Crop straw biochar immobilized microbial agent and application thereof in improvement of acid soil of solanaceae crops

Abstract

The invention discloses a crop straw biochar immobilized microbial agent and application thereof in improving solanaceae crop acid soil, and relates to the technical field of biology. According to the invention, biochar and a microbial agent are combined for use, a crop straw biochar immobilized microbial agent is developed, and the microbial agent takes biochar as a carrier and loads various microorganisms including aspergillus tubingensis, rhodococcus ruber, bacillus megaterium and zygosaccharomyces rouxii. Through the combined action of the biochar and the immobilized microbial agent, the soil acidity is remarkably improved. The combined action can effectively relieve soil acidification, improve the fertility and biological activity of soil, and finally achieve the purpose of relieving continuous cropping obstacles of crops.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a crop straw biochar immobilization bacterial agent and application thereof in improving acidic soil of Solanaceae crops. Background Art

[0002] Solanaceae crops (such as tomatoes, peppers, eggplants, etc.) are important economic crops widely grown around the world. However, long-term continuous planting of Solanaceae crops on the same plot of land (i.e. continuous cropping) will lead to a series of serious continuous cropping problems, which not only affect the yield and quality of crops, but may also lead to soil degradation and ecological balance disruption, specifically manifested in:

[0003] 1. Soil acidification: Solanaceae crops release organic acids during their growth, which gradually accumulate in the soil, causing the soil pH to drop and soil acidification to occur. Soil acidification not only affects the growth and development of crop roots, but also reduces the effectiveness of nutrients in the soil, especially the absorption and utilization of key elements such as phosphorus, calcium and magnesium.

[0004] 2. Imbalance of soil microbial communities: Under continuous cropping conditions, specific pathogenic microorganisms will multiply in large numbers in the soil, while the number and diversity of beneficial microorganisms will be significantly reduced. This imbalance in microbial communities not only increases the incidence of diseases, but also weakens the soil's self-repair ability.

[0005] 3. Nutrient depletion: Solanaceae crops have a large demand for certain nutrients during their growth. Long-term continuous cropping will cause these nutrients to be depleted in the soil, thus affecting the growth of subsequent crops.

[0006] 4. Accumulation of root secretions: The root system of plants is an important place for plants to exchange substances and energy with the external environment. The main function of the root system is to fix and support the plant body, and it also has absorption and storage functions, but it will secrete some substances that are toxic to the plant itself. Studies have found that long-term continuous cropping will lead to the accumulation of phenolic acids secreted by plant roots, which will have a self-toxic effect on the plants. The inhibitory effect of phenolic acids is mainly manifested in inhibiting the soil nitrification process and affecting the transformation of nitrogen forms; inhibiting the absorption of soil nutrients by plant roots; reducing crop photosynthetic products and lowering chlorophyll content; inhibiting the activity of catalase and peroxidase, and destroying cell integrity.

[0007] At present, the methods to alleviate the continuous cropping obstacles of Solanaceae crops mainly include crop rotation, fallow, application of organic fertilizers and biological agents, etc. The present invention intends to develop a crop straw biochar immobilized agent to achieve the purpose of improving the acidic soil of Solanaceae crops and alleviating the continuous cropping obstacles of Solanaceae crops. Summary of the invention

[0008] The purpose of the present invention is to provide a crop straw biochar immobilized bacterial agent and its application in improving acidic soil of Solanaceae crops to solve the problems existing in the above-mentioned prior art. The crop straw biochar immobilized bacterial agent can effectively alleviate soil acidification, improve soil fertility and biological activity, and ultimately achieve the purpose of alleviating the obstacle of crop continuous cropping.

[0009] To achieve the above object, the present invention provides the following solutions:

[0010] The invention provides a microbial combination for improving acidic soil of Solanaceae crops, comprising Aspergillus tubingensis, Rhodococcus ruber, Bacillus megaterium and Zygosaccharomyces rouxii.

[0011] Furthermore, the Aspergillus tubingensis is Aspergillus tubingensis ACCC 31494; the Rhodococcus erythrorhizium is Rhodococcus erythrorhizium CICC23623; the Bacillus megaterium is Bacillus megaterium CICC 20611; and the Zygosaccharomyces rouxii is Zygosaccharomyces rouxii CICC 32899.

[0012] The present invention also provides the use of the above-mentioned microbial combination in preparing a microbial agent for improving acidic soil of Solanaceae crops.

[0013] The present invention also provides a microbial agent for improving acidic soil of Solanaceae crops, wherein the active ingredients include the above-mentioned microbial combination.

[0014] Furthermore, the microbial agent is a crop straw biochar immobilization agent; the crop straw biochar immobilization agent also includes a carrier; the carrier is biochar.

[0015] The present invention also provides a method for preparing a crop straw biochar immobilized bacterial agent for improving acidic soil of Solanaceae crops, comprising the following steps:

[0016] The Aspergillus tubingensis suspension, the Rhodococcus erythrorhizium suspension, the Bacillus megaterium suspension and the Zygosaccharomyces rouxii suspension are uniformly mixed to obtain a mixed bacterial agent;

[0017] The biochar and the mixed bacterial agent are mixed evenly, and ventilated and dried until the moisture content is less than 10%, thereby obtaining the crop straw biochar immobilized bacterial agent.

[0018] Furthermore, the Aspergillus tubingensis is Aspergillus tubingensis ACCC 31494; the Rhodococcus erythrorhizium is Rhodococcus erythrorhizium CICC23623; the Bacillus megaterium is Bacillus megaterium CICC 20611; and the Zygosaccharomyces rouxii is Zygosaccharomyces rouxii CICC 32899.

[0019] Furthermore, the concentrations of the Aspergillus tubingensis suspension, the Rhodococcus erythrorhizium suspension, the Bacillus megaterium suspension and the Zygosaccharomyces rouxii suspension are all 10 8 CFU / mL; and / or

[0020] The volume ratio of the Aspergillus tubingensis suspension, the Rhodococcus erythrorhizium suspension, the Bacillus megaterium suspension and the Zygosaccharomyces rouxii suspension is 1:2:1:1; and / or

[0021] The mass ratio of the biochar to the mixed bacterial agent is 1:1.

[0022] The present invention also provides application of the microbial agent in improving acidic soil of Solanaceae crops.

[0023] The present invention also provides a method for improving acidic soil of Solanaceae crops, comprising the step of applying the above-mentioned microbial agent to continuously cropped acidic soil.

[0024] The present invention discloses the following technical effects:

[0025] The present invention combines biochar and microbial agents to develop a crop straw biochar immobilization agent, which uses biochar as a carrier and loads a variety of microorganisms, including Aspergillus tubingensis, Rhodococcus erythrorhizium, Bacillus megaterium and Zygosaccharomyces rouxii. Among them, the surface of biochar is rich in alkaline functional groups, such as hydroxyl (-OH), etc. These functional groups can react with acidic ions in the soil to neutralize, thereby increasing the soil pH value and reducing soil acidification; biochar has a highly developed pore structure and a large specific surface area, which can effectively adsorb acidic ions and harmful substances in the soil. Through adsorption, biochar can reduce free H in the soil. + concentration, reducing soil acidity and at the same time reducing the toxic effects of acidic substances on plant roots; at the same time, biochar provides a stable microenvironment for microorganisms, which is conducive to the growth, reproduction and metabolic activities of microorganisms, and the metabolic activities of microorganisms can further enhance the adsorption and neutralization effects of biochar. The present invention significantly improves soil acidity through the combined action of biochar and immobilized bacterial agents. The synergistic effect produced by biochar and immobilized bacterial agents can effectively alleviate soil acidification, improve soil fertility and biological activity, and ultimately achieve the purpose of alleviating obstacles to continuous cropping. DETAILED DESCRIPTION

[0026] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0027] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0028] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0029] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0030] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0031] Cherry tomato (Lycopersicon esculentum var.cerasiforme A.Gray) is a cultivated variety of the genus Lycopersicon in the Solanaceae family. It is an annual or perennial vine herb with a strong odor on its branches. The plant grows vigorously, with vine stems, short yellow hairs on the body surface, and large leaves. The inflorescence is mostly simple racemose, with yellow corolla; the ovary is spherical, with a short stigma or the same length as the stamen; the fruit is nearly round, with a skin color of fiery red, pink, etc., and the fruit is smooth. The seeds are heart-shaped, yellow, and have hairs. Cherry tomatoes are sown in April, mature in June-July, and grow fast. Cherry tomatoes are named "cherry tomatoes" because of their appearance similar to cherries.

[0032] Tomato wilt is a disease of tomatoes caused by the tomato-specific form of Fusarium oxysporum. It mainly harms the rhizomes of tomatoes and usually begins to develop during the flowering and fruiting period. In the early stages of the disease, only the lower leaves of the plant turn yellow, then turn brown and wilt and dry up, but do not fall off. Symptoms sometimes only appear on one side of the stem, or one side of a leaf turns yellow while the other side is normal. When the stem, petiole and fruit stalk are dissected, the vascular bundles are all brown. In a humid environment, pink mold appears at the base of the stem of the diseased plant.

[0033] Tomato early blight, also known as ring rot and summer blight, is a plant disease caused by infection of Alternaria solanacearum, which mainly affects the leaves, stems and fruits of tomatoes. It is characterized by the formation of obvious ring rots on the lesions, so it is also called ring rot. The symptoms of tomato early blight include: Leaves: Initially, water-soaked dark green lesions appear, gradually expanding to round or irregular shapes, with light green or yellow halos on the edges, and black mold-like substances appear on the diseased parts when it is wet. Stems: The lesions are oval to oblong, brown to dark brown, and the lesions on the branches are irregularly round, slightly concave, and gray-black mold-like substances grow on the surface. Fruits: The lesions mostly occur near the fruit pedicles, initially oval or amorphous brown or black spots, concave, and the fruits crack in the later stage. The diseased parts are hard and densely covered with black mold. The pathogen of tomato early blight mainly overwinters in the soil through hyphae and conidia on the diseased residues, and can also overwinter on seeds. The pathogen infects tomatoes under suitable temperature and humidity conditions (such as 23-30℃ and high humidity), usually starting from the lower leaves and gradually spreading upwards.

[0034] The strain information used in the following examples is as follows:

[0035] Rhodococcus ruber was purchased from China Industrial Culture Collection Center (CICC) with the strain number CICC 23623; Aspergillus tubingensis was purchased from China Agricultural Culture Collection Center (ACCC) with the strain number ACCC 31494; Bacillus megaterium was purchased from China Industrial Culture Collection Center with the strain number CICC 20611; Zygosaccharomyces rouxii was purchased from China Industrial Culture Collection Center with the strain number CICC32899; Bacillus subtilis and Trichoderma harzianum were purchased from Shandong Dayi Biotechnology Group Co., Ltd.

[0036] The culture medium formula used in the following examples is as follows:

[0037] PDA liquid culture medium: 200 g / L potato and 20 g / L sucrose.

[0038] PDA solid culture medium: potato 200 g / L, sucrose 20 g / L and agar 20 g / L.

[0039] LB liquid medium: 10 g / L tryptone, 5 g / L yeast extract and 10 g / L sodium chloride.

[0040] LB solid medium: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L and agar 20 g / L.

[0041] Fermentation medium: composed of the following components, by mass: 60 parts of rice husk, 10 parts of wheat straw powder, 10 parts of corn powder, 4 parts of soybean meal, 1 part of dipotassium hydrogen phosphate, 1 part of potassium dihydrogen phosphate, 1 part of magnesium sulfate and 100 parts of water.

[0042] YPD liquid medium: yeast extract 10 g / L, peptone 20 g / L and glucose 20 g / L.

[0043] YPD solid medium: yeast extract 10 g / L, peptone 20 g / L, glucose 20 g / L and agar 20 g / L.

[0044] Example 1 Preparation of mixed bacterial agent

[0045] 1. Preparation of Aspergillus tubingensis suspension

[0046] (1) Solid slant culture: Aspergillus tubingensis was inoculated into PDA solid culture medium and cultured at 30°C for 3 days to activate the strain.

[0047] (2) Primary seed culture: The strain activated in step (1) was inoculated into PDA liquid culture medium using an inoculation loop and cultured at 30° C. and 150 rpm for 3 days to obtain a primary seed solution.

[0048] (3) Secondary seed culture: The primary seed solution obtained in step (2) was inoculated into a 5 L shake flask (containing PDA liquid culture medium) at a 5% inoculation rate and cultured at 30° C. and 150 rpm for 3 days to obtain the secondary seed solution.

[0049] (4) The secondary seed liquid was inoculated into a container containing a fermentation medium at an inoculum rate of 3%, stirred once every 48 hours, and cultured at 28° C. for 7 days. After the culture was completed, sterile physiological saline was added and microwaved for 20 minutes to obtain a spore suspension.

[0050] (5) The concentration of the spore suspension obtained in step (4) was adjusted to 10 8 CFU / mL, for future use.

[0051] 2. Preparation of Rhodococcus erythrocyte suspension

[0052] (1) Solid slant culture: Inoculate Rhodococcus erythrorhizium into PDA solid culture medium and culture at 28°C for 3 days to activate the strain.

[0053] (2) Primary seed culture: The strain activated in step (1) was inoculated into PDA liquid culture medium using an inoculation loop and cultured at 28° C. and 150 rpm for 3 days to obtain a primary seed solution.

[0054] (3) Secondary seed culture: The primary seed solution obtained in step (2) was inoculated into a 5 L shake flask (containing PDA liquid culture medium) at a 5% inoculation rate and cultured at 30° C. and 150 rpm for 3 days to obtain the secondary seed solution.

[0055] (4) The secondary seed liquid was inoculated into a container containing PDA liquid culture medium at an inoculum rate of 3%, and fermented at 37°C and 180 rpm for 3 days. The fermented liquid was then centrifuged to obtain bacterial cells, which were resuspended in sterile saline to 10 8 CFU / mL, for future use.

[0056] 3. Preparation of Bacillus megaterium suspension

[0057] (1) Solid slant culture: Bacillus megaterium was inoculated into LB solid medium and cultured at 37°C for 1 day to activate the strain.

[0058] (2) Primary seed culture: The strain activated in step (1) was inoculated into LB liquid culture medium and cultured at 37° C. and 160 rpm for 1 day to obtain a primary seed solution.

[0059] (3) Secondary seed culture: The primary seed solution obtained in step (2) was inoculated into a 5 L shake flask (containing LB liquid culture medium) at an inoculum size of 8%, and cultured at 37° C., 160 rpm for 1 day to obtain the secondary seed solution.

[0060] (4) The secondary seed liquid obtained in step (3) was inoculated into a fermentation medium (i.e., LB liquid medium) at an inoculum rate of 10%, and fermented at 37° C. and 180 rpm for 2 days. The fermentation liquid was then centrifuged to obtain bacterial cells, which were resuspended in sterile saline to 10 8 CFU / mL, for future use.

[0061] 4. Preparation of Zygosaccharomyces rouxii Suspension

[0062] (1) Solid slant culture: Zygosaccharomyces rouxii was inoculated into YPD solid medium and cultured at 35°C for 1 day to activate the strain.

[0063] (2) Primary seed culture: The strain activated in step (1) was inoculated into YPD liquid culture medium and cultured at 35° C. and 150 rpm for 1 day to obtain a primary seed solution.

[0064] (3) Secondary seed culture: The primary seed solution obtained in step (2) was inoculated into a 5 L shake flask (containing YPD liquid culture medium) at an inoculum size of 8%, and cultured at 35° C., 150 rpm for 1 day to obtain the secondary seed solution.

[0065] (4) The secondary seed liquid obtained in step (3) was inoculated into a fermentation medium (i.e., YPD liquid medium) at an inoculum rate of 10%, and fermented at 35° C. and 160 rpm for 2 days. The fermentation liquid was then centrifuged to obtain bacterial cells, which were resuspended in sterile saline to 10 8 CFU / mL, for future use.

[0066] 5. Preparation of mixed bacterial agents

[0067] The Aspergillus tubingensis suspension, the Rhodococcus erythrorhizium suspension, the Bacillus megaterium suspension and the Zygosaccharomyces rouxii suspension were uniformly mixed in a volume ratio of 1:2:1:1 to obtain a mixed bacterial agent.

[0068] Example 2 Preparation of biochar immobilized bacterial agent

[0069] 1. Preparation of Biochar

[0070] (1) Straw pretreatment: The collected wheat straw was cut into small segments (about 1-2 cm) for subsequent processing.

[0071] (2) Preparation of biochar by pyrolysis: The pretreated straw is placed in a pyrolysis furnace and pyrolyzed at 500°C for 1 hour under anoxic conditions. After the pyrolysis is completed, the straw is cooled to room temperature to obtain biochar.

[0072] 2. Preparation of biochar immobilized bacteria

[0073] The biochar and the mixed bacterial agent prepared in Example 1 were uniformly mixed in a mass ratio of 1:1, and then naturally dried in a well-ventilated environment until the moisture content was less than 10%, thereby obtaining a biochar-immobilized bacterial agent.

[0074] Comparative Example 1

[0075] Same as Example 2, except that Bacillus megaterium is replaced by Bacillus subtilis.

[0076] Comparative Example 2

[0077] Same as Example 2, except that Aspergillus tubingensis is replaced by Trichoderma harzianum.

[0078] Example 3

[0079] 1. Materials and Methods

[0080] The planting experiment of this embodiment was carried out in a field where cherry tomatoes were planted for three consecutive years.

[0081] The cherry tomato planting field that had been planted for three consecutive years was randomly divided into five experimental areas, and the mixed inoculant prepared in Example 1 and the biochar immobilized inoculant prepared in Example 2 and Comparative Examples 1-2 were applied respectively, and the experimental area without the application of the biochar immobilized inoculant was used as a control. Among them, each biochar immobilized inoculant was evenly applied to the soil surface, and the application amount was 2kg / mu. The mixed inoculant applied in the experimental area of ​​Example 1 was equal to the mixed inoculant used in the preparation of the biochar immobilized inoculant in Example 2. Cherry tomato seeds were planted in each experimental area at the same planting density, and then conventional field management was carried out according to local management methods; at the same time, after 4 months of planting, the cherry tomato yield, survival rate, incidence of wilt and early blight, and soil pH of each experimental area were counted.

[0082] 2. Experimental results

[0083] After the planting experiment, the yield, survival rate, incidence of wilt and early blight, and soil pH of cherry tomatoes in each experimental area were counted, and the results are shown in Table 1. As can be seen from Table 1, the mixed bacterial agent and biochar immobilized bacterial agent developed by the present invention can effectively improve soil acidification, increase the single plant yield and survival rate of cherry tomatoes, and reduce the incidence of continuous cropping diseases, thereby effectively alleviating soil continuous cropping obstacles.

[0084] Table 1 Number of plants infected with root rot, root weight per plant and survival rate in each experimental area

[0085]

[0086] Note: Compared with the control group, * P<0.05, ** P<0.01.

[0087] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A microbial combination for improving acidic soil of Solanaceae crops, characterized in that: These include Aspergillus tubingensis, Rhodococcus ruber, Bacillus megaterium and Zygosaccharomyces rouxii.

2. The microbial combination according to claim 1, characterized in that The Aspergillus tubingensis is Aspergillus tubingensis ACCC31494; the Rhodococcus erythrorhizium is Rhodococcus erythrorhizium CICC 23623; the Bacillus megaterium is Bacillus megaterium CICC20611; and the Zygosaccharomyces rouxii is Zygosaccharomyces rouxii CICC 32899.

3. Use of the microbial combination as claimed in claim 1 or 2 in the preparation of a microbial agent for improving acidic soil of Solanaceae crops.

4. A microbial agent for improving acidic soil of Solanaceae crops, characterized in that: The active ingredient comprises the microbial combination according to claim 1 or 2.

5. The microbial agent according to claim 4, characterized in that: The microbial agent is a crop straw biochar immobilization agent; the crop straw biochar immobilization agent also includes a carrier; the carrier is biochar.

6. A method for preparing a crop straw biochar immobilized bacterial agent for improving acidic soil of Solanaceae crops, characterized in that: The steps include: The Aspergillus tubingensis suspension, the Rhodococcus erythrorhizium suspension, the Bacillus megaterium suspension and the Zygosaccharomyces rouxii suspension are uniformly mixed to obtain a mixed bacterial agent; The biochar and the mixed bacterial agent are mixed evenly, and ventilated and dried until the moisture content is less than 10%, thereby obtaining the crop straw biochar immobilization bacterial agent.

7. The preparation method according to claim 6, characterized in that: The Aspergillus tubingensis is Aspergillus tubingensis ACCC31494; the Rhodococcus erythrorhizium is Rhodococcus erythrorhizium CICC 23623; the Bacillus megaterium is Bacillus megaterium CICC20611; and the Zygosaccharomyces rouxii is Zygosaccharomyces rouxii CICC 32899.

8. The preparation method according to claim 6, characterized in that: The concentrations of the Aspergillus tubingensis suspension, the Rhodococcus erythrorhizium suspension, the Bacillus megaterium suspension, and the Zygosaccharomyces rouxii suspension were all 10 8 CFU / mL; and / or The volume ratio of the Aspergillus tubingensis suspension, the Rhodococcus erythrorhizium suspension, the Bacillus megaterium suspension and the Zygosaccharomyces rouxii suspension is 1:2:1:1; and / or The mass ratio of the biochar to the mixed bacterial agent is 1:

1.

9. Use of the microbial agent as claimed in claim 4 or 5 in improving acidic soil of Solanaceae crops.

10. A method for improving acidic soil of Solanaceae crops, characterized in that: The method comprises the step of applying the microbial agent according to claim 4 or 5 to continuously cropped acidic soil.

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