Ferrophilic biocontrol bacterium pseudomonas strain and application thereof

Abstract

The invention discloses a siderophore biocontrol bacterium pseudomonas strain and application thereof, the classification name of the strain is pseudomonas T-16, the strain is preserved in the China Center for Type Culture Collection (CCTCC), the preservation number of the strain is CCTCC NO: M 2025817, and the preservation date is April 18, 2025. The Pseudomonas T-16 disclosed by the invention belongs to rhizosphere siderophore bacteria, and has a siderophore effect; the compound has a remarkable inhibition effect on different pathogenic fungi such as rhizoctonia solani and fusarium verticillium, and plays an important role in biological control of diseases such as wheat sharp eyespot; meanwhile, the strain has a growth promoting effect and can promote wheat growth. The strain provided by the invention has the advantages of simple culture conditions, easiness in preservation, easiness in industrial production and good development and application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant protection, and specifically relates to an iron-loving biocontrol bacterium Pseudomonas strain and its application. Background Art

[0002] Wheat is one of the main crops in China and is widely planted in China. However, it is damaged by pathogenic bacteria at all stages of its growth, resulting in a decrease in yield and quality and toxin contamination. At present, the prevention and control of wheat diseases mainly rely on chemical control. However, with the extensive use of a large number of chemical pesticides, the disadvantages of chemical control have gradually emerged. Chemical pesticides are not easily degraded, are prone to residues and environmental biological hazards, and damage the ecological balance. In recent years, biological control has received extensive attention due to its safety, and new biocontrol agents have been continuously discovered.

[0003] Biocontrol agents have many advantages in the prevention and control of wheat diseases. Since they are made of natural microorganisms, they are environmentally friendly and do not pollute the soil, water source or surrounding ecological environment. Biocontrol bacteria can inhibit the growth of pathogenic bacteria by secreting other metabolites, such as siderophores, antibiotics, etc., which helps to reduce the occurrence of diseases. In addition, some biocontrol bacteria can promote plant growth through various ways while controlling pathogenic bacteria. One of the most important mechanisms is the production of plant growth hormones. For example, some biocontrol bacteria can synthesize plant hormones such as indole-3-acetic acid (IAA), gibberellin (GA), cytokinin (CK), etc. These hormones can directly regulate the growth and development process of plants, such as promoting cell division, elongation and root development, thereby enhancing the plant's ability to absorb nutrients and improving crops. Summary of the Invention

[0004] Aiming at the problem that the existing biocontrol bacteria applied to 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 isolated a biocontrol strain Pseudomonas from the wheat rhizosphere, which has an iron-loving effect and promotes the growth of wheat. Pseudomonas sp. T-16, this strain has been deposited in the China Center for Type Culture Collection (CCTCC), and its deposit number is: CCTCC NO: M 2025817, deposit date: April 18, 2025.

[0006] The Pseudomonas of the present invention Pseudomonas sp. The optimal fermentation conditions for the production of siderophores by Pseudomonas

[0007] In one aspect of the present invention, the present invention provides the application of Pseudomonas sp. T-16 in preventing and controlling plant fungal diseases, where the fungal diseases are diseases caused by the infection of Rhizoctonia solani ( Rhizoctonia solani ). Preferably, the disease is sharp eyespot of wheat.

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

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

[0010] During the plant growth period, the bacterial agent containing Pseudomonas sp. T-16 can be applied to the roots of the plant. Preferably, the bacterial liquid containing Pseudomonas sp. T-16 is applied. In the present invention, plant growth mainly involves the underground and above-ground parts, and indicators such as root length and leaf length can be used to measure the influence of the bacterial agent on plant growth.

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

[0012] In the present invention, "Pseudomonas sp. T-16", "Pseudomonas sp. ( Pseudomonas sp. ) T-16", and " Pseudomonas sp. T-16" all refer to the siderophilic biocontrol bacterium Pseudomonas strain with the preservation number of CCTCC NO: M2025817 of the present invention. Beneficial effects

[0013] (1) Pseudomonas sp. T-16 of the present invention is a kind of plant growth-promoting rhizobacteria, with strong inhibitory effects on bacteria such as Rhizoctonia solani, and plays a very important role in the biological control of plant diseases. It has the advantages of broad-spectrum antagonism against pathogenic bacteria and promoting plant growth.

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

[0015] Figure 1 It is the colony morphology diagram and Gram staining result diagram of the strain numbered T-16 in Example 1.

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

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

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

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

[0020] Figure 6 It is the positive experimental result diagram of the physiological and biochemical properties of the strain 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 It is the study on the biological characteristics of Pseudomonas sp. T-16 in Example 3, where A phosphorus-solubilizing ability test; B potassium-solubilizing ability test; C nitrogen-fixing ability test; D-F iron-chelating ability and fluorescent siderophore test (CK is the blank test).

[0022] Figure 8 It is that Pseudomonas sp. T-16 has broad-spectrum antibacterial effects in Example 4, A Rhizoctonia solani; B Verticillium dahliae; C Neocosmospora vasinfecta; D Fusarium oxysporum.

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

[0024] Figure 10 , For the study of the control effect of Pseudomonas sp. T-16 on wheat sharp eyespot in Example 6. Detailed implementation manners

[0025] In order to make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below in conjunction with embodiments. The equipment and reagents used in each embodiment and test example can be obtained from commercial channels without special instructions. Unless otherwise specified, the reagents used in the present invention are all analytical grade reagents. The specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0026] The biochemical reagents and culture media of the present invention are all from commercially available reagents without special instructions. Example 1 Isolation and screening of Pseudomonas sp.

[0027] A strain of wheat was collected from the northern part of Anhui Province, and then its roots were gently shaken to remove most of the loose and easily detachable soil. Using a sterile brush, these rhizosphere samples were taken away from the roots of the plants. Rhizosphere samples of multiple plants of the same species were collected and placed in a sterile bag.

[0028] For the various microbial communities existing in the rhizosphere, the dilution coating method was adopted for isolation in the experiment. 15 grams of naturally air-dried soil was selected as the experimental sample. The specific operation was as follows: First, 150 mL of sterile water was prepared in a 250 mL Erlenmeyer flask for standby. Secondly, 15 grams of soil was transferred into the Erlenmeyer flask and shaken at 180 r.min-1 for half an hour. Then the mixed liquid was allowed to stand for 20 s to obtain a solution diluted 10 times. Finally, 10-fold gradient dilution was performed on the 10-fold diluted solution to obtain a series of diluted solutions. 100 μL of the liquid of each gradient was evenly dropped at the center of the LB plate. The coating operation should be carried out using sterile equipment to ensure uniformity. After coating, it was placed in the ultra-clean bench to dry for 5 min. Before the LB medium completely absorbed the liquid substance, the culture dish cover was covered, and it was placed in a biochemical incubator for cultivation in the dark environment at 28°C. After about 24 hours, single colony isolates would form on the LB plate. According to the different morphologies and colors of the colonies, the required single colonies could be screened out from the new LB plate by using a bacterial inoculation loop, and a series of operations such as isolation and purification of the colonies could be carried out accordingly. The screened single colony bacteria were suspended in 15% glycerol and stored at -80°C ultra-low temperature.

[0029] Colonies with strong siderophore-producing ability were screened and extracted by the CAS plate method, numbered T-16. It was inoculated onto LB medium and its colony morphology was observed. When the T-16 colony was cultured for 24 h, its surface was smooth and the edge was neat, (see Figure 1 ), and it was subjected to Gram staining. The result of its Gram staining was red, indicating negative (see Figure 1 ).

[0030] Based on the 16S rDNA sequence (SEQ ID NO.1) of strain T-16, two conserved housekeeping genes gyrB (SEQ ID NO.2), rpoB (SEQ ID NO.3), and the multilocus sequence analysis (Multilocus Sequence Analysis, MLSA) after concatenating the three genes (16S rDNA, gyrB 、 rpoB ), phylogenetic trees were constructed respectively to identify the specific genus and species of this strain. As can be seen from Figures 2 - 5 , the identification results of the 4 phylogenetic trees could not clarify the specific species of this strain, suggesting that this strain is a new species. Therefore, in the present invention, the strain was temporarily identified to the genus, and this bacterium belongs to the genus Pseudomonas. Thus, strain T-16 was named Pseudomonas ( Pseudomonas sp. ), deposited in the China Center for Type Culture Collection, with the deposit number CCTCC NO: 2025817, the deposit date being April 18, 2025, and the deposit address being Wuhan University, Wuhan, China. Example 2 Physiological and biochemical experimental tests of Pseudomonas T-16

[0031] (1) Methyl red test: The strain cultured for 24 h was inoculated into the methyl red assay medium, and after incubation at an appropriate temperature for 2 d and 6 d, a drop of methyl red detection reagent was added to the medium. The result was observed: If the medium turned red, it was positive; otherwise, it was negative.

[0032] (2) Voges-Proskauer test: Strain T-16 cultured for 24 h was inoculated into the Voges-Proskauer assay medium, incubated at 28 °C for 2 d and 6 d, an equal volume of 40% sodium hydroxide solution was mixed with the culture solution, a little creatine was added, and after reacting for 10 min, the result was observed: If the medium turned red, it was positive; otherwise, it was negative.

[0033] (3) Catalase test: A single colony of strain T-16 was picked with an inoculation needle and evenly smeared on a glass slide with 10% hydrogen peroxide on the surface. The result was observed: If bubbles appeared, it was positive; otherwise, it was negative.

[0034] (4)Starch hydrolysis test: Inoculate the strain T-16 cultured for 24 h into the starch hydrolysis medium, and culture it at an appropriate temperature for 2 - 5 d. After the colonies appear, add iodine solution dropwise on the plate. Result observation: If a clear zone appears around the colonies, it is positive; otherwise, it is negative.

[0035] (5)Lipase test: Use an inoculation needle to pick a single colony of the strain T-16 and inoculate it into the lipase medium, then culture it in an incubator at 28 °C for 24 h. Result observation: If a white turbid zone appears around the colonies, it indicates that the lipase reaction is positive; otherwise, it is negative.

[0036] (6)Nitrate reduction test: Inoculate the strain T-16 cultured for 24 h into the nitrate reduction medium, and culture it at an appropriate temperature for 1 d, 3 d, and 5 d respectively. Add solution A and solution B dropwise into the medium respectively. Result observation: If the medium turns red, orange, or brown, it is positive; otherwise, it is negative.

[0037] (7)Nitrite reduction test: Inoculate the strain T-16 cultured for 24 h into the nitrite reduction medium, and culture it at an appropriate temperature for 1 d, 3 d, and 5 d respectively. Add solution A and solution B dropwise into the medium respectively. Result observation: If the red color of the medium disappears, indicating ammonia production, it is positive; otherwise, it is negative.

[0038] (8)Malonate utilization test: Use an inoculation needle to pick a single colony of the strain T-16 and inoculate it into the malonate medium, then culture it in an incubator at 28 °C for 2 - 4 d. If the medium changes color, it indicates that the malonate reaction is a positive reaction; otherwise, it is negative.

[0039] (9)Citrate utilization test: Inoculate the strain T-16 cultured for 24 h into the citrate utilization medium, and culture it at 28 °C for 2 - 4 d. Result observation: If the medium turns pink, it is positive; otherwise, it is negative.

[0040] (10)Ammonia production test: Inoculate the strain T-16 cultured for 24 h into the ammonia production medium, and culture it at 28 °C for 5 d. Add Nessler's reagent dropwise into the medium. Result observation: If a red precipitate appears, it is positive; otherwise, it is negative.

[0041] (11)Fluorescent pigment determination test: Inoculate the strain T-16 cultured for 24 h into the King B medium, and culture it at 30 °C for 1 d, 3 d, and 5 d respectively. Result observation: Observe under ultraviolet light for fluorescence. If there is fluorescence, it is positive; otherwise, it is negative.

[0042] From Table 1 and Figure 6It can be seen that the methyl red test is negative; the V-P test is negative; the catalase test is positive, indicating that the bacterium can react with hydrogen peroxide and release gas; the nitrate reduction test is positive, indicating that the bacterium can perform nitrate reduction. The nitrite reduction test is positive, indicating that it can perform nitrite reduction; the starch hydrolysis test is negative, indicating that the strain cannot hydrolyze starch; the citrate reduction test is positive, indicating that it can utilize sodium citrate; the lipase test is negative; the ammonia production test is negative, indicating that it cannot produce ammonium ions; the malonate utilization test is positive, indicating that it can be decomposed to form sodium carbonate; the fluorescent pigment test is positive, indicating that it can produce fluorescent pigments. Using the strain Pseudomonas A3 as a control, the physiological and biochemical results are similar to those of the genus Pseudomonas. Further confirmation shows that strain T-16 belongs to the genus Pseudomonas ( Pseudomonas sp. ).

[0043] Table 1 Partial physiological and biochemical test results of strain T-16 ; Example 3 Biological characteristic test of Pseudomonas T-16

[0044] (1) Determination of phosphorus-solubilizing ability of Pseudomonas T-16 Inoculate the Pseudomonas T-16 strain on a bacterial medium containing specific phosphorus-solubilizing components, and observe whether there is a transparent phosphorus-solubilizing zone around the colony. If a transparent phosphorus-solubilizing zone appears, it preliminarily indicates that the strain has the ability to solubilize phosphorus.

[0045] (2) Determination of potassium-solubilizing ability of Pseudomonas T-16 Inoculate the Pseudomonas T-16 strain on a bacterial medium containing specific potassium-solubilizing components. Observe whether there are oil-drop-like substances around the colony. If oil-drop-like substances appear, it preliminarily indicates that the strain has the ability to solubilize potassium.

[0046] (3) Determination of nitrogen-fixing ability of Pseudomonas T-16 Inoculate the Pseudomonas T-16 strain on a specific nitrogen-fixing bacterial sucrose medium. After cultivation, observe whether it can survive after being transferred three generations on the medium. If it can survive, it proves that Pseudomonas T-16 has nitrogen-fixing ability.

[0047] (4) Determination of iron-chelating ability and fluorescent siderophore of Pseudomonas T-16 Inoculate Pseudomonas T-16 on a specific CAS medium. After cultivation, observe whether there is a yellow halo around the colony. If there is a yellow halo, it preliminarily indicates that the strain has the ability to secrete siderophores.

[0048] Inoculate Pseudomonas T-16 in a specific modified SM liquid medium. After cultivation, the color change of the culture solution can be observed with the naked eye. If the color turns yellowish-green, it indicates that the strain can produce fluorescent siderophores.

[0049] From Figure 7 the results, it can be seen that Pseudomonas sp. T-16 does not have the ability to dissolve inorganic phosphorus and potassium, but has the abilities to dissolve organic phosphorus, fix nitrogen, and secrete siderophores. Pseudomonas )Broad-spectrum antibacterial ability test of T-16

[0050] Select a fresh single colony of Pseudomonas sp. ( Pseudomonas )T-16, inoculate and culture it. Take a fresh mycelial block at the edge of the fungal colony and transfer it to the center of a clean PDA medium plate. Place four sterile filter papers (Φ = 6 mm) around each mycelial block, and then pipette 2 μL of the bacterial solution onto the filter papers. At the same time, use 2 μL of sterile water as a blank control. Incubate these plates in an incubator at 25°C. When the mycelium in the control group is close to filling the entire plate, measure the colony diameter. Repeat each experiment 3 times and calculate the antibacterial rate. The results are shown in the following antagonistic effect diagram as Figure 8 .

[0051] Calculation formula for the antibacterial rate of Pseudomonas sp. T-16 in wheat root soil: ; In the formula: A refers to the diameter of the pathogen colony in the control group; B refers to the diameter of the pathogen colony in the treatment group; Table 2 Antibacterial rates of different pathogenic fungi ; Example 5 Study on the effect of Pseudomonas sp. T-16 on the growth of wheat seedlings

[0052] Select one strain from the selected single colony of Pseudomonas sp. T-16, inoculate and culture it. Select the cultured single colony of Pseudomonas sp. T-16 and inoculate it again into a new LB medium for culture. Subsequently, transfer the cultured bacterial solution into a centrifuge tube and centrifuge it in a low-temperature high-speed centrifuge to remove the supernatant and leave the cell pellet. Finally, resuspend the cell pellet with sterile water and then use it for inoculation. Add 100 mL of LB liquid medium to each 250 mL Erlenmeyer flask, sterilize it by high temperature, and inoculate 20 mL of the bacterial suspension. Finally, culture it for 2 - 3 days at 37°C and 160 r / min to obtain the inoculant of Pseudomonas sp. T-16. Then, before wheat planting, perform disinfection and germination acceleration treatments. After 3 days of wheat seedling emergence, pour the inoculant of Pseudomonas sp. T-16 onto the wheat roots. As a control group, irrigate with sterile water using the same steps.

[0053] See Figure 9The results showed that the bacterial suspension of Pseudomonas sp. T-16 had a growth-promoting effect on the growth of wheat seedlings during the growth process of wheat. For the wheat irrigated with the bacterial suspension, the average root length of wheat increased by 17.9%, and there were also significant differences in plant height compared with the CK. Under the same sample conditions, during the growth process of wheat, when irrigated with a certain amount of bacterial suspension, there was a certain growth-promoting effect. The roots of the wheat plants irrigated with the bacterial suspension were thicker than those of the wheat without irrigation, and the number of lateral roots increased significantly. After treatment with Pseudomonas sp. T-16 strain, it had a growth-promoting effect on wheat seedlings. Example 6 Study on the control effect of Pseudomonas sp. T-16 against sharp eyespot of wheat

[0054] The tested wheat variety was AK 58, and the experimental site was Anhui Agricultural University. A single colony of the tested strain was picked and inoculated into LB medium, and cultured in a shaker at 28 °C and 180 r / min for 48 h. Wheat seeds with healthy and plump grains were taken, added with an appropriate amount of sterile water and incubated overnight in the dark. After the seeds germinated, they were sown in small potted plants filled with sterile nutrients, and watered appropriately during this period. When the wheat grew to 4 - 5 cm, 5 mL of the cultured biocontrol bacterial suspension was used for root irrigation. After 7 d, the wheat without root irrigation with the biocontrol bacterial suspension was used as the control. The wheat grains infected by the pathogen (the cooked wheat seeds were spread on the PDA plate inoculated with the pathogen and co-cultured with the pathogen Rhizoctonia solani until the hyphae completely infected the wheat grains, which were the wheat grains with the pathogen) were evenly scattered on the roots of the wheat. The wheat without receiving the biocontrol bacterial suspension and without receiving the wheat grains with the pathogen was used as the blank control. Then, the growth and disease incidence of the wheat under each treatment were observed.

[0055] To further verify the biocontrol effect of strain T-16, the pot control effect of strain T-16 against Rhizoctonia solani was detected by the method of in vivo inoculation and infection of coleoptiles. The results of the pot control test of wheat are as Figure 10 shown. After 30 d, the wheat in the sterile water control grew well, as shown in A in Figure 10 , and the lodging rate was about 20%; the wheat inoculated with bacterial suspension T-16 (root irrigation) and then with Rhizoctonia solani grew well compared with the sterile water control, as shown in the left figure of B in Figure 10 ; the wheat only inoculated with Rhizoctonia solani withered and wilted yellow, as shown in the right figure of B in Figure 10 , and the lodging rate was 100%. This result indicates that strain T-16 can effectively prevent wheat from lodging.

[0056] The above content further elaborates on the present invention in combination with specific embodiments. It cannot be determined that the specific implementation of the present invention is only limited to these descriptions. For those of ordinary skill in the technical field to which the present invention pertains, without departing from the concept of the present invention, several simple deductions or substitutions can still be made, which should all be regarded as falling within the protection scope determined by the claims submitted for the present invention.

Claims

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

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

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

4. The microbial agent according to claim 2, characterized in that, The liquid preparation is a fermentation broth containing Pseudomonas siderophila T-16; the powder preparation is a freeze-dried powder containing Pseudomonas sp. T-16.

5. Use of the iron-oxidizing biocontrol bacterium according to claim 1 or the microbial agent according to claim 2 in the prevention and control of plant diseases, characterized in that, The disease is a disease caused by the infection of Rhizoctonia solani ); the disease is wheat sharp eyespot.

6. Use of the siderophilic biocontrol bacterium described in claim 1 or the bacterial agent described in claim 2 in promoting plant growth.

7. The application according to claim 6, wherein The plants include wheat, rice, corn, rapeseed, barley; preferably, the plant is wheat.

8. The iron-oxidizing biocontrol bacterium according to claim 1, characterized in that, The optimal fermentation conditions for the production of siderophore by Pseudomonas sp. T-16 are: shake culture is carried out using a modified SM medium, the shake culture speed is 180 r / min, the culture temperature is 28 °C, the initial pH is 8.0, and the fermentation time is 48 h.

Images