Endophytic pantoea aglomerase and application thereof

Abstract

The application discloses a salt-tolerant endophytic Pantoea and application thereof, and relates to the technical field of microorganisms. Pantoea endophytica A salt-tolerant endophytic Pantoea (TH1) is isolated from grape young fruits, and the preservation number of the strain is CCTCC NO: M 2025960. The strain can promote grape growth, improve grape quality, increase the content of vanillin in the grape, increase the content of anthocyanin, flavonoids and flavanone in the grape, and reduce the content of tannin and total phenol in the grape. In addition, the strain has the abilities of high-efficiency potassium solution, nitrogen fixation, phosphorus dissolution and IAA production, and salt tolerance, can improve plant photosynthesis, improve soil environment and promote plant growth under salt stress, and can also be used for preventing and treating grape powdery mildew.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a salt-tolerant endophytic pantothecin strain and its applications. Background Technology

[0002] Vanillin (4-hydroxy-3-methoxybenzoic acid, C8H8O3), also known as vanillin or vanillin aldehyde, is an aromatic aldehyde compound extracted from vanilla beans and widely used in wine, spices, and other fields. In wine production, vanillin, as a key aromatic compound, directly affects the aroma characteristics of wine, significantly influencing its sensory appeal. However, the vanillin content in wine grapes is currently low. Therefore, increasing the vanillin content in grapes is crucial for improving the taste and aroma of wine.

[0003] Currently, vanillin preparation methods include plant extraction, chemical synthesis, and microbial transformation. Among these, plant extraction results in low yields and high production costs; chemical synthesis causes environmental pollution, and the enantiomers produced can affect product purity; while microbial transformation overcomes the geographical limitations of original plant material production, allowing for localized production and synthesis, thus conserving resources to the greatest extent, making it a current research hotspot. In the microbial transformation process, the substrates for vanillin production include ferulic acid, eugenol, and isoeugenol. Among these, ferulic acid, as one of the main phenolic acids in wine grapes, has low toxicity and high yield, making it a popular choice for preparing vanillin via microbial transformation.

[0004] Plant endophytes are bacteria or fungi that colonize within plant tissues and do not negatively impact plant growth. Their applications mainly include: crop stress regulation, development of medicinal secondary metabolites, bioremediation of polluted environments, and promoting nutrient absorption by plants. However, there are no reports of using plant endophytes to increase vanillin content in grapes. Summary of the Invention

[0005] In view of the above-mentioned prior art, the purpose of this invention is to provide a salt-tolerant endophytic pantothecin strain and its applications. This invention isolates an endophytic pantothecin strain ( ) from young grape berries. Pantoea endophytica TH1, this strain can increase the vanillin content in grape berries, promote grape growth, improve grape quality, increase the content of anthocyanins, flavonoids, and flavanones in grapes, and decrease the content of tannins and total phenols in grapes. In addition, this strain can also promote plant growth, improve plant photosynthesis, reduce disease occurrence, and improve plant stress resistance under salt stress.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides an endophytic pantothecin strain ( Pantoea endophytica TH1, this strain was deposited at the China Center for Type Culture Collection (CCTCC) on May 6, 2025, at Wuhan University, Bayi Road, Wuchang District, Wuhan, Hubei Province; its accession number is: CCTCC NO: M 2025960.

[0008] The endophytic pantothecin of the present invention ( Pantoea endophytica TH1 was isolated from young Cabernet Sauvignon grapes and has the following characteristics:

[0009] This strain can increase vanillin content in grapes, promote grape growth, improve grape quality, increase anthocyanin, flavonoid, and flavanone content, and decrease tannin and total phenol content. In addition, this strain has highly efficient potassium solubilization, nitrogen fixation, phosphorus solubilization, and IAA production capabilities, as well as salt tolerance. Under salt stress conditions, it can enhance plant photosynthesis, improve the soil environment, and promote plant growth. It can also be used to control grape powdery mildew.

[0010] A second aspect of the present invention provides a microbial agent, said microbial agent being the above-mentioned endophytic pantothecin ( Pantoea endophytica TH1 is the active ingredient.

[0011] Preferably, the endophytic pantothecin in the bacterial agent ( Pantoea endophytica TH1 exists in the form of cultured live bacteria, live bacterial fermentation broth, or bacterial suspension.

[0012] Furthermore, endophytic pantothecin ( Pantoea endophytica The TH1 bacterial suspension was prepared by the following method:

[0013] Endogenous pantothecin ( Pantoea endophytica TH1 was inoculated into LB liquid medium and cultured at 37°C and 180 rpm for 24 h. After centrifugation, the bacterial cells were collected and the bacterial suspension was obtained by distilling the bacterial cells with distilled water.

[0014] Furthermore, endophytic pantothecin ( Pantoea endophytica The inoculum size of TH1 was 1% of the volume of LB liquid medium; the centrifugation speed was 10,000 rpm and the centrifugation time was 10 min.

[0015] A third aspect of the present invention provides the above-mentioned endophytic pantothecin ( Pantoea endophytica TH1 or the inoculant may be used in any of the following: 1)-4)

[0016] 1) Increase the vanillin content in grape berries;

[0017] 2) Promotes grape growth and improves grape quality;

[0018] 3) Increase the content of anthocyanins, flavonoids, and flavanones in grape skins;

[0019] 4) Reduce the content of tannins and total phenols in grape skins.

[0020] In a fourth aspect, the present invention provides the above-mentioned endophytic pantothecin ( Pantoea endophytica The application of TH1 or fungicides in the control of grape powdery mildew and / or in the preparation of products for the control of grape powdery mildew.

[0021] In a fifth aspect, the present invention provides the aforementioned endophytic pantothecin ( Pantoea endophytica TH1 or the inoculant may be used in any of the following: 1)-4)

[0022] 1) Promotes plant growth under salt stress;

[0023] 2) Improve the photosynthetic capacity of plants under salt stress;

[0024] 3) Enhance the antioxidant capacity of plants under salt stress;

[0025] 4) Improve the soil environment under salt stress by increasing enzyme activity and soil nutrient content.

[0026] Preferably, the plant is grape or corn.

[0027] Preferably, the soil environment includes soil enzyme activity and soil nutrient content.

[0028] Preferably, the soil enzymes include sucrase, FDA hydrolase, and alkaline phosphatase.

[0029] Preferably, the soil nutrients include soil organic carbon, soil organic matter, soil available potassium, soil available phosphorus, soil available nitrogen, soil total potassium, soil total sodium, soil total nitrogen, and soil total phosphorus.

[0030] The beneficial effects of this invention are:

[0031] 1. This invention isolates and screens an endophytic pantothecin strain from young grape berries. Pantoea endophytica TH1, this strain can significantly increase the vanillin content in grapes, promote grape growth, improve grape quality, increase the content of anthocyanins, flavonoids and flavanones in grapes, and reduce the content of tannins and total phenols in grapes.

[0032] 2. The strain of this invention, as a plant endophyte, has excellent salt tolerance, nitrogen fixation, phosphorus solubilization, potassium solubilization, and IAA production capabilities.

[0033] 3. The endophytic pantothecin of the present invention ( Pantoea endophyticaTH1 can not only promote plant growth under salt stress, improve the photosynthetic capacity and antioxidant capacity of plants under salt stress, and increase soil enzyme activity and soil nutrient content under salt stress, but also improve the plant's resistance to powdery mildew under salt stress, thereby achieving effective control of grape powdery mildew. Attached Figure Description

[0034] Figure 1 Gram staining image of the TH1 strain of the present invention;

[0035] Figure 2 Phylogenetic tree based on 16S rRNA sequence;

[0036] Figure 3 Colony diagram of endophytic pantothenic bacterium TH1 cultured on combined nitrogen-fixing medium for 1 day;

[0037] Figure 4 Colony diagram of endophytic pantothenic bacterium TH1 cultured on inorganic phosphorus bacteria medium for 4 days;

[0038] Figure 5 Image of endophytic pantothecin TH1 colonies cultured on an organophosphate bacteria medium for 4 days;

[0039] Figure 6 Image of endophytic pantothenic acid bacterium TH1 colonies cultured on silicate medium for 4 days;

[0040] Figure 7 : The color development of endophytic pantothenic acid TH1 using the Salksowski staining method;

[0041] Figure 8 : Graph showing the quantitative determination results of IAA production by endophytic pantothenic bacteria TH1;

[0042] Figure 9 Figure 1 shows the growth characteristics of grape seedlings in a pot experiment.

[0043] Figure 10 Figure 1 shows the photosynthetic capacity results of a potted grape seedling experiment.

[0044] Figure 11 Figure 1 shows the results of the reactive oxygen species scavenging system measurement in a potted grape seedling experiment.

[0045] Figure 12 Figure 1 shows the soil enzyme measurement results of a potted grape seedling experiment.

[0046] Figure 13 Figure 2 shows the effect of endophytic pantothecin TH1 on grape fruit growth performance.

[0047] Figure 14Figure 2 shows the effect of endophytic pantothecin TH1 on vanillin content.

[0048] Figure 15 Figure 2 shows the effect of endophytic pantothecin TH1 on the content of secondary metabolites in grape skins.

[0049] Figure 16 Figure 3 shows the results of a potted corn seedling experiment.

[0050] Figure 17 Figure 4 shows the antagonistic effect against grape powdery mildew. Detailed Implementation

[0051] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0052] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0053] The experimental materials used in the embodiments of this invention are all conventional experimental materials in the art and can be purchased through commercial channels.

[0054] In this invention, the M9 glucose culture medium consists of 1g ammonium chloride, 0.13g magnesium sulfate, 3g potassium dihydrogen phosphate, 6g disodium hydrogen phosphate, and 0.338g glucose, dissolved in 1L of distilled water and sterilized at 115°C for 20 minutes.

[0055] The preparation method of FA selective liquid culture medium was based on Wu Fenghui (2020). The combined nitrogen-fixing bacteria culture medium, silicate bacteria culture medium, organic phosphorus bacteria culture medium and inorganic phosphorus bacteria culture medium were purchased from Qingdao Haibo Biotechnology Co., Ltd., with product numbers HB8541, HB8548-1, HB8549-1 and HB8549-2, respectively.

[0056] Example 1: Isolation and purification of strains

[0057] The surface of young Cabernet Sauvignon grapes from the experimental field (116.16'N, 36.17'E) of the Horticultural Experiment Station of Shandong Agricultural University was washed with running water to remove surface attachments. One g of grape sample was weighed, disinfected with 75% alcohol for 30 seconds, washed 3-5 times with sterile distilled water, rinsed in 0.1% mercuric chloride solution for 1 minute, and then washed 5 times with sterile distilled water. The disinfected and cleaned young Cabernet Sauvignon grapes were placed in a high-temperature sterilized mortar, and an appropriate amount of sterilized quartz sand and 9 mL of sterile water were added. The plant sample was ground, and after grinding, it was allowed to stand for 30 minutes. Then, 100 µL of the supernatant was spread onto M9 glucose medium and incubated at 28℃.

[0058] Single colonies with different morphological characteristics were selected for purification. The salt tolerance of the purified strains was determined using LB medium containing different concentrations of NaCl. The strain with the best salt tolerance (which could still grow on LB medium with 5% NaCl) was selected and named TH1, and stored in glycerol tubes.

[0059] Example 2: Identification of the strain

[0060] 1. Morphological identification of the strain

[0061] Gram staining of stable strain TH1 yielded a purple color ( ). Figure 1 This confirms that it is a Gram-negative bacterium. Under an optical microscope, it appears as a rod-shaped bacterium. The growth temperature of strain TH1 is 23–37°C, the pH range is 4–9, the optimal growth temperature is 28°C, and the optimal pH is 7.

[0062] 2. Identification of the strain's 16S rDNA:

[0063] Single colonies were picked from plates using the three-zone streak method and activated on LB agar. The bacterial culture was then sent to Shanghai Sangon Biotech for sequencing. After sequencing, the sequencing assembly results were submitted to NCBI for BLAST sequence alignment. 16S rRNA sequences of strains from closely related species were obtained from GenBank. Six to eight sequences with over 98% homology were selected, and a phylogenetic tree was constructed using MEGA64 with neighbor-joining. Phylogenetic analysis revealed that this strain is related to *Ubiquitin*. Pantoea endophytica The sequence similarity is the highest, such as Figure 2 As shown.

[0064] Based on the results of morphological, physiological and biochemical identification and 16S rDNA identification of the strain, strain TH1 was identified as an endophyte pantothenic bacterium. Pantoea endophytica (and carried out biological preservation of the patented technology, with the preservation information as follows:)

[0065] Referenced biological material (strain): TH1;

[0066] Suggested classification and nomenclature: Endophytic pantothecin Pantoea endophytica ;

[0067] Accession number: CCTCC NO: M 2025960.

[0068] Example 3: Growth-promoting properties

[0069] 1. Test method:

[0070] For endophytic pantothecin ( Pantoea endophytica The growth-promoting ability of TH1 was tested. The growth-promoting indicators included nitrogen fixation capacity, potassium solubilization capacity, phosphorus solubilization capacity, phosphorus production capacity, and IAA production capacity. The results are as follows: Figures 3-8 As shown. The specific detection method is as follows:

[0071] The methods for determining nitrogen fixation capacity, potassium solubilization capacity, phosphorus solubilization capacity, and phosphorus dissolution capacity are as follows: Purified endophytic pantothenia (… Pantoea endophytica TH1 single colonies were inoculated into LB liquid medium and cultured with shaking at 37 ℃ and 180 rpm for 24-48 h to activate them. Then, single colonies were inoculated into the corresponding medium for observation.

[0072] Among them, nitrogen fixation capacity was determined using a combined nitrogen-fixing bacteria culture medium, potassium solubilization capacity was determined using a silicate bacteria culture medium, phosphorus solubilization capacity was determined using an organic phosphorus bacteria culture medium, and phosphorus dissolving capacity was determined using an inorganic phosphorus bacteria culture medium.

[0073] The method for determining the IAA production capacity was as follows: qualitative analysis was performed using the Salkowski colorimetric method, with LB liquid medium containing 200 mg / L L-tryptophan and no bacteria used as a blank control, and IAA used as a positive control; quantitative analysis was performed using ultraviolet spectrophotometry.

[0074] 2. Experimental Results:

[0075] Depend on Figure 3 It can be seen that endophytic pantothecin ( Pantoea endophytica TH1 grew well on the combined nitrogen-fixing medium, indicating that it has good nitrogen-fixing ability. Figure 4 and Figure 5 It can be seen that endophytic pantothecin ( Pantoea endophytica TH1 grew on both inorganic and organic phosphorus-containing bacterial cultures, and the colonies exhibited distinct phosphate-solubilizing zones, demonstrating good phosphate-solubilizing and dissolving effects. Figure 6 It can be seen that endophytic pantothecin ( Pantoea endophyticaWhen TH1 was inoculated into silicate medium, the colony morphology was transparent oil droplets, indicating that it had a good potassium solubilizing effect.

[0076] Depend on Figure 7 As can be seen, the solution is pink in color, indicating the presence of endophytic pantothecin (…). Pantoea endophytica TH1 has the capability to produce IAA. (By...) Figure 8 It can be seen that after 72 hours, the amount of IAA secreted by strain TH1 was approximately 55.414 mg / L.

[0077] Example 4: Preparation of endophytic pantothecin TH1 bacterial suspension

[0078] The purified endophytic pantothenic acid TH1 was inoculated into LB liquid medium at a 1% (v / v) inoculation rate and cultured in a constant temperature shaking incubator at 37°C and 180 rpm for 24 h. The fermentation broth was then transferred to sterile centrifuge tubes and centrifuged at 10,000 rpm for 10 min at room temperature. The supernatant was carefully discarded, and the cells were resuspended three times in sterile distilled water. The OD was then measured. 600 The absorbance value at 600 nm was adjusted to 1 to prepare an endophytic pantothenic bacterium TH1 suspension.

[0079] Experiment 1: Growth-promoting experiment on Cabernet Sauvignon grape seedlings under salt stress

[0080] 1. Test method:

[0081] Vermiculite and vineyard soil were mixed in a 1:1 mass ratio and sterilized to obtain mixed soil. One-year-old Cabernet Sauvignon seedlings were planted in the mixed soil. When the seedlings grew to 5-6 functional leaves, Cabernet Sauvignon seedlings of uniform size and growth were selected for further treatment.

[0082] The experiment consisted of four treatment groups:

[0083] Single-inoculation treatment (TH1): Water each seedling pot with 500mL of clean water once every 5 days; 3 days after watering, apply 50mL of the endophytic pantothecin TH1 bacterial suspension prepared in Example 4 to each pot once a week for a total of two applications of bacterial suspension.

[0084] Single salt stress treatment (NaCl): Water each seedling pot with 500 mL of 250 mM NaCl solution once every 5 days; 3 days after watering with NaCl solution, apply 50 mL of clean water to each pot once a week, for a total of two waterings.

[0085] Salt stress + bacterial treatment (NaCl + TH1): 500 mL of 250 mM NaCl solution was applied to each pot of seedlings every 5 days; 3 days after applying the NaCl solution, 50 mL of the endophytic pantothecin TH1 bacterial suspension prepared in Example 4 was applied to each pot once a week for a total of two applications of bacterial suspension.

[0086] Control treatment (CK): Water each seedling with 500mL of clean water once every 5 days; 3 days after watering, add 50mL of clean water to each pot once a week, for a total of two waterings.

[0087] Water each seedling with 50 mL of clean water once a week for a total of two weeks. Each treatment group has 14 biological replicates, and the treatment lasts for 28 days.

[0088] Sampling and measurement of various indicators, including: growth indicators (plant height, stem diameter, root length, aboveground fresh weight, root fresh weight, aboveground dry weight, root dry weight), photosynthetic indicators (net photosynthetic rate, stomatal conductance, intercellular CO2 concentration, transpiration rate, water use efficiency, chlorophyll a content, chlorophyll b content, total chlorophyll content, carotenoid content), MDA content, hydrogen peroxide content, superoxide anion content, antioxidant enzyme (ascorbate peroxidase activity, peroxidase activity, superoxide dismutase activity) activity, and antioxidant substance (reduced glutathione) content.

[0089] In addition, the following parameters were measured in the treated soil: sucrase activity, FDA hydrolase activity, alkaline phosphatase, soil organic carbon content, soil organic matter content, soil available potassium content, soil available phosphorus content, soil available nitrogen content, soil total potassium content, soil total sodium content, soil total nitrogen content, and soil total phosphorus content.

[0090] 2. Experimental Results

[0091] Depend on Figure 9 It can be seen that under salt stress conditions, the application of endophytic pantothecin ( Pantoea endophytica Cabernet Sauvignon seedlings treated with TH1 bacterial suspension showed a significant increase in new leaves, with darker green foliage. Plant height, stem diameter, and root length were also significantly improved, as were fresh and dry weights. Figure 10 It can be seen that endophytic pantothecin ( Pantoea endophytica TH1 not only significantly increases the content of chlorophyll a, chlorophyll b, total chlorophyll, and carotenoids, but also improves net photosynthetic rate and reduces intercellular CO2 concentration. Therefore, strain TH1 can not only significantly increase the photosynthetic pigment content of Cabernet Sauvignon seedlings, but also enhance plant photosynthesis. Figure 11 It can be seen that endophytic pantothecin ( Pantoea endophytica TH1 application can significantly improve the reactive oxygen species scavenging system. Figure 12 It can be seen that the inoculated strains significantly improved the soil environment under salt stress conditions.

[0092] Experimental Example 2: Experiment on the growth and secondary metabolites of Cabernet Sauvignon fruit / Field trial

[0093] 1. Test method:

[0094] This experiment was conducted at the Horticultural Experiment Station of Shandong Agricultural University, using the fruit of the perennial "Cabernet Sauvignon" grape variety as the research object. On July 25, 2023, before the veraison stage, 14 bunches of fruit with uniform growth were selected for each treatment.

[0095] This experiment consisted of four treatment groups, as detailed below:

[0096] Control group (CK): 20 mL of sterile water per bunch of grapes was sprayed evenly onto the surface of the grapes, once a week, for a total of three treatments.

[0097] Group C1: Concentration was 1×10 6 Endophytic pantothenic bacterium TH1 suspension at a concentration of CFU / mL;

[0098] Group C2: Concentration was 1×10 8 Endophytic pantothenic bacterium TH1 suspension at a concentration of CFU / mL;

[0099] Group C3: Concentration was 1×10 9 An endophytic pantothenic bacterium TH1 suspension at a concentration of CFU / mL.

[0100] The preparation method of the endophytic pantothecin TH1 bacterial suspension is the same as in Example 4. The inoculation solution was sprayed evenly onto the surface of the grape berries at a rate of 20 mL per bunch, once a week, for a total of three treatments.

[0101] After the grapes reach maturity on August 31, 2023, the grapes will be collected, and various indicators of the grapes will be measured, including: single berry weight and 100-berry weight, transverse and longitudinal diameter of the grape, anthocyanin content in the grape skin, tannin content in the grape skin, total phenolic, flavonoid, and flavanol content in the grape, and vanillin content. Among these:

[0102] The method for detecting anthocyanin content in grape skins was based on the experimental methods of Lee J et al. (2017) and Ribéreau-Gayon et al. (1965).

[0103] The method for determining the tannin content in grape skins is as follows: Weigh 1 g of grape skin into a 50 mL centrifuge tube, add 20 mL of water, boil in a water bath for 30 min, and filter through double-layer filter paper into a 50 mL beaker. Take 1 mL of the extract into a 10 mL centrifuge tube, add 0.5 mL of Fc-Folin-Ciocalteu reagent and 6 mL of water, mix well, immediately add 1 mL of 20% Na2CO3 solution, shake well, and dilute to 10 mL with deionized water. After standing at room temperature for 30 min, measure the absorbance at a UV wavelength of 680 nm (Geng Nana et al., 2013).

[0104] The determination procedures for total phenols, flavonoids, and flavanols in grapes were as follows: 1 g of grape peel was weighed into a 10 mL centrifuge tube, 4 mL of 1% hydrochloric acid-methanol solution was added, and the mixture was extracted by sonication under sealed conditions for 30 min. Water was then added to bring the volume to 8 mL, and the mixture was incubated overnight at 4°C for 24 hours in the dark. The solution was then centrifuged at 10,000 rpm for 10 min at 4°C. The stock solution was stored in a refrigerator at 4°C in the dark. The determination of total phenols and flavonoids was based on Petra et al., 2019; the determination of flavanols was based on Wang Yanping et al., 2017.

[0105] Vanillin content: The vanillin content was determined using high performance liquid chromatography (HPLC). The specific steps were as follows:

[0106] (1) Preparation of vanillin standard solution:

[0107] Weigh the vanillin standard separately, dissolve it in methanol (HPLC grade), and prepare a standard solution with a concentration of 0.01 mg / mL.

[0108] (2) Sample pretreatment:

[0109] Take 0.5 g of grape pulp and peel samples respectively, grind them with liquid nitrogen, transfer them to pre-cooled centrifuge tubes, add 5 mL of methanol, and shake in the dark for 24 h. After removal, centrifuge at 10000 rpm for 10 min, filter and retain the supernatant. Take 400 μL of the supernatant, dilute with distilled water to 2 mL, add an equal volume of dichloromethane, shake in the centrifuge for 9 h, centrifuge, aspirate the lower extract, evaporate in a metal bath at 50℃, add 300 μL of methanol for reconstitution, and take 200 μL for sample loading. Before loading, extract the sample by sonication for 30 min.

[0110] (3) The vanillin content was detected by SHIMADZU Nexera UHPLC LC-30A high performance liquid chromatograph. The detection wavelength of vanillin was 280 nm. The chromatographic column was a Hypersil BDS C18 column (250 mm*4.6 mm, 5 μm). The column temperature was 25℃, the flow rate was 1 mL / min, the injection volume was 20 μL, and the mobile phase was methanol:1% glacial acetic acid = 35:65 (V / V).

[0111] 2. Experimental conclusions:

[0112] Depend on Figure 13 It can be seen that the bacterial suspension of strain TH1 can significantly increase the single fruit weight, 100-fruit weight, and transverse and longitudinal diameter of grape fruits.

[0113] Vanillin plays a crucial role in the growth, ripening, and fruit quality formation of grapes. This invention utilizes high-performance liquid chromatography (HPLC) to determine the vanillin content in grape pulp and skin. The residence time of the vanillin standard solution was 6.94 min, and the content was calculated based on peak area. Figure 14 It can be seen that, compared with the control (CK), the vanillin content in grape skins and pulp increased significantly after treatment with endophytic pantothecin TH1.

[0114] Tannins, anthocyanins, flavanones, and total phenols in grape skins are key components determining fruit quality. Figure 15 It can be seen that, compared with the control group, the spraying concentration of 1×10 8 A CFU / mL suspension of endophytic pantothenic bacteria TH1 can significantly increase the content of anthocyanins, flavonoids, and flavanones, while decreasing the content of tannins and total phenols.

[0115] Experimental Example 3: Growth-promoting experiment on maize under salt stress

[0116] 1. Test method:

[0117] Vermiculite and cornfield soil were mixed in a 1:1 mass ratio and then sterilized to obtain mixed soil. Corn seedlings were planted in the mixed soil, and when the seedlings grew to two leaves and a bud, corn seedlings of uniform size and growth were selected for further treatment.

[0118] The experiment consisted of four treatment groups:

[0119] Single-inoculation treatment (TH1): At the beginning, thoroughly water the soil with clean water. After 3 days, water each seedling with 20 mL of the endophytic pantothecin TH1 bacterial suspension prepared in Example 4, once a week for a total of three times.

[0120] Single salt stress treatment (NaCl): At the beginning, thoroughly water the soil with the same amount of NaCl solution (250mM) as the single inoculation treatment. After 3 days, water each seedling with 20mL of clean water once a week for a total of three times.

[0121] Salt stress + bacterial treatment (NaCl + TH1): At the beginning, the soil was thoroughly watered with the same amount of NaCl solution (250mM) as the single bacterial treatment. After 3 days, each seedling was watered with 20mL of the endophytic pantothecin TH1 bacterial suspension prepared in Example 4, once a week for a total of three times.

[0122] Control treatment (CK): At the beginning, the soil was thoroughly watered with the same amount of water as the single-inoculation treatment. After 3 days, each seedling was watered with 20mL of water once a week for a total of three times.

[0123] Eight biological replicates were set up for each treatment group. Samples were taken on day 27 after treatment to measure various indicators, including: plant height, stem diameter, aboveground fresh weight, underground fresh weight, root length, dry weight, chlorophyll a content, chlorophyll b content, total chlorophyll content, and carotenoid content.

[0124] 2. Test Results:

[0125] like Figure 16 As shown, endophytic pantothenic bacteria were introduced under salt stress conditions. Pantoea endophytica The biomass (total fresh weight, aboveground fresh weight, underground fresh weight, dry weight, plant height, and root length) of maize seedlings inoculated with TH1 bacteria suspension was significantly increased compared to uninoculated maize seedlings. Furthermore, the inoculation of the strain significantly increased the content of photosynthetic pigments in maize seedlings under salt stress conditions.

[0126] Experiment Example 4: Antagonistic Experiment of Grape Powdery Mildew under Salt Stress

[0127] 1. Test method:

[0128] Vermiculite and vineyard soil were mixed in a 1:1 mass ratio and sterilized to obtain mixed soil. One-year-old Cabernet Sauvignon seedlings were planted in the mixed soil. When the seedlings grew to 5-6 functional leaves, Cabernet Sauvignon seedlings of uniform size and growth were selected for further treatment.

[0129] Preparation of powdery mildew pathogen spore suspension: The pathogen causing grape powdery mildew is *Hylocereus undatus*. Infected grape leaves were collected, and powdery mildew spores were gently brushed into sterilized water. After mixing, a spore suspension was obtained. The spore concentration in the suspension was adjusted to ≥ 3 × 10⁻⁶ using a hemocytometer method. 6 / mL, ready for use.

[0130] The experiment consisted of four treatment groups:

[0131] Pathogen + fungicide treatment (powdery mildew + TH1): Water each seedling pot with 500mL of clean water, spray 20mL of powdery mildew pathogen spore suspension on both sides of the grape leaves, and 3 days later, apply 50ml of endophytic pantothecin TH1 fungal suspension prepared in Example 4 to each pot.

[0132] Salt stress + pathogen treatment (NaCl + powdery mildew): Apply 500mL of 250mM NaCl solution to each seedling pot, spray 20mL of powdery mildew pathogen spore suspension on both sides of the grape leaves, and after 3 days, apply 50ml of water to each pot.

[0133] Salt stress + pathogen + fungal treatment (NaCl + powdery mildew + TH1): Apply 500 mL of 250 mM NaCl solution to each seedling pot, spray 20 mL of powdery mildew pathogen spore suspension on both sides of the grape leaves, and after 3 days, apply 50 mL of endophytic pantothecin TH1 fungal suspension prepared in Example 4 to each pot.

[0134] Control treatment (CK + pathogen): Water each seedling with 500mL of clean water, spray 20mL of powdery mildew pathogen spore suspension on both sides of the grape leaves, and 3 days later, apply 50ml of clean water to each seedling.

[0135] Fourteen biological replicates were set up for each treatment group to observe the growth of plants in each treatment and their resistance to powdery mildew.

[0136] 2. Test Results:

[0137] The results are as follows Figure 17 As shown, Cabernet Sauvignon seedlings inoculated with endophytic pantothecin TH1 suspension under pathogen stress exhibited significantly increased plant height compared to uninoculated seedlings, indicating a reduction in powdery mildew stress. Furthermore, under salt stress, the damage caused by powdery mildew pathogens to Cabernet Sauvignon seedlings increased. However, even under the dual stress of salt stress and pathogen stress, inoculation with endophytic pantothecin TH1 suspension still significantly reduced powdery mildew stress, and plant height also increased significantly. These results indicate that endophytic pantothecin TH1 can enhance plant resistance to powdery mildew under salt stress conditions.

[0138] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A strain of endophytic pantoea (Pantoea endophytica) TH1, with accession number CCTCC NO: M 2025960, characterized in that, The endophytic pantoea TH1 was isolated from young Cabernet Sauvignon grapes and is a plant endophyte; it is a Gram-negative bacterium with a growth temperature of 23-37℃ and a pH range of 4-9. The endophytic pantoea TH1 has the following functions: (1) Increase the vanillin content in grape berries, increase the anthocyanin, flavonoid and flavanone content in grape skins, and decrease the tannin and total phenol content in grape skins; (2) Improve the resistance of grapes to powdery mildew under salt stress; (3) Promote the growth of grapes under salt stress.

2. A microbial agent, characterized in that, The microbial agent uses Pantoea endophytica TH1 as the active ingredient as described in claim 1.

3. The microbial agent as described in claim 2, characterized in that, In the bacterial agent, Pantoea endophytica TH1 exists in the form of cultured live bacteria, live bacterial fermentation broth, or bacterial suspension.

4. The microbial agent as described in claim 3, characterized in that, The bacterial suspension was prepared by the following method: Pantoea endophytica TH1 was inoculated into LB liquid medium and cultured at 37°C and 180 rpm for 24 h. After centrifugation, the bacterial cells were collected and resuspended in distilled water to obtain the bacterial suspension.

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