Biocontrol pseudomonas fluorescens FH and application thereof

Abstract

The invention belongs to the technical field of plant disease and insect pest control, and particularly relates to biocontrol pseudomonas fluorescens FH and application thereof, and the preservation number of the pseudomonas fluorescens FH is CCTCC NO: M 2025096. The pseudomonas fluorescens FH not only can effectively inhibit pyricularia grisea, but also can promote germination of the millet seeds, and the strain can promote germination of the millet seeds on the premise that normal growth of crops is not affected, so that the dual effects of resisting diseases and promoting growth are achieved. A new technical approach is provided for solving the problem of valley blast, and efficient, economical, safe and environment-friendly prevention and control are achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of plant disease and insect pest control, and particularly relates to a biocontrol Pseudomonas fluorescens FH strain and an application thereof. Background Art

[0002] Millet belongs to the genus Setaria of the Poaceae family. It is an annual herb with rich nutritional value and is widely cultivated as an important food crop. Magnaporthe oryzae The disease can occur at all stages of millet growth. The grain blast pathogen has numerous physiological races, which are prone to variation. Its pathogenicity to millet varieties is clearly differentiated, and millet varieties from different regions exhibit varying resistance to it. Existing control measures for grain blast primarily rely on chemical agents and the breeding of disease-resistant varieties. However, the extensive use of chemical pesticides can easily cause environmental pollution and pesticide residues, and can easily lead to the development of drug resistance in pathogens, hindering the sustainable development of agriculture. Breeding disease-resistant varieties is also costly and has a low success rate. Therefore, new control strategies are urgently needed to prevent and control grain blast.

[0003] Biocontrol agents use antagonistic interactions between microorganisms to inhibit the growth, development, and reproduction of pathogens, thereby achieving the purpose of disease prevention and control. In recent years, research progress in microbial ecology and plant-microbe interactions has shown that some specific rhizosphere microorganisms play a key role in regulating crop growth and development and promoting crop disease resistance. Bacillus , Flavobacterium Flavobacterium , Arthrobacter Arthrobacter , Escherichia coli Enterobacter It is a common rhizosphere growth-promoting bacteria genus. Bacillus cereus DW019 is effective against cherry tomatoes. Solanumlycopersicum var. cerasiforme It has excellent growth-promoting effect on lettuce and has a good inhibitory effect on a variety of soil-borne pathogens; Bacillus velez Bacillus velezensis GZA12 has a good inhibitory effect on the pathogen of konjac soft rot and has a growth-promoting effect. Therefore, in order to provide a biocontrol agent that can prevent and control grain blast, further research on other types of microorganisms is needed. Summary of the Invention

[0004] In order to solve the above technical problems, the present invention provides a biocontrol Pseudomonas fluorescens FH and its application. The biocontrol Pseudomonas fluorescens FH can not only effectively inhibit the grain blast pathogen, but also promote the germination of millet seeds, providing a new technical approach to solving the problem of grain blast.

[0005] The first aspect of the present invention provides a biocontrol Pseudomonas fluorescens FH. The biocontrol Pseudomonas fluorescens FH is deposited in the China Center for Type Culture Collection on January 13, 2025, with a deposit number of CCTCC NO: M2025096. The depository address is Wuhan University, Wuhan, China, and the classification name is Pseudomonas fluorescens FH.

[0006] The present invention screened out a biocontrol strain of Pseudomonas fluorescens FH from millet rhizosphere soil samples taken in Taigu area. This strain can not only effectively inhibit the grain blast pathogen, but also promote the germination of millet seeds. The strain can also promote the germination of millet seeds without affecting the normal growth of crops, thereby achieving the dual effects of both disease resistance and growth promotion, providing a new technical approach to solving the problem of grain blast.

[0007] The second aspect of the present invention provides the use of the biocontrol Pseudomonas fluorescens FH in the preparation of millet disease control products.

[0008] In another preferred embodiment, the millet disease is millet blast.

[0009] In another preferred embodiment, the product comprises a biocontrol agent.

[0010] In another preferred embodiment, the biocontrol agent is used to inhibit grain blast fungus.

[0011] In another preferred embodiment, the biocontrol agent includes the Pseudomonas fluorescens FH or the fermentation broth of the Pseudomonas fluorescens FH.

[0012] The third aspect of the present invention provides the use of the biocontrol Pseudomonas fluorescens FH in the preparation of a millet growth regulator.

[0013] In another preferred embodiment, the millet growth regulating bacterial agent is used to improve the germination rate of millet seeds.

[0014] Compared with the prior art, the present invention has the following beneficial effects.

[0015] The present invention screened out a biocontrol Pseudomonas fluorescens FH strain from millet rhizosphere soil samples taken in Taigu area. The strain can not only effectively inhibit the grain blast pathogen, but also promote the germination of millet seeds without affecting the normal growth of crops, thereby achieving the dual effects of both disease resistance and growth promotion. This provides a new technical approach to solving the problem of grain blast and realizes efficient, economical, safe and environmentally friendly prevention and control. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the fluorescence result after plate rescreening of biocontrol Pseudomonas fluorescens FH.

[0017] Figure 2 Phylogenetic tree of the biocontrol strain Pseudomonas fluorescens FH.

[0018] Figure 3 Graph showing the inhibitory effect of the biocontrol agent Pseudomonas fluorescens FH on grain blast fungus; wherein, A is the growth result of grain blast fungus in the control group, and B is the inhibitory effect of the biocontrol agent Pseudomonas fluorescens FH on grain blast fungus in the treatment group.

[0019] Figure 4 This is a diagram showing the effect of the biocontrol agent Pseudomonas fluorescens FH on the germination rate of millet seeds. DETAILED DESCRIPTION

[0020] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0021] Unless otherwise specified, the experimental methods described in the following examples are all conventional methods; the reagents and materials described are all commercially available unless otherwise specified.

[0022] The grain blast fungus in the following examples was provided by the Plant Pathology Laboratory of the College of Plant Protection, Shanxi Agricultural University. The published article is: Identification of Pathogenicity of Physiological Races of Grain Blast Fungus and Establishment of Genetic Transformation System, Science China Magazine, Issue 4, 2023.

[0023] The biocontrol strain Pseudomonas fluorescens FH described in the present invention was deposited with the China Center for Type Culture Collection on January 13, 2025, with the deposit number CCTCC NO: M 2025096. The depository address is Wuhan University, Wuhan, China, and the strain is classified as Pseudomonas fluorescens FH. The biocontrol strain Pseudomonas fluorescens FH is referred to herein as FH strain.

[0024] The 16S rDNA sequence of the FH bacteria is shown in SEQ ID NO.1.

[0025]

[0026] Example 1: Isolation, identification and biological characteristics analysis of FH bacteria.

[0027] 1. Isolation and culture of FH bacteria.

[0028] The preparation process of hexadecyltrimethylammonium bromide agar medium is as follows: 3g beef extract, 10g peptone, 5g sodium chloride, 0.3g hexadecyltrimethylammonium bromide, and 1000mL distilled water are mixed and heated to dissolve, and the pH is adjusted to 7.6. Then 20g agar is added, and the culture medium is sterilized at 121℃ for 20min. The plate is prepared and used for later use.

[0029] The preparation process of KB medium is as follows: 20 g of peptone, 10 mL of glycerol, 1.5 g of K2HPO4, 1.5 g of MgSO4·7H20, 15 g of agar and 1 L of distilled water are mixed and heated, and sterilized at 121°C for 20 min.

[0030] The LB liquid medium was prepared as follows: 5 g of yeast extract, 10 g of peptone, and 10 g of NaCl were mixed with 1000 mL of distilled water, and the pH value was adjusted to 7. The mixture was sterilized at 121°C for 20 min and set aside.

[0031] Separation and screening: 10 g of millet rhizosphere soil was added to 90 mL of sterile water to prepare a soil suspension. The suspension was thoroughly shaken and diluted in steps of 10 to 10. -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , obtain soil suspensions with different dilution ratios; use a spreading rod to evenly spread the soil suspensions with different dilution ratios on hexadecyltrimethylammonium bromide agar medium and KB medium in turn, repeat each dilution ratio three times, culture in a constant temperature incubator at 28°C, and observe the growth of the strain every 24 hours.

[0032] Purification and rescreening: Irradiate the KB culture medium plate with colonies with a 365nm ultraviolet lamp, mark the colonies with yellow-green fluorescence, pick out the marked colonies and place them in LB liquid culture medium for overnight culture, and then streak culture on the KB culture medium plate. After streaking on the KB culture medium plate for purification three times, a single colony is obtained, which is rescreened and observed again with an ultraviolet lamp. Finally, the purified strain is stored in a 4°C refrigerator for later use. FH bacteria are picked from single colonies and purified multiple times and streaked onto KB culture medium for rescreening. After culture on the KB plate, FH bacteria form off-white, flat, slightly raised colonies with neat edges. The picked colonies are sticky and will emit yellow-green fluorescence under 365nm ultraviolet light. Figure 1 shown.

[0033] 2. Identification and phylogenetic analysis of FH bacteria.

[0034] (1) Molecular biological identification: The genome of FH bacteria was extracted using a kit and PCR amplified using primers 27F / 1492R. The PCR reaction system and amplification reaction procedure are shown in Table 1 and Table 2, respectively. A single bright band was obtained and sent for sequencing. After BLAST sequence alignment of FH bacteria, the 16S rDNA amplified sequence is shown in SEQ ID NO.1. The phylogenetic tree Figure 2 As shown. After identification, FH bacteria are Pseudomonas fluorescens .

[0035] Table 1: PCR reaction system Table 2: PCR amplification reaction program Note: The number of denaturation, annealing and extension cycles is 40.

[0036] (2) Physiological and biochemical characteristics: The physiological and biochemical characteristics of FH bacteria were identified using Haibo Bio's patented HBI microbial biochemical identification strips. The specific procedures were described in the identification strip instructions. The identification results are shown in Table 3.

[0037] Table 3: Physiological and biochemical identification results of FH bacteria Note: “+” indicates positive, “-” indicates negative, and VP indicates acetyl methyl carbinol test.

[0038] Example 2: Inhibitory effect of Pseudomonas fluorescens FH on grain blast.

[0039] FH bacteria were placed in 5 mL of fresh LB liquid medium and activated at 180 rpm and 37 °C for 48 h to obtain seed bacteria. The seed bacteria were then inoculated into 150 mL of fresh LB liquid medium at a volume ratio of 1:100 and cultured at 180 rpm and 37 °C for 48 h to obtain a concentration of 1 × 10 8 The bacterial solution of FH bacteria with a CFU / mL was used for the plate colony confrontation test.

[0040] The FH bacterial solution was centrifuged at 10,000 rpm at room temperature for 5 minutes, and the supernatant was aspirated with a syringe and filtered through a disposable filter membrane for sterilization. The supernatant was the original fermentation solution of FH bacteria and was set aside.

[0041] The inhibitory effect of FH on P. gracilis was determined using a plate-to-plate culture method. A 5 mm diameter activated P. gracilis cake was inoculated in the center of the PDA medium. The FH fermentation broth was then streaked symmetrically 2.5 cm from the cake onto the plate. This served as the treatment group. A plate inoculated only with the P. gracilis cake served as the control group. Both the treatment and control groups were incubated inverted in a constant-temperature incubator set at 16 h light / 8 h dark, 75% humidity, and 25°C for 10 days, with three replicates. Observations and photographs were taken. The PDA medium consisted of 200 g of potatoes, 20 g of glucose, 15 g of agar, and 1 L of distilled water.

[0042] After 10 days of culture, the growth of grain blast fungus in the control group and the treatment group was observed. Figure 3 As shown, from Figure 3 It can be seen that compared with the control group, the fermentation broth of FH bacteria in the treatment group has a significant inhibitory effect on the growth of grain blast fungus, indicating that FH bacteria can be used as a biocontrol agent to prevent and control grain blast fungus.

[0043] Example 3: Effect of FH bacteria on the germination of Jingu 21 seeds.

[0044] Seed disinfection: Take uniform-sized and plump millet seeds of Jingu 21 variety and put them into a 10mL centrifuge tube. First, soak them in 10wt% sodium hypochlorite with shaking for 30 minutes, then wash them thoroughly with sterile water for 5 times, then soak them in 75wt% ethanol with shaking for 1 minute, and finally wash them thoroughly with sterile water for 5 times and set aside.

[0045] Preparation of 1 wt% bacterial suspension: Centrifuge the FH bacterial solution at 10,000 rpm at room temperature for 5 min, collect the supernatant, and filter it through a 0.22 μm sterile filter membrane to obtain a sterile FH bacterial fermentation broth stock solution. Take 0.2 mL of the sterile FH bacterial fermentation broth stock solution and add 19.8 mL of distilled water to prepare a 1 wt% bacterial suspension.

[0046] Seed spotting: Place two sterilized filter papers in a disposable culture dish, use a pipette to draw 4 mL of the prepared 1 wt% bacterial suspension, and soak the filter paper. The bacterial suspension treatment group is recorded as FH, and the control group is recorded as CK by drawing 4 mL of sterile water to soak the filter paper. Then, the sterilized Jingu 21 is connected to the soaked filter paper, and 30 seeds are spotted on each plate. Repeat three times. Observe and record the germination of millet seeds, and calculate the germination rate. The results are as follows: Figure 4 shown.

[0047] from Figure 4 As can be seen, the germination rate of millet seeds in the control group was 73.33% ± 0.02%, while the germination rate of millet seeds in the treated group after bacterial suspension treatment was 82.22% ± 0.01%. Compared with the control group, the germination rate of millet seeds treated with bacterial suspension significantly increased by 8.89%. The experimental results show that FH bacteria can significantly increase the germination rate of millet seeds and can be used as a growth regulator to promote millet seed germination.

[0048] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A biocontrol agent for Pseudomonas fluorescens ( Pseudomonas fluorescens )FH, characterized in that The biocontrol Pseudomonas fluorescens FH was deposited in the China Center for Type Culture Collection on January 13, 2025, with a deposit number of CCTCC NO: M 2025096.

2. Use of the biocontrol Pseudomonas fluorescens FH strain according to claim 1 in preparing a product for controlling millet diseases.

3. The use of the biocontrol Pseudomonas fluorescens FH according to claim 2 in the preparation of a millet disease control product, characterized in that: The millet disease is millet blight.

4. The use of the biocontrol Pseudomonas fluorescens FH according to claim 2 in preparing a millet disease control product, characterized in that: The product is a biocontrol agent.

5. The use of the biocontrol Pseudomonas fluorescens FH according to claim 4 in preparing a millet disease control product, characterized in that: The biocontrol agent is used for inhibiting grain blast fungus.

6. The use of the biocontrol Pseudomonas fluorescens FH according to claim 4 in preparing a millet disease control product, characterized in that: The biocontrol agent includes the Pseudomonas fluorescens FH or the fermentation liquid of the Pseudomonas fluorescens FH.

7. Use of the biocontrol Pseudomonas fluorescens FH strain according to claim 1 in the preparation of a millet growth regulator.

8. The use of the biocontrol Pseudomonas fluorescens FH in the preparation of a millet growth regulator according to claim 7, characterized in that: The millet growth regulating bacterial agent is used for improving the germination rate of millet seeds.

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