A strain of Pseudomonas palustris and a microbial agent and preparation method and application thereof

Abstract

The present invention discloses a strain of Pseudomonas fildesensis, named as Pseudomonas fildesensis CY58, which was deposited in China Culture Collection on September 27, 2024, with a deposit number of CCTCC NO: M 20242104. The present invention also discloses a microbial agent containing the Pseudomonas fildesensis CY58, and a preparation method and application thereof. The microbial agent containing Pseudomonas fildesensis CY58 or its fermentation liquid disclosed in the present invention can effectively prevent and treat tea anthracnose, pepper anthracnose, apple anthracnose and sorghum anthracnose, significantly slow down the incidence of plant diseases, has good prevention and treatment effects, especially has the best prevention and treatment effect on pepper anthracnose, and finds a new direction for the prevention and treatment of plant anthracnose.

Description

Technical Field

[0001] The invention relates to the technical field of microbial strains and applications thereof, and in particular to a strain of Pseudomonas palustris and a microbial agent and a preparation method and application thereof. Background Art

[0002] Pseudomonas is one of the most widely distributed microorganisms in biocontrol microorganisms and is also an important biocontrol group. Many strains have the function of inhibiting plant diseases and promoting plant growth. Pseudomonas can effectively interfere with the normal physiological metabolism of pathogens by producing antimicrobial active substances to dissolve the cell wall of pathogens or inhibit hyphae growth and spore germination, thereby inhibiting their growth and achieving effective control of diseases.

[0003] Colletotrichum is a globally distributed plant pathogenic fungus. It has a wide range of hosts, from gymnosperms to angiosperms, from monocots to dicots. It often harms the roots, stems, leaves, flowers, fruits and seedlings of fruit trees, vegetables, trees, flowers, medicinal plants and field crops, causing plant wilt, fruit rot, leaf spots and other symptoms, resulting in serious economic losses.

[0004] At present, the main Pseudomonas used for biological control are Pseudomonasfluorescens, Pseudomonasaeruginosa, and Pseudomonaschlororaphis. There are no reports on the use of Pseudomonas fildesensis to control plant diseases. Based on the above background, it is of great significance to actively carry out the screening of Pseudomonas fildesensis strains, enrich the Pseudomonas strain resource library, and discover Pseudomonas fildesensis with biocontrol potential, high inhibitory activity and stable function for the prevention and control of plant anthrax. Summary of the invention

[0005] One of the purposes of the present invention is to provide a strain of Pseudomonas fildesensis CY58, which has significant and stable performance in preventing and controlling plant anthracnose.

[0006] The second object of the present invention is to provide a microbial agent containing the Pseudomonas palustris CY58.

[0007] The third object of the present invention is to provide a method for preparing the microbial agent.

[0008] A fourth object of the present invention is to provide an application of the microbial agent.

[0009] To achieve the above object, the present invention provides the following technical solutions:

[0010] The present invention first provides a strain of Pseudomonas palustris CY58, which was deposited in the China Culture Collection on September 27, 2024, with a deposit number of CCTCC NO: M 20242104.

[0011] The Pseudomonas palustris CY58 and the fermentation liquid of Pseudomonas palustris CY58 both have antibacterial activity against anthrax bacteria.

[0012] The present invention also provides a microbial agent containing the Pseudomonas palustris CY58, wherein the microbial agent contains the Pseudomonas palustris CY58 or a fermentation liquid of the Pseudomonas palustris CY58.

[0013] In some specific embodiments, the fermentation broth concentration of Pseudomonas palustris CY58 in the microbial agent is 2×10 6 ~2×10 8 CFU / mL.

[0014] The present invention also provides a method for preparing the microbial agent, comprising the following steps:

[0015] S1. inoculating Pseudomonas CY58 from the Paldzia Gulf into LB solid medium for activation to obtain activated Pseudomonas CY58 from the Paldzia Gulf;

[0016] S2. The activated Pseudomonas CY58 of the Falz Bay was inoculated into LB liquid medium for a fermentation to obtain a seed solution of Pseudomonas CY58 of the Falz Bay;

[0017] S3. Inoculate the seed liquid of Pseudomonas palustris CY58 into LB liquid culture medium for secondary fermentation to obtain the microbial agent.

[0018] Furthermore, in step S1, the formula of the LB solid culture medium is: 8-12 g / L peptone, 4-6 g / L yeast extract, 9-11 g / L sodium chloride, 14-16 g / L agar powder, and pH 6.3-6.7.

[0019] Furthermore, in step S2 and step S3, the formula of the LB liquid culture medium is: peptone 8-12 g / L, yeast extract 4-6 g / L, sodium chloride 9-11 g / L, and pH 6.3-6.7.

[0020] The process conditions for activating Pseudomonas palustris CY58 are as follows: temperature 28-35° C., time 1-3 days.

[0021] Furthermore, in step S2, the process conditions of the primary fermentation are: liquid volume 40-60%, inoculation amount 1-3%, culture temperature 28-35°C, and shaking culture at 180-200 rpm for 16-20 hours.

[0022] Furthermore, in step S3, the process conditions of the secondary fermentation are: liquid volume 50% to 70%, inoculation amount 1 to 3%, culture temperature 28 to 35° C., and shaking culture at 180 to 200 rpm for 46 to 50 hours.

[0023] The present invention also provides an application of the Pseudomonas palustris CY58 or the microbial agent as described above or the microbial agent prepared by the preparation method as described above in preventing and controlling plant anthracnose pathogens, wherein the plant anthracnose pathogens are one or more of tea anthracnose pathogens, pepper anthracnose pathogens, apple anthracnose pathogens and sorghum anthracnose pathogens.

[0024] The use of the Pseudomonas Falzia CY58 or the above-mentioned microbial agent or the microbial agent prepared by the above-mentioned preparation method in preventing and controlling plant anthracnose pathogens comprises spraying and inoculating the Pseudomonas Falzia CY58 or the above-mentioned microbial agent on detached leaves of plants.

[0025] Furthermore, the plant is one or more of tea, pepper, apple and sorghum.

[0026] Furthermore, when the plant is pepper, when the pepper grows to 4 true leaves, the Pseudomonas Falzia CY58, the fermentation liquid of Pseudomonas Falzia CY58 or the microbial agent is inoculated by root irrigation.

[0027] Furthermore, the root irrigation inoculation volume is 30 ml / plant each time, applied in the early and early stages of the disease, with an interval of 7-8 days, and applied continuously for 3-4 times;

[0028] The concentration of the Pseudomonas CY58, the fermentation broth of Pseudomonas CY58 or the microbial agent is 1.5×10 8 ~2×10 8 CFU / mL.

[0029] Beneficial effects of the present invention:

[0030] The invention provides a strain of Pseudomonas fildesensis CY58. Through a plate confrontation experiment, it is found that the strain has a good antagonistic effect on tea anthracnose pathogens, pepper anthracnose pathogens, apple anthracnose pathogens and sorghum anthracnose pathogens. Through a plant detached leaf protection effect determination, it is found that the strain CY58 and a microbial agent prepared by the strain can effectively prevent and control tea anthracnose, pepper anthracnose, apple anthracnose and sorghum anthracnose, significantly slow down the disease of plants, have a good prevention and control effect, and find a new direction for the prevention and control of plant anthracnose. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a plate colony morphology of Pseudomonas fildesensis CY58;

[0032] Figure 2 Phylogenetic tree analysis of 16S rDNA sequence of Pseudomonas fildesensis CY58 constructed by the neighbor-joining method.

[0033] Figure 3 This is a diagram showing the antagonistic effect of Pseudomonas fildesensis CY58 on tea anthrax pathogens, wherein A: tea anthrax pathogens; B: confrontation culture effect diagram of Pseudomonas fildesensis CY58 and tea anthrax pathogens.

[0034] Figure 4 This is a diagram showing the antagonistic effect of Pseudomonas fildesensis CY58 on pepper anthrax pathogen, wherein A: pepper anthrax pathogen; B: confrontation culture effect diagram of Pseudomonas fildesensis CY58 and pepper anthrax pathogen.

[0035] Figure 5 This is a diagram showing the antagonistic effect of Pseudomonas fildesensis CY58 on apple anthracnose pathogens, wherein A: apple anthracnose pathogens; B: confrontation culture effect diagram of Pseudomonas fildesensis CY58 and apple anthracnose pathogens.

[0036] Figure 6This is a diagram showing the antagonistic effect of Pseudomonas fildesensis CY58 on sorghum anthracnose pathogen, wherein A: sorghum anthracnose pathogen; B: confrontation culture effect of Pseudomonas fildesensis CY58 and sorghum anthracnose pathogen. DETAILED DESCRIPTION

[0037] In order to facilitate the understanding of the present invention, the present invention will be described more comprehensively and meticulously below in conjunction with the accompanying drawings and preferred embodiments of the present invention, but the protection scope of the present invention is not limited to the following specific embodiments.

[0038] Unless otherwise defined, all professional terms used below have the same meanings as those generally understood by those skilled in the art. The professional terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention.

[0039] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0040] Example 1 Isolation and screening of bacteria

[0041] The separation and screening method of Pseudomonas fildesensis CY58 of the present invention comprises the following steps: fresh and symptom-free tea leaves are collected from Gaoqiao Demonstration Base in Changsha City, Hunan Province, washed with tap water, then soaked in 5% sodium hypochlorite for 1 minute and 75% alcohol for 30 seconds for disinfection, washed with sterile water for 2-3 times, put the tea leaves into a sterile mortar, add a little sterile water for grinding, separate the endophytic bacteria of the tea leaves by a plate dilution coating method, absorb 100 μL of the dilution solution, evenly coat it on LB culture medium, place it in a 30°C constant temperature incubator for cultivation and purification, and finally preserve the strain at -70°C with a 25% glycerol suspension to obtain the Pseudomonas fildesensis CY58 strain. Figure 1 This is a plate colony morphology of Pseudomonas fildesensis CY58.

[0042] Example 2 Classification and Identification of Bacteria

[0043] According to the Manual of Identification of Common Bacteria Systems, the morphological characteristics and physiological and biochemical characteristics of strain CY58 were identified. Strain CY58 grew well on LB solid medium, with a colony morphology that was approximately round, with irregular edges, a dry and wrinkled surface, small protrusions, and was opaque and milky white.

[0044] The strain CY58 was identified by 16S rDNA identification technology. The 16S rRNA gene was amplified using bacterial universal primers (sequences shown in SEQ ID: 2 and SEQ ID: 3). After PCR amplification, the PCR product was detected by electrophoresis using 1% agarose gel; the gene product obtained by PCR was sequenced, and the obtained sequence was entered into the NCBI database and compared and analyzed using BLAST software. Strains with higher homology to the strain were selected, and multiple sequence alignment was performed using MEGA7.0 software to establish a phylogenetic tree, see Figure 2 As shown. Comparison analysis found that the similarity between strain CY58 and Pseudomonas fildesensis reached 99.86%. Phylogenetic tree analysis and comparison found that strain CY58 and Pseudomonas fildesensis were clustered on the same branch. Combined with the characteristics of the plate colony, the strain was identified as Pseudomonas fildesensis and named Pseudomonas fildesensis CY58. It was deposited in the China Center for Type Culture Collection, Wuhan (Wuhan University), with the strain collection number of CCTCC NO: M 20242104, and the preservation time was September 27, 2024.

[0045] The results of 16S rRNA gene sequence determination are shown in SEQ ID NO.1.

[0046] SEQ ID: 1:

[0047]

[0048] SEQ ID: 2: 27F 5'-AGAGTTTGATCCTGGCTCAG-3';

[0049] SEQ ID: 3: 1492R 5'-TACGGCTACCTTGTTACGACTT-3';

[0050] Example 3 Antagonism experiment of Pseudomonas aeruginosa CY58 against anthrax pathogens

[0051] The plate confrontation method was used: tea anthracnose pathogens, pepper anthracnose pathogens, apple anthracnose pathogens and sorghum anthracnose pathogens were inoculated in the center of the PDA culture medium plate, and Pseudomonas fildesensis CY58 was inoculated 2.5 cm away from the center of the culture medium by the cross method; the control group was not inoculated with Pseudomonas fildesensis CY58, and was cultured at 30°C for 7 days. The diameter of the pathogen colony was measured, and each treatment was repeated 3 times. The results are shown in Figure 3-5 And as shown in Table 1.

[0052] In Table 1, the inhibition rate (%) = (control colony diameter - treated colony diameter) / control colony diameter * 100% Table 1 Inhibitory effect of Pseudomonas fildesensis CY58 on tea anthracnose pathogens, pepper anthracnose pathogens, apple anthracnose pathogens and sorghum anthracnose pathogens

[0053]

[0054] The results are as follows Figure 3-5 As shown in Table 1, Pseudomonas fildesensis CY58 has significant antagonistic effects on tea anthracnose pathogens, pepper anthracnose pathogens, apple anthracnose pathogens, and sorghum anthracnose pathogens. Among them, the inhibition rate against pepper anthracnose was the highest, at 78.60%; followed by tea anthracnose, sorghum anthracnose, and apple anthracnose, with inhibition rates of 75.21%, 54.29%, and 65.00%, respectively, indicating that Pseudomonas fildesensis CY58 has a good antagonistic effect on tea anthracnose pathogens, pepper anthracnose pathogens, apple anthracnose pathogens, and sorghum anthracnose pathogens.

[0055] Example 4 Preparation of Pseudomonas fildesensis CY58 bacterial agent

[0056] After activation of Pseudomonas fildesensis CY58 on LB solid medium, it was inoculated into a 150 mL conical flask containing 50 mL LB liquid medium and cultured at 28°C, 180 r / min for 18 h to prepare seed solution. Then, it was inoculated into LB liquid medium at a 1% inoculum and cultured at 28°C, 180 r / min for 48 h to obtain the stock solution. Before use, the fermentation solution concentration was adjusted to 2×10 6 CFU / mL, 2×10 7 CFU / mL, 2×10 8 CFU / mL, stored for future use.

[0057] Example 5 Determination of the efficacy of the in vitro leaf protection agent CY58 against Pseudomonas fildesensis

[0058] Select fresh and healthy leaves of tea, pepper, apple and sorghum, disinfect the surface with 75% alcohol, rinse with sterile water three times and dry, put them in the inoculation basin, moisten them with wet absorbent cotton, pierce the leaves with a sterile needle, and spray the fermentation liquid of strain CY58 with three treatments: T1 (2×10 6 CFU / mL), T2(2×10 7 CFU / mL), T3(2×10 8 CFU / mL), the spraying amount is appropriate to moisten the leaves without dripping, and after drying, inoculate the wound with tea anthracnose, pepper anthracnose, apple anthracnose, and sorghum anthracnose cakes with a diameter of 5 mm, and use the leaves not treated with the antagonistic bacteria fermentation liquid as the control (CK). Each treatment has 3 leaves, repeated 5 times, and finally covered with plastic wrap, placed in a 28℃ incubator for alternating light and dark, and observed the disease regularly. The diameter of the lesion was measured after 5 days. The results are shown in Table 2.

[0059] Among them, the control effect (%) in Table 2 = (lesion diameter of control group - lesion diameter of treatment group) / lesion diameter of control group × 100%.

[0060] Table 2 The control effect of Pseudomonas fildesensis CY58 on anthracnose of detached leaves of tea, pepper, apple and sorghum

[0061]

[0062]

[0063] According to the analysis in Table 2, after treatment with the fermentation liquid of Pseudomonas fildesensis CY58, the diameter of the lesions on the leaves of tea, pepper, apple and sorghum was significantly smaller than that of the control group. The fermentation liquid of Pseudomonas fildesensis CY58 at different concentrations had a certain control effect on tea anthracnose, pepper anthracnose, apple anthracnose and sorghum anthracnose, and the control effect increased with the increase of the fermentation liquid concentration, that is, the concentration of Pseudomonas fildesensis CY58 fermentation liquid was 2×10 8 CFU / mL, Pseudomonas falzia CY58 had the best control effect on tea anthracnose, pepper anthracnose, apple anthracnose and sorghum anthracnose.

[0064] Example 6 Determination of the efficacy of the fermentation liquid of Pseudomonas CY58 against anthrax in potted peppers

[0065] S1. Preparation of pepper anthracnose spore suspension: The fungus cake of pepper anthracnose was inoculated into PDB, and after shaking culture at 28°C and 180 r / min for 7 days, the mycelium was filtered through 4 layers of sterile gauze to obtain the spore suspension. The concentration of the spore suspension was counted using a hemocytometer, and the concentration of the spore suspension was adjusted to 1×10 8 CFU / mL.

[0066] S2. When the peppers have grown 4 true leaves, transplant them into small plastic flower pots filled with substrate soil, with one plant per pot, and place them in a (25±4)℃ greenhouse for routine management. After 5 days of seedling acclimatization, inoculate 30mL of 2×10 8 CFU / mL of CY58 fermentation broth, with water as the blank control. After the CY58 strain was colonized, 30 mL of 1×10 8 CFU / mL pepper anthracnose spore suspension. CY58 fermentation liquid treatment group and water control group were 5 plants each, with 3 replicates, and the disease status of the plants was observed and recorded after 15 days.

[0067] S3. Pepper plants were graded according to DB43 / T 1000-2015 "Technical Rules for Monitoring and Forecasting of Anthracnose in Peppers" and the incidence of peppers in each treatment group was statistically analyzed according to the following formula. The results are shown in Table 3.

[0068] Disease index = (number of diseased plants at each level × corresponding disease level value) / (total number of leaves × highest level value) × 100;

[0069] The control effect (%) = (disease index of the control group - disease index of the treatment group) / disease index of the control group × 100.

[0070] Table 3 The control effect of CY58 fermentation liquid on anthracnose of potted pepper

[0071]

[0072] The disease index was calculated based on the lesion classification to evaluate the control effect of CY58 fermentation liquid. The results are shown in Table 3. The disease index of peppers treated with CY58 fermentation liquid was 21.92, while the disease index of peppers treated with water control was 50.35, indicating that CY58 fermentation liquid had a certain control effect on potted peppers, with a control efficiency of 59.78%.

Claims

1. A strain of Pseudomonas palustris, characterized in that The Pseudomonas palindica was named Pseudomonas palindica ( Pseudomonas fildesensis )CY58, deposited in China Culture Collection on September 27, 2024, with the deposit number of CCTCC NO: M 20242104; The Pseudomonas palustris CY58 and the fermentation liquid of Pseudomonas palustris CY58 both have antibacterial activity against anthrax bacteria.

2. A microbial agent comprising the Pseudomonas palustris according to claim 1, characterized in that: The microbial agent comprises Pseudomonas palustris CY58 or the fermentation broth of Pseudomonas palustris CY58.

3. The microbial agent according to claim 2, characterized in that: In the microbial agent, the fermentation broth concentration of Pseudomonas falzia CY58 is 2×10 6 ~2×10 8 CFU / mL.

4. A method for preparing the microbial agent according to any one of claims 2 to 3, characterized in that: The following steps are involved: S1. The Pseudomonas CY58 of the Falz Bay was inoculated into LB solid medium for activation to obtain the activated Pseudomonas CY58 of the Falz Bay; S2. The activated Pseudomonas CY58 was inoculated into LB liquid medium for a fermentation to obtain a seed solution of Pseudomonas CY58; S3. The seed liquid of Pseudomonas palustris CY58 was inoculated into LB liquid culture medium for secondary fermentation to obtain the microbial agent.

5. The preparation method according to claim 4, characterized in that: In step S1, the formula of the LB solid culture medium is: peptone 8-12 g / L, yeast extract 4-6 g / L, sodium chloride 9-11 g / L, agar powder 14-16 g / L, and pH 6.3-6.7; the process conditions for activating Pseudomonas aeruginosa CY58 are: temperature 28-35°C, time 1-3 days; In step S2 and step S3, the formula of the LB liquid culture medium is: 8-12 g / L peptone, 4-6 g / L yeast extract, 9-11 g / L sodium chloride, and pH 6.3-6.7; In step S2, the process conditions of the primary fermentation are: liquid volume 40-60%, inoculation amount 1-3%, culture temperature 28-35°C, shaking culture at 180-200 rpm for 16-20 hours; In step S3, the process conditions of the secondary fermentation are: liquid volume 50%-70%, inoculation amount 1-3%, culture temperature 28-35°C, and shaking culture at 180-200 rpm for 46-50 hours.

6. Use of the Pseudomonas palustris described in claim 1, the microbial agent described in any one of claims 2 to 3, or the microbial agent prepared by the preparation method described in any one of claims 4 to 5 in preventing and controlling plant anthrax pathogens, characterized in that: The plant anthracnose pathogens are one or more of tea anthracnose pathogens, pepper anthracnose pathogens, apple anthracnose pathogens and sorghum anthracnose pathogens.

7. The use according to claim 6, characterized in that: The application comprises spraying and inoculating the Pseudomonas Falz Bay CY58 or the microbial agent on detached leaves of plants; the plants are one or more of tea, pepper, apple and sorghum.

8. The use according to claim 6, characterized in that: When the plant is pepper, when the pepper grows to 4 true leaves, root inoculation of the Pseudomonas Falzia CY58, the fermentation liquid of the Pseudomonas Falzia CY58 or the microbial agent is performed; The root irrigation inoculation volume is 30 ml / plant each time, applied in the early stage and early stage of the disease, with an interval of 7-8 days, and applied continuously for 3-4 times; The concentration of the Pseudomonas CY58, the fermentation broth of Pseudomonas CY58 or the microbial agent is 1.5×10 8 ~2×10 8 CFU / mL.

Images