Pseudomonas and application thereof in preparation of prevention and control preparation for tobacco black shank and bacterial wilt

Abstract

The invention discloses a pseudomonas strain and application thereof in preparation of a prevention and control preparation for tobacco black shank and bacterial wilt, and belongs to the field of microorganism application. The strain is preserved in the China Center for Type Culture Collection (CCTCC), the preservation number is CCTCC NO: M 20252496, the address is Luojia Hill, Luojia Hill, eight road, Wuchang District, Wuhan City, Hubei Province, and the preservation date is November 10, 2025. The strain is sampled from soil around a tobacco diseased plant, and the screened and purified strain can play an obvious role in antagonizing and inhibiting pathogenic bacteria of black shank and bacterial wilt, and can effectively reduce the incidence rate of the black shank and the bacterial wilt of the tobacco plant when being put into a planting process for actual use.

Description

Technical Field

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

[0002] Both black shank and bacterial wilt are highly pathogenic and prevalent. The incidence rate in the field can usually reach 20%-30%, and under suitable conditions (high temperature and high humidity), the incidence rate in the affected area can be as high as 80% or more, even leading to the complete loss of the entire tobacco field.

[0003] Blackleg disease attacks the roots and stems, causing plants to wilt and die, directly reducing yield per unit area. Bacterial wilt damages the vascular bundles, disrupting water and nutrient transport, causing tobacco leaves to wither prematurely and drastically reducing yield. Furthermore, diseased tobacco leaves are of poor quality after curing, with poor color, low oil content, and weak elasticity, severely lowering their commercial grade and economic value.

[0004] Both pathogens can overwinter or oversummer in the soil and diseased plant debris, surviving for a long time (up to several years), making the soil in the diseased area a continuous source of infection and difficult to eradicate. The pathogens can also be spread through various means such as irrigation water, rainwater splash, agricultural operations (such as tilling and transplanting), contaminated fertilizers, and the transportation of diseased seedlings, making them extremely easy to spread in the field and to new areas.

[0005] Biological control is increasingly demonstrating numerous advantages in current plant disease management, especially the use of antagonistic bacteria. Compared with traditional chemical control, antagonistic bacteria control has the advantages of being residue-free and protecting the ecological environment. Antagonistic bacteria inhibit pathogens through multiple mechanisms rather than acting on a single target, making it difficult for pathogens to develop drug resistance. Furthermore, antagonistic bacteria are harmless to non-target organisms and do not disrupt the ecological balance. These two points stand in stark contrast to the drawbacks of chemical pesticides, such as accidentally killing natural enemies and polluting water bodies. Summary of the Invention

[0006] In view of the above-mentioned problems in the prior art, the purpose of this invention is to provide a strain of Pseudomonas and its application in the preparation of control agents for tobacco black shank and bacterial wilt.

[0007] To solve the above problems, the technical solution adopted by the present invention is as follows: A type of Pseudomonas, characterized in that it is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO:M 20252496, located at Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and deposited on November 10, 2025.

[0008] Furthermore, the nucleotide sequence of the Pseudomonas is shown in SEQ ID NO:1.

[0009] An antimicrobial agent comprising the aforementioned Pseudomonas.

[0010] Furthermore, the above-mentioned Pseudomonas and its agents are referenced in any of the following: (1) Used to prevent and treat tobacco black shank disease; (2) Used to prevent and control tobacco bacterial wilt.

[0011] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention obtained a Pseudomonas 3A4 strain through isolation, screening, and purification. This strain has an extremely good antagonistic effect on tobacco black shank pathogens and tobacco wilt fungus. Irrigating tobacco plants with a bacterial solution containing Pseudomonas 3A4 can effectively prevent and control tobacco black shank and wilt. Attached Figure Description

[0012] Figure 1 This is a diagram showing the antagonistic effect of Pseudomonas 3A4 on Ralstonia solanacearum. Figure 2 This is a diagram showing the antagonistic effect of Pseudomonas 3A4 against the pathogen of black shank. Figure 3 An indoor potted plant experiment using Pseudomonas 3A4 to prevent bacterial wilt; Figure 4 An indoor potted plant experiment using Pseudomonas 3A4 to prevent black shank disease. Detailed Implementation

[0013] The present invention will be further described below with reference to specific embodiments.

[0014] Soil sample preparation: Take 5-10g of soil sample (collected from soil surrounding diseased plants in the Hunan tobacco region), place it in a sterile 50ml centrifuge tube, add 30-40ml of sterile water, vortex for 2-3 minutes, let stand for 30-60 minutes, vortex again for 2-3 minutes, and centrifuge at 1000rpm for 1 minute. Mix the prepared soil suspension with 50% glycerol at a volume ratio of 1:1, divide into five portions, 2ml each, and store at -80℃. Dilute the prepared soil suspension to 10... -3 10 -4 10 -5 To isolate bacteria present in the soil, 100 μL of each sample from each concentration gradient was spread onto plates, two plates per group.

[0015] Initial screening of antagonistic bacteria: Inoculate pathogenic bacteria onto PDA and R2A agar plates, and spread the bacterial spots obtained from the plating around the edges. Incubate at 30°C for 5-7 days. Select bacteria exhibiting antagonistic effects from the plates and streak them for purification, purifying 1-3 strains per plate.

[0016] Secondary screening of antagonistic bacteria: Select colonies with antagonistic effects, add them to 10 μl of sterile water, mix well, amplify by PCR using universal bacterial 16S primers, and then send the samples for sequencing.

[0017] The nucleotide sequence of the bacterial strain is shown in SEQ ID NO.1. Physiological and biochemical experiments were also performed on the strain, and the results are shown in the table below.

[0018] The bacteria were identified as Pseudomonas. (Pseudomonas adaceae) The strain was named Pseudomonas 3A4.

[0019] Storage: Transfer the strain to a sterile 50ml Erlenmeyer flask, add 10ml (or 5-7ml to a 10ml centrifuge tube) of 1 / 2LB liquid culture medium and shake overnight (large shaker: 180rpm, 30℃; 1.5-2ml shaker: 900rpm, 30℃). Then mix with 50% glycerol at a 1:1 ratio, divide into 9 portions, each 1.5-2ml, and store at -80℃.

[0020] Add the activated Ralstonia solanacearum to NA medium (1L: 3.0g beef extract, 5.0g peptone, 2.5g glucose, 18.0g agar) at approximately 50℃, mix well, pour into petri dishes, and allow to solidify before use. Make wells in the agar medium and add 10ul of 10... 8 A concentration of Pseudomonas 3A4 was used. The culture dishes were incubated at 28°C, and the formation of inhibition zones was observed. The size of the inhibition zones was used to determine the effectiveness of the antibacterial agent. A blank control group without Pseudomonas 3A4 was also incubated at a constant temperature of 28°C. The effect of this control group on the experimental results was as follows: Figure 1 As shown in the figure, (a) is the blank control group and (b) is the experimental group inoculated with Pseudomonas 3A4.

[0021] The pathogen of tobacco black shank was activated by inoculating oat medium. On well-grown plates, 5mm diameter mycelial discs were taken from the edge of each colony. Pseudomonas 3A4 and the pathogen were symmetrically inoculated 2.5cm from the center of each PDA plate. The mycelial discs were inverted and incubated in the dark at 28℃. The growth of pathogens in the control plate was observed. After the entire plate was covered, the radius of the pathogen colonies was measured and the inhibition rate was calculated. This was repeated three times. A blank control group without Pseudomonas 3A4 was also incubated at 28℃. The effect of this control group on the experimental results was as follows: Figure 2 As shown in the figure, (a) is the blank control group and (b) is the experimental group inoculated with Pseudomonas 3A4.

[0022] Based on experimental data comparison, its inhibition rate against Ralstonia solanacearum reached 68.23%, and its inhibition rate against the pathogen of black shank reached 71.88%. The inhibition rate was calculated using the following formula: Inhibition rate = (Control diameter - Treatment diameter) / Control diameter - Mycelium cake diameter.

[0023] Then, an indoor pot experiment was conducted to test the effectiveness of the fungal strains in controlling tobacco bacterial wilt and tobacco black shank.

[0024] First, the fermentation broth of Pseudomonas 3A4 was prepared: Take out the 3A4 bacterial culture from the -80℃ freezer, use a pipette to aspirate 10ul of bacterial culture and add it to 1ml of LB medium that has been sterilized at high temperature. Then transfer it to a 10ml centrifuge tube and place it in a shaker at 28℃ for 8-10h to activate.

[0025] After activation, the bacterial suspension was purified and cultured in LA medium using the streak plate method for 5-7 days. Finally, a single colony was picked and transferred to LB medium to reactivate the required volume of bacterial suspension.

[0026] The bacterial suspension concentration was adjusted to 1.0 × 10⁸ CFU / mL. After activation, the suspension was centrifuged at 6000 rpm for 5 minutes. The supernatant was then used as the bacterial agent.

[0027] Root drenching treatment: Apply 20 ml of fermentation broth to the roots of tobacco plants with uniform growth, repeating the application after one day, for a total of two applications. Inoculate the plants with pathogens 24 hours after the initial application.

[0028] Inoculation with pathogens first requires the preparation of a pathogen suspension. The preparation method for a *Ralstonia solanacearum* bacterial suspension is as follows: The *Ralstonia solanacearum* strain preserved in sterile water was streaked on a TTC plate and cultured for 48 hours. Then, highly pathogenic single colonies were picked and cultured on NA medium for 48 hours. After that, a bacterial suspension was prepared with sterile water and the bacterial concentration was adjusted to an OD600 of about 0.3 for later use.

[0029] The inoculation method for Ralstonia solanacearum is as follows: Before inoculation, the roots of the tobacco seedlings should be injured by cutting off one-third of the fibrous roots with sterile scissors. Each plant should be watered with 20 mL of bacterial suspension. The inoculated tobacco plants should be placed in a greenhouse for light cultivation, and the plants should be watered regularly and the disease status of the plants should be observed and recorded.

[0030] Indoor pot experiment on the control of tobacco bacterial wilt by Pseudomonas 3A4: Figure 3 As shown in the figure, mock is the blank control group, and the other groups are experimental groups inoculated with Pseudomonas 3A4. The results are shown in Table 1: Incidence rate Disease index Inhibition rate Mock 100% 31.26 - <![CDATA[3A4]]> 20% 4.43 85.83% Table 1 The calculation methods for each data point in the table are as follows: Disease index = ∑(Number of plants at a certain disease level × Disease level) / (Total number of plants surveyed × Highest disease level) × 100% Inhibition rate = (Control disease index - Treatment disease index) / Control disease index × 100% The preparation method of the bacterial suspension of tobacco black shank pathogen is as follows: Inoculate the preserved inoculum onto oat medium. Incubate at 25-28°C for 5-7 days, until the mycelium covers the plate and forms sporangia.

[0031] 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 conical flask, and gently shake to clean the hyphae.

[0032] Filter the solution with sterile gauze or filter paper to remove mycelial fragments and obtain a pure spore suspension.

[0033] Take 10 μl of spore suspension and place it in a hemocytometer. Count the number of zoospores under a microscope. Adjust the concentration to 1 × 10⁸ spores / mL with sterile water.

[0034] The inoculation method for the pathogen of black shank is as follows: Before inoculation, the roots of the tobacco seedlings should be injured by using sterile scissors to cut off 1 / 3 of the fibrous roots. Each plant should be watered with 20mL of bacterial suspension. The inoculated tobacco plants should be placed in a greenhouse for light cultivation, and the plants should be watered regularly and the disease status of the plants should be observed and recorded.

[0035] Indoor pot experiment on the control of tobacco black shank by Pseudomonas 3A4. Figure 4 As shown in Table 2: Incidence rate Disease index Inhibition rate Mock 80% 35.74 - <![CDATA[3A4]]> 30% 9.14 74.43% Table 2 Based on the above experimental results, it can be concluded that *Pseudomonas 3A4* exhibits extremely good antagonistic effects against the pathogens of tobacco black shank and *Ralstonia solanacearum*. Irrigating tobacco plants with an agent containing *Pseudomonas 3A4* can effectively control tobacco black shank and bacterial wilt. Furthermore, in the experiment, the inhibition rate against bacterial wilt reached 85.83%, and the inhibition rate against black shank reached 74.43%. This strain shows broad application prospects and excellent application effects in the biological control of tobacco black shank and bacterial wilt.

Claims

1. A type of Pseudomonas, characterized in that, It is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO:M 20252496.

2. The *Pseudomonas* strain according to claim 1, characterized in that, The nucleotide sequence of the Pseudomonas is shown in SEQ ID NO:

1.

3. A microbial agent, characterized in that, Includes the Pseudomonas as described in claim 1.

4. The use of the *Pseudomonas* strain according to claim 1 or 2, or the inoculum according to claim 3, in any of the following: (1) Used to prevent and treat tobacco black shank disease; (2) Used to prevent and control tobacco bacterial wilt.

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