Plant salt tolerance promoting metabolite isolated from rhizosphere growth promoting pseudomonas putida and application thereof

Abstract

The application discloses a plant salt-tolerance promoting active metabolite separated from a rhizosphere growth-promoting Pseudomonas proteus and application thereof. The metabolite is dibutyl phthalate (DBP) with a molecular formula of C 16 H 22 O4, has the effect of improving the salt tolerance of rice, and obviously increases the plant height, root length, aboveground fresh weight and underground fresh weight of the rice, is stable in effect, and has a good application prospect; and can be used as a preparation for improving the salt tolerance of crops in agricultural green production.

Description

Technical Field

[0001] This invention belongs to the field of microbiology, specifically a plant salt-tolerant active metabolite isolated from the rhizosphere-promoting Pseudomonas prolifera and its application. Background Technology

[0002] In recent years, soil salinization has become a prominent problem, affecting nearly 1 billion square kilometers of soil globally. Rational improvement of saline soils through scientific and technological means can enhance the comprehensive utilization of land and is of practical significance for alleviating the conflict between human and land resources and ensuring food security.

[0003] *Pseudomonas* is a genus of Gram-negative aerobic or microaerophilic bacteria belonging to the phylum *Proteobacteria*. This genus encompasses a diverse range of bacteria widely distributed in nature, including soil, water, food, and air. Over a long evolutionary process, *Pseudomonas* has developed varied metabolic capabilities and a broad potential for environmental adaptation. This adaptability allows *Pseudomonas* to survive and reproduce in various extreme environments, including sediments, clinical samples, plant rhizosphere, and the bodies of diseased animals. *Pseudomonas prevoyense*, as a member of the genus *Pseudomonas*, has an evolutionary history closely related to the entire genus, exhibiting similar adaptability and survival strategies. Current research on *Pseudomonas prevoyense* in plants mainly focuses on biocontrol of plant diseases, promoting plant growth, the structure and function of rhizosphere microbial communities, and the discovery and functional analysis of novel lipopeptide plant immune elicitors. However, research on the identification of bioactive substances from *Pseudomonas prevoyense* in the rice rhizosphere is relatively limited. Previous laboratory studies screened a strain of *Pseudomonas proteus* from the rhizosphere of rice plants grown in saline-alkali soil. This strain can significantly improve the germination rate of rice seeds and plant growth indicators under salt stress, whether applied directly or used as a seed coating. However, the substances in the strain that enhance rice salt tolerance are still unclear. Developing functional microbial preparations that enhance crop salt stress tolerance and improve crop growth in saline-alkali soil by utilizing the secondary metabolites produced by the fermentation of this strain has strong application prospects. Summary of the Invention

[0004] The purpose of this invention is to provide a salt-tolerant active substance fermented by Pseudomonas protozoa, and to provide a method for extracting and separating the salt-tolerant active substance and its application.

[0005] The strain provided by this invention has been deposited at the China General Microbiological Culture Collection Center (CGMCC; address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; postcode: 100101). The *Pseudomonas preibryophyte* strain described herein has the accession number CGMCC No. 28061.

[0006] To address the aforementioned technical problems, this invention first provides the application of dibutyl phthalate (I) in improving crop salt tolerance.

[0007]

[0008] (I).

[0009] Preferably, the dibutyl phthalate is extracted from the supernatant of Pseudomonas proteoglycans fermentation broth or chemically synthesized.

[0010] Preferably, the dibutyl phthalate is extracted from the supernatant of the fermentation broth of *Pseudomonas proteus* Pse_ST, with accession number CGMCC No. 28061.

[0011] The method for isolating the active metabolite for improving crop salt tolerance from *Pseudomonas protozoa* includes: fermenting *Pseudomonas protozoa* in Landy medium and collecting the supernatant of the fermentation broth; extracting the supernatant with n-hexane; collecting the n-hexane organic phase and removing the solvent by rotary evaporation; then separating the metabolite by silica gel column chromatography, using a petroleum ether-n-hexane mixture and a n-hexane-methanol mixture for elution; and finally preparing the active metabolite for improving crop salt tolerance by HPLC under the following conditions: an Ultimate® XB-C18 column (250 mm × 4.6 mm, 5 μm, Yuexu Technology (Shanghai) Co., Ltd.); a mobile phase of acetonitrile:water = 35:65 (v:v); a detection wavelength of 278 nm; a flow rate of 1 ml / min; and an injection volume of 20 ml.

[0012] The Landy medium contains 5 g of L-glutamic acid, 0.5 g of magnesium sulfate heptahydrate, 0.5 g of potassium chloride, 1 g of potassium dihydrogen phosphate, 0.15 mg of ferrous sulfate heptahydrate, 5 mg of manganese sulfate tetrahydrate, 0.16 mg of copper sulfate heptahydrate, 1 g of yeast extract, 2 mg of L-phenylalanine, and 20 g of glucose per liter. After adjusting the pH to 7.0, it is sterilized at 115°C for 30 min.

[0013] The method for preparing the Pseudomonas proteobacterium fermentation broth is as follows: 1% of the Pseudomonas proteobacterium seed culture is transferred to an Erlenmeyer flask containing fresh Landy medium and cultured at 30°C and 170 rpm for 2 days; then the cultured broth is inoculated into a 100L fermenter, which is filled with 70L of Landy medium. After the broth enters the stationary phase, it begins to produce secondary metabolites in large quantities and continues fermentation for 3 days.

[0014] Preferably, the application of dibutyl phthalate of formula (I) in promoting rice growth under salt stress.

[0015] The application of dibutyl phthalate, as shown in formula (I), in the preparation of formulations that improve crop salt tolerance.

[0016]

[0017] (I).

[0018] Preferably, the use of dibutyl phthalate of formula (I) in the preparation of formulations that promote the growth of rice under salt stress.

[0019] A method for extracting dibutyl phthalate from the fermentation broth of *Pseudomonas protozoa* includes fermenting *Pseudomonas protozoa* and collecting the supernatant of the fermentation broth. The supernatant is extracted with n-hexane, and the hexane organic phase is collected and the solvent is removed by rotary evaporation. Separation is then performed using silica gel column chromatography, successively using a petroleum ether-n-hexane mixture and a hexane-methanol mixture. Finally, the salt-tolerant active substance is prepared by HPLC using an Ultimate® XB-C18 column (250 mm × 4.6 mm, mobile phase: acetonitrile:water = 35:65, detection wavelength: 278 nm, flow rate: 1 ml / min, injection volume: 20 ml). The *Pseudomonas protozoa* strain Pse_ST is preferably preserved under the CGMCC No. 28061.

[0020] Preferred fermentation method for *Pseudomonas proteus* includes: first, inoculating the strain stored at -80℃ into LB solid medium and culturing overnight at 30℃; then, selecting a single colony and inoculating it into LB liquid medium, culturing overnight at 30℃ and 170 rpm to obtain a seed culture; transferring the *Pseudomonas proteus* seed culture at an inoculation rate of 1% into an Erlenmeyer flask containing fresh Landy medium, culturing at 30℃ and 170 rpm for 2 days; then, inoculating the cultured culture into a 100L fermenter, filling the fermenter with 70L of Landy medium, and after the culture enters the stationary phase, starting to produce secondary metabolites in large quantities, and continuing fermentation for 3 days.

[0021] Beneficial effects:

[0022] This invention, through hydroponic experiments during the seedling stage, verified that the fermentation broth of *Pseudomonas protozoa* enhances the salt tolerance activity of rice, explored the optimal culture medium and conditions for the fermentation of secondary metabolites, and isolated and purified a benzene ring-based salt-tolerant growth-promoting substance, C. 16 H 22 O4 was used to identify the molecular structure of the substance. Verification showed that this salt-tolerant active substance can promote rice plant height, root length, and both above-ground and underground fresh weight. Attached Figure Description

[0023] Figure 1To investigate the salt tolerance and life-promoting effects of Pseudomonas procymidone fermentation broth on rice seedlings under salt stress.

[0024] A represents the effect of Pseudomonas protozoa fermentation broth on the growth phenotype of rice seedlings under salt stress.

[0025] The effect of strain B, *Pseudomonas protozoa* fermentation broth, on the plant height of rice seedlings under salt stress.

[0026] C represents the effect of Pseudomonas protozoa fermentation broth on the root length of rice seedlings under salt stress.

[0027] D represents the effect of Pseudomonas protozoa fermentation broth on the aboveground fresh weight of rice seedlings under salt stress.

[0028] E represents the effect of Pseudomonas protozoa fermentation broth on the fresh weight of the underground parts of rice seedlings under salt stress.

[0029] Figure 2 Salt tolerance and life-promoting properties of rice seedlings under relative salt stress were determined by extracting various strains of Pseudomonas protozoa fermentation broth.

[0030] A represents the effect of various extracts from the fermentation broth of *Pseudomonas proteus* strain on the growth phenotype of rice seedlings under salt stress.

[0031] B represents the effect of various extracts from the fermentation broth of strain *Pseudomonas proteus* on the relative height of rice seedlings under salt stress.

[0032] C represents the effect of various extracts from the fermentation broth of *Pseudomonas proteus* strain on the root length of rice seedlings under salt stress.

[0033] D represents the effect of various extracts from the fermentation broth of *Pseudomonas protozoa* strain on the relative fresh weight of aboveground parts of rice seedlings under salt stress.

[0034] E represents the effect of various extracts from the fermentation broth of *Pseudomonas proteobacterium* strain on the relative fresh weight of the underground parts of rice seedlings under salt stress.

[0035] Figure 3 The sixth segment of the hexane phase separation from the fermentation broth of *Pseudomonas protozoa* strain demonstrates its salt tolerance and life-promoting properties in rice seedlings under salt stress.

[0036] A represents the effect of the sixth hexane-phase separation of the fermentation broth from strain *Pseudomonas protozoa* on the growth phenotype of rice seedlings under salt stress.

[0037] The effect of the sixth hexane phase separation of the fermentation broth of strain B, *Pseudomonas protozoa*, on the plant height of rice seedlings under salt stress.

[0038] C represents the effect of the sixth hexane phase separation of the fermentation broth from strain *Pseudomonas protozoa* on the root length of rice seedlings under salt stress.

[0039] D represents the effect of the sixth hexane phase separation of the fermentation broth from strain *Pseudomonas protozoa* on the aboveground fresh weight of rice seedlings under salt stress.

[0040] E represents the effect of the 6th-4th fraction of the hexane phase separation from the fermentation broth of strain *Pseudomonas protozoa* on the fresh weight of the underground parts of rice seedlings under salt stress.

[0041] Figure 4 The salt tolerance and life-promoting properties of rice seedlings under salt stress were determined by separating the 6th-4th fraction of the n-hexane phase from the fermentation broth of *Pseudomonas protozoa* strain.

[0042] A represents the effect of the 6th-4th fractions of the hexane phase separation from the fermentation broth of strain *Pseudomonas protozoa* on the growth phenotype of rice seedlings under salt stress.

[0043] The effect of the 6th-4th fraction of the hexane phase separation from the fermentation broth of strain B, *Pseudomonas proteus*, on the plant height of rice seedlings under salt stress.

[0044] C represents the effect of the 6th-4th fraction of the hexane phase separation from the fermentation broth of strain *Pseudomonas protozoa* on the root length of rice seedlings under salt stress.

[0045] D represents the effect of the 6th-4th fraction of the hexane phase separation from the fermentation broth of strain *Pseudomonas protozoa* on the aboveground fresh weight of rice seedlings under salt stress.

[0046] E represents the effect of the 6th-4th fraction of the hexane phase separation from the fermentation broth of strain *Pseudomonas protozoa* on the fresh weight of the underground parts of rice seedlings under salt stress.

[0047] Figure 5 To promote the salt tolerance and survival of rice seedlings under salt stress using DBP

[0048] A represents the effect of DBP on the growth phenotype of rice seedlings under salt stress.

[0049] B represents the effect of DBP on the plant height of rice seedlings under salt stress.

[0050] C represents the effect of DBP on the root length of rice seedlings under salt stress.

[0051] D represents the effect of DBP on the aboveground fresh weight of rice seedlings under salt stress.

[0052] E represents the effect of DBP on the fresh weight of the underground part of rice seedlings under salt stress.

[0053] Biological sample preservation information

[0054] Pseudomonas prednisone Pseudomonas punonensis It is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, on July 28, 2023, with accession number CGMCC No. 28061. Detailed Implementation

[0055] This invention discloses an active substance for improving the salt tolerance of rice. Those skilled in the art can refer to the content of this document and appropriately modify the parameters to achieve the desired result. To enable those skilled in the art to better understand the technical solution of this invention, the invention will be further described in detail below with reference to specific implementation examples.

[0056] The rice variety used in the experiment was Nipponbare.

[0057] Example 1: Verification of the effect of the Pseudomonas proteobacterium fermentation broth of the present invention on promoting salt tolerance and life of rice.

[0058] A 1% inoculum of *Pseudomonas proteobacterium* seed culture was transferred to an Erlenmeyer flask containing fresh Landy medium and incubated at 30°C and 170 rpm for 2 days. The cultured culture was then inoculated into a 100L fermenter, which was filled with 70L of Landy medium. After the culture reached the stationary phase, secondary metabolites were produced in large quantities, and fermentation continued for 3 days. The culture was centrifuged at 7000 rpm and 4°C for 10 minutes to remove bacterial cells, and the supernatant was collected and stored at 4°C for later use.

[0059] After surface disinfection and germination, seedlings of Nipponbare rice were selected and transplanted into hydroponic containers containing 1L of Hogrange nutrient solution. Each liter of Hogrange nutrient solution contained 0.115 g of ammonium dihydrogen phosphate, 0.242 g of magnesium sulfate heptahydrate, 0.605 g of potassium nitrate, 1.359 g of calcium nitrate tetrahydrate, 0.181 g of calcium chloride dihydrate, 30.78 mg of disodium ethylenediaminetetraacetate and 27.75 mg of ferrous sulfate heptahydrate, 7.2 mg of boric acid, 0.2 mg of copper chloride dihydrate, 4.5 mg of manganese chloride tetrahydrate, 0.6 mg of zinc chloride, and 0.098 mg of ammonium molybdate monohydrate. The pH of the Hogrange nutrient solution was 6.0. The nutrient solution was changed every three days, and the seedlings were cultured until they reached the three-leaf stage for hydroponic growth under salt stress.

[0060] Rice seedlings with uniform growth were selected and their roots were immersed in the supernatant for 24 hours. Seedlings immersed only in Hoagland's nutrient solution served as a control. After immersion, the seedlings were transplanted into Hoagland's nutrient solution containing 100 mM NaCl for a salt stress growth experiment. The nutrient solution was changed every 3 days, and various indicators of the rice plants were measured after one week of cultivation.

[0061] The testing indicators include plant height, above-ground and underground fresh weight. Plant height was measured using a measuring tape, and the above-ground and underground fresh weights were measured using an electronic balance.

[0062] Results and Analysis

[0063] like Figure 1As shown, CK represents the treatment without salt or bacterial strain, 100 mM NaCl represents the treatment with salt but without bacterial strain, and the others represent the treatment with different concentrations of bacterial strain.

[0064] like Figure 1 As shown in Figure A, the fermentation broth of Pseudomonas proteus strain significantly improved the hydroponic growth of rice seedlings under salt stress. The growth of rice seedlings soaked in the fermentation broth of Pseudomonas proteus strain was significantly better than that of the control treatment with added salt but without the fermentation broth.

[0065] like Figure 1 As shown in Figure B, the fermentation broth of Pseudomonas proteus strain significantly improved the hydroponic growth of rice seedlings under salt stress. The plant height of rice seedlings soaked in the fermentation broth of Pseudomonas proteus strain was significantly higher than that of the control treatment with added salt but without the fermentation broth of strain.

[0066] like Figure 1 As shown in Figure C, the fermentation broth of Pseudomonas proteus strain significantly improved the hydroponic growth of rice seedlings under salt stress. The root length of rice seedlings soaked in the fermentation broth of Pseudomonas proteus strain was significantly higher than that of the control treatment with added salt but without the fermentation broth of strain.

[0067] like Figure 1 As shown in Figure D, the fermentation broth of Pseudomonas proteus strain significantly improved the hydroponic growth of rice seedlings under salt stress. The fresh weight of the aboveground parts of rice seedlings soaked in the fermentation broth of Pseudomonas proteus strain was significantly higher than that of the control treatment with added salt but without the fermentation broth of strain.

[0068] like Figure 1 As shown in Figure E, the fermentation broth of Pseudomonas proteus strain significantly improved the hydroponic growth of rice seedlings under salt stress. The fresh weight of the underground parts of rice seedlings soaked in the fermentation broth of Pseudomonas proteus strain was significantly higher than that of the control treatment with added salt but without the fermentation broth of strain.

[0069] Example 2: Verification of the relative salt tolerance and life-promoting effects of different extraction methods of the fermentation broth of the strain described in this invention on rice.

[0070] The rice germination and three-leaf stage seedling cultivation were carried out in the same manner as in Example 1.

[0071] The supernatant of the strain was collected in the same manner as in Example 1.

[0072] The supernatant of the bacterial strain was poured into a separatory funnel, ethyl acetate was added and shaken well, and after standing for 30 minutes, the ethyl acetate organic phase was collected. The separated aqueous phase was then mixed thoroughly with n-hexane, and after standing for 30 minutes, the n-hexane organic phase was collected. The separated aqueous phase was then mixed thoroughly with petroleum ether and allowed to stand for 30 minutes, and the petroleum ether organic phase was collected. Finally, the separated aqueous phase was mixed thoroughly with chloroform and allowed to stand for 30 minutes, and the chloroform organic phase was collected. All collected organic phases were then evaporated to dryness using a rotary evaporator and stored at -80°C for later use.

[0073] Rice seedlings of uniform growth were subjected to salt stress growth experiments in a mixture containing 100 mM NaCl in Hogrange solution and dimethyl sulfoxide (DMSO) solutions of all components. DMSO served as a control group. The mixture was changed every 3 days, and after one week of cultivation, various indicators of the rice plants were measured. The measured indicators included plant height, aboveground and underground fresh weight. Plant height was measured using a measuring tape, and aboveground and underground fresh weights were measured using an electronic balance.

[0074] Results and Analysis

[0075] like Figure 2 As shown, CK represents the treatment without salt or added bacterial fermentation broth extract phase material, 100 mM NaCl represents the treatment with salt but without added bacterial fermentation broth extract phase material, and 0.1% DMSO represents the treatment with only added DMSO solution.

[0076] like Figure 2 As shown in Figure A, the addition of hexane extract from the fermentation broth of *Pseudomonas protozoa* significantly improved the hydroponic growth of rice seedlings under salt stress. The rice seedlings treated with hexane extract from the fermentation broth of *Pseudomonas protozoa* showed significantly better growth than the control treatment without added salt and without the fermentation broth. The rice seedlings treated with other extracts also showed better growth.

[0077] like Figure 2 As shown in Figure B, the addition of hexane extract phase material from the fermentation broth of Pseudomonas protozoa significantly improved the hydroponic growth of rice seedlings under salt stress. The plant height of rice seedlings treated with hexane extract phase from the fermentation broth of Pseudomonas protozoa was significantly better than that of the control treatment without added salt and without the fermentation broth of Pseudomonas protozoa, and the plant height of rice seedlings treated with other extract phase materials was also longer.

[0078] like Figure 2 As shown in Figure C, the addition of hexane extract phase material from the fermentation broth of Pseudomonas protozoa significantly improved the hydroponic growth of rice seedlings under salt stress. The root length of rice seedlings treated with hexane extract phase from the fermentation broth of Pseudomonas protozoa was significantly better than that of the control treatment without added salt and without the fermentation broth of Pseudomonas protozoa, and the root length of rice seedlings treated with other extract phase materials was also longer.

[0079] like Figure 2 As shown in Figure D, the addition of hexane extract phase material from the fermentation broth of Pseudomonas protozoa significantly improved the hydroponic growth of rice seedlings under salt stress. The aboveground fresh weight of rice seedlings treated with hexane extract phase from the fermentation broth of Pseudomonas protozoa was significantly better than that of the control treatment without added salt and without the fermentation broth of Pseudomonas protozoa, and was even better than that of rice seedlings treated with other extract phase materials.

[0080] like Figure 2 As shown in Figure E, the addition of hexane extract phase material from the fermentation broth of Pseudomonas protozoa significantly improved the hydroponic growth of rice seedlings under salt stress. The fresh weight of the underground part of rice seedlings treated with hexane extract phase from the fermentation broth of Pseudomonas protozoa was significantly better than that of the control treatment without added salt and without the fermentation broth of Pseudomonas protozoa, and was even better than that of rice seedlings treated with other extract phase materials.

[0081] Example 3: Preparation and purification of the salt-tolerant active substance described in this invention

[0082] The supernatant of the strain was collected in the same manner as in Example 1. The supernatant was extracted in the order of ethyl acetate, n-hexane, petroleum ether, and chloroform. Each extract phase was collected and the solvent was removed by rotary evaporation. The substance of the n-hexane extract phase was separated by column chromatography using a 4.0×70cm glass column. The crude product was mixed with silica gel (100-200 mesh) for column chromatography separation. A petroleum ether-n-hexane mixed system was used, with a gradient elution ratio from 10:1 to 1:1. One flask was collected for every 250ml. After HPLC analysis, similar fractions were combined and divided into 6 segments. The fractions were collected sequentially to verify the growth-promoting effect on rice development (method referred to Example 2). The 6th segment showed strong activity. The substance of the 4th segment was separated by column chromatography using a 2.5×60cm glass column. The crude product was mixed with silica gel (100-200 mesh) for column chromatography separation. A hexane-methanol mixture was used for gradient elution at ratios ranging from 20:1 to 10:1. One 100ml sample was collected for HPLC analysis. HPLC conditions were: Ultimate® XB-C18 column (250 mm × 4.6 mm, 5 μm, Yuexu Technology (Shanghai) Co., Ltd.), mobile phase: acetonitrile:water = 35:65 (V:V), detection wavelength: 278nm, flow rate: 1 ml / min, injection volume: 20ml. Similar fractions were combined and further divided into 5 segments. Segments 6-4 showed good growth-promoting activity in rice (method referred to Example 2), ultimately yielding the pure active component. Mass spectrometry and NMR (including 2-position NMR structure analysis) were performed on the active component, ultimately yielding a salt-tolerant active substance with the molecular structure shown below:

[0083]

[0084] The collected components were freeze-dried to obtain active substance powder, which was then dissolved in 50 ml of 10% DMSO solvent as active substance stock solution. Further gradient dilution was performed to obtain diluents of 0.01‰, 0.1‰, 1‰, 5‰ and 10‰.

[0085] Results and Analysis

[0086] like Figure 3 As shown, CK represents the treatment of the sixth stage of hexane phase separation in the fermentation broth without salt or bacterial strain addition, while 100 mM NaCl represents the treatment of the sixth stage of hexane phase separation in the fermentation broth with salt but without bacterial strain addition. The treatment groups are: 0.01‰ (0.01‰ of the total hydroponic volume), 0.1‰ (0.1‰ of the total hydroponic volume), 1‰ (1‰ of the total hydroponic volume), 5‰ (5‰ of the total hydroponic volume), and 10‰ (10‰ of the total hydroponic volume).

[0087] like Figure 3 As shown in Figure A, the addition of the sixth segment of the hexane extract phase of the Pseudomonas protozoa fermentation broth significantly improved the hydroponic growth of rice seedlings under salt stress. The rice seedlings treated with the sixth segment of the hexane extract phase of the Pseudomonas protozoa fermentation broth showed significantly better growth than the control treatment without added salt and the fermentation broth, and also showed better growth than the rice seedlings treated with other extract phases.

[0088] like Figure 3 As shown in Figure B, the addition of the sixth segment of the hexane extract of the fermentation broth of *Pseudomonas protozoa* significantly improved the hydroponic growth of rice seedlings under salt stress. The rice seedlings treated with 0.01‰ of the sixth segment of the hexane extract of the fermentation broth of *Pseudomonas protozoa* showed significantly better plant height than the control treatment without added salt and without the fermentation broth, and were also longer than the rice seedlings in other treatments.

[0089] like Figure 3 As shown in Figure C, the addition of the sixth segment of the hexane extract of the fermentation broth of *Pseudomonas protozoa* significantly improved the hydroponic growth of rice seedlings under salt stress. The root length of rice seedlings treated with the 1‰ treatment group containing the sixth segment of the hexane extract of the fermentation broth of *Pseudomonas protozoa* was significantly better than that of the control treatment without added salt and without the fermentation broth of the strain, and the root length of rice seedlings in other treatments was also longer.

[0090] like Figure 3As shown in Figure D, the addition of the sixth segment of the hexane extract of the fermentation broth of *Pseudomonas protozoa* significantly improved the hydroponic growth of rice seedlings under salt stress. The aboveground fresh weight of rice seedlings treated with 0.01‰ of the sixth segment of the hexane extract of the fermentation broth of *Pseudomonas protozoa* was significantly better than that of the control treatment without added salt and without the fermentation broth of the strain, and was also better than that of rice seedlings in other treatments.

[0091] like Figure 3 As shown in Figure E, the addition of the sixth segment of the hexane extract of the fermentation broth of *Pseudomonas protozoa* significantly improved the hydroponic growth of rice seedlings under salt stress. The fresh weight of the underground part of rice seedlings treated with 0.01‰ of the sixth segment of the hexane extract of the fermentation broth of *Pseudomonas protozoa* was significantly better than that of the control treatment without added salt and without the fermentation broth of the strain, and was also better than that of rice seedlings under other treatments.

[0092] like Figure 4 As shown, CK represents the treatment of substances in the hexane phase separation stage 6-4 of the fermentation broth without salt or bacterial strain addition, while 100mM NaCl represents the treatment of substances in the ethyl acetate phase separation stage 6-4 of the fermentation broth with salt but without bacterial strain addition. The treatment groups are: 0.01‰ (0.01‰ of the total hydroponic volume), 0.1‰ (0.1‰ of the total hydroponic volume), 1‰ (1‰ of the total hydroponic volume), 5‰ (5‰ of the total hydroponic volume), and 10‰ (10‰ of the total hydroponic volume).

[0093] like Figure 4 As shown in Figure A, the addition of the 6th-4th fraction of the hexane extract phase of the Pseudomonas protozoa fermentation broth significantly improved the hydroponic growth of rice seedlings under salt stress. The rice seedlings treated with the 6th-4th fraction of the hexane extract phase of the Pseudomonas protozoa fermentation broth showed significantly better growth than the control treatment without added salt and the control treatment without added fermentation broth. The rice seedlings treated with other extract phases also showed better growth.

[0094] like Figure 4 As shown in Figure B, the addition of the 6th-4th fraction of the hexane extract from the fermentation broth of *Pseudomonas protozoa* significantly improved the hydroponic growth of rice seedlings under salt stress. The rice seedlings treated with 0.01‰ of the 6th-4th fraction of the hexane extract from the fermentation broth of *Pseudomonas protozoa* showed significantly better plant height than the control treatment without added salt and without the fermentation broth, and were also longer than the rice seedlings in other treatments.

[0095] like Figure 4As shown in Figure C, the addition of the 6th-4th fraction of the hexane extract from the fermentation broth of *Pseudomonas protozoa* significantly improved the hydroponic growth of rice seedlings under salt stress. The root length of rice seedlings treated with the 1‰ treatment group containing the 6th-4th fraction of the hexane extract from the fermentation broth of *Pseudomonas protozoa* was significantly better than that of the control treatment without added salt and without the fermentation broth, and the root length of rice seedlings was also longer than that of other treatments.

[0096] like Figure 4 As shown in Figure D, the addition of the 6th-4th fraction of the hexane extract from the fermentation broth of *Pseudomonas protozoa* significantly improved the hydroponic growth of rice seedlings under salt stress. The aboveground fresh weight of rice seedlings treated with 0.01‰ of the 6th-4th fraction of the hexane extract from the fermentation broth of *Pseudomonas protozoa* was significantly better than that of the control treatment without added salt and without the fermentation broth, and was also better than that of other treatments.

[0097] like Figure 4 As shown in Figure E, the addition of the 6th-4th fraction of the hexane extract from the fermentation broth of *Pseudomonas protozoa* significantly improved the hydroponic growth of rice seedlings under salt stress. The fresh weight of the underground parts of rice seedlings treated with 0.01‰ of the 6th-4th fraction of the hexane extract from the fermentation broth of *Pseudomonas protozoa* was significantly better than that of the control treatment without added salt and without the fermentation broth, and was also better than that of rice seedlings under other treatments.

[0098] As shown in Table 1, through 13 C-NMR and DEPT spectra revealed a total of 16 valid signal peaks, including 4 methine peaks (δC 132.0, 132.0, 129.1, 129.0), 6 methylene peaks (δC 65.5, 65.5, 30.5, 30.5, 19.1, 19.1), 2 methyl peaks (δC 14.0, 14.0), and 4 quaternary carbon signals (δC 167.4, 167.4, 132.2, 132.2). The compound exhibits a symmetrical structure, and its corresponding NMR signals are completely overlapping. Detailed structural analysis is as follows:

[0099] (1) Through 1 H-NMR coupling information and 1H-1H COSY spectrum can be used to determine that H-2, H-1, H-1', and H-2' are coupled to each other sequentially in the structure. Combined with HSQC spectrum, the carbon chemical shifts of C-2, C-1, C-1', and C-2' can be determined to be δC 129.1, 132.0, 132.0, and 129.1, respectively.

[0100] (2) Through 1 H-NMR coupling information and 1 H- 1¹H COSY spectroscopy confirms that H-5 through H-8 are sequentially coupled within the structure. Combined with HSQC spectroscopy, the carbon chemical shifts for C-5, C-6, C-7, and C-8 are determined to be δC 65.5, 30.5, 19.1, and 14.0, respectively. Similarly, the carbon chemical shifts for the remaining C-5', C-6', C-7', and C-8' are determined to be δC 65.5, 30.5, 19.1, and 14.0, respectively.

[0101] (3) In the HMBC spectrum, the long-range correlation points of H-1 / 1' with C-2, 3, 2', 3', H-2 / 2' with C-1, 1', 4, 4', and H-5 / 5' with C-4, 4' can be used to determine the carbon chemical shifts of the relevant quaternary carbons C-3, C-3', C-4, C-4' as δC 132.2, 132.2, 167.4, 167.4.

[0102] (4) In addition, the signal in the NOESY spectrum also confirms the correctness of the above attribution.

[0103] The final separation, purification, and identification determined that the molecular formula of this salt-tolerant active substance is C0. 16 H 22 O4.

[0104] Table 1: Mass spectrometry and NMR analysis results

[0105]

Claims

1. The application of dibutyl phthalate (I) in improving crop salt tolerance. 。 2. The application according to claim 1, characterized in that, The dibutyl phthalate mentioned above is extracted from the supernatant of Pseudomonas proteoglycans fermentation broth or chemically synthesized.

3. The application according to claim 1, characterized in that, The dibutyl phthalate was extracted from the supernatant of the fermentation broth of *Pseudomonas proteus* Pse_ST, with accession number CGMCC No. 28061.

4. A method for isolating the active metabolite for improving crop salt tolerance as described in claim 1 from *Pseudomonas protozoa*, characterized in that, The *Pseudomonas proteus* strain with preservation number CGMCC No. 28061 was fermented, and the supernatant of the fermentation broth was collected. The supernatant was extracted sequentially with ethyl acetate and n-hexane. The n-hexane organic phase was collected, and the solvent was removed by rotary evaporation. Separation was then performed by silica gel column chromatography, using a petroleum ether-n-hexane mixture and a n-hexane-methanol mixture for elution. Finally, the salt-tolerant active substance was prepared by HPLC under the following conditions: mobile phase: acetonitrile:water = 35:65, detection wavelength: 278 nm, flow rate: 1 ml / min, and injection volume: 20 ml.

5. The method according to claim 4, characterized in that... The fermentation medium for *Pseudomonas proteus* with fermentation preservation number CGMCC No. 28061 was Landy medium, with the following composition per liter: 5 g L-glutamic acid, 0.5 g magnesium sulfate heptahydrate, 0.5 g potassium chloride, 1 g potassium dihydrogen phosphate, 0.15 mg ferrous sulfate heptahydrate, 5 mg manganese sulfate tetrahydrate, 0.16 mg copper sulfate heptahydrate, 1 g yeast extract, 2 mg L-phenylalanine, and 20 g glucose. After adjusting the pH to 7.0, the medium was sterilized at 115°C for 30 min.

6. The method according to claim 4, characterized in that... The fermentation method of *Pseudomonas proteus* includes: first, inoculating the strain stored at -80°C into LB solid medium and culturing overnight at 30°C; then, selecting a single colony and inoculating it into LB liquid medium, culturing overnight at 30°C and 170 rpm to obtain a seed culture; transferring the *Pseudomonas proteus* seed culture at a 1% inoculation rate into an Erlenmeyer flask containing fresh Landy medium, culturing for 2 days at 30°C and 170 rpm; then inoculating the cultured culture into a 100L fermenter, filling the fermenter with 70L of Landy medium, and after the culture enters the stationary phase, starting to produce secondary metabolites in large quantities, and continuing fermentation for 3 days.

7. The application of the salt-tolerant growth-promoting substance described in claim 1 in promoting rice growth under salt stress.

8. The use of the salt-tolerant growth-promoting substance described in claim 1 in the preparation of a formulation that promotes the growth of rice under salt stress.

Images