Bacillus velezensis and fermentation liquor and application thereof

Abstract

The application provides a bacillus velezensis strain and a fermentation liquor and application thereof, and belongs to the technical field of microbiology and agricultural application thereof.A bacillus strain is obtained by screening and separation, and its classification position is identified by morphological, physiological and biochemical characteristics and gene sequence analysis, is determined as the bacillus velezensis, and is preserved, and the preservation number is CGMCC NO.24988.The bacillus velezensis is used to prepare the fermentation liquor, is further prepared into a bacterial fertilizer or is mixed with a root base-mineral source black gold life liquid to prepare a compound fertilizer, the bacterial fertilizer or the compound fertilizer is applied to the planting of tomatoes, has an obvious promoting effect on the growth of the tomatoes, and has very obvious prevention and control effects on tomato botrytis and tomato anthracnose, and has great significance for the planting of the tomatoes.

Description

Technical Field

[0001] This invention belongs to the field of microbiology and its agricultural applications, and particularly relates to a strain of Bacillus belye and its fermentation broth and applications. Background Technology

[0002] Tomato, also known as Japanese tomato, is an annual or perennial herbaceous plant belonging to the Solanaceae family and the *Solanum* genus. It grows to a height of 0.6-2 meters, with the entire plant covered in sticky glandular hairs and a strong odor. The stems are prone to lodging. The leaves are pinnately compound or deeply pinnately lobed. The inflorescence peduncle is 2-5 cm long, usually bearing 3-7 flowers. The calyx and corolla are radiate. The berry is oblate or nearly spherical, fleshy and juicy, with yellow seeds. Flowering and fruiting occur in summer and autumn. Tomatoes originated in South America and are widely cultivated in my country. The fruit is rich in nutrients and has a unique flavor. Studies have shown that tomato fruit has the effects of quenching thirst, aiding digestion, clearing heat and relieving summer heat, and tonifying the kidneys and promoting urination. It can treat symptoms such as thirst due to heat-related illnesses, loss of appetite, and internal damp-heat. Furthermore, tomatoes have significant hemostatic, antihypertensive, and cholesterol-lowering effects, and are particularly effective in treating hemophilia and pellagra.

[0003] However, gray mold is a serious and common disease affecting tomatoes. Besides tomatoes, it can also infect more than 20 other crops, including eggplant, peppers, cucumbers, and bottle gourds. It is prevalent in protected cultivation in the north and open fields in the south, and can cause complete crop failure in severe cases. It can damage stems, leaves, flowers, and fruits, but primarily affects the fruit, with unripe fruits usually being more severely affected.

[0004] Anthracnose is also one of the most common diseases of tomatoes. It thrives in high temperature and humidity conditions, is widely distributed, has a complex range of species, a wide host range, and a strong ability to spread. It generally affects mature fruits. The lesions on the fruit are round or nearly round. The diseased area initially develops water-soaked transparent spots, 5-10 mm in diameter, which are slightly sunken. As they enlarge, they turn black and have concentric rings. Small black dots are densely distributed on them. When the humidity is high, the lesions secrete a light red sticky substance, which eventually leads to fruit rot or drop, directly affecting the yield and quality of tomatoes.

[0005] Currently, there is an urgent need in the market for a tomato-specific fertilizer that not only has a significant growth-promoting effect on tomatoes, but also has a significant control effect on tomato gray mold and tomato anthracnose. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a strain of *Bacillus belyssiensis*, its fermentation broth, and its applications. This invention isolates and screens a *Bacillus* strain, identifies its taxonomic position through morphological, physiological, and biochemical characteristics and gene sequence analysis, confirming it as *Bacillus belyssiensis*, and preserves it under the accession number CGMCC NO. 24988. This invention uses *Bacillus belyssiensis* to produce a fermentation broth, which can be further processed into microbial fertilizer or mixed with Root-Based Mineral-Based Black Gold Life Liquid to produce compound fertilizer. Applying this microbial fertilizer or compound fertilizer to tomato cultivation not only significantly promotes tomato growth but also demonstrates remarkable control over tomato gray mold and anthracnose, making it of great significance for tomato cultivation.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] This invention provides a strain of Bacillus velezensis, with accession number CGMCCNO.24988.

[0009] The present invention also provides a fermentation broth containing the aforementioned Bacillus belye.

[0010] The present invention also provides a microbial fertilizer made from the fermentation broth, wherein the effective viable count of Bacillus berberis is ≥1 billion / ml.

[0011] Preferably, the microbial fertilizer is diluted with water 90 to 300 times before application.

[0012] Preferably, the microbial fertilizer is diluted 100 times with water before application.

[0013] The present invention also provides a compound fertilizer containing the fermentation liquid, wherein the compound fertilizer further includes Root Essence - Mineral Source Black Gold Life Liquid;

[0014] Preferably, the mixing volume ratio of the root-source black gold life liquid to the fermentation liquid is 70%–30%:30%–70%.

[0015] Preferably, the mixing volume ratio of the root-source black gold life liquid to the fermentation liquid is 50%:50%.

[0016] The present invention also provides the application of the Bacillus vesiculosus, the fermentation liquid, the microbial fertilizer, or the compound fertilizer in promoting tomato growth.

[0017] The present invention also provides the application of the Bacillus vesiculosus, the fermentation broth, the microbial fertilizer, or the compound fertilizer in the prevention and control of tomato gray mold and / or tomato anthracnose.

[0018] Compared with the prior art, the present invention has the following technical effects:

[0019] (1) The compound fertilizer prepared by mixing the root-mineral-source black gold life liquid and fermentation liquid of the present invention showed better performance in all indicators of seedling growth than the single treatments—water, Bacillus berberis fermentation liquid alone, or Root-mineral-source black gold life liquid alone; at the same time, under the condition that the initial gray mold lesion area was similar, the lesion area growth in the later stage was significantly reduced after applying the compound fertilizer of the present invention. This indicates that the compound fertilizer of the present invention has a good promoting effect on the growth of tomato seedlings, and at the same time has a good inhibitory effect on the incidence of gray mold in tomato seedlings.

[0020] (2) The field control efficacy of the microbial fertilizer made from the fermentation liquid of this invention against tomato gray mold reached over 78%, and was superior to the control agent—50% carbendazim wettable powder. Furthermore, the microbial fertilizer diluted 100 times was superior to the microbial fertilizer diluted 300 times, with a maximum field control efficacy of 83.50% against tomato gray mold. This indicates that the microbial fertilizer made from the fermentation liquid of this invention can effectively control tomato gray mold, reduce the application of pesticides for tomato gray mold, and promote the rapid development of the tomato industry.

[0021] (3) The field control efficacy of the microbial fertilizer made from the fermentation liquid of this invention against tomato anthracnose reached over 82%, and was superior to the control agent—10% difenoconazole water-dispersible granules diluted 2000 times; at the same time, the microbial fertilizer diluted 100 times was superior to the microbial fertilizer diluted 300 times, with the highest field control efficacy against tomato anthracnose reaching 83.28%. This indicates that the microbial fertilizer made from the fermentation liquid of this invention can also effectively control tomato anthracnose, protect tomato fruits, reduce the application of pesticides for tomato anthracnose, and promote the rapid development of the tomato industry.

[0022] Preservation certificate statement:

[0023] Preservation institution: China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Cultures;

[0024] Accession number: CGMCCNO.24988;

[0025] Date of deposit: May 30, 2022;

[0026] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing;

[0027] Taxonomic name: Bacillus velezensis. Attached Figure Description

[0028] Figure 1 This is a morphological diagram of Bacillus velezensis in Example 1 of the present invention;

[0029] Figure 2 This is a phylogenetic tree of Bacillus velezensis in Example 1 of the present invention. Detailed Implementation

[0030] The following examples are for illustrative purposes only and are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the invention without departing from its spirit and essence are within the scope of the invention. The reagents, kits, and instruments used in the following examples are commercially available, and the methods used in the examples, unless otherwise specified, are consistent with conventional methods.

[0031] The technical solution of the present invention will be further described in detail below with reference to the embodiments.

[0032] Example 1: Isolation and Identification of Strains

[0033] 1. Strains Collection and Indoor Screening

[0034] Take 200 μL of liquid sample from the plate and frame filter press production line of Yuantaifeng Biotechnology Co., Ltd., spread it evenly on a PDA plate, pick Botrytis cinerea to inoculate in the center of the plate, incubate at 25℃ for 5 days, then pick colonies of different morphologies that inhibit the growth of pathogens, isolate and purify them, number them, and store them in a refrigerator at 4℃.

[0035] 2. Identification of the strain through physiological and biochemical tests

[0036] The morphology of the isolated strains was observed using Gram staining. Physiological and biochemical tests were conducted according to the *Handbook of Systematic Identification of Common Bacteria*, including sugar fermentation experiments, nitrite reduction, gelatin liquefaction, milk peptonization, starch hydrolysis, oxidase, catalase, salt tolerance, and salinity testing. Salt tolerance and salinity testing was performed by adding NaCl to NA medium to alter the salinity, using normal NA medium as a positive control. Micro-biochemical identification tubes were used to study the biochemical characteristics of the strains, including nitrate reduction, gelatin liquefaction, and sugar fermentation, following the methods described for *HiBacillus*. TM Instructions for use of the Identification Kit for Bacillus (Catalog No.: KB013, Distributor: Beijing Diyi Biotechnology Co., Ltd.)

[0037] The results showed that strain G + It is rod-shaped, non-motile, ferments lactose to produce acid and gas, can peptone milk, can liquefy gels, and can decompose starch. It is negative in the nitrate reduction test and positive in the oxidase test. Its optimal NaCl concentration is 4%, which is consistent with the physiological and biochemical characteristics of Bacillus belyssus.

[0038] 3. Molecular biological identification of the strain

[0039] The specific steps for extracting Bacillus belyss DNA were performed according to the TaKaRa MiniBEST DNA Fragment Purification Kit Ver.4.0 (Takara Bio Engineering (Dalian) Co., Ltd.). Using genomic DNA as a template, the strain's genome was amplified by PCR using universal 16S rDNA primers, specifically primers 27F and 1492R, with the specific sequences shown in SEQ ID No. 1–SEQ ID No. 2. The length of the PCR amplification product was detected by 1% agarose gel electrophoresis, and the amplified product was promptly sent to Rongxu Biotechnology (Shanghai) Co., Ltd. for gene sequencing. The sequencing results were assembled using Bioedit software, and the assembled results were compared for homology using blastnsuite software on the BLAST website (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). The type strain sequence was then searched for and downloaded from the GenBank database. After multiple sequence alignment using Bioedit, the relevant files were run using the phylogenetic analysis software MEGA* (Molecular Evolutionary Genetics Analysis) X, and a phylogenetic tree was constructed using the Neighbor-Joining algorithm. The assembled sequences are shown in SEQ ID No. 3, and the phylogenetic tree is shown below. Figure 2 As shown.

[0040] SEQ ID No. 1: 27F (5′-AGAGTTTGATCCTGGCTCAG-3′);

[0041] SEQ ID No. 2: 1492R (5′-GGTTACCTTGTTACGACTT-3′);

[0042] SEQ ID No. 3: 16S rDNA sequence

[0043]

[0044] Based on the comparison results, it can be inferred that the collected strain is Bacillus velezensis.

[0045] Example 2: Flat Plate Standoff Test

[0046] After culturing the gray mold and anthracnose pathogens used in Example 1 for one day, mycelial cakes were punched at the edge of the hyphae using a 5mm diameter punch and inoculated onto the center of a PDA plate. A cross pattern with a radius of 2.5cm was drawn on the back of the plate, centered on the pathogen. A *Bacillus belyssioides* plate placed in a 4℃ incubator was used to inoculate a suitable amount of bacterial solution onto the four points marked on the cross pattern. The inoculated PDA plate was then placed in a 25℃ constant temperature biochemical incubator for cultivation. A blank control was used, without inoculation with *Bacillus belyssioides*. After 3 days of cultivation, the presence of antagonistic zones (inhibition zones) on the plate was used to determine whether the *Bacillus belyssioides* strain had an antagonistic effect on anthracnose and tomato gray mold, and the diameter of the inhibition zone was recorded. Each treatment was replicated in triplicate.

[0047] Antibacterial rate formula:

[0048] Results of the confrontation experiment: After 3 days, the pathogens of gray mold and anthracnose in the blank control had completely covered the PDA plates, and inhibition zones had appeared on the plates used in the confrontation experiment. Specifically, the inhibition zone of *Bacillus belliferus* in this invention was 26.54 mm against the anthracnose pathogen and 28.69 mm against the gray mold pathogen. Based on the inhibition rate formula, the inhibition rate of *Bacillus belliferus* in this invention reached 65.32% against the anthracnose pathogen and 81.65% against the gray mold pathogen.

[0049] Example 3: Pot Experiment

[0050] Test reagents: Root-Mineral Black Gold Life Liquid (Yuantaifeng (Baotou) Biotechnology Co., Ltd., Y010103007); Bacillus vesiculosus fermentation broth (effective viable count ≥1 billion / ml).

[0051] Bacillus berberis fermentation medium: 3% soybean meal (80 mesh), 2% corn starch, 1% sucrose, 2‰ sodium chloride, 3‰ potassium dihydrogen phosphate, 3‰ dipotassium hydrogen phosphate, 1‰ magnesium sulfate.

[0052] Preparation of Bacillus vesiculosus fermentation broth: 10% liquid volume in Erlenmeyer flasks, 5% inoculum, fermentation at 37℃ and 200 rpm for 48 h. The fermentation broth was centrifuged at 8000 rpm for 10 min, and the supernatant was collected, diluted 100 times, and used for pot experiments.

[0053] Soil for the experiment: The soil and vermiculite were mixed evenly at a volume ratio of 2:1, and then sterilized by dry heat in an electric heating drying oven for 4 hours. After cooling, the mixture was used for the experimental treatment.

[0054] Select pink tomato seeds (Diana) of similar size and plumpness, disinfect them with a 3% sodium hypochlorite solution for 2 minutes, rinse three times with sterile water for 5 seconds each time, soak them in sterile water for 12 hours, and then cultivate seedlings. When the tomato seedlings have grown to "two leaves and one bud", select seedlings of similar growth for transplanting. One seedling per pot. During the experiment, other management and cultivation measures were carried out according to conventional tomato cultivation methods.

[0055] The experiment included eight treatments:

[0056] T1 is the water control;

[0057] T2 was administered only with Bacillus vesiculosus fermentation broth;

[0058] T3 only uses the Root Essence - Mineral Source Black Gold Life Liquid;

[0059] T4 is the foundation of life - 70% mineral-derived black gold life liquid and 30% Bacillus vesiculosus fermentation liquid;

[0060] T5 is the foundation - mineral-derived black gold life liquid 60%, Bacillus vesiculosus fermentation liquid 40%;

[0061] T6 is the foundation of life - mineral-derived black gold life liquid 50%, Bacillus vesiculosus fermentation liquid 50%;

[0062] T7 is the foundation of life - mineral-derived black gold life liquid 40%, Bacillus vesiculosus fermentation liquid 60%;

[0063] T8 is the foundation – 30% mineral-derived black gold life liquid and 70% Bacillus vesiculosus fermentation liquid.

[0064] All percentages mentioned above are mixed volume ratios.

[0065] Experimental Procedure: After transplanting the tomato seedlings, continue cultivating them for 3 weeks. Select 5-8 tomato leaves from each plant, treat the wounds, and spray the surface of the tomato leaves with the suspension of the gray mold pathogen. Three days after inoculation, 100ml of the liquid from each treatment was added to the water and used to irrigate the roots of the tomato seedlings. Irrigation was carried out once every 7 days, and after 5 consecutive irrigations, experimental data were collected.

[0066] Table 1. Effects of each treatment on tomato seedling growth and disease incidence.

[0067]

[0068] Pot experiments showed that the treatment using both RootRoot-Mineral Source Black Gold Life Liquid and Bacillus venetum fermentation liquid was superior to single treatments—water, Bacillus venetum fermentation liquid alone, or RootRoot-Mineral Source Black Gold Life Liquid alone—in all indicators of seedling growth. Furthermore, when the initial gray mold lesion area was similar, the application of the mixture of RootRoot-Mineral Source Black Gold Life Liquid and Bacillus venetum fermentation liquid significantly reduced the subsequent lesion area growth. This indicates that the mixture of RootRoot-Mineral Source Black Gold Life Liquid and Bacillus venetum fermentation liquid has a good promoting effect on the growth of tomato seedlings and also has a good inhibitory effect on the incidence of gray mold in tomato seedlings.

[0069] Example 4 Field efficacy trial

[0070] A. Field control efficacy trial for tomato gray mold

[0071] The test crop was greenhouse tomato, specifically the pink tomato variety (Diana).

[0072] Test reagents: Bacillus vesiculosus fermentation broth (effective viable count ≥ 1 billion / ml); 50% iprodione wettable powder (Zibo Meishou Agricultural Technology Co., Ltd.)

[0073] Experimental treatment:

[0074] T1: Water control;

[0075] T2: 100-fold dilution of Bacillus vesiculosus fermentation broth;

[0076] T3: 300-fold dilution of Bacillus vesiculosus fermentation broth;

[0077] T4: 50% styrene wettable powder (Zibo Meishou Agricultural Technology Co., Ltd.) diluted 500 times.

[0078] Small-plot experiment: A single-factor randomized block design was used to divide the greenhouse into small plots of 20m each. 2 Each treatment was repeated 3 times. For the specific block design, please refer to Table 2.

[0079] Table 2 Randomized block design of field control efficacy against tomato gray mold

[0080] Bin 2 T1 T4 T2 Bin 3 T4 T3 T4 Bin 4 T2 T1 T1

[0081] Greenhouses that had experienced tomato gray mold in the past three years were selected. Treatment began after flowering, with a 10-day interval between applications, for a total of three applications. During the trial, tomatoes were managed according to normal greenhouse practices. Any phytotoxic effects on tomato growth were observed during the treatment period.

[0082] Two disease surveys were conducted in this experiment: one before application of the pesticide to assess the baseline disease incidence, and another 10 days after the last application to assess the control efficacy. A five-point sampling method was used for sampling in each plot, with three plants randomly selected from each point. The disease index of all leaves was investigated, and the control efficacy was calculated. The results are shown in Table 3.

[0083] Grading standards for tomato gray mold (based on leaf):

[0084] Grade 0: No lesions; Grade 1: 1-3 lesions on a single leaf; Grade 3: 4-6 lesions on a single leaf; Grade 5: 7-10 lesions on a single leaf; Grade 7: 11-20 lesions on a single leaf, some of which are densely clustered; Grade 9: Dense lesions on a single leaf covering more than one-quarter of the leaf area.

[0085] Methods for calculating the effectiveness of prevention

[0086] Disease index = [(∑(number of diseased leaves at each level) × relative level value) / (total number of leaves surveyed × 9)] × 100

[0087] Prevention and control efficacy (%) = (1 - CK1 × T1 / (CK2 × T0)) × 100

[0088] In the formula: CK1 and CK2 are the disease indices before and after drug application in the blank control area, respectively; T0 and T1 are the disease indices before and after drug application in the drug-treated area, respectively.

[0089] Table 3. Field control efficacy of Bacillus vesiculosus fermentation broth against tomato gray mold.

[0090]

[0091] The results showed that the field control efficacy of the *Bacillus vesiculosus* fermentation broth from this invention against tomato gray mold reached over 78%, and was superior to the control agent—50% iprodione wettable powder. Furthermore, the 100-fold diluted broth was more effective than the 300-fold diluted broth, with the highest field control efficacy reaching 83.50%. This indicates that the *Bacillus vesiculosus* fermentation broth from this invention can effectively control tomato gray mold, reduce pesticide application, and promote the rapid development of the tomato industry.

[0092] B. Field control efficacy trial of tomato anthracnose

[0093] The test crop was greenhouse tomato, specifically the pink tomato variety (Diana).

[0094] Test reagents: Bacillus vesiculosus fermentation broth (effective viable count ≥ 1 billion / ml); Test reagents: 10% difenoconazole water-dispersible granules (Qingdao Dongsheng Pharmaceutical Co., Ltd., Shandong Province).

[0095] Experimental treatment:

[0096] T1: Water control;

[0097] T2: 100-fold dilution of Bacillus vesiculosus fermentation broth;

[0098] T3: 300-fold dilution of Bacillus vesiculosus fermentation broth;

[0099] T4: 10% difenoconazole water-dispersible granules diluted 2000 times.

[0100] Plot Experiment: A single-factor randomized block design was used to divide the experimental greenhouse into plots, each plot being 15m². 2 Each treatment was repeated 4 times. For the specific block design, please refer to Table 4.

[0101] Table 4 Randomized block design for field control efficacy against tomato anthracnose

[0102]

[0103]

[0104] The experiment was conducted in greenhouses that had experienced tomato anthracnose outbreaks in the past two years. The pesticide was applied starting at the green fruit stage of the tomatoes, with 10-day intervals between applications, for a total of three sprays. During the experiment, the tomatoes were managed according to normal greenhouse practices. Any phytotoxic effects on tomato growth were observed during the pesticide application period.

[0105] This experiment included two disease surveys: one before application of pesticides to assess the baseline disease incidence, and another 10 days after the last application to assess the control efficacy. Each plot used a 5-point sampling method, randomly selecting 3 plants at each point to investigate the disease index of all tomato fruits and calculate the control efficacy.

[0106] Grading standards for tomato anthracnose (based on the number of fruits):

[0107] Grade 0: No lesions; Grade 1: Diseased petals or stigma; Grade 3: Sepals rot or stigma disease spreading to the navel; Grade 5: Water-soaked, chlorotic spots on the fruit surface; Grade 7: Chlorotic spots on the fruit surface, some densely clustered; Grade 9: Chlorotic spots on the fruit surface covering one-quarter of the fruit's surface area.

[0108] Methods for calculating the effectiveness of prevention

[0109] Disease index = [(∑(number of diseased fruits at each level) × relative level value) / (total number of fruits surveyed × 9)] × 100

[0110] Prevention and control efficacy (%) = (1 - CK1 × T1 / (CK2 × T0)) × 100

[0111] In the formula: CK1 and CK2 are the disease indices before and after drug application in the blank control area, respectively; T0 and T1 are the disease indices before and after drug application in the drug-treated area, respectively.

[0112] Table 5. Field control efficacy of Bacillus vesiculosus fermentation broth against tomato anthracnose.

[0113]

[0114] The results showed that the Bacillus venetianus fermentation broth of this invention achieved a field control efficacy of over 82% against tomato anthracnose, which was superior to the control agent—a 2000-fold dilution of 10% difenoconazole water-dispersible granules. Furthermore, the 100-fold dilution of the broth was more effective than the 300-fold dilution, with the highest field control efficacy reaching 83.28%. This indicates that the Bacillus venetianus fermentation broth of this invention can effectively control tomato anthracnose, protecting tomato fruits and reducing the need for pesticide application, thus promoting the rapid development of the tomato industry.

[0115] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of a strain of Bacillus velezensis in promoting tomato growth, characterized in that, The preservation number of the Bacillus vesicularis is CGMCC NO.24988; the application specifically includes the application of the Bacillus vesicularis, fermentation broth containing the Bacillus vesicularis, microbial fertilizer made from the fermentation broth, or compound fertilizer containing the fermentation broth in promoting tomato growth.

2. The application according to claim 1, characterized in that, The effective viable count of Bacillus vesiculosus in the microbial fertilizer is ≥1 billion / ml.

3. The application according to claim 2, characterized in that, The microbial fertilizer should be diluted with water 90 to 300 times before application.

4. The application according to claim 3, characterized in that, The microbial fertilizer is diluted 100 times with water before application.

5. The application according to claim 1, characterized in that, The volume ratio of the fermentation liquid in the compound fertilizer is 30% to 70%.

6. The application according to claim 5, characterized in that, The volume ratio of the fermentation liquid in the compound fertilizer is 50%.

7. The application of Bacillus vesiculosus, fermentation broth, microbial fertilizer or compound fertilizer as described in claim 1 in the prevention and control of tomato gray mold and / or tomato anthracnose.

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