), Address: Wuhan University, Wuhan, China, CCTCC No. M 20251099, Date of deposit: May 19, 2025. The lysinic Bacillus HA-B2 has high biological activity against major rice pests and diseases, namely, rice planthoppers and rice blast, indicating that it has control effects against both rice planthoppers and rice blast.

[0029] The occurrence phases of rice pests and diseases overlap. During the same growth period, multiple pests and diseases may occur simultaneously or around the same time. For example, rice planthoppers and rice blast can occur at both the seedling and heading stages. Therefore, applying Bacillus lysinicola HA-B2, which controls both rice planthoppers and rice blast, can effectively reduce the number of pest and disease control visits and the investment in control products.

[0030] In some embodiments, the lysinibacillus HA-B2 is an endophytic lysinibacillus of rice.

[0031] In this embodiment, the lysinibacillus HA-B2 is an endophytic Bacillus of rice. In addition to having the advantages of strong stress resistance and environmental friendliness of Bacillus, it is isolated from rice tissue and is superior to strains isolated from soil and other environments in terms of colonization and safety. It can coexist with rice for a long time and is safer for rice consumption.

[0032] Furthermore, the lysinibacillus HA-B2 is derived from rice stems and can effectively colonize within rice plants, thereby providing a sustained control effect against rice planthoppers and rice blast. Rice stems are the feeding site of rice planthoppers. Lysinibacillus HA-B2 colonized in rice stems can enter the planthoppers' bodies as they feed, exerting its insecticidal activity. This effectively prevents most bacterial biocontrol agents from only attaching or surviving on the crop surface, resolving the problem of other biocontrol agents being less effective due to their difficulty reaching the planthoppers' bodies as they feed.

[0033] In some embodiments, the lysinic Bacillus HA-B2 is cultured on a plate at 28-37° C. for 24-48 hours, and the colonies have neat edges, are creamy yellow or white, flat, round, slightly raised, opaque, and have a smooth and moist surface.

[0034] The lysinibacillus HA-B2 of the present application was identified by 16S rDNA. The 16S rDNA was amplified and sequenced using universal primers 8F and 1492R, and then compared with the NCBI GenBank nucleic acid database to identify the HA-B2 strain as lysinibacillus. Lysinibacillus sp. .

[0035] The present application also provides a biological preparation, comprising the lysinic Bacillus HA-B2 or a product prepared based on the lysinic Bacillus HA-B2.

[0036] In one embodiment, the biological preparation can be prepared by liquid fermentation of the lysinophilic Bacillus HA-B2 provided in the present application.

[0037] Specifically, the fermentation conditions are as follows: transfer the seeds of Bacillus lysininus HA-B2 to a liquid culture medium and culture for 16 to 20 hours at a pH of 6.5 to 7.5, a temperature of 28 to 37°C, and a shaking speed of 150 to 250 rpm. The liquid culture medium may be LB medium, TB medium, or NA medium.

[0038] In one embodiment, the biological agent may include a fermentation product of Bacillus lysininii HA-B2. Specifically, the fermentation product may include active proteins and secondary metabolites isolated after fermentation, proteins and secondary metabolites screened and developed using Bacillus lysininii HA-B2 as a source, and protein gene products with insecticidal or antibacterial activity screened or modified using Bacillus lysininii HA-B2 as a basis. Protein gene products include gene sequences and active proteins expressed based on these sequences.

[0039] The lysinic Bacillus HA-B2 and biological preparation provided in the present application are used for the prevention and control of rice diseases and insect pests, and are specifically used for the prevention and control of rice planthoppers and rice blast.

[0040] Among them, rice planthoppers are specifically one or more of gray planthoppers, brown planthoppers, white-backed planthoppers, etc.

[0041] The rice blast disease is one or more of seedling blast, leaf blast, leaf pillow blast, node blast, panicle blast, branch blast or grain blast.

[0042] The causative agent of rice blast is Pyricularia oryzae Magnaporthe oryzae .

[0043] In other embodiments, the lysinic acid Bacillus HA-B2 and the biological preparation can also be applied to the prevention and treatment of sheath blight. Specifically, the lysinic acid Bacillus HA-B2 and the biological preparation are effective against sheath blight. Rhizoctonia solani It also has an inhibitory effect on the growth of pathogenic bacteria.

[0044] In other embodiments, the lysinic Bacillus HA-B2 and biological agents are also active against Hemiptera pests such as aphids and whiteflies, and can be applied to the prevention and control of Hemiptera pests such as aphids and whiteflies on other crops.

[0045] In some embodiments, when the lysinic Bacillus HA-B2 and its biological preparation are applied to control rice diseases and insect pests, the lysinic Bacillus HA-B2 and its biological preparation can be used as seed treatment or sprayed on the plant surface.

[0046] The technical solutions and technical effects of the present application are described in detail below through specific examples and comparative examples. The following examples are only some examples of the present application and are not intended to limit the present application. It is understood that the reagents and materials used in the examples and comparative examples are commercially available products unless otherwise specified.

[0047] Example 1 Isolation and Purification of Lysinibacillus HA-B2

[0048] Rice plants were collected from well-grown rice fields in Huai'an. After being rinsed with tap water, the rice stems were cut into small sections. The surface was then disinfected with 5% sodium hypochlorite and 70% ethanol, and then rinsed with sterile water for more than 4 times. After being dried with sterile filter paper, the rice stems were transferred to a mortar and ground thoroughly. The grinding liquid was collected and diluted 10 3 , 10 4 , 10 5 After doubling, 100 μL was aspirated and plated (LB plate preparation: weigh 10 g of tryptone (BBI), 5 g of yeast extract (BBI), 10 g of sodium chloride, and 15 g of agar, add water to make up to 1000 mL and stir evenly, sterilize at 121°C for 20 min, turn the plate over when cooled to 50-60°C, seal with sealing film after cooling and solidifying, and store upside down in a refrigerator at 4°C until use), and culture at 37°C overnight.

[0049] The rinsing liquid test method was used to test the disinfection effect of the tissue surface. 100 μL of the rinsing liquid from the last rinsing of the disinfected material was spread on the LB plate and cultured simultaneously with the separation plate. After incubation at 37°C overnight, observe whether there are any colonies growing. If no colonies appear, it means that the material surface is thoroughly disinfected. The colonies grown on the separation plate can be used for the next test. Otherwise, the plate should be discarded and re-isolated.

[0050] Observe the characteristics of the colonies on the plate under a stereo microscope, pick colonies of different morphologies for liquid culture, and then smear the plate again for purification until the colonies on the plate have the same morphology. Figure 1 The colonies are characterized by neat edges, creamy yellow or white color, flat, round, slightly raised, opaque, and smooth, moist surface.

[0051] Example 2 Identification of Lysinibacillus HA-B2

[0052] The preserved HA-B2 bacteria were taken out and activated, and then stained with Gram staining solution and Scharffer-Fulton spore staining kit. The staining results are shown in Figure 2 and Figure 3 , Figure 2 For Gram stain, Figure 3 Stain for spores.

[0053] At the same time, 16S rDNA PCR amplification was performed using 8F / 1492R. Tks Gflex DNA polymerase (Takara, Beijing) was used for amplification. The system was as follows: HA-B2 bacterial solution, 1 μL; 2× Gflex PCR Buffer (mg 2+ , dNTP plus), 25 μL; TKS Gflex DNA polymerase, 1 μL; 8F (10 mM), 2 μL; 1492R (10 mM), 2 μL; ddH2O; 19 μL. Amplification was performed using a PCR amplification instrument, and the program was as follows: 94℃ pre-denaturation for 1 min, 98℃ denaturation for 10 s, 55℃ annealing for 15 s, 68℃ extension for 2 min, for a total of 30 cycles, and 72℃ extension for 10 min after the end of the cycle. After the PCR was completed, agarose gel electrophoresis was performed, and then a single band of about 1500 bp was purified and recovered and sequenced. It was then compared with the NCBI GenBank nucleic acid database, combined with the colony morphology characteristics of the bacteria on LB culture medium plates and related physiological and biochemical characteristics tests, it was confirmed that the HA-B2 strain was classified and named Bacillus lysinicola. Lysinibacillus sp. , named Bacillus lysinicola Lysinibacillus sp. HA-B2.

[0054] The sequence of the 16s rDNA base sequence fragment of Bacillus lysinicus HA-B2 is shown in SEQ ID NO: 1, and is as follows:

[0055]

[0056] Example 3 Growth curve of Bacillus lysinicus HA-B2

[0057] Dilute the stored HA-B2 bacteria and plate them. Pick a single colony and transfer it to 5 mL of LB liquid medium. Shake and culture at 200 rpm and 37°C until the OD600 reaches 1.0 as a seed. Then add fresh medium at a ratio of 1:40 and measure the OD600 value every hour. The growth curve is shown in Figure 2. Figure 4 , with culture time as the horizontal axis (unit: hours) and OD600 as the vertical axis. Figure 4 It can be seen that HA-B2 grows extremely fast and a large number of bacteria can be obtained in a very short time. Its OD600 value reaches above 1.0 3 hours after transfer and reaches above 2.0 after 5 hours.

[0058] Example 4 Toxicity of Bacillus lysinicola HA-B2 to Rice Planthoppers

[0059] The toxicity of HA-B2 to rice planthoppers was determined by feeding. The specific steps were as follows: one end of a two-way tube was sealed with 100-mesh gauze to maintain air circulation, and a second-instar nymph of rice planthopper was introduced from the other end. After the test insect was introduced, the other end was sealed with parafilm. 50 μL of feed mixture (containing 5 μL HA-B2 bacteria, OD600 of 1.0) was added to the center of the membrane, and the membrane was sealed again with parafilm. The feed was changed daily, and the number of surviving test insects was counted. The toxicity of Bacillus lysinicola HA-B2 to second-instar nymphs of Laodelphax striatellus can be found in the table. Figure 5 The toxicity to the second instar nymphs of brown planthopper is shown in Figure 6 .

[0060] Depend on Figure 5 and Figure 6 It can be seen that Bacillus lysinicola HA-B2 has good biological activity against rice planthoppers such as gray planthopper and brown planthopper. On the 6th day after feeding, the corrected mortality rates of gray planthopper and brown planthopper can reach 88% or above.

[0061] Example 5 Inhibitory Effect of Lysinibacillus HA-B2 on Rice Blast Fungus

[0062] Fold a 9cm diameter piece of paper in half and then in half again to mark the center of the culture dish. Inoculate a 0.5cm diameter rice blast fungus cake onto the center. Use an inoculation loop to dip into Lysinibacillus HA-B2 seeds (OD600 = 1.0) and apply it 1.5cm from the center. Use a plate inoculated with only Rice blast fungus as a blank control. After 7 days, observe the inhibitory effect of Lysinibacillus HA-B2 on Rice blast fungus. Results are shown in the table. Figure 7 Lysinic Bacillus HA-B2 has a good inhibitory effect on the growth of rice blast fungus, with an inhibition rate of about 77%. Figure 7 The left side is a blank control chart, and the right side is a chart showing the inhibitory effect of Bacillus lysinin HA-B2 on rice blast fungus. In this example, the rice blast fungus is a laboratory-preserved strain.

[0063] Example 6 Inhibitory Effect of Lysinibacillus HA-B2 on Rhizoctonia solani

[0064] Fold a 9cm diameter piece of paper in half and then in half again to mark the center of the culture dish. Inoculate a 0.5cm diameter cake of Rhizoctonia solani at the center. Use an inoculation loop to dip into Lysinibacillus HA-B2 seeds (OD600 = 1.0) and apply it 1.5cm from the center. Use a plate inoculated only with Rhizoctonia solani as a blank control. After 4 days, observe the inhibitory effect of Lysinibacillus HA-B2 on Rhizoctonia solani. Results are shown in the table. Figure 8 Lysinic Bacillus HA-B2 has a good inhibitory effect on the growth of Rhizoctonia solani, with an inhibition rate of about 84%. Figure 8 The left side is a blank control graph, and the right side is a graph showing the inhibitory effect of Bacillus lysininus HA-B2 on Rhizoctonia solani. The Rhizoctonia solani in this example is a laboratory-preserved strain.

[0065] The above is a detailed introduction to the lysinophilic Bacillus and its biological preparations and applications provided in the examples of the present application. Specific examples are used herein to illustrate the principles and implementation methods of the present application. The description of the above embodiments is only intended to help understand the method and core ideas of the present application. At the same time, for those skilled in the art, according to the ideas of the present application, there may be changes in the specific implementation methods and application scopes. In summary, the content of this specification should not be understood as limiting the present application.

Claims

1. A lysinic Bacillus, characterized in that The Latin name of the lysinic Bacillus is Lysinibacillus sp., It is deposited in China Center for Type Culture Collection with the accession number: CCTCC NO: M20251099.

2. A biological agent, characterized in that The biological preparation comprises the lysinic Bacillus according to claim 1.

3. Use of the lysinibacillus according to claim 1 or the biological preparation according to claim 2 in the prevention and treatment of rice planthoppers and rice blast.

4. The use according to claim 3, characterized in that The rice planthoppers include one or more of the group consisting of the brown planthopper, the brown planthopper, and the white-backed planthopper.

5. The use according to claim 3, characterized in that The causative agent of rice blast is Pyricularia oryzae Magnaporthe oryzae .

6. The lysinic Bacillus according to claim 1 or the biological preparation according to claim 2 in Rhizoctonia solani Rhizoctonia solani Application in prevention and control.

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