Method for preparing extract of fermentation broth of plant endophytic fungi and its application

Abstract

The invention discloses a preparation method of an extract of plant endophytic fungal fermentation broth, belonging to the technical field of agricultural preparations. After activating and culturing the plant endophytic fungi, they are inoculated into a potato dextrose liquid medium for culture. The obtained fermentation broth is concentrated to one-fifth of the original volume, and then absolute ethanol is added until the ethanol content in the mixed solution is 75%. It is left standing at 4°C for more than 2 h, and the precipitate is removed by centrifugation. The supernatant is concentrated to one-fourth of the original volume to obtain a supernatant concentrate. Then, sevage reagent is added, and after mixing, it is centrifuged, and the upper layer solution is collected, concentrated under reduced pressure, and freeze-dried to obtain the product. This method is simple to operate. The prepared extract of plant endophytic fungal fermentation broth can improve the salt tolerance of plants, and is green and environmentally friendly. It can be used to prepare a plant salt-resistant coating agent and applied to various seed plants. After use, it can induce plants to produce salt-resistant effects, promote plant growth, and improve the yield and quality of crops, and has good application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of agricultural preparations, and particularly relates to a preparation method of an extract of a plant endophytic fungal fermentation broth and its application for preparing a plant anti-salt coating agent. Background Art

[0002] The problem of cultivated land salinization in China is becoming increasingly serious. At present, the saline-alkali land area in China accounts for about 10.9% of the total land area, and it is distributed in the northwest, north China, the western part of northeast China and the coastal areas. The formation of alkali soil and alkalized soil in China is mostly related to the accumulation of carbonates in the soil. In severely saline-alkali soil areas, plants can hardly survive. A large number of crops are difficult to grow and develop normally due to varying degrees of salt damage, resulting in a significant reduction in yield, which seriously restricts the development of agriculture. The harm of salinity stress to plants includes causing ion toxicity, inhibiting photosynthesis, leading to lipid peroxidation, excessive oxygen free radicals, nutrient imbalance in plants, and imbalance in endogenous hormone levels, ultimately affecting crop yields. Compared with normal conditions, the bud length of rice seeds germinated in a salt environment is significantly reduced; salt stress also causes the leaves of naked oats to wither and turn yellow, and the chlorophyll content to decrease; when Arabidopsis thaliana grows in a medium containing 120 mM NaCl compared with plants in a salt-free medium, the leaves are smaller and withered and yellow, and the whole plant is smaller. At present, in addition to the method of flooding to reduce the salt content on the soil surface, transgenic salt-tolerant plants can also be planted to increase the yield of crops growing under salt stress. However, these methods have disadvantages such as high cost and being unfavorable for environmental sustainable development.

[0003] In recent years, many reports have confirmed that root microorganisms symbiotic with plants can assist plants in improving their ability to resist salt stress. Assisting plants in resisting salt through root microorganisms has the characteristics of low toxicity, easy degradation, and no pollution to the environment, and has become one of the research directions for improving the salt tolerance of crops in saline-alkali land. However, there is currently no report on using an extract of a plant endophytic fungal fermentation broth to prepare a plant anti-salt coating agent. Summary of the Invention

[0004] The purpose of the present invention is to solve the deficiencies of the prior art, and provide a preparation method of an extract of a plant endophytic fungal fermentation broth, which is simple to operate, and the prepared extract of the plant endophytic fungal fermentation broth has the effect of improving plant salt tolerance and is environmentally friendly.

[0005] Another purpose of the present invention is also to provide the application of the above-mentioned extract of the plant endophytic fungal fermentation broth for preparing a plant anti-salt coating agent.

[0006] Technical Solution

[0007] A preparation method of an extract of a plant endophytic fungal fermentation broth includes the following steps:

[0008] (1) After activating and culturing the endophytic fungi of plants, inoculate them into a potato dextrose liquid medium for culture to obtain a fermentation broth;

[0009] (2) Concentrate the fermentation broth to one-fifth of its original volume, then add absolute ethanol to the mixture until the ethanol content in the mixture is 75%, let it stand at 4°C for more than 2 h, centrifuge to remove the precipitate, and concentrate the obtained supernatant to one-fourth of its original volume to obtain a concentrated supernatant;

[0010] (3) Add sevage reagent to the concentrated supernatant, mix evenly, then centrifuge and collect the upper layer solution;

[0011] (4) Concentrate the upper layer solution under reduced pressure and then lyophilize it to obtain an extract of the endophytic fungal fermentation broth of plants.

[0012] In step (1), the endophytic fungi of plants are Paecilomyces variotii.

[0013] Furthermore, in steps (2) and (3), the conditions for centrifugation are: 4000 - 5000 r / min, 15 - 25 min.

[0014] Furthermore, in step (3), the sevage reagent is composed of chloroform and n-butanol mixed in a volume ratio of 5:1.

[0015] Furthermore, in step (3), the dosage of the sevage reagent accounts for 1 / 4 of the volume of the concentrated supernatant.

[0016] The application of the extract of the endophytic fungal fermentation broth of plants prepared by the above method in the preparation of a plant salt-resistant coating agent is as follows: Add 3 - 5 parts of agarose and 1 - 5 parts of hydroxypropyl starch to 100 parts of deionized water, heat to complete boiling, and completely dissolve the agarose and hydroxypropyl starch to obtain a mixed solution. Cool it to 40°C, then add 1 - 5 parts of the extract of the endophytic fungal fermentation broth of plants, 0.5 - 1 part of carbendazim, and 0.5 - 1 part of scarlet red powder, mix evenly to obtain a plant salt-resistant coating agent; the above parts are all in parts by mass.

[0017] The usage method of the plant salt-resistant coating agent: Mix the seeds with the plant salt-resistant coating agent preheated to 40°C at a mass ratio of 1:10 evenly, and naturally cool to room temperature to obtain the coated seeds.

[0018] The beneficial effects of the present invention:

[0019] 1) The present invention provides a method for preparing an extract of an endophytic fungal fermentation broth of plants, which is simple to operate. The prepared extract of the endophytic fungal fermentation broth of plants is a secondary metabolite of endophytic fungi, non-toxic, pollution-free, and environmentally friendly.

[0020] 2) The extract of the fermentation broth of the endophytic fungi of the present invention has the effect of improving the salt tolerance of plants. It is a green and environmentally friendly bio-source induced anti-salt substance, which can be used to prepare a plant anti-salt coating agent and applied to various seed plants, such as wheat, rice, sorghum, corn, etc. After use, it can induce plants to produce anti-salt effects, promote plant growth, improve the yield and quality of crops, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Photographs of sorghum seedlings in the salt stress control group and the experimental group treated with the anti-salt coating agent;

[0022] Figure 2 Photographs of rice seedlings in the normal group, the salt stress control group and the experimental group treated with the anti-salt coating agent;

[0023] Figure 3 Photographs of mint plants in the normal group, the salt stress control group and the experimental group treated with the anti-salt coating agent. DETAILED DESCRIPTION OF THE INVENTION

[0024] The technical solutions of the present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0025] Example 1

[0026] A method for preparing an extract of the fermentation broth of endophytic fungi of plants, comprising the following steps:

[0027] (1) After activating and culturing Paecilomyces variotii, inoculate it into 1000 mL of potato dextrose liquid medium (preparation method: take 200 g of peeled potatoes, put them into 1 liter of deionized water and boil until soft and rotten, then filter, take the filtrate and make up to 1 liter, add 20 g of glucose, and sterilize at 121 °C for 30 min.), and shake and culture at 28 °C and 120 r / min for 7 d to obtain the fermentation broth;

[0028] (2) Concentrate the fermentation broth to 200 mL, then add 600 mL of absolute ethanol, let it stand at 4 °C for more than 2 h, centrifuge at 5000 r / min for 20 min, remove the precipitate, and concentrate the obtained supernatant to 200 mL to obtain a concentrated supernatant;

[0029] (3) Add 50 mL of sevage reagent (chloroform: n-butanol (5:1 v / v)) to the concentrated supernatant, shake for 20 min, centrifuge at 5000 r / min for 20 min, collect the upper layer solution, and repeat until there is no protein emulsion in the middle layer;

[0030] (4) Concentrate the upper layer solution under reduced pressure and then freeze-dry to obtain the extract of the endophytic bacteria fermentation broth.

[0031] Preparation of a plant salt - resistant coating agent using the above - mentioned extract of the endophytic bacteria fermentation broth: Add 5 g of agarose and 5 g of hydroxypropyl starch to 100 mL of deionized water, heat to complete boiling, dissolve the agarose and hydroxypropyl starch completely in water to obtain a mixed solution, cool to 40 °C, add 5 g of the extract of the endophytic bacteria fermentation broth, 1 g of carbendazim and 1 g of scarlet powder, stir and mix evenly to obtain the plant salt - resistant coating agent.

[0032] Application test: Select 60 g of sorghum seeds of uniform size, disinfect with 75% ethanol for 30 s, wash repeatedly with deionized water, air - dry, and divide them evenly into 3 parts, namely the normal group (Normal), the salt - stress control group (CK), and the experimental group (Treatment). Among them, the seeds in the experimental group are coated with the plant salt - resistant coating agent of Example 1 (mix the seeds with the plant salt - resistant coating agent pre - heated to 40 °C at a mass ratio of 1:10, and cool naturally to room temperature to obtain the coated seeds), the seeds in the normal group are not treated, and the seeds in the salt - stress control group (CK) are coated with a coating agent without adding the extract of the endophytic bacteria fermentation broth, and the coating method is the same as that of the experimental group. Mix peat soil: loess: nutrient soil in a ratio of 3:1:1 as the experimental soil. Among them, the soil in the normal group does not contain salt, and the salt content of the soil in the control group and the experimental group is 5‰. Sow the three groups of sorghum seeds into the experimental soil respectively, and place them in a plant light incubator. The growth conditions are: 14 h of light / 10 h of darkness, humidity 35%, and temperature 25 °C. After they grow to 3 cm in height, select seedlings with consistent growth trends and uniform sizes among groups and transplant them into large flowerpots to continue growing. After 45 days, count the plant height, stem diameter, and total root length of the sorghum seedlings.

[0033] Figure 1 Figure [X] shows the photos of sorghum seedlings in the salt - stress control group and the experimental group treated with the salt - resistant coating agent. Table 1 shows the statistical test results of plant height, stem diameter, and total root length:

[0034] Table 1 Effects of the plant salt - resistant coating agent on the growth of sorghum seedlings

[0035]

[0036] From Figure 1 and Table 1, it can be seen that compared with the salt - stress control group, the plant height of the experimental group treated with the salt - resistant coating agent increased by 25%, the stem diameter increased by 25%, and the root length increased by 90%. Thus, it can be seen that the use of the salt - resistant coating agent can significantly improve the growth state of sorghum under salt stress.

[0037] Example 2

[0038] The preparation of the extract of the endophytic bacteria fermentation broth is the same as that in Example 1.

[0039] Preparation of a plant salt-resistant coating agent using the above-mentioned extract of the endophytic bacteria fermentation broth: Add 4 g of agarose and 3 g of hydroxypropyl starch to 100 mL of deionized water, heat to complete boiling, dissolve the agarose and hydroxypropyl starch completely in the water to obtain a mixed solution, cool to 40 °C, add 3 g of the extract of the endophytic bacteria fermentation broth, 0.8 g of carbendazim and 0.8 g of scarlet powder, stir and mix evenly to obtain the plant salt-resistant coating agent.

[0040] Application test: Select 30 g of rice seeds of uniform size, disinfect them with 75% ethanol for 30 s, rinse them thoroughly with deionized water repeatedly, and after drying, divide them evenly into 3 parts by weight, namely the normal group (Normal), the salt stress control group (CK), and the experimental group (Treatment). Among them, the seeds in the experimental group are coated with the plant salt-resistant coating agent of Example 2 (mix the seeds with the plant salt-resistant coating agent preheated to 40 °C at a mass ratio of 1:10, and cool naturally to room temperature to obtain the coated seeds). The seeds in the normal group are not treated, and the seeds in the salt stress control group (CK) are coated with a coating agent without adding the extract of the endophytic bacteria fermentation broth, and the coating method is the same as that of the experimental group. Peat soil: Loess: Nutrient soil are mixed evenly at a ratio of 3:1:1 as the experimental soil. Among them, the soil in the normal group does not contain salt, and the salt content of the soil in the control group and the experimental group is 5‰. Sow the three groups of rice seeds into the experimental soil respectively and place them in a plant light incubator. The growth conditions are: 14 h of light / 10 h of darkness, humidity 40%, and temperature 25 °C. After culturing for 60 days, count the plant height and total root length of the rice seedlings.

[0041] Figure 2 Photographs of rice seedlings in the normal group, the salt stress control group, and the experimental group treated with the salt-resistant coating agent. The statistical test results of plant height and total root length are shown in Table 2:

[0042] Table 2 Effects of the plant salt-resistant coating agent on the growth of rice seedlings

[0043]

[0044] From Figure 2 and Table 2, it can be seen that after adding the salt-resistant coating agent, the plant height and root system of rice have been significantly improved compared with the control group.

[0045] Example 3

[0046] The preparation of the extract of the endophytic bacteria fermentation broth is the same as that in Example 1.

[0047] Preparation of a plant salt-resistant coating agent using the extract of the above-mentioned endophytic bacteria fermentation broth: Add 3 g of agarose and 1 g of hydroxypropyl starch to 100 mL of deionized water, heat to complete boiling to completely dissolve the agarose and hydroxypropyl starch in water to obtain a mixed solution, cool to 40 °C, add 1 g of the extract of the endophytic bacteria fermentation broth, 0.5 g of carbendazim, and 0.5 g of scarlet red powder, stir and mix evenly to obtain the plant salt-resistant coating agent.

[0048] Application test: Select 15 g of mint seeds of uniform size, disinfect with 75% ethanol for 30 s, rinse repeatedly with deionized water, dry, and divide them into 3 equal parts by weight, namely the normal group (Normal), the salt stress control group (CK), and the experimental group (Treatment). Among them, the seeds in the experimental group are coated with the plant salt-resistant coating agent of Example 3 (mix the seeds with the plant salt-resistant coating agent preheated to 40 °C at a mass ratio of 1:10, and naturally cool to room temperature to obtain the coated seeds), the seeds in the normal group are not treated, and the seeds in the salt stress control group (CK) are coated with a coating agent without adding the extract of the endophytic bacteria fermentation broth, and the coating method is the same as that of the experimental group. Peat soil: Loess: Nutrient soil are mixed evenly at a ratio of 3:1:1 as the experimental soil. Among them, the soil in the normal group does not contain salt, and the salt content of the soil in the control group and the experimental group is 5‰. Sow the three groups of mint seeds into the experimental soil respectively and place them in a plant light incubator. The growth conditions are: 14 h of light / 10 h of darkness, humidity 40%, and temperature 25 °C. After culturing for 60 days, count the plant height and total root length of the mint seedlings.

[0049] Figure 3 Photos of mint plants in the normal group, the salt stress control group, and the experimental group treated with the salt-resistant coating agent. The statistical test results of plant height and total root length are shown in Table 3:

[0050] Table 3 Effects of the plant salt-resistant coating agent on the growth of mint seedlings

[0051]

[0052] As can be seen from Figure 3 and Table 3, after adding the salt-resistant coating agent, the growth of mint has been significantly improved.

Claims

1. Method for preparing extract of fermentation broth of plant endophytic fungi capable of inducing salt resistance in plants, characterized in that, it comprises the following steps: (1) After activating and culturing the plant endophytic fungi, inoculate them into a potato dextrose liquid medium for culture to obtain a fermentation broth; (2) Concentrate the fermentation broth to one-fifth of its original volume, then add absolute ethanol to make the ethanol content in the mixture 75%, let it stand at 4°C for more than 2 h, centrifuge to remove the precipitate, and concentrate the obtained supernatant to one-fourth of its original volume to obtain a concentrated supernatant; (3) Add sevage reagent to the concentrated supernatant, mix evenly, then centrifuge and collect the upper layer solution; (4) Concentrate the upper layer solution under reduced pressure and then freeze-dry to obtain the extract of the fermentation broth of plant endophytic fungi; In step (1), the plant endophytic fungi are Paecilomyces variotii.

2. Method for preparing extract of fermentation broth of plant endophytic fungi capable of inducing salt resistance in plants according to claim 1, characterized in that, In steps (2) and (3), the centrifugation conditions are: 4000 - 5000 r / min, 15 - 25 min.

3. Method for preparing extract of fermentation broth of plant endophytic fungi capable of inducing salt resistance in plants according to claim 1, characterized in that, In step (3), the sevage reagent is composed of chloroform and n-butanol mixed in a volume ratio of 5:

1.

4. Method for preparing extract of fermentation broth of plant endophytic fungi capable of inducing salt resistance in plants according to claim 1 or 2 or 3, characterized in that, In step (3), the dosage of the sevage reagent accounts for 1 / 4 of the volume of the concentrated supernatant.

5. Application of the extract of the fermentation broth of plant endophytic fungi prepared by the method according to claim 1 or 2 or 3 or 4 in the preparation of a plant salt-resistant coating agent.

6. Application according to claim 5, characterized in that, The application method is: add 3 - 5 parts of agarose and 1 - 5 parts of hydroxypropyl starch to 100 parts of deionized water, heat to complete boiling and completely dissolve the agarose and hydroxypropyl starch to obtain a mixed solution, cool to 40°C, then add 1 - 5 parts of the extract of the fermentation broth of plant endophytic fungi, 0.5 - 1 part of carbendazim, and 0.5 - 1 part of scarlet powder, mix evenly to obtain a plant salt-resistant coating agent; the above parts are all by mass.

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