A Pseudomonas bacterium, a preparation method for producing non-ribosomal peptides therefrom, and applications thereof

Abstract

The present invention discloses a strain of Pseudomonas and a preparation method and application of the non-ribosomal peptide produced thereby. The Pseudomonas HN8-3 of the present invention is isolated from the rhizosphere soil of Lens culinaris in Gansu Province and can produce pseudophomins A-D through liquid fermentation. These substances can be separated and purified into monomeric compounds by solid-phase extraction technology combined with high-performance liquid chromatography technology. Under in vitro experimental conditions, pseudophomins A-D at a concentration of 1 μM can well lyse the zoospores of Phytophthora capsici ( Phytophthora capsici ). On the living body of plants (cucumber leaves), the compounds at a concentration of 10 μM can well prevent and control the occurrence of diseases caused by Phytophthora capsici. Compared with the control group, this concentration can achieve a 100% effect in preventing and controlling Phytophthora capsici. Thus, it can be seen that pseudophomins A-D have a good effect in preventing and controlling Phytophthora capsici. Such lead compounds have the potential to be used as biological agents and biological drugs and can be used as the parent compounds of Pseudomonas-derived biological pesticides, having important potential application values in the fields of agricultural production and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial pesticides, and particularly relates to a strain of Pseudomonas. The present invention also relates to a method for preparing non-ribosomal peptides from the Pseudomonas, and the application of the isolated non-ribosomal peptides in preventing and controlling Phytophthora capsici as a biological pesticide parent body. Background Art

[0002] The Ministry of Agriculture stipulates that after 2020, the use of chemical fertilizers and pesticides in the whole country will achieve "zero growth". Therefore, the development of biological pesticides for preventing and controlling crop diseases is of great significance.

[0003] Biological pesticides include biological fungicides and the like, and have the characteristics of small environmental pollution, low toxicity, easy degradation, and good prevention and control effects on soil-borne diseases. Biological pesticides are developed using natural active substances or living organisms, and can be used to prevent, control, or inhibit harmful organisms. The existing types of biological pesticides in China mainly include three categories: biochemical pesticides, microbial pesticides, and plant-derived pesticides. As one of the important categories of biological pesticides, microbial pesticides use living organisms such as bacteria, fungi, viruses, protozoa, or genetically modified microorganisms as active ingredients.

[0004] Commercially available bacterial-source biological pesticides are developed using microorganisms such as Bacillus spp., Pseudomonas spp., and Streptomyces spp. as biological pesticide parent bodies. As an important biological pesticide parent body, Pseudomonas can produce a variety of secondary metabolites for preventing and controlling crop diseases. These secondary metabolites can directly serve as the parent compound resources for the development of microbial pesticides. Bacillus spp.), Pseudomonas spp. Pseudomonas spp.), and Streptomyces spp. Streptomyces spp.)

[0005] Pseudomonas can produce a class of secondary metabolites of non-ribosomal peptides. The chemical structure of these substances consists of a parent body formed by an amino acid ring connected to a fatty acid chain. So far, more than 13 major classes of Pseudomonas non-ribosomal peptides have been reported, including non-ribosomal peptides of families such as bananamide, viscosin, orfamide, tension, putisolvin, tolaasin, etc. Each major class of non-ribosomal peptides contains several homologues, and these homologues either differ in the length of the fatty acid chain or in the types of amino acids. Under laboratory conditions, the non-ribosomal peptides in some Pseudomonas have quite high yields, reaching nearly 1 g / L of the fermentation broth. In addition, the non-ribosomal peptide molecules produced by this type of Pseudomonas have the characteristic of amphiphilicity. Therefore, they can interact better with a variety of biological membranes and thus can exhibit multiple biological activities. Through research, these non-ribosomal peptides have good biological control effects on a variety of crop pathogens and thus have good potential to become the parent body of biological pesticides.

[0006] Phytophthora capsici Phytophthora capsici is an important plant disease, and the pathogen belongs to one of the oomycetes ( Phytophthora spp.). In the asexual reproduction stage of Phytophthora capsici, the zoospores released by the pathogen's sporangia are one of the important virulence factors. Zoospores can adhere to the surface of plant tissues, further germinate and enter the disease cycle. When the pathogen transforms from the parasitic type to the saprophytic type, sporangia are produced. Phytophthora capsici can infect a variety of crops including the Chinese medicinal material Codonopsis pilosula, cucurbits such as cucumbers, and peppers. When it occurs, it can quickly cause the decay of crop leaves, roots and other parts, thus seriously affecting the production of vegetables and other crops and the post-harvest preservation process. There are research reports that the non-ribosomal peptide pultisolvin produced by Pseudomonas has biological activity against Phytophthora capsici. In addition, there are few reports at home and abroad on the prevention and control of Phytophthora capsici by other Pseudomonas non-ribosomal peptides.

[0007] Therefore, exploring new-structured non-ribosomal peptides with good biological control effects on crop pathogens is of great significance for the development of biological pesticides for controlling crop pathogens such as Phytophthora capsici. Summary of the Invention

[0008] Based on the above, the object of the present invention is to provide a class of non-ribosomal peptides with a new structure and good biological control effects on crop pathogens.

[0009] Another object of the present invention is to provide a preparation method of the above non-ribosomal peptides.

[0010] To achieve its object, the technical solution adopted by the present invention is as follows:

[0011] A class of non-ribosomal peptide compounds, named pseudophomins A-D, with the following chemical structure:

[0012]

[0013] The above non-ribosomal peptide compounds were obtained by liquid fermentation culture, solid-phase extraction and chromatographic separation of Pseudomonas sp. HN8-3 isolated from the rhizosphere soil of Lens culinaris in Gansu Province, China. The chemical structures of these compounds were determined by high-resolution mass spectrometry, nuclear magnetic resonance spectroscopy and single-crystal diffraction techniques; the Pseudomonas sp. HN8-3 Pseudomonas sp. HN8-3) has been deposited in the Guangdong Microbial Culture Collection Center, abbreviated as GDMCC, address: Guangzhou, China, deposit date: October 8, 2022, deposit number GDMCC No. 62849; it was detected to be viable on October 8, 2022.

[0014] Furthermore, the parameters and operation process of the preparation method of the above non-ribosomal peptide compounds are as follows:

[0015] The weight composition of the liquid fermentation medium of Pseudomonas sp. HN8-3 is as follows: 1-5% protease peptone, 0.1-1.0% KH2PO4, 0.1-0.5% MgSO4, 0.5-5.0% glycerol, 0.1-2.0% L-leucine, pH value 5.0-9.0, and the balance is water; after fermentation, centrifuge at 10000 rpm for 5 min at room temperature, and collect the fermentation supernatant;

[0016] Load the fermentation supernatant onto a solid-phase extraction column, and perform rapid elution under reduced pressure successively with 50% acetonitrile / 50% water and acetonitrile by volume percentage. After drying the acetonitrile elution fraction, a crude extract is obtained;

[0017] Dissolve the crude extract with methanol and perform reverse-phase high-performance liquid chromatography separation. The chromatographic column uses a YMC ODS-A semi-preparative column of 250 × 10 mm. The chromatographic mobile phase is acetonitrile: water = 80:20 by volume ratio, the flow rate is 2.5 mL / min, and the detection wavelength is 210 nm. Four pure compounds pseudophomins A-D are prepared.

[0018] Furthermore, in the above preparation method, the fermentation temperature is 20-35 °C and the fermentation time is 25-75 hours.

[0019] Further, in the above preparation method, the concentration of acetonitrile in the eluent for solid-phase extraction column chromatography is 20-100% by volume ratio, and the concentration of acetonitrile in the mobile phase of high-performance liquid chromatography is 50-100% by volume ratio.

[0020] The above non-ribosomal peptide compounds pseudophomins A-D have good prevention and control effects on Phytophthora capsici Phytophthora capsici under in vitro conditions and in in vivo plant experiments, and can be used as the parent compound of biopesticides derived from Pseudomonas.

[0021] The beneficial effects of the present invention are as follows:

[0022] In the present invention, Pseudomonas HN8-3 was collected from the rhizosphere soil of Lens culinaris in Gansu Province, China. After liquid fermentation culture, solid-phase extraction and chromatographic separation, a class of non-ribosomal peptide compounds pseudophomins A-D was obtained; under in vitro experimental conditions, pseudophomins A-D at a concentration of 1 μM can well lyse the zoospores of Phytophthora capsici Phytophthora capsici . On living plants (cucumber leaves), this class of compounds at a concentration of 10 μM can well prevent and control the diseases of Phytophthora capsici. Compared with the control group, this concentration can achieve a 100% prevention and control effect on Phytophthora capsici.

[0023] It can be seen that pseudophomins A-D have good prevention and control effects on Phytophthora capsici. This class of lead compounds has the potential to be used as biological agents and biological drugs, and can be used as the parent compound of biopesticides derived from Pseudomonas. Description of the Drawings

[0024] Figure 1 is the typical two-dimensional nuclear magnetic resonance correlation signal diagram of compounds A and C;

[0025] Figure 2 is the crystal diffraction three-dimensional diagram of compound A;

[0026] Figure 3 is the time curve diagram of the lysis of zoospores by compounds A-D at different concentrations;

[0027] Figure 4 is the disease index obtained after treating cucumber leaves with compounds pseudophomins A-D at different concentrations. Detailed Embodiments

[0028] The present invention will be further described below with reference to specific embodiments.

[0029] Example 1: Preparation and Characterization of Pseudophomins A-D

[0030] Twenty 250 mL Erlenmeyer flasks were used, each filled with 50 mL of fermentation medium (by weight percentage, containing 2% protease peptone, 0.15% KH2PO4, 0.15% MgSO4, 1% glycerol, 0.5% L-leucine, natural pH, and the balance being water). Sterilize at 121 °C for 30 minutes. Inoculate one loop of fresh and activated Pseudomonas sp. HN8-3 into the cooled liquid medium and place it on a shaker at 28 °C and 180 rpm for 48 hours.

[0031] After fermentation, centrifuge the fermentation broth at 10000 rpm for 10 min at room temperature, and collect approximately 800 mL of fermentation supernatant. Under reduced pressure, directly pass the fermentation supernatant through a solid-phase extraction column (60 × 100 mm, containing 5 g of YMC ODS-A gel with a particle size of 50 μm), and elute it successively with 200 mL each of acetonitrile / water eluents with a volume ratio of 50 / 50 and 100 / 0. Dry the 100% acetonitrile eluate to obtain approximately 918.2 mg of crude substance.

[0032] Use a high-performance liquid chromatograph (Agilent 1100 series quaternary gradient system) and a YMC ODS-A semi-preparative column (250 × 10 mm) to separate and prepare the compounds. The mobile phase is acetonitrile:water = 80:20 ( v / v ), the flow rate is 2.5 mL / min, and the detection wavelength is 210 nm. Four pure compounds were prepared, namely 30.2 mg (A, retention time 30.7 min), 56.5 mg (B, retention time 57.6 min), 26.3 mg (C, retention time 25.9 min), and 16.8 mg (D, retention time 48.0 min).

[0033] This type of target compound is a white powdery solid, poorly soluble in methanol, acetonitrile, water, chloroform, etc., and soluble in dimethyl sulfoxide and dimethylformamide. After determination, the physicochemical properties of these compounds are shown in Table 1 below.

[0034] Table 1 Physicochemical properties of compounds pseudophomins A-D

[0035]

[0036] Measure the 1D and 2D nuclear magnetic resonance (NMR) spectra of the pure compounds respectively, such as 11H NMR (600 MHz, dimethylformamide- d 7), 13 13C NMR (150 MHz, dimethylformamide- d 7), 1 1H- 1 HCOSY, 1 1H- 13 13C HSQC, 1 1H- 13 13C HMBC, 1 1H- 1 1H TOCSY, 1 1H- 1 1H ROESY and high-resolution mass spectrometry (HR-MS) data were used to analyze the planar structures of these substances.

[0037] From the 1 1H-NMR spectrum, resonance signals typical of peptide substances (including fatty acids, peptide bonds, carbonyl groups, amino acid H α , etc.) could be seen. 13 In the 1 13C-NMR spectrum, amino acid signals and fatty acid signals could be further confirmed. By 1 1H- 1 1H TOCSY spectrum, the spin system of peptide substances could be confirmed. Further, through 13 1H- 1 13C HSQC, 1 1H- 1 1H COSY and 13 1H- α 13CHMBC, the number and types of amino acids in peptide substances could be determined. The connection order of amino acids was obtained by analyzing the 1 1H- 1 1H ROESY correlation signals of 13CH and -NH-. By comprehensively analyzing 1D and 2D NMR spectra and HR-MS data, planar structure information such as the number, types, and connection order of amino acids and the length of fatty acids could be obtained, and the planar structure of this class of target compounds was resolved as follows:

[0038]

[0039] Among them, compounds C and D are different from compounds A and B in that the former two have rich leucine (Leu) in their polypeptide chains. The NMR chemical shift signal assignments of these compounds are shown in Tables 2 and 3.

[0040] Table 2 Assignment of 1 H- and 13 C-NMR data for Pseudophomin A and B (dimethylformamide- d 7)

[0041]

[0042]

[0043] Note: a or b indicates overlapping chemical shift signals in the same column.

[0044] Table 3 Assignment of 1 H- and 13 C-NMR data for Pseudophomin C and D (dimethylformamide- d 7)

[0045]

[0046]

[0047] Note: a or b indicates overlapping chemical shift signals in the same column.

[0048] Among them, the typical two-dimensional nuclear magnetic resonance correlation spectra of compounds A and C are as Figure 1 shown.

[0049] After Figure 1 analyzing the planar structures of compounds A and C, X-ray single crystal diffraction technology was used in this experiment to determine the stereochemical structures of these substances. Compound A (6.0 mg) was dissolved in 1 mL of methanol / acetonitrile / water (5:5:1, v / v / v ), and the solvent was slowly evaporated at room temperature to form visible needle-like crystals. A crystal of appropriate size (0.15 × 0.12 × 0.11 mm) was subjected to crystal diffraction using an X-ray diffractometer to collect the corresponding data. The stereostructure of compound A was analyzed as Figure 2 shown. The crystallographic data of compound A are shown in Table 4.

[0050] Table 4 X-ray diffraction crystallographic data of compound A

[0051]

[0052] From the above results, it can be seen that compound A is the non-ribosomal peptide pseudophomin A. Since compounds B-D are all biosynthesized by Pseudomonas sp. HN8-3, they have the same stereochemical structure as pseudophomin A. So far, literature search shows that pseudophomins C and D are non-ribosomal peptides with novel structures, while pseudophomins A and B are known non-ribosomal peptide substances.

[0053] Example 2: Applications of pseudophomins A-D

[0054] Phytophthora capsici Phytophthora capsici pathogens were cultured on potato dextrose agar (PDA) at 28 °C in the dark for 7 days, and then the Petri dishes were placed on the laboratory bench for 5 days. 10 mL of sterile water was added to the Petri dishes, and they were placed at 4 °C for 30 min, and then the Petri dishes were placed on the laboratory bench to collect zoospores. The concentration of the spore suspension was adjusted to 1 × 10 5 CFU / mL for subsequent experiments. Pseudophomins A-D were prepared into a 50 mM stock solution in dimethyl sulfoxide, with dimethyl sulfoxide as the control. 1 mL of the zoospore suspension was taken in a sterile tube respectively, and the concentrations of compounds A-D in the zoospore suspension were prepared into 1 μM, 10 μM, and 50 μM. The effects of compounds A-D at different concentrations (1 μM, 10 μM, and 50 μM) on zoospores were tested, and the lysis time (s) of zoospores under different treatments was calculated. The results are shown in Figure 3 .

[0055] Figure 3 The results in show that under in vitro experimental conditions, the non-ribosomal peptide pure compounds pseudophomins A-D at a concentration of 1 μM or higher can lyse the zoospores of Phytophthora capsici; moreover, with the increase of the substance concentration, the speed of lysing zoospores accelerates, and the time required to lyse zoospores becomes less and less.

[0056] Plant seeds (cucumbers) were treated with 1% ( w / v)Surface sterilize with sodium hypochlorite solution for 1 min, then rinse 4 - 5 times with sterile water, pour into a sterile petri dish, and plant in a plastic box (21.5 × 14.5 × 8.2 cm) after drying. Cultivate in a plant growth chamber (25 °C, 12 h light / 12 h dark). Water the plants with an appropriate amount of tap water every 3 - 4 days to maintain a moist growth environment. After 3 weeks, conduct in - vivo plant tests when the seedlings grow up. Add 1 mL of sterile distilled water to each well of a sterile 24 - well plate to create a moist environment. Select the second young leaf of the plant (cucumber), cut a leaf disc with a diameter of 12 mm on the plant leaf, and quickly float the leaf disc in the 24 - well plate containing sterile distilled water with small forceps, with one leaf disc placed in each well. Prepare 4 24 - well plates for each experiment (a total of 96 plant leaf discs are added), and repeat the experiment independently 3 times (a total of 12 24 - well plates and 288 plant leaf discs are required for the experiment). Collect zoospores of Phytophthora capsici ( Phytophthora capsici )using the same method as in the in - vitro experiment, and adjust the concentration of zoospores to 1 × 10 5 CFU / mL for subsequent experiments. Prepare stock solutions of compounds A - D in DMSO using the same method as in the in - vitro experiment, and use DMSO as a negative control. Add 1 mL of zoospore suspension to 1.5 mL sterile tubes respectively, add a certain volume of the compound stock solution to the sterile tubes, and adjust the concentrations of the compounds to 1 μM, 10 μM, and 50 μM respectively. Drop 10 μL of zoospores of Phytophthora capsici pathogens treated with pseudophomins A - D onto the surface of the leaf discs to conduct in - vivo plant biocontrol experiment tests. Incubate the 24 - well plates in the dark at 25 °C for 48 hours, calculate the disease index (disease index = 100 × number of leaf discs with pathogen spread / total number of leaf discs), take the average value of the disease indices of 3 independent repeated experiments, obtain the average disease indices of different compounds at different concentrations, and use SPSS 26 statistical software to perform multiple comparisons between the average disease indices using Tukey test ( p <0.05). Different lowercase letters represent significant differences between the average disease indices corresponding to different treatments. The disease indices obtained after treating cucumber leaves with different concentrations of compound pseudophomins A - D are as shown in Figure 4 .

[0057] Figure 4 The results show that in vivo on plants (cucumber leaves), non - ribosomal peptide compounds of this type at a concentration of 10 μM or higher (50 μM) can effectively control Phytophthora capsici. Compared with the control group, non - ribosomal peptide compounds at a concentration of 10 μM or 50 μM can achieve a 100% control effect on Phytophthora capsici.

[0058] In summary, the non-ribosomal peptide compounds pseudophomins A-D can be used as good parent compounds for biopesticides and are used for the biological control of diseases such as Phytophthora capsici.

Claims

1. A class of non-ribosomal peptide compounds, characterized in that, This class of compounds is named pseudophomins C-D, and their chemical structures are as follows: 。 2. The method for preparing a class of non-ribosomal peptide compounds as claimed in claim 1, characterized in that, The non-ribosomal peptide compounds are obtained by liquid fermentation culture, solid-phase extraction technology and high-performance liquid chromatography separation of Pseudomonas sp. HN8-3. The weight composition of the liquid fermentation medium is as follows: 1-5% protease peptone, 0.1-1.0% KH2PO4, 0.1-0.5% MgSO4, 0.5-5.0% glycerol, 0.1-2.0% L-leucine, with a pH value of 5.0-9.0, and the balance is water; Pseudomonas sp. HN8-3 has been deposited in the Guangdong Microbial Culture Collection Center, with the deposit number GDMCC No. 62849.

3. The preparation method of a class of non-ribosomal peptide compounds as described in claim 2, characterized in that, After the fermentation is completed, centrifuge at 10,000 rpm for 5 min at room temperature, and collect the fermentation supernatant. Load the fermentation supernatant onto a solid-phase extraction column, and perform rapid decompression elution successively with 50% acetonitrile / 50% water and acetonitrile by volume percentage. The acetonitrile elution fraction is dried to obtain a crude extract. Dissolve the crude extract in methanol and perform reverse-phase high-performance liquid chromatography separation. The chromatographic column uses a 250×10 mm semi-preparative column of YMC ODS-A. The chromatographic mobile phase is acetonitrile:water = 80:20 by volume ratio, the flow rate is 2.5 mL / min, and the detection wavelength is 210 nm. Two pure compounds, pseudophomins C-D, are prepared.

4. The preparation method of a class of non-ribosomal peptide compounds according to claim 3, characterized in that, The fermentation temperature is 20-35 °C, and the fermentation time is 25-75 hours.

5. The preparation method of a class of non-ribosomal peptide compounds as claimed in claim 3, wherein The concentration of acetonitrile in the eluent used for the solid-phase extraction column is 20-100% by volume ratio, and the concentration of acetonitrile in the high-performance liquid chromatography mobile phase is 50-100% by volume ratio.

6. Use of the non-ribosomal peptide compound pseudophomins C-D according to claim 1 in the preparation of a biological drug for controlling Phytophthora capsici.

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