Corn endophytic pseudomonas putida strain and application thereof

Abstract

The invention discloses a corn endophytic pseudomonas putida strain and application thereof. The corn endophytic pseudomonas putida strain is named as Pseudomonas putida SF07 and is preserved in the China Center for Type Culture Collection, the preservation starting date is September 26, 2024, and the preservation number is M 20242084; the preservation address is Wuhan University, Wuhan, China. The corn endophytic pseudomonas putida strain disclosed by the invention can be used for effectively inhibiting the growth of bipolaris maydis and setosphaeria turcica, has relatively good chitinase activity and relatively low cellulase and pectinase activity, is difficult to degrade plant cell walls, is relatively friendly to plants, and can be used as a biopesticide for preventing and treating corn diseases.

Description

Technical Field

[0001] The invention relates to the field of biological control of corn diseases, and in particular to an endophytic Pseudomonas putida strain of corn and application thereof. Background Art

[0002] In the past decade, fresh corn has been favored by the market for its rich nutrition, excellent taste, short growth period and high planting efficiency. The annual planting area in China has stabilized at about 25 million mu. However, there are still some challenges in the production of fresh corn, such as the decline in commercial value caused by untimely prevention and control of pests and diseases, and environmental pollution, increased costs and reduced quality caused by excessive use of chemical pesticides. These problems not only affect the economic value of fresh corn, but also pose a threat to the sustainable development of agriculture. At present, the prevention and control methods of corn diseases are still mainly chemical control, which is not conducive to environmental protection and food safety. Summary of the invention

[0003] In order to overcome the above-mentioned shortcomings and deficiencies of the prior art, the object of the present invention is to provide an endophytic Pseudomonas putida strain of corn, which can effectively inhibit the growth of corn leaf blight pathogen and corn leaf blight pathogen, with an inhibition rate of 60.37% for corn leaf blight pathogen and 46.48% for corn leaf blight pathogen; at the same time, it has good chitinase activity and low cellulase and pectinase activity, is difficult to degrade plant cell walls, and is relatively friendly to plants.

[0004] The purpose of the present invention is achieved through the following technical solutions:

[0005] The present invention provides a corn endophytic Pseudomonas putida strain, named Pseudomonas putidaSF07, which is deposited in China Center for Type Culture Collection, with a deposit start date of September 26, 2024 and a deposit number of M20242084; the deposit address is: Wuhan University, Wuhan, China.

[0006] Specifically, the sequence of 16S rDNA of the corn endophytic Pseudomonas putida strain is as follows:

[0007]

[0008] The present invention also provides application of the endophytic Pseudomonas putida strain in corn for preventing and controlling corn diseases.

[0009] Specifically, the corn endophytic Pseudomonas putida strain is used to inhibit U. maydis and U. maydis.

[0010] Specifically, the corn leaf blight pathogen is Exserohilum turcicum.

[0011] Specifically, the corn leaf blight is caused by Bipolaris maydis.

[0012] The present invention also provides a biological pesticide for preventing and controlling corn diseases, comprising the corn endophytic Pseudomonas putida strain.

[0013] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0014] The corn endophytic Pseudomonas putida strain disclosed in the present invention is identified and named as Pseudomonas putida SF07, and is deposited in the China Center for Type Culture Collection with a deposit number of M 20242084. The strain of the present invention has an inhibitory effect on the growth of corn leaf blight pathogen and corn leaf blight pathogen under culture conditions, and the inhibition rate of corn leaf blight pathogen is 60.37%, and the inhibition rate of corn leaf blight pathogen is 46.48%, which has not been reported by predecessors; at the same time, it has good chitinase activity, can effectively degrade the cell wall of pathogens, and achieve the effect of killing pathogens, and at the same time has low cellulase and pectinase activity, is difficult to degrade plant cell walls, is relatively friendly to plants, and can be used as a biopesticide for corn. Since the main component of the insect exoskeleton is chitin, the strain of the present invention has a high chitinase activity and can be used to prepare insecticides. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a schematic diagram of the antibacterial activity test of Pseudomonas putida SF07 in the examples of the present invention against Exserohilum turcicum and Bipolaris maydis; in the figure, A is Exserohilum turcicum and B is Bipolaris maydis.

[0016] Figure 2 These are the chitinase activity test results of Pseudomonas putida SF07 and Paenibacillus polymyxa SF05 in the examples of the present invention.

[0017] Figure 3Schematic diagram of the cellulase activity test of Pseudomonas putida SF07 and Paenibacillus polymyxa SF05 in the examples of the present invention, wherein A is Pseudomonas putida SF07 and B is Paenibacillus polymyxa SF05.

[0018] Figure 4 Schematic diagram of the pectinase activity test of Pseudomonas putida SF07 and Paenibacillus polymyxa SF05 in the examples of the present invention, wherein A is Pseudomonas putida SF07 and B is Paenibacillus polymyxa SF05.

[0019] Figure 5 These are the test results of cellulase activity and pectinase activity of Pseudomonas putida SF07 and Paenibacillus polymyxa SF05 in the examples of the present invention. DETAILED DESCRIPTION

[0020] The present invention will be further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto.

[0021] Example 1: Isolation and identification of Pseudomonas putida SF07

[0022] The strain Pseudomonas putida SF07 of the present invention is isolated from corn leaf sheaths, and the specific operation method is as follows:

[0023] (1) Take the corn sheath tissue showing sheath blight symptoms and cut it into 3×3 mm pieces using a scalpel;

[0024] (2) Place the tissue in 70% alcohol for 5 seconds for surface disinfection, rinse with sterile water for 15 seconds, and repeat three times;

[0025] (3) Use sterile absorbent paper to absorb moisture from the tissue surface;

[0026] (4) Place the leaf sheath tissue in the center of the PDA plate and place the plate in an incubator at 28°C for 2 days;

[0027] (5) White hyphae grow around the tissue, but inhibition zones appear in some culture media. Bacteria in the center of the inhibition zones are picked and purified on a new LB plate;

[0028] (6) The isolated biocontrol colonies were picked and placed in fresh LB liquid medium and cultured at 28°C with shaking until the concentration was about 1×10 8 CFU / ml;

[0029] (7) Place a 5 mm diameter corn sheath blight fungus block in the center of the PDA plate, take 10 μl of bacterial liquid and drop it on the four corners 2 cm away from the fungus block, and incubate it at 28 °C for 7 days;

[0030] (8) The strain with the largest inhibition zone was selected for further antagonism experiments. The colony morphology and biological characteristics of the strain that still maintained good antibacterial activity after five consecutive generations were identified.

[0031] The main biological characteristics of the strain isolated above are as follows: SF07 is positive for nitrate reduction to nitrite, positive for arginine dihydratase, positive for hydrolysis of esculin (β-glucosidase), positive for hydrolysis of gelatin (protease), negative for urease, and can assimilate glucose, mannose, mannitol, gluconate, capric acid, malic acid, citric acid and phenylacetic acid. BIOLOG results showed that SF07 could grow at pH = 6, and could use carbon sources such as α-D-glucose, sodium lactate, fusidic acid, D-aspartic acid, troleandomycin, rifamycin SV, minocycline, L-aspartic acid, L-glutamic acid, L-histamine, L-pyroglutamic acid, L-serine, lincomycin, guanidine hydrochloride, sodium tetradecyl sulfate, L-galacturonolactone, D-gluconic acid, D-glucuronic acid, mucic acid, quinic acid, saccharic acid, vancomycin, tetrazolium violet, tetrazolium blue, L-lactic acid, citric acid, D-malic acid, L-malic acid, nalidixic acid, potassium tellurite, acetic acid, and sodium bromate for growth.

[0032] Combined with the following 16S rDNA sequencing, the bacterium was identified as Pseudomonas putida, named SF07, and deposited in the China Center for Type Culture Collection. The deposit start date is September 26, 2024, the deposit number is M20242084, and the deposit address is Wuhan University, Wuhan, China.

[0033] The sequence of Pseudomonas putida SF07 16S rDNA is as follows:

[0034] TAGACTAGCTACTTCTGGTGCAACCCACTCCCATGGTGTGACGGGC

[0035] GGTGTGTACAAGGCCCGGGAACGTATTCACCGCGACATTCTGATTCGCG

[0036] ATTACTAGCGATTCCGACTTCACGCAGTCGAGTTTGCAGACTGCGATCCG

[0037] GACTACGATCGGTTTTGTGAGATTAGCTCCACCTCGCGGCTTGGCAACC

[0038] CTCTGTACCGACCATTGTAGCACGTGTGTAGCCCAGGCCGTAAGGGCCA

[0039] TGATGACTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCT

[0040] CCTTAGAGTGCCCACCATAACGTGCTGGTAACTAAGGACAAGGGTTGCG

[0041] CTCGTTACGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGC

[0042] CATGCAGCACCTGTGTCAGAGTTCCCGAAGGCACCAATCCATCTCTGGA

[0043] AAGTTCTCTGCATGTCAAGGCCTGGTAAGGTTCTTCGCGTTGCTTCGAAT

[0044] TAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCATTTGAGT

[0045] TTTAACCTTGCGGCCGTACTCCCCAGGCGGTCAACTTAATGCGTTAGCTG

[0046] CGCCACTAAAATCTCAAGGATTCCAACGGCTAGTTGACATCGTTTACGG

[0047] CGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCACC

[0048] TCAGTGTCAGTATCAGTCCAGGTGGTCGCCTTCGCCACTGGTGTTCCTTC

[0049] CTATATCTACGCATTTCACCGCTACACAGGAAATTCCACCACCCTCTACC

[0050] ATACTCTAGCTCGCCAGTTTTGGATGCAGTTCCCAGGTTGAGCCCGGGG

[0051] CTTTCACATCCAACTTAACGAACCACCTACGCGCGCTTTACGCCCAGTAA

[0052] TTCCGATTAACGCTTGCACCCTCTGTATTACCGCGGCTGCTGGCACAGAG

[0053] TTAGCCGGTGCTTATTCTGTCGGTAACGTCAAAACAGCAAGGTATTAACT

[0054] TACTGCCCTTCCTCCAACTTAAAGTGCTTTACAATCCGAAGACCTTCTT

[0055] CACACACGCGGCATGGCTGGATCAGGCTTTCGCCCATTGTCCAATATTCC

[0056] CCACTGCTGCCTCCCGTAGGAGTCTGGACCGTGTCTCAGTTCCAGTGTG

[0057] ACTGATCATCCTCTCAGACCAGTTACGGATCGTCGCCTTGGTGAGCCATT

[0058] ACCTCACCAACTAGCTAATCCGACCTAGGCTCATCTGATAGCGCAAGGC

[0059] CCGAAGGTCCCCTGCTTTCTCCCGTAGGACGTATGCGGTATTAGCGTTCC

[0060] TTTCGAAACGTTGTCCCCACTACCAGGCAGATTCCTAGGCATTACTCAC

[0061] CCGTCCGCCGCTGAATCAAGGAGCAAGCTCCCGTCATCCGCTCGACTTG

[0062] CATGTGTTAGGCCTGCCGCCAGCGTTCAATCTGAGCAG.

[0063] Example 2: Determination of antibacterial activity of Pseudomonas putida SF07

[0064] The antibacterial activity of Pseudomonas putida SF07 of the present invention is determined by antagonistic culture method, and the specific operation is as follows:

[0065] (1) Pick fresh Pseudomonas putida SF07 colonies and place them in fresh LB liquid medium. Cultivate them at 28°C with shaking until the concentration is about 1×10 8 CFU / ml;

[0066] (2) Place a 5 mm diameter pathogenic fungus block in the center of the PDA plate, and drop 10 μl of the bacterial solution on the four corners 2 cm away from the block. The pathogenic fungi used in this study mainly refer to Exserohilum turcicum and Bipolaris maydis.

[0067] (3) A control experiment was conducted using sterile water instead of Pseudomonas putida SF07;

[0068] (4) After culturing at 28°C for 7 days, the colony diameters of the treatment group and the control group were measured and the inhibition rate was calculated. Inhibition rate (%) = 100 × (colony diameter of the treatment group - bacterial plate diameter) / (colony diameter of the control group - bacterial plate diameter).

[0069] The results showed that the inhibition rate of Pseudomonas putida SF07 on corn leaf blight was 60.37% ( Figure 1 A), the inhibition rate of corn leaf blight was 46.48% ( Figure 1 (B), it can be seen that Pseudomonas putida SF07 has good antibacterial activity against the above two pathogenic fungi.

[0070] Example 3: Chitinase, pectinase and cellulase activity test of Pseudomonas putida SF07

[0071] The chitinase activity of Pseudomonas putida SF07 of the present invention is determined using a chitinase activity detection kit (BC0825), and the specific operation is as follows:

[0072] (1) Pick fresh Pseudomonas putida SF07 colonies and place them in fresh LB liquid medium. Cultivate them at 28°C with shaking until the concentration is about 1×10 8 CFU / ml;

[0073] (2) The chitinase activity of Pseudomonas putida SF07 was tested using a chitinase activity detection kit (BC0825), and the laboratory-stored Peanibacillus polymyxa SF05 was used as a control.

[0074] The chitinase activity of SF07 was not significantly different from that of Paenibacillus polymyxa SF05 ( Figure 2 ).

[0075] The cellulase activity of Pseudomonas putida SF07 of the present invention is determined by a staining method, and the specific operation is as follows:

[0076] (1) Pick a fresh single colony of pear rust pathogen and inoculate it into LB liquid medium without antibiotics. Incubate the culture in a shaking incubator at 28°C overnight. Adjust the OD600 of the bacterial suspension to about 0.3 (1×10 8 CFU / ml).

[0077] (2) Add 2 μl of the bacterial suspension onto a cellulase test plate. After the plate is fully dried, place it in a 28°C incubator for 96 hours.

[0078] (3) Then, add a sufficient amount of 0.2% Congo red dye solution to the plate and stain for 20 min. Then, decolorize with 1 mol / L NaCl solution for 15 min, and stain with 1 mol / L HCl for 5 min. Peanibacillus polymyxa SF05 stored in the laboratory was used as a control.

[0079] The measurement found that the cellulase of SF07 was not detected and was significantly lower than that of Bacillus polymyxa SF05 ( Figure 3 , Figure 5 ).

[0080] The Pseudomonas putida SF07 pectinase activity was determined by staining method, and the specific operation was as follows:

[0081] (1) Pick a fresh single colony of pear rust pathogen and inoculate it into LB liquid medium without antibiotics. Incubate the culture in a shaking incubator at 28°C overnight. Adjust the OD600 of the bacterial suspension to about 0.3 (1×10 8 CFU / ml).

[0082] (2) Add 2 μl of the bacterial suspension onto a pectinase activity test plate. After the plate is dry, place it in a 28°C incubator for 96 hours.

[0083] (3) Add sufficient 7.5% copper acetate solution to the pectinase detection plate, stain for 1 hour, wash twice with water, and use Peanibacillus polymyxa SF05 stored in the laboratory as a control.

[0084] The results showed that no pectinase activity was detected in either SF07 or Paenibacillus polymyxa SF05 strains ( Figure 4 , Figure 5 ).

[0085] The above results show that the Pseudomonas putida SF07 of the present invention has good chitinase activity and low cellulase and pectinase activities, is difficult to degrade plant cell walls, is relatively friendly to plants, and can be used as a biopesticide for corn.

[0086] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A strain of endogenous Pseudomonas putidae, It is characterized in that Named Pseudomonas putida SF07, it is deposited in the China Model Culture Collection Center. The collection starts on September 26, 2024, and the collection number is M20242084; collection address: Wuhan University, Wuhan, China.

2. The endogenous Pseudomonas putida strain of corn according to claim 1, It is characterized in that The sequence of its 16S rDNA is as follows: TAGACTAGCTACTTCTGGTGCAACCCACTCCCATTCCCATGGTGTGACGGCGGTGTGTACAAGGCCGGGAACGTATTCACCGCGACATTCTGATTCGCGATTACTAGCGATTCCGACTTCACGCAGTCGAGTTGCAGACTGCGATCCGGACTACGATCGGTTTTGTGAGATTAGCTCCACC TCGCGGCTTGGCAACCCCTCTGTACCGACCATTGTAGCACGTGTGTAGCCCAGGCCGTAAGGGCCATGATGACTTGACGTCATCCCCACCTTCCTCTCTCCCGGTTGTCACCGCAGTCTCCTTAGAGTGCCACCATAACGTGCTGGTAACTAAGGACAAGGGTTGCGCTCGTTACGGGACT TAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCAGCACCTGTGTCAGAGTTCCCGAAGGCACCAATCCATCTCTGGAAAGTTCTCTGCATGTCAAGGCCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGCCCCGTCAATTCAT TTGAGTTTTAACCTTGCGCCGTACTCCCCCAGGCGGTCAACTTAATGCGTTAGCTGCGCCACTAAAATCTCAAGGATTCCAACGGCTAGTTGACATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCACCTCAGTTCAGTATCAGTCCAGTCCAGGT GGTCGCCTTCGCCACTGGTGTTCCTTCCTATATCTACGCATTTCACCGCTACACAGGAAATTCCACCACCCTCTACCATACTCTAGCTCGCCAGTTTGGATGCAGTTCCCCAGGTTGAGCCGGGCTTTCACATCCAACTTAACGAACCACCTACGCGCTTTACGCCCAGTAATT CCGATTAACGCTTGCACCCTCTGTATTACCGCGGCTGCTGGCACAGAGTTAGCCGGTGCTTATTCTGTCGGTAACGTCAAAACAGCAAGGTATTAACTTACTGCCCTTCCTCCCAACTTAAAGTGCTTTACAATCCGAAGACCTTCTTCACACACGCGGCATGGCTGGATCAGGCTTT CGCCATTGTCCAATATTTCCCCACTGCTGCCTCCCGTAGGAGTCTGGACCGTGTCTCAGTTCCAGTGTGACTGATCATCCTCTCTCCAGACCAGTTACGGATCGTCGCCTTGGTGAGCCATTACCTCACCAACTAGCTAATCCGACCTAGGCTCATCTGATAGCGCAAGGCCGAAGGTCC CCTGCTTTCCCGTAGGACGTATGCGGTATTAGCGTTCCTTTCGAAACGTTGTCCCCCACTACCAGGCAGATTCCTAGGCATTACTCACCGTCCGCCGCTGAATCAAGGAGCAAGCTCCCCGTCATCCGTCATCCGCTCGACTTGCATGTGTTAGGCCTGCCGCCAGCGTTCAATCTGAGCAG.

3. The application of the endogenous Pseudomonas putida strain of corn as claimed in claim 1, It is characterized in that Used for corn disease prevention and control.

4. The application of claim 3, It is characterized in that The Pseudomonas putida strain is used to inhibit corn spot bacteria and corn spot bacteria.

5. The application of claim 4, It is characterized in that The corn spot bacteria are Exerohilum turcicum.

6. The application of claim 4, It is characterized in that The corn spot disease is Bipolaris maydis.

7. A biological pesticide used for corn disease prevention and control, It is characterized in that The Pseudomonas putida strain of corn as claimed in claim 1 or 2 is included.

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