Staphylococcus strain and its use in the preparation of a control agent for tobacco black shank and bacterial wilt

Abstract

The application discloses a staphylococcus and application of the staphylococcus in preparation of a control agent for tobacco black shank and tobacco bacterial wilt, relates to the technical field of microorganisms, and the staphylococcus 3C obtained by the application has obvious inhibiting effect on tobacco phytophthora and ralstonia solanacearum, and the fermentation liquor prepared from the staphylococcus 3C has obvious prevention and treatment effect on tobacco black shank and tobacco bacterial wilt.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a strain of Staphylococcus and its application in the preparation of control agents for tobacco black shank and bacterial wilt. Background Technology

[0002] Tobacco bacterial wilt is a devastating soil-borne disease worldwide, caused by Rhesperidone (a member of the Solanaceae family). Ralstonia solanacearum This bacterial disease, caused by [unspecified pathogen], is prevalent in tobacco-growing areas south of the Yangtze River in my country. It is characterized by rapid onset, rapid spread, and difficulty in control. Severely affected fields can suffer yield losses exceeding 80%, and the incidence rate in continuously cropped tobacco fields can reach over 60%. The disease progresses rapidly, often causing devastating losses in a short period. Typical symptoms include sudden wilting of plants, browning of vascular bundles, and the exudation of milky white bacterial ooze upon cross-section. The utilization of varietal resistance remains limited by several factors. First, the breeding cycle for resistant varieties is long, and the resistance level is not high. Second, the irrational distribution of resistant varieties and large-scale monoculture often lead to the rapid loss of varietal resistance, significantly shortening their effective lifespan. Cultivation management measures also face many difficulties in practical application. Traditional agricultural control methods have limited effectiveness in controlling soil-borne diseases, and the implementation of scientific crop rotation systems is challenging due to limitations in arable land and water resources. Chemical control also has significant shortcomings. Currently, the number of effective pesticides registered for controlling bacterial wilt is limited, and long-term use of single pesticides not only easily induces drug resistance in pathogens but also has a negative impact on the ecological environment. In contrast, numerous studies have shown that biological control methods for bacterial wilt control have significant application prospects. Therefore, in-depth development of biological control technologies will become an important direction for future research on bacterial wilt control. Summary of the Invention

[0003] To achieve the above objectives, the present invention provides the following technical solution:

[0004] A strain of Staphylococcus, specifically Staphylococcus pasteurellii ( Staphylococcus pasteuri Staphylococcus 3C is deposited at the China Center for Type Culture Collection (CCTCC), located at Luojia Mountain, Bayi Road, Wuchang District, Wuhan, Hubei Province, on November 10, 2025, with accession number CCTCC NO: M 20252495. In this paper, it is referred to as "Staphylococcus 3C".

[0005] Furthermore, the morphological characteristics of Staphylococcus 3C are: forming round, raised, smooth, moist, and opaque colonies on agar plates.

[0006] Furthermore, the physiological and biochemical characteristics of Staphylococcus 3C are as follows: Gram-positive cocci arranged in a grape-like pattern; non-motile and without flagella; non-spore-forming; facultatively anaerobic, with no special nutritional requirements, and capable of growing in 10% NaCl conditions; catalase-positive and oxidase-negative.

[0007] The present invention also provides the application of Staphylococcus 3C as described above in the preparation of control agents for tobacco black shank and bacterial wilt.

[0008] Furthermore, the causative agent of tobacco black shank is Phytophthora indicum (…). Phytophthora nicotianae ).

[0009] Furthermore, the causative agent of bacterial wilt is Ralstonia solanacearum (Ralstonia solanacearum). Ralstonia solanacearum ).

[0010] The present invention has the following beneficial effects:

[0011] The Staphylococcus 3C provided by this invention has a significant inhibitory effect on Phytophthora indicum and Ralstonia solanacearum, and the fermentation broth prepared by Staphylococcus 3C has a significant preventive and control effect on tobacco black shank disease and tobacco bacterial wilt. Attached Figure Description

[0012] Figure 1 This is a control diagram of Ralstonia solanacearum without the addition of Staphylococcus 3C in this invention;

[0013] Figure 2 This is a diagram illustrating the antagonistic effect of Staphylococcus 3C on Ralstonia solanacearum in this invention.

[0014] Figure 3 This is a control diagram of Phytophthora intoxin without the addition of Staphylococcus 3C in this invention;

[0015] Figure 4 This is a diagram illustrating the antagonistic effect of Staphylococcus 3C on Phytophthora intoxin in this invention.

[0016] Preservation status description:

[0017] Staphylococcus 3C is deposited at the China Center for Type Culture Collection (CCTCC), located at Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province. The deposit date is November 10, 2025, and the accession number is CCTCC NO: M 20252495. Detailed Implementation

[0018] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. 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 skilled in the art without creative effort are within the scope of protection of the present invention.

[0019] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments.

[0020] Example 1: Isolation and Identification of Strains:

[0021] I. Soil Sample Processing:

[0022] Take 5-10g of soil sample (collected from the soil surrounding diseased Yunyan 87 tobacco plants in Linjia'ya, Pantang Town, Taoyuan County, Changde, Hunan Province) and place it in a sterile 50ml centrifuge tube. Add 30-40ml of sterile water to the centrifuge tube, vortex for 2-3 minutes, let stand for 30-60 minutes, vortex again for 2-3 minutes, and centrifuge at 1000rpm for 1 minute to obtain a soil suspension. Mix 5ml of the prepared soil suspension with 5ml of 50% glycerol (volume percentage concentration), divide into five portions, and store each portion (2ml) at -80℃. Dilute the remaining soil suspension (not mixed with glycerol) to a concentration of 10. -3 10 -4 10 -5 Take 100 μL of each concentration gradient sample and spread it on a plate. Spread two plates for each concentration gradient sample.

[0023] II. Initial screening of antagonistic bacteria:

[0024] The pathogen of black shank disease, namely Phytophthora indicum ( Phytophthora nicotianae The bacteria obtained from the biocontrol sample preparation (i.e., the bacteria obtained from the soil sample preparation) were spotted around the plate and incubated at 30°C for 5–7 days. Using a sterile inoculation loop, bacteria with antagonistic effects were scraped from the plate (forming an inhibition zone larger than 2 cm in diameter around the biocontrol bacteria, with no pathogenic bacteria growing within the inhibition zone) and streaked for purification. One to three types of bacteria were purified on each plate.

[0025] III. Secondary screening of antagonistic bacteria:

[0026] Using a sterile inoculation loop, scrape colonies with antagonistic effects from a plate (i.e., a plate used to purify 1-3 bacteria during the initial screening of antagonistic bacteria) into 10 μl of sterile water and mix well. Use the obtained antagonistic bacterial solution as a template and perform PCR amplification using universal bacterial 16S primers (universal primers: 27F: 5'-AGAGTTTGATCCTGGCTCAG-3', 1492R: 5'-GGTTACCTTGTTACGACTT-3'). The PCR amplification reaction conditions are as follows: pre-denaturation at 94℃ for 5 min; denaturation at 94℃ for 30 s; annealing at 55℃ for 30 s; extension at 72℃ for 90 s; total number of cycles: 35; termination at 72℃ for 10 min; and storage at 4℃.

[0027] The PCR reaction system is as follows:

[0028] System components reaction volume / μL Tag-Mix 12.5 27F 0.5 1492R 0.5 DNA template 1 <![CDATA[ddH2O]]> 10.5

[0029] PCR amplification products were sent to Shanghai Bioengineering Co., Ltd. for sequencing, yielding 16S rDNA sequences. BLAST homology alignment was performed using NCBI, and the obtained sequences were compared with databases. The results showed the highest similarity (99%) to *Staphylococcus pasteurellii*. Based on this identification result and combined with physiological and biochemical characteristics, 3C was confirmed as *Staphylococcus pasteurellii*. *Staphylococcus pasteurellii* 3C has been deposited at the China Center for Type Culture Collection (CCTCC), located at Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province, on November 10, 2025, with accession number CCTCC NO: M 20252495.

[0030] IV. Preservation:

[0031] Add 5 ml of activated biocontrol bacterial culture (containing LB medium) to a 10 ml sterilized centrifuge tube, then add 5 ml of 50% glycerol (volume percentage concentration), mix well, divide into 5 portions, each 2 ml, and store at -80℃.

[0032] V. Morphological characteristics of Staphylococcus aureus 3C:

[0033] The colonies form circular raised areas on agar plates, with neat edges, smooth surfaces, and are moist and opaque.

[0034] VI. Physiological and biochemical characteristics of Staphylococcus aureus 3C:

[0035] Gram-positive cocci, arranged in a grape-like pattern; non-flagellated and non-motile; non-spore-forming; facultatively anaerobic, with no special nutritional requirements, and can grow in 10% NaCl; catalase-positive and oxidase-negative.

[0036] The gene sequence of Staphylococcus 3C is shown in SEQ ID NO:1, specifically:

[0037] GGGCTGGGCGGGTGCCTATACTGCAAGTCGAGCGAACAGATAAGGAGCTTGCTCCTTTGACGTTAGCGGCGGACGGGTGAGTAACACGTGGATAACCTACCTATAAGACTGGGATAACTTCGGGAAACCGGAGCTAATACCGGATAACATATTGAACCGCATGGTTCAATAGTGAAAGGCGGCTTTGCTGTCACTTATAGATGGATCCGCGCCGTATTAGCTAGTTGGTAAGGTAACGGCTTACCAAGGCAACGATACGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGGCGAAAGCCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTCTTCGGATCGTAAAACTCTGTTATCAGGGAAGAACAAATGTGTAAGTAACTGTGCACATCTTGACGGTACCTGATCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGCGCGTAGGCGGTTTTTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGAAAACTTGAGTGCAGAAGAGGAAAGTGGAATTCCATGTGTAGCGGTGAAATGCGCAGAGATATGGAGGAACACCAGTGGCGAAGGCGACTTTCTGGTCTGTAACTGACGCTGATGTGCGAAAGCGTGGGGATCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGGAGTACGACCGCAAGGGTTGAAACTCAAAGGAATTGACGGGG。

[0038] Example 2: I. Preparation of fermentation broth:

[0039] Remove the 3C bacterial strain from the -80℃ freezer. Use a pipette to extract 10 μL of the bacterial suspension and add it to 1 ml of autoclaved LB medium. Transfer both to a 2 ml centrifuge tube and incubate at 28℃ for 8–10 hours. After activation, purify the bacterial suspension on solid LB medium (LB medium formula (per liter): yeast extract 5 g; tryptone 10 g; sodium chloride 10 g; agar powder 15 g) using the streak plate method for 5–7 days to obtain single colonies of the 3C strain. Pick single colonies and transfer them to LB medium, incubate at 28℃ for 8–10 hours to prepare the required volume of bacterial suspension (the volume depends on the number of potted plants; 20 ml of fermentation broth is needed for root irrigation per pot). Adjust the bacterial suspension concentration to 1.0 × 10⁻⁶. 8 The concentration of cfu / mL (calculated from an OD value of 600) was increased. After activation, the mixture was centrifuged at 6000 rpm at room temperature for 5 minutes. The supernatant was then collected as the fermentation broth.

[0040] II. Preparation of bacterial suspension of the pathogen of tobacco bacterial wilt:

[0041] Ralstonia solanacearum preserved in sterile water ( Ralstonia solanacearum (EF Smith) Yabuuhi et al. strain was streaked on TTC plates and cultured for 48 h. Single colonies of Ralstonia solanacearum (Ralstonia solanacearum appears pink on TTC selective medium) were picked and transferred to NA medium. After culturing for 48 h, a bacterial suspension was prepared with sterile water and the bacterial concentration was adjusted to an OD600 value of about 0.3 for later use.

[0042] III. Indoor potted plant experiment on the control of tobacco bacterial wilt by Staphylococcus aureus 3C:

[0043] Fermentation broth root irrigation treatment: Take 20ml of fermentation broth and irrigate the roots of tobacco plants with uniform growth. Repeat the irrigation after 1 day, for a total of two irrigations. After the root irrigation, inoculate with pathogen (Ralstonia solanacearum) 24 hours later.

[0044] Inoculation method for the pathogen of tobacco bacterial wilt: Before inoculation, the roots of the tobacco seedlings were first injured by cutting off 1 / 3 of the fibrous roots with sterile scissors. Each plant was then irrigated with 20 mL of bacterial suspension. The inoculated tobacco plants were placed in a greenhouse under light and watered regularly. The disease status of the plants was observed and recorded. The disease status is shown in Table 1, where "mock" is the control group that did not receive root irrigation with the fermentation solution.

[0045] Table 1:

[0046] Incidence rate Disease index Inhibition rate Mock 80% 28.68 - 3C 20% 4.84 83.12%

[0047] in:

[0048] Disease index = ∑(Number of plants with a certain disease level × Disease level) / (Total number of plants surveyed × Highest disease level) × 100%;

[0049] Inhibition rate = (Control disease index - Treatment disease index) / Control disease index × 100%.

[0050] As shown in Table 1, compared with the control group (i.e., mock) cultured under the same conditions, the disease incidence of plants treated with the 3C fermentation broth through root irrigation was significantly reduced, demonstrating a clear control effect against tobacco bacterial wilt. Figure 1 and Figure 2 The inhibition zone diameter of Staphylococcus 3C is 2.35 cm, and it has a significant inhibitory effect on Ralstonia solanacearum.

[0051] Example 3: I. Preparation of bacterial suspension of the pathogen of tobacco black shank:

[0052] Preserved tobacco Phytophthora ( Phytophthora nicotianae Inoculate onto oat medium (1L oat medium: 30g oats (crushed and filtered), 18g agar, add water to bring the volume to 1L). Incubate at 25–28℃ for 5–7 days until the hyphae cover the plate and sporangia form. Add an appropriate amount of sterile water to the petri dish, scrape off a block of hyphae with a sterile glass slide, transfer it to a sterile Erlenmeyer flask, and gently shake to wash the hyphae. Filter with sterile gauze or filter paper to remove hyphae fragments and obtain a pure spore suspension. Take 10μl of the spore suspension and count the number of zoospores under a microscope using a hemocytometer. Adjust the concentration to 1×10⁸ spores / mL with sterile water.

[0053] II. Indoor potted experiment on the prevention and control of tobacco black shank disease by Staphylococcus aureus 3C:

[0054] Fermentation broth root irrigation treatment: Take 20ml of fermentation broth and irrigate the roots of tobacco plants with uniform growth. Repeat the irrigation after 1 day, for a total of two irrigations. Inoculate with pathogen (Phytophthora indica) 24 hours after the root irrigation.

[0055] Black shank pathogen inoculation: Before inoculation, the roots of the tobacco seedlings were first injured by using sterile scissors to cut off 1 / 3 of the fibrous roots. Each plant was then irrigated with 20 mL of the bacterial suspension. The inoculated tobacco plants were placed in a greenhouse under light, and the plants were watered regularly to observe and record the disease status. The disease status is shown in Table 2, where "mock" represents the control group that did not receive root irrigation with the fermentation solution.

[0056] Table 2:

[0057] Incidence rate Disease index Inhibition rate Mock 90% 25.58 - 3C 30% 5.41 78.85%

[0058] in:

[0059] Disease index = ∑(Number of plants with a certain disease level × Disease level) / (Total number of plants surveyed × Highest disease level) × 100%;

[0060] Inhibition rate = (Control disease index - Treatment disease index) / Control disease index × 100%.

[0061] As shown in Table 2, compared with the control group (i.e., mock) cultured under the same conditions, the disease incidence of plants treated with the 3C fermentation broth through root irrigation was significantly reduced, demonstrating a clear control effect against tobacco black shank disease. Figure 3 and Figure 4 The inhibition rate of Staphylococcus 3C was 84.57%, showing a significant inhibitory effect on Phytophthora indica. The inhibition rate was calculated using the following formula: Inhibition rate = (Control diameter - Treatment diameter) / Control diameter - Mycelial cake diameter.

[0062] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A strain of Staphylococcus, characterized in that, The Staphylococcus species specifically refers to Staphylococcus pasteurellum (Staphylococcus pasteurellum). Staphylococcus pasteuri )3C, deposited at the China Center for Type Culture Collection (CCTCC), on November 10, 2025, with accession number CCTCC NO: M 20252495.

2. The application of Staphylococcus aureus according to claim 1 in the preparation of control agents for tobacco black shank and bacterial wilt.

3. The application according to claim 2, characterized in that, The pathogen causing tobacco black shank is Phytophthora indicum.

4. The application according to claim 2, characterized in that, The pathogen causing bacterial wilt is Ralstonia solanacearum.

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