A strain of Bacillus subtilis and its application in plant disease control

Abstract

This invention belongs to the field of microbial technology and discloses a strain of *Bacillus zeylanus* and its application in the control of plant diseases, particularly its application in the control of root rot in wolfberry. The *Bacillus zeylanus* strain (… Paenibacillus zeae This strain, isolated from the rhizosphere soil of wolfberry plants suffering from root rot, is designated R2 and has been preserved under the CGMCC No. 36137. It inhibits the growth of various fungal pathogens, including *Lycium chinense*, the causal agent of wolfberry root rot, produces cellulase and amylase, and can induce plant... PR1 , CAT2 The strain expressed defensive genes. Pot experiments showed that it achieved a 66.66% control effect against wolfberry root rot, significantly reducing the incidence and disease index, and providing an effective biological resource for the green control of plant diseases, especially wolfberry root rot.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology and relates to a strain of Bacillus subtilis and its application in the prevention and control of plant diseases. Background Technology

[0002] Traditional chemical control methods have led to a series of derivative problems, including increased pathogen resistance, soil microecological imbalance, environmental pollution, and excessive residues in agricultural products. Against this backdrop, developing green, safe, and sustainable plant disease control technologies has become an urgent need for modern agricultural development. Microbial inoculants, as a core means of biological control, have gradually become a research focus in the field of plant protection due to their advantages such as good environmental compatibility, diverse mechanisms of action, and low likelihood of inducing resistance.

[0003] Goji berries ( Lycium barbarum Lycium barbarum (L.) is a distinctive and advantageous economic crop in Northwest my country, possessing both ecological restoration and high-value utilization potential. Its dried fruit and processed products are widely used in health foods and traditional Chinese medicine. However, with extended cultivation years and widespread continuous cropping, the incidence and severity of root rot in Lycium barbarum have been steadily increasing, becoming a key bottleneck restricting the industry's quality and efficiency improvement. Lycium barbarum diseases are mainly caused by Fusarium oxysporum (…). F. oxysporum Fusarium solani () Fusarium solani Rhizoctonia solani ( ) Rhizoctonia solani This disease is caused by a complex infection of various soil-borne pathogenic fungi, such as α, β, and γ. The pathogens can invade the vascular system through micro-wounds in the roots or directly, disrupting the transport of water and nutrients, leading to browning and rotting of the roots and wilting and death of the above-ground parts. It is characterized by a long incubation period, high infectivity, and difficulty in prevention and control.

[0004] Currently, field control mainly relies on chemical pesticides for root drenching. However, long-term use not only leads to drug resistance in pathogens but also causes soil compaction, decreased enzyme activity, and decline in beneficial microbial communities, exacerbating the risk of disease outbreaks. Biological control, with its green and safe approach, has become an important strategy for controlling wolfberry root rot. Microbial agents exhibit multiple synergistic mechanisms to antagonize root rot, including: inhibiting pathogen colonization through nutrient competition and ecological site occupation; secreting antimicrobial active substances that directly act on the pathogen's cell membrane or cell wall; and inducing systemic resistance (ISR) in plants, enhancing overall immunity. Furthermore, some growth-promoting bacteria can improve plant nutrition by secreting growth hormones, fixing nitrogen, and solubilizing phosphorus, achieving a dual effect of "disease prevention and growth promotion."

[0005] In recent years, numerous studies have been conducted on the biological control of root rot in wolfberry. Researchers have isolated several highly effective antagonistic bacterial strains from the rhizosphere soil and endophytic microbial communities of healthy wolfberry plants. For example, *Bacillus amyloliquefaciens* (… Bacillus amyloliquefaciensSN16-1 inhibits Fusarium mycelial growth by producing lipopeptide antibiotics such as surfactantin and iturin, and can induce upregulation of related defense genes in wolfberry plants, thereby enhancing systemic resistance. In addition, Bacillus spp. (…) Paenibacillus Trichoderma strains have attracted much attention in recent years due to their ability to produce abundant antibacterial metabolites and their strong rhizosphere colonization ability. Trichoderma ) and Aspergillus terreus ( Aspergillus terreus It has also been shown that it can effectively antagonize the root rot fungus of wolfberry through reparasitism and the activity of cell wall degrading enzymes (chitinase, glucanase).

[0006] Despite some successes in biological control, existing biocontrol agents still suffer from problems such as unstable field efficacy, limited mechanisms of action, and limited growth-promoting effects. Most strains exhibit poor environmental adaptability, making sustained colonization in the rhizosphere difficult, and the synergistic mechanism of compound agents remains unclear. Therefore, discovering novel strains possessing broad-spectrum antibacterial activity, high rhizosphere adaptability, and growth-promoting functions, and thoroughly analyzing their metabolites and mechanisms of action, are crucial for developing highly efficient and stable microbial agents. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention has screened a Bacillus-like bacterium that exhibits significant preventative efficacy against root rot in wolfberry, and systematically evaluated its antibacterial activity and growth-promoting potential, providing new microbial resources and theoretical basis for the green control of wolfberry diseases. To achieve this technical objective, this invention provides the following technical solution.

[0008] First, this invention provides a strain of Bacillus zeiformis R2, which was isolated from the rhizosphere soil of wolfberry plants suffering from root rot in Nuomuhong Farm, Qinghai Province. Its preservation information is as follows:

[0009] Bacterial strain name: Bacillus zeylindrica

[0010] Latin name: Paenibacillus zeae

[0011] Strain number: R2

[0012] Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee

[0013] Collection institution abbreviation: CGMCC

[0014] Address of depository: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing

[0015] Deposit date: October 9, 2025

[0016] Accession number: CGMCC No.36137.

[0017] Furthermore, the 16S rDNA nucleotide sequence of the Bacillus zeylindrica R2 is shown in SEQ ID NO.1.

[0018] Furthermore, the Bacillus zeylinum R2 colonies are relatively large, with a diameter of 2-5 mm. They are approximately circular, off-white, and opaque. The colony surface is relatively rough, with a slightly convex center and irregular edges, forming petal-shaped or serrated protrusions.

[0019] Secondly, the present invention provides a microbial preparation for the prevention and control of plant diseases, the microbial preparation comprising the above-mentioned Bacillus zeylindrica R2 or its metabolites.

[0020] Furthermore, the viable count of Bacillus zeylans R2 in the microbial preparation is 1 × 10⁻⁶. 7 ~1×10 9 CFU / mL.

[0021] Thirdly, this invention seeks protection for the application of the aforementioned Bacillus zeylans R2 in the control of plant fungal diseases, wherein the fungus includes: Fusarium oxysporum. Fusarium oxysporum Tomato gray mold Botrytis cinerea Sclerotinia sclerotiorum var. ... Sclerotinia sclerotiorum Apple anthracnose bacteria Colletotrichum gloeosporioides Pear tree dieback fungus Phomopsis fukushii、 cotton damping-off fungus Rhizoctonia solani、 Tobacco star bacterium Alternaria alternata Apple rot bacteria Valsa mali Or Lycium barbarum wilt fungus Verticillium dahliae At least one of them.

[0022] Furthermore, in the above applications, the application method includes: applying the fermentation broth of Bacillus zeylindrica R2 or its preparation to the roots of plants or rhizosphere soil.

[0023] Furthermore, in the above applications, the dilution factor of the fermentation broth is 5 to 20 times.

[0024] Fourthly, this invention claims protection for the application of the aforementioned Bacillus rhizogenes R2 in inducing plant disease resistance, wherein inducing plant disease resistance includes: inducing intracellular... PR1 Gene, CAT2 Gene, NPR1 Gene or RBOHD (C) Gene expression is upregulated.

[0025] Compared with existing technologies, the present invention, "a strain of Bacillus zeylansium and its application in the prevention and control of plant diseases," has at least the following beneficial effects:

[0026] Firstly, it has a broad antibacterial spectrum, which can inhibit the growth of various plant fungal pathogens such as Lycium barbarum root rot fungus, tomato gray mold fungus, and rapeseed sclerotinia rot fungus, providing biological resources for the prevention and control of various plant diseases;

[0027] Secondly, it has the ability to produce enzymes, including cellulase and amylase, as well as a certain ability to produce protease, which can help improve the rhizosphere environment of plants and enhance biocontrol.

[0028] Third, it can induce plant disease resistance, enabling plants such as wolfberry to develop disease resistance. PR1 , CAT2 , NPR1 , RBOHD (C) Upregulation of defense gene expression activates the plant's own defense system;

[0029] Fourth, the strain has outstanding control effect on wolfberry root rot. Pot experiments show that the strain can reduce the incidence of wolfberry root rot from 100% to 62.5%, significantly reduce the disease index, and achieve a control effect of 66.66%. It can effectively reduce the damage of the disease to wolfberry plants and provide an effective technical means for the green control of plant diseases, especially wolfberry root rot, avoiding the problems of drug resistance and environmental pollution caused by traditional chemical control. Attached Figure Description

[0030] Figure 1 This is a streak culture result of Bacillus zeylans R2 on R2A medium.

[0031] Figure 2 It is Bacillus zeiformis R2 and Fusarium oxysporum, the pathogen causing root rot of wolfberry. Fusarium oxysporum The results of the in-plate confrontation experiment (H0) include the treatment group (R2) inoculated with Bacillus zeiformis R2 and the control group (CK) not inoculated with this strain.

[0032] Figure 3 This is the result of an experiment on the inhibitory effect of sterile fermentation broth of Bacillus zeiformis R2 on the mycelial growth of Fusarium oxysporum (H0), the pathogen causing root rot of Lycium barbarum. CK represents the control group (blank PDA medium inoculated only with Fusarium oxysporum mycelium), and R2 represents the treatment group (mixed PDA medium containing 10% sterile fermentation broth of R2).

[0033] Figure 4 The image shows the colony morphology (left) and Gram staining results of Bacillus zeiformis R2 (right).

[0034] Figure 5 This is a phylogenetic tree constructed from the 16S rRNA gene sequence of Bacillus zeylanus R2.

[0035] Figure 6This is a graph showing the antibacterial spectrum test results of Bacillus subtilis R2 in maize. "CK" represents the control group without R2 inoculation, and "R2" represents the treatment group inoculated with R2. The horizontal arrangement illustrates different target pathogens, including: Botrytis cinerea (Tomato gray mold). Botrytis cinerea ), rapeseed sclerotinia pathogen ( Sclerotinia sclerotiorum Apple anthracnose bacteria ( Colletotrichum gloeosporioides ), pear tree dieback fungus ( Phomopsis fukushii ) 、 cotton damping-off fungus ( Rhizoctonia solani ) 、 Tobacco star bacterium ( Alternaria alternata ), apple rot bacteria ( Valsa mali ), Lycium barbarum wilt pathogen ( Verticillium dahliae ).

[0036] Figure 7 This is the result of the test on the siderophore-producing ability of Bacillus zeylindrica R2.

[0037] Figure 8 This is the result of the test on the cellulase production ability of Bacillus zeiformis R2.

[0038] Figure 9 This is the result of the test for the protease production ability of Bacillus zeylindrica R2.

[0039] Figure 10 This is the result of the test on the amylase production ability of Bacillus zeylindrica R2.

[0040] Figure 11 These are the test results for disease resistance in wolfberry induced by Bacillus zeiformis R2, which includes four defense-related genes. PR1 (A) CAT2 (B) NPR1 (C) RBOHD Line graphs showing the relative expression levels of (C) and (D). The horizontal axis represents the time points after treatment (0h, 3h, 6h, 12h, 24h), and the vertical axis represents the relative gene expression level.

[0041] Figure 12 This is the result of a pot experiment on the effect of Bacillus zeiformis R2 fermentation broth on the prevention and control of root rot in wolfberry. The experiment was divided into aboveground and underground parts. CK was the control group irrigated with diluted LB medium, and R2 was the treatment group irrigated with Bacillus zeiformis R2 fermentation broth. Detailed Implementation

[0042] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0043] The experimental materials, reagents, culture media, and instruments used in the examples are summarized below:

[0044] I. Experimental Materials

[0045] Bacillus zeiformis R2: isolated from soil samples taken from the rhizosphere soil of wolfberry plants suffering from wolfberry root rot in the wolfberry field of Nuomuhong Farm, Qinghai Province.

[0046] Test pathogen: Fusarium oxysporum, the pathogen causing root rot of wolfberry ( Fusarium oxysporum H0, Tomato gray mold ( Botrytis cinerea ), rapeseed sclerotinia pathogen ( Sclerotinia sclerotiorum Apple anthracnose bacteria ( Colletotrichum gloeosporioides ), pear tree dieback fungus ( Phomopsis fukushii ) 、 cotton damping-off fungus ( Rhizoctonia solani ) 、 Tobacco star bacterium ( Alternaria alternata ), apple rot bacteria ( Valsa mali ), Lycium barbarum wilt pathogen ( Verticillium dahliae All of these strains were isolated and preserved in the laboratory in the early stages.

[0047] Test plant materials: one-year-old wolfberry seedlings (greenhouse seedlings, selected healthy plants with a height of 30-50cm, uniform growth, and no diseases or pests, for the induction of disease resistance test and potted plant control efficacy test).

[0048] II. Test Reagents

[0049] 0.1% Congo red staining solution, Golu's iodine solution (13g iodine and 35g potassium iodide dissolved in 100mL deionized water, diluted with distilled water to a final volume of 1000mL), sterile water; plant RNA extraction kit (Beijing Huayueyang Biotechnology Co., Ltd.), reverse transcription kit (Thermo Fisher Scientific (China) Co., Ltd.), real-time PCR kit (Nanjing Novizan Biotechnology Co., Ltd.); bacterial genomic DNA extraction kit (Thermo Fisher Scientific (China) Co., Ltd.); common bacterial biochemical identification tubes (Guangdong Huankai Microbial Technology Co., Ltd., used for identifying the physicochemical properties of bacterial strains).

[0050] III. Culture medium formulation

[0051] R2A medium (for strain isolation and purification): 0.5g yeast extract, 0.5g peptone, 0.5g casein hydrolysate, 0.5g glucose, 0.5g soluble starch, 0.3g sodium pyruvate, 0.3g dipotassium hydrogen phosphate (K2HPO4), 0.05g magnesium sulfate heptahydrate (MgSO4·7H2O), 15.0g agar, add distilled water to 1000mL, autoclave at 121℃ for 20min.

[0052] LB medium (used for strain activation and fermentation broth preparation):

[0053] Solid LB medium: 10.0 g tryptone, 5.0 g yeast extract, 10.0 g sodium chloride (NaCl), 15.0 g agar, add distilled water to 1000 mL, adjust pH to 7.0, autoclave at 121℃ for 20 min;

[0054] Liquid LB medium: 10.0 g tryptone, 5.0 g yeast extract, 10.0 g sodium chloride (NaCl), add distilled water to 1000 mL, adjust pH to 7.0, and autoclave at 121 °C for 20 min.

[0055] PDA medium (for pathogen culture and plate confrontation test): 200.0g potato (peeled and diced, boiled for 30min and filtered through gauze to obtain the filtrate), 20.0g glucose, 15.0g agar, add distilled water to 1000mL, adjust pH to 7.0, autoclave at 121℃ for 20min.

[0056] PDB medium (for preparing pathogen spore suspension): 200.0g potato (peeled and diced, boiled for 30 minutes and filtered through gauze to obtain the filtrate), 20.0g glucose, add distilled water to 1000mL, adjust pH to 7.0, autoclave at 121℃ for 20 minutes.

[0057] CAS medium (for siderophore production capacity assay): 1 mL 20% sucrose solution, 3 mL 10% acid-hydrolyzed casein, 100 μL 1 mmol / L calcium chloride (CaCl2) solution, 2 mL 1 mmol / L magnesium sulfate (MgSO4) solution, 5 mL 10×MM9 salt solution, 5 mL CAS staining solution, 1.8 g agar, add 83.9 mL distilled water, autoclave at 121℃ for 20 min (CAS staining solution was sterilized separately and then aseptically mixed).

[0058] Cellulase screening medium (for cellulase production capacity testing): 20.0 g sodium carboxymethyl cellulose, 5.0 g yeast extract, 10.0 g tryptone, 10.0 g sodium chloride, 15.0 g agar, add distilled water to 1000 mL, autoclave at 121 °C for 20 min.

[0059] Casein culture medium (for protease production capacity assay): 5.0g yeast extract, 10.0g tryptone, 10.0g sodium chloride, 10.0g casein, 15.0g agar, add distilled water to 1000mL, adjust pH to 7.2, autoclave at 121℃ for 20min.

[0060] Amylase screening medium (for amylase production capacity detection): 10.0g soluble starch, 5.0g peptone, 5.0g sodium chloride, 5.0g beef extract, 20.0g agar, add distilled water to 1000mL, adjust pH to 7.0, and autoclave at 121℃ for 20min.

[0061] IV. Test Instruments

[0062] Incubator, shaker, clean bench, liquid nitrogen tank, -80℃ freezer, quantitative PCR instrument, high-speed centrifuge, pipettes (10μL, 100μL, 1000μL), sterile culture dishes (90mm), sterile centrifuge tubes (10mL, 50mL), sterile hole punch (5mm diameter), electronic balance (accuracy 0.001g), pH meter, autoclave.

[0063] Example 1

[0064] This embodiment describes the isolation and purification of Bacillus zeatus R2.

[0065] I. Experimental Methods

[0066] Preparation of soil suspension: 10g of soil sample and 90mL of sterile water are placed in an Erlenmeyer flask, and 10 sterile glass beads are added. The mixture is then placed in a shaker and shaken for 30 minutes to prepare the soil suspension.

[0067] Strain isolation and purification: Take 1 mL of soil suspension, add it to another centrifuge tube containing 9 mL of sterile water, and shake well to obtain a concentration of 10. -1 Soil suspension; then diluted sequentially to a concentration of 10. -2 10 -3 10 -4 10 -5 10 -6 The suspension; 10 μL of each suspension was taken. -2 10 -3 10 -4 10 -5 10 -6 100 μL of each concentration of suspension was spread on R2A medium; the spread medium was placed in an incubator at 28°C for 2 days; the growth of colonies was observed, and colonies of different shapes and colors were picked for isolation and purification culture; repeated streak culture was performed to obtain pure culture.

[0068] II. Test Results

[0069] Through the above separation and purification process, several purified bacterial strains were obtained from the rhizosphere soil sample of Lycium barbarum. Among them, the streak culture results of strain R2 on R2A medium plates are shown below. Figure 1 As shown.

[0070] Example 2

[0071] This embodiment describes the initial screening of the antagonistic ability of Bacillus zeatus R2.

[0072] Following the experiment in Example 1, several purified strains were obtained. In this example, the biological control of wolfberry root rot was used as the target for strain screening. The purified strains obtained in Example 1 were initially screened for pathogenic antagonism to obtain strains with biocontrol potential.

[0073] I. Experimental Methods

[0074] 1. Activation of the strain

[0075] The biocontrol strains stored at -80℃ were taken out and activated by streaking on LB medium for subsequent experiments.

[0076] 2. Antagonistic ability test of biocontrol bacteria

[0077] The target pathogen was *Fusarium oxysporum*, the pathogen causing root rot of wolfberry, which was previously isolated in the laboratory. Fusarium oxysporum H0: Take a 5mm diameter mycelial cake from an activated Fusarium oxysporum H0 colony and invert it in the center of a PDA medium. Separately, pick an activated single colony of R2 and streak it 2.5cm from both sides of the H0 mycelial cake. Use the uninoculated R2 treatment as a control. After sealing, incubate at 28℃ for 7 days. After the control has filled the petri dish, observe and calculate the antibacterial ability of the R2 strain against Fusarium oxysporum H0 using the cross-cross method.

[0078] II. Test Results

[0079] The results of the in-plate confrontation experiment between strain R2 and Fusarium oxysporum H0 are as follows: Figure 2 As shown, the measured width of the inhibition zone was approximately 14 mm. Based on the cross-multiplication method, the inhibition rate of strain R2 against *Fusarium oxysporum* H0 reached 68.18%. These results indicate that strain R2 has a significant inhibitory effect on *Fusarium oxysporum*, which causes root rot in wolfberry, and therefore it was selected as the target strain for initial screening.

[0080] Example 3

[0081] This embodiment describes a secondary screening of the ability of Bacillus zeylans R2 to inhibit the mycelial growth of pathogenic bacteria (Fusarium oxysporum H0).

[0082] I. Experimental Methods

[0083] 1. Preparation of sterile fermentation broth

[0084] Select a single colony of activated R2 strain and inoculate it into LB liquid medium. Incubate at 28°C and 150 rpm for 72 h to obtain the fermentation broth of R2 strain. Centrifuge the fermentation broth at 12000 r for 10 min to remove the bacterial precipitate. Filter the obtained fermentation supernatant through a 0.22 μm filter membrane to obtain the sterile fermentation broth of R2.

[0085] 2. Mycelial growth inhibition test

[0086] The obtained R2 sterile fermentation broth was mixed with PDA medium at approximately 40°C to prepare a mixed PDA medium containing 10% R2 sterile fermentation broth. Activated Fusarium oxysporum H0 edge hyphae were separately prepared into 5mm diameter mycelial discs. These mycelial discs were inverted and placed in the center of the mixed PDA medium as the experimental group (R2 treatment), while the PDA medium inoculated only with Fusarium oxysporum H0 served as the control group (CK). After sealing, the discs were incubated upside down at 28°C. When the Fusarium oxysporum H0 hyphae in the CK group had grown to cover the entire culture dish, the diameter of the mycelial discs in both the CK and R2 treatment groups was measured using the cross-sectional method. The pathogen inhibition rate was calculated using the following formula:

[0087] Pathogen inhibition rate = (diameter of mycelium in CK group - diameter of mycelium in R2 treatment) / diameter of mycelium in CK group × 100%

[0088] II. Test Results

[0089] The test results are as follows Figure 3 As shown, on mixed PDA medium containing 10% R2 sterile fermentation broth ( Figure 3 The mycelial growth of Fusarium oxysporum H0 in the R2 group was inhibited to a certain extent, with an inhibition rate of 19.32%. This result indicates that the antagonistic substances of the R2 strain are not present in large quantities in the fermentation filtrate.

[0090] Example 4

[0091] This embodiment describes the identification of Bacillus zeatus R2.

[0092] I. Experimental Methods

[0093] 1. Identification of strain morphology

[0094] The R2 strain was cultured on solid LB medium at 37°C for 48 hours. The colony morphology was observed, and Gram staining was performed to observe the staining results.

[0095] 2. Physicochemical property identification

[0096] After activating the R2 strain on solid LB medium, it was inoculated into liquid LB medium and cultured at 37°C for 48 h. The culture concentration was then adjusted to OD. 600 =0.1 for backup; The nitrate reducing ability, fructose, xylose, glucose, maltose, galactose, cellobiose, sorbitol, mannitol, urea, glycerol, gelatin liquefaction, nitrite gas production, and nitrite reducing ability of the R2 strain were tested using ordinary bacterial biochemical identification tubes. The operation steps are as described in the instruction manual of the identification tubes.

[0097] 3. Biological identification

[0098] DNA was extracted from strain R2 using a bacterial genomic DNA extraction kit, and the gene sequence of 16S rRNA was amplified by PCR using universal bacterial primers.

[0099] PCR reaction mixture (25 μL): 12.5 μL 2×Taq MasterMix, 1 μL each of forward and reverse primers, 1 μL template, 9.5 μL ddH2O. PCR reaction program: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 1 min 30 s, 72℃ final extension for 2 min, 35 cycles.

[0100] The PCR products obtained from the above amplification were detected by 1% agarose gel electrophoresis. PCR products with the correct target band were recovered and sent to Beijing Qingke Biotechnology Co., Ltd. for purification and sequencing. The sequencing results were compared with the NCBI database. A phylogenetic tree was constructed using the Neighbor-Joining method with MAGEX software.

[0101] II. Test Results

[0102] 1. Strain morphology and Gram staining results

[0103] The colony morphology of strain R2 is as follows: Figure 4 As shown in the left image, the colonies of strain R2 on solid LB medium are generally round or nearly round, off-white to light yellow, opaque, with a relatively smooth and moist surface, a slightly convex center, and irregularly shaped petal-like or serrated edges. The Gram staining results of strain R2 are shown below. Figure 4 As shown in the right figure, this strain is a Gram-positive bacterium with rod-shaped cells.

[0104] 2. Physicochemical properties

[0105] The identification results are shown in Table 1.

[0106] Table 1. Biochemical identification results of strain R2

[0107]

[0108] Note: "+" indicates positive; "-" indicates negative.

[0109] The results in Table 1 show that strain R2 can decompose and utilize fructose, xylose, glucose, maltose, galactose, cellobiose, and mannitol and gelatin, reflecting its strong energy utilization capacity, but it lacks the ability to utilize sorbitol and reduce nitrate.

[0110] 3. Biological identification

[0111] Sequencing revealed that the nucleotide sequence of the 16S rRNA of strain R2 is shown in SEQ ID NO.1, and the constructed phylogenetic tree is as follows. Figure 5 As shown, the results indicate that strain R2 is related to Bacillus maize (…). Paenibacillus maysiensis The similarity between strain 1-49 and NR_165764.1 reached 99.09%, therefore strain R2 was identified as *Bacillus zeylindrica*. Paenibacillus zeae The Bacillus zeatans R2 is currently deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 36137.

[0112] Example 5

[0113] This embodiment describes the comprehensive biocontrol potential evaluation of Bacillus zeatus R2.

[0114] This embodiment evaluates the comprehensive biocontrol potential of Bacillus zeylindrica R2 from two aspects: the range of its antibacterial spectrum and its enzyme production capacity (side carriers, cellulase, protease, and amylase), providing data support for the feasibility of this strain in the biological control of plant diseases.

[0115] I. Experimental Methods

[0116] 1. Preparation of bacterial suspension

[0117] Bacillus rhizogenes R2, stored at -80℃, was retrieved, streaked onto LB solid medium, and activated in a 37℃ incubator for 48 hours. Single colonies of the activated bacteria were picked and inoculated into LB liquid medium, and cultured at 37℃ and 150 rpm for 24 hours to obtain OD (Oxygen Demand). 600 Prepare a 0.1% R2 bacterial suspension for later use.

[0118] 2. Antibacterial spectrum test (plate confrontation method)

[0119] Activated cultures of the tested pathogens (see the "Experimental Materials" section for details) were collected. Using a sterile punch (5mm diameter), mycelial cakes were created at the edge of the pathogen colonies and placed upside down in the center of PDA medium. *Bacillus zeiformis* R2 was symmetrically streaked 2.5cm from the center of the mycelial cake (activated single colonies were directly streaked). A control group (CK) was used, consisting only of pathogen mycelial cakes and no *Bacillus zeiformis* R2. Each treatment was replicated three times. The culture dishes were incubated at 28℃ for 7 days. When the pathogens in the CK group had completely covered the culture dish, the width of the inhibition zone between *Bacillus zeiformis* R2 and the pathogens was observed and measured, and the antagonistic effect of each pathogen was recorded. For *Verticillium wilt* of wolfberry, since mycelial cakes could not grow normally on PDA medium, antagonistic activity was observed by spreading pathogen spores and inoculating R2 solution in the center of the PDA medium. The size of the clear zone reflected the resistance of R2 to the pathogen.

[0120] 3. Enzyme production capacity detection

[0121] 1) Iron production capacity testing

[0122] Take 5 μL of R2 bacterial suspension and spot it in the center of CAS medium. Each treatment is repeated 3 times. After incubation at 28℃ for 2 days, observe whether a yellow halo appears around the colony. If a yellow halo appears, it indicates that the strain has the ability to produce siderophores; otherwise, it does not.

[0123] 2) Cellulase production capacity test

[0124] Take 5 μL of R2 bacterial suspension and spot it in the center of the cellulase selection medium. Each treatment is repeated 3 times. After incubation at 28℃ for 3 days, spray 0.1% Congo red staining solution evenly onto the surface of the medium and stain for 30 min. Pour off the staining solution and decolorize with physiological saline 3 times (5 min each time). Observe whether a clear zone is formed around the colony. If a clear zone is formed, it indicates that the strain has the ability to produce cellulase. The larger the diameter of the clear zone, the stronger the enzyme production ability.

[0125] 3) Protease production capacity test

[0126] Take 5 μL of R2 bacterial suspension and spot it in the center of the casein agar medium. Each treatment is repeated 3 times. After incubation at 28℃ for 3 days, observe whether a clear zone is formed around the colony (principle: protease decomposes casein in the culture medium to form a clear zone), and record the size of the clear zone.

[0127] 4) Amylase production capacity test

[0128] Take 5 μL of R2 bacterial suspension and spot it in the center of the amylase selection medium. Each treatment is repeated 3 times. After incubating at 28℃ for 3 days, spray the surface of the medium evenly with Grünster's iodine solution and stain in the dark for 20 min. Pour off the iodine solution and observe whether a clear zone is formed around the colony. Record the size of the clear zone.

[0129] II. Test Results

[0130] 1. Antibacterial spectrum test results

[0131] Bacillus zeiformis R2 showed antagonistic activity against all tested pathogens, with the specific results as follows:

[0132] like Figure 6 As shown, Bacillus zeylinus R2 exhibits significant antagonistic effects against apple anthracnose fungus, tomato gray mold fungus, rapeseed sclerotinia stem rot fungus, and cotton damping-off fungus, with an inhibition zone width of 10-15 mm; it also shows good antagonistic effects against tobacco scab fungus, apple rot fungus, and pear wilt fungus, with an inhibition zone width of 8-11 mm; and it demonstrates good resistance to Verticillium wilt of wolfberry, with a transparent zone diameter of 19 mm.

[0133] 2. Results of enzyme production capacity test

[0134] 1) Iron production capacity: such as Figure 7 As shown, Bacillus zeiformis R2 could hardly grow on CAS medium, and no yellow halo was observed around the colony, indicating that this strain does not have the ability to produce siderophores.

[0135] 2) Cellulase production capacity: such as Figure 8 As shown, a clear and large transparent zone (approximately 27 mm in diameter) appears around the Bacillus zeylindrica R2 colony, indicating that this strain has a strong ability to produce cellulase.

[0136] 3) Protease production capacity: such as Figure 9 As shown, only a small clear zone (approximately 18 mm in diameter) appears around the Bacillus zeylindrica R2 colony, indicating that this strain has a weak protease production capacity.

[0137] 4) Ability to produce amylase: such as Figure 10 As shown, a clear and large transparent zone (approximately 21 mm in diameter) appears around the Bacillus zeylindrica R2 colony, indicating that this strain has a good ability to produce amylase.

[0138] III. Experimental Conclusions

[0139] Bacillus rhizogenes R2 has broad-spectrum antibacterial activity and has the potential for application in the biological control of various plant diseases.

[0140] Although this strain does not have the ability to produce siderophores, it has a strong ability to produce cellulase and a good ability to produce amylase, and also has a certain ability to produce protease. Cellulase can degrade pathogen cell wall components (such as cellulose), and amylase can improve soil carbon source utilization efficiency. These enzyme-producing characteristics can not only directly inhibit the growth of pathogens, but also indirectly improve the plant rhizosphere microenvironment, further enhancing its biocontrol potential.

[0141] In summary, Bacillus zeylinus R2 exhibits excellent comprehensive biocontrol potential in terms of both antibacterial range and functional metabolism (enzyme production), making it a superior strain resource for developing green control agents for plant diseases.

[0142] Example 6

[0143] This embodiment describes an experiment to detect the disease resistance of wolfberry induced by Bacillus zeiformis R2.

[0144] To clarify whether Bacillus zeiformis R2 can enhance the resistance of Lycium barbarum to root rot by inducing the upregulation of defense-related genes in Lycium barbarum plants, this example uses... PR1 , CAT2 , NPR1 , RBOHD (C) As the gene to be tested, the biocontrol mechanism of Bacillus zeylindrica R2 was analyzed.

[0145] I. Experimental Methods

[0146] 1. Preparation of R2 fermentation broth

[0147] Select a single colony of activated Bacillus zeiformis R2 and inoculate it into LB liquid medium. Incubate at 28°C and 150 rpm for 72 h to obtain R2 fermentation broth for later use.

[0148] 2. Treatment of wolfberry plants and sample collection

[0149] The experiment was divided into two groups: blank control group (CK): watered with LB liquid medium; R2 treatment group (R2): watered with R2 fermentation broth diluted 10 times; each group was set up with 3 pots for replication, and each pot was planted with 1 wolfberry seedling.

[0150] Apply the corresponding treatment solution to the roots of the two groups of wolfberry plants at a dosage of 200 mL per plant.

[0151] At 0h, 3h, 6h, 12h and 24h after treatment, 1-2g of root samples were taken from each plant in each group (avoiding rotten or damaged parts), immediately flash-frozen in liquid nitrogen, and then transferred to a -80℃ freezer for storage for subsequent RNA extraction.

[0152] 3. Gene expression detection

[0153] RNA extraction and cDNA synthesis: Total RNA was extracted from wolfberry root samples according to the instructions of the plant RNA extraction kit; then, the RNA was reverse transcribed into cDNA according to the reverse transcription kit operation steps for later use.

[0154] Real-time PCR detection: using cDNA as a template, EF-1α The gene is an internal reference gene, and detection was performed using a real-time PCR kit. PR1 , CAT2 , NPR1 , RBOHD (C) Relative gene expression levels. PCR reaction system (25 μL): 12.5 μL of 2× real-time PCR Mix, 1 μL each of forward and reverse primers (10 μmol / L), 1 μL of cDNA template, and 9.5 μL of enzyme-free water. Reaction program: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 40 cycles, with a final extension at 72℃ for 5 min. Each sample was tested in triplicate, using 2... -ΔΔCt The method calculates the relative expression level of genes.

[0155] Defense genes used for PCR detection ( PR1 , CAT2 , NPR1 , RBOHD (C)) and internal reference gene ( EF-1α The specific primer sequences for ) are shown in Table 2.

[0156] Table 2. Primer sequence list for defense gene detection

[0157]

[0158] II. Test Results

[0159] Internal reference gene EF-1α The expression was stable across all time points and sample groups, and can be used as a reference for gene expression normalization. Compared with the blank control group (CK), the expression of four defense-related genes in the roots of Lycium barbarum in the R2 treatment group was upregulated to varying degrees:

[0160] PR1 Genes: such as Figure 11 As shown in Figure A, the expression level reached its peak 3 hours after treatment, with a relative expression level approximately 6 times that of the CK group, and then gradually decreased over time.

[0161] CAT2 Genes: such as Figure 11 As shown in Figure B, the expression was significantly upregulated 3 hours after treatment, with a relative expression level approximately 3 times that of the CK group, and the expression level decreased after 6 hours.

[0162] NPR1 Genes: such as Figure 11 As shown in Figure C, the expression peaked 3 hours after treatment, with the relative expression level increasing by 2.5 times compared to the CK group. It then gradually decreased to below the control, but after 24 hours of treatment, it was significantly higher than the control again, with the relative expression level being about 3 times that of the CK group.

[0163] RBOHD (C) Genes: such as Figure 11 As shown in Figure D, the expression level was significantly higher than that of the CK group 3 hours after treatment, with a relative expression level approximately 3 times that of the CK group. After 12 hours, it recovered to a level close to that of the CK group, and then showed significant expression again at 24 hours.

[0164] III. Experimental Conclusions

[0165] Bacillus rhamnoides R2 can effectively induce defense-related genes in wolfberry plants. PR1 , CAT2 , NPR1 , RBOHD The upregulation of (C) expression, with the gene expression peak concentrated 3 hours after treatment, indicates that this strain can enhance the disease resistance of wolfberry by activating its own defense system. This is one of the important mechanisms by which Bacillus zeylindrica R2 controls wolfberry root rot.

[0166] Example 7

[0167] This embodiment describes a pot experiment on the efficacy of Bacillus zeiformis R2 fermentation broth for disease control.

[0168] This embodiment verifies the actual control effect of Bacillus zeylindrica R2 fermentation broth on wolfberry root rot, clarifies its ability to reduce the incidence and disease index of wolfberry root rot, and provides pot experiment data for the application of this strain in the green control of wolfberry root rot.

[0169] I. Experimental Methods

[0170] 1. Preparation of R2 fermentation broth and Fusarium oxysporum spore suspension

[0171] R2 fermentation broth preparation: Activated Bacillus zeiformis R2 was picked and inoculated into LB liquid medium. The culture was carried out at 28℃ and 150 rpm for 72 h on a shaker to obtain the R2 fermentation broth. The bacterial concentration of the fermentation broth was adjusted to 6 × 10⁻⁶ cells / mL with sterile water. 8 CFU / mL, for later use.

[0172] Preparation of Fusarium oxysporum spore suspension: Mycelial cakes were collected from the edge of activated Fusarium oxysporum H0 colonies using a sterile punch and inoculated into PDB medium. The culture was incubated at 25°C on a shaker until abundant sporulation occurred. Mycelia were removed by filtration through gauze, and the spore suspension was collected. The spore concentration was adjusted to 1×10⁻⁶ with sterile water. 6 CFU / mL, for later use.

[0173] 2. Potted plant treatment and cultivation

[0174] Experimental Groups: The experiment was divided into 2 groups, with 6 pots in each group and 1 wolfberry seedling planted in each pot.

[0175] Blank control group (CK): Irrigated with diluted LB liquid culture medium (10-fold dilution);

[0176] R2 treatment group: irrigation concentration was 6×10 8 R2 fermentation broth, CFU / mL.

[0177] Root irrigation treatment: Apply the corresponding treatment solution slowly to the soil around the roots of the two groups of wolfberry plants at a dosage of 100 mL per plant; after treatment, place the plants in a well-ventilated and well-lit place in the greenhouse, maintain a high temperature and humidity environment throughout the process, and add water as needed according to soil moisture.

[0178] Pathogen inoculation: One day after the treatment solution was applied, small amounts of Fusarium oxysporum spore suspension were repeatedly poured onto the soil around the roots of the two groups of plants, 50 mL per plant, and cultured for 20 days.

[0179] 3. Disease survey and efficacy calculation

[0180] Disease grading criteria: Grading is set based on the disease characteristics of wolfberry root rot.

[0181] Grade 0: No root rot, healthy above-ground parts of the plant;

[0182] Grade 1: The root rot area is ≤1 / 4, the main root is slightly damaged, and there are no obvious symptoms in the above-ground parts or only slight yellowing of the leaves;

[0183] Level 2: 1 / 4 < root rot area ≤ 1 / 2, main root disease begins to appear, above-ground growth is slow, lower leaves are yellowing and wilting;

[0184] Level 3: 1 / 2 < root rot area ≤ 3 / 4, the main root disease is more obvious, the above-ground parts are obviously stunted, the leaves are yellowed and fall off in large areas, and some branches die.

[0185] Level 4: Root rot area > 3 / 4, main root severely damaged, plant near death or dying.

[0186] Investigation and Calculation: After 20 days of cultivation, count the number of diseased plants in each group and the number of diseased plants at each level. Calculate the incidence rate, disease index, and control effect using the following formulas:

[0187] Incidence rate (%) = (Number of diseased plants / Total number of plants surveyed) × 100;

[0188] Disease index = [∑(number of diseased plants at each level × representative value of that level) / (total number of plants surveyed × highest level representative value)] × 100;

[0189] Prevention and control effect (%) = [(disease index of control area - disease index of treatment area) / disease index of control area] × 100.

[0190] II. Test Results

[0191] The blank control group (CK) showed that the incidence of root rot in wolfberry was 100.00% and the disease index was 84.38. The above-ground parts of the plant showed severe wilting and a large number of leaves falling off, while the underground parts showed a large area of ​​root rot and severe damage to the main root.

[0192] R2 treatment group: The incidence of root rot in wolfberry was reduced to 62.5%, and the disease index was reduced to 28.13; the wilting symptoms of the above-ground parts of the plant were greatly reduced, the yellowing and shedding of leaves were significantly reduced, and the root rot rate and rot area of ​​the underground parts were significantly reduced; it was calculated that the control effect of R2 fermentation broth on root rot of wolfberry reached 66.66%.

[0193] III. Experimental Conclusions

[0194] The fermentation broth of Bacillus zeylinum R2 can significantly reduce the incidence and disease index of root rot in wolfberry, effectively alleviate the degree of root rot and the symptoms of damage to the above-ground parts, and achieve a control effect of 66.66% on root rot in wolfberry, showing potential for practical application in the green control of root rot in wolfberry.

[0195] The embodiments described above are only some, not all, of the embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. All other embodiments obtained by those skilled in the art through related deductions and substitutions based on the inventive concept, without inventive effort, are within the scope of protection of the present invention.

Claims

1. A strain of Bacillus zeylanus ( Paenibacillus zeae R2, characterized in that, The Bacillus zeatans R2 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 36137.

2. The Bacillus rhizogenes R2 according to claim 1, characterized in that, The 16S rDNA nucleotide sequence of the Bacillus zeylanus R2 is shown in SEQ ID NO.

1.

3. A microbial preparation for the prevention and control of plant diseases, characterized in that, It includes the Bacillus rhizobium R2 as described in claim 1.

4. The microbial preparation according to claim 3, characterized in that, The viable count of Bacillus zeiformis R2 in the microbial preparation is 1×10⁻⁶. 7 ~1×10 9 CFU / mL.

5. The application of Bacillus rhizogenes R2 as described in claim 1 in inhibiting plant fungi, characterized in that, The plant fungus is: Fusarium oxysporum. Fusarium oxysporum Tomato gray mold Botrytis cinerea Sclerotinia sclerotiorum var. ... Sclerotinia sclerotiorum Apple anthracnose bacteria Colletotrichum gloeosporioides Pear tree dieback fungus Phomopsis fukushii、 cotton damping-off fungus Rhizoctonia solani Tobacco star bacterium Alternaria alternata Apple rot bacteria Valsa mali Or Lycium barbarum wilt fungus Verticillium dahliae At least one of them.

6. The application of Bacillus rhizogenes R2 as described in claim 1 in the prevention and control of root rot in wolfberry.

7. The application according to claim 6, characterized in that, The application method includes: applying the fermentation broth containing Bacillus zeylindrica R2 to the roots or rhizosphere soil of wolfberry.

8. The application according to claim 7, characterized in that, The dilution ratio of the bacterial fermentation broth is 5 to 20 times.

9. The application of Bacillus rhizogenes R2 as described in claim 1 in inducing resistance to root rot in Lycium barbarum plants, characterized in that, The method of inducing resistance to root rot in wolfberry plants includes: inducing resistance within wolfberry plants... PR1 Gene, CAT2 Gene or NPR1 Gene expression is upregulated.

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