A siderophilic biocontrol bacterium pseudomonas strain and application thereof

Abstract

The application discloses a kind of siderophore biocontrol bacteria pseudomonas strain and application thereof, the classification designation of the strain is pseudomonas (Pseudomonas) Pseudomonas ) T-16, has been preserved in China typical culture preservation center CCTCC, and its preservation number is: CCTCC NO: M 2025817, and the preservation date is April 18, 2025.The pseudomonas Pseudomonas T-16 of the application belongs to rhizosphere siderophore bacteria, has siderophore action; it has significant inhibitory effect on different pathogenic fungi such as rhizoctonia solani and fusarium pseudograminearum, plays an important role in biological control of wheat sheath blight and other diseases; meanwhile, the strain has growth-promoting effect and can promote wheat growth.The strain of the application has simple culture condition, is easy to preserve, is easy to industrial production, and has good development and application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of plant protection, and in particular relates to a Pseudomonas iron-loving biocontrol bacterium strain and application thereof. Background Art

[0002] Wheat is a staple crop widely cultivated in my country. However, it is susceptible to pathogens at every stage of its growth, leading to yield and quality decline and toxin contamination. Currently, wheat disease control relies primarily on chemical control. However, with the widespread use of chemical pesticides, these drawbacks have become increasingly apparent. Chemical pesticides are difficult to degrade, are prone to residues, and pose environmental biohazards, disrupting the ecological balance. In recent years, biological control has garnered widespread attention due to its safety, and new biocontrol agents are constantly being discovered.

[0003] Biocontrol agents offer numerous advantages for wheat disease control. Because they are produced from naturally occurring microorganisms, they are environmentally friendly and do not pollute soil, water sources, or the surrounding ecosystem. Biocontrol agents can inhibit the growth of pathogens by secreting other metabolites, such as siderophores and antibiotics, helping to reduce the occurrence of diseases. Furthermore, while biologically controlling pathogens, some biocontrol agents can also promote plant growth through various pathways, one of the most important of which is the production of plant growth hormones. For example, some biocontrol agents can synthesize plant hormones such as indoleacetic acid (IAA), gibberellins (GA), and cytokinins (CK). These hormones directly regulate plant growth and development, such as promoting cell division, elongation, and root development, thereby enhancing the plant's ability to absorb nutrients and improving crop yields. Summary of the Invention

[0004] In view of the fact that the existing biocontrol bacteria used for crops such as wheat are still insufficient, the present invention provides a Pseudomonas through extensive screening and testing, which can act on a variety of plant fungi and promote plant growth, thereby completing the present invention.

[0005] The present invention has isolated a biocontrol strain Pseudomonas from the wheat rhizosphere that has an iron-loving effect and promotes wheat growth. Pseudomonas sp. T-16, the bacteria has been deposited in the China Center for Type Culture Collection CCTCC, its deposit number is: CCTCC NO: M 2025817, deposit date: April 18, 2025.

[0006] Pseudomonas of the present invention Pseudomonas sp. The optimal fermentation conditions for T-16 to produce siderophore were as follows: using modified SM medium for shaking culture, shaking culture speed of 180r / min, culture temperature of 28℃, initial pH of 8, and fermentation time of 48h.

[0007] In one aspect of the present invention, the present invention provides the use of Pseudomonas sp. T-16 in preventing and treating plant fungal diseases caused by Rhizoctonia solani ( Rhizoctonia solani ) is infected by a disease caused by wheat. Preferably, the disease is wheat sheath blight.

[0008] In one aspect, the present invention also provides the use of Pseudomonas T-16, and fermentation broth and fermentation supernatant containing metabolites, for promoting plant growth. The fermentation broth is a bacterial liquid containing a large amount of Pseudomonas T-16 obtained by fermenting and culturing the Pseudomonas T-16 strain using a culture medium; the fermentation supernatant is a supernatant obtained by centrifuging the fermentation broth obtained after fermenting and culturing the Pseudomonas T-16 strain. Those skilled in the art will appreciate that both the fermentation broth and the fermentation supernatant contain various metabolites produced by the strain during the fermentation process, and these metabolites are the main components that enable the strain to perform its functions.

[0009] In one aspect of the present invention, a bacterial agent containing Pseudomonas T-16 is also disclosed. The bacterial agent can be a liquid preparation, a powder, or a solid granule. For the liquid preparation, it is a bacterial liquid containing Pseudomonas T-16. The powder is prepared by fermenting the Pseudomonas T-16 strain to obtain a fermentation liquid and then freeze-drying it. During the freeze-drying process of the fermentation liquid, substances such as freeze-drying protectants and buffers well known in the art can also be added. Solid granules are formed by immobilizing Pseudomonas T-16 in a porous carrier. The porous carrier includes porous silica, biochar, porous ceramics, and chitosan.

[0010] During plant growth, a bacterial agent containing Pseudomonas T-16 can be applied to the plant roots. Preferably, a bacterial solution containing Pseudomonas T-16 is applied. In the present invention, plant growth primarily involves both underground and aboveground parts, and the effect of the bacterial agent on plant growth can be measured using indicators such as root length and leaf length.

[0011] In one aspect of the present invention, due to its broad-spectrum antibacterial effect, the Pseudomonas sp. T-16 can also be used to prevent and control fungal diseases of one or more of various other plants including wheat, rice, corn, rapeseed, and barley.

[0012] In the present invention, "Pseudomonas T-16", "Pseudomonas ( Pseudomonas sp. )T-16"," Pseudomonas sp. "T-16" refers to the iron-loving biocontrol bacteria Pseudomonas strain with the deposit number of CCTCC NO: M2025817 of the present invention. Beneficial effects

[0013] (1) The Pseudomonas sp. T-16 of the present invention is a type of growth-promoting soil bacterium that has a strong inhibitory effect on bacteria such as Rhizoctonia solani and plays a very important role in the biological control of plant diseases. It has a broad spectrum of antagonistic effects on pathogens and also has the advantages of promoting plant growth.

[0014] (2) The Pseudomonas T-16 of the present invention can be artificially cultured, has simple culture conditions, is easy to preserve, is easy to industrialize, and has good development and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 , are the colony morphology and Gram staining results of the bacteria numbered T-16 in Example 1.

[0016] Figure 2 , is the phylogenetic tree of the bacterium numbered T-16 constructed based on the 16S rDNA sequence in the present invention.

[0017] Figure 3 , is the phylogenetic tree of the bacterium numbered T-16 constructed based on the rpoB sequence in the present invention.

[0018] Figure 4 , is the phylogenetic tree of the bacterium numbered T-16 constructed based on the gyrB sequence in the present invention.

[0019] Figure 5 , is the phylogenetic tree of the bacterium numbered T-16 constructed based on the 16S rDNA-rpoB-gyrB tandem sequence in the present invention.

[0020] Figure 6 , are the positive test results of the physiological and biochemical properties of the bacteria numbered T-16 in Example 2, A citrate test, B fluorescent pigment determination test, C catalase test, D malonate utilization test, E nitrate reduction test, F nitrite reduction test.

[0021] Figure 7 , is a study on the biological characteristics of Pseudomonas T-16 in Example 3, wherein A is a test for phosphate solubilization ability; B is a test for potassium solubilization ability; C is a test for nitrogen fixation ability; DF is a test for siderophilic ability and fluorescent siderophilin (CK is a blank test).

[0022] Figure 8 , is the Pseudomonas T-16 in Example 4 with a broad-spectrum antibacterial effect, A Rhizoctonia solani; B Fusarium oxysporum; C Fusarium solani; D Fusarium oxysporum.

[0023] Figure 9, is a study on the effect of Pseudomonas T-16 on wheat seedling growth in Example 5, A is a wheat morphological control using Pseudomonas T-16 bacterial suspension and a blank control (CK), B is a comparison of root length; C is a comparison of plant height.

[0024] Figure 10 , is a study on the protective effect of Pseudomonas T-16 against wheat sheath blight in Example 6. DETAILED DESCRIPTION

[0025] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to the following examples. Unless otherwise specified, the equipment and reagents used in each example and test example can be obtained from commercial sources. Unless otherwise specified, the reagents used in the present invention are all analytical grade reagents. The specific examples described herein are only used to illustrate the present invention and are not intended to limit the present invention.

[0026] Unless otherwise specified, the biochemical reagents and culture medium of the present invention are all commercially available reagents.

[0027] Example 1 Isolation and Screening of Pseudomonas

[0028] A wheat plant was collected from northern Anhui Province. Its roots were gently shaken to remove most of the loose, easily detached soil. These rhizosphere samples were removed from the plant's roots using a sterile brush. Rhizosphere samples were collected from multiple plants of the same species and placed in sterile bags.

[0029] The various microbial communities present in the rhizosphere were isolated using the dilution and spreading method. 15 g of naturally air-dried soil was selected as the experimental sample. The specific procedure was as follows: First, 150 mL of sterile water was prepared in a 250 mL Erlenmeyer flask. Next, 15 g of soil was transferred to the flask and shaken at 180 rpm for half an hour. The mixture was then allowed to stand for 20 seconds to obtain a 10-fold diluted solution. Finally, the 10-fold diluted solution was subjected to a 10-fold gradient dilution to obtain a series of dilutions. 100 μL of each gradient was evenly dropped onto the center of the LB plate. The spreading operation was performed using sterile equipment to ensure uniformity. After spreading, the plate was placed in a clean bench to air dry for 5 minutes. Before the LB medium completely absorbed the liquid, the culture dish was covered and placed in a biochemical incubator for incubation in the dark at 28°C. After approximately 24 hours, single colonies will form on the LB plate. Using a bacterial inoculation loop, the desired single colony can be screened from the new LB plate based on its morphology and color. This allows for a series of operations such as isolation and purification. The selected single colony is suspended in 15% glycerol and stored at -80°C.

[0030] The CAS plate method was used to separate and screen a colony with strong siderophore production ability, which was numbered T-16. It was inoculated on LB medium and its colony morphology was observed. After culturing for 24 hours, the surface of T-16 colony was smooth and the edges were neat (see Figure 1 ) and gram staining was performed on it, and the gram staining result was red and negative (see Figure 1 ).

[0031] The 16S rDNA sequence of strain T-16 (SEQ ID NO.1), two conserved housekeeping genes gyrB (SEQ ID NO.2), rpoB (SEQ ID NO.3), and the concatenated three genes (16S rDNA, gyrB 、 rpoB ) were used to construct a phylogenetic tree to identify the specific genus of the strain. Figure 2-Figure 5 It can be seen that the identification results of the four phylogenetic trees failed to clearly identify the specific species of the strain, suggesting that the strain is a new species. Therefore, the present invention temporarily identified the strain to the genus, and the bacteria belonged to the genus Pseudomonas. Therefore, the T-16 strain was named Pseudomonas ( Pseudomonas sp. ) T-16, deposited in the China Center for Type Culture Collection, with the deposit number CCTCCNO: 2025817, the deposit date is April 18, 2025, and the deposit address is Wuhan University, Wuhan, China.

[0032] Example 2 Physiological and biochemical test of Pseudomonas T-16

[0033] (1) Methyl red test: The strain cultured for 24 hours was inoculated into the methyl red assay medium. After culturing at an appropriate temperature for 2 days and 6 days, a drop of methyl red detection reagent was added to the culture medium. The results were observed: if red appeared in the culture medium, it was positive, otherwise it was negative.

[0034] (2) Voges-Proskauer test: The strain T-16 cultured for 24 h was inoculated into the Voges-Proskauer assay medium and cultured at 28°C for 2 and 6 days. An equal amount of 40% sodium hydroxide solution was taken and mixed evenly, and a small amount of creatine was added dropwise. After reacting for 10 min, the results were observed: if the culture medium turned red, it was positive; otherwise, it was negative.

[0035] (3) Catalase test: Use an inoculation needle to pick up a single colony of strain T-16 and evenly smear it on a glass slide with 10% hydrogen peroxide on the surface. Observe the results: if bubbles appear, it is positive, otherwise it is negative.

[0036] (4) Starch hydrolysis test: The strain T-16 cultured for 24 hours was inoculated into starch hydrolysis medium and cultured at an appropriate temperature for 2-5 days. After the colonies were formed, iodine solution was added to the plate and the results were observed: if a transparent circle appeared around the colonies, it was positive, otherwise it was negative.

[0037] (5) Lipase test: Use an inoculation needle to pick up a single colony of the T-16 strain and inoculate it into the lipase culture medium. Place it in a 28°C incubator and culture it for 24 hours. Observe the results: If a white turbid circle appears around the colony, it indicates that the lipase reaction is positive, otherwise it is negative.

[0038] (6) Nitrate reduction test: The strain T-16 cultured for 24 h was inoculated into nitrate reduction medium and cultured at appropriate temperature for 1 day, 3 days, and 5 days respectively. Liquid A and liquid B were added dropwise to the culture medium respectively. The results were observed: if the culture medium turned red, orange, or brown, it was positive; otherwise, it was negative.

[0039] (7) Nitrite reduction test: The strain T-16 cultured for 24 h was inoculated into nitrite reduction medium and cultured at appropriate temperature for 1 d, 3 d, and 5 d, respectively. Liquid A and liquid B were added dropwise to the culture medium, and the results were observed: if the red color of the culture medium disappeared, it was ammonia production, which was positive; otherwise, it was negative.

[0040] (8) Malonate utilization test: Use an inoculation needle to pick up a single colony of strain T-16 and inoculate it into a malonate medium. Place it in a 28°C incubator and culture it for 2-4 days. If the culture medium changes color, it means that the malonate reaction is positive, otherwise it is negative.

[0041] (9) Citrate utilization test: Inoculate strain T-16 cultured for 24 h into citrate utilization medium and culture at 28°C for 2-4 days. Observe the results: if the culture medium turns pink, it is positive; otherwise, it is negative.

[0042] (10) Ammonia production test: The strain T-16 cultured for 24 h was inoculated into an ammonia production medium and cultured at 28°C for 5 days. Nessler reagent was added to the medium and the results were observed: if a red precipitate appeared, it was positive; otherwise, it was negative.

[0043] (11) Fluorescent pigment determination test: The strain T-16 cultured for 24 h was inoculated into King B medium and cultured at 30°C for 1 day, 3 days, and 5 days. The results were observed under ultraviolet light. If there was fluorescence, it was positive, otherwise it was negative.

[0044] From Table 1 and Figure 6As can be seen, the methyl red test is negative; the V-P test is negative; the contact enzyme test is positive, indicating that the bacteria can react with hydrogen peroxide and release gas; the nitrate reduction test is positive, indicating that the bacteria can reduce nitrate; the nitrite reduction test is positive, which can reduce nitrite; the starch hydrolysis test is negative, indicating that the strain cannot hydrolyze starch; the citrate reduction test is positive, which can utilize sodium citrate; the lipase test is negative; the ammonia production test is negative, which cannot produce ammonium ions; the malonate utilization test is positive, indicating that it can be decomposed to generate sodium carbonate; the fluorescent pigment test is positive, which can produce fluorescent pigment. With strain Pseudomonas A3 as a control, the physiological and biochemical results of Pseudomonas are similar. It is further determined that strain T-16 is Pseudomonas Pseudomonas sp. )。

[0045] Table 1 Partial physiological and biochemical test results of strain T-16

[0046] ;

[0047] Example 3 Biological property test of Pseudomonas T-16

[0048] (1) Determination of the phosphorus solubilizing ability of Pseudomonas T-16

[0049] The Pseudomonas T-16 strain is inoculated on a specific bacterial culture medium containing phosphorus-solubilizing bacteria. It is observed whether a transparent phosphorus-solubilizing circle appears around the colony. If a transparent phosphorus-solubilizing circle appears, it is initially indicated that the strain has the ability to solubilize phosphorus.

[0050] (2) Determination of the potassium solubilizing ability of Pseudomonas T-16

[0051] The Pseudomonas T-16 strain is inoculated on a specific bacterial culture medium containing potassium-solubilizing bacteria. It is observed whether oil droplet-like substances appear around the colony. If oil droplet-like substances appear, it is initially indicated that the strain has the ability to solubilize potassium.

[0052] (3) Determination of the nitrogen fixation ability of Pseudomonas T-16

[0053] The Pseudomonas T-16 strain is inoculated on a specific nitrogen-fixing bacterial sucrose medium. After cultivation, it is observed whether the subculture can survive on the medium for three generations. If it survives, it proves that Pseudomonas T-16 has nitrogen fixation ability.

[0054] (4) Determination of the iron-solubilizing ability and fluorescent siderophore of Pseudomonas T-16

[0055] The Pseudomonas T-16 is inoculated on a specific CAS medium. After cultivation, it is observed whether a yellow halo appears around the colony. If a yellow halo appears, it is initially indicated that the strain has the ability to secrete iron carriers.

[0056] Pseudomonas T-16 is inoculated into a modified SM liquid medium. After incubation, the color of the culture medium can be observed visually. If the color changes to yellow-green, it indicates that the strain is capable of producing fluorescent siderophore.

[0057] Depend on Figure 7 The results showed that Pseudomonas T-16 did not have the ability to degrade inorganic phosphorus and potassium, but had the ability to degrade organic phosphorus, fix nitrogen and secrete iron carriers.

[0058] Pseudomonas ) Test of the broad-spectrum antibacterial ability of T-16

[0059] Select fresh Pseudomonas Pseudomonas ) T-16 single colony, inoculate it and culture it. Take fresh mycelium blocks at the edge of the fungal colony and transfer them to the center of a clean PDA culture medium plate. Place four sterile filter paper pieces (Φ=6 mm) around each fungal block, and then draw 2 µL of bacterial solution onto the filter paper pieces. At the same time, use 2 µL of sterile water as a blank control. These plates were cultured in an incubator at 25°C. When the mycelium of the control group almost filled the entire plate, the colony diameter was measured. Each group of experiments was repeated 3 times, and the inhibition rate was calculated. The results are shown in the antagonistic effect figure below. Figure 8 .

[0060] The calculation formula of the inhibition rate of Pseudomonas T-16 in wheat root soil is:

[0061] ;

[0062] Where: A refers to the diameter of the pathogen colony in the control group;

[0063] B refers to the diameter of the pathogen colony in the treatment group;

[0064] Table 2 Inhibition rate of different pathogenic fungi

[0065] ;

[0066] Example 5 Study on the Effect of Pseudomonas T-16 on Wheat Seedling Growth

[0067] From the selected Pseudomonas T-16 single colonies, a single strain was selected, inoculated, and cultured. The cultured single colony of Pseudomonas T-16 was then re-inoculated into fresh LB medium for culture. The culture was then transferred to a centrifuge tube and centrifuged in a low-temperature high-speed centrifuge to remove the supernatant and retain the bacterial pellet. Finally, the bacterial pellet was resuspended in sterile water and used for inoculation. Each 250 mL Erlenmeyer flask was filled with 100 mL of LB liquid medium, sterilized at high temperature, and inoculated with 20 mL of the bacterial suspension. Finally, the inoculum of Pseudomonas T-16 was obtained by incubating the flask at 37°C and 160 rpm for 2-3 days. Next, the wheat seedlings were disinfected and germinated before planting. Three days after the wheat seedlings emerged, the Pseudomonas T-16 inoculum was applied to the roots. A control group was watered with sterile water using the same procedure.

[0068] See also Figure 9 The results showed that the Pseudomonas T-16 bacterial solution promoted the growth of wheat seedlings. Wheat treated with the bacterial solution had an average root length increase of 17.9%, and plant height was significantly different from that of wheat treated with the bacterial solution. Under the same sample conditions, wheat treated with a certain amount of bacterial solution during growth had a certain growth-promoting effect. Plants had stronger roots and a significantly increased number of lateral roots compared to wheat not treated with the bacterial solution. Treatment with the Pseudomonas T-16 strain has a growth-promoting effect on wheat seedlings.

[0069] Example 6 Study on the Control Effect of Pseudomonas sp. T-16 on Wheat Sharp Eye Blight

[0070] The wheat strain used was AK 58, and the experiment was conducted at Anhui Agricultural University. A single colony of the test strain was inoculated into LB medium and incubated at 28°C, 180 rpm, in a shaker for 48 hours. Healthy, plump wheat seeds were taken and incubated overnight in the dark with an appropriate amount of sterile water. After germination, the seeds were planted in small pots filled with sterile nutrients and watered moderately. When the wheat grows to 4-5 cm, take 5 mL of the cultured biocontrol fungus solution and irrigate the roots. After 7 days, use the wheat that is not treated with the biocontrol fungus solution as the control, and evenly sprinkle the wheat kernels infected with the pathogen (the cooked wheat seeds are spread on the PDA plate infected with the pathogen and co-cultured with the pathogen Rhizoctonia solani until the mycelium completely infects the wheat kernels, that is, the wheat kernels with the pathogen) on the roots of the wheat. The wheat that is neither inoculated with the biocontrol fungus solution nor with the pathogen-infected wheat kernels serves as the blank control. Afterwards, observe the growth and disease of wheat under each treatment.

[0071] In order to further verify the biocontrol effect of strain T-16, the potted control effect of strain T-16 on Rhizoctonia solani was tested by inoculating coleoptiles. Figure 10After 30 days, the wheat in the sterile water control group grew well, as shown in the figure below. Figure 10 As shown in A, the lodging rate is about 20%; the wheat and Rhizoctonia solani pathogens inoculated with bacterial solution T-16 (irrigation) and then inoculated with Rhizoctonia solani grew well compared with the sterile water control. Figure 10 As shown in the left picture of B; the wheat inoculated with only Rhizoctonia solani becomes yellow and wilts. Figure 10 As shown in the right figure of B, the lodging rate is 100%. This result shows that strain T-16 can effectively prevent and control wheat lodging.

[0072] The above content is a further detailed description of the present invention in conjunction with specific implementation methods. It cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, they can make several simple deductions or substitutions without departing from the concept of the present invention, which should be regarded as falling within the scope of protection determined by the claims submitted for the present invention.

Claims

1. A ferroxine biocontrol bacterium, characterized in that: The iron-loving biocontrol bacteria is Pseudomonas ( Pseudomonas sp. ) T-16, deposited in China Center for Type Culture Collection, with the deposit number CCTCC NO: M 2025817, the deposit date is April 18, 2025, and the deposit address is Wuhan University, Wuhan, China.

2. A bacterial agent, characterized in that The bacterial agent contains the Pseudomonas T-16 according to claim 1.

3. The microbial agent according to claim 2, characterized in that The bacterial agent is a liquid preparation or a powder.

4. The microbial agent according to claim 3, characterized in that The liquid preparation is a fermentation broth containing the iron-loving bacterium Pseudomonas T-16; and the powder is a freeze-dried powder containing Pseudomonas T-16.

5. Use of the iron-loving biocontrol bacterium according to claim 1 or the bacterial agent according to claim 2 in the prevention and treatment of plant diseases, characterized in that: The disease is caused by Rhizoctonia solani ( Rhizoctonia solani ) infection; the disease is wheat sheath blight.

6. Use of the iron-loving biocontrol bacterium according to claim 1 or the bacterial agent according to claim 2 in promoting plant growth.

7. The use according to claim 6, characterized in that The plant is wheat.

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