A Pseudomonas with the functions of degrading phenolic acids and promoting growth and its application

Abstract

The present invention provides a Pseudomonas sp. with the functions of degrading phenolic acids and promoting growth. The Pseudomonas sp. is Pseudomonas putida strain FS-20, which is deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms with the deposit number of CGMCC No. 30971. This strain can degrade phenolic acids well and promote the growth of crops at the same time.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms and relates to a Pseudomonas bacterium capable of degrading phenolic acid substances and promoting growth and an application thereof. Background Art

[0002] Continuous cropping causes severe crop failure and soil degradation, necessitating an urgent need for reliable and stable soil bioremediation technologies to maintain soil health. Phenolic acids are important root secretions of plants. Continuous cropping leads to their accumulation, a major contributing factor to soil-borne diseases and the frequent occurrence of continuous cropping failure. Phenolic acids inhibit plant absorption of mineral nutrients. As phenolic acids accumulate in the rhizosphere, the balance of available nutrients such as nitrogen, phosphorus, and potassium in the soil gradually shifts, leading to soil degradation. Phenolic acids provide a rich carbon source for soil microorganisms, thus affecting microbial community distribution and potentially promoting the proliferation of harmful soil microorganisms. Phenolic acids are a major factor in plant allelopathic autotoxicity. When their concentrations reach a certain level, they can become autotoxic to plants, hindering their growth and development. Therefore, effectively and safely degrading phenolic acids to improve soil quality and alleviate continuous cropping failure offers a key development direction for improving crop yield and quality.

[0003] Microbial fertilizers produced using rhizosphere growth-promoting bacteria are a trend in the development of environmentally friendly agriculture. Phenolic acid-degrading bacteria are a type of plant rhizosphere growth-promoting bacteria. These bacteria degrade autotoxic allelopathic substances, inhibit the growth of harmful plant pathogens, promote plant growth, increase the number of beneficial microorganisms in the soil microbiome, and are environmentally friendly. Phenolic acid-degrading bacteria can degrade phenolic acids, an autotoxic allelopathic substance that accumulates in the soil, effectively alleviating crop problems caused by continuous cropping. This, in turn, promotes plant absorption of nutrients and improves crop yield and quality. Summary of the Invention

[0004] In order to solve the problems existing in the prior art, the present invention provides a Pseudomonas strain that has the functions of degrading phenolic acids and promoting growth. The strain can effectively degrade phenolic acids and promote crop growth.

[0005] The first object of the present invention is to provide a Pseudomonas sp. having the effect of degrading phenolic acids and promoting growth. The Pseudomonas sp., as determined by molecular biological identification, is a strain of Pseudomonas putida FS-20, which is deposited in the General Microbiology Center of the China Culture Collection Administration with a deposit number of CGMCC No. 30971.

[0006] The present invention isolated Pseudomonas putida FS-20 from the rhizosphere soil of continuously planted Lanzhou lily, and the strain was identified and named as Pseudomonas sp., and was deposited in the General Microbiology Center of China Culture Collection of Microorganisms (CGMCC) on June 17, 2024, with the deposit address being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number being CGMCC No. 30971.

[0007] The second object of the present invention is to provide a bacterial solution of Pseudomonas sp., which is obtained by expanding and culturing the above-mentioned Pseudomonas sp. CGMCC No. 30971 strain.

[0008] Preferably, the expanded culture is specifically: inoculating the Pseudomonas sp. CGMCC No. 30971 strain into a culture medium, and culturing it at 28±1° C. on a shaking table; preferably, the culture medium is LB liquid culture medium.

[0009] The third object of the present invention is to provide the use of the above-mentioned Pseudomonas sp. or Pseudomonas sp. bacterial solution in the preparation of products for degrading phenolic acid substances.

[0010] Preferably, the phenolic acid substance includes at least one of cinnamic acid and p-coumaric acid.

[0011] Preferably, the concentration of the cinnamic acid is 24-240 mg / L; the concentration of the p-coumaric acid is 1-4 g / L, preferably the concentration of the p-coumaric acid is 1-3 g / L.

[0012] A fourth object of the present invention is to provide the use of the aforementioned Pseudomonas sp. or Pseudomonas sp. bacterial solution in the preparation of products for overcoming continuous cropping obstacles or preventing and treating plant wilt diseases.

[0013] Preferably, the continuous cropping obstacle includes at least one of a plant pathogenic continuous cropping obstacle and a plant allelopathic autotoxicity-induced continuous cropping obstacle;

[0014] Preferably, the pathogen comprises Fusarium oxysporum.

[0015] A fifth object of the present invention is to provide the use of the aforementioned Pseudomonas sp. or Pseudomonas sp. bacterial solution in the preparation of products for improving soil physical and chemical properties and microbial environment and promoting crop growth.

[0016] Preferably, the crops include at least one of root vegetables, nightshade vegetables, leafy vegetables and lilies.

[0017] Preferably, the root vegetables include radish;

[0018] The nightshade vegetables include tomatoes;

[0019] The leafy vegetables include bok choy.

[0020] The Pseudomonas putida FS-20 strain of the present invention can effectively degrade cinnamic acid and p-coumaric acid, can effectively colonize in soil using cinnamic acid or p-coumaric acid as the sole carbon source, and can alleviate the autotoxicity stress caused by cinnamic acid and p-coumaric acid treatment on Lanzhou lilies. In addition, watering the root vegetable white radish, the nightshade vegetable tomato, the leafy vegetable bok choy, and the Lanzhou lily with a suspension of the Pseudomonas putida FS-20 strain has a plant growth-promoting effect.

[0021] The Pseudomonas putida FS-20 strain of the present invention has the function of degrading phenolic acids secreted by plant roots, thereby reducing the inhibitory effect of continuous cropping on plant growth, alleviating plant autotoxicity stress, promoting plant growth and absorption of nutrients, and improving crop yield and quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0023] Figure 1 This is the colony morphology of Pseudomonas putida FS-20.

[0024] Figure 2 The growth of Pseudomonas putida FS-20 on different solid culture media.

[0025] Figure 3 This is the molecular phylogenetic tree of Pseudomonas putida strain FS-20.

[0026] Figure 4 The growth of Pseudomonas putida FS-20 strain in different concentrations of cinnamic acid.

[0027] Figure 5 The growth of Pseudomonas putida FS-20 strain in different concentrations of p-coumaric acid.

[0028] Figure 6 is the degradation rate of Pseudomonas putida FS-20 strain in different concentrations of cinnamic acid.

[0029] Figure 7is the degradation rate of Pseudomonas putida FS-20 strain in different concentrations of p-coumaric acid.

[0030] Figure 8 Effects of different treatments on the soil colonization ability of Pseudomonas putida FS-20 strain.

[0031] Figure 9 Effects of different treatments on the growth of TOM tomato plants.

[0032] Figure 10 The results show the effects of different treatments on the growth of Lilium lanzhouensis plants.

[0033] Figure 11 Effects of different treatments on root activity of Lilium lanzhouensis.

[0034] Figure 12 Effects of different treatments on the activities of antioxidant enzymes in Lilium lanzhouense leaves.

[0035] Figure 13 The results show the effects of different treatments on the osmotic substances in the leaves of Lilium lanzhouensis.

[0036] Figure 14 Effects of different treatments on substrate enzyme activities in Lilium lanzhouense cultivation.

[0037] Figure 15 This is the effect of different treatments on the nutrient content of the cultivation medium of Lanzhou lily.

[0038] Figure 16 This is the effect of the Pseudomonas putida FS-20 strain of the present invention on the plant height and stem diameter of different plants.

[0039] Figure 17 This is the effect of the Pseudomonas putida FS-20 strain of the present invention on the leaf morphology of different plants.

[0040] Figure 18 This is the effect of the Pseudomonas putida FS-20 strain of the present invention on the root growth of different plants.

[0041] Figure 19 This is the effect of the Pseudomonas putida FS-20 strain of the present invention on the antioxidant enzyme activities of different plants.

[0042] Figure 20 This is the effect of the Pseudomonas putida FS-20 strain of the present invention on the osmotic substances of different plants. DETAILED DESCRIPTION

[0043] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples, unless otherwise specified, are conventional methods. The experimental materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent companies.

[0044] Example 1

[0045] 1. Preparation of culture medium:

[0046] LB solid medium: 10 g yeast powder, 20 g tryptone, 20 g sodium chloride, 28 g agar powder, 2000 mL distilled water, adjust the pH to 7.0-7.2, and sterilize in a high-pressure steam sterilizer at 121°C for 20 min.

[0047] PDA solid medium: Peel and weigh 20g of Atlantic potatoes, cut them into small pieces, place them in a pot, and add 100ml of distilled water. Cook until the potatoes are soft (easily crushed by a glass rod). Filter the cooked mixture through eight layers of cheesecloth. Add 1.5-2.0g of agar to the filtrate and stir until completely dissolved. Then, add 2g of glucose and mix thoroughly. After cooling, add distilled water to 100ml. Stir in an autoclave at 121°C for 20 minutes.

[0048] MSM liquid culture medium: K2HPO4 5.8 g, (NH4)2SO4 2.0 g, KH2PO4 4.5 g, CaCl2 0.02 g, MgCl2·6H2O 0.16 g, FeCl3 0.0018 g, Na2MoO4·2H2O 0.0024 g, MnCl2·2H2O 0.0015 g, distilled water 1000 mL, pH 7.0-7.2, autoclave at 121°C for 20 min.

[0049] Beef extract peptone agar medium: 6 g beef extract, 10 g peptone, 36 g agar, 2000 mL distilled water, pH 7.0-7.2, autoclave at 121°C for 20 min.

[0050] Indole test medium: 10 g / L tryptone aqueous solution, pH 7.2-7.6, sterilized in a high-pressure steam sterilizer at 110°C for 30 min.

[0051] Methylation (MR) test and acetylmethyl carbinol (VP) culture medium: 5 g glucose, 5 g peptone, 5 g K2HPO4 (or NaCl), 1000 mL distilled water, pH 7.0-7.2, autoclave at 110°C for 20 min.

[0052] p-Coumaric acid inorganic salt liquid culture medium: p-coumaric acid 0.5 g, K2HPO4 5.8 g, (NH4)2SO4 2.0 g, KH2PO4 4.5 g, CaCl2 0.02 g, MgCl2·6H20 0.16 g, FeCl3 0.0018 g, Na2MoO4·2H2O 0.0024 g, MnCl2·2H2O 0.0015 g, distilled water 1000 mL, pH 7.0-7.2, sterilization at 121°C for 20 min.

[0053] p-Coumaric acid inorganic salt solid culture medium: Add 15g / L agar powder to p-Coumaric acid inorganic salt liquid culture medium.

[0054] 2. Obtaining strains and preparing bacterial suspension

[0055] 2.1 Strain Acquisition: Weigh 5 g of rhizosphere soil sample from a continuously cropped field of Lilium chinense in Zhongbao Town, Lintao County, Dingxi City, Gansu Province, add it to 45 mL of sterile water, shake and mix thoroughly, and dilute it 10-fold to prepare a bacterial suspension. Take 100 μL of the bacterial suspension and spread it evenly onto a plate containing an inorganic salt solid medium containing p-coumaric acid. Place the plate in a 28°C incubator and observe the growth of the bacteria daily. Once clearly visible colonies have grown, isolate and purify them by plate streak method. This isolates and screens for strains that grow using p-coumaric acid as their sole carbon and energy source. The isolated and screened single colony of the target strain was transferred into the p-coumaric acid inorganic salt liquid culture medium (same as above), and cultured at 28°C and 170r / min with shaking for 7 days. The blank culture medium without bacteria was used as a control, and the content of p-coumaric acid was detected by high performance liquid chromatography (HPLC), thereby screening out the target strain Pseudomonas putida FS-20 (Pseudomonas putida) that can degrade p-coumaric acid. After identification, the strain was named Pseudomonas sp. and deposited in the General Microbiology Center of China Microorganism Culture Collection Committee (CGMCC) on June 17, 2024. The deposit address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is: CGMCC No. 30971.

[0056] 2.2 Preparation of bacterial suspension: Pick a single colony of Pseudomonas putida FS-20 and add it to LB liquid medium. After overnight culture in a constant temperature incubator at 28°C and 180 rpm, centrifuge at 11000 rpm for 5 minutes, pour out the supernatant, and resuspend the bacteria in sterile water to an OD value of 600 = 1 bacterial suspension for later use.

[0057] 3 The Pseudomonas putida FS-20CGMCC No.30971 of the present invention has the following biological characteristics:

[0058] 3.1 Morphological characteristics: The Gram stain test, starch hydrolysis test, methyl red reaction, acetylmethyl carbinol test, and indole reaction test of Pseudomonas putida FS-20 strain were all negative, and the hydrogen peroxide reaction test was positive.

[0059] 3.2 Culture characteristics: Pseudomonas putida FS-20 strain can be observed to have slightly yellow colonies with round protrusions, neat edges, and a foul odor on LB culture medium; while on beef extract peptone culture medium, the colonies are slightly yellow with irregular protrusions, sparse growth, and a foul odor.

[0060] Figure 1 Figure 1 shows the colony morphology of Pseudomonas putida FS-20. Figure A shows the colony morphology of Pseudomonas putida FS-20 on LB medium; Figure B shows the colony morphology of Pseudomonas putida FS-20 on beef extract peptone medium.

[0061] Figure 2 Figure 2 shows the growth of Pseudomonas putida FS-20 on different solid media. Figure A shows beef extract peptone medium, and Figure B shows LB solid medium.

[0062] 3.3 Genetic characteristics (strain 16srRNA sequence): The present invention measured the 16srRNA sequence of Pseudomonas putida FS-20 strain, which is as follows, with a full length of 1437 bp.

[0063]

[0064] AGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGCCTTGACATGCAGAGAAC TTCCAGAGATGGATTGGTGCCTTCGGGAACTCTGACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGT CGTGAGATGTTGGGTTAAGTCCCGTAACGAGCGCAACCCTTGTCCTTAGTTACCAGCACGTTATGGTGG GCACTCTAAGGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTT ACGGCCTGGGCTACACACGTGCTACAATGGTCGGTACAGAGGGTTGCCAAGCCGCGAGGTGGAGCTAAT CTCACAAAACCGATCGTAGTCCGGATCGCAGTCTGCAACTCGACTGCGTGAAGTCGGAATCGCTAGTAA TCGCGAATCAGAATGTCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAG TGGGTTGCACCAGAAGTAGCTAGTCTAACCTTCGGGAGGACGGTACCACGGATGATTTAGAGA

[0065] 3.4 Molecular phylogenetic identification of Pseudomonas putida strain FS-20

[0066] After extracting the genomic DNA of Pseudomonas putida FS-20 strain, PCR reaction was performed using primers 27F and 1492R. The result was amplification of a PCR product of approximately 1.5 kb, which was sent to the Chengdu branch of Beijing Qingke Biotechnology Co., Ltd. for first-generation sequencing.

[0067] The DNA sequence of Pseudomonas putida strain FS-20 was compared with sequences in the Gen-Bank database. The results showed that the 16S rDNA gene of the FS-20 strain was highly similar to that of multiple members of the genus Pseudomonas. The 16S rDNA gene sequences of related strains were further downloaded, and the molecular phylogenetic tree was constructed using MEGA7 after alignment analysis using ClustalX 2.1.

[0068] Figure 3 This is the molecular phylogenetic tree of Pseudomonas putida strain FS-20.

[0069] Example 2 Phenolic acid degradation function of Pseudomonas putida FS-20 strain CGMCC No. 30971 of the present invention under in vitro conditions

[0070] 1 Growth of Pseudomonas putida FS-20 strain in different concentrations of cinnamic acid and p-coumaric acid

[0071] 1 mL of Pseudomonas putida FS-20 suspension was added to MSM culture medium containing cinnamic acid (0.24 mg / L, 2.4 mg / L, 24 mg / L, 240 mg / L) and p-coumaric acid (1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L), and the OD values ​​were measured at 0 h, 12 h, 24 h, 36 h, 48 h, and 60 h, respectively. 600The absorbance value is used to indicate the growth of Pseudomonas putida FS-20. MSM medium without carbon source (pure MSM) was used as the control, and three replicates were set for each treatment.

[0072] Figure 4 The growth of Pseudomonas putida FS-20 strain in different concentrations of cinnamic acid.

[0073] Figure 5 The growth of Pseudomonas putida FS-20 strain in different concentrations of p-coumaric acid.

[0074] It has been determined that the Pseudomonas putida FS-20 strain of the present invention can effectively grow in a culture medium containing 24 mg / L and 240 mg / L cinnamic acid, or in a culture medium containing 1 g / L, 2 g / L, and 3 g / L p-coumaric acid, and can also grow slowly in a culture medium containing 4 g / L p-coumaric acid.

[0075] 2 Degradation efficiency of cinnamic acid and p-coumaric acid by Pseudomonas putida FS-20 strain

[0076] 1 mL of a Pseudomonas putida FS-20 suspension was added to MSM culture medium containing cinnamic acid (240 mg / L) and p-coumaric acid (1 g / L, 2 g / L, and 3 g / L). 2 mL samples were taken at 24, 36, 48, and 60 hours. The 2 mL suspension was centrifuged at 12,000 rpm for 5 minutes. 1 mL of the supernatant was collected and mixed with 1 mL of methanol at a 1:1 ratio. 0.1 mL of this mixture was then added to 1.9 mL of 50% methanol solution and sterilized by filtration through a 0.22 μm organic syringe filter. Residual phenolic acids were determined by HPLC. Phenolic acid degradation rates were determined using controls containing cinnamic acid and p-coumaric acid without the addition of FS-20 culture medium at varying concentrations.

[0077] Degradation rate of autotoxic substances = [1-(treated value / control value)] × 100%.

[0078] It was determined that the degradation rate of Pseudomonas putida FS-20 of the present invention for 24 mg / L of cinnamic acid was the highest at 48 hours, which was 44.83%; the degradation rates for 240 mg / L of cinnamic acid and 1 g / L and 2 g / L of p-coumaric acid were the highest at 60 hours, which were 85.76%, 96.60%, and 96.90%, respectively; and the degradation rate for 3 g / L of p-coumaric acid was the highest at 36 hours, which was 96.63%.

[0079] Figure 6 is the degradation rate of Pseudomonas putida FS-20 strain in different concentrations of cinnamic acid.

[0080] Figure 7is the degradation rate of Pseudomonas putida FS-20 strain in different concentrations of p-coumaric acid.

[0081] 3. Colonization ability of Pseudomonas putida FS-20 strain in substrate, and colonization ability of substrate after simultaneous application of cinnamic acid and FS-20, or simultaneous application of p-coumaric acid and FS-20.

[0082] Depend on Figure 7 It can be seen that the degradation rate of 1-3 g / L p-coumaric acid by Pseudomonas putida FS-20 is above 96%. Therefore, the colonization ability of the soil is similar within this range. The following uses 1 g / L p-coumaric acid as an example to illustrate the colonization ability after p-coumaric acid and FS-20 are simultaneously irrigated into the substrate.

[0083] Three 150g portions of substrate (vermiculite:perlite volume ratio = 3:1) were placed in 500mL beakers and sterilized at 121°C for 20 min. FS (FS-20 bacterial solution), FR (FS-20 bacterial solution + 240mg / L cinnamic acid), and FD (FS-20 bacterial solution + 1g / L p-coumaric acid) treatment solutions were then poured into the sterilized substrates. After mixing, 10g of the solution was placed in sealed Petri dishes and incubated in a 28°C incubator. Twelve dishes were used per treatment. Samples were collected at 0, 2, 4, 6, 9, 12, 16, 20, 25, 32, 37, and 40 days to determine fertility. For sampling, 1g of substrate was aseptically diluted (the dilution factor was the same for each group) from the center of the substrate using a sterile spatula. 100μL of the solution was then spread onto LB medium. The culture plates were placed at 28°C for 2 to 3 days, and the bacterial count on each plate was recorded. Finally, the bacterial count per gram of rhizosphere substrate (CFU / g) was calculated. The colonization density was used to represent the colonization capacity of treatments FS, FR, and FD in the substrate.

[0084] Pseudomonas putida FS-20 strain was found to be able to effectively and persistently colonize in soil using phenolic acids. Pseudomonas putida FS-20 strain was able to grow in soil using both cinnamic acid and p-coumaric acid as nutrients. Colonization density increased in treatments FS, FR, and FD on days 6, 12, and 32.

[0085] Figure 8 Effects of different treatments on the soil colonization ability of Pseudomonas putida FS-20 strain.

[0086] Therefore, the Pseudomonas putida FS-20 strain of the present invention can effectively colonize in the soil using 240 mg / L cinnamic acid solution or 1-3 g / L p-coumaric acid solution as the sole carbon source, and has a persistent effect.

[0087] Example 3 Application of Pseudomonas putida FS-20 strain CGMCC No.30971 of the present invention in vegetable production

[0088] Case 1: Promoting tomato growth by overcoming pathogenic continuous cropping barriers

[0089] The technical principle behind this case study is as follows: phenolic acids are a high-quality carbon source for rotting fungi. Continuous cropping causes root-secreted phenolic acids to accumulate in the soil, leading to an increase in the number of pathogenic fungi. Fusarium oxysporum is the primary fungus responsible for plant wilt. Root-secreted phenolic acids increase the number of Fusarium oxysporum, causing plant wilt and exacerbating continuous cropping problems. Therefore, it's highly likely that phenolic acid-degrading bacteria (such as FS-20) will alleviate pathogenic continuous cropping problems by degrading phenolic acids.

[0090] This case study used a two-factor design for tomatoes (TOM cherry tomatoes). Factor A: Tomato plants were inoculated with Fusarium oxysporum (a recognized wilt pathogen), causing wilt (a typical soil-borne disease); Factor B: FS-20 bacterial solution was irrigated into the tomato cultivation soil to degrade phenolic acids. There were four treatments, 15 plants per treatment, and three replicates.

[0091] (1) Treatment 1: Inoculation of Fusarium oxysporum. The inoculation method is as follows: culture the Fusarium oxysporum strain in PDA medium at 28°C in a constant temperature incubator for 7 days, place 12 cakes in each triangular flask, add glass beads, and shake at 28°C and 180 rpm for 24 hours. Filter and collect the filtrate, dilute the filtrate with sterile water, count the number of spores by hemocytometer, and dilute the spore solution to a concentration of 10 7 When the tomato has three leaves and one heart, water the tomato pot with 50 mL of Fusarium oxysporum solution. Water each pot once every five days for a total of eight times.

[0092] (2) Treatment 2: Inoculate with Fusarium oxysporum, irrigate each pot with 50 mL, irrigate once every 5 days, and irrigate 4 times. After the plant becomes ill, irrigate with the FS-20 bacterial solution of the present invention, irrigate each pot with 50 mL, irrigate once every 5 days, and irrigate 4 times. The method of inoculating Fusarium oxysporum is the same as that of treatment (1). The preparation method of the FS-20 bacterial solution of Pseudomonas putida is the same as that in step 2.2 of Example 1. Suspend the FS-20 bacterial solution with sterile water and adjust the concentration to OD 600 =1.

[0093] (3) Treatment 3: Clean water treatment: Water the pots with 50 mL of clean water, once every 5 days, for a total of 8 times.

[0094] (4) Treatment 4: Pseudomonas putida FS-20 bacterial solution of the present invention was poured in. The preparation method of Pseudomonas putida FS-20 bacterial solution was the same as that of treatment (2). 50 mL of the solution was poured into each pot, once every 5 days, for a total of 8 times.

[0095] The TOM cherry tomato seeds were sown in nutrient pots (8cm×10cm), with one seed sown per pot. The seeds were placed in an artificial climate chamber and cultured at 25°C, 85% relative humidity, and a photoperiod of 12h daytime / 12h nighttime. The treatment began when the plants grew to three leaves and one heart. Each plant was observed and recorded 5 days after the last treatment. Table 1 shows the classification standard for the disease level of TOM tomato wilt, Table 2 shows the incidence of TOM tomato wilt after treatment with Fusarium oxysporum and FS-20, and Table 3 shows the disease index after treatment with Fusarium oxysporum and FS-20. The experimental results are shown in Table 1. Figure 9 .

[0096] Figure 9 Figure 3 shows the effects of different treatments on the growth of TOM tomato plants. Figure A shows treatment group 1 (the Fusarium oxysporum group); Figure B shows treatment group 2 (the Fusarium oxysporum + FS-20 group); Figure C shows treatment group 3 (the control group); and Figure D shows treatment group 4 (the FS-20 group).

[0097] Depend on Figure 9 It can be seen that after the tomato was inoculated with Fusarium oxysporum, the plant showed typical symptoms of wilt disease: the tomato showed yellowing leaves, wilting, and stunted growth from the lower part of the plant to the upper part ( Figure 9 A), after watering the plants inoculated with the pathogen with FS-20 bacteria, the symptoms of yellowing and wilting of tomatoes were improved ( Figure 9 B). Compared with water irrigation ( Figure 9 C), tomatoes grow better after being watered with FS-20 bacterial solution ( Figure 9 D). The disease index of treatment 2 was 0.3667, which was significantly lower than that of treatment 1.

[0098] Table 1 TOM tomato wilt disease classification standards

[0099]

[0100] Note: The survey results of the number of diseased plants include treatment 1 (Fusarium oxysporum) and treatment 2 (Fusarium oxysporum + FS-20).

[0101] Table 2 Incidence of Fusarium oxysporum and FS-20 in TOM tomatoes after treatment

[0102]

[0103] Table 3 Disease index of Fusarium oxysporum after treatment with FS-20

[0104]

[0105] Among them: Disease index formula:

[0106]

[0107] In the formula: DI - disease index; s - representative value of each disease level; n - number of plants at each disease level; N - total number of plants surveyed; S - representative value of the highest disease level.

[0108] Conclusion: The Pseudomonas putida FS-20 strain of the present invention is capable of degrading phenolic acids, thereby competing with pathogenic microorganisms for carbon sources and inhibiting their proliferation. Simultaneously, the allelopathic autotoxic effects of phenolic acids on the plant itself are weakened. These two factors synergistically overcome pathogenic continuous cropping barriers and promote plant growth. Treatment with the phenolic acid-degrading bacteria (i.e., the present Pseudomonas putida FS-20 strain) improved tomato growth to varying degrees, with overall growth being promoted.

[0109] Case 2: Alleviating lily autotoxicity stress by reducing phenolic acid, optimizing soil physical and chemical properties, and promoting plant growth

[0110] Six treatment groups were designed: CK (sterile water), FS (FS-20 bacterial solution of the present invention), R (240 mg / L cinnamic acid), D (1 g / L p-coumaric acid), FR (FS-20 bacterial solution of the present invention + 240 mg / L cinnamic acid), and FD (FS-20 bacterial solution of the present invention + 1 g / L p-coumaric acid). Each treatment consisted of 15 strains, with three replicates.

[0111] The preparation method of the FS-20 bacterial solution of the present invention is the same as that in step 2.2 of Example 1. The concentration of the FS-20 bacterial solution in each treatment group is OD 600 =1.

[0112] Lily bulbs were planted in nutrient pots (8 cm x 10 cm) containing 250 g of sterilized peat:vermiculite in a 3:1 volume ratio) in an artificial climate chamber. The pots were incubated at 25°C, 85% relative humidity, and a 12-hour day / 12-hour night photoperiod. Treatments were initiated when the plants reached 10 cm in height. The plants were irrigated with 50 mL of the corresponding treatment solution per pot every five days for a total of eight irrigations. Sampling and measurement of relevant parameters were performed five days after the final treatment.

[0113] 1. Determination of plant growth and physiological indicators

[0114] Determination of plant growth indicators: plant height was measured with a tape measure (from the root collar to the growth point of the plant); stem diameter (diameter at the root collar of the plant) was measured with an electronic vernier caliper; leaf length, leaf width, leaf circumference, and leaf area were measured with a YMJ-C leaf area meter (leaves 5 cm below the growth point of the plant were selected); the dry and fresh weight of the aboveground and underground parts of the plant were measured with a 1 / 1000 electronic balance, and the total plant growth was the sum of the fresh weight of the aboveground and underground parts of the plant.

[0115] Plant physiological index measurements: Root activity, carotenoid content, chlorophyll content, superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbic acid peroxidase (APX) activities were measured. Proline (Pro) content was measured using the acid ninhydrin colorimetric method, malondialdehyde (MDA) content was measured using the thiobarbituric acid colorimetric method, and soluble protein content was measured using the Coomassie brilliant blue method.

[0116] The results of plant growth physiological indexes are shown in Tables 4-6 and Figure 10-13 .

[0117] Table 4 Effects of different treatments on growth indicators of Lilium lanzhouensis plants

[0118] deal with Plant height / cm Stem diameter / mm Leaf length / mm Leaf width / mm Leaf circumference / mm <![CDATA[Leaf area / mm 2 > CK 33.03±1.52ab 5.98±0.21ab 65.39±2.77a 6.90±0.89ab <![CDATA[139.51±6.21 a ]]> <![CDATA[198.71±14.81 a ]]> FS <![CDATA[34.79±1.78 a ]]> <![CDATA[6.23±0.31 a ]]> <![CDATA[70.05±5.14 a ]]> <![CDATA[9.40±1.66 a ]]> <![CDATA[152.04±10.34 a ]]> <![CDATA[210.44±12.06 a ]]> R <![CDATA[28.16±1.05 c ]]> <![CDATA[5.45±0.24 b ]]> <![CDATA[47.26±2.01 b ]]> <![CDATA[5.61±0.62 b ]]> <![CDATA[101.04±4.20 b ]]> <![CDATA[139.06±11.80 b ]]> D <![CDATA[30.22±1.30 bc ]]> <![CDATA[5.59±0.27 ab ]]> <![CDATA[46.99±1.57 b ]]> <![CDATA[5.54±0.34 b ]]> 100.25±3.41b 137.82±7.02b FR 35.44±1.33a 5.89±0.17ab <![CDATA[63.84±2.03 a ]]> 8.06±0.95ab 136.93±4.84a 203.95±7.75a FD <![CDATA[35.35±1.26 a ]]> <![CDATA[6.07±0.20 ab ]]> <![CDATA[64.09±2.69 a ]]> <![CDATA[6.69±1.36 ab ]]> 136.94±5.57a 199.16±9.56a

[0119] Table 5 Effects of different treatments on the fresh and dry weight of aboveground and underground parts of Lilium lanzhouensis plants

[0120]

[0121]

[0122] Table 6 Effects of different treatments on chlorophyll content of Lilium lanzhouensis plants

[0123]

[0124] Figure 10 The results show the effects of different treatments on the growth of Lilium lanzhouensis plants.

[0125] Figure 11 Effects of different treatments on root activity of Lilium lanzhouensis.

[0126] Figure 12 Effects of different treatments on the activities of antioxidant enzymes in Lilium lanzhouense leaves.

[0127] Figure 13 The results show the effects of different treatments on the osmotic substances in the leaves of Lilium lanzhouensis.

[0128] Conclusion: The Pseudomonas putida FS-20 strain of the present invention can alleviate the stress caused by cinnamic acid and p-coumaric acid treatment on plants, increasing the biomass, root activity, and chlorophyll content of Lanzhou lily. Physiological indicators of the plants, such as SOD, POD, CAT, malondialdehyde, and proline content, also improved to varying degrees. The Pseudomonas putida FS-20 strain of the present invention can enhance plant stress resistance and promote plant growth by altering the activity of key enzymes in secondary metabolic pathways or the biosynthesis of secondary metabolites.

[0129] 2 Physical and chemical properties of matrix:

[0130] 2.1 Determination of substrate physical and chemical properties and nutrient content

[0131] The matrix bulk density and porosity were determined by the ring knife method; the specific gravity was determined by the drainage weighing method; the soil moisture content was determined by the drying method; the soil pH was determined by a pH meter (HANNAHI8314 portable pH meter), and the soil conductivity was determined by a conductivity meter (Shanghai Leici Conductivity Meter DDS-307A); the alkaline nitrogen was determined by the alkaline diffusion method; and the available phosphorus was detected by the molybdenum antimony colorimetric method.

[0132] 2.2 Determination of matrix enzyme activity

[0133] Sucrase activity was determined by the nitro salicylic acid (DNS) colorimetric method, urease activity was determined by the indophenol blue colorimetric method, catalase activity was determined by the potassium permanganate calibration method, and alkaline phosphatase activity was determined by the disodium phenyl phosphate colorimetric method.

[0134] The results of the physical and chemical properties of the matrix are shown in Table 7 and Figure 14 、 15 .

[0135] Table 7 Physicochemical properties of cultivation substrates under different treatments

[0136]

[0137] Figure 14 Effects of different treatments on substrate enzyme activities in Lilium lanzhouense cultivation.

[0138] Figure 15 This is the effect of different treatments on the nutrient content of the cultivation medium of Lanzhou lily.

[0139] Conclusion: Phenolic acids interact with soil physicochemical properties. Phenolic acids in soil can activate insoluble nutrients in the rhizosphere, increasing soil nutrient levels. The Pseudomonas putida FS-20 strain, through its phenolic acid degradation, disrupts soil phenolic acid content and composition, optimizing soil physicochemical and biological properties, thereby promoting nutrient absorption and utilization and boosting vegetable growth.

[0140] In summary, the Pseudomonas putida FS-20 strain of the present invention can alleviate the stress caused by cinnamic acid treatment and p-coumaric acid treatment on plants, increase the plant height, leaf area, plant growth, and root length of Lanzhou lily, enhance root activity, increase chlorophyll content, and enhance the activities of soil sucrase, urease, and alkaline phosphatase, thereby improving the physical and chemical properties of the soil and the microbial environment.

[0141] Case 3 Growth-promoting effect of the Pseudomonas putida FS-20 strain of the present invention on different plants

[0142] This case further studied the growth-promoting effect of Pseudomonas putida FS-20 strain on root vegetables such as white radish, tomato and Shanghai green.

[0143] Two treatment groups were designed for each plant: CK group (sterile water) and FS group (FS-20 bacterial solution of the present invention), with 15 plants in each treatment and 3 replicates.

[0144] The preparation method of the FS-20 bacterial solution of the present invention is the same as that in step 2.2 of Example 1. The concentration of the FS-20 bacterial solution in each treatment group is OD 600 =1.

[0145] White water radish, Tom tomato and Shanghai green were planted in nutrient pots (a substrate with a volume ratio of sterilized peat: vermiculite = 3:1 was placed in a nutrient pot (8cm×10cm, 250g per pot), one seed was sown in each pot, and the pot was placed in an artificial climate chamber and cultured at 25°C, 85% relative humidity and a photoperiod of 12h day / 12h night. Treatment began when the Tom tomato plants grew to three leaves and one heart, and treatment began when the white water radish and Shanghai green plants grew to two leaves and one heart. Each pot was irrigated with 50mL of bacterial suspension, and watered once every 3 days (Tom tomato and white water radish were watered 9 times, and Shanghai green was watered 8 times). The control group CK can be replaced by an equal amount of sterilized water. Sampling and measuring relevant indicators began 5 days after the last treatment. The method for measuring relevant indicators is the same as in Case 2. The experimental results are shown in Tables 8-10 and Figure 16-20 .

[0146] Table 8 Effects of FS-20 on plant leaf growth

[0147]

[0148] Table 9 Effect of FS-20 on plant growth

[0149]

[0150] Table 10 Effect of FS-20 on chlorophyll content in plant leaves

[0151]

[0152] Figure 16 This is the effect of the Pseudomonas putida FS-20 strain of the present invention on the plant height and stem diameter of different plants.

[0153] Figure 17 The figure shows the effect of the Pseudomonas putida FS-20 strain of the present invention on the leaf morphology of different plants. Figure A shows Tom tomato, Figure B shows white radish, and Figure C shows bok choy.

[0154] Figure 18 This is the effect of the Pseudomonas putida FS-20 strain of the present invention on the root growth of different plants.

[0155] Figure 19 This is the effect of the Pseudomonas putida FS-20 strain of the present invention on the antioxidant enzyme activities of different plants.

[0156] Figure 20 This is the effect of the Pseudomonas putida FS-20 strain of the present invention on the osmotic substances of different plants.

[0157] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art will be able to modify the technical solutions described in the aforementioned embodiments or substitute equivalents for some of the technical features. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A Pseudomonas bacterium with the ability to degrade phenolic acids and promote growth ( Pseudomonas sp. ), characterized in that: The Pseudomonas is Pseudomonas putida ( Pseudomonas putida ) FS-20 strain, deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC No.30971.

2. A Pseudomonas putida bacterial liquid, characterized in that: The Pseudomonas strain described in claim 1 is obtained by expanding and culturing the strain with the deposit number of CGMCC No. 30971.

3. The Pseudomonas putida bacterial liquid according to claim 2, characterized in that: The expanded culture specifically includes: inoculating the Pseudomonas strain with a preservation number of CGMCC No. 30971 into a culture medium, and culturing at 28±1° C. on a shaking table.

4. The Pseudomonas putida bacterial liquid according to claim 3, characterized in that: The culture medium is LB liquid culture medium.

5. Use of the Pseudomonas according to claim 1 or the Pseudomonas putida bacterial solution according to any one of claims 2 to 4 in the preparation of products for degrading phenolic acid substances; the phenolic acid substances are: cinnamic acid with a concentration of 24-240 mg / L or p-coumaric acid with a concentration of 1-4 g / L.

6. The use according to claim 5, characterized in that: The concentration of p-coumaric acid is 1-3 g / L.

7. Use of the Pseudomonas according to claim 1 or the Pseudomonas putida bacterial solution according to any one of claims 2 to 4 in the preparation of products for overcoming continuous cropping obstacles or preventing and controlling tomato wilt; The continuous cropping obstacle is Fusarium oxysporum ( Fusarium oxysporum ) or the continuous cropping problem caused by the allelopathic autotoxic effects of cinnamic acid and p-coumaric acid.

8. Use of the Pseudomonas according to claim 1 or the Pseudomonas putida bacterial solution according to any one of claims 2 to 4 in the preparation of products for improving soil physical and chemical properties and microbial environment and promoting crop growth; the crops are radish, tomato, bok choy or lily.

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