A root-promoting active substance isolated from the metabolites of the plant probiotic Trichoderma and its application

Abstract

The present invention discloses a root-promoting active substance separated from the metabolites of the plant probiotic Trichoderma and its application. The benzene-ring type root-promoting active substance has the molecular formula C7H7NO2, has the effect of promoting plant root development, significantly increases the total root length, total surface area and total root number of Arabidopsis thaliana, tomatoes and rice, has a stable effect, and has good application prospects; it can be used as a root-promoting agent in agricultural green production.

Description

Technical Field

[0001] The present invention belongs to the field of microorganisms, specifically a benzene-ring type root-promoting active substance produced by Trichoderma guizhouense NJAU4742 and its application. Technical Background

[0002] Providing a healthy food source for the growing global population has become one of the greatest challenges of this century. Therefore, the development of new technologies and products with low energy consumption and low pollution is crucial for the green and sustainable development of agriculture. Fungi of the genus Trichoderma spp., as one of the microbial groups that benefit human production and life, related developed microbial agents have been widely used in agricultural production and shown excellent plant growth promotion and biocontrol effects. In recent decades, the number of related research and patent applications has increased exponentially. Currently, the identified Trichoderma spp. beneficial to agricultural production are mainly distributed within the populations of T. atroviride, T. viride, T. asperellum, T. asperelloides, T. harzianum, T. guizhouense, and T. virens; some of these species (T. atroviride, T. harzianum, T. guizhouense, T. virens) have the effects of plant protection (biological fungicide) and plant growth promotion (biological fertilizer), and have been widely used in the biological control of fungal pests in sustainable agricultural production.

[0003] Rhizosphere-colonizing Trichoderma can produce more than 120 different types of secondary metabolites, and the most common chemical structures include terpenes, pyranones, polyketones, and non-ribosomal peptides. Currently, the identified hormones and signaling substances that regulate plant root architecture and promote root nutrient uptake include phytohormones, siderophores, koninginins, 6-pentyl-α-pyrone (6PP), trichocaranes A-D, harzianopyridone, cyclonerodiol, harzianic acid, swollen, etc. Summary of the Invention

[0004] The purpose of the present invention is to provide a benzene-ring type root-promoting active substance produced by fermentation of Trichoderma guizhouense NJAU4742.

[0005] Another purpose of the present invention is to provide a method for extracting and separating the above-mentioned root-promoting active substance.

[0006] Another purpose of the present invention is to provide the application of the above-mentioned root-promoting active substance.

[0007] The purpose of the present invention can be achieved by the following technical solutions:

[0008] A benzene ring-based root-promoting active substance, which is a compound with the following structure:

[0009]

[0010] The method for extracting and separating the above-mentioned benzene ring-based root-promoting active substance: ferment and culture Trichoderma guizhouense NJAU4742 with the preservation number of CGMCC NO.12166 and collect the supernatant of the fermentation broth. The supernatant is loaded onto a macroporous resin column and eluted with industrial alcohol in a gradient manner. The eluate with 50% concentration of industrial alcohol is collected. After the collected eluate is rotary evaporated to remove the solvent, it is dissolved in acetonitrile, and the above-mentioned benzene ring-based root-promoting active substance is further separated and purified by HPLC method.

[0011] As a preferred technical solution, the separation and purification are carried out under the conditions of acetonitrile-water 60:40, flow rate 2 ml / min, detection wavelength 280 nm, and Rt = 26.6 to obtain the above-mentioned benzene ring-based root-promoting active substance.

[0012] As a preferred technical solution, the medium used for the fermentation culture is an oligosaccharide inorganic salt medium. The formula of 1 L of the oligosaccharide inorganic salt medium is: 5 g of ammonium sulfate, 0.6 g of magnesium sulfate heptahydrate, 15 g of potassium dihydrogen phosphate, 1 g of calcium chloride, 5 mg of ferric sulfate heptahydrate, 1.6 mg of manganese sulfate monohydrate, 1.4 mg of zinc sulfate heptahydrate, 2 mg of cobalt chloride hexahydrate, and 10 g of glucose.

[0013] As a preferred technical solution, the macroporous resin column is eluted with industrial alcohol in gradients of 30%, 50%, and 80% concentrations respectively, and the eluate with 50% concentration is collected.

[0014] As a preferred technical solution, the process of fermenting and culturing Trichoderma guizhouense NJAU4742 with the preservation number of CGMCC NO.12166 is as follows: transfer the NJAU4742 seed liquid to the oligosaccharide inorganic salt medium at an inoculation amount of 1%, and culture at 28 °C and 170 rpm for 2 d; then inoculate the cultured bacterial liquid into a fermenter filled with the oligosaccharide inorganic salt medium for fermentation culture. At the beginning, the temperature is 28 °C, the rotation speed is 200 rpm, and the dissolved oxygen value is 75. When the dissolved oxygen value starts to drop below 30, gradually increase the rotation speed and at the same time increase the oxygen supply amount to keep the dissolved oxygen value above 10. After culturing for 2 d, adjust the temperature to 20 °C, the rotation speed to 200 rpm, and the oxygen supply amount to the lowest level. The bacterial liquid enters the stationary phase and starts to produce a large amount of secondary metabolites, and continue to ferment for 3 d.

[0015] The application of the above-mentioned benzene ring-based root-promoting active substance in promoting plant root development or preparing a preparation for promoting plant root development.

[0016] The plant described is a dicotyledon or a monocotyledon. The dicotyledon is Arabidopsis thaliana or tomato; the monocotyledon is rice.

[0017] Application of the Guizhou Trichoderma strain NJAU4742 with the preservation number of CGMCC NO.12166 in the production of the above-mentioned benzene-ring type root-promoting active substance.

[0018] The present invention screens and verifies the root-promoting active substance through the following method: Knock out the auxin synthesis-related genes and 6PP synthesis genes of Guizhou Trichoderma NJAU4742 with the preservation number of CGMCC NO.12166 to construct the Guizhou Trichoderma NJAU4742-ΔiaaH2&ald1&ald2&nit1-Δctf1 mutant strain. Ferment the mutant strain. After the fermentation ends, filter the mycelium from the fermentation broth with four layers of sterile gauze, collect the supernatant, load the supernatant onto a macroporous resin column of about 500 g with 100-200 mesh, and elute it with industrial alcohol at concentrations of 30%, 50% and 80% in gradient. Collect the eluate with 50% concentration of industrial alcohol. After rotary evaporation to remove the solvent from the 50% concentration of industrial alcohol eluate, dissolve it with acetonitrile, and further separate and purify the growth-promoting substance by HPLC method. The above-mentioned benzene-ring type growth-promoting substance is obtained at acetonitrile-water 60:40, flow rate 2 ml / min, detection wavelength 280 nm, and Rt = 26.6. Track the biological activity function of the active components separated by HPLC with about 500 g of 100-200 mesh, and finally obtain the pure product of the above-mentioned benzene-ring type root-promoting active substance.

[0019] Advantages of the present invention:

[0020] This application verifies the growth-promoting activity of the fermentation broth of Guizhou Trichoderma NJAU4742 through gene knockout experiments and growth-promoting experiments, explores the optimal culture medium and culture conditions for substance fermentation, separates and purifies a benzene-ring type root-promoting active substance C7H7NO2, and identifies the molecular structure of the substance. After verification, this root-promoting active substance can promote the total root length, total surface area and total root number of Arabidopsis thaliana, tomato and rice.

[0021] Biological preservation information:

[0022] NJAU4742, classified and named as Trichoderma guizhouense, is preserved in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms. The preservation address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The preservation number is CGMCC NO.12166, and the preservation date is April 11, 2016. Description of the drawings

[0023] Figure 1Promoting effects of wild-type NJAU4742 and mutant ΔiaaH2&ald1&ald2&nit1-Δctf1 on the root development of Arabidopsis thaliana.

[0024] Figure 2 Promoting effects of benzene ring compound C7H7NO2 on the root development of tomatoes.

[0025] Figure 3 Function of benzene ring compound C7H7NO2 in promoting the root development of rice. Specific implementation manners

[0026] The present invention discloses a benzene ring type root-promoting active substance, and those skilled in the art can refer to the content herein and appropriately modify the parameters to implement it. In order to enable those skilled in the art to better understand the technical solution of the present invention, the following further details the present invention in combination with specific implementation cases.

[0027] Example 1: Verification of the root-promoting activity of the benzene ring compound described in the present invention on Arabidopsis thaliana:

[0028] Trichoderma guizhouense NJAU4742 extracellularly secretes auxin (IAA) and its analogs and 6-pentyl-α-pyrone, which have the activity of promoting plant root development. In order to explore whether there are other active substances in the extracellular secretions of NJAU4742 with growth-promoting functions, the genes related to auxin synthesis and 6PP synthesis genes of Trichoderma were knocked out successively by the three-fragment fusion method replaced by hygromycin B resistance to construct the mutant strain Trichoderma guizhouense NJAU4742-ΔiaaH2&ald1&ald2&nit1-Δctf1 【1】 . First, the gene of the hygromycin B resistance screening marker was fused with the upstream and downstream homologous arms of the gene to be knocked out by the method of fusion PCR. The fusion fragment was transferred into the competent NJAU4742. 200 μl of the above mixture was pipetted onto a PDA plate containing 1 M sucrose, evenly spread, and cultured in the dark at 28 °C for 12 - 16 h. The next day, a PDA (without sucrose) medium containing 200 μg / ml hygromycin B with a thickness of about 2 mm was slowly poured onto the PDA medium, and continued to be cultured in the dark at 28 °C for 24 - 48 h until small NJAU4742 transformants grew on the PDA plate containing hygromycin B. The transformants were selected and verified to obtain the correct mutant ΔiaaH2&ald1&ald2&nit1-Δctf1.

[0029] NJAU4742 and ΔiaaH2&ald1&ald2&nit1-Δctf1 were inoculated into an oligosaccharide inorganic salt medium (the formula for 1 L of the medium was: 5 g of ammonium sulfate, 0.6 g of magnesium sulfate heptahydrate, 15 g of potassium dihydrogen phosphate, 1 g of calcium chloride, 5 mg of ferric sulfate heptahydrate, 1.6 mg of manganese sulfate monohydrate, 1.4 mg of zinc sulfate heptahydrate, 2 mg of cobalt chloride hexahydrate, 10 g of glucose). First, they were cultured at 28 °C and 170 rpm for 2 d, then at 20 °C and 200 rpm for 3 d. Finally, the fermentation broth was filtered through four layers of sterile gauze to remove the mycelium. The supernatant was loaded onto a macroporous resin column and eluted with industrial alcohol in a gradient (the elution sequence was 30%, 50%, and 80% in turn). The effluent of 50% industrial alcohol was taken, rotary evaporated, and extracted with dimethyl sulfoxide (DMSO) to obtain the crude extract. The crude extract was filtered through a 0.22-μm sterile organic phase filter membrane to remove the precipitate, impurities, and mycelium, and stored at -20 °C. DMSO was used as the control group, and the crude extracts of NJAU4742 and ΔiaaH2&ald1&ald2&nit1-Δctf1 were used as the experimental groups.

[0030] The model plant Arabidopsis thaliana in root development research was selected, and a root growth promotion experiment was carried out on 1 / 2 MS medium (the formula for 1 L of the medium was: 2.154 g of MS salt, 0.5 g of MES, 0.1 g of inositol, 10 g of sucrose, 8 - 10 g of agar, pH 5.7). First, the crude extract was added to the 1 / 2 MS medium at an addition amount of 4‰ (v / v) and poured into plates (13 * 13 cm). After the plates cooled, 5-day-old Arabidopsis thaliana seedlings were transplanted into the culture dishes (4 plants / dish). The culture dishes were placed in an artificial climate chamber (16 hr:8 hr, day:night), at a temperature of 22 °C, and a light intensity of 100 μmol m -2 s -1 , and after being vertically placed and cultured for 7 d, the experimental results were observed.

[0031] The experimental results of the growth promotion effects of wild-type NJAU4742 and mutant ΔiaaH2&ald1&ald2&nit1-Δctf1 on the root development of Arabidopsis thaliana were as Figure 1 shown. Both wild-type NJAU4742 and mutant ΔiaaH2&ald1&ald2&nit1-Δctf1 had promoting effects on the root development of Arabidopsis thaliana, indicating that there were other unknown growth-promoting substances in the fermentation broth of mutant ΔiaaH2&ald1&ald2&nit1-Δctf1.

[0032] Example 2: Preparation and separation and purification of the benzene ring type root growth promoting active substance of the present invention:

[0033] The fermentation broth of the mutant ΔiaaH2&ald1&ald2&nit1-Δctf1 still has the ability to promote the root development of Arabidopsis thaliana. Therefore, we can isolate and purify the bioactive substances contained in the fermentation broth of ΔiaaH2&ald1&ald2&nit1-Δctf1 by means of bioactivity tracking, that is, verify the promoting activity on the root development of Arabidopsis thaliana at each step of separation, and further isolate and purify the active components.

[0034] The NJAU4742 seed liquid was transferred to a fresh conical flask of oligosaccharide inorganic salt medium at an inoculation amount of 1% (v / v), and cultured at 28 °C and 170 rpm for 2 days; then the cultured bacterial liquid was inoculated into a 50 L small fermenter filled with 30 L of oligosaccharide inorganic salt medium. The initial temperature was 28 °C, the rotation speed was 200 rpm, and the dissolved oxygen value was 75. When the dissolved oxygen value began to drop below 30, the rotation speed was gradually increased, and at the same time, the oxygen supply was increased to keep the dissolved oxygen value above 10. After 2 days of culture, the temperature was adjusted to 20 °C, the rotation speed was adjusted to 200 rpm, and the oxygen supply was maintained at the lowest level. The bacterial liquid entered the stationary phase and began to produce a large amount of secondary metabolites, and continued to ferment for 3 days.

[0035] After the fermentation was completed, the fermentation broth was filtered and sterilized with four layers of sterile gauze, and the supernatant was collected. The supernatant was loaded onto a macroporous resin column of about 500 g with a mesh size of 100-200, and gradient elution was carried out with industrial alcohol (the elution sequence was 30%, 50% and 80% in turn). 100 grams of extract was obtained after rotary evaporation of the solvent from the effluent of 50% concentration industrial alcohol. An appropriate amount of acetonitrile was added to dissolve it completely and filtered through a 0.22 μm sterile organic phase filter membrane to remove precipitates, impurities and mycelia. The obtained crude extract was further isolated and purified using an HPLC chromatographic column, and gradient elution was carried out with an acetonitrile-water concentration gradient (20:80; 30:70; 40:60; 50:50; 60:40; 70:30; 80:20; 90:10, v:v), the flow rate was 2 ml / min, and the detection wavelength was 280 nm. The crude substance was divided into 8 segments, and the promoting bioactivity on the root development of Arabidopsis thaliana was verified for each segment collected in turn (the method refers to Example 1). The 5th segment had strong activity. The 5th segment (1.5 grams) was dissolved in acetonitrile completely and further isolated and purified using an HPLC chromatographic column. Gradient elution was carried out with an acetonitrile-water concentration gradient (60:40), the flow rate was 2 ml / min, and the detection wavelength was 280 nm. Compound 5-(3) with relatively significant activity was obtained at Rt = 26.6. 5-(3) has good promoting bioactivity on the root development of Arabidopsis thaliana (the method refers to Example 1), and finally the pure product of the active component was obtained. The active component was tested by mass spectrometry and NMR (including two-dimensional NMR structure analysis). The final active compound was a benzene ring compound, and the molecular structure is shown as follows:

[0036]

[0037] By using a 600 MHz nuclear magnetic resonance spectrometer to conduct proton nuclear magnetic resonance (1H-NMR), carbon-13 nuclear magnetic resonance (13C-NMR), 1 H- 1 H COSY, HSQC, and HMBC tests on 5-(3), the analysis results are shown in Table 1:

[0038] Table 1 Identification Results of the Molecular Structure of Compound 5-(3)

[0039]

[0040] Through 1 H-NMR coupling information and 1 H- 1 H COSY spectrum, it can be determined that H-3 (δ H 7.68), H-4 (δ H 6.49), H-5 (δ H 7.20), and H-6 (δ H 6.72) in 5-(3) are mutually coupled. Combining with the HSQC spectrum, their carbon-hydrogen assignments can be determined, that is, the chemical shifts of C-3, C-4, C-5, and C-6 are 131.7, 115.0, 134.1, and 116.8 respectively. In the HMBC spectrum: the long-range carbon-hydrogen correlations of H-3 to C-1, C-5, and C-7, H-5 to C-3 and C-7, and H-6 to C-2 and C-4 can confirm the above speculation and determine that the chemical shifts of the quaternary carbons C-1, C-2, and C-7 in the structure are 170.2, 110.3, and 152.0 respectively. Finally, through separation, purification, and identification, it is determined that the root-promoting active substance is a benzene-ring compound with the molecular formula C7H7NO2.

[0041] Example 3: Verifying the promoting activity of the benzene-ring substance described in the present invention on plant root development:

[0042] One dicotyledonous crop and one monocotyledonous crop were selected to verify the root-promoting activity of the isolated benzene-ring substance. The two selected crops are:

[0043] Tomato (Solanum lycopersicum), variety 903;

[0044] Rice (Oryza sativa), variety Nipponbare;

[0045] For the plant root growth promotion experiment, first, the isolated benzene ring substances were prepared into a 40 mM stock solution and serially diluted to final concentrations of 0 μM, 5 μM, 15 μM, 30 μM, and 45 μM. These were added to 1 / 2 MS medium and poured into plates (13 * 13 cm). After the plates cooled, the disinfected tomato and rice seeds were sown separately in petri dishes (10 seeds per dish). The petri dishes were placed in an artificial climate chamber (16 hr:8 hr, day:night), at a temperature of 22 °C, and a light intensity of 100 μmol m -2 s -1 . After vertical cultivation for 10 days, when the seeds germinated and the root tips touched the bottom of the petri dish, the petri dishes were collected and the experimental results were observed.

[0046] The experimental results of the growth promotion effect of benzene ring compound C7H7NO2 on tomato root development are as Figure 2 shown. The isolated benzene ring substances had good growth promotion activity on the total root length, total root surface area, and total root number of tomato plants.

[0047] The experimental results of the effect of benzene ring compound C7H7NO2 on promoting rice root development are as Figure 3 shown. The isolated benzene ring substances had good promoting activity on the total root length, total root surface area, and total root number of rice plants.

[0048] 1. Zhu, H. et al. Intracellular kynurenine promotes acetaldehyde accumulation, further

[0049] inducing the apoptosis in soil beneficial fungi Trichoderma guizhouense NJAU4742

[0050] under acid stress. Environ. Microbiol. (2022) doi:10.1111 / 1462 - 2920.16286.

Claims

1. A method for extracting and separating benzene ring-based root-promoting active substances, characterized in that, Ferment and culture Trichoderma guizhouense NJAU4742 with the preservation number of CGMCC NO. 12166 and collect the supernatant of the fermentation broth. The supernatant is loaded onto a macroporous resin column and eluted with a gradient of industrial alcohol. Collect the eluate with 50% concentration of industrial alcohol. After rotary evaporation to remove the solvent from the collected eluate, dissolve it with acetonitrile, and further separate and purify to obtain the benzene ring type root-promoting active substance by HPLC method; the benzene ring type root-promoting active substance is a compound with the following structure: 。 2. The method according to claim 1, wherein The above-mentioned separation and purification is to obtain the benzene ring type root-promoting active substance under the conditions of acetonitrile-water 60:40, flow rate 2 ml / min, detection wavelength 280 nm, and Rt = 26.

6.

3. The method according to claim 1, wherein The medium used for the above-mentioned fermentation and culture is an oligosaccharide inorganic salt medium.

4. The method according to claim 1, characterized in that The above-mentioned macroporous resin column is eluted with a gradient of industrial alcohol with concentrations of 30%, 50% and 80% respectively, and the eluate with 50% concentration is collected.

5. The method according to claim 1, wherein The process of fermenting and culturing Trichoderma guizhouense NJAU4742 with the preservation number of CGMCC NO. 12166 is as follows: Transfer the NJAU4742 seed liquid to the oligosaccharide inorganic salt medium at an inoculation amount of 1%, and culture at 28 °C and 170 rpm for 2 d; then inoculate the cultured bacterial liquid into a fermenter filled with the oligosaccharide inorganic salt medium for fermentation and culture. At the beginning, the temperature is 28 °C, the rotation speed is 200 rpm, and the dissolved oxygen value is 75. When the dissolved oxygen value starts to drop below 30, gradually increase the rotation speed and at the same time increase the oxygen supply amount to keep the dissolved oxygen value above 10. After culturing for 2 d, adjust the temperature to 20 °C, the rotation speed to 200 rpm, and keep the oxygen supply amount at the lowest level. The bacterial liquid enters the stationary phase and starts to produce a large amount of secondary metabolites, and continue to ferment for 3 d.

6. Use of Trichoderma guizhouense NJAU4742 strain with preservation number CGMCC NO. 12166 in the production of benzene ring-based root-promoting active substances, characterized in that, The above-mentioned benzene ring type root-promoting active substance is a compound with the following structure: 。

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