Soil conditioner capable of degrading phenolic acid and application of soil conditioner

Abstract

The invention belongs to the technical field of biological fertilizers, and discloses a phenolic acid degrading soil conditioner and application thereof, and the phenolic acid degrading soil conditioner is prepared by using robinia pseudoacacia roots as a raw material. The active ingredient of the soil conditioner is robinia pseudoacacia root exudates, and the preparation method of the soil conditioner comprises the following steps: performing water culture on robinia pseudoacacia plants in a container for 3-5 days to obtain a collection solution containing the robinia pseudoacacia root exudates. Another preparation method of the soil conditioner comprises the following steps: S1, mixing robinia pseudoacacia roots and water in a mass ratio of 1: (1-2), and standing for 3-5 days under an illumination condition to obtain a mixed solution; s2, homogenizing the mixed solution, adding cellulose degrading bacteria, and fermenting for 5-7 days to obtain a root fermentation product; and S3, mixing the root fermentation product with a carrier to prepare the phenolic acid degradation fertilizer. The soil conditioner is simple in preparation method, low in cost and safe and environment-friendly to use, the content of phenolic acid substances in continuous cropping soil of crops can be effectively reduced, harm of the phenolic acid substances to growth and development of konjak is relieved, and konjak planting is facilitated.

Description

Technical Field

[0001] The invention relates to the technical field of biological fertilizers, and in particular to a soil conditioner for degrading phenolic acid and application thereof. Background Art

[0002] At present, the problems of reduced crop yield and quality, aggravated diseases, etc. caused by long-term continuous cropping are becoming more and more serious. Continuous cropping of konjac also greatly affects the development of konjac planting industry. Studies have found that among the many factors leading to continuous cropping obstacles, the accumulation of phenolic acid autotoxic allelopathic substances is an important factor, which can be released into the soil environment through plant volatilization, root secretion, leaching, decomposition of litter and its residues; studies have shown that phenolic acid can significantly change the physical and chemical properties of the soil and the structure of microbial populations, and also have an important impact on the enzyme content and activity, cell membrane permeability, absorption of mineral elements, chlorophyll content, etc. in crops, thereby inhibiting the growth of crops; in addition, under continuous cropping conditions, phenolic acid accumulated in the soil will promote the reproduction of soil-borne pathogens, leading to an increase in the incidence of plant diseases. The accumulation characteristics and hazards of phenolic acid substances in continuous cropping soil have been confirmed in vegetables, forests, Chinese medicinal materials and many other plants.

[0003] However, at present, there are no products on the market that can effectively degrade phenolic acids in the soil of continuously cropped crops and are safe and environmentally friendly. Therefore, the existing technology needs to be further improved. Summary of the invention

[0004] In view of the above problems, the present invention provides a soil conditioner for degrading phenolic acid and its application. The soil conditioner is prepared with Robinia pseudoacacia root as raw material, is safe and environmentally friendly to use, can effectively degrade phenolic acid substances in the rhizosphere soil of crops, and is used to alleviate the impact of crop continuous cropping obstacles.

[0005] In a first aspect, the present application provides a soil conditioner for degrading phenolic acid, the active ingredient of which is: Robinia pseudoacacia root secretions.

[0006] Optionally, in the soil conditioner, the Robinia pseudoacacia root exudates are prepared by the following method: The Robinia pseudoacacia plants are hydroponically cultivated in containers at a temperature of 25-30°C with alternating light and dark periods. The water in the container is collected after cultivation, which is the collection liquid containing Robinia pseudoacacia root secretions.

[0007] Optionally, the hydroponic culture time is 3 to 5 days.

[0008] Optionally, when hydroponically growing, the amount of water used is 1 / 3 to 2 / 3 of the container capacity.

[0009] Optionally, the ratio of dark light culture time to light light culture time is 12h:12h.

[0010] In a second aspect, the present application provides a use of the above-mentioned soil conditioner in degrading phenolic acid substances in soil.

[0011] Optionally, the soil is rhizosphere soil of konjac intercropping.

[0012] Optionally, the application method is: perform root irrigation treatment on crops during the crop growth period, apply 100-200 ml of phenolic acid-degrading soil conditioner per hole once every 7 to 10 days.

[0013] The crops include konjac, pepper, cucumber and the like.

[0014] In a third aspect, the present application also provides another soil conditioner for degrading phenolic acid, the preparation method of which is as follows: S1. Preparation of a mixed solution of roots and root extracts: Mix Robinia pseudoacacia roots and water in a mass ratio of 1:1-2, place at a temperature of 30-35° C. under light conditions for 3-5 days without stirring, to obtain a mixed solution of roots and root extracts; S2. Fermentation treatment: placing the mixed liquid of the above roots and root extracts in a homogenizer to obtain a slurry mixture, adding fiber-degrading bacteria to the slurry mixture, the mass ratio of the slurry mixture to the fiber-degrading bacteria powder with an effective live bacteria count of ≥5 billion / g is (3000-4000):1, placing the above materials in a constant temperature fermentation box for constant temperature anaerobic fermentation at 60-75°C, fermenting for 5-7 days, stirring every 4-6 hours during the fermentation, to obtain a root fermentation product.

[0015] S3. The root fermentation product is fully mixed with a carrier to prepare a soil conditioner for degrading phenolic acid, wherein the carrier is one or both of wood ash and humus.

[0016] Optionally, in step S3 of the method for preparing a soil conditioner for degrading phenolic acid, the mass ratio of the root fermentation product to the carrier is 1:(2-4).

[0017] In a fourth aspect, the present application also provides the use of the above-mentioned improver in degrading phenolic acid substances in soil, and the application method is: Before sowing or planting crops, the improver is applied to the soil and the tillage layer is ploughed, water is sprayed until the soil moisture is 75-85%, and then a film is used to cover the soil for 10 days. After the film is removed and aired for 5-10 days, the crops can be sown normally.

[0018] Preferably, the application amount of the soil conditioner for degrading phenolic acid is 30-60 kg / mu.

[0019] In a fifth aspect, the present application also provides the use of the above-mentioned soil conditioner for degrading phenolic acid in inhibiting konjac soft rot fungi.

[0020] The present invention has the following beneficial effects: The soil conditioner for degrading phenolic acid provided by the invention has few raw materials and a simple preparation method, is not only low in cost, but also safe and environmentally friendly to use, can effectively degrade the content of phenolic acid substances in the soil of continuous cropping, alleviate continuous cropping obstacles, reduce the harm to the growth and development of konjac, and at the same time improve the physical and chemical properties of the soil, which is conducive to the cultivation of konjac. DETAILED DESCRIPTION

[0021] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention. In the present invention, unless otherwise specified, the equipment and raw materials used can be purchased from the market or are commonly used in the art. The methods in the following embodiments are conventional methods in the art unless otherwise specified.

[0022] Example 1 Preparation of Robinia pseudoacacia root exudate collection solution In this embodiment, Robinia pseudoacacia plants planted in pots for 3 months after germination of Robinia pseudoacacia seeds are used as the object to prepare the Robinia pseudoacacia root secretion collection liquid according to the above scheme. In other embodiments, the Robinia pseudoacacia plants are not limited to plants of 3 months.

[0023] 1. This embodiment provides a soil conditioner for degrading phenolic acid, which is a collected solution of Robinia pseudoacacia root exudates. The preparation method of the collected solution of Robinia pseudoacacia root exudates is as follows: The rooted Robinia pseudoacacia plant was used as the object, and the impurities of the Robinia pseudoacacia root system were washed with water. The Robinia pseudoacacia plant was placed in a container for hydroponics, and the mass ratio of the root to the water was 1: (1-2), the culture temperature was 25°C, the ratio of the dark light culture to the light culture time was 12h:12h, and the water in the container was collected after 4 days of hydroponics to obtain the collected liquid containing the secretions of the Robinia pseudoacacia root system. The amount of water in the container was 1 / 3-2 / 3 of the container capacity.

[0024] The collected liquid containing Robinia pseudoacacia root exudates prepared in this example was used as a soil conditioner in the experiment of Example 2.

[0025] Example 2 Application of the collected root exudates of Robinia pseudoacacia in the degradation of phenolic acid substances in konjac soil In this example, the collected solution containing root secretions of Robinia pseudoacacia prepared in Example 1 was used as a soil conditioner to perform root irrigation treatment on konjac seedlings, and the degradation effect of the collected solution on phenolic acid substances in the rhizosphere soil of the konjac seedlings was measured.

[0026] 1. Experimental location: Experimental base of Jiangbei campus of Ankang University; 2. Test period: April to October 2023; 3. Experimental subjects: Potted konjac seedlings; 4. Test methods: The konjac soil collected from the experimental base of Jiangbei Campus of Ankang University, which had been planted for many years, was used as the experimental soil, and 5 kg of soil was placed in each pot. The konjac seeds with healthy and white top buds were planted in pots, one plant per pot, and water and fertilizer management was carried out according to conventional methods.

[0027] This example sets up two treatment groups, the experimental group and the control group, with 30 repetitions for each treatment group. The treatments of the two treatment groups are as follows: Experimental group: During the growth period of konjac, the roots of konjac in each pot were irrigated with fertilizer once every 10 days, and the root irrigation amount for each plant was 100 ml.

[0028] Control group: Root irrigation with tap water was used as the control treatment, and the treatment method was the same as that of the experimental group.

[0029] 5. Determination of phenolic acid content in soil During the maturity period of konjac, the five-point method was used for sampling. The soil sample at 5 cm from the surface of the cultivated layer was removed, and the rhizosphere soil (within 1 cm from the root system) was taken. The soil on the root system was collected into a sterilized ziplock bag by the shaking soil method. Then, the fresh soil samples were fully mixed and naturally air-dried. The soil phenolic acid substances were extracted and determined according to the method of Dalton et al. (1987). The soil phenolic acid substances include: p-hydroxybenzoic acid, vanillic acid, syringic acid and ferulic acid.

[0030] 6. Test results and analysis From the results in Table 1, we can see that compared with the control group, the contents of p-hydroxybenzoic acid, vanillic acid, syringic acid and ferulic acid in the experimental group were reduced by 71.6%, 73.4%, 73.8% and 69.2%, respectively. These four common phenolic acids are allelopathic substances in the soil that have an inhibitory effect on crops. The reduction in the content of these phenolic acids will inevitably reduce their harm to the growth and development of konjac, and at the same time improve the physical and chemical properties of the soil, which is conducive to the cultivation of konjac.

[0031] The above results of this example show that the root exudate collected from Robinia pseudoacacia for root irrigation of konjac can significantly reduce the content of various phenolic acid substances in the soil, thereby achieving the effect of improving the soil.

[0032] Table 1 Phenolic acid content in soils under different treatments (μg∙g -1 )

[0033] Note: Different lowercase letters after the data in the same column indicate significant differences ( P <0.05), the same below.

[0034] Example 3 Preparation of phenolic acid degrading soil conditioner This embodiment provides a soil conditioner for degrading phenolic acid, which is prepared by fermentation of Robinia pseudoacacia roots and root extracts. The method for preparing the soil conditioner for degrading phenolic acid specifically comprises the following steps: S1. Preparation of mixed solution of Robinia pseudoacacia roots and root extracts Robinia pseudoacacia roots were mixed with water in a mass ratio of 1:2, the temperature was 30-35°C, and the mixture was placed under light conditions for 4 days without stirring to obtain a mixed solution of roots and root extracts.

[0035] S2. Fermentation The mixed solution of the Robinia pseudoacacia root and the root extract was placed in a homogenizer to obtain a slurry mixture. Fiber-degrading bacteria (purchased from Guangzhou Weiyuan Biotechnology Co., Ltd., the product name is crude fiber-degrading bacteria-Weiyuan Biotechnology) were added to the slurry mixture, so that the mass ratio of the slurry mixture to the fiber-degrading bacteria was 3500:1, and then placed in a constant temperature fermentation box for constant temperature fermentation at 65°C for 6 days, during which stirring was performed every 6 hours to obtain a root fermentation product.

[0036] S3, Load The root fermentation product and humus soil are mixed and stirred at a weight ratio of 1:3 to obtain a soil conditioner for degrading phenolic acid.

[0037] The prepared improver is used for degrading phenolic acid substances in konjac soil in the field.

[0038] Example 4 Application of soil conditioner in field degradation of phenolic acid substances in konjac soil In this example, the soil conditioner prepared in Example 3 was used as a research object to determine the degradation effect of the soil conditioner on phenolic acid substances in the rhizosphere soil of konjac continuous cropping in the field.

[0039] 1. Test time: 2023 2. Test location: Konjac Test Base, Dahekan Town, Nanzheng District 3. Experimental crops: Konjac 4. Continuous cropping period: 3 years 5. Test methods: This embodiment sets the following groups: Experimental group 1: The soil conditioner for degrading phenolic acid prepared in Example 3 was used to treat the soil. The specific method was as follows: before sowing or planting crops, the soil conditioner was applied at a dosage of 50 kg / mu, and a 20 cm cultivated layer was plowed, water was sprayed until the soil moisture was 75-85%, and then covered with a film for 10 days.

[0040] Experimental group 2: using the collected solution of Robinia pseudoacacia root exudates prepared in Example 1, and following the method of experimental group 1; Control group: normal water and fertilizer management was adopted as blank control.

[0041] After removing the film and drying for 7 days, the phenolic acid content in the soil was determined.

[0042] 6. Method for determining the content of phenolic acid in soil: refer to the method in Example 2.

[0043] 7. Effects of soil conditioners on phenolic acid content in continuously cropped soils From the results in Table 2, it can be seen that the contents of p-hydroxybenzoic acid, vanillic acid, syringic acid and ferulic acid in the experimental group 1 treated with the phenolic acid-degrading soil conditioner of Example 3 were reduced by 73.7%, 67.9%, 87.2% and 67.7%, respectively, compared with the control group. The contents of p-hydroxybenzoic acid, vanillic acid, syringic acid and ferulic acid in the experimental group 2 treated with the collected liquid of Robinia pseudoacacia root exudates prepared in Example 1 were reduced by 60.4%, 49.9%, 71.3% and 54.8%, respectively, compared with the control group. This shows that both phenolic acid-degrading soil conditioners can be effectively used to degrade phenolic acids in soils with continuous cropping and alleviate continuous cropping obstacles.

[0044] Table 2 Phenolic acid content in soils under different treatments (μg∙g -1 )

[0045] Example 5 Stability determination of soil conditioner for degrading phenolic acid 1. Experimental methods The collected liquid of Robinia pseudoacacia root exudates (Example 1) was filtered through a 0.22 μm bacterial filter and then sealed and sterilely stored in a greenhouse away from light, as Experimental Group 2; the soil conditioner for degrading phenolic acid (Example 3) was stored at room temperature away from light, as Experimental Group 1. After the two experimental groups were stored for 15 d, 30 d, 60 d and 90 d, respectively, the soil was treated according to the operating method of Example 4, and a control group was set up at the same time. After the treatment, samples were taken to detect the phenolic acid degradation rate of each group of soil. 2. Experimental results and analysis The results are shown in Table 3, which shows that the soil conditioner for degrading phenolic acid can still maintain a high degradation rate after 90 days of storage. After 15 days of storage, the degradation rates of p-hydroxybenzoic acid, vanillic acid, syringic acid and ferulic acid in the experimental group 1 using the soil conditioner were 73.6%, 67.5%, 85.1% and 67.3%, respectively; the degradation rates of p-hydroxybenzoic acid, vanillic acid, syringic acid and ferulic acid in the experimental group 2 using the collected solution of Robinia pseudoacacia root secretions were 64.4%, 50.4%, 75.8% and 57.9%, respectively. When the storage time was 30 days and 60 days, the degradation rate of phenolic acid in the experimental group 2 treated with the collected solution of Robinia pseudoacacia root secretions decreased, but the degradation rates of p-hydroxybenzoic acid, vanillic acid, syringic acid and ferulic acid in the experimental group 1 treated with the soil conditioner were always significantly higher than those in the experimental group 2, and the activity was relatively stable. When the storage time was 90 days, the experimental group 2 treated with the collected solution of Robinia pseudoacacia root secretions had been contaminated and the degradation rate could not be determined. The above results indicate that the soil conditioner has good long-term storage stability, stable activity and a long shelf life.

[0046] Table 3 Degradation rate of phenolic acid in different treatments%

[0047] Example 6 Inhibition of konjac soft rot by collecting liquid from Robinia pseudoacacia root system secretions 1. Experimental methods (1) Extraction of Robinia pseudoacacia root exudates The following operations were performed using the collected solution of Robinia pseudoacacia root exudates prepared in Example 1 as a raw material: The collected liquid of Robinia pseudoacacia root exudates was extracted with ethyl acetate in a volume ratio of 1:2, separated by a separatory funnel, the ethyl acetate layer was collected, and the aqueous phase was extracted with ethyl acetate in a volume ratio of 1:2. After extraction three times, the three ethyl acetate layers were combined, and the ethyl acetate layers were dried over anhydrous Na2SO4, freeze-evaporated at -50°C, and the concentrate was dissolved in 5 mL of methanol, filtered with a 0.22 μm ultrafiltration membrane, and stored aseptically at 4°C.

[0048] (2) Co-culture different volumes of Robinia pseudoacacia root secretions with konjac soft rot bacteria to observe their effects on the growth of konjac soft rot bacteria. The steps are as follows: Dilute LB liquid medium 8 times and dispense into glass test tubes, 4 mL per test tube, sterilize at 121°C for 15 min, and when the medium cools down to a level that is not too hot, add an appropriate amount of root exudate extract filtered through a 0.22 μm ultrafiltration membrane (double distilled water as control), the volume of exudate extract added is 0.5 mL, 1 mL, 2 mL, and double distilled water is added to 2 mL, double distilled water is added to the control group, and 4 replicates are set for each treatment. 30 μL of activated cultured konjac soft rot pathogens were inoculated into each test tube, and placed in a constant temperature shaker at 28°C and 200 rpm for 10 h, and then 200 μL of bacterial solution was placed in a 96-well ELISA plate, and the absorbance value OD600 nm was measured using an ELISA reader.

[0049] The konjac soft rot pathogen in this embodiment is: Dichella dauricum ( Dickeya Fangzhongdai ) strain CZSB4 (accession number: CCTCCAB2015171, purchased from China Center for Type Culture Collection (CCTCC)).

[0050] 2. Experimental results and analysis As shown in Table 3, the Robinia pseudoacacia root secretions have excellent antibacterial effects on the konjac soft rot pathogenic bacteria. When the root secretions are added in an amount of 2 mL, the inhibitory effect on the konjac soft rot pathogenic bacteria CZSB4 reaches 84.93%. Therefore, the soil conditioner prepared by the Robinia pseudoacacia root secretions collected liquid can not only improve the soil and degrade phenolic acid, but also has an antibacterial effect on the konjac soft rot pathogenic bacteria in the soil, which is helpful for the cultivation of konjac, especially for the planting of konjac in continuous cropping soil.

[0051] Table 3 Inhibitory effect of root secretions of Robinia pseudoacacia seedlings cultured for 30 days on konjac soft rot pathogenic bacteria (%)

[0052] Note: Different lowercase letters in the same row indicate significant differences ( P <0.05).

[0053] It is understandable that those skilled in the art can make equivalent substitutions or changes based on the technical solution and concept of the present invention, and all these changes or substitutions should fall within the protection scope of the claims attached to the present invention.

Claims

1. A soil conditioner for degrading phenolic acid, characterized in that: Its active ingredient is: Robinia pseudoacacia root exudates.

2. The soil conditioner for degrading phenolic acid according to claim 1, characterized in that: The Robinia pseudoacacia root exudates are prepared by the following method: The Robinia pseudoacacia plants are hydroponically cultivated in containers at a temperature of 25-30°C with alternating light and dark periods. The water in the container is collected after cultivation, which is the collection liquid containing Robinia pseudoacacia root secretions.

3. The soil conditioner for degrading phenolic acid according to claim 1, characterized in that: The hydroponic time is 3~5 days.

4. The soil conditioner for degrading phenolic acid according to claim 1, characterized in that: Its preparation method is: S1. Preparation of a mixed solution of roots and root extracts: Mix Robinia pseudoacacia roots and water in a mass ratio of 1:(1-2), place at a temperature of 30-35°C and under light conditions for 3-5 days to obtain a mixed solution of roots and root extracts; S2, fermentation treatment: placing the mixed liquid of the above roots and root extracts in a homogenizer to obtain a slurry mixture, adding fiber-degrading bacteria to the slurry mixture, the mass ratio of the slurry mixture to the fiber-degrading bacteria powder with an effective viable count of ≥5 billion / g is 3000-4000:1, placing the above materials in a constant temperature fermentation box for constant temperature anaerobic fermentation at 60-75° C. for 5-7 days, stirring every 4-6 hours during the fermentation, to obtain a root fermentation product; S3. The root fermentation product is fully mixed with a carrier to prepare a solid soil conditioner for degrading phenolic acid, wherein the carrier is plant ash or humus soil, or a mixture of the two.

5. The soil conditioner for degrading phenolic acid according to claim 4, characterized in that: In step S3 of the preparation method, the mass ratio of the root fermentation product to the carrier is 1:(2-4).

6. Use of the soil conditioner for degrading phenolic acid as claimed in claim 2 or 4 in inhibiting konjac soft rot fungi.

7. Use of the soil conditioner according to claim 2 or 4 in degrading phenolic acid substances in soil; optionally, the soil is rhizosphere soil of konjac intercropping.

8. The use according to claim 7, characterized in that: The soil conditioner for degrading phenolic acid is root secretions of Robinia pseudoacacia, and the application method is: root irrigation treatment is performed on crops during the crop growth period, and 100-200 ml of the soil conditioner for degrading phenolic acid is applied to each hole once every 7-10 days.

9. Use of the soil conditioner according to claim 7 in degrading phenolic acid substances in soil, characterized in that: The soil conditioner for degrading phenolic acid is a solid preparation, and the application method is as follows: Before sowing or planting crops, the improver is applied to the soil and the tillage layer is ploughed, water is sprayed until the soil moisture is 75-85%, and then a film is used to cover the soil for 8-12 days. After the film is removed and aired for 5-10 days, the crops can be sown normally.

10. The use according to claim 9, characterized in that: The application amount of the soil conditioner for degrading phenolic acid is 30-60 kg / mu.

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