A method for isolating plant salt tolerance promoting metabolites from planomicrobium sp. and use thereof

Abstract

The application discloses a method for separating plant salt tolerance promoting active metabolites from Planomonospora pulmonaria and application thereof. The application discloses application of the plant salt tolerance promoting active metabolite xanthine separated from Planomonospora pulmonaria in promoting growth of rice under salt stress. The substance is obviously increased in rice plant height, root length, fresh weight of aboveground part and fresh weight of underground part, is stable in effect, has a good application prospect, and can be used as a preparation for improving salt tolerance of crops in agricultural green production.

Description

Technical Field

[0001] This invention belongs to the field of microbiology, specifically a method for isolating plant salt-tolerant active metabolites from Alkaloidobacterium panlonga and its application. Background Technology

[0002] Globally, nearly 932 million hectares of land are affected by salinization, leading to land degradation, decreased agricultural productivity, and even food security issues. Soil salinization not only damages soil structure and reduces soil fertility but also alters soil fauna structure and affects plant absorption and metabolism. It can be said that salinization has become one of the major factors restricting and threatening agricultural development, impacting food yield and food security. Therefore, the current state of soil salt stress cannot be ignored, and effective remediation measures are needed to improve the quality of saline soils, increase crop yields and resilience, and address this global ecological problem.

[0003] *Pannonibacter* is a genus in bacterial taxonomy that includes various bacteria, such as *Pannonibacter pannonibacter*, a species of alkaline plant. The growth environment of this genus is typically associated with specific aquatic environments, particularly alkaline ones. Current research on *Pannonibacter* Pan_ST focuses primarily on its potential ability to degrade organic pollutants, showing potential application value in environmental protection and pollution control. However, research on the identification of active substances in the rhizosphere of rice from *Pannonibacter* Pan_ST is relatively limited. Previous laboratory studies screened a strain of *Pannonibacter* Pan_ST from the rhizosphere of rice plants grown in saline-alkali soil. This strain significantly improved rice seed germination rate and plant growth indicators under salt stress when applied directly or used as a seed coating. However, the substances in the strain that enhance rice salt tolerance remain unclear. Developing functional microbial agents that enhance crop salt stress tolerance and improve crop growth in saline-alkali soils using the secondary metabolites produced by the strain's fermentation has strong application potential. Summary of the Invention

[0004] The purpose of this invention is to provide an application of a salt-tolerant active substance fermented by Alkaloidobacterium pan_ST.

[0005] Another object of the present invention is to provide a method for the extraction and separation of the salt-resistant active substance.

[0006] The objective of this invention can be achieved through the following technical solutions:

[0007] Application of xanthine, an active metabolite isolated from the saline bacterium Pannonibacter phragmitetus Pan_ST, in promoting rice growth under salt stress. The structural formula of xanthine is shown below:

[0008] .

[0009] The aforementioned Pan_ST alkaloid bacterium is deposited at the China General Microbiological Culture Collection Center (CGMCC; address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; postcode: 100101), on July 28, 2023, with accession number CGMCC No. 28063.

[0010] The method for isolating xanthine, the active metabolite of claim 1, from *Alkaliidae Pan_ST* comprises: fermenting *Alkaliidae Pan_ST* in Landy medium and collecting the supernatant of the fermentation broth; extracting the supernatant with dichloromethane; collecting the dichloromethane organic phase and removing the solvent by rotary evaporation; then separating the metabolite by silica gel column chromatography, using a petroleum ether-dichloromethane mixture and a dichloromethane-methanol mixture for elution; and finally preparing the salt-tolerant active substance 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; a detection wavelength of 278 nm; a flow rate of 1 ml / min; and an injection volume of 20 ml.

[0011] Preferably, similar fractions separated by HPLC are combined and then divided into three segments, with the second segment being used to obtain the pure xanthine active component.

[0012] Preferably, 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] Preferably, the method for preparing the fermentation broth of Alkaloidobacterium pan_ST is as follows: the seed culture of Alkaloidobacterium pan_ST is transferred to an Erlenmeyer flask containing fresh Landy medium at an inoculation rate of 1%, and cultured at 30 ℃ and 170 rpm for 2 days; then the cultured broth is inoculated into a 100 L fermenter, which is filled with 70 L of Landy medium. After the broth enters the stationary phase, it begins to produce secondary metabolites in large quantities and continues to ferment for 3 days.

[0014] The application of the salt-tolerant growth-promoting substance xanthine in the preparation of formulations that promote the growth of rice under salt stress.

[0015] Beneficial effects:

[0016] This invention, through hydroponic experiments during the seedling stage, verified the activity of the fermentation broth of *Alkalibium pantalis* (Pan_ST) in enhancing the salt tolerance of rice. It determined the optimal culture medium and conditions for the fermentation of secondary metabolites and isolated and purified a purine-cyclic salt-tolerant growth-promoting substance, C5H4N4O2, and identified its molecular structure. Verification of the pure substance showed that it can promote the growth of rice under salt stress, increasing plant height, root length, aboveground fresh weight, and underground fresh weight. Attached Figure Description

[0017] Figure 1 The effects of fermentation broth of *Alkaliobacterium pantalis* on the salt tolerance and growth promotion of rice seedlings under salt stress. (A) Effect of fermentation broth of *Alkaliobacterium pantalis* on the growth phenotype of rice seedlings under salt stress; (B) Effect of fermentation broth of *Alkaliobacterium pantalis* on the plant height of rice seedlings under salt stress; (C) Effect of fermentation broth of *Alkaliobacterium pantalis* on the root length of rice seedlings under salt stress; (D) Effect of fermentation broth of *Alkaliobacterium pantalis* on the aboveground fresh weight of rice seedlings under salt stress; (E) Effect of fermentation broth of *Alkaliobacterium pantalis* on the underground fresh weight of rice seedlings under salt stress.

[0018] Figure 2 The effects of different extracts of the fermentation broth of *Alkaliobacterium panlonga* (Pan_ST) on the salt tolerance and survival rate of rice seedlings under salt stress. (A) Effects of different extracts of the fermentation broth of *Alkaliobacterium panlonga* (Pan_ST) on the growth phenotype of rice seedlings under salt stress; (B) Effects of different extracts of the fermentation broth of *Alkaliobacterium panlonga* (Pan_ST) on the plant height of rice seedlings under salt stress; (C) Effects of different extracts of the fermentation broth of *Alkaliobacterium panlonga* (Pan_ST) on the root length of rice seedlings under salt stress; (D) Effects of different extracts of the fermentation broth of *Alkaliobacterium panlonga* (Pan_ST) on the aboveground fresh weight of rice seedlings under salt stress; (E) Effects of different extracts of the fermentation broth of *Alkaliobacterium panlonga* (Pan_ST) on the underground fresh weight of rice seedlings under salt stress.

[0019] Figure 3The study investigated the effect of the fourth segment of the fermentation broth of *Alkaliobacterium pan_ST* on the salt tolerance and growth promotion of rice seedlings under salt stress. (A) Effect of the fourth segment of the fermentation broth of *Alkaliobacterium pan_ST* on the growth phenotype of rice seedlings under salt stress; (B) Effect of the fourth segment of the fermentation broth of *Alkaliobacterium pan_ST* on the plant height of rice seedlings under salt stress; (C) Effect of the fourth segment of the fermentation broth of *Alkaliobacterium pan_ST* on the root length of rice seedlings under salt stress; (D) Effect of the fourth segment of the fermentation broth of *Alkaliobacterium pan_ST* on the aboveground fresh weight of rice seedlings under salt stress; (E) Effect of the fourth segment of the fermentation broth of *Alkaliobacterium pan_ST* on the underground fresh weight of rice seedlings under salt stress.

[0020] Figure 4 The effect of the fourth-second segment of the dichloromethane phase separation of the fermentation broth of strain *Alkaliobacterium panlonga* (Pan_ST) on the salt tolerance and growth promotion of rice seedlings under salt stress. (A) Effect of the fourth-second segment of the dichloromethane phase separation of the fermentation broth of strain *Alkaliobacterium panlonga* (Pan_ST) on the growth phenotype of rice seedlings under salt stress; (B) Effect of the fourth-second segment of the dichloromethane phase separation of the fermentation broth of strain *Alkaliobacterium panlonga* (Pan_ST) on the plant height of rice seedlings under salt stress; (C) Effect of the fourth-second segment of the dichloromethane phase separation of the fermentation broth of strain *Alkaliobacterium panlonga* (Pan_ST) on the root length of rice seedlings under salt stress; (D) Effect of the fourth-second segment of the dichloromethane phase separation of the fermentation broth of strain *Alkaliobacterium panlonga* (Pan_ST) on the aboveground fresh weight of rice seedlings under salt stress; (E) Effect of the fourth-second segment of the dichloromethane phase separation of the fermentation broth of strain *Alkaliobacterium panlonga* (Pan_ST) on the underground fresh weight of rice seedlings under salt stress.

[0021] Table 1: Hydrogen and Carbon Spectra of Salt-Tolerant Active Substances 1 H- 1 Analysis results of H COSY spectrum, HSQC spectrum, and HMBC spectrum tests.

[0022] Biological sample preservation information

[0023] Alkaloidea pan_ST is deposited at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, on July 28, 2023, with accession number CGMCC No. 28063. Detailed Implementation

[0024] This invention discloses an active substance that promotes salt tolerance in rice. Those skilled in the art can refer to the content of this document and appropriately modify the parameters to achieve the desired effect. 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.

[0025] The rice variety used in the experiment was Nipponbare.

[0026] Example 1: Verification of the salt tolerance-promoting effect of the Pan_ST fermentation broth of Alkaloidobacterium panloniformis described in this invention on rice.

[0027] The *Alkalibium pan_ST* seed culture was transferred at a 1% inoculum 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, it began to produce secondary metabolites in large quantities and fermented for another 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.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] Results and Analysis

[0032] like Figure 1As shown, CK represents the treatment without salt or added bacterial fermentation broth, 100 mM represents the treatment with salt but without added bacterial fermentation broth, and 1‰ treatment group means that the content of added bacterial fermentation broth accounts for 1‰ of the total hydroponic volume.

[0033] As shown in Figure A, the fermentation broth of Alkaloidobacterium pan_ST significantly improved the hydroponic growth of rice seedlings under salt stress. The rice seedlings soaked in the fermentation broth of Alkaloidobacterium pan_ST showed significantly better growth than the control treatment with added salt but no fermentation broth.

[0034] As shown in Figure B, the fermentation broth of Alkaloidobacterium pan_ST significantly improved the hydroponic growth of rice seedlings under salt stress. The plant height of rice seedlings soaked in the fermentation broth of Alkaloidobacterium pan_ST was significantly higher than that of the control treatment with added salt but without the addition of the fermentation broth.

[0035] As shown in Figure C, the fermentation broth of Alkaloidobacterium pan_ST significantly improved the hydroponic growth of rice seedlings under salt stress. The root length of rice seedlings soaked in the fermentation broth of Alkaloidobacterium pan_ST was significantly higher than that of the control treatment with added salt but without the addition of the fermentation broth.

[0036] As shown in Figure D, the fermentation broth of strain Pan_ST 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 strain Pan_ST was significantly higher than that of the control treatment with added salt but without the addition of the fermentation broth.

[0037] As shown in Figure E, the fermentation broth of strain Pan_ST significantly improved the hydroponic growth of rice seedlings under salt stress. The fresh weight of the underground part of rice seedlings soaked in the fermentation broth of strain Pan_ST was significantly higher than that of the control treatment with added salt but without the addition of the fermentation broth.

[0038] 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.

[0039] The rice germination and three-leaf stage seedling cultivation were carried out in the same manner as in Example 1.

[0040] The supernatant of the strain was collected in the same manner as in Example 1.

[0041] The supernatant of the bacterial strain was poured into a separatory funnel, dichloromethane was added and shaken well, and after standing for 30 minutes, the dichloromethane 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.

[0042] 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.

[0043] Results and Analysis

[0044] like Figure 2 As shown, CK represents the treatment without salt or added bacterial fermentation broth extract, 100 mM represents the treatment with salt but without added bacterial fermentation broth extract, and 0.1% DMSO represents the treatment with only added DMSO solution.

[0045] As shown in Figure A, the addition of dichloromethane extract from the fermentation broth of Alcaligenes panlongensis significantly improved the hydroponic growth of rice seedlings under salt stress. The rice seedlings treated with the dichloromethane extract from the fermentation broth of Alcaligenes panlongensis showed significantly better growth than the control treatment without added salt and the fermentation broth of Alcaligenes panlongensis, and also showed better growth than the rice seedlings treated with other extracts.

[0046] As shown in Figure B, the addition of dichloromethane extract phase material from the fermentation broth of strain Pan_ST significantly improved the hydroponic growth of rice seedlings under salt stress. The plant height of rice seedlings treated with dichloromethane extract phase from the fermentation broth of strain Pan_ST was significantly better than that of the control treatment without added salt and without the strain's fermentation broth, and the plant height of rice seedlings treated with other extract phase materials was also longer.

[0047] As shown in Figure C, the addition of dichloromethane extract phase material from the fermentation broth of strain Pan_ST significantly improved the hydroponic growth of rice seedlings under salt stress. The root length of rice seedlings treated with dichloromethane extract phase from the fermentation broth of strain Pan_ST was significantly better than that of the control treatment without added salt and without the strain's fermentation broth, and the root length of rice seedlings treated with other extract phase materials was also longer.

[0048] As shown in Figure D, the addition of dichloromethane extract phase material from the fermentation broth of strain Pan_ST significantly improved the hydroponic growth of rice seedlings under salt stress. The aboveground fresh weight of rice seedlings treated with the dichloromethane extract phase of the fermentation broth of strain Pan_ST was significantly better than that of the control treatment without added salt and without the strain's fermentation broth. The aboveground fresh weight of rice seedlings treated with other extract phase materials was also better.

[0049] As shown in Figure E, the addition of dichloromethane extract from the fermentation broth of strain Pan_ST significantly improved the hydroponic growth of rice seedlings under salt stress. The fresh weight of the underground part of rice seedlings treated with the dichloromethane extract from the fermentation broth of strain Pan_ST was significantly better than that of the control treatment without added salt and without the fermentation broth. The fresh weight of the underground part of rice seedlings treated with other extracts was also better.

[0050] Example 3: Preparation and purification of the salt-tolerant active substance described in this invention

[0051] The supernatant of the strain was collected in the same manner as in Example 1. The supernatant was extracted in the order of dichloromethane, n-hexane, petroleum ether, and chloroform. Each extract phase was collected and the solvent was removed by rotary evaporation. The substance of the dichloromethane 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-dichloromethane mixture 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 their growth-promoting effect on rice development (method referred to Example 2). The 4th 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 dichloromethane-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 three segments. Segment 4-2 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:

[0052]

[0053] 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‰.

[0054] Results and Analysis

[0055] like Figure 3 As shown, CK represents the fourth stage of dichloromethane phase separation in the fermentation broth without salt or added strains, while 100mM represents the fourth stage of dichloromethane phase separation in the fermentation broth with salt but without added strains. 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).

[0056] As shown in Figure A, the fourth segment of the substance separated by the dichloromethane extract of the fermentation broth of Alkaloidobacterium pan_ST significantly improved the hydroponic growth of rice seedlings under salt stress. The rice seedlings treated with the fourth segment of the dichloromethane extract of the fermentation broth of Alkaloidobacterium pan_ST showed significantly better growth than the control treatment without added salt and the fermentation broth of the strain. The rice seedlings treated with other extracts also showed better growth.

[0057] As shown in Figure B, the fourth segment of the substance separated by the dichloromethane extract of the fermentation broth of Alkaloidobacterium pan_ST significantly improved the hydroponic growth of rice seedlings under salt stress. The rice seedlings treated with the 1‰ treatment group containing the fourth segment separated by the dichloromethane extract of the fermentation broth of Alkaloidobacterium pan_ST were significantly taller than the control treatment without added salt and the rice seedlings of other treatments were also longer.

[0058] As shown in Figure C, the fourth segment of the substance separated by the dichloromethane extract of the fermentation broth of Alkaloidobacterium pan_ST significantly improved the hydroponic growth of rice seedlings under salt stress. The root length of rice seedlings treated with the 10‰ treatment group containing the fourth segment separated by the dichloromethane extract of the fermentation broth of Alkaloidobacterium pan_ST 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.

[0059] As shown in Figure D, the addition of the fourth segment of the dichloromethane extract from the fermentation broth of strain Pan_ST significantly improved the hydroponic growth of rice seedlings under salt stress. The aboveground fresh weight of rice seedlings treated with the 1‰ treatment group containing the fourth segment of the dichloromethane extract from the fermentation broth of strain Pan_ST 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 in other treatments.

[0060] As shown in Figure E, the addition of the fourth segment of the dichloromethane extract from the fermentation broth of strain Pan_ST significantly improved the hydroponic growth of rice seedlings under salt stress. The fresh weight of the underground part of rice seedlings treated with the 10‰ treatment group containing the fourth segment of the dichloromethane extract from the fermentation broth of strain Pan_ST 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.

[0061] like Figure 4 As shown, CK represents the treatment of the 4-2 stage of dichloromethane phase separation in the fermentation broth without salt or added strains, while 100mM represents the treatment of the 4-2 stage of dichloromethane phase separation in the fermentation broth with salt but without added strains. 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).

[0062] As shown in Figure A, the addition of the substance from the dichloromethane extract phase of the fermentation broth of Alkaloidobacterium pan_ST significantly improved the hydroponic growth of rice seedlings under salt stress. The rice seedlings treated with the substance from the dichloromethane extract phase of the fermentation broth of Alkaloidobacterium pan_ST showed significantly better growth than the control treatment without added salt and the fermentation broth of the strain. The rice seedlings treated with other extract phases also showed better growth.

[0063] As shown in Figure B, the addition of the 4-2 fraction of the fermentation broth from the Pan_ST strain of Alkaloidobacterium panloni significantly improved the hydroponic growth of rice seedlings under salt stress. The rice seedlings treated with the 1‰ treatment group containing the 4-2 fraction of the fermentation broth from the Pan_ST strain of Alkaloidobacterium panloni significantly had a greater plant height than the control treatment without added salt and without the strain of Alkaloidobacterium panloni, and were also longer than the rice seedlings in other treatments.

[0064] As shown in Figure C, the addition of the 4-2 fraction of the fermentation broth of Alkaloidobacterium pan_ST significantly improved the hydroponic growth of rice seedlings under salt stress. The root length of rice seedlings treated with the 10‰ treatment group containing the 4-2 fraction of the fermentation broth of Alkaloidobacterium pan_ST was significantly better than that of the control treatment without added salt and without the fermentation broth, and was also longer than that of rice seedlings in other treatments.

[0065] As shown in Figure D, the addition of the 4-2 fraction of the fermentation broth from strain Pan_ST significantly improved the hydroponic growth of rice seedlings under salt stress. The aboveground fresh weight of rice seedlings treated with 1‰ of the 4-2 fraction of the fermentation broth from strain Pan_ST 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 in other treatments.

[0066] As shown in Figure E, the addition of the 4-2 segment of the dichloromethane extract phase of the fermentation broth of Alkaloidobacterium pan_ST significantly improved the hydroponic growth of rice seedlings under salt stress. The fresh weight of the underground part of rice seedlings treated with 10‰ of the 4-2 segment of the dichloromethane extract phase of the fermentation broth of Alkaloidobacterium pan_ST was significantly better than that of the control treatment without added salt and without the addition of the fermentation broth, and was also better than that of rice seedlings under other treatments.

[0067] As shown in Table 1, through 1 H-NMR coupling information and 1 H- 1 The 1H COSY spectrum reveals relatively simple proton signals in the structure. These signals primarily consist of a single isolated aromatic proton signal [δH 7.92, s] and three active hydrogen signals [δH 13.3, s; 11.5, s; 10.8, s]. The 13C-NMR and DEPT spectra show a total of five valid signal peaks, including one methine (δC 141.1) and four quaternary carbon signals (δC 155.9, 151.8, 149.5, 107.0). Detailed structural analysis is as follows:

[0068] (1) The chemical shift of the C-8 carbon spectrum can be determined to be δC141.1 by HSQC spectrum.

[0069] (2) In the HMBC spectrum, the long-range correlation points of H-8 with C-1, 5, 6, the long-range correlation points of H-4 with C-5, and the long-range correlation points of H-7 with C-6 can determine the carbon chemical shifts of the relevant carbons C-1, C-5, and C-6 as δC155.9, 149.5, and 107.0, respectively.

[0070] (3) At this point, only one carbon spectrum (δC151.8) and proton signal (δH7.92, s) remain unassigned, and they are assigned to the C-3 and H-2 positions respectively.

[0071] (4) In addition, the correlation signals of H-7 / H-8 in the NOESY spectrum also indicate the correctness of the above attribution.

[0072] Finally, the structure of the compound was confirmed by high-resolution mass spectrometry. Ultimately, the molecular formula of this salt-resistant active substance was determined to be C5H4N4O2 through separation, purification, and identification.

[0073] Table 1: Mass spectrometry and NMR analysis results

[0074]

Claims

1. A species of alkaline lake bacterium that lives on the plant Panlonia Pannonibacter phragmitetus The application of xanthine, an active metabolite isolated from Pan_ST, in promoting rice growth under salt stress is characterized by... The structural formula of xanthine is as follows: .

2. A method for isolating the active metabolite xanthine as claimed in claim 1 from the Pan ST of the bacterium Panionia, characterized in that, The salt-tolerant active substance xanthine was prepared by fermenting *Alkaloidea pan_ST* with the fermentation preservation number CGMCC No. 28063 and collecting the supernatant of the fermentation broth. The supernatant was extracted with dichloromethane, and the dichloromethane organic phase was collected and the solvent was removed by rotary evaporation. Then, the separation was carried out by silica gel column chromatography. Elution was performed successively with a petroleum ether-dichloromethane mixture and a dichloromethane-methanol mixture. The salt-tolerant active substance xanthine was prepared by HPLC under the following conditions: Ultimate® XB-C18 column, mobile phase acetonitrile:water = 35:65, detection wavelength 278 nm, flow rate 1 ml / min, and injection volume 20 ml.

3. The method according to claim 2, characterized in that, Similar fractions separated by HPLC were combined and then divided into three segments. The second segment was used to obtain the pure xanthine, the active component.

4. The method according to claim 2, characterized in that, The fermentation medium for Pan_ST Alkaloidobacterium pan-ST, with fermentation preservation number CGMCC No. 28063, was Landy medium. The composition was as follows: per liter, L-glutamic acid 5 g, magnesium sulfate heptahydrate 0.5 g, potassium chloride 0.5 g, potassium dihydrogen phosphate 1 g, ferrous sulfate heptahydrate 0.15 mg, manganese sulfate tetrahydrate 5 mg, copper sulfate heptahydrate 0.16 mg, yeast extract 1 g, L-phenylalanine 2 mg, and glucose 20 g. After adjusting the pH to 7.0, it was sterilized at 115℃ for 30 min.

5. The method according to claim 2, characterized in that... The fermentation method of Alkaloidobacterium pan_ST 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 Pan_ST seed culture of Alkaloidobacterium pan_ST at an inoculation rate of 1% into an Erlenmeyer flask containing fresh Landy medium, culturing at 30°C 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.

6. The use of xanthine, the salt-tolerant growth promoter described in claim 1, in the preparation of a formulation that promotes the growth of rice under salt stress.

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