CN113943671A

CN113943671A - Improved compound microbial agent and application thereof in yield increase of angelica sinensis

Info

Publication number: CN113943671A
Application number: CN202111236953.4A
Authority: CN
Inventors: 杨涛; 牛永艳; 王治业; 赵疆; 毛婷; 姚阳阳; 李鑫
Original assignee: Institute of Biology of Gansu Academy of Sciences
Current assignee: Gansu Lvneng Agricultural Science And Technology Co ltd
Priority date: 2021-10-24
Filing date: 2021-10-24
Publication date: 2022-01-18
Anticipated expiration: 2041-10-24
Also published as: CN113943671B

Abstract

The invention belongs to the technical field of microorganisms and biology, and particularly relates to an improved compound microbial agent and application thereof in yield increase of angelica sinensis. The compound microbial agent is prepared by compounding pseudomonas fluorescens, pseudomonas alcaligenes, pseudomonas psychrophila and zoogloea cladinosa; the compound microbial agent not only keeps the functional characteristics of the original microbial agent, but also improves the conversion, absorption and utilization of soil nutrients; the compound microbial agent can promote the conversion of ammonium nitrogen in the rhizosphere soil into nitrate nitrogen, reduce the toxic action of soil ammonium salt on plants and improve the quick-acting nitrogen content of the rhizosphere soil; but also can enhance the activity of nitrate reductase in plants, promote nitrate nitrogen in plants to be converted into ammonium nitrogen, promote the absorption and enrichment of nitrogen by plants and improve the yield of plants.

Description

Improved compound microbial agent and application thereof in yield increase of angelica sinensis

Technical Field

The invention belongs to the technical field of microorganisms and biology, and particularly relates to an improved compound microbial agent and application thereof in yield increase of angelica sinensis.

Background

Angelica sinensis (Oliv.) Diels, a dried root of Angelica sinensis (Oliv.) Diels, a plant belonging to the family Umbelliferae, is a traditional bulk Chinese medicine, and is listed in the homology of medicine and food. Gansu is the region of Angelica sinensis production in the field, and the planting area of the whole province reaches 400km²Yield 1.2X 10⁵kg, which accounts for more than 80% of the total supply quantity of the national market, and plays a decisive role in the national market price of Chinese angelica. The angelica can be cultivated at the altitude of 1500-3000m, is fond of high and cold climate, is fond of deep soil layer, loose soil layer, good drainage, rich sandy loam rich in humus, is not suitable for cultivation in low-lying accumulated water or clay easy to harden and poor sandy soil, and is forbidden for continuous cropping. At present, the problems of heavy input of chemical fertilizers and pesticides, poor stability of biological systems, low diversity of soil microorganisms, continuous cropping obstacles and the like of the artificial cultivation traditional Chinese medicines such as angelica and the like generally exist, and the development of the functional microbial agent of the angelica is imperative.

The rhizosphere is the micro-area environment where plants contact with soil, and is the main area where plants obtain nutrients. The enzymes secreted by the plant root system and the remains thereof, the soil animals and the remains thereof and the microorganisms can catalyze the complex organic matters in the soil to be converted into simple inorganic matters for the utilization of the plants. Soil enzyme activity, such as: phosphatase, urease, beta-glucosidase and the like can reflect the strength of the soil nutrient conversion capacity, and the activity is related to the crop yield. PGPR is a soil-beneficial bacterium existing in plant rhizosphere, and has great influence on soil ecological environment such as soil microorganisms, enzyme activity, nutrients and the like. Researches show that wheat treated by PGPR (straw returning soil improvement fertilization matrix and compound microbial inoculum preparation have influence on soil ecology), tartary buckwheat (influence of organic matters and microbial agents on tartary buckwheat continuous cropping agronomic characters and soil enzyme activity), cantaloupe (influence of different microbial agent treatments on cantaloupe quality and soil nutrient and enzyme activity), hot pepper (influence of compound microbial agents on hot pepper growth and rhizosphere soil microbial structure) and the like are remarkably improved in soil nutrient nitrogen and phosphorus content, urease and phosphatase activity and yield without application, and the compound microbial inoculum is inoculated to soil urease, sucrase and protease activity by medicinal plant Paris polyphylla (influence of arbuscular mycorrhizal fungi on physicochemical properties of Paris polyphylla rhizosphere soil) to improve absorption of soil urease, sucrase and protease activity and mineral nutrient elements quick-acting nitrogen, phosphorus and potassium and improve yield.

The inventor researches the influence of the pseudomonas compound microbial agent on the physiology, biochemistry and quality of the continuous cropping Chinese angelica, applies for a national invention patent (the pseudomonas compound microbial agent and the application thereof in the disease-resistant production-increasing and quality-improving of the Chinese angelica) of related microbial agent products according to research results, does not consider the problems of seedling burning and ammonium salt poison caused by the large and unreasonable use of urea in the planting process of farmers, and has the effect of promoting flowering, so the problem of phosphorus dissolution is also considered in the development of the related microbial agent for Chinese angelica planting.

Based on the compound microbial inoculum CBSB, CBS5 and CBS7 of pseudomonas in the early stage, the invention deeply studies the influence of different microorganisms on rhizosphere soil enzyme activity and mineral nutrient activation utilization, and further compounds and optimizes the compound microbial inoculum from the angle of regulating and utilizing the soil nutrient of angelica.

Disclosure of Invention

Aiming at the technical problems, the invention aims to provide an improved compound microbial agent and application thereof in yield increase of angelica, wherein the compound microbial agent can promote ammonium nitrogen in rhizosphere soil to be converted into nitrate nitrogen, reduce the toxic action of ammonium salt on plants and improve the quick-acting nitrogen content of the rhizosphere soil; but also can enhance the activity of nitrate reductase in plants, promote nitrate nitrogen in plants to be converted into ammonium nitrogen, promote the absorption and enrichment of nitrogen by plants and improve the yield of plants. The method specifically comprises the following steps:

in a first aspect, the present invention provides an improved complex microbial inoculant comprising pseudomonas fluorescens, pseudomonas alcaligenes, pseudomonas psychrophila and zoogloea cladinosa.

Preferably, the compound microbial agent consists of pseudomonas fluorescens CBS5, pseudomonas alcaligenes CBS7, pseudomonas psychrophila CBSB and zoogloea cladinosa CBS 4; the 16S rDNA sequence of the pseudomonas fluorescens CBS5 is shown in SEQ ID NO. 1; the 16S rDNA sequence of the pseudomonas alcaligenes CBS7 is shown as SEQ ID NO.2, and the 16S rDNA sequence of the pseudomonas psychrophila CBSB is shown as SEQ ID NO. 3; the 16S rDNA sequence of the zoogloea cladosporium CBS4 is shown in SEQ ID No. 4. The pseudomonas fluorescens CBS5(NCBI No. MW981369.1), the pseudomonas alcaligenes CBS7(NCBI No. MW981370.1), the pseudomonas psychrophila CBSB (NCBI No. MW981371.1) and the zoogloea CBS4(NCBI No. MW981368.1) are purchased from the Gansu center of China center for preservation and management of industrial microbial strains.

Preferably, the compound microbial agent is prepared by mixing the strains in equal proportion.

Preferably, the microbial inoculum contains 10 CFU of the microbial strains⁶～10⁹/ml。

In a second aspect, the present invention provides the use of the complex microbial inoculant described in the first aspect for promoting plant growth.

Preferably, the plant is angelica.

Preferably, the compound microbial agent can promote the growth of underground medicinal organ roots of the angelica, increase the diameter of the reed head, the length of the reed head and the average single plant weight, increase the yield and improve the appearance of the commercial angelica.

In a third aspect, the invention provides an application of the complex microbial agent described in the first aspect in improving the activity of nitric acid reductase in plants.

Preferably, the plant is angelica.

In a fourth aspect, the present invention provides the use of the complex microbial inoculant described in the first aspect for promoting plants to absorb and enrich ammonium nitrogen.

Preferably, the plant is angelica.

In a fifth aspect, the present invention provides an application of the complex microbial agent of the first aspect in promoting conversion of soil ammonium nitrogen into nitrate nitrogen.

In a sixth aspect, the invention provides an application of the compound microbial agent of the first aspect in improving the content of quick-acting nitrogen in soil.

In a seventh aspect, the present invention provides an application of the complex microbial agent of the first aspect in improving soil enzyme activity.

Preferably, the compound microbial agent can improve the rhizosphere soil sucrase and urease of angelica.

In an eighth aspect, the invention provides an application of the compound microbial agent described in the first aspect in preparation of a nitrogen-fixing bacterial fertilizer.

In a ninth aspect, the invention provides a special microbial fertilizer for angelica sinensis, which comprises the compound microbial agent of the first aspect.

The invention has the beneficial effects that: firstly, on the basis of the original pseudomonas bacteria (pseudomonas fluorescens CBS5, pseudomonas alcaligenes CBS7 and pseudomonas psychrophila CBSB), the invention adds the rhizobiaceae zoogloea strain CBS4 to obtain an improved compound microbial agent; the compound microbial agent not only keeps the functional characteristics of the original microbial agent, but also can promote the conversion, absorption and utilization of soil nutrients; the compound microbial inoculum can promote the ammonium nitrogen in the rhizosphere soil to be converted into nitrate nitrogen, reduce the toxic action of ammonium salt on plants and improve the quick-acting nitrogen content of the rhizosphere soil; but also can enhance the activity of nitrate reductase in plants, promote nitrate nitrogen to be converted into ammonium nitrogen, promote the absorption and enrichment of the plants on nitrogen and improve the yield of the plants.

Drawings

FIG. 1 is a main component analysis of enzyme activity indexes of angelica rhizosphere soil at the S2 period;

FIG. 2 is the main component analysis of enzyme activity index of angelica rhizosphere soil at the S3 period;

FIG. 3 is the analysis of the principal components of nutrient indexes in the rhizosphere soil and roots of angelica at the time of S2;

and 4, analyzing main components of nutrient indexes in rhizosphere soil and roots of the angelica in the period of S3 in figure 4.

Detailed Description

The present invention will be further described with reference to the following examples, but the scope of the present invention is not limited to the following examples, and modifications or substitutions of the methods, steps or conditions of the present invention can be made without departing from the spirit and substance of the present invention.

The test plants described in the following examples were 1-year-old seedlings of Angelica regale, variety Mingui No.1, provided by Minxian county Chinese medicinal crop production technology guide station. The test strain is obtained by separating from Chinese medicinal materials at the early stage of the subject group member. The test is carried out on a land plot of angelica in an ecological planting demonstration base of Gansu Tianzhu Chinese medicinal materials for 3 years in 4-10 months in 2020. The soil type is the millet calcareous soil, and the physicochemical property of the soil to be tested is quick-acting potassium of 6.65 mg.kg^-1Quick-acting phosphorus 15.99 g.kg^-1Alkaline hydrolyzable nitrogen 35.05. mu.g.g^-12.9 percent of organic matter and 7.85 of pH value. Preparing soil at the bottom of 4 months, applying organic fertilizer (organic matter is more than or equal to 45 percent, and total nutrient N + P)₂O₅+K₂O≥5％)3000kg·hm^-2. The reagents and culture medium used in the test are all chemically pure.

Nitrite nitrogen (LH-NO2-100) and nitrate nitrogen (LH-NO3-100) kits were purchased from Yinchuan Lianhua scientific and technological development Inc.; the BCA protein concentration determination kit is purchased from Ku Laibobu technologies, Inc. of Beijing; an enzyme activity detection kit nitrate reductase (Lot.No.20200930), soil catalase (Lot.No.20200810), soil sucrase (Lot.No.20200831), soil urease (Lot.No.20200806), soil FDA hydrolase (Lot.No.20200903), soil beta-glucosidase (Lot.No.20200805), soil alkaline protease (Lot.No.20200716) and soil alkaline phosphatase (Lot.No. 20200811); plant sucrose (lot.no.20201012), plant ammonia nitrogen (lot.no.20200927), plant nitrate nitrogen (lot.no.20201012), plant nitrate reductase (lot.no.20201034), plant soluble protein (lot.no.20200927), soil ammonium nitrogen (lot.no.20200903), soil nitrate nitrogen (lot.no.20200903), soil rapid-acting potassium (lot.no.20200907), and alkaline soil rapid-acting phosphorus (lot.no.20200907) content detection kits are all purchased from beijing sojourn rice treasure science and technology ltd; ethanol, toluene, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, magnesium chloride and ammonium chloride are analytically pure and purchased from Tianjin Damao chemical reagent company; the water is ultrapure water.

PDB culture medium: 200g/L of potato, 20g/L of glucose and natural pH;

aberra nitrogen-free medium: KH (Perkin Elmer)₂PO₄ 0.2g/L，MgSO₄·7H₂O 0.2g/L，NaCl 0.2g/L，CaCO₃5.0g/L, mannitol 10.0g/L, CaSO₄·2H₂O 0.1g/L，pH 7.0±0.1；

Nitrifying the culture medium: k₂HPO₄ 1.0g/L，KH₂PO₄ 1.0g/L，MgCl₂ 0.4g/L，A5 1.0ml/L，NH₄0.3g/L of Cl, 10.0g/L of mannitol and 7.0 +/-0.2 of pH;

NBRIP medium: glucose 10g/L, MgCl₂·6H₂O 5.0g/L，MgSO₄·H₂O 0.25g/L，KCl 0.2g/L，(NH₄)₂SO₄ 0.1g/L，Ca₃(PO₄)₂ 5.0g/L，pH 7.0。

According to the invention, on the basis of the original compound microbial inoculum (pseudomonas fluorescens CBS5, pseudomonas alcaligenes CBS7 and pseudomonas psychrophila CBSB), screening research experiments show that the compound microbial inoculum obtained by adding zoogloea zoogloeosporium CBS4 can promote ammonium nitrogen in rhizosphere soil to be converted into nitrate nitrogen, reduce the toxic action of ammonium salt on plants and improve the quick-acting nitrogen content of rhizosphere soil; but also can enhance the activity of nitrate reductase in plants, promote nitrate nitrogen in plants to be converted into ammonium nitrogen, promote the absorption and enrichment of nitrogen by the plants and improve the yield of the plants. The bacterial agents and experimental design involved in the following examples are therefore as follows:

experimental group complex microbial inoculant (T1): mixing Pseudomonas fluorescens CBS5(NCBI No. MW981369.1), Pseudomonas alcaligenes CBS7(NCBI No. MW981370.1), Pseudomonas psychrophila CBSB (NCBI No. MW981371.1) and Achillea rubber bacteria CBS4(NCBI No. MW981368.1) which are respectively cultured by a PDB culture medium in equal proportion; adjusting to total bacteria concentration of 1 × 10⁷CFU·mL^-1；

Positive control group complex inoculant (T2): mixing Pseudomonas fluorescens CBS5(NCBI No. MW981369.1), Pseudomonas alcaligenes CBS7(NCBI No. MW981370.1) and Pseudomonas psychrophila CBSB (NCBI No. MW981371.1) respectively cultured in PDB culture medium in equal proportion, and adjusting to total bacterial concentration of 1 × 10⁷CFU·mL^-1；

Positive control group single bacterium CBS4(CBS 4): acidocella mobilis CBS4(NCBI No. MW981368.1) cultured in PDB medium and adjusted to total bacteria concentration of 1 × 10⁷CFU·mL^-1；

Negative control group (CK): PDB medium was not inoculated.

The test adopts single-factor completely random design, and 4 treatments of T1, T2, CBS4 and CK are set, and different strains are treated at 28 ℃ and 200 r.min^-1Shake culturing with PDB culture medium for 2d under the condition, adjusting to the same OD value, mixing at equal ratio, adjusting to 1 × 10 with sterile water⁷CFU·mL^-1The application rates for the different treatments were equal in volume. Each treatment was repeated 3 times, and the cell area was 60m²(6 m.times.10 m). Planting the angelica sinensis without covering a film, irrigating roots and spraying leaf surfaces once every 3 weeks for 4 times in 7-9 months, and performing field management according to conventional measures.

Sampling 3 times respectively at 7/8 days (S1 period) in 2020, 1 day (S2 period) after 3 rd application in 8/25 days in 2020, 1 day after 4 th application in 10/9 days in 2020 (S3 period), repeating the treatment for 3 times, collecting 10 plants in each cell, lightly shaking off the soil around the root system, lightly brushing the soil attached to the surface of the root system with a brush, sieving, removing residual roots mixed in the soil, naturally drying, sealing with a sterile sealing bag, and storing at 4 ℃ for later use. After the roots of the Chinese angelica are cleaned, the roots of the Chinese angelica are cut into small sections with the length of 1cm, the small sections are evenly mixed, and relevant indexes are measured on fresh samples. Finally, the yield is measured after harvesting.

Example 1 microbial phosphorus removal, Nitrogen fixation, nitration assays

1. Qualitative detection of phosphate solubilizing ability

Respectively inoculating different strains on an NBRIP inorganic phosphorus solid culture medium, culturing at the constant temperature of 28 ℃ for 2-5 days, observing the existence and size of a phosphorus-solubilizing ring, and determining the phosphorus-solubilizing effect of the strains on inorganic phosphorus according to the size of the phosphorus-solubilizing ring.

2. Quantitative detection of nitrogen fixation activity

Inoculating 5ml of purified bacterial liquid into sterilized 50ml of nitrogen-free culture medium of arbuscular mycorrhizal fritillary bulb for 200r min^-1Shaking culture at 30 deg.C for 72h, and determining turbidity OD₆₀₀。

The azotase activity of the strain is determined by an acetylene reduction method: transferring 5ml of the fresh bacterial liquid into a 20ml headspace bottle, closing the cap, pumping 2ml of gas out of the bottle, injecting equal volume of acetylene, continuing culturing for 24h, and detecting the content of the generated ethylene by using a gas chromatograph. Gas chromatography conditions: the capillary column adopts PLOT/Q packed capillary chromatographic column [30m (length) × 0.53mm (inner diameter) × 40m (film thickness)]The carrier gas is nitrogen, and the flow rate of the carrier gas is 2.0 mL/min^-1(ii) a Sample inlet temperature: the split ratio is 10:1 at 200 ℃. Temperature rising procedure: the temperature was kept constant at 60 ℃ for 6 min. FID detector: the temperature is 250 ℃, and the hydrogen is 40 mL/min^-1Air 450 mL. min^-1Tail gas blowing (helium) 30 mL. min^-1. The nitrogenase activity was calculated as follows and finally divided by OD₆₀₀Values, normalized to the azotase activity of bacteria in unit turbidity.

3. Quantitative determination of nitration Capacity

Collecting the above activated bacteria solution, 5000 r.min^-1Centrifuging for 10min, collecting thallus, and adjusting OD by resuspending in sterile water₆₀₀Inoculating the bacterial liquid into a nitrifying culture medium at a volume ratio of 1 ‰, and culturing at 28 deg.C for 200r min^-1Shake culturing for 2d, determining OD₆₀₀Concentration of the bacteria detected by the method of the kitThe nitrifying power of the liquid nitrite nitrogen and the nitrate nitrogen is calculated according to the following formula, and the calculated result is divided by the turbidity of the corresponding bacterial liquid and is standardized to the nitrifying power of the bacterial liquid with unit turbidity. Each strain was replicated 3 times and the average was calculated.

4. Results

The results of the phosphorus-dissolving, nitrogen-fixing and nitrifying forces of different strains are shown in Table 1. The results show that CBS7 has weak phosphate solubilizing capability, and other strains have no phosphate solubilizing activity; each strain has nitrogen fixation and nitrification capacity, and the nitrogen fixation and nitrification capacity of the strain CBS4 are higher than those of other strains.

TABLE 1 determination of phosphorus-dissolving and nitrogen-fixing and nitrifying power of different strains

Note: "+" has this function, "-" does not; each column of different lower case letters respectively represents multiple comparisons of corresponding indexes.

Example 2 Effect of different treatments on yield-related indices of Angelica sinensis

After harvesting, respectively calculating the number of the adventitious roots by a conventional method, measuring the reed head diameter, the reed head length and the body length of the angelica by a vernier caliper, weighing the fresh weight of each plant by an electronic scale, measuring 300 plants each time, and calculating the average value.

As shown in Table 2, compared with CK, the number of adventitious roots, the diameter of reed heads, the length of bodies and the average fresh weight of single plants of angelica sinensis treated by CBS4 single bacteria are basically unchanged, and the number of adventitious roots, the diameter of reed heads, the length of bodies and the average fresh weight of single plants of angelica sinensis treated by the T1 and T2 complex microbial agents are increased. Compared with CK group, after treatment by the composite bacterial agent (T1), the indefinite number, the reed head diameter, the reed head length, the body length and the average fresh weight of each plant of the Chinese angelica are respectively increased by 14.30%, 19.74%, 9.45%, 10.59% and 33.52%; while the indefinite number, the diameter, the length, the body length and the average fresh weight of the single plant of the angelica after the treatment of the contrast fungicide (T2) are respectively increased by 26.74 percent, 14.04 percent, 5.26 percent, 6.88 percent and 20.12 percent (P is less than 0.05); compared with the T2 group, after the treatment by the compound microbial inoculum (T1) disclosed by the invention, the indefinite number of the angelica sinensis is reduced by 9.8%, and the diameter of the reed rhizome, the length of the body and the average fresh weight of a single plant are respectively increased by 5.0%, 4.0%, 3.5% and 11.1%. The compound bacterial agent (T1) provided by the invention is shown to remarkably promote the growth of angelica sinensis.

TABLE 2 Effect of different treatments on the yield index of Angelica sinensis

Note: each column of different lower case letters respectively represents multiple comparisons of corresponding indexes

Example 3 Angelica sinensis rhizosphere soil enzyme Activity assay

And (3) measuring rhizosphere soil urease (S-UE), soil sucrase (S-SC), soil FDA hydrolase, soil beta-glucosidase (S-beta-GC), soil alkaline protease (S-ALPT), soil alkaline phosphatase activity (S-AKP/ALP) and soil catalase (S-CAT) activity of the Chinese angelica at different periods according to the method of the kit, repeating the steps for 3 times for each sample, and calculating the average value.

The effect of the different treatments on the activity of the rhizosphere soil enzymes is shown in table 3. Compared with CK group, after treatment by the T1 and T2 complex microbial inoculum (in S2 and S3 periods), the enzyme activities of Angelica sinensis rhizosphere soil FDA hydrolase, soil sucrase, soil-beta-glucosidase, soil urease and soil alkaline protease are improved, and the alkaline phosphatase activity is reduced; the results show that the two complex microbial agents can improve the microbial activity of rhizosphere soil and the conversion capacity of organic matters, promote carbon and nitrogen metabolism and inhibit phosphorus metabolism. Specifically, the method comprises the following steps: in the S1 period, the activity of the enzyme of each soil treated by the method is not obviously different; after the compound microbial inoculum is applied, from S2 in the vigorous growth phase of the underground part to S3 in the early stage of harvest, the enzymatic activities of FDA hydrolase, soil sucrase, soil-beta-glucosidase and soil alkaline protease are reduced, the activity of soil alkaline phosphatase is increased, and the activity of soil urease is unchanged after the two microbial inoculants are treated, which shows that the influence of different microbial inoculum treatments on rhizosphere soil nitrogen metabolism is long in time and deep in degree. Compared with a CK group, the soil catalase is gradually increased from the S1 stage to the S3 stage, the enzyme activity of the catalase is reduced in the S2 stage (1 day after the 3 rd application) and is increased in the S3 stage (25 days after the 4 th application) by the treatment of the T1 and T2 composite bacteria, so that the catalase is increased in the growth vigorous stage after the two composite bacteria are applied, the catalase activity is reduced due to short-term interference, the hydrogen peroxide content is increased, the catalase activity is increased in different treatments and in contrast with the growth continuation, and the long-term influence is represented by improving the catalase activity and eliminating the influence of active oxygen on the growth. Compared with the compound microbial inoculum T2, the compound microbial inoculum provided by the invention has the advantages that the activities of soil sucrase and urease of angelica sinensis in the S2 stage are obviously improved.

TABLE 3 Effect of different treatment periods on the enzyme activity of the rhizosphere soil of Angelica sinensis

Note: different lower case letters in each row respectively represent multiple comparisons of corresponding indexes at different periods

Example 4 measurement of nutrient content in Angelica sinensis rhizosphere soil and root and nitrate reductase Activity in root

And (3) determining the contents of rhizosphere soil nitrate nitrogen, soil ammonium nitrogen, soil quick-acting potassium, alkaline soil quick-acting phosphorus, plant sucrose, plant soluble protein, plant ammonia nitrogen, plant nitrate nitrogen and plant nitrate reductase activity of the angelica at different periods according to the method of the kit, repeating the steps for 3 times for each sample, and calculating the average value.

The results are shown in Table 4, and the change of the quick-acting nitrogen, phosphorus and potassium nutrients in the rhizosphere soil has different rules. The quick-acting phosphorus of the soil treated in different periods has no obvious difference, and the quick-acting potassium, the ammonium nitrogen, the nitrate nitrogen and the quick-acting nitrogen (the sum of the ammonium nitrate nitrogen) have obvious difference. Compared with the CK group, after treatment by the T1 and T2 composite microbial agents, the content of quick-acting potassium is improved in the S2 and S3 periods, and no obvious difference exists between the T1 and the T2 groups in different periods; the quick-acting potassium content is reduced from the stage S2 to the stage S3 by the treatment of T1, but no obvious difference exists, and in the stages S2 and S3, compared with a CK group, the quick-acting potassium content is respectively increased by 108 percent and 25.5 percent after the treatment of the composite microbial agent T1; the quick-acting potassium content is reduced from the stage S2 to the stage S3 by the treatment of T2, but the quick-acting potassium content is not obviously different, and in the stages S2 and S3, compared with a CK group, the quick-acting potassium content is only increased by 97.3% and 13.6% after the treatment of the composite microbial agent T2; namely, the compound microbial inoculum T1 of the invention obviously improves the content of quick-acting potassium in rhizosphere soil.

Compared with the CK group, after treatment by the T1 and T2 composite bacteria, the content of nitrate nitrogen is increased and the content of ammonium nitrogen is reduced in S2 and S3 periods; in the stage S2, compared with the compound microbial inoculum T2, after the compound microbial inoculum T1 is treated, nitrate nitrogen has no obvious difference, and ammonium nitrogen is obviously improved by 16.2 percent; in the stage S3, compared with the compound microbial inoculum T2, the ammonium nitrogen treated by the compound microbial inoculum T1 has no obvious difference, and the nitrate nitrogen is obviously improved by 9.1 percent; the treatment of the compound microbial inoculum T1 disclosed by the invention is from S2 stage to S3 stage, the ammonium nitrogen is obviously reduced by 32.2%, and the nitrate nitrogen is obviously reduced by 12.1%; compared with a CK group, after the compound bacteria T1 is treated, ammonium nitrogen in S2 and S3 periods is reduced by 37.5% and 29.4% respectively, and nitrate nitrogen is increased by 83.8% and 70.1% respectively. After the treatment of the compound bacteria T2, from the S2 stage to the S3 stage, the ammonium nitrogen is obviously reduced by 24.8%, the nitrate nitrogen is obviously reduced by 18.3%, compared with the CK group, after the treatment of the compound bacteria T2, the ammonium nitrogen is respectively reduced by 46.2% and 32.7% in the S2 and S3 stages, and the nitrate nitrogen is respectively increased by only 81.2% and 55.8%. Comprehensively analyzing the content of the quick-acting nitrogen, phosphorus and potassium elements in the rhizosphere soil, treating by the compound bacteria T1 and T2 improves the content of the quick-acting potassium, promotes the conversion of ammonium nitrogen into nitrate nitrogen, changes the proportion of different nitrogen elements, and reduces the nitrogen loss caused by the volatilization of the ammonium nitrogen and the ammonium salt toxicity caused by high-content ammonium nitrogen under the condition of alkaline soil; compared with a treatment group of a complex microbial inoculum T2, the treatment of the complex microbial inoculum T1 provided by the invention obviously increases the content of ammonium nitrogen in S2 stage, nitrate nitrogen in S3 stage and quick-acting nitrogen (the sum of nitrate ammonium nitrogen) in S2 and S3 stage.

TABLE 4 influence of different treatments on soil nutrients in the rhizosphere of Angelica sinensis at different times

The nutrient contents and enzyme activities of the roots of Chinese angelica at different treatment periods are shown in Table 5. Compared with a CK group, the compound microbial inoculum T1 and T2 treated by the compound microbial inoculum obviously improve the content of protein and sucrose in roots in different periods. Compared with the compound microbial inoculum T2, the compound microbial inoculum T1 has no significant difference in the S2 stage of the protein in roots after treatment, and the S3 stage is significantly increased; there was no significant difference between the two periods of sucrose in the roots; the T1, T2 and CK groups showed a gradual decrease in root protein and sucrose from S2 to S3. Compared with a CK group, the contents of ammonium nitrogen and nitrate nitrogen in roots in different periods are obviously improved after treatment by the compound bacteria T1 and T2. Compared with the compound microbial inoculum T2, the compound microbial inoculum T1 has the advantages that the ammonium nitrogen in S2 and S3 at different periods is obviously improved after treatment, the nitrate nitrogen in S2 period has no obvious difference, and the ammonium nitrogen in S3 period is obviously improved; ammonium and nitrate nitrogen declined gradually in roots from S2 to S3 in T1, T2 treatment and CK. The nitrate reductase is the first rate-limiting enzyme for catalyzing nitrate nitrogen to be reduced into ammonium nitrogen in plants, and compared with a CK group, the nitrate reductase activity in S2 and S3 stages is improved after treatment by using the compound bacteria T1 and T2. Compared with the complex inoculant T2, the complex inoculant T1 has no significant difference in the S2 period and is significantly improved in the S3 period after treatment.

TABLE 5 different times, different placesInfluence of physiological effects on nutrients in the root of Angelica sinensis

The ratios of different forms of nitrogen in the soil and plants at the S2 and S3 periods are shown in Table 6. Compared with the CK group, the compound bacteria T1 and T2 effectively convert soil ammonium nitrogen into nitrate nitrogen at different periods after treatment, and the ratio of the soil nitrate nitrogen/the ammonium nitrogen is changed from less than 1 to more than 1. The proportion of the soil nitrate nitrogen to the total quick-acting nitrogen of the soil after the treatment of the compound bacteria T1 and T2 is larger than that of the ammonium nitrogen, and the proportion of the soil nitrate nitrogen is gradually increased from S2 to S3; the proportion of CK soil nitrate nitrogen in the total quick-acting nitrogen of the soil is less than that of ammonium nitrogen, and the proportion of the CK soil nitrate nitrogen is gradually increased from S2 to S3. The proportion of plant ammonium nitrogen in total quick-acting nitrogen of the plants in different periods of time of T1, T2 and CK is obviously higher than that of nitrate nitrogen, and compared with CK group, the ratio of the plant nitrate nitrogen to the plant ammonium nitrogen is obviously improved after the treatment of the compound bacteria T1 and T2. The enrichment absorption degree of the plants on nitrate nitrogen and ammonium nitrogen is respectively expressed by the ratio of the plant nitrate nitrogen to the soil nitrate nitrogen and the ratio of the plant ammonium nitrogen to the soil ammonium nitrogen, compared with a CK group, the composite bacteria T1 and T2 can convert the soil ammonium nitrogen into the nitrate nitrogen after treatment, but also can obviously improve the activity of the plant nitrate reductase, so that the enrichment absorption of the nitrate nitrogen in the S2 stage is slightly lower than that of the CK group, the S3 stage is higher than that of the CK group, but the enrichment absorption of the ammonium nitrogen is obviously higher than that of the CK group; in the S2 stage, the enrichment absorption of the compound bacterial agent T1 on ammonium nitrogen is 2.12 times of CK, and the enrichment absorption of the compound bacterial agent T2 is 2.10 times of CK; the enrichment and absorption of the compound bacteria T1 to the ammonium nitrogen at the S3 stage is 1.91 times of CK, the enrichment and absorption of the compound bacteria T2 to the ammonium nitrogen is 1.74 times of CK, and the enrichment and absorption of the compound bacteria T1 to the ammonium nitrogen is improved by 9.8% compared with that of the compound bacteria T2. The results show that the compound microbial inoculum T1 can effectively promote the conversion of soil ammonium nitrogen into nitrate nitrogen and the conversion of plant nitrate nitrogen into ammonium nitrogen, and the effect is obviously better than that of the compound microbial inoculum T2.

TABLE 6 ratio of nitrogen in different forms

Note: different lower case letters in each row respectively represent multiple comparisons of corresponding indexes at different periods (P <0.05)

The results show that the compound microbial inoculum T1 can promote the conversion of ammonium nitrogen in rhizosphere soil into nitrate nitrogen, improve the quick-acting nitrogen content of the rhizosphere soil, and relieve the toxic action of high-concentration ammonium salt in the soil on plants through nitrification; the activity of nitrate reductase in the plant can be enhanced, the nitrate nitrogen in the plant can be promoted to be converted into ammonium nitrogen, the total value of the nitrate nitrogen and the ammonium nitrogen in the plant can be improved, the absorption and utilization of nitrogen can be promoted by regulating and controlling the conversion of nitrogen with different forms, and the yield of the plant can be improved.

Example 5 different processing data analysis and summary

The main component analysis of enzyme activity indexes of angelica rhizosphere soil at S2 and S3 periods is shown in figures 1 and 2, the load of a main component 1 at S2 period is 90.14%, and the load of a main component 2 is 8.21%; soil catalase contributes more to the main component 2, and other indexes contribute more to the main component 1; CK is mainly distinguished from T1 and T2 by main component 1, high soil alkaline phosphatase activity is the main characteristic of CK, and low soil catalase activity is the main characteristic of T2; in the S3 stage, the load of the main component 1 is 96.87%, and the load of the main component 2 is 2.12%; soil catalase, soil alkaline protease and soil FDA hydrolase contribute more to the main component 2, and other indexes contribute more to the main component 1; CK is mainly distinguished from T1 and T2 by a main component 1, high soil alkaline phosphatase activity is the main characteristic of CK, high soil alkaline protease activity is the main characteristic of T1, and high soil catalase and soil FDA hydrolase activity is the main characteristic of T2, but the load of the main component 2 is low, which shows that the difference between T1 and T2 is not very obvious.

The principal component analysis of nutrient indexes in the angelica rhizosphere soil and roots in the S2 and S3 periods is shown in FIGS. 3 and 4, the load of the principal component 1 in the S2 period is 73.23 percent, and the load of the principal component 2 is 22.72 percent; the cumulative variance contribution rate of the

principal components

1 and 2 is 95.95%; ammonium nitrogen in roots, nitrate nitrogen in roots, soil quick-acting potassium, soil nitrate nitrogen and soil ammonium nitrogen contribute more to the main component 1, and soil quick-acting phosphorus contributes more to the main component 2; CK is mainly distinguished from T1 and T2 by a main component 1, and the high content of soil ammonium nitrogen is the main characteristic of CK; t1 and T2 are mainly distinguished by principal component 2, the high ammonium nitrogen in roots being the main feature of T1. In the S3 period, the load of the principal component 1 is 74.92%, the load of the principal component 2 is 19.18%, and the cumulative variance contribution rate of the

principal components

1 and 2 is 94.1%; the contribution of different indexes to the principal component is in stage S2. CK is mainly distinguished from T1 and T2 by a main component 1, and the high content of soil ammonium nitrogen is the main characteristic of CK; t1 and T2 are mainly distinguished by main component 2, and the high content of ammonium nitrogen and soil quick-acting potassium in roots is the main characteristic of T1.

In conclusion, the treatment effect of the compound microbial agent in the experimental group T1 is better than that of the compound microbial agent in the positive control group T2 and better than that of the control group CK. On the basis of the original strain, the zoogloea cladosporium CBS4 is added, and the improved composite microbial inoculum has obvious yield increase effect on plant growth.

Sequence listing

<110> institute of biological research of science institute of Gansu province

<120> improved compound microbial agent and application thereof in yield increase of angelica sinensis

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 886

<212> DNA

<213> Pseudomonas sp (Pseudomonas sp)

<400> 1

cgctaactgc actctagcgg tcagaaactt gcttctcttg aagcggcgga cgggtgaata 60

atgcctacga atctgcctgg tagtggggga taacgttccg aaaccgacgc taataccgca 120

tacgtcctac gggagaaagc aggggacctt cgggccttgc gctatcagat gagcctaggt 180

cggattagct aggtggtgag gtaatggctc accaaggcga cgatccgtaa ctggtctgat 240

aggatgatca gtcacgctgg aactgagaca cggtccagac tcctacagga ggcaccagtg 300

gggaatattg gacgatgggc gaaagcctga tccagccatg ccgcgtgtgt gaagaaggtc 360

ttcggattgt aaagcacttt aagttgggag gaagggcagt tacctaatac gtgattgttt 420

tgacgttacc gacagaataa tcaccggcta actctgtgcc agcaaccgcg gtaatacaga 480

gggtgcgagc gttaatcgga attactgggc gtaaagcgcg cgtaggtggt ttgttaagtt 540

ggatgtgaaa tccccgggct caacctggga actgcattcg aaactgactg actacagtat 600

ggtagagggt ggtggaattt cctgtgtagc ggtgaaatgc ggaaatatat gaaggagcac 660

aagtggcaaa agcgagcacc tggactgata ctgacactga ggtgcgaaag cgtggggagc 720

aaactggatt agataccgtg gaagtccacg ccgtaaacga tgtcgactag gacgttggga 780

gccgtgagct cttagtggcg catctaacgc attgagttga cggcctgcgg agaacgggcc 840

gcaaggctag aaatcgaatg aattgactgg ggccggcaca agcgga 886

<210> 2

<211> 894

<212> DNA

<213> Pseudomonas sp (Pseudomonas sp)

<400> 2

gggtgtcacg ctacctgcag tcgagcggat gagtggagct tgctccatga ttcagcggcg 60

gacgggtgag taatgcctag gaatctgcct ggtagtgggg gacaacgttt cgaaaggaac 120

gctaataccg catacgtcct acgggagaaa gcaggggacc ttcgggcctt gcgctatcag 180

atgagcctag gtcggattag ctagttggtg aggtaaaggc tcaccaaggc gacgatccgt 240

aactggtctg agaggatgat cagtcacact ggaactgaga cacggtccag actcctacgg 300

gaggcagcag tggggaatat tggacaatgg gcgaaagcct gatccagcca tgccgcgtgt 360

gtgaagaagg tcttcggatt gtaaagcact ttaagttggg aggaagggca gtaagttaat 420

accttgctgt tttgacgtta ccgacagaat aagcaccggc taacttcgtg ccagcagccg 480

cggtaatacg aagggtgcaa gcgttaatcg gaattactgg gcgtaaagcg cgcgtaggtg 540

gttcagcaag ttggatgtga aagccccggg ctcaacctgg gaactgcatc caaaactact 600

gagctagagt acggtagagg gtggtggaat ttcctgtgta gcggtgaaat gcgtagatat 660

aggaaggaac accagtggcg aaggcgacca cctggactga tactgacact gaggtgcgaa 720

agcgtgggga gcaaacagga ttagataccc tggtagtcca cgccgtaaac gatgtcgact 780

agccgttggg atccttgaga tcttagtggc gaagctaacg cgataagtcg accgcctggg 840

gagtacggcc gcaaggttaa aactcaaatg aattgacggg ggcccgcaca agcg 894

<210> 3

<211> 939

<212> DNA

<213> Pseudomonas sp (Pseudomonas sp)

<400> 3

ggggttcacg cttcctgcac tcgagcggta gagagaagct tgcttctctt gagagcggcg 60

gacgggtgag taatgcctac gaatctgcct ggtagtgggg gataacgttc ggaaacggac 120

gctaataccg catacgtcct acgggagaaa gcaggggacc ttcgggcctt gcgctatcag 180

atgagcctag gtcggattag ctagttggtg aggtaatggc tcaccaaggc gacgatccgt 240

aactggtctg agaggatgat cagtcacgct ggaactgaga cacggtccaa actcctacgg 300

gaggcaccag tggggaatat tggacgatgg gcgaaagcct gatccagcca tgccgcgtgt 360

gtgaagaagg tcttcggatt gtaaagcact ttaagttggg aggaagggca gttacctaat 420

acgtgattgt tttgacgtta ccgacagaat aatcaccggc taactctgtg ccagcagccg 480

cggtaataca gagggtgcaa gcgttaatcg gaattactgg gcgtaaagcg cgcgtaggtg 540

gtttgttaag ttggatgtga aatccccggg ctcaacctgg gaactgcatt cgaaactgac 600

tgactacagt atggtagagg gtgatggaat ttcctgtgta ccggtgaaat gcgtacatat 660

atgaaggagc accagtggcg aaggcgacca cctggactga tactgacact gaggtgcgaa 720

agcgtgggga gcaacacgat tagataccgt ggaagtccac gccgtaaacg atgccaacta 780

cccggtggga gccgtgagct cttagtggcg cagctaacgc attatctcac cgcctgggga 840

gaacggccgc aggctagaac tcacatgaat tgacggggcc cgcacaagcg atgtaggatg 900

ggggtagttc taagcacgcg aagaccttac cggggttga 939

<210> 4

<211> 1202

<212> DNA

<213> Zoogloea sp

<400> 4

cgtactacgg ggtcgactgc ctccttgcgg ttagcgcatc gccttcgggt aaaccaactc 60

ccatggtgtg acgggcggtg tgtacaaggc ccgggaacgt attcaccgcg gcatgctgat 120

ccgcgattac tagcgattcc aacttcatgc actcgagttg cagagtgcaa tccgaactga 180

gatggctttt ggagattagc tcgacatcgc tgtctcgctg cccactgtca ccaccattgt 240

agcacgtgtg tagcccagcc cgtaagggcc atgaggactt gacgtcatcc ccaccttcct 300

ctcggcttat caccggcagt ccccttagag tgcccaacct aatgctggca actaagggcg 360

agggttgcgc tcgttgcggg acttaaccca acatctcacg acacgagctg acgacagcca 420

tgcagcacct gtgttcggtc cagcctaact gaaggaaaac atctctgtaa tccgcgaccg 480

acatgtcaag ggctggtaag gttctgcgcg ttgcttcgaa ttaaaccaca tgctccaccg 540

cttgtgcggg cccccgtcaa ttcctttgag ttttaatctt gcgaccgtac tccccaggcg 600

gaatgtttaa tgcgttagct gcgccaccga catgcatgca tgccgacggc taacattcat 660

cgtttacggc gtggactacc agggtatcta atcctgtttg ctccccacgc tttcgcacct 720

cagcgtcagt aatggaccag tgagccgcct tcgccactgg tgttcctccg aatatctacg 780

aatttcacct ctacactcgg aattccactc acctcttcca tactctaggt acccagtatc 840

aaaggcagtt ccagagttga gctctgggat ttcacccctg acttaaatac ccgcctacgt 900

gcgctttacg cccagtaatt ccgaacaacg ctagccccct tcgtattacc gcggctgctg 960

gcacgaagtt agccggggct tcttctccgg ttaccgtcat tatcttcacc ggtgaaagag 1020

ctttacaacc ctagggcctt catcactcac gcggcatggc tggatcaggc ttgcgcccat 1080

tggccatatt ccccactgct gcctcccgga ggagtttggg ccgggctcag ccccatgggg 1140

ctgtcatcct ctcagcccag tttgggtcgg cgccttggta ggcctttacc caccacttag 1200

ta 1202

Claims

1. An improved compound microbial agent, which is characterized by comprising pseudomonas fluorescens, pseudomonas alcaligenes, pseudomonas psychrophila and zoogloea cladinosa.

2. The complex microbial inoculant according to claim 1, which consists of pseudomonas fluorescens CBS5, pseudomonas alcaligenes CBS7, pseudomonas psychrophila CBSB and zoogloea cladinosa CBS 4; the 16S rDNA sequence of the pseudomonas fluorescens CBS5 is shown in SEQ ID NO. 1; the 16S rDNA sequence of the pseudomonas alcaligenes CBS7 is shown as SEQ ID NO.2, and the 16S rDNA sequence of the pseudomonas psychrophila CBSB is shown as SEQ ID NO. 3; the 16S rDNA sequence of the zoogloea cladosporium CBS4 is shown in SEQ ID No. 4.

3. Use of the complex microbial inoculant of claim 1 or 2 for promoting plant growth.

4. The use of the complex microbial inoculant of claim 1 or 2 for increasing the nitrate reductase activity of a plant.

5. The use of the complex microbial inoculant according to claim 1 or 2 for promoting the absorption of enriched ammonium nitrogen by plants.

6. The use according to any one of claims 3 to 5, wherein the plant is Angelica sinensis.

7. The use of the complex microbial inoculant according to claim 1 or 2 for promoting the conversion of soil ammonium nitrogen into nitrate nitrogen.

8. The use of the complex microbial inoculant according to claim 1 or 2 for increasing the content of available nitrogen in soil.

9. The use of the complex microbial inoculant according to claim 1 or 2 for the preparation of a nitrogen-fixing bacterial fertilizer.

10. A microbial fertilizer specially used for angelica sinensis, which is characterized by comprising the compound microbial agent of claim 1 or 2.