CN117800794B - Microbial fertilizer for promoting growth of purple perilla and accumulation of medicinal components and application thereof - Google Patents
Microbial fertilizer for promoting growth of purple perilla and accumulation of medicinal components and application thereof Download PDFInfo
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- CN117800794B CN117800794B CN202410217890.5A CN202410217890A CN117800794B CN 117800794 B CN117800794 B CN 117800794B CN 202410217890 A CN202410217890 A CN 202410217890A CN 117800794 B CN117800794 B CN 117800794B
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- 238000009825 accumulation Methods 0.000 title claims abstract description 21
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05D—INORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C; FERTILISERS PRODUCING CARBON DIOXIDE
- C05D9/00—Other inorganic fertilisers
-
- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
- C05G3/00—Mixtures of one or more fertilisers with additives not having a specially fertilising activity
- C05G3/80—Soil conditioners
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
- C12R2001/10—Bacillus licheniformis
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
- C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/785—Mucor
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
- Y02A40/22—Improving land use; Improving water use or availability; Controlling erosion
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- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Virology (AREA)
- Microbiology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Medicinal Chemistry (AREA)
- Biomedical Technology (AREA)
- General Engineering & Computer Science (AREA)
- Mycology (AREA)
- Botany (AREA)
- Soil Sciences (AREA)
- Pest Control & Pesticides (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Inorganic Chemistry (AREA)
- Fertilizers (AREA)
Abstract
The invention relates to a microbial fertilizer for promoting growth of purple perilla and accumulation of medicinal components and application thereof, wherein the microbial fertilizer for promoting growth of purple perilla and accumulation of medicinal components comprises the following components: biochar, pine needle powder, turfy soil, polyglutamic acid, organic fertilizer and composite microbial inoculum; the strain in the composite microbial inoculum consists of bacillus licheniformis, bacillus amyloliquefaciens, bacillus subtilis and mucor circinelloides. The microbial fertilizer has simple constitution and simple and convenient preparation process, has obvious effect on promoting the growth of medicinal and edible plants of perilla, greatly promotes the increase of the content of medicinal components of perilla, improves the soil fertility, reduces the content of lead in the soil, and has higher application value in promoting the ecological cultivation and sustainable development of medicinal plants.
Description
Technical Field
The invention belongs to the technical field of plant fertilizers, and relates to a microbial fertilizer for promoting growth of purple perilla and accumulation of medicinal components and application thereof.
Background
The medicinal plant is an important medicinal resource and is the material basis for the development of the traditional Chinese medicine industry and the prevention and treatment of diseases. In the cultivation production process, various chemical fertilizers are often applied to improve the yield of medicinal plants in order to meet the increasing medicinal demands. However, long-term and excessive use of the fertilizer can cause a series of problems such as soil hardening, acidification, salinization, environmental pollution and the like, so that the soil fertility is reduced, the fertilizer efficiency of the fertilizer is reduced, the occurrence of plant diseases and insect pests is aggravated, and the yield and the medicinal quality of medicinal plants are doubly reduced. Therefore, the development of new green high-efficiency fertilizers has important significance for promoting the sustainable development of medicinal plant production.
Research shows that the microbial fertilizer improves the soil environment, activates soil nutrients, inhibits plant diseases and insect pests, enhances the stress resistance of plants and the like by introducing beneficial microorganisms into the soil and utilizing the physiological metabolism of the microorganisms, thereby improving the yield and quality of plants. As an emerging green biological fertilizer, the biological fertilizer has the unique advantages of being harmless to the environment and nontoxic to organisms, can effectively reduce the use of chemical fertilizers, and has important significance for soil restoration and ecological cultivation promotion.
For example, CN113968761A discloses a microbial fertilizer containing a composite fermentation microbial agent and a composite functional microbial agent, the product has stable fertilizer efficiency, can reduce the application amount of chemical fertilizer, can promote plant growth, reduce the morbidity and mortality of plants, improve the vegetable yield, increase economic income, and is beneficial to promoting the green, organic and nuisanceless development of modern agriculture when applied to vegetable cultivation.
For example, CN116676238A discloses a high-efficiency biocontrol compound bacterial agent for tomato bacterial wilt, which consists of streptomyces NJAU-J78 and streptomyces NJAU-J82, can effectively promote the growth of tomatoes, reduce pathogenic bacteria in soil or effectively prevent and control the occurrence of the tomato bacterial wilt after pathogenic bacteria invade, and has remarkable value for the biological control of the tomato bacterial wilt.
However, most of the existing microbial fertilizers are aimed at grain and vegetable crops, and microbial fertilizer products special for medicinal plants are freshly reported. The medicinal plants are various in variety and complex in species, and the requirements of different medicinal species on growth environments, soil conditions, nutrient absorption and the like are different. In addition, while the yield of the medicinal plants is pursued to be improved, the medicinal quality of the medicinal plants is ensured. Therefore, the development of the microbial fertilizer special for the medicinal plants has important significance for promoting the ecological cultivation and sustainable development of the medicinal plants.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a microbial fertilizer for promoting the growth of purple perilla and the accumulation of medicinal components and application thereof.
In order to achieve the aim of the invention, the invention adopts the following technical scheme:
In a first aspect, the present invention provides a microbial fertilizer for promoting growth and accumulation of a pharmaceutically effective ingredient of perilla, the microbial fertilizer for promoting growth and accumulation of a pharmaceutically effective ingredient of perilla comprising: biochar, pine needle powder, turfy soil, polyglutamic acid, organic fertilizer and composite microbial inoculum;
The strain in the composite microbial inoculum consists of bacillus licheniformis, bacillus amyloliquefaciens, bacillus subtilis and mucor circinelloides.
The invention creatively uses bacillus licheniformis, bacillus amyloliquefaciens, bacillus subtilis and mucor circinelloides as fertilizers, and the four specific strains are matched and synergistic mutually and matched with biochar, pine needle powder, turfy soil, polyglutamic acid and organic fertilizers mutually to jointly exert excellent effects of promoting the growth of purple perilla and accumulating the medicinal effect components, and is specifically characterized in that: promoting the growth and photosynthesis of medicinal and edible plant Perilla frutescens, improving the content of perillaldehyde and rosmarinic acid in Perilla frutescens leaves with antibacterial, antiinflammatory and antioxidant effects, improving soil fertility, promoting the increase of organic matter content in soil, and reducing lead content in soil.
Most strains of the genus bacillus have the ability to rapidly colonise plants and are highly tolerant and can adapt to different types of environmental conditions. The bacillus licheniformis can secrete organic acid, protease, lipase, amylase and other enzymes, promote the decomposition of soil organic matters and the dissolution of minerals, enhance the nutrient absorption of plants, and has an inhibition effect on various pathogenic bacteria such as fusarium oxysporum, botrytis cinerea and the like, so that the bacillus licheniformis is an important biocontrol microorganism; the bacillus subtilis can secrete various antibacterial substances such as iturin, chitinase, hydrolase, fengycin and the like through improving the antioxidant enzyme activity of plants, so that the plants are protected from being affected by pathogenic bacteria, and secretion of the bacillus subtilis can promote release of nutrient elements such as nitrogen, phosphorus and potassium in soil and improve soil fertility; the bacillus amyloliquefaciens has the capabilities of fixing nitrogen and dissolving phosphorus, can promote the conversion of effective states of nitrogen, phosphorus and potassium, and activates soil nutrients; regulate the structure of rhizosphere microbial community, recruit beneficial bacteria and increase the diversity of rhizosphere microorganisms. It can synthesize indoleacetic acid, gibberellin, abscisic acid, etc. to promote plant growth and raise disease resistance of plant.
The composite microbial inoculum has obvious synergistic effect between Mucor circinelloides and bacillus or between three kinds of bacillus, and the synergistic effect promotes the activation of soil nutrients and the utilization rate of fertilizer, thereby enhancing the nutrient absorption and growth and development ability of plants and finally obviously promoting the growth of perilla and the accumulation of medicinal components (perilla aldehyde and rosmarinic acid).
The organic fertilizer, the biochar and the pine needle powder in the microbial fertilizer have a synergistic effect in providing nutrients necessary for growth and metabolism for plants and microorganisms. Meanwhile, the biochar has a large specific surface area and rich pore structures, and a proper habitat is provided for microorganisms in the composite microbial agent. The polyglutamic acid and the turfy soil in the microbial fertilizer also have synergistic effect in improving soil properties, improving soil water and fertilizer retention capacity, promoting purple perilla root system development and enhancing purple perilla stress resistance. These substances are matched with the composite microbial inoculum, so that the microorganism can play a better role, the fertilizer efficiency of the microbial fertilizer is improved, and the excellent effects of promoting the growth of purple perilla and accumulating the medicinal components are finally obtained.
Preferably, the ratio of the viable count of the bacillus licheniformis to the viable count of the bacillus amyloliquefaciens to the viable count of the bacillus subtilis to the viable count of the mucor circinelloides is (10-500): 1.
Based on the potential mutual coordination relation of the four microorganisms, the purple perilla compound has more excellent effects of promoting the growth of purple perilla and accumulating the medicinal components when the compound satisfies the specific proportion.
The specific point values in (10-500) may be selected independently from 10, 30, 50, 80, 100, 120, 150, 180, 200, 220, 250, 270, 300, 320, 350, 400, 450, 500, etc., and other specific point values in the numerical range may be selected, which will not be described in detail herein.
Preferably, the ratio of the viable count of the bacillus licheniformis to the viable count of the bacillus amyloliquefaciens to the viable count of the bacillus subtilis to the viable count of the mucor circinelloides is (10-100): 1.
Preferably, the content of the strain in the compound microbial agent in the microbial fertilizer is (1-10) multiplied by 10 8 CFU, such as 1×108CFU、2×108CFU、3×108CFU、4×108CFU、5×108CFU、6×108CFU、7×108CFU、8×108CFU、9×108CFU、1×109CFU, etc., and other specific values in the numerical range can be selected, so that the detailed description is omitted.
Preferably, the biochar is bamboo charcoal fired at 500-700 ℃ (e.g. 500 ℃, 550 ℃, 600 ℃, 630 ℃, 650 ℃, 670 ℃, 680 ℃, 700 ℃, etc.). Other specific point values within the numerical range can be selected, and will not be described in detail herein.
Preferably, the organic matter content in the organic fertilizer is more than or equal to 30%, N is more than or equal to 1.3%, and P 2O5≥1.1%,K2 O is more than or equal to 1.6%.
Preferably, in the microbial fertilizer, the mass ratio of the biochar, pine needle powder, turfy soil, polyglutamic acid and organic fertilizer is (100-150): (30-40): (540-580): (0.1-1.0): (250-300).
Based on the potential mutual coordination relation of the organic fertilizer, the biochar and the pine needle powder and the potential mutual coordination relation of the turfy soil and the polyglutamic acid, the purple perilla compound fertilizer has more excellent effects of promoting the growth of the purple perilla and accumulating the medicinal effect components when meeting the specific proportion.
The specific point values in (100-150) may be selected from 100, 110, 120, 130, 140, 150, etc.; the specific point values in (30-40) may be selected from 30, 32, 33, 35, 36, 37, 38, 40, etc.; the specific point values in the (540-580) may be selected from 540, 550, 560, 570, 580, etc.; the specific point value in (0.1-1.0) may be selected from 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, etc.; the specific point values in the (250-300) may be selected from 250, 260, 270, 280, 290, 300, etc.; other specific point values within the above numerical ranges are all selectable, and will not be described in detail herein.
Preferably, the Mucor circinelloides is a strain with a preservation number of CGMCC No.40842 and classified name of Mucor circinelloides Mucor circinelloides.
The invention also creatively discovers that the self-separated Mucor circinelloides plays a better effect in microbial fertilizers than Mucor circinelloides of other sources.
Preferably, the composite microbial inoculum is prepared by a preparation method comprising the following steps:
Mixing bacterial suspensions of bacillus licheniformis, bacillus amyloliquefaciens, bacillus subtilis and mucor circinelloides in proportion to obtain the composite microbial inoculum;
The bacterial suspension is obtained by dissolving bacterial powder in a solvent, or is obtained by sequentially activating and amplifying culturing bacterial strains.
Preferably, the mucor circinelloides bacterial suspension is prepared by a preparation method comprising the following steps:
Activating Mucor circinelloides in PDA culture medium, selecting single colony, inoculating to PDB culture medium, culturing to obtain Mucor circinelloides seed solution, inoculating to PDB culture medium, and culturing to obtain;
Wherein the formula of the PDA culture medium is as follows: potato soaked powder 2-6 g, glucose 15-25 g, agar 10-20 g, distilled water 1000-mL; the formula of the PDB culture medium is as follows: potato soaked powder 2-6 g, glucose 15-25 g, distilled water 1000-mL.
The specific point values selected from the 2-6 g can be 2g, 2.5 g, 3g, 3.5 g, 4 g, 4.5 g, 5g, 5.5 g, 6g, etc.; the specific point values selected from the 15-25 g values can be 15 g, 18 g, 20g, 22 g, 24 g, 25 g, etc.; the specific point values selected from the 10-20 g can be 10 g, 12 g, 13 g, 14 g, 15 g, 16 g, 17 g, 18 g, 20g and the like; other specific point values within the above numerical ranges are all selectable, and will not be described in detail herein.
In a second aspect, the invention provides the application of the microbial fertilizer for promoting growth of perilla and accumulation of medicinal components in cultivation of perilla, which is a plant for both medicine and food.
Compared with the prior art, the invention has the following beneficial effects:
The invention creatively uses bacillus licheniformis, bacillus amyloliquefaciens, bacillus subtilis and mucor circinelloides as fertilizers, and the four specific strains are matched and synergistic mutually and matched with biochar, pine needle powder, turfy soil, polyglutamic acid and organic fertilizers mutually to jointly exert excellent effects of promoting the growth of perilla and accumulating medicinal components (perilla aldehyde and rosmarinic acid).
The microbial fertilizer has simple constitution and simple and convenient preparation process, has obvious effect on promoting the growth of medicinal and edible plants of perilla, greatly promotes the increase of the content of medicinal components of perilla, improves the soil fertility, reduces the content of lead in soil, and has higher application value in promoting the ecological cultivation and sustainable development of medicinal plants.
Drawings
FIG. 1 is a diagram showing the appearance of a colony form of Mucor circinelloides zh on PDA medium, which is obtained by self-isolation;
FIG. 2 is a diagram showing the form of mycelia of Mucor circinelloides zh isolated by itself under an optical microscope at 20X 10 times;
FIG. 3 is a diagram showing the form of mycelia of Mucor circinelloides zh isolated by itself under an optical microscope at 40X 10 times;
FIG. 4 is an agarose gel electrophoresis of the amplification product of the ITS gene sequence of Mucor circinelloides zh;
FIG. 5 is a graph of a Perilla frutescens growth-promoting test (wherein A is a test layout graph, B is a Perilla frutescens seedling growth graph, and C is a pre-harvest Perilla frutescens growth graph);
FIG. 6 is a graph showing the statistical results of plant heights of various groups of purple perilla;
FIG. 7 is a graph showing the results of stem thickening statistics of each group of perilla;
FIG. 8 is a graph showing the above-ground biomass statistics of each group of perilla frutescens;
FIG. 9 is a graph showing the statistical result of leaf chlorophyll content of each group of perilla;
FIG. 10 is a graph showing the statistical results of the content of rosmarinic acid and perillaldehyde in the leaves of each group of perilla;
FIG. 11 is a graph showing the statistical result of the organic matter content in the soil planted by each group of purple perilla;
Fig. 12 is a graph showing statistical results of lead content in soil planted with each group of perilla.
The classification of the mucor circinelloides zh strain related to the invention is named mucor circinelloides
Mucor circinelloides the preservation unit is China general microbiological culture Collection center, the preservation time is 2023, 10 months and 9 days, the preservation number is CGMCC No.40842, and the addresses are: the korean district North Star, beijing city, part No. 1, no. 3.
Detailed Description
The technical scheme of the invention is further described by the following specific embodiments. It will be apparent to those skilled in the art that the examples are merely to aid in understanding the invention and are not to be construed as a specific limitation thereof.
Example 1
Isolation and identification of Mucor circinelloides zh:
(1) Separation and purification of Mucor circinelloides zh: taking healthy honeysuckle plant roots, cleaning the roots with clear water, cutting the roots into uniform small pieces, and respectively soaking the small pieces in 75% ethanol and 3% sodium hypochlorite solution for disinfection. After the disinfection, the surface is washed by sterile water, and the surface water is wiped by sterile absorbent paper. The plant pieces were inoculated on PDA medium and cultured at 30.+ -. 0.2 ℃ under dark conditions. After the colony grows out, single colonies with different morphological structures are picked up, streaked and separated on a PDA culture medium, and cultured under the same condition. The separated strains were frozen in a-80℃refrigerator. The formula of the PDA culture medium is as follows: 3.0 g parts of potato soaked powder, 20.0 g parts of glucose, 16.0 parts of agar g parts of agar and 1000 mL parts of distilled water.
(2) Morphological and molecular biological identification of Mucor circinelloides zh: inoculating the obtained flos Lonicerae endophyte into PDA culture medium, culturing in dark at 30+ -0.2deg.C for 5 days, observing colony morphological characteristics, picking mycelium, tabletting on clean glass slide, and observing mycelium morphology under microscope.
The strain is filamentous fungi, the colony is flocculent, the initial stage is grey-white, the later stage is light grey-brown (shown in figure 1), the hyphae grow luxuriantly, no transverse diaphragm is generated, multiple cores are generated, and no rhizoid and creeping hyphae are generated. The tube shape was observed under a microscope, and the terminal was enlarged to form sporangia, as shown in FIG. 2. The sporangium has oval shape and smooth surface, as shown in figure 3.
(3) The strain was inoculated into PDB medium (potato extract 6.0 g, glucose 20.0 g, distilled water 1000 mL) and cultured in the dark at 30.+ -. 0.2 ℃ and 180 rpm for 12 h. Taking 1mL bacterial liquid in a 1.5 mL centrifuge tube, centrifuging 12000 rpm for 2 min, and collecting bacterial precipitate. The strain DNA was extracted using a genomic DNA extraction kit (Tiangen Biochemical technologies Co., ltd.). The ITS gene sequence of the strain was amplified using the extracted DNA as a template and the universal primer pair ITS1F (5'-TTCCGTAGGTGAACCTGCGG-3')/ITS 4R (5'-TCCTCCGCTTATTGATATGC-3'). The PCR amplification system consisted of: 2X TAQ PCR MASTER Mix 12.5. Mu.L, 1. Mu.L each of primer ITS1F/ITS4R, 2. Mu.L of DNA template, and 25. Mu.L total of ddH 2 O8.5. Mu.L. The reaction procedure is: pre-denaturation at 94 ℃ 5 min; denaturation at 94℃for 30 s, annealing at 56℃for 30 s, extension at 72℃for 45 s, and circulation 40 times; incubation at 72℃was 10 min. The amplified products were subjected to agarose gel electrophoresis (120V, 30 min) as shown in FIG. 4. The purified amplified product is sequenced by major engineering laboratory of China national academy of agricultural sciences as shown in SEQ ID NO. 1. Removing low-quality areas at two ends of a sequencing result sequence, selecting a section with clear peaks and troughs in the middle of the sequence and no overlapping peaks, performing sequence alignment analysis on a national center for biotechnology information (NCBI, https:// www.ncbi.nlm.nih.gov /) website, identifying the strain as a Mucor circinelloides Mucor circinelloide strain, and naming the strain as zh.
Example 2
The embodiment provides a microbial fertilizer which consists of a composite microbial agent and the following components in parts by weight: 138 parts of biochar, 33 parts of pine needle powder, 551 parts of turfy soil, 5.7 parts of polyglutamic acid stock solution (with the concentration of 3.5%) and 276 parts of organic fertilizer; wherein the biochar is bamboo charcoal burned at 700 ℃; wherein the organic fertilizer is a product purchased from Beijing Dongxiang environmental science and technology Co., ltd (organic matter is more than or equal to 30%, N is more than or equal to 1.3%, and P 2O5≥1.1%,K2O≥1.6%,N+P2O5+K2 O is more than or equal to 4.0%); wherein the pine needle powder is a product purchased from special business of Pingjiang PingMingtang in Anji county; wherein the turfy soil is a product purchased from the danish tupe group (PINDSTRUP); wherein the polyglutamic acid stock solution is a product purchased from Shandong Qianfeng agricultural technologies Co.
The composite microbial inoculum is prepared by the following preparation method:
(1) Respectively weighing water-soluble bacillus licheniformis, water-soluble bacillus amyloliquefaciens and water-soluble bacillus subtilis bacterial powder (both purchased from Shandong and Mingxing biotechnology Co., ltd.) and dissolving in sterile water, and fully and uniformly mixing to obtain bacterial liquids of bacillus licheniformis, bacillus amyloliquefaciens and bacillus subtilis.
(2) Activating the mucor circinelloides zh separated in the example 1 in a PDA culture medium, picking single bacterial colony, inoculating the single bacterial colony in a PDB culture medium, culturing the single bacterial colony in a dark environment at 30+/-0.2 ℃ under 180 rpm to obtain mucor circinelloides zh seed liquid, inoculating the seed liquid in a new PDB culture medium, and culturing the single bacterial colony under the same condition to obtain mucor circinelloides zh bacterial liquid. Wherein the formula of the PDA culture medium is as follows: 3.0 g parts of potato soaked powder, 20.0 g parts of glucose, 16.0 parts of agar g parts of distilled water 1000 mL parts; the formula of the PDB culture medium is as follows: potato starch 6.0 g, glucose 20.0 g, distilled water 1000 and mL.
(3) The bacillus licheniformis, the bacillus amyloliquefaciens, the bacillus subtilis and the mucor circinelloides zh bacterial liquid are mixed to obtain a composite bacterial agent, the ratio of the viable count of the bacillus licheniformis to the viable count of the bacillus amyloliquefaciens to the viable count of the bacillus subtilis to the viable count of the mucor circinelloides zh in the composite bacterial agent are 74:74:74:1, and the consumption of the mucor circinelloides zh in the microbial fertilizer is 1.62X10 6 CFU per g of biochar.
Example 3
The embodiment provides a microbial fertilizer which consists of a composite microbial agent and the following components in parts by weight: 138 parts of biochar, 33 parts of pine needle powder, 551 parts of turfy soil, 5.7 parts of polyglutamic acid stock solution (with the concentration of 3.5%) and 276 parts of organic fertilizer; wherein the biochar is bamboo charcoal burned at 700 ℃; wherein the organic fertilizer is a product purchased from Beijing Dongxiang environmental science and technology Co., ltd (organic matter is more than or equal to 30%, N is more than or equal to 1.3%, and P 2O5≥1.1%,K2O≥1.6%,N+P2O5+K2 O is more than or equal to 4.0%). Wherein the pine needle powder is a product purchased from special business of Pingjiang PingMingtang in Anji county; wherein the turfy soil is a product purchased from the danish tupe group (PINDSTRUP); wherein the polyglutamic acid stock solution is a product purchased from Shandong Qianfeng agricultural technologies Co.
The preparation method of the composite microbial agent is different from that of the embodiment 2 only in that in the step (3), the ratio of the viable count of bacillus licheniformis to bacillus amyloliquefaciens to bacillus subtilis to mucor circinelloides zh in the composite microbial agent is 148:148:148:1, and the dosage of mucor circinelloides zh in the microbial fertilizer is 8.1 multiplied by 10 5 CFU per g of biochar.
Comparative example 1
The comparative example provides a microbial fertilizer which consists of a composite microbial agent and the following components in parts by weight: 138 parts of biochar, 33 parts of pine needle powder, 551 parts of turfy soil, 5.7 parts of polyglutamic acid stock solution (with the concentration of 3.5%) and 276 parts of organic fertilizer; wherein the biochar is bamboo charcoal burned at 700 ℃; wherein the organic fertilizer is a product purchased from Beijing Dongxiang environmental science and technology Co., ltd (organic matter is more than or equal to 30%, N is more than or equal to 1.3%, and P 2O5≥1.1%,K2O≥1.6%,N+P2O5+K2 O is more than or equal to 4.0%). Wherein the pine needle powder is a product purchased from special business of Pingjiang PingMingtang in Anji county; wherein the turfy soil is a product purchased from the danish tupe group (PINDSTRUP); wherein the polyglutamic acid stock solution is a product purchased from Shandong Qianfeng agricultural technologies Co.
The composite microbial inoculum is prepared by the following preparation method:
(1) Respectively weighing water-soluble bacillus licheniformis, water-soluble bacillus amyloliquefaciens and water-soluble bacillus subtilis bacterial powder (both purchased from Shandong and Mingxing biotechnology Co., ltd.) and dissolving in sterile water, and fully and uniformly mixing to obtain bacterial liquids of bacillus licheniformis, bacillus amyloliquefaciens and bacillus subtilis.
(2) The bacillus licheniformis, the bacillus amyloliquefaciens and the bacillus subtilis bacterial liquid are mixed to obtain a composite bacterial agent, the ratio of the viable count of the bacillus licheniformis to the viable count of the bacillus amyloliquefaciens to the viable count of the bacillus subtilis in the composite bacterial agent is 1:1:1, and the consumption of the bacillus licheniformis in the microbial fertilizer is 1.2 multiplied by 10 8 CFU per g biochar.
Comparative example 2
The comparative example provides a microbial fertilizer which comprises the following components in parts by weight: 138 parts of biochar, 33 parts of pine needle powder, 551 parts of turfy soil, 5.7 parts of polyglutamic acid stock solution (with the concentration of 3.5%) and 276 parts of organic fertilizer; wherein the biochar is bamboo charcoal burned at 700 ℃; wherein the organic fertilizer is a product purchased from Beijing Dongxiang environmental science and technology Co., ltd (organic matter is more than or equal to 30%, N is more than or equal to 1.3%, and P 2O5≥1.1%,K2O≥1.6%,N+P2O5+K2 O is more than or equal to 4.0%). Wherein the pine needle powder is a product purchased from special business of Pingjiang PingMingtang in Anji county; wherein the turfy soil is a product purchased from the danish tupe group (PINDSTRUP); wherein the polyglutamic acid stock solution is a product purchased from Shandong Qianfeng agricultural technologies Co.
Test case
The microbial fertilizers prepared in examples 2-3 and comparative examples 1-2 were verified for their effect of promoting growth of Perilla frutescens:
District tests were carried out in a laboratory shed of the institute of medical and plant laboratory of the national academy of medical science, the sea lake area of Beijing, at 2023, 6 to 8 months, with the test material being PF40 type Perilla frutescens (Perilla frutescens (L.) Britt.). Four treatments were set up for the trial, 21 purple perilla strains each: the microbial fertilizers prepared in examples 2-3 and comparative examples 1-2 were used, respectively.
And (3) at 2023, 4 and 19 days, selecting a plurality of purple perilla seeds with full seeds and uniform size, sowing the purple perilla seeds in a seedling tray, and germinating and culturing the purple perilla seeds in a climatic chamber under the conditions of 25 ℃ and light intensity of 1000 Lx (photoperiod 16 h/8 h) and relative humidity of 65%. After the seedlings grow out in 2023 and 6 months, the biochar, pine needle powder, turfy soil and organic fertilizer in each group of fertilizer are mixed with the soil matrix and uniformly stirred, and 10 kg of the solid matrix is applied per square meter. Selecting Perilla seedlings with consistent growth vigor, transplanting into test soil (shown in figure 5, wherein A is a test layout diagram, B is a Perilla seedling growth diagram, C is a pre-harvest Perilla growth diagram), and irrigating polyglutamic acid stock solution and composite microbial inoculum in each group of fertilizers with water. And during the test, the purple perilla is treated for three times by using the polyglutamic acid stock solution and the compound microbial inoculum in each group of fertilizers on the 20 th day, the 30 th day and the 40 th day respectively, the use amount of the polyglutamic acid stock solution and the compound microbial inoculum treated each time is the same, the purple perilla is harvested after 54 days of planting, and meanwhile, the data such as the plant height, the stem thickness, the fresh weight of the overground part, the chlorophyll content, the drug effect component content (perillaldehyde, rosmarinic acid), the lead element content and the organic matter content in the cultivated soil and the like are measured and counted.
As shown in fig. 6, the plant height statistics results of each group of perilla were shown, and the microbial fertilizer according to the present invention was able to significantly increase the plant height of perilla, and the effect of example 2 was better than that of a fertilizer without using the composite microbial inoculum.
As shown in fig. 7, the stem thickness statistics result of each group of perilla was remarkably improved by using the microbial fertilizer according to the present invention, and the effect of example 2 was better than that of the fertilizer without using the composite microbial inoculum.
As shown in FIG. 8, the above-ground biomass statistics of each group of purple perilla were shown to be better in the effect of example 2, since the microbial fertilizer according to the present invention was used to significantly increase the above-ground biomass of purple perilla than the fertilizer without using the composite microbial inoculum.
The statistical results of leaf chlorophyll content of each group of purple perilla are shown in figure 9, and compared with the fertilizer without the composite microbial inoculum, the microbial fertilizer can obviously improve the chlorophyll content of purple perilla.
The statistical results of the content of rosmarinic acid and perillaldehyde in the leaves of each group of purple perilla are shown in figure 10, compared with the fertilizer without the composite microbial inoculum, the microbial fertilizer can remarkably improve the content of rosmarinic acid and perillaldehyde in the leaves of purple perilla, and the effect of the embodiment 2 is better.
The statistical results of the organic matter content in the soil planted by each group of purple perilla are shown in fig. 11, and compared with the fertilizer without the composite microbial inoculum, the microbial fertilizer can provide rich organic matters for the soil, and the effect of the embodiment 2 is better.
The statistical results of the lead content in the soil planted by each group of purple perilla are shown in figure 12, and compared with the fertilizer without the composite microbial inoculum, the microbial fertilizer can obviously reduce the lead content in the soil.
The applicant states that the technical solution of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above embodiments, i.e. it does not mean that the present invention must be implemented by the above embodiments. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure.
The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.
In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.
Claims (10)
1. A microbial fertilizer for promoting growth of perilla and accumulation of a pharmaceutically effective ingredient, wherein the microbial fertilizer for promoting growth of perilla and accumulation of a pharmaceutically effective ingredient comprises: biochar, pine needle powder, turfy soil, polyglutamic acid, organic fertilizer and composite microbial inoculum;
The strain in the composite microbial inoculum consists of bacillus licheniformis, bacillus amyloliquefaciens, bacillus subtilis and mucor circinelloides; the Mucor circinelloides is a strain with a preservation number of CGMCC No.40842 and a classification name of Mucor circinelloides Mucor circinelloides.
2. The microbial fertilizer for promoting growth of Perillae herba and accumulation of pharmaceutically effective components according to claim 1, wherein the ratio of the number of viable bacteria of Bacillus licheniformis, bacillus amyloliquefaciens, bacillus subtilis and Mucor circinelloides is (10-500): 1.
3. The microbial fertilizer for promoting growth of Perillae herba and accumulation of pharmaceutically effective components according to claim 1, wherein the ratio of the number of viable bacteria of Bacillus licheniformis, bacillus amyloliquefaciens, bacillus subtilis and Mucor circinelloides is (10-100): 1.
4. The microbial fertilizer for promoting growth of perilla and accumulation of effective components according to claim 1, wherein the content of the strain in the composite microbial agent in the microbial fertilizer is (1-10) x 10 8 CFU per g biochar.
5. The microbial fertilizer for promoting the growth of perilla and the accumulation of effective components according to claim 1, wherein the biochar is bamboo charcoal fired at 500-700 ℃.
6. The microbial fertilizer for promoting growth of perilla and accumulation of effective components according to claim 1, wherein the organic matters content in the organic fertilizer is more than or equal to 30%, N is more than or equal to 1.3%, and P 2O5≥1.1%,K2 O is more than or equal to 1.6%.
7. The microbial fertilizer for promoting growth of perilla and accumulation of effective components according to claim 1, wherein the mass ratio of the biochar, pine needle powder, turfy soil, polyglutamic acid and organic fertilizer in the microbial fertilizer is (100-150): (30-40): (540-580): (0.1-1.0): (250-300).
8. The microbial fertilizer for promoting growth of perilla and accumulation of effective components according to claim 1, wherein the composite microbial agent is prepared by a preparation method comprising the following steps:
Mixing bacterial suspensions of bacillus licheniformis, bacillus amyloliquefaciens, bacillus subtilis and mucor circinelloides in proportion to obtain the composite microbial inoculum;
The bacterial suspension is obtained by dissolving bacterial powder in a solvent, or is obtained by sequentially activating and amplifying culturing bacterial strains.
9. The microbial fertilizer for promoting growth of perilla frutescens and accumulation of pharmaceutically effective components according to claim 8, wherein the bacterial suspension of mucor circinelloides is prepared by a preparation method comprising the following steps:
Activating Mucor circinelloides in PDA culture medium, selecting single colony, inoculating to PDB culture medium, culturing to obtain Mucor circinelloides seed solution, inoculating to PDB culture medium, and culturing to obtain;
Wherein the formula of the PDA culture medium is as follows: potato soaked powder 2-6 g, glucose 15-25 g, agar 10-20 g, distilled water 1000-mL; the formula of the PDB culture medium is as follows: potato soaked powder 2-6 g, glucose 15-25 g, distilled water 1000-mL.
10. Use of a microbial fertilizer for promoting growth of perilla and accumulation of pharmaceutically effective components according to any one of claims 1 to 9 in cultivation of perilla, a plant for both medicine and food.
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