A strain of Bacillus belyssus 2020111906-1 GUCCTB302, inoculant and its application
By using the bacterial agent prepared from Bacillus belysus 2020111906-1GUCCTB302, the problem of biological control of yellow water disease in Dictyophora rubrum was solved, achieving efficient, environmentally friendly, and stable control of a variety of plant diseases, which is suitable for industrial application.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-02
- Publication Date
- 2026-04-07
AI Technical Summary
Existing technologies lack efficient, environmentally friendly, and stable biological control methods for the prevention and control of yellow water disease in red-topped bamboo fungus. Chemical control poses environmental pollution risks, physical control is difficult to eradicate infected diseases, and biological control technology resources are scarce and unstable, making it difficult to meet the needs of industrialization.
Bacillus berberis 2020111906-1GUCCTB302 was used as a biocontrol strain to prepare an inoculum for the control of yellow water disease in Dictyophora indica var. rubrum. The inoculum was also extended to other plant diseases, including tobacco black shank and maize leaf spot.
It has achieved effective control of yellow water disease in red-topped bamboo fungus, and its application has been expanded to a variety of plant diseases. It has environmentally friendly, safe and stable biological control effects, and is suitable for industrial promotion.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of microorganisms, in particular to a bacillus velezensis 2020111906-1GUCCTB302, a microbial inoculant and an application thereof. BACKGROUND
[0002] Dictyophora rubrocyannis is a mesophilic saprophytic fungus with high economic and nutritional value. Its cultivation industry has attracted much attention due to its broad market prospects. However, during the cultivation of Dictyophora rubrocyannis, yellow water disease, a serious fungal disease, has a significant impact on yield and quality, causing economic losses to the industry. The pathogen of yellow water disease is Saccharomycopsis phalluae sp. nov., which has a complex infection mechanism and is difficult to control.
[0003] Currently, the prevention and control methods for yellow water disease of Dictyophora rubrocyannis mainly include chemical control and physical control. Chemical control uses fungicides and other chemical agents to inhibit the growth and reproduction of pathogenic fungi. Although it can achieve certain results in the short term, long-term use can lead to environmental pollution, increased drug resistance, and residue problems, which do not meet the development requirements of green agriculture. Physical control mainly relies on improving the cultivation environment, controlling temperature and humidity, etc. Although it can reduce the occurrence of some diseases, it is difficult to fundamentally treat infected fungi and has limited effect in practical application.
[0004] In recent years, biological control, as a new disease control approach, has received widespread attention due to its environmental, safe, and sustainable characteristics. Biological control uses the antagonistic effect between microorganisms and their metabolites to inhibit the infection of pathogenic fungi and stimulate the immune mechanisms of plants or fungi, thereby reducing disease occurrence. However, current research on biological control of yellow water disease of Dictyophora rubrocyannis is still in its infancy, lacking systematic and efficient solutions. On the one hand, there is a lack of high-efficiency antagonistic bacterial strains for preventing and controlling yellow water disease; on the other hand, existing biological control techniques lack stability and adaptability in practical application, making it difficult to meet the needs of large-scale industrialization.
[0005] In summary, there is an urgent need to develop an efficient, environmentally friendly, and stable biological control method for yellow water disease of Dictyophora rubrocyannis to address the shortcomings of existing technologies and promote the healthy and sustainable development of the Dictyophora rubrocyannis industry. SUMMARY
[0006] The present application aims to provide a Bacillus velezensis 2020111906-1GUCCTB302, a microbial agent and an application thereof, so as to solve the problems existing in the prior art. The present application finds through experiments that the strain 2020111906-1GUCCTB302 can antagonize the pathogenic fungus Saccharomycopsis phalli of Phallus impudicus yellow water disease. The strain can be used as a biocontrol strain for preventing and treating Phallus impudicus yellow water disease and has application potential in the field of Phallus impudicus cultivation.
[0007] To achieve the above-mentioned purpose, the present application provides the following solutions.
[0008] The present application provides a Bacillus velezensis 2020111906-1GUCCTB302, which was preserved in the China General Microbiological Culture Collection Center on April 27, 2025, and the preservation number is CGMCC No.34374.
[0009] The present application also provides an application of the Bacillus velezensis 2020111906-1GUCCTB302 in preparing a biocontrol microbial agent.
[0010] Optionally, the biocontrol microbial agent is used for preventing and treating Phallus impudicus yellow water disease.
[0011] Optionally, the Phallus impudicus yellow water disease is caused by Saccharomycopsis phalli.
[0012] Optionally, the biocontrol microbial agent is also used for preventing and treating tobacco black foot disease, corn large spot disease, pepper anthracnose disease, radix pseudostellariae root rot disease, rice rice blast disease, rice rice smut disease, black skin chicken gong green mold disease, hairy ear fungus cobweb disease, black fungus cobweb disease, pilose antler fungus cobweb disease, morel white hair disease and pleurotus brown spot disease.
[0013] The present application also provides a product for preventing and treating Phallus impudicus yellow water disease, and the effective component comprises the Bacillus velezensis 2020111906-1GUCCTB302.
[0014] Optionally, the product comprises a microbial agent.
[0015] The present application discloses the following technical effects:
[0016] The present application finds a Bacillus velezensis 2020111906-1GUCCTB302 from the root system soil of healthy K. aethalica fruiting bodies. Experiments show that the strain can antagonize the pathogenic fungus of the red to bamboo green water disease, i.e. the ghost pen complex membrane yeast. The strain can be used as a biocontrol strain for preventing and treating the red to bamboo green water disease, and has application potential in the red to bamboo cultivation field. BRIEF DESCRIPTION OF DRAWINGS
[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.
[0018] Figure 1 Fig. 1 is the morphological characteristics of the strain GUCCTB302; wherein, A is the growth state of the strain GUCCTB302 on LB medium; B is the colony morphology of GUCCTB302; C is the result of gram staining; D is the bacterial body under scanning electron microscope;
[0019] Figure 2 Fig. 2 is the ML phylogenetic tree of the strain GUCCTB302 based on 16S rDNA and gyrA sequence;
[0020] Figure 3 Fig. 3 is the antibacterial spectrum experiment of the strain GUCCTB302; SGB9 is the root rot pathogen of Radix Pseudoginseng, YCYM is the pathogen of tobacco black foot disease, YMDB is the pathogen of corn large spot disease, LJTJ is the pathogen of pepper anthracnose disease, HS2 is the pathogen of rice bunt disease, 629-19 is the pathogen of rice blast disease, GUCCTB620 is the pathogen of velvet disease of Cornus rubra mushroom, GUCCTB621 is the pathogen of velvet disease of Auricularia auricula, GUCCTB623 is the pathogen of velvet disease of Auricularia polytricha, GUCCTB386 is the pathogen of white hair disease of Morchella esculenta, GUCCTB619 is the pathogen of green mold disease of K. aethalica, GUCCTB176 is the pathogen of red to bamboo green water disease, and GUCCTB35 is the pathogen of brown spot disease of Pleurotus ostreatus. DETAILED DESCRIPTION
[0021] The various exemplary embodiments of the present application will now be described in detail, which should not be considered as limiting the present application, but should be understood as a more detailed description of some aspects, characteristics and embodiments of the present application.
[0022] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.
[0023] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.
[0024] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.
[0025] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.
[0026] The tested strains of this invention are: *GUCCTB619*, *GUCCTB176*, *GUCCTB622*, *GUCCTB621*, *GUCCTB620*, *GUCCTB386*, *GUCCTB35*, *SGB9*, *YCYM*, *YMDB*, and *LJTJ*. All strains were provided by the Plant Protection and Culture Collection Center of Guizhou University. *HS2*, *629-19*, were provided by the Agricultural Product Quality and Safety Laboratory of Guizhou University. The strain numbers and specific information are shown in Table 1.
[0027] Table 1. Strain IDs and Specific Information
[0028]
[0029]
[0030] Example 1: Screening and Identification of Strains
[0031] 1. Isolation of bacteria
[0032] Soil samples were collected from the roots of healthy fruiting bodies of black-skinned matsutake mushrooms at the Hongtuo bamboo fungus cultivation base in Bijie City, Guizhou Province. Microorganisms in the collected soil samples were isolated using the dilution spread method. The specific steps are as follows:
[0033] Weigh 10g of soil sample and place it in 90mL of sterile water. Shake at 25℃ for 10 minutes, then dilute to 10000g. -2 10 -3 10 -4 10 -5 Diluted the culture medium by 100 μL and spread it onto PDA medium in triplicate for each concentration. Incubate at 25°C. After a period of time, different colonies will appear. Select individual colonies and transfer them to a new medium for further purification. For fungal colonies, select only the edge hyphae of a single colony and perform multiple purifications until a homogeneous pure culture is obtained. For bacteria, select single colonies and streak them in a "Z" pattern until a single colony is obtained. Store the obtained pure cultures at 4°C. A total of 51 isolates were obtained.
[0034] 2. Screening of strains
[0035] (1) Initial screening of antagonistic bacteria:
[0036] The isolated pure culture was subjected to an antagonistic experiment with the pathogen causing yellow water disease of *Dictyophora indica*. The specific procedure was as follows:
[0037] ① The isolate is bacteria. The activated pathogen is inoculated in the center of PDA medium. Single colonies are picked and streaked on both sides 2 cm away from the pathogen block. No inoculation is used as a control.
[0038] ② The isolate was a fungus. The fungal isolate was inoculated 1.5 cm from the edge of the PDA medium culture dish, and the pathogen of yellow water disease of *Dictyophora indica* was inoculated 1.5 cm from the edge of the other end of the culture dish. The PDA medium inoculation was used as a control.
[0039] All of the above were incubated at 25℃, and the isolates were observed daily to see if they inhibited the growth of pathogens.
[0040] (2) Secondary screening of antagonistic bacteria:
[0041] Based on the initial screening results of antagonistic bacteria, a secondary screening of antagonistic bacteria was conducted. The plate confrontation method was used to screen for antagonistic bacteria. If the antagonistic bacteria were bacteria, the pathogen of *Dictyophora indica* yellow water disease was inoculated in the center of a PDA plate. Two μL of overnight-cultured bacterial isolate was spot-inoculated at three points 2.5 cm from the inoculated pathogen block. The plates were incubated at 25°C. Each strain represented one treatment, with an equal volume of LB as a control. Each treatment was repeated in triplicate. If the antagonistic bacteria were fungi, a 5 mm pathogenic fungal disc was inoculated 1.5 cm from the edge of the PDA medium plate, and the antagonistic fungus was inoculated 1.5 cm from the other edge. PDA medium inoculation served as a control. The experiment was repeated in triplicate. All confrontation experiments were incubated at a constant temperature of 25°C. Observations were made daily, and the colony diameter was measured using the cross-cross method to calculate the inhibition rate.
[0042] Inhibition rate = (Coronary diameter of control group - Colony diameter of treatment group) / Colony diameter of control group × 100%.
[0043] Finally, an antagonistic bacterium that inhibits the pathogen of yellow water disease in *Dictyophora indicum* was screened out, and it was designated as strain 2020111906-1GUCCTB302 (hereinafter referred to as strain GUCCTB302).
[0044] 3. Identification of strains
[0045] (1) Morphological and physiological biochemical identification
[0046] The antagonistic bacteria were streaked onto LB agar and cultured for 48 hours. Colony morphology was observed, including colony color, shape, and edge characteristics. Cell morphology was also examined using a HITACHI scanning electron microscope.
[0047] The physiological and biochemical characteristics of the antagonistic strains were determined with reference to Bergey's Manual of Bacterial Identification and the Manual of Systematic Identification of Common Bacteria.
[0048] The results are as follows Figure 1 As shown, after culturing strain GUCCTB302 on LB plates for 48 hours, the bacterial colony was pale yellow with irregular edges and Gram staining was positive. Electron microscopy showed that the GUCCTB302 cells were initially rod-shaped with blunt ends and a smooth surface. As the growth time increased, the cell surface became wrinkled, accompanied by the death of a small number of cells.
[0049] (2) Molecular biological identification
[0050] DNA from antagonistic bacteria was extracted using the BioTeke Bacterial Genomic DNA Extraction Kit DP2001 (Beijing BioTeke Biotechnology Co., Ltd.). Using the obtained DNA as a template, PCR amplification of the 16S rDNA was performed using the universal 16S rDNA primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') / 1492R (5'-GGTTACCTTGTTACGACTT-3'). PCR amplification of the gyrase A subunit gene was performed using primers F (5'-CAGTCAGGAAATGCGTACGTCCTT-3') and R (5'-CAAGGTAATGCTCCAGGCATTGCT-3'). After verification by gel electrophoresis, the PCR products were sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. Sequence alignment was performed using NCBI, multiple sequence comparisons were performed using BioEdit, and a phylogenetic tree was constructed using the CIPRES SCIENCE GATEWAY website to determine the taxonomic position.
[0051] The strain GUCCTB302 was constructed using a multi-gene phylogenetic tree consisting of 16S rDNA and gryA genes, such as... Figure 2 As shown, this bacterium clustered with two other strains, Bacillus velezensis SRCM102744 and Bacillus velezensis SRCM102747, with a support rate of 100%. Based on morphological and physiological biochemical assays, strain 2020111906-1GUCCTB302 was identified as Bacillus velezensis.
[0052] This strain was deposited on April 27, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 34374.
[0053] Example 2: Determination of the antibacterial spectrum of the strain
[0054] The confrontation culture method was used to conduct plate confrontation on common pathogens of characteristic economic crops and edible fungi in Guizhou Province (Table 1). The pathogens were cultured at 25℃ for 4 days and then made into mycelial cakes using a 5 mm punch. The mycelial cakes were inoculated in the center of PDA plates, and 2 μL of antagonistic bacteria GUCCTB302 cultured overnight was spot-inoculated 2.5 cm away from the periphery of the mycelial cake. An equal volume of LB medium was spot-inoculated as a control. The plates were incubated at 25℃. Each pathogen was a treatment, and each treatment was replicated 3 times. The plates were incubated until the control reached two-thirds of the plate. The colony radius of the control and each treatment was measured, and the inhibition rate was calculated. Bacteria were subjected to plate confrontation. The pathogenic bacteria that had been cultured overnight were spread evenly on the surface of the culture medium using a sterile spreader, ensuring that the bacterial film was uniform and free of air bubbles. After the surface of the plate was slightly dry, 2 μL of the antagonistic bacteria GUCCTB302 that had been cultured overnight was inoculated in the center of the plate. An equal volume of LB medium was used as a control. The plates were incubated at 25°C. Each pathogenic bacterium was a treatment, and each treatment was repeated 3 times. The plates were incubated until the control bacteria were evenly distributed in the plate, and the diameter of the inhibition zone was measured.
[0055] Inhibition rate = (Coronary diameter of control group - Colony diameter of treatment group) / Colony diameter of control group × 100%.
[0056] The antibacterial results are shown in Table 2 and Figure 3 As shown, the results indicate that strain GUCCTB302 possesses broad-spectrum antibacterial activity, exhibiting inhibitory effects against various diseases including tobacco black shank (Phytophthora nicotianae), maize large leaf spot (Exserohilum turcicum), pepper anthracnose (Colletotrichum scovillei), root rot of *Fusarium oxysporum*, rice blast (Magnaporthe oryzae), rice false smut (Ustilaginoidea virens), green mold of *Trichoderma* sp., yellow water disease of *Saccharomycopsis sphalli*, spider web disease of *Auricularia auricula-judae* sp., spider web disease of *Auricularia auricula-judae* sp., spider web disease of *Auricularia auricula-judae* sp., white hair disease of *Morchella esculenta* sp., and brown spot disease of *Pseudomonas oyster mushroom* sp.
[0057] Table 2. Determination of the antibacterial spectrum of strain GUCCTB302
[0058] Pathogenic bacteria Disease caused Taxonomic status Inhibition rate / diameter of inhibition zone YCYM Tobacco black shank Phytophthora nicotianae 49.84％ YMDB Corn large spot Exserohilum turcicum 43.28％ LJTJ Pepper anthracnose Colletotrichum scovillei 56.90％ SGB9 Radish root rot Fusarium oxysporum 33.20％ 629-19 Rice blast Magnaporthe oryzae 34.12％ HS2 Rice smut Ustilaginoidea virens 31.34％ GUCCTB619 Green mold of black-bark chicken staghorn Trichoderma sp. 86.16％ GUCCTB176 Yellow water disease of red-toe bamboo staghorn Saccharomycopsis phalli 15.86 mm GUCCTB622 Cladobotryum disease of hairy wood ear Cladobotryum sp. 58.55％ GUCCTB621 Cladobotryum disease of black fungus Cladobotryum sp. 80.89％ GUCCTB620 Cladobotryum disease of pilose antler fungus Cladobotryum sp. 71.38％ GUCCTB386 White hair disease of morel Diploospora sp. 40.09％ GUCCTB35 Brown spot disease of Pleurotus ostreatus Pseudomonas sp. 10.86 mm
[0059] 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. A strain of Bacillus belye ( Bacillus velezensis The application of 2020111906-1GUCCTB302 in the preparation of biocontrol agents, characterized in that, The Bacillus belyssus 2020111906-1GUCCTB302 was deposited at the China General Microbiological Culture Collection Center on April 27, 2025, with accession number CGMCC No. 34374; The biocontrol agent is used to control yellow water disease in *Dictyophora indica*. The yellow water disease of *Dictyophora indica* is caused by *Phallus spp.* (… Saccharomycopsis phalli )cause.
2. The application according to claim 1, characterized in that, The biocontrol agent is also used to control tobacco black shank disease, corn leaf blight, pepper anthracnose, ginseng root rot, rice blast, rice false smut, black-skinned chicken mushroom green mold, hairy fungus spider web disease, black fungus spider web disease, deer antler mushroom spider web disease, morel white hair disease, and oyster mushroom brown spot disease.