Compound microbial agent as well as preparation method and application thereof

Abstract

The invention discloses a compound microbial agent as well as a preparation method and application thereof. The compound microbial agent comprises a compound fungal agent and a compound bacterial agent, the compound fungus fungicide is prepared from Cunninghamella echinacea, Lachnum sp., Acremonium sp., Saccharomyces cerevisiae, Saccharomyces cerevisiae, Saccharomyces cerevisiae, Saccharomyces cerevisiae, Saccharomyces cerevisiae, Saccharomyces cerevisiae, Saccharomyces cerevisiae, Saccharomyces cerevisiae, Saccharomyces cerevisiae and Saccharomyces cerevisiae. The composite bacterial agent is prepared from Serratia sp., Pseudomonas fluorescens, Bacillus spp., Streptomyces ardesiacus, and Streptomyces sp., and the composite bacterial agent is prepared from the following raw materials: Serratia sp., Pseudomonas fluorescens, Bacillus spp., Streptomyces ardesiacus, and Streptomyces spp., and the composite bacterial agent is prepared from the following raw materials: Saccharomyces cerevisiae, Saccharomyces cerevisiae, Saccharomyces cerevisiae, Saccharomyces cerevisiae, Saccharomyces cerevisiae and Saccharomyces cerevisiae. The prepared compound microbial agent has a remarkable promoting effect on growth of artificially cultivated rhizoma atractylodis and accumulation of medicinal active ingredients, and the effectiveness of the compound microbial agent is proved in pot experiments and field experiments. The method is low in operation cost, has universality and is easy for large-scale production.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms and biofertilizers, and specifically relates to a composite microbial agent and a preparation method and application thereof. Background Art

[0002] Atractylodes is a perennial herb of the genus Atractylodes in the Asteraceae family. Its dried rhizomes are used as medicine. It has the effects of drying dampness and strengthening the spleen, dispelling wind and cold, and improving eyesight. It is mainly used to treat abdominal distension, diarrhea, edema, rheumatism, indigestion and other symptoms. In recent years, with the development of the traditional Chinese medicine industry, the market demand for Atractylodes has continued to grow. Due to the slow growth of wild Atractylodes and excessive excavation and habitat destruction, wild Atractylodes resources have become exhausted, but the market demand for Atractylodes medicinal materials is increasing, and the gap between supply and demand is widening year by year. Although artificial cultivation can alleviate the demand pressure of wild resources to a certain extent, the content and diversity of medicinal active ingredients in artificially cultivated Atractylodes are lower than those of authentic medicinal materials. The artificial cultivation system that guarantees both yield and quality needs further exploration and practice.

[0003] In agricultural production, microbial fertilizers have the characteristics of promoting growth and increasing production, and being green and environmentally friendly compared to traditional chemical fertilizers and agents. They are currently widely used in food and cash crops. Microbial agents are divided into single-bacteria agents and compound agents. Single-bacteria agents are made from one functional microorganism; compound agents are composed of two or more functional microorganisms. Traditional single-bacteria agents have problems such as single function and limited effect. Therefore, the development of compound agents has become a hot direction in the scientific development of biofertilizers. However, most of the current compound agents are composed of a single bacterial or fungal species, and still have problems such as poor actual effect and unstable performance. Summary of the invention

[0004] Purpose of the invention: The purpose of the present invention is to provide a composite microbial agent with good growth-promoting effect and significant accumulation of active ingredients; another purpose of the present invention is to provide a preparation method and application of the composite microbial agent.

[0005] Technical solution: The composite microbial agent of the present invention includes a composite fungal agent and a composite bacterial agent; the composite fungal agent includes Cunninghamella echinulate, Lachnum sp., and Acremonium sp.; the composite bacterial agent includes Serratia sp., Pseudomonas fluorescens, Bacillus sp., Streptomyces ardesiacus, and Streptomyces sp.

[0006] Preferably, in the composite microbial agent, the mass ratio of Cunninghamella echinulate: Lachnum sp.: Acremonium sp.: Serratia sp.: Pseudomonas fluorescens: Bacillus sp.: Streptomyces ardesiacus: Streptomyces sp. is (0.7-1.3): (0.7-1.3): (0.7-1.3): (0.8-1.2): (0.8-1.2): (0.8-1.2): (0.8-1.2): (0.8-1.2).

[0007] Preferably, Cunninghamella echinulate, Lachnum sp., Acremonium sp., Serratia sp., Pseudomonas fluorescens, Bacillus sp., Streptomyces ardesiacus, and Streptomyces sp. are isolated from the roots of Atractylodes lancea.

[0008] The method for preparing the composite microbial agent of the present invention comprises preparing the composite fungal agent and the composite bacterial agent separately:

[0009] (1) inoculating Lachnum sp. into a culture medium, inoculating Acremonium sp. after culturing for 72 to 96 hours, inoculating Cunninghamella echinulata after culturing for 24 to 48 hours, washing and resuspending the cells after culturing for 12 to 24 hours, and obtaining a composite fungal agent;

[0010] (2) Serratia sp., Pseudomonas fluorescens, and Bacillus sp. were inoculated into the culture medium respectively and cultured until the absorbance at OD600nm was 0.6 to 1.0. The cells were washed and resuspended to make the OD of each strain 600The value is maintained at a level of 0.8 to 1.0 to obtain a single bacterial suspension; marine Streptomyces (Streptomyces ardesiacus) and Streptomyces (Streptomyces sp.) are inoculated into the culture medium respectively, and after culturing for 36 to 48 hours, the bacteria are washed and resuspended to obtain a single bacterial suspension; the above single bacterial suspensions are mixed to obtain a composite bacterial agent.

[0011] Preferably, the specific method of step (1) is: inoculating Lachnum sp. into a PDA medium containing 1% bran by weight of the culture medium, inoculating Acremonium sp. after culturing in a shake flask at 28°C for 72 to 96 hours, inoculating Cunninghamella echinulata after culturing in a shake flask at 28°C for 24 to 48 hours, and washing and resuspending the bacteria with PBS after culturing in a shake flask at 28°C for 12 to 24 hours to obtain a composite fungal agent.

[0012] Preferably, the specific method of step (2) is: Serratia sp., Pseudomonas fluorescens, and Bacillus sp. are inoculated into LB medium respectively, and cultured in a shaking flask at 30°C until the absorbance at OD600nm is 0.6 to 1.0, and the bacteria are washed and resuspended with PBS to make the OD of each bacterial strain 600 The value is maintained at 0.8 to 1.0 to obtain a single bacterial suspension; Streptomyces ardesiacus and Streptomyces sp. are inoculated into LB medium respectively, and after 36 to 48 hours of shaking flask culture at 30°C, the bacteria are washed and resuspended with PBS to obtain a single bacterial suspension. The above single bacterial suspensions are mixed to obtain a composite bacterial agent.

[0013] Application of the composite microbial agent of the present invention in the cultivation of Atractylodes lancea.

[0014] Preferably, the composite microbial agent is applied to the roots of Atractylodes lancea.

[0015] Preferably, the specific application method is: applying the composite fungal agent 3 to 5 days after applying the composite bacterial agent.

[0016] Preferably, the cultivation of Atractylodes lancea includes the production of Atractylodes lancea seedlings or field planting;

[0017] During seedling production, the composite microbial agent is irrigated at the root, and the irrigating amount is 15-25 mL / plant; wherein, the wet weight of bacterial cells is 0.1-0.5 g / mL, and the wet weight of fungal hyphae is 0.01-0.03 g / mL; the composite microbial agent is irrigated again after 15-20 days, and the irrigating amount is 15-25 mL / plant;

[0018] During field planting, the composite microbial agent is irrigated at the roots at a rate of 90 to 110 mL per plant; wherein the wet weight of the bacterial cells is 0.1 to 0.5 g / mL, and the wet weight of the fungal hyphae is 0.01 to 0.03 g / mL; the composite microbial agent is irrigated again after 15 to 20 days at a rate of 90 to 110 mL per plant.

[0019] Further preferably, in seedling production, 6-month-old Atractylodes lancea tissue culture seedlings are selected for transplanting, and are further cultivated indoors for 1 to 2 months, and potted seedlings with similar growth are selected for testing, and the roots are watered with the composite microbial agent, and the irrigation amount is 15 to 25 mL / plant; wherein, the bacterial cells are 0.1 to 0.5 g / mL wet weight, and the fungal mycelium is 0.01 to 0.03 g / mL wet weight; the composite microbial agent is watered again every 15 to 20 days, and the irrigation amount is 15 to 25 mL / plant.

[0020] Further preferably, in field planting, germinated Atractylodes tubers are selected and planted in field soil, cultivated for 2 to 3 months, and the roots are irrigated with the composite microbial agent at a rate of 90 to 110 mL / plant; wherein the bacterial cells are 0.1 to 0.5 g / mL wet weight, and the fungal mycelium is 0.01 to 0.03 g / mL wet weight; the composite microbial agent is irrigated again every 15 to 20 days, and the rate of irrigation is 90 to 110 mL / plant.

[0021] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: the composite microbial agent of the present invention can significantly improve the growth and active ingredient accumulation of artificially cultivated Atractylodes lancea, overcoming the problems of poor effect and unstable performance of simple compounding of functional strains in plants in previous methods, and provides an effective composite microbial agent for improving the problem of low quality of artificially cultivated Atractylodes lancea; the present invention has low operating cost, universal applicability, and is easy to mass produce. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a diagram of the mutual antagonism of the strains in Example 1 of the present invention;

[0023] Figure 2 The growth of the Atractylodes lancea potted plants treated with a single bacterium in Example 1 of the present invention;

[0024] Figure 3 This is a diagram showing the accumulation of medicinal active ingredients in potted Atractylodes lancea treated with a single bacterium according to Example 1 of the present invention;

[0025] Figure 4 The phylogenetic tree constructed based on 16SrRNA and 18SrRNA gene sequences of each strain in Example 1 of the present invention is as follows: A is Cunninghamella echinulata; B is Lachnum sp.; C is Acremonium sp.; D is Serratiasp.; E is Pseudomonasfluorescens; F is Bacillus sp.; G is Streptomyces ardesiacus; H is Streptomyces sp.;

[0026] Figure 5 This is a graph showing the ability of each strain in Example 2 of the present invention to fix nitrogen, solubilize phosphorus, solubilize potassium, produce siderophore, produce ACC deaminase, and produce IAA;

[0027] Figure 6 The growth and accumulation of active medicinal ingredients of potted Atractylodes lancea treated with different combinations of composite microbial agents in Example 3 of the present invention are shown in FIG.

[0028] Figure 7 This is a diagram showing the accumulation of primary metabolites and important precursor substances in potted Atractylodes lancea treated with different groups of composite microbial agents in Example 3 of the present invention;

[0029] Figure 8 This is a diagram showing the photosynthesis of field Atractylodes lancea treated with the composite microbial agent in Example 4 of the present invention;

[0030] Fig. 9 This is a graph showing the growth of the medicinal parts and the accumulation of medicinal active ingredients of the field Atractylodes lancea treated with the composite microbial agent in Example 4 of the present invention. DETAILED DESCRIPTION

[0031] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0032] The present invention combines microbiome sequencing technology with pure culture separation and screening of fungi and bacteria, selects strains with functional advantages for combination, and screens them through pot experiments to obtain a highly efficient and stable composite microbial agent. Finally, pot and field experiments are used to verify the actual growth-promoting and active ingredient accumulation effects of the composite microbial agent.

[0033] The composite microbial agent of the present invention includes composite fungal agents and composite bacterial agents; the composite fungal agent includes Cunninghamella echinulate, Lachnum sp., and Acremonium sp.; the composite bacterial agent includes Serratia sp., Pseudomonas fluorescens, Bacillus sp., Streptomyces ardesiacus, and Streptomyces sp. It can be separated from the root of Atractylodes lancea.

[0034] Example 1

[0035] Isolation, screening and identification of test strains

[0036] 1. Isolation, purification and cultivation of test strains

[0037] The test strain was isolated from the root tissue of Atractylodes lancea in the Botanical Garden of Nanjing Normal University. It is a key species in the endogenous microbial community of healthy Atractylodes lancea and is used for the screening of functional strains.

[0038] Test medium

[0039] Starch casein solid culture medium (SCA): 5.0 g glucose, 5.0 g soluble starch, 2.0 g acid hydrolyzed casein, 2.0 g calcium carbonate, 1.0 g yeast powder, 15-20 g agar, distilled water to 1 L, pH = 7.2±0.2.

[0040] TWYE solid culture medium: 0.5 g potassium dihydrogen phosphate, 0.25 g yeast extract, 15-20 g agar, distilled water to 1 L, pH = 7.2±0.2.

[0041] R2A solid culture medium: 0.5 g yeast extract powder, 0.5 g peptone, 0.5 g casein hydrolysate, 0.5 g glucose, 0.5 g soluble starch, 0.3 g dipotassium hydrogen phosphate, 0.3 g sodium pyruvate, 0.024 g anhydrous magnesium sulfate, 15-20 g agar, distilled water to 1 L, pH = 7.2±0.2.

[0042] Gao's solid medium No. 1 (GS): soluble starch 20.0g, potassium nitrate 1.0g, sodium chloride 0.5g, potassium dihydrogen phosphate trihydrate 0.5g, magnesium sulfate heptahydrate 0.5g, ferrous sulfate heptahydrate 0.01g, agar 15-20g, distilled water to 1L, pH = 7.2±0.2.

[0043] LB medium: 10 g of tryptone, 5 g of yeast extract, 5 g of sodium chloride, 1 mL of 1 mol / L sodium hydroxide. If preparing a solid medium, add 15-20 g of agar, and distilled water to 1 L, pH = 7.0 ± 0.2.

[0044] PDA culture medium: 200 g potato, 20 g glucose. If preparing solid culture medium, add 15-20 g agar, distilled water to 1 L, pH = 7.0 ± 0.2.

[0045] 2. Evaluation of strain antagonism

[0046] An important criterion for preparing composite microbial agents is that there is no obvious antagonism between strains and that they can establish a stable coexistence relationship. Therefore, in order to verify whether the isolated endophytic strains can coexist in a friendly manner, the bacterial strains were subjected to a plate antagonism experiment based on the filter paper method, and the fungal strains were subjected to a bacterial cake plate antagonism experiment.

[0047] The bacterial strains were activated and cultured in LB medium until the absorbance at OD600nm was 0.6-1.0, and the cells were washed and resuspended in 0.9% sterile saline to make the OD of each bacterial strain 600 The value is maintained at 0.8-1.0 to obtain a single bacterial suspension. Use a sterilized cotton swab to dip the single bacterial suspension and spread it on the LB solid plate. Rotate 120° each time you spread it. Repeat 3 times for 3 plates. After the plate is dried, use sterile tweezers to clamp the sterile filter paper, absorb the bacterial suspensions other than the bacteria spread on the plate, spread the filter paper flat on the plate, and culture at 30℃ for 48-72h to observe whether there is an inhibition zone formed around the filter paper.

[0048] Use a hole puncher to punch the fungal strains on the PDA solid culture medium, divide the plate equally, place the punched fungal cakes in their respective areas, repeat on 3 plates, culture at 28℃ for 7-14 days, and observe whether there is an inhibition zone formed between the fungal cakes.

[0049] 3. Effect of single bacteria treatment on Atractylodes macrocephala potted plants

[0050] According to the results of strain antagonism experiments, strains without obvious antagonistic effects were selected as candidate strains for the preparation of composite microbial agents, and the effect of single-strain treatment on Atractylodes lancea potted plants was first investigated.

[0051] (1) Preparation and application of single bacterial inoculum

[0052] Bacteria: Inoculate the bacterial strains into LB medium, shake flask culture at 30℃ until the absorbance at OD600nm is 0.6-1.0, centrifuge at 8000rpm, discard the supernatant, wash and resuspend the bacteria with sterile PBS, and adjust the OD of each bacterial strain to 600The value was maintained at 0.8-1.0. For bacteria that produced flocculent substances in shake flasks, the flasks were cultured uniformly for 36-48 hours, centrifuged at 8000 rpm, the supernatant was discarded, and the bacteria were washed and resuspended with sterile PBS to obtain a single bacterial suspension.

[0053] Fungi: The fungal strains were inoculated into PDA medium containing 1% bran by mass of the culture medium (in order to prevent the mycelium from forming balls in the shaking flask, which would make the prepared composite fungal agent uneven, an appropriate amount of bran was added to the PDA medium. The bran served as a dispersion medium and could cut the mycelium into mycelium balls of uniform size). After culturing in shaking flasks at 28°C for 2 to 7 days, the mycelium was centrifuged at 8000 rpm, the supernatant was discarded, and the mycelium was washed and resuspended with sterile PBS to obtain a single fungal suspension.

[0054] The 6-month-old Atractylodes lancea tissue culture seedlings were selected for transplanting, and continued to be cultivated indoors for 1-2 months. Potted seedlings with similar growth were selected for the test, and the composite microbial agent was irrigated at the roots, with an irrigation amount of 15-25 mL / plant; among them, the bacterial cells were 0.1-0.5 g / mL wet weight, and the fungal hyphae were 0.01-0.03 g / mL wet weight; the composite microbial agent was irrigated again after 15-20 days, with an irrigation amount of 15-25 mL / plant. The same watering amount was maintained during the later management process, and watering was performed every 3-4 days. After 30 days, the growth-promoting effect of Atractylodes lancea and the accumulation of medicinal active ingredients were counted.

[0055] (2) Determination of agronomic characteristics and content of medicinal active ingredients in Atractylodes lancea

[0056] Three Atractylodes lancea plants were randomly selected from each treatment group to measure their agronomic traits such as fresh weight and root fresh weight, as well as the contents of medicinal active ingredients such as sesquiterpenes and atractylodesin. Agronomic traits were measured manually, by plant phenotyping system, and by plant root scanner; medicinal active ingredients were measured by gas chromatography (GC).

[0057] 4. Identification of test strains

[0058] Finally, three strains of endophytic fungi and five strains of endophytic bacteria were determined as functional strains for preparing composite microbial agents. In order to molecularly identify the eight strains, their 16SrRNA and 18SrRNA genes were PCR amplified, and the successfully amplified PCR products were sent to General Biotechnology (Anhui) Co., Ltd. for sequencing. The obtained sequences were compared with the homologous sequences in Genbank by NCBI-Blast. The 16SrRNA and 18SrRNA gene sequences of typical strains with relatively high homology were downloaded from Genbank, and sequence comparison analysis was performed using MEGA11.0 software. Finally, the phylogenetic tree was constructed and analyzed using the Neighbor-Joining (NJ) method.

[0059] 5. Results and Analysis

[0060] (1) Evaluation of strain antagonism

[0061] Figure 1 Figure 2 is a diagram of the antagonistic effects of various strains. Figure 1 As shown in the figure, through the plate confrontation experiment, it was found that no obvious inhibition zone was observed between the three fungi and the 10 bacteria, indicating that there was no obvious antagonism between them, and the subsequent co-inoculation experiment could be carried out. The 13 strains were named: S609001, S609002, S609003, S609005, S609006, S609007, S609008, S609009, S609010, S609011, S609012, S609013, S609014.

[0062] (2) Effects of single bacterial treatment on the growth of potted Atractylodes lancea and the accumulation of medicinal active ingredients

[0063] Figure 2 , Figure 3 The growth and accumulation of active medicinal ingredients of Atractylodes lancea potted plants treated with single bacteria are shown respectively. Figure 2 , Figure 3 As shown, 3 fungi (S609009, S609010, S609011) and 5 bacteria (S609001, S609005, S609006, S609007, S609008) can effectively promote the growth of Atractylodes lancea potted plants and the accumulation of medicinal active ingredients to varying degrees. Therefore, these 8 strains were used as candidate strains for the preparation of composite microbial agents.

[0064] (3) Phylogenetic analysis of the tested strains

[0065] Figure 4 The phylogenetic trees of each strain were constructed based on the 16SrRNA and 18SrRNA gene sequences. S609009 was identified as Cunninghamella spp. Figure 4 A), and its taxonomic name is Cunninghamella echinulata; S609010 was identified as Trichophyton Figure 4 B), classified as Lachnum sp.; S609011 was identified as Acremonium sp. Figure 4 C), and its classification name is Acremonium sp.; S609001 was identified as Serratia ( Figure 4 D), and the classification name is Serratia sp.; S609005 was identified as Pseudomonas fluorescens ( Figure 4E), and its classification name is Pseudomonas fluorescens; S609006 was identified as Bacillus ( Figure 4 F), and the classification name is Bacillus sp.; S609008 was identified as marine Streptomyces ( Figure 4 G), and its classification name is Streptomyces ardesiacus; S609007 was identified as Streptomyces ( Figure 4 H), the classification name is Streptomycess p.

[0066] Example 2

[0067] Capacity determination of test strains

[0068] 1. Determination of nitrogen fixation ability of test strains

[0069] Ashuber solid culture medium: mannitol 20.0 g, calcium carbonate 5.0 g, potassium dihydrogen phosphate 0.2 g, magnesium sulfate 0.2 g, sodium chloride 0.2 g, potassium sulfate 0.1 g, agar 15.0 g, distilled water to 1 L, pH = 7.4 ± 0.2.

[0070] 2. Determination of the solubility of organic phosphorus and inorganic phosphorus of the test strains

[0071] Inorganic phosphorus solid culture medium: glucose 10.0 g, calcium phosphate 5.0 g, magnesium sulfate heptahydrate 0.25 g, potassium chloride 0.2 g, ammonium sulfate 0.1 g, agar 15.0 g, distilled water to 1 L, pH = 7.0 ± 0.2.

[0072] Organophosphorus solid culture medium: glucose 10.0 g, sodium phytate 5.0 g, magnesium sulfate heptahydrate 0.25 g, potassium chloride 0.2 g, ammonium sulfate 0.1 g, agar 15.0 g, distilled water to 1 L, pH = 7.0 ± 0.2.

[0073] 3. Determination of potassium solubilization ability of test strains

[0074] Potassium-dissolving solid culture medium: 5.0 g glucose, 2.0 g disodium hydrogen phosphate, 0.5 g ammonium sulfate, 0.5 g yeast powder, 0.3 g magnesium sulfate, 2.0 g disodium hydrogen phosphate, 0.03 g ferrous sulfate, 0.03 g manganese sulfate, 2.0 g potassium feldspar, 15 g agar, distilled water to 1 L, pH = 7.2 ± 0.2.

[0075] 4. Determination of the ability of the test strain to produce siderophore

[0076] CAS solid culture medium: CAS 0.06 g, hexadecyltrimethylammonium bromide 0.07 g, ferric chloride hexahydrate 0.003 g, potassium dihydrogen phosphate dihydrate 0.3 g, disodium hydrogen phosphate dodecahydrate 1.2 g, ammonium chloride 0.13 g, potassium dihydrogen phosphate 0.04 g, sodium chloride 0.06 g, agar 9 g, distilled water to 1 L, pH = 6.8 ± 0.1.

[0077] The strain culture method for testing the ability of nitrogen fixation, phosphorus solubilization, potassium solubilization and iron carrier production of the test strain is:

[0078] Bacteria: Inoculate the bacteria into LB medium and culture in shake flasks at 30℃ until the absorbance at OD600nm is 0.8-1.0; for bacteria that produce flocculent substances in shake flasks, culture in shake flasks for 36-48h, pipette 2μL of bacterial liquid and drop it on the medium, repeat for 3 plates, culture at 30℃ for 7-14d, and observe whether there is a transparent circle formed around the colony.

[0079] Fungi: Use a hole puncher to take the fungal strains on the PDA solid culture medium and place them on the culture medium. Repeat for 3 plates and culture at 28°C for 7 to 14 days. Observe whether there is a transparent circle formed around the colony.

[0080] 5. Determination of the ability of the test strain to produce ACC deaminase

[0081] The assay was performed according to the instructions of the ACC deaminase enzyme-linked immunosorbent assay kit (Jingmei Biotechnology Co., Ltd, China).

[0082] 6. Determination of IAA production ability of test strains

[0083] Bacteria: Inoculate bacteria into LB medium, culture in a shake flask at 30°C until the logarithmic growth phase, centrifuge at 8000rpm, discard the supernatant, wash and resuspend the bacteria with sterile PBS, and adjust the OD of each strain to 600 The value was maintained at 0.5-0.8; 100 μL of the bacterial suspension was added to 10 mL of LB medium containing L-tryptophan (100 mg / L), and cultured in a shake flask at 30°C for 24 hours. The culture was centrifuged at 8000 rpm, and 100 μL of the supernatant was placed in a 96-well plate. An equal volume of Salkowski colorimetric solution was added, and the mixture was mixed and incubated in the dark for 30 minutes. The color turned red, indicating that the strain could produce IAA.

[0084] Fungi: Inoculate the fungi into PDA medium containing L-tryptophan (100 mg / L), culture in a shake flask at 28°C for 2 to 7 days, centrifuge at 8000 rpm, take 100 μL of the supernatant into a 96-well plate, add an equal volume of Salkowski colorimetric solution, mix well, incubate in the dark for 30 minutes, and a red color indicates that the strain can produce IAA.

[0085] 7. Results and Analysis

[0086] Figure 5 The results are the test results of the ability of each strain to fix nitrogen, dissolve phosphorus, dissolve potassium, produce iron carriers, produce ACC deaminase, and produce IAA. Figure 3 As shown in the figure, they have more than three growth-promoting phenotypes. Cunninghamella echinulata has the ability to dissolve organic phosphorus, produce siderophores, produce ACC deaminase, and produce IAA. Serratia sp. has the ability to fix nitrogen, dissolve organic phosphorus, produce siderophores, and produce ACC deaminase. Lachnum sp., Acremonium sp., and Pseudomonas fluorescens have the ability to dissolve organic phosphorus, produce siderophores, and produce ACC deaminase. Bacillus sp., Streptomyces ardesiacus, and Streptomyces sp. have the ability to fix nitrogen, dissolve organic phosphorus, and produce ACC deaminase.

[0087] Example 3

[0088] Construction and screening of composite microbial agents

[0089] 1. Potted experiment design

[0090] The potted plant experiment selected untreated control group (Mock), compound bacterial agent group (Bacteria), compound fungal agent group (Fungi), compound microbial agent group 1 (Mixture1), compound microbial agent group 1-1

[0091] (Mixture 1-1), compound microbial agent group 1-2 (Mixture 1-2) and compound microbial agent group 2

[0092] (Mixture2), a total of 7 treatment groups, see Table 1 for details.

[0093] Table 1 Treatment group

[0094]

[0095] Composite fungal agent group (Fungi): Lachnum sp. was inoculated into PDA medium containing 1% bran of the mass of culture medium (in order to prevent the mycelium from forming balls in the shaking flask, which would cause the prepared composite fungal agent to be uneven, an appropriate amount of bran was added to the PDA medium, and the bran was used as a dispersion medium to cut the mycelium into mycelium balls of uniform size.), and then Acremonium sp. was inoculated after 72 to 96 hours of shaking flask culture at 28°C, and then Cunninghamella echinulata was inoculated after 24 to 48 hours of co-culture at 28°C, and then the mycelium was washed and resuspended with PBS after 12 to 24 hours of co-culture at 28°C to obtain the composite fungal agent. Among them, the mass ratio of Cunninghamella echinulate: Lachnum sp.: Acremonium sp. was 1:1:1.

[0096] Composite bacterial agent group (Bacteria): Serratia sp., Pseudomonas fluorescens, and Bacillus sp. were inoculated into LB medium respectively, and cultured in a shaking flask at 30°C until the absorbance at OD600nm was 0.6-1.0. The bacteria were washed and resuspended with PBS to make the OD of each bacterial strain 600 The value was maintained at 0.8-1.0 to obtain a single bacterial suspension; two endophytic Streptomyces, Streptomyces ardesiacus and Streptomyces sp., were inoculated into LB medium respectively, and after 36-48 hours of shaking flask culture at 30°C, the bacteria were washed and resuspended with PBS to obtain a single bacterial suspension. The above single bacterial suspensions were mixed in a certain mass ratio to obtain a composite bacterial agent. Among them, the mass ratio of Serratia sp.: Pseudomonasfluorescens: Bacillus sp.: Streptomyces ardesiacus: Streptomyces sp. was 1:1:1:1:1.

[0097] Mixture 1: 3 to 5 days after the application of the composite fungal agent (Fungi), the composite bacterial agent (Bacteria) was applied. Among them, the mass ratio of Cunninghamella echinulate: Lachnum sp.: Acremonium sp.: Serratia sp.: Pseudomonas fluorescens: Bacillus sp.: Streptomyces ardesiacus: Streptomyces sp. was 1:1:1:1:1:1:1:1:1.

[0098] Mixture 2: 3 to 5 days after the application of the compound fungal agent (Fungi), a single bacterial suspension of Pseudomonas fluorescens was applied. The mass ratio of Cunninghamella echinulate: Lachnum sp.: Acremonium sp.: Pseudomonas fluorescens was 1:1:1:5.

[0099] Mixture 1-1: 3 to 5 days after the application of the compound fungal agent (Fungi1-1), the compound bacterial agent (Bacteria1-1) was applied. The preparation method of the composite fungal agent (Fungi1-1) is the same as that of the composite fungal agent group (Fungi), and the preparation method of the composite bacterial agent (Bacteria1-1) is the same as that of the composite bacterial agent group (Bacteria), wherein the mass ratio of Cunninghamella echinulate: Lachnum sp.: Acremonium sp.: Serratia sp.: Pseudomonasfluorescens: Bacillus sp.: Streptomyces ardesiacus: Streptomyces sp. is 0.7:1.1:1.3:1.2:0.8:0.9:1.2:1.1.

[0100] Mixture 1-2: 3 to 5 days after the application of the compound fungal agent (Fungi 1-2), the compound bacterial agent (Bacteria 1-2) was applied. The preparation method of the composite fungal agent (Fungi1-2) is the same as that of the composite fungal agent group (Fungi), and the preparation method of the composite bacterial agent (Bacteria1-2) is the same as that of the composite bacterial agent group (Bacteria), wherein the mass ratio of Cunninghamella echinulate: Lachnum sp.: Acremonium sp.: Serratia sp.: Pseudomonasfluorescens: Bacillus sp.: Streptomyces ardesiacus: Streptomyces sp. is 1.3:1.3:0.7:0.8:1.2:1.1:0.9:1.0.

[0101] The 6-month-old Atractylodes lancea tissue culture seedlings were selected for transplanting, and continued to be cultivated indoors for 1-2 months. Potted seedlings with similar growth were selected for the test, and the roots were irrigated with the composite microbial agent at a rate of 15-25 mL / plant; wherein the bacterial cells were 0.1-0.5 g / mL wet weight, and the fungal hyphae were 0.01-0.03 g / mL wet weight; the composite microbial agent was irrigated again after 15-20 days, and the irrigating amount was 15-25 mL / plant. The control group was irrigated with an equal amount of sterile PBS.

[0102] 2. Determination of agronomic characteristics and active ingredient content of Atractylodes lancea

[0103] Three Atractylodes lancea plants were randomly selected from each treatment group to measure their agronomic traits such as fresh weight and root fresh weight, as well as the contents of medicinal active ingredients such as sesquiterpenes and atractylodesin. Agronomic traits were measured manually, by plant phenotyping system, and by plant root scanner; medicinal active ingredients were measured by gas chromatography (GC).

[0104] 3. Determination of primary metabolites and important precursors of Atractylodes lancea

[0105] Three Atractylodes lancea plants were randomly selected from each treatment group to determine the contents of primary metabolites such as soluble sugar and chlorophyll, as well as important precursor substances such as pyruvate and acetyl-CoA.

[0106] (1) Determination of soluble sugar content

[0107] Weigh 0.1g of fresh tissue of Atractylodes lancea, add 2mL of double distilled water, grind, boil in water bath for 30min, cool and centrifuge at 8000rpm for 10min to obtain supernatant. Take 0.05mL of supernatant, add 0.95mL of double distilled water, then add 5mL of anthrone reagent, shake well, boil in water bath for 10min, immediately cool to room temperature with cold water, detect absorbance at OD625nm wavelength, and calculate soluble sugar content per unit mass according to glucose standard curve.

[0108] (2) Determination of chlorophyll content

[0109] Take fresh leaves of Atractylodes lancea, wash them, cut them into pieces, remove the veins, weigh 0.1g, add a small amount of calcium carbonate powder, silicon dioxide, and 2-3mL 95% anhydrous ethanol, grind them into a homogenate, add 10mL ethanol and continue grinding until the tissue turns white, let it stand for 3-5min, filter with qualitative filter paper, rinse the mortar and filter paper with ethanol several times, wash as much chlorophyll as possible into a volumetric flask, make up to 25mL in a brown volumetric flask, shake well, detect the absorbance at OD649nm and 665nm wavelengths, and calculate the chlorophyll content per unit mass according to the formula.

[0110] (3) Determination of pyruvic acid content

[0111] Weigh 0.1 g of fresh tissue of Atractylodes lancea, add 2.5 mL of 8% trichloroacetic acid, grind thoroughly, let stand for 30 min, centrifuge at 4°C 10000 rpm for 5 min to obtain the supernatant. Take 1.0 mL of the supernatant, add 2.0 mL of 8% trichloroacetic acid and 1.0 mL of 0.1% 2,4-dinitrophenylhydrazine, shake well, and then add 5.0 mL of 1.5 mol L -1 Sodium hydroxide solution, shake well, color reaction for 10 minutes, detect the absorbance at OD520nm wavelength, and calculate the pyruvic acid content per unit mass according to the pyruvic acid standard curve.

[0112] (4) Determination of acetyl-CoA content

[0113] The assay was performed according to the instructions of the acetyl-CoA enzyme-linked immunosorbent assay kit (Jingmei Biotechnology Co., Ltd, China).

[0114] 4. Results and Analysis

[0115] Figure 6 The growth and accumulation of active medicinal ingredients of potted Atractylodes lancea treated with different groups of composite microbial agents. Figure 6As shown, the compound microbial agent group 1 (Mixture1), the compound microbial agent group 1-1 (Mixture1-1), and the compound microbial agent group 1-2 (Mixture1-2) can simultaneously promote the growth of Atractylodes lancea potted plants and the accumulation of medicinal active ingredients. Therefore, the compound microbial agent group 1 (Mixture1) is selected as the best option.

[0116] Figure 7 The accumulation of primary metabolites and important precursor substances in potted Atractylodes lancea treated with different groups of composite microbial agents. Figure 7 As shown, the composite microbial agent group 1 (Mixture 1) can effectively promote the accumulation of soluble sugar and chlorophyll in potted Atractylodes lancea, which is beneficial to improve the stress resistance and photosynthetic capacity of Atractylodes lancea and promote plant growth; on the contrary, the content of pyruvate and acetyl-CoA, important precursor substances required for the synthesis of medicinal active ingredients of Atractylodes lancea, is significantly reduced, which indicates that the composite microbial agent group 1 (Mixture 1) can effectively promote Atractylodes lancea to utilize pyruvate and acetyl-CoA to produce medicinal active substances, which is beneficial to the accumulation of medicinal active ingredients of Atractylodes lancea.

[0117] Example 4 Application of composite microbial inoculants in field planting of Atractylodes lancea

[0118] 1. Field Experiment Design of Atractylodes lancea

[0119] The potted experiment selected two treatment groups: an untreated control group (Mock) and a composite microbial agent group 1 (Mixture 1), as shown in Table 2 for details.

[0120] Table 2 Treatment group grouping

[0121]

[0122] The germinated tubers of Atractylodes lancea were selected and planted in field soil, cultivated for 2 to 3 months, and the roots were irrigated with the composite microbial agent at a rate of 90 to 110 mL / plant; wherein the bacterial cells were 0.1 to 0.5 g / mL wet weight, and the fungal hyphae were 0.01 to 0.03 g / mL wet weight; the composite microbial agent was irrigated again after 15 to 20 days at a rate of 90 to 110 mL / plant. The control group was irrigated with an equal amount of sterile PBS.

[0123] 2. Determination of photosynthetic indexes of Atractylodes lancea

[0124] (1) Determination of chlorophyll content

[0125] g Take fresh leaves of Atractylodes lancea, wash, cut into pieces, remove the veins, weigh 0.1g, add a small amount of calcium carbonate powder, silicon dioxide, 2-3mL95% anhydrous ethanol, grind into a homogenate, add 10mL of ethanol and continue grinding until the tissue turns white, let it stand for 3-5min, filter with qualitative filter paper, rinse the mortar and filter paper with ethanol several times, wash as much chlorophyll as possible into a volumetric flask, make up to 25mL in a brown volumetric flask, shake well, detect the absorbance at OD649nm and 665nm wavelengths, and calculate the chlorophyll content per unit mass according to the formula.

[0126] (2) Determination of leaf photosynthetic parameters

[0127] The CIRAS-4 portable photosynthesis measurement system was used to measure the leaf photosynthetic parameters of Atractylodes lancea, including A (net photosynthetic rate), E (transpiration rate), and gs (stomatal conductance).

[0128] 3. Growth of medicinal parts of Atractylodes lancea and determination of active ingredient content

[0129] Six Atractylodes lancea plants were randomly selected from each treatment group, and their root fresh weight and the content of medicinal active ingredients such as sesquiterpenes and atractylodesin were determined. The growth of medicinal parts was measured based on the root fresh weight, and the medicinal active ingredients were determined by gas chromatography (GC).

[0130] 4. Results and Analysis

[0131] Figure 8 This is the photosynthesis of Atractylodes lancea in the field treated with compound microbial agents. Figure 8 As shown, the composite microbial agent group 1 (Mixture 1) can significantly improve A (net photosynthetic rate), E (transpiration rate) and gs (stomatal conductance) of Atractylodes lancea in the field, which is beneficial to improving the photosynthetic capacity of Atractylodes lancea and promoting plant growth.

[0132] Fig. 9 The growth of medicinal parts and accumulation of medicinal active ingredients of Atractylodes lancea in the field treated with composite microbial agents. Fig. 9 As shown, the composite microbial agent group 1 (Mixture 1) can significantly promote the growth of medicinal active parts of Atractylodes lancea in the field and the accumulation of medicinal active ingredients.

Claims

1. A composite microbial agent, characterized in that: The composite microbial agent includes a composite fungal agent and a composite bacterial agent; the composite fungal agent includes Cunninghamella echinulate, Lachnum sp., and Acremonium sp.; the composite bacterial agent includes Serratia sp., Pseudomonas fluorescens, Bacillus sp., Streptomyces ardesiacus, and Streptomyces sp.

2. The composite microbial agent according to claim 1, characterized in that: In the composite microbial agent, the mass ratio of Cunninghamella echinulate: Lachnum sp.: Acremonium sp.: Serratia sp.: Pseudomonas fluorescens: Bacillus sp.: Streptomyces ardesiacus: Streptomyces cess p. is (0.7-1.3): (0.7-1.3): (0.7-1.3): (0.8-1.2): (0.8-1.2): (0.8-1.2): (0.8-1.2): (0.8-1.2).

3. The composite microbial agent according to claim 1, characterized in that: Cunninghamella echinulate, Lachnum sp., Acremonium sp., Serratia sp., Pseudomonas fluorescens, Bacillus sp., Streptomyces ardesiacus, and Streptomyces sp. were isolated from the roots of Atractylodes lancea.

4. A method for preparing the composite microbial agent according to claim 1, characterized in that: The method comprises preparing a composite fungal inoculant and a composite bacterial inoculant respectively: (1) inoculating Lachnum sp. into a culture medium, inoculating Acremonium sp. after culturing for 72 to 96 hours, inoculating Cunninghamella echinulata after culturing for 24 to 48 hours, washing and resuspending the cells after culturing for 12 to 24 hours, and obtaining a composite fungal agent; (2) Serratia sp., Pseudomonas fluorescens, and Bacillus sp. were inoculated into the culture medium respectively and cultured until the absorbance at OD600nm was 0.6 to 1.

0. The cells were washed and resuspended to make the OD of each strain 600 The value is maintained at a level of 0.8 to 1.0 to obtain a single bacterial suspension; marine Streptomyces (Streptomyces ardesiacus) and Streptomyces (Streptomyces sp.) are inoculated into the culture medium respectively, and after culturing for 36 to 48 hours, the bacteria are washed and resuspended to obtain a single bacterial suspension; the above single bacterial suspensions are mixed to obtain a composite bacterial agent.

5. The method for preparing the composite microbial agent according to claim 4, characterized in that: The specific method of step (1) is as follows: Lachnum sp. is inoculated into a PDA medium containing 1% bran by weight of the culture medium, and then Acremonium sp. is inoculated after culturing in a shaking flask at 28° C. for 72 to 96 hours, and then Cunninghamella echinulata is inoculated after culturing in a shaking flask at 28° C. for 24 to 48 hours, and then the bacterial cells are washed with PBS and resuspended after culturing in a shaking flask at 28° C. for 12 to 24 hours to obtain a composite fungal agent.

6. The method for preparing the composite microbial agent according to claim 4, characterized in that: The specific method of step (2) is: Serratia sp., Pseudomonas fluorescens, and Bacillus sp. are inoculated into LB medium respectively, and cultured in a shaking flask at 30°C until the absorbance at OD600nm is 0.6-1.0, and the bacteria are washed and resuspended with PBS to make the OD of each bacterial strain 600 The value is maintained at 0.8 to 1.0 to obtain a single bacterial suspension; Streptomyces ardesiacus and Streptomyces sp. are inoculated into LB medium respectively, and after 36 to 48 hours of shaking flask culture at 30°C, the bacteria are washed and resuspended with PBS to obtain a single bacterial suspension. The above single bacterial suspensions are mixed to obtain a composite bacterial agent.

7. Use of the composite microbial agent according to claim 1 in cultivating Atractylodes lancea.

8. The use according to claim 7, characterized in that: Apply the composite microbial agent to the roots of Atractylodes lancea.

9. The use according to claim 8, characterized in that: The specific application method is: 3 to 5 days after applying the composite fungal agent, apply the composite bacterial agent.

10. The use according to claim 7, characterized in that: The cultivation of Atractylodes lancea includes the production of Atractylodes lancea seedlings or field planting; During seedling production, the composite microbial agent is irrigated at the root, and the irrigating amount is 15-25 mL / plant; wherein, the wet weight of bacterial cells is 0.1-0.5 g / mL, and the wet weight of fungal hyphae is 0.01-0.03 g / mL; the composite microbial agent is irrigated again after 15-20 days, and the irrigating amount is 15-25 mL / plant; During field planting, the composite microbial agent is irrigated at the roots at a rate of 90 to 110 mL per plant; wherein the wet weight of the bacterial cells is 0.1 to 0.5 g / mL, and the wet weight of the fungal hyphae is 0.01 to 0.03 g / mL; the composite microbial agent is irrigated again after 15 to 20 days at a rate of 90 to 110 mL per plant.

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