Bacillus megaterium YBZ13 for preventing and treating walnut leaf scorch and application thereof
By screening and applying Bacillus megaterium YBZ13 and its preparations from the walnut rhizosphere, the problem of controlling walnut leaf scorch was solved, achieving efficient and environmentally friendly control, and enhancing the salt resistance and growth capacity of walnut seedlings.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- NORTHEAST FORESTRY UNIV
- Filing Date
- 2023-11-29
- Publication Date
- 2026-06-05
AI Technical Summary
Existing technologies have not yet developed a large-scale, systematic, and standardized method for preventing and controlling walnut leaf scorch, and the control measures are costly and have limited effectiveness.
By using Bacillus megaterium YBZ13 isolated from the rhizosphere of walnut trees and its microbial preparations, the plant can be promoted to absorb nutrients and enhance its ability to resist abiotic stresses by directly or indirectly participating in the soil nutrient cycle.
It significantly reduces the area of scorched leaves in walnut seedlings, enhances root vitality, improves soil, and prevents walnut leaf scorch disease. It is also non-toxic, residue-free, and does not pollute the environment.
Smart Images
Figure CN117701422B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of microbial technology, and more specifically, to a strain of Bacillus megaterium YBZ13 for the prevention and treatment of walnut leaf scorch and its application. Background Technology
[0002] Walnut leaf scorch was first reported in the main walnut-producing areas of southern Xinjiang in 2012. Its symptoms mainly manifest as follows: in the early stages, chlorosis and scorching begin at the leaf margins or tips, gradually extending towards the center and eventually affecting the entire leaf. In recent years, the affected area of walnut leaf scorch has shown a trend of increasing severity year by year, seriously threatening the healthy development of the walnut industry in Xinjiang.
[0003] Walnut leaf scorch can be caused by a variety of factors: (1) Severe soil salinization, with excessively high levels of Na and Cl ions, and arid climates making it difficult for surface salts to leach out. Intense evaporation causes salt water to rise through capillaries and accumulate on the surface, leading to a large accumulation of Na and Cl ions in plant leaves, directly affecting photosynthesis and causing leaf scorch; (2) High temperatures, drought, and insufficient soil moisture prevent the roots from absorbing enough water, and the leaves also lack sufficient water supply; (3) Strong sunlight and hot, dry winds lead to high transpiration rates, causing severe physiological water loss in the leaves and resulting in leaf scorch; (4) Soil compaction, poor aeration, poor water and fertilizer retention capacity, weak tree vigor, and nutrient deficiencies in the plants can lead to severe leaf scorch in some walnut orchards, resulting in underdeveloped, blackened, and rotten fruit, seriously affecting walnut yield and quality. In conclusion, leaf scorch has become a major bottleneck restricting the healthy development of the Xinjiang walnut industry.
[0004] Currently, the main measures used to control walnut leaf scorch are: applying substances that promote the leaching of Na and Cl ions in the soil; using surface water for irrigation; applying fertilizers scientifically to improve tree resistance; and spraying foliar physiological regulators and antitranspirants. While these measures have achieved some success in controlling walnut leaf scorch, the costs are high, and a large-scale, systematic, and standardized control technology system has not yet been established, resulting in limited benefits.
[0005] Beneficial microorganisms can enhance the host plant's tolerance to high salt stress and effectively alleviate the negative impact of soil salinization on plant growth and development. The development of beneficial microbial preparations for controlling walnut leaf scorch caused by high soil salinity through the isolation and screening of salt-tolerant and growth-promoting microbial strains has attracted widespread attention.
[0006] In view of this, the present invention is hereby proposed. Summary of the Invention
[0007] The purpose of this invention is to provide a strain of Bacillus megaterium YBZ13 for the prevention and treatment of walnut leaf scorch and its application. The Bacillus megaterium (… Bacillus megaterium YBZ13, isolated from the rhizosphere of walnut plants suffering from leaf scorch, has the function of enhancing plant salt tolerance and promoting plant growth.
[0008] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted:
[0009] One aspect of the present invention relates to Bacillus megaterium (Beta vulgaris) Bacillus megaterium YBZ13 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 28098.
[0010] This invention isolates salt-tolerant bacteria from the rhizosphere of walnut trees suffering from severe leaf scorch. Under greenhouse conditions, the degree to which these bacterial isolates alleviate walnut leaf scorch is investigated, and their growth-promoting effects on walnuts under high salt stress are evaluated. Finally, a strain of Bacillus megaterium that can effectively control walnut leaf scorch is screened out. Bacillus megaterium YBZ13.
[0011] This invention utilizes plate culture technology combined with a live seedling screening experimental strategy under greenhouse conditions to scientifically evaluate the effectiveness of beneficial microorganisms in controlling walnut leaf scorch by investigating leaf damage and root vigor. It establishes an efficient and convenient method for screening microbial strains for controlling walnut leaf scorch.
[0012] Another aspect of the invention relates to a microbial preparation comprising the aforementioned Bacillus megaterium (Beta vulgaris). Bacillus megaterium YBZ13 cells and its suspension.
[0013] The microbial preparation includes Bacillus megaterium YBZ13, which was isolated from the rhizosphere of walnut plants exhibiting leaf scorch. Therefore, this microbial preparation is an environmentally friendly preparation, characterized by rapid effectiveness, low cost, no pollution, and sustainable application. When applied to the plant itself or its surrounding environment, this microbial preparation promotes nutrient absorption and enhances the plant's ability to resist abiotic stresses by directly or indirectly participating in soil nutrient cycling.
[0014] Preferably, the microbial preparation comprises a solid preparation or a liquid preparation.
[0015] Preferably, when the microbial preparation is a solid dosage form, the number of viable Bacillus megaterium YBZ13 in the microbial preparation is 10. 8 CFU / g or higher.
[0016] Preferably, when the microbial preparation is a liquid preparation, the number of viable Bacillus megaterium YBZ13 in the microbial preparation is 10. 8 CFU / mL or higher.
[0017] Another aspect of the invention relates to a method for preventing and treating walnut leaf scorch by applying the Bacillus megaterium YBZ13 and / or the microbial preparations described herein to walnuts.
[0018] Another aspect of the invention relates to a method for promoting walnut growth under high salt stress by applying the Bacillus megaterium YBZ13 and / or the microbial preparations described herein to the walnut.
[0019] Another aspect of the invention relates to Bacillus megaterium (Betaminae) Bacillus megaterium The application of YBZ13 or the microbial preparations described herein in the prevention and control of non-infectious plant diseases and / or in promoting plant growth.
[0020] Preferably, the non-infectious plant diseases include: plant diseases caused by high salt stress.
[0021] Preferably, the plant diseases caused by high salt stress include: walnut leaf scorch.
[0022] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0023] (1) The Bacillus megaterium YBZ13 of the present invention is a rhizosphere symbiotic bacterium of walnut. Its bacterial suspension has a significant effect in preventing and controlling leaf scorch in walnut seedlings. It can effectively reduce the scorched area of seedling leaves, alleviate the damage of high salt stress to walnut seedling leaves, increase the fresh weight of the main root of walnut seedlings and improve the root vitality of seedlings.
[0024] (2) The microbial preparation of the present invention is an environmentally friendly preparation that can effectively prevent and control leaf scorch in walnut seedlings. It is non-toxic, leaves no residue, does not cause secondary pollution to the environment, and also improves the soil. Attached Figure Description
[0025] To more clearly illustrate the specific embodiments or technical solutions of the present invention, the accompanying drawings used in the description of the specific embodiments or prior art will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.
[0026] Figure 1 The plant morphology of walnut seedlings at different growth stages;
[0027] Figure 2 Grading of leaf symptoms of leaf scorch in walnut seedlings;
[0028] Figure 3 To alleviate the symptoms of leaf scorch in walnut seedlings using strain YBZ13;
[0029] Figure 4 The effect of strain YBZ13 on reducing the severity of leaf scorch in walnut seedlings;
[0030] Figure 5 To investigate the effect of strain YBZ13 on root growth in scorched leaf walnut seedlings;
[0031] Figure 6 To investigate the effect of strain YBZ13 on the root vigor of scorched leaf walnut seedlings;
[0032] Figure 7 Colony morphology of Bacillus megaterium YBZ13 (scale bar: 2 mm). Detailed Implementation
[0033] The technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings and specific embodiments. However, those skilled in the art will understand that the embodiments described below are some embodiments of the present invention, but not all embodiments, and are only used to illustrate the present invention, and should not be regarded as limiting the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall be followed. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially.
[0034] Example 1 Sample processing and strain isolation
[0035] The tested bacterial strain was isolated in April 2021 from the root system and surrounding soil of walnut trees in Aksu Prefecture, Xinjiang Uygur Autonomous Region, which were suffering from severe leaf scorch. When collecting root and soil samples, the leaf litter layer was first removed, and the top layer of soil was dug away with a shovel. The roots were then removed along their growth direction, and samples of the surrounding soil, rhizosphere soil, and root system were collected and transported back to the laboratory for bacterial isolation and screening experiments.
[0036] (1) Take 0.25g of soil from the periphery of walnut roots and transfer it to a 15mL centrifuge tube. Add 10mL of 1×PBS buffer to prepare the original sample solution, and then serially dilute it 10-fold to 10 ... 9The diluted sample solution was vortexed and mixed thoroughly. 20 µL of bacterial solution was then evenly dropped onto the culture medium plate and spread evenly using a spreader. The sample solutions for each gradient should be spread separately on LB medium (sodium chloride 10 g / L, yeast extract 5 g / L, tryptone 10 g / L, pH 7.0), R2A medium (yeast extract 0.5 g / L, peptone 0.5 g / L, casein hydrolysate 0.5 g / L, glucose 0.5 g / L, soluble starch 0.5 g / L, potassium dihydrogen phosphate 0.5 g / L, sodium pyruvate 0.3 g / L, anhydrous magnesium sulfate 0.025 g / L, pH 7.0 to 7.4), 1 / 10 TSA medium (tryptone 1.5 g / L, soybean peptone 0.5 g / L, sodium chloride 0.5 g / L, pH 7.1 to 7.5), and TYG medium (tryptone 3.0 g / L, yeast extract 3.0 g / L, glucose 3.0 g / L, dipotassium hydrogen phosphate 1.0 g / L, pH 7.0 to 7.5). 7.4) and nitrifying bacteria selective medium (ammonium sulfate 0.5 g / L, sodium chloride 0.3 g / L, ferrous sulfate 0.03 g / L, sodium dihydrogen phosphate 1.0 g / L, magnesium sulfate 0.03 g / L, calcium chloride 7.5 g / L, pH 7.5). Incubate overnight at 30°C. Observe colony morphology the next day, pick single colonies, streak to purify until no contaminating bacteria grow. Pick purified single colonies into 5 mL of LB liquid medium and incubate at 28°C and 160 rpm for 12 to 14 hours. When the strain is in the logarithmic growth phase, transfer 1 mL to a preservation tube, add 200 µL of 87% glycerol solution, vortex to mix, and store long-term at -80°C.
[0037] (2) Using a sterile scalpel, cut 20 root segments, each 2 cm long and approximately 2 mm in diameter, and transfer them to 1.5 mL centrifuge tubes. Add 1 mL of 1×PBS buffer and 3 to 5 sterile glass beads to the centrifuge tubes. Vortex to wash away the soil adhering to the root surface, and then remove the root samples and glass beads with sterile forceps. Centrifuge at 4°C and 13000 rpm for 15 min to collect the precipitate. Discard the supernatant and add 1 mL of 1×PBS buffer to the centrifuge tubes to prepare the stock solution. Dilute 10-fold to 10 ... 9 The dilution procedure is the same as (1).
[0038] (3) Transfer the roots washed in (2) to a sterile culture dish, add 20 mL of 1% sodium hypochlorite solution for surface sterilization; after soaking for 1 min, transfer the roots to a culture dish containing sterile water for washing (this step should be repeated at least 3 times to ensure the removal of residual sodium hypochlorite from the root surface). After the roots are air-dried under sterile conditions, transfer them to a sterile mortar, add an appropriate amount of 1×PBS buffer, and grind thoroughly into a homogenate. Transfer the homogenate to a 15 mL centrifuge tube, and make up to 10 mL with 1×PBS buffer to prepare the original sample solution, and then serially dilute it 10-fold to 10 mL. 9 The dilution procedure is the same as (1).
[0039] Example 2 Activation of the strain
[0040] Streaking the bacteria onto LB agar plates to activate them, picking a single colony and inoculating it into 5 mL of LB liquid medium. Incubate overnight at 30°C and 200 rpm, adjusting the bacterial concentration to OD0.05. 600 =1.0. A small amount of bacterial culture was streaked onto an LB agar plate containing 3% NaCl (hereinafter referred to as 3% NaCl-LB plate) and incubated at 30℃ for 24 hours. Bacterial growth was then observed.
[0041] Example 3 Preparation of bacterial suspension
[0042] A single colony from the LB plate in Example 2 was transferred to 10 mL of LB liquid medium and incubated overnight at 30°C and 200 rpm. The next day, 10 mL of the bacterial culture was transferred to an Erlenmeyer flask containing 90 mL of LB liquid medium and incubated overnight at 30°C and 200 rpm. On the third day, 100 mL of the bacterial culture was centrifuged at 8000 rpm for 3 min, the supernatant was discarded, and the bacterial pellet was retained. The bacterial pellet was resuspended in sterile water and the OD was adjusted. 600 Value, obtained as 100mL with a concentration of 10 9 A bacterial suspension of CFU / mL was diluted with 900mL of sterile distilled water to a final concentration of 10. 8 CFU / mL bacterial suspension.
[0043] Example 4: Experiment on the control of leaf scorch in greenhouse walnut seedlings
[0044] Walnut seedlings from germination to stage III seedlings (see...) Figure 1During this period, water with 200mL of clean water every 3 days to maintain a suitable moisture content in the pot. When the seedlings reach stage IV, water with 200mL of 25% Hoagland's nutrient solution every 5 days. When the seedlings reach stage VI, stop watering with nutrient solution and begin inoculating with bacteria. Inoculate with 200mL / pot of the bacterial suspension from Example 3 every 5 days via root drenching, for a total of 3 inoculations. When the seedlings reach stage VIII, water with 200mL of 125mM NaCl solution every 5 days in both the NaCl treatment group and the NaCl+ strain YBZ13 treatment group, for a total of 8 waterings. During this period, water with an equal amount of clean water in the clean water control group.
[0045] Walnut seedling leaf scorch symptoms are classified into 5 levels based on the area of scorched leaves, such as... Figure 2 As shown in (A). The number of leaves at different disease levels was investigated and counted to calculate the severity of walnut seedling leaf scorch. The leaf symptom grading criteria are shown in Table 1.
[0046] Table 1 Grading Criteria for Leaf Scorch in Walnut Seedlings
[0047]
[0048] The severity of leaf scorch in walnut seedlings can be calculated using the following formula:
[0049] Severity of leaf scorch = 100 × ∑ (number of leaves at each disease level × representative value of each level) / (total number of leaves investigated × highest representative value).
[0050] Walnut root vigor was assessed using the Plant Root Vigor Assay Kit - TTC Method (Solarbio).
[0051] This invention screened a bacterial strain, YBZ13, for controlling leaf scorch in walnut seedlings through NaCl plate culture and greenhouse walnut seedling leaf scorch control experiments. When strain YBZ13 was inoculated into the roots of walnut seedlings via root irrigation, the incidence of leaf scorch was significantly reduced compared to the NaCl treatment group (see...). Figure 3 The severity decreased by 33.27 (see) Figure 4 The fresh weight of the primary root in the NaCl+ treated group was significantly increased by 4.95 g compared to the NaCl-treated group (see...). Figure 5 The root activity of the seedlings increased significantly to 1645.21 µg / gh (see...). Figure 6 Therefore, strain YBZ13 has shown promising application prospects in the control of walnut leaf scorch.
[0052] Example 5 Identification of strain YBZ13
[0053] (1) Colony morphology of strain YBZ13
[0054] When strain YBZ13 was inoculated onto LB solid medium and incubated at 30°C for 24 hours, nearly circular colonies with smooth edges were observed. The colony surface was moist, smooth, and wrinkle-free, and appeared pale pink (see...). Figure 7 ).
[0055] (2) Amplification and sequencing analysis of the 16S rRNA gene of strain YBZ13
[0056] Genomic DNA was extracted from strain YBZ13 and used as a template. The 16S rRNA gene fragment of this strain was amplified using universal primer pairs 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'-TACGGYTACCTTGTTACGACTT-3'). The amplification program was as follows: 95℃ pre-denaturation for 5 min; 30 cycles, each cycle consisting of 95℃ denaturation for 30 s, 55℃ annealing for 30 s, and 72℃ extension for 2 min; total extension at 72℃ for 5 min. The reaction system is shown in Table 2.
[0057] Table 2 PCR reaction system
[0058]
[0059] After the PCR products obtained from the amplification were detected by gel electrophoresis (1% agarose), they were sent to Ruiboxingke Biotechnology Co., Ltd. for sequencing analysis.
[0060] The 16S rRNA gene sequencing results are as follows:
[0061] SEQ ID No. 1:
[0062]
[0063] The obtained sequences were subjected to a BLAST search on the National Center for Biotechnology Information (NCBI) website (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). The results showed that the sequence with the highest similarity to the 16S rRNA gene fragment of strain YBZ13 was... Bacillus megaterium The similarity reached 99.92%. Therefore, strain YBZ13 can be identified as Bacillus megaterium and named Bacillus megaterium (B. megaterium). Bacillus megaterium YBZ13 was deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 28098 on August 4, 2023.
[0064] Although the present invention has been illustrated and described with specific embodiments, it should be understood that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; those skilled in the art should understand that modifications can be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein, without departing from the spirit and scope of the present invention; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention; therefore, this means that all such substitutions and modifications that fall within the scope of the present invention are included in the appended claims.
Claims
1. Bacillus megaterium ( Bacillus megaterium YBZ13 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 28098.
2. A microbial preparation, characterized in that, The microbial preparation includes the bacterial cells and suspension of Bacillus megaterium YBZ13 as described in claim 1.
3. The microbial preparation according to claim 2, characterized in that, The microbial preparations include solid or liquid preparations.
4. The microbial preparation according to claim 3, characterized in that, When the microbial preparation is a solid dosage form, the number of viable Bacillus megaterium YBZ13 in the microbial preparation is 10. 8 CFU / g or higher.
5. The microbial preparation according to claim 3, characterized in that, When the microbial preparation is a liquid preparation, the number of viable Bacillus megaterium YBZ13 in the microbial preparation is 10. 8 CFU / mL or higher.
6. A method for preventing and controlling walnut leaf scorch, characterized in that, Apply Bacillus megaterium YBZ13 as described in claim 1 and / or the microbial preparation as described in any one of claims 2 to 5 to walnuts.
7. A method for promoting walnut growth under high salt stress, characterized in that, Apply Bacillus megaterium YBZ13 as described in claim 1 and / or the microbial preparation as described in any one of claims 2 to 5 to walnuts.
8. The application of the microbial preparation of Bacillus megaterium YBZ13 as described in claim 1 or any one of claims 2 to 5 in the prevention and control of non-infectious plant diseases and / or the promotion of walnut growth; The non-infectious plant disease mentioned is walnut leaf scorch.