CN115558615A

CN115558615A - Stress-resistant growth-promoting compound microbial agent and application thereof

Info

Publication number: CN115558615A
Application number: CN202210552623.4A
Authority: CN
Inventors: 张新慧; 李小康; 郎多勇; 周丽; 王兰梦
Original assignee: Ningxia Medical University
Current assignee: Ningxia Medical University
Priority date: 2022-05-19
Filing date: 2022-05-19
Publication date: 2023-01-03

Abstract

The invention belongs to the technical field of microbial agent preparation, and particularly relates to a stress-resistant growth-promoting compound microbial agent and application thereof. The stress-resistant growth-promoting compound microbial agent comprises streptomyces and bacillus cereus, and the compound microbial agent formed by compounding the streptomyces and the bacillus cereus can enhance the capability of plants to resist abiotic stress such as acid and alkali, high temperature, high salt and ultraviolet irradiation; meanwhile, the plant growth promoter can also enhance the capability of the plant to resist biotic stress and has the effect of promoting the growth of the plant; and the capability of resisting stress and promoting the growth of plants is more prominent compared with that of a single microbial agent.

Description

Stress-resistant growth-promoting compound microbial agent and application thereof

Technical Field

The invention belongs to the technical field of microbial agent preparation, and particularly relates to a stress-resistant growth-promoting compound microbial agent and application thereof.

Background

In nature, plants are continually challenged by adverse environmental conditions such as plant disease, drought, soil salinization, nutrient deficiency, etc., limiting the widespread use of arable land worldwide and negatively impacting crop productivity. Chemical treatments using chemical fertilizers and pesticides have long been the most common global strategy for controlling plant diseases and promoting plant growth, but have had limited effect on solving the above problems due to chemical substances and also have adverse effects on the environment and human health associated with the use of chemical substances. Therefore, in order to comply with the development trend of "pure natural, pollution-free and green organic products", the development of biological control by beneficial microorganisms is considered to be a promising sustainable comprehensive management method.

As a novel green pesticide/fertilizer, the compound microbial agent has the effects of enhancing plant disease resistance, promoting plant growth and the like, has the characteristics of high quality, high efficiency, low toxicity, low residue, environmental safety, environmental friendliness, remarkable stability superior to that of a single microbial agent and the like, and gradually replaces the original chemical pesticide and chemical fertilizer. The actinomycetes are widely distributed in nature, can generate various effective metabolites, can prevent and treat plant fungal diseases, and are extremely important microbial pesticide/fertilizer resources. The bacteria are the main microorganism group which can be used for producing microbial pesticides/fertilizers, have outstanding growth promoting effect and are the most applied and best in effect. Therefore, the screening of environment-friendly actinomycetes and bacteria resources with disease-resistant growth-promoting activity and the development of novel stress-resistant growth-promoting compound microbial agents capable of gradually replacing chemical pesticides and chemical fertilizers are of great significance to the field.

Disclosure of Invention

The invention aims to provide a stress-resistant growth-promoting compound microbial agent and application thereof.

The invention provides a stress-tolerant growth-promoting compound microbial agent, which comprises one or more of Streptomyces sp and Bacillus cereus, bacterial liquid and metabolite of effective components.

Preferably, the streptomyces comprises streptomyces SF1, and the preservation number is CGMCC No.23417; the bacillus cereus comprises bacillus cereus G2 with the preservation number of CGMCC No.16671.

Preferably, the concentrations of streptomyces SF1 and bacillus cereus G2 in the stress-tolerant growth-promoting microbial agent are (1-6). Times.10 respectively ⁸ CFU/mL。

Preferably, the effective concentration of the stress-resistant growth-promoting compound microbial agent is (1-6) multiplied by 10 ⁸ CFU/mL。

The invention also provides application of the streptomycete SF1 or the stress-tolerant growth-promoting compound microbial agent in the technical scheme in one or more of enhancing abiotic stress tolerance of plants, enhancing biotic stress tolerance of plants and promoting plant growth.

Preferably, the abiotic stress includes one or more of high acid-base, drought, high salt, high temperature and ultraviolet stress.

Preferably, the biotic stress includes plant diseases.

Preferably, the plant disease comprises one or more of plant root rot, plant gray mold, plant leaf spot, plant anthracnose and plant leaf spot.

Preferably, the promoting plant growth comprises promoting seed germination and/or seedling growth.

Preferably, the plant comprises a medicinal plant.

Has the advantages that:

the invention provides a stress-resistant growth-promoting compound microbial agent, which comprises streptomyces and bacillus cereus, wherein the compound microbial agent formed by compounding the streptomyces and the bacillus cereus can enhance the capability of plants to resist abiotic stress such as acid and alkali, high temperature, high salt and ultraviolet irradiation; meanwhile, the plant biological stress resistance can be enhanced, such as various diseases including plant root rot, plant gray mold, plant leaf spot, plant anthracnose and plant leaf spot can be effectively controlled; furthermore, the compound microbial agent can be metabolized to generate indoleacetic acid and NH ₃ ACC deaminase, nitrogen fixation, iron production carrier, inorganic phosphorus dissolving, organic phosphorus dissolving, protease producing, potassium dissolving, cellulose and other growth promoting substances, and has the function of promoting plant growth; and the capability of resisting stress and promoting plant growth is more outstanding compared with that of a single microbial agent.

Biological preservation information

Bacillus cereus G2 is preserved in China general microbiological culture Collection center (CGMCC) in 31.10.2018 at the No.3 Xilu-1 of Beijing market facing Yang district, and the preservation number is CGMCC No.16671 at the institute of microbiology of China academy of sciences.

Streptomyces sp.SF 1 is preserved in China general microbiological culture Collection center (CGMCC) at 9.15.2021, with the preservation address of No.3 of the national institute of microbiology, no.1 of Western Lu, north Cheng, the Korean district, beijing, and the preservation number of CGMCC No.23417.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments will be briefly described below.

FIG. 1 is a colony morphology of Streptomyces SF 1;

FIG. 2 is a diagram showing the co-culture medium of Bacillus cereus G2 and Streptomyces SF1 in example 2;

FIG. 3 is a graph showing the examination of the affinity between complex bacteria in example 2;

FIG. 4 is a diagram of antagonistic pathogenic bacteria colonies in the investigation of the fermentation form of the stress-tolerant growth-promoting complex microbial inoculant in example 3;

FIG. 5 is a graph showing the results of the dry weight of the cells in the fermentation pattern of the stress-tolerant growth-promoting complex microorganism bacterium agent in example 3;

FIG. 6 is a diagram of antagonistic colonies in the antibiogram of the stress-tolerant growth-promoting complex microbial inoculant of example 3;

FIG. 7 is a graph showing the safety evaluation of the stress-tolerant growth-promoting complex microbial agent of example 4;

FIG. 8 is a graph showing the results of the in vitro disease prevention test of the stress-tolerant growth-promoting complex microbial inoculant of example 4;

FIG. 9 is a comparison chart of experimental colonies planted in the test root tissues by the stress-tolerant growth-promoting compound microbial agent in example 4;

FIG. 10 is a graph showing the growth promoting characteristics of the stress-tolerant growth-promoting complex microbial inoculant of example 5;

FIG. 11 is a colony map of antagonistic pathogenic bacteria in the study of the stability (pH value) of different bacterial agents in example 7;

FIG. 12 is a chart of antagonistic pathogen colonies in the stability (heat) examination of different microbial agents in example 7;

FIG. 13 is a chart of antagonistic pathogen colonies in the stability (UV irradiation) examination of different bacterial agents in example 7;

FIG. 14 is a graph of the experimental results of the stress-tolerant growth-promoting compound microbial inoculant of example 8 on the germination of licorice seeds.

In FIGS. 7 to 8, A represents Codonopsis pilosula, B represents Angelica sinensis, and C represents Astragalus membranaceus.

In the above drawings, SF1 and G2 represent different single microbial agents, respectively, and SF1+ G2 represent a stress-resistant growth-promoting compound microbial agent, which is based on the description of the embodiment corresponding to the drawings.

Detailed Description

The streptomycete preferably comprises streptomycete SF1 with the preservation number of CGMCC No.23417; the bacillus cereus preferably comprises bacillus cereus G2, and the preservation number is CGMCC No.16671.

The streptomyces SF1 is preferably separated from healthy and plump liquorice seeds. The ITS sequence of the streptomycete SF1 is preferably shown as SEQ ID No. 1: <xnotran> ATGGCTCAGGACGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAACGATGAACCA CTTCGGTGGGGATTAGTGGCGAACGGGTGAGTAACACGTGGGCAATCTGCCCTTCACTC TGGGACAAGCCCTGGAAACGGGGTCTAATACCGGATAACACTCTCGCAGGCATCTGTGG GGGTTGAAAGCTCCGGCGGTGAAGGATGAGCCCGCGGCCTATCAGCTTGTTGGTGAGGT AGTGGCTCACCAAGGCGACGACGGGTAGCCGGCCTGAGAGGGCGACCGGCCACACTG GGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATG GGCGAAAGCCTGATGCAGCGACGCCGCGTGAGGGATGACGGCCTTCGGGTTGTAAACC TCTTTCAGCAGGGAAGAAGCGAAAGTGACGGTACCTGCAGAAGAAGCGCCGGCTAACT ACGTGCCAGCAGCCGCGGTAATACGTAGGGCGCAAGCGTTGTCCGGAATTATTGGGCGT AAAGAGCTCGTAGGCGGTCTGTCGCGTCGGATGTGAAAGCCCGGGGCTTAACCCCGGG TCTGCATTCGATACGGGCAGACTAGAGTGTGGTAGGGGAGATCGGAATTCCTGGTGTAG CGGTGAAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGATCTCTGGGCCA TTACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGT CCACGCCGTAAACGGTGGGCACTAGGTGTTGGCGACATTCCACGTCGTCGGTGCCGCAG CTAACGCATTAAGTGCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAA TTGACGGGGGCCCGCACAAGCAGCGGAGCATGTGGCTTAATTCGACGCAACGCGAAGA ACCTTACCAAGGCTTGACATCGCCCGGAAAGCATCAGAGATGGTGCCCCCCTTGTGGTC GGGTGACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCC CGCAACGAGCGCAACCCTTGTTCTGTGTTGCCAGCATGCCCTTCGGGGTGATGGGGACT CACAGGAGACTGCCGGGGTCAACTCGGAGGAAGGTGGGGACGACGTCAAGTCATCATG CCCCTTATGTCTTGGGCTGCACACGTGCTACAATGGCAGGTACAATGAGCTGCGATACCG CAAGGTGGAGCGAATCTCAAAAAGCCTGTCTCAGTTCGGATTGGGGTCTGCAACTCGA CCCCATGAAGTCGGAGTTGCTAGTAATCGCAGATCAGCATTGCTGCGGTGAATACGTTCC CGGGCCTTGTACACACCGCCCGTCACGTCACGAAAGTCGGTAACACCCGAAGCCGGTG GCCCAA. </xnotran> The similarity between the Streptomyces SF1 and Streptomyces sp.strain A217 in the NCBI database is 99.57%, which shows that the strain has high similarity with the Streptomyces in the database.

The colony morphology of the streptomycete SF1 is as follows: the bacterial colony is light yellow, nearly circular, and has more regular edge, smaller bacterial colony without extensive extension, smooth and opaque surface, and the bacterial colony is combined with the culture medium tightly and is not easy to pick up, as shown in figure 1.

The streptomycete SF1 has the characteristics of producing citric acid, esterase, oxidase, catalase, urease and indoleacetic acid; the acid and alkali resistance range is as follows: the pH value is 5.0-9.0, and the pH value of the optimum growth is 7.0; the temperature tolerance range is 15-37 ℃, and the optimal growth temperature is 28 ℃; the carbon source can be D-mannitol, D-galactose, D-xylose, D-glucose, D-maltose, D-fructose, D-sorbitol and sucrose; nitrogen sources which can be utilized are urea, glutamine, glycine, ammonium sulfate and histidine.

The invention also provides a stress-resistant growth-promoting compound microbial agent, and the effective components of the stress-resistant growth-promoting compound microbial agent comprise one or more of the thallus, bacterial liquid and metabolite of the streptomycete SF1 and the Bacillus cereus G2 in the technical scheme.

The preservation number of the bacillus cereus G2 is CGMCC No.16671. In the present invention, the effective component of the stress-tolerant growth-promoting compound microbial agent is preferably any one or more of the bacteria, bacteria liquid and metabolites of the streptomycete SF1 and bacillus cereus G2, more preferably a bacteria liquid obtained by co-fermentation of the streptomycete SF1 and bacillus cereus, and even more preferably a seed liquid obtained by co-fermentation. The concentration of the streptomycete SF1 in the stress-tolerant growth-promoting compound microbial agent is preferably (1-6) multiplied by 10 ⁸ CFU/mL, more preferably (1 to 4.5). Times.10 ⁸ CFU/mL; the concentration of the Bacillus cereus G2 is preferably (1-6). Times.10 ⁸ CFU/mL, more preferably (1 to 4.5). Times.10 ⁸ CFU/mL. The effective concentration of the stress-resistant growth-promoting compound microbial agent is preferably (1-6). Times.10 ⁸ CFU/mL, more preferably (1 to 4.5). Times.10 ⁸ CFU/mL, more preferably (1-4). Times.10 ⁸ CFU/mL。

The invention also provides the hypochondriac resistanceThe preparation method of the forced growth promoting compound microbial agent comprises the following steps: respectively inoculating activated streptomycete SF1 and activated bacillus cereus G2 into liquid culture media, and respectively culturing for 7d and 2d to obtain streptomycete SF1 seed liquid and bacillus cereus G2 seed liquid; mixing the streptomycete SF1 seed solution and the bacillus cereus G2 seed solution in equal volume, and performing co-fermentation culture for 7 days to obtain the stress-resistant growth-promoting compound microbial agent; the concentrations of streptomyces SF1 and bacillus cereus G2 in the stress-tolerant growth-promoting compound microbial agent are preferably (1-6) multiplied by 10 ⁸ CFU/mL. The prepared streptomycete SF1 seed solution and the prepared bacillus cereus G2 seed solution are preferably mixed and then fermented, and compared with the preparation method that the streptomycete SF1 seed solution and the bacillus cereus G2 seed solution are respectively fermented and then mixed, the compound microbial agent prepared by the preparation method has higher bacteriostatic rate. The liquid culture medium is preferably beef extract peptone liquid culture medium, which is illustrated in the examples by taking the beef extract peptone liquid culture medium as an example, and comprises the following components: 3g of beef extract, 10g of peptone, 5g of NaCl, 17g of agar and 1000mL of distilled water, and the pH value is natural.

In the present invention, preferably, the Streptomyces SF1 and Bacillus cereus G2 are cultured under the same conditions. The temperature for the culture according to the present invention is preferably 15 to 37 ℃ and more preferably 28 ℃. The culture of the invention is preferably shaking culture, and the rotation speed of the shaking culture is preferably 140 to 240r/min, and more preferably 180r/min. The culture time of the streptomyces SF1 is preferably 7d, and the culture time of the bacillus cereus G2 is preferably 2d. The fermentation culture is preferably shaking culture after mixing, and the rotation speed of the fermentation culture is preferably 140-240 r/min, and more preferably 180r/min; the temperature of the fermentation culture is preferably 15 to 37 ℃, and more preferably 28 ℃. The present invention preferably further comprises inoculating the activated Streptomyces sp 1 and Bacillus cereus G2 separately into a liquid medium after dividing into cake-shaped fungus cakes. The diameter of the blocky fungus cake is preferably 0.6cm. The activation method of the present invention is not particularly limited, and a conventional activation method in the art may be used. The stress-resistant growth-promoting compound microbial agent prepared by the preparation method of the invention contains live bacteriaThe effective concentration is preferably (1-6) x 10 ⁸ CFU/mL. Any one or more of the thalli, bacterial liquid and metabolites prepared by the preparation method can be used as the effective components of the stress-resistant growth-promoting compound microbial agent.

The invention also provides application of the streptomycete SF1 or the stress-tolerant growth-promoting compound microbial agent in the technical scheme in one or more of enhancing abiotic stress tolerance of plants, enhancing biotic stress tolerance of plants and promoting plant growth. The streptomyces SF1 and the bacillus cereus G2 are compounded, so that the abiotic stress and biotic stress resistance of the streptomyces SF 2 can be obviously improved, and the streptomyces SF 2 has better tolerance to various abiotic stresses including acid-base stress, drought stress, high temperature stress, high salt stress and the like; meanwhile, various plant diseases including plant root rot, plant gray mold, plant leaf spot, plant anthracnose and plant leaf spot can be effectively controlled; in addition, indole acetic acid and NH generated by compound metabolism of streptomyces SF1 and bacillus cereus G2 ₃ ACC deaminase, nitrogen fixation, siderophore production, inorganic phosphorus dissolution, protease production, potassium dissolution, cellulase and other growth promoting substances can obviously promote the growth of plants, the corresponding technical effects are obviously higher than those of a single strain, and the effects are especially more prominent under the conditions of strain concentration and the like provided by the invention.

The abiotic stress of the present invention preferably includes one or more of high acid-base, drought, high salt, high temperature and ultraviolet stress, and more preferably includes high acid-base, drought and high salt.

The biotic stress according to the present invention preferably includes plant diseases, more preferably includes plant diseases caused by pathogenic bacteria; the plant diseases caused by the pathogenic bacteria preferably comprise one or more of plant root rot, plant gray mold, plant leaf spot, plant anthracnose and plant leaf spot, and more preferably comprise one or more of rhizoma anemarrhenae gray mold, radix astragali leaf spot, fructus Lycii anthracnose, rhizoma Dioscoreae leaf spot, radix Codonopsis root rot, radix Angelicae sinensis root rot and radix astragali root rot.

The pathogenic bacteria of the plant diseases preferably comprise fungal pathogenic bacteria, more preferably comprise one or more of anemarrhena gray mold pathogenic bacteria, alternaria alternate, colletotrichum oxysporum, yam rhizoctonia solani, sclerotinia sclerotiorum, rhizoctonia solani and fusarium oxysporum, and further preferably are sclerotinia sclerotiorum, rhizoctonia solani, fusarium oxysporum, alternaria alternate, colletotrichum oxysporum and yam coniothyrium.

The promotion of plant growth according to the present invention preferably comprises promotion of seed germination and/or seedling growth, the promotion of seed germination preferably comprises improvement of seed germination rate, germination vigor and germination index; the promoting seedling growth comprises increasing seedling radicle length, promoting radicle thickening, improving seedling vigor and increasing seedling dry weight.

The plant disclosed by the invention preferably comprises a medicinal plant, and more preferably comprises rhizoma anemarrhenae, astragalus membranaceus, medlar and Chinese yam.

When the stress-resistant growth-promoting compound microbial agent is applied to actual production, the use mode of the stress-resistant growth-promoting compound microbial agent is not particularly limited, and the use mode of the conventional microbial agent in the field can be adopted.

In order to further illustrate the present invention, the following detailed description of the technical solutions provided by the present invention is made with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of the present invention.

The rhizoctonia solani, fusarium oxysporum, sclerotinia sclerotiorum, alternaria alternate, colletotrichum oxysporum and Chinese yam leaf spot mold are all conventional strains, and DG-13, HQI-5, DS-8, HQ-6, TJD, TJA, TJCE and WHN7 are named only for distinguishing infection pathogenic bacteria on different plants.

In the following examples of the present invention, SF1 represents Streptomyces SF1 and G2 represents Bacillus cereus G2, unless otherwise specified.

Example 1

Separation and identification of streptomycete SF1

1) Selecting healthy and plump liquorice seeds, soaking the seeds for 45min by using a concentrated sulfuric acid solution with the mass percentage of 85%, stirring at random, washing the seeds with sterile water for 3 times, then soaking the seeds with a hydrogen peroxide solution with the mass percentage of 0.1% for 10min, washing the seeds with the sterile water for 5 times, then soaking the seeds with the sterile water for 3h, and then transferring the seeds into an ultra-clean workbench to perform surface disinfection on the seeds. Under aseptic condition, soaking in 75% ethanol solution for 5min, soaking in 1% hydrogen peroxide solution for 30s, soaking in 5% sodium hypochlorite solution for 10s, washing with sterile water for 10 times, placing the surface sterilized Glycyrrhrizae radix seed in a sterile culture dish with sterile absorbent paper, and placing in a sterile super clean bench for naturally drying. (to ensure thorough sterilization, 200. Mu.L of the washing water from the last washing of the licorice seeds was applied to NA solid medium (peptone 10.0g, beef extract 3.0g, sodium chloride 5.0g, agar 17g, and distilled water 1000 mL), and the plate was incubated at 28 ℃ for one week to find no growth of the cells). The treated licorice seeds are put into a sterile mortar, and 10mL of sterile water is added for grinding. And uniformly coating 200 mu L of the suspension in a Gao's I solid culture medium, culturing in an artificial climate box at 28 ℃ for 5d, selecting a single bacterial colony of the strain, and streaking and purifying to obtain a pure cultured strain, which is named as SF1.

Inoculating the separated strains on different culture media by streaking, culturing at the constant temperature of 28 ℃ for 5-7 days, and recording the colors and growth conditions of aerial hyphae, intrabasal hyphae and soluble pigments of colonies, wherein the results are shown in Table 1.

TABLE 1 growth of SF1 in different media

As can be seen from Table 1, the SF1 strain of the invention can grow well in different culture media, and the colors of aerial hyphae and intrabasal hyphae of bacterial colonies in different culture media are different, but the culture media mainly take yellow brown and yellow white, and the conditions of soluble pigments are different, which shows that SF1 is a single microorganism and has good stability.

2) The physiological and biochemical characteristics of the separated SF1 strain are determined by referring to Bergey's Manual of bacteria identification and Manual of general bacteria System identification, and the results are shown in Table 2.

TABLE 2 physiological and biochemical characteristics of SF1 strains

Note: "+" represents positive; "-" indicates negative.

As shown in Table 2, SF1 has the characteristics of producing citric acid, esterase, oxidase, catalase, urease and indoleacetic acid; the acid and alkali resistance pH range is 5.0-9.0, and the optimum growth pH value is 7.0; the temperature tolerance range is 15-37 ℃, and the optimal growth temperature is 28 ℃; the carbon source can be D-mannitol, D-galactose, D-xylose, D-glucose, D-maltose, D-fructose, D-sorbitol, and sucrose; the nitrogen source can be urea, glutamine, glycine, ammonium sulfate, histidine. Based on the above basic experiments, SF1 was shown to belong to the actinomycete Streptomyces genus.

3) The strain DNA was extracted using a DNA extraction kit (purchased from Kogyo Biotech Co., ltd., product number SK8 255), and the strain DNA was purified using a 16S rDNA bacterial universal primer set (SEQ ID No.2 forward primer 7F-1540R:5' CAGAGTTTGATCCTGGCCTAGGAGGTGATCCAGCCGCA-: 5 '-AGTTTGATCTMTGGCTCAGGGTTACTTGTTACCTGTTACTGACTT-3') DNA was subjected to PCR amplification. The PCR reaction system is as follows: template DNA 0.5. Mu.L, 10 XBuffer (with Mg) ²⁺ ) 2.5. Mu.L, 1. Mu.L of dNTP (10. Mu.M), 0.2. Mu.L of Taq enzyme (5U/. Mu.L), 0.5. Mu.L each primer (10. Mu.M); reaction procedures are as follows: pre-denaturing at 95 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 57 ℃ for 30s, extension at 72 ℃ for 90s, and 30 cycles; final extension at 72 ℃ for 10min. After PCR amplification, detecting a PCR product by 1% agarose gel electrophoresis, sending the amplification product to a company Limited (Shanghai) for sequencing, and performing BLAST comparison analysis by an NCBI database, wherein the similarity of the strain and Streptomyces sp.strain A217 reaches 9.57%, so that the strain is identified as Streptomyces sp.and named as SF1.

Example 2

Screening of streptomycete SF1 and bacillus cereus G2 co-culture medium and investigation of composite bacteria affinity

1) Screening of the co-culture medium: beef extract peptone (beef extract 3g, peptone 10g, naCl 5g, agar 17g, distilled water 1000mL, natural pH) culture medium, gao's I (soluble starch 20g, KNO) ₃ 1g、K ₂ HPO ₄ 0.5g、NaCl 0.5g、MgSO ₄ 0.5g、FeSO ₄ 0.01g, agar 17g, and distilled water 1000mL, natural pH) as a target medium. The cells of G2 and SF1 were picked up by a sterile inoculating loop, inoculated into the target medium, sealed, cultured at 28 ℃ for 2d in G2 and 7d in SF1, and the co-medium was selected depending on whether or not G2 and SF1 grew well in the target medium, and the results are shown in FIG. 2.

The results in FIG. 2 show that: the strains G2 and SF1 grow well in a beef extract peptone culture medium, so the beef extract peptone culture medium is selected as a co-culture medium.

2) Preparation of seed solutions of bacillus cereus G2 and streptomycete SF 1: respectively activating the G2 and SF1 strains, beating the strains into fungus cakes with diameters of 0.6cm and respectively containing G2 and SF1 bacterial colonies by using a sterilization puncher, respectively picking the fungus cakes by using a sterilization toothpick, respectively inoculating the fungus cakes into 250mL triangular bottles containing 100mL beef extract peptone liquid culture medium, carrying out shake culture at 28 ℃ and 180r/min, carrying out G2 culture for 2d and SF1 culture for 7d until the concentrations of G2 and SF1 seed solutions are (1-6) multiplied by 10 ⁸ CFU/mL to obtain G2, SF1 seed solution.

3) Investigation of composite intersomycete affinity: and (3) inspecting whether antagonism exists between the strains G2 and SF1 by adopting an Oxford cup method. Taking the co-culture medium obtained by screening in the step 1) as a basic culture medium, coating the G2 seed solution prepared in the step 2) in the whole culture medium, punching a hole in the center of a flat plate by using a puncher with the diameter of 0.6cm, inoculating an equal amount of SF1 seed solution in the hole, sealing, placing in dark for 5 days at 28 ℃, observing whether a transparent bacteriostatic ring exists around the hole center, and if the transparent bacteriostatic ring does not exist, indicating that the affinity between the two is good, wherein the result is shown in figure 3.

As can be seen from FIG. 3, there is no transparent zone of inhibition at the contact edge between the SF colony 1 and the G2 colony, which indicates that the affinity between the strains G2 and SF1 is good, and the strains can be compounded.

Example 3

Preparation of stress-resistant growth-promoting compound microbial agent and inhibition effect thereof on plant pathogenic bacteria

1) The determination method of the bacteriostatic rate comprises the following steps: the method comprises the steps of selecting pathogenic bacteria thallus (slant culture and preservation) by using a sterilization inoculating loop, inoculating the thallus into a potato glucose agar (PDA) culture medium (potato extract powder 12.0g, glucose 20.0g, agar 14.0g, distilled water 1000mL, natural PH), sealing, and culturing in a constant temperature box at 28 ℃ for 5 days, wherein the whole culture medium is basically full of the fungal pathogenic bacteria and the activity state is good. Taking the fungal pathogenic bacteria cultured for 5 days, preparing into a bacterial cake by a 0.6cm puncher, inversely inoculating to the center of a Potato Dextrose Agar (PDA) solid culture medium, flatly pasting a sterilization filter paper sheet with the diameter of 0.6cm on four points of the same cross line at a position 2cm away from the center of the bacterial cake, inoculating 5 mu L of stress-resistant growth-promoting microbial agent (test group) on the sterilization filter paper sheet, and dropwise adding equal amount of sterilization distilled water as a control group (CK). Repeating the treatments for 3 times, sealing, culturing at 28 deg.C until each pathogenic bacteria colony of the control group does not grow, measuring the diameter of the pathogenic bacteria colony by cross method, and calculating the antibacterial rate.

Bacteriostasis rate (%) = (D1-D2)/(D1-D3). Times.100%;

wherein D1 represents the diameter of the colony of the pathogenic bacteria of the control group (inoculated with the same amount of sterilized distilled water); d2 represents the colony diameter of pathogenic bacteria in a test group (inoculated with the stress-resistant growth-promoting compound microbial agent); d3 represents the diameter of the beaten cake of pathogenic bacteria (0.6 cm).

2) Investigation of the fermentation form of the stress-resistant growth-promoting microbial agent and preparation of the stress-resistant growth-promoting microbial agent: the optimal fermentation form (mixed fermentation and mixing after fermentation) of the compound microbial agent is inspected by taking the bacteriostasis rate of pathogenic bacteria (radix codonopsis root rot pathogenic bacteria sclerotinia sclerotiorum DS-8, angelica root rot pathogenic bacteria Rhizoctonia solani DG-13 and astragalus root rot pathogenic bacteria Fusarium oxysporum HQI-5) and the dry weight of the disease-resistant growth-promoting compound bacteria as measurement indexes. Mixing and fermenting: g2, SF1 seed solutions prepared in step 2) of example 2 were mixed at a ratio of 1:1 is inoculated in a 250mL conical flask containing 100mL liquid co-culture medium in equal quantity, and is fermented for 7 days at 28 ℃ and 180r/min to obtain the stress-resistant growth-promoting microbial inoculum. Mixing after fermentation: inoculating the G2 and SF1 seed solution prepared in the step 2) of the example 2 into the seed solution containing the 1-amino-acid-containing substance according to the inoculation amount of 1%Respectively fermenting in 250mL conical flasks of 00mL liquid co-culture medium to obtain bacterial liquids, and mixing in equal volume to obtain the stress-resistant growth-promoting compound microbial agent, wherein the bacterial liquids of SF1 and G2 have the concentrations of (1-6) multiplied by 10 ⁸ CFU/mL, the results are shown in Table 3 and FIGS. 4 to 5.

The results in table 3 and fig. 4 to 5 show that the stress-tolerant growth-promoting microbial agent prepared by mixed fermentation has higher bacteriostatic ratio and dry thallus weight than the stress-tolerant growth-promoting microbial agent prepared by mixing after fermentation, so the stress-tolerant growth-promoting microbial agent is prepared by selecting a mixed fermentation mode.

TABLE 3 results of the bacteriostatic rate of stress-tolerant growth-promoting microbial agents in different fermentation forms

Note: different lower case letters indicate significant difference (P < 0.05).

2) Investigation of the antibacterial spectrum of the stress-resistant growth-promoting microbial agent: the method in the step 1) is adopted to measure the antibacterial spectrums (pathogenic bacteria of common anemarrhena gray mold, pathogenic bacteria of astragalus leaf spot disease, pathogenic bacteria of alternaria alternate HQ-6 of astragalus root leaf spot disease, pathogenic bacteria of lycium bararum, pathogenic bacteria of yam leaf spot disease, pathogenic bacteria of lycium bararum, and pathogenic bacteria of yam leaf spot disease, and the pathogenic bacteria of lycium bararum, i.e., anthrax TJCE), and the results are shown in table 4 and fig. 6.

TABLE 4 results of the stress-resistant growth-promoting microbial inoculum antibiogram test

The results of table 4 and fig. 6 show that the stress-resistant growth-promoting microbial agent has strong inhibition rates on botrytis cinerea, alternaria solani HQ-6, colletotrichum oxysporum TJA, colletotrichum oxysporum TJD, piyama dioscoreae leaf spot mildew and colletotrichum oxysporum TJCE, and the composite microbial agent has a wide antibacterial spectrum.

Example 4

Evaluation of safety and disease prevention efficacy of stress-resistant growth-promoting microbial agent

Preparation of test materials: washing healthy plant roots (radix Codonopsis, radix Angelicae sinensis, and radix astragali) with flowing water for 30min, sequentially soaking in 75% ethanol for 20s, 3% sodium hypochlorite solution for 3min, and washing with sterilized distilled water for 5 times; the resulting sheets were cut into 5mm thick sheets with a sterile scalpel blade.

Preparation of pathogen spore suspension: inoculating pathogenic bacteria DS-8, DG-13 and HQI-5 to be tested on a fresh PDA solid culture medium, culturing at 28 deg.C for 5 days, pouring 100mL of sterilized distilled water, gently shaking to wash spores, filtering the washing liquid with sterile gauze into a sterile triangular flask, measuring the concentration of spore suspension with a blood counting plate, and making into 10 ⁶ Suspension per mL for use.

1) Safety evaluation of stress-tolerant growth-promoting microbial agent

The roots of the test plants, which had been sliced, were placed in a sterile petri dish containing sterilized filter paper (1 mL of sterile water was added for moisture retention), and 20 μ L of the stress-tolerant growth-promoting microbial agent of example 3 (prepared by mixed fermentation and designated as SF1+ G2) was inoculated by spot inoculation with the same amount of distilled water as a Control (CK). Sealing, culturing at 28 deg.C in dark, calculating disease index of each treatment according to severity grading standard (see Table 5) after 7d, and counting incidence, wherein each treatment is repeated for 9 times.

Disease index = Σ (number of diseased plants at each level × severity progression)/(total number of tested plants × highest progression) × 100%; the highest order is 5.

Incidence rate = (number of diseased plants/total number of test plants) × 100%

The results in table 6 and fig. 7 show that the morbidity and incidence rate of each test plant after inoculation of the stress-tolerant growth-promoting microbial agent of the invention are not significantly different from those of the CK group, which indicates that the stress-tolerant growth-promoting microbial agent of the invention is non-pathogenic and can be continuously developed and utilized.

TABLE 5 severity grading Standard

Severity grading	Grading standards
		Grade
0	Healthy root tissue
		Level
1	The root area of less than or equal to 5 percent shows symptoms
		Stage 2	>5 percent and less than or equal to 25 percent of root area presents symptoms
Grade
	3	>25 percent and less than or equal to 50 percent of root area presents symptoms
4 stage			>50 percent and less than or equal to 75 percent of root area presents symptoms
	Grade
5		>75% of the root area presents symptoms

TABLE 6 evaluation results of safety of stress-resistant growth-promoting microbial agents

2) In-vitro disease prevention test of stress-resistant growth-promoting microbial agent

The roots of the test plants which had been sectioned were placed in sterile petri dishes containing sterile filter paper (1 mL sterile water added for moisturizing). In the test, CK (only equivalent sterilized distilled water is inoculated), pathogen spore suspension (respectively marked as DS-8, DG-13 and HQI-5) and stress-resistant growth-promoting microbial agent and pathogen spore suspension (marked as SF1+ G2+ pathogen) are inoculated for three treatments, wherein the stress-resistant growth-promoting microbial agent and pathogen spore suspension comprises the following components: inoculating 20 mu L of stress-resistant growth-promoting microbial agent in a bacterial liquid point inoculation mode, inoculating 20 mu L of pathogenic bacteria spore suspension in the same mode after 24h of point inoculation, sealing, placing at 28 ℃ for dark culture, calculating the disease condition index of each treatment and the relative prevention effect of the stress-resistant growth-promoting microbial agent according to a severity grading standard (shown in table 5) after 7d, counting the morbidity, and repeating each treatment for 9 times.

Relative control effect (%) = (disease index of inoculated pathogenic bacteria isolated root-inoculated stress-resistant growth-promoting microbial agent and pathogenic bacteria isolated root)/disease index of inoculated stress-resistant growth-promoting microbial agent and pathogenic bacteria isolated root x 100%; the results are shown in table 7 and fig. 8.

TABLE 7 in vitro disease prevention test results for stress-tolerant growth-promoting microbial agents

3) Establishment experiment of stress-resistant growth-promoting microbial agent in test root tissue

Preserving the test tissue samples obtained in the step 2) of the example 4 (the test root tissue samples CK and SF1+ G2+ pathogenic bacteria are treated), separating endophytes, and comparing the colonization of the compound bacterial strains in the test root tissues after different treatments.

And (3) separating endophytes in the root tissues to be tested: collecting experimental root tissue, pulverizing in sterile stirring cup, collecting 2g of the experimental root tissue, adding liquid nitrogen until the root tissue just submerges the root tissue, immediately grinding for 2-3min, repeating twice, collecting supernatant 1mL, placing in sterile test tube, and diluting with sterile water to 10 gradient ^-3 And 10 ^-4 And (4) respectively sucking 200 mu L of the two gradients, coating the two gradients on a beef extract peptone solid culture medium, and placing the beef extract peptone solid culture medium in a thermostat for culturing for 5-7 days at 28 ℃. According to the presence or absence of colonies of the Complex StrainThe number of the strains was measured to determine whether the strains colonized the root tissues. The results are shown in fig. 9, and it can be seen from fig. 9 that the number of endophytes in root tissues of codonopsis pilosula, angelica sinensis and astragalus membranaceus of the test plants is significantly increased after the disease-resistant growth-promoting composite bacteria are inoculated compared with the CK group, which indicates that the field planting of the composite microbial inoculum is the basis for the disease-resistant effect of the composite microbial inoculum.

Example 5

Research on growth promoting characteristics of stress-resistant growth promoting microbial agent

Ability to produce IAA (indoleacetic acid): the stress-tolerant growth-promoting microbial inoculum (prepared by mixed fermentation) of example 3 was inoculated into a beef extract peptone liquid medium containing tryptophan at 0.5g/L in an amount of 1mL, subjected to shaking culture at 180r/min at 28 ℃ for 5d, centrifuged at 9000r/min for 10min, and 2mL of the supernatant was added with 4mL of Salkowski reagent (0.5 mM FeCl) ₃ And 50mLH ₂ SO ₄ Mixing), standing in dark at 28 deg.C for 30min, and repeating 3 times for each experiment if the color turns red to indicate that the complex bacteria has IAA producing property. The results are shown in table 8 and fig. 10.

The result shows that the stress-tolerant growth-promoting microbial agent has the capability of producing the indoleacetic acid.

Capacity to produce siderophores: 5 μ L of the stress tolerant growth-promoting microbial inoculum (prepared by mixed fermentation) of example 3 was pipetted and inoculated in a medium (60.5 mg of Chromel, 72.9mg of cetyltrimethylammonium bromide, feCl) in a patch on CAS ₂ ·6H ₂ O2.645 mg, peptone 4.5g, glucose 9g, beef extract powder 2.7g, naCl 4.5g, agar 20g, distilled water 1000 mL) on a sterilized filter paper sheet with the diameter of 0.6cm, culturing for 5D at 28 ℃, if a transparent ring appears around a bacterial colony, determining the diameter D (cm) of the transparent ring and the diameter D (cm) of the bacterial colony, and calculating the D/D value, wherein the larger the ratio is, the higher the activity of the produced siderophore is, and each experiment is repeated for 3 times. The results in Table 8 and FIG. 10 show that the stress-tolerant growth-promoting microbial agent has siderophore production ability.

Nitrogen fixation: absorbing 5 mu L of stress-resistant growth-promoting microbial agent (prepared by mixed fermentation) and inoculating the stress-resistant growth-promoting microbial agent on an Ashby nitrogen-free solid culture medium (10 g of mannitol, 0.2g of NaCl, caCO) ₃ 5g、KH ₂ PO ₄ 0.2g、FeSO ₄ ·7H ₂ O 0.1g、CaSO ₄ ·2H ₂ 0.1g of O, 20g of agar and 1000mL of distilled water) on a sterilized filter paper sheet with the diameter of 0.6cm, culturing at 28 ℃ for 5 days to observe the growth of the complex bacteria, and repeating each experiment for 3 times. The results are shown in table 8 and fig. 10.

The results show that the composite bacteria can grow on a nitrogen-free culture medium, which indicates that the composite bacteria have nitrogen fixation activity.

Producing ACC deaminase activity: absorbing 5 mu L of stress-resistant growth-promoting microbial agent (prepared by mixed fermentation) and inoculating the stress-resistant growth-promoting microbial agent on a solid culture medium (KH) which is flatly attached to ADF ₂ PO ₄ 4g、Na ₂ HPO ₄ 6g、MgSO ₄ ·7H ₂ O 0.2g、FeSO ₄ ·7H ₂ O 0.1g、H ₃ BO ₃ 10μg、 MnSO ₄ 10μg、ZnSO ₄ 70μg、CuSO ₄ 50μg、MoO ₃ 10 ug, 2g of glucose, 2g of gluconic acid, 2g of citric acid, 20g of agar and 1000mL of distilled water) on a sterilized filter paper sheet with a diameter of 0.6cm, culturing at 28 ℃ for 5d, observing the growth of the complex bacteria, and repeating each experiment for 3 times. If the complex bacteria can grow on a culture medium with ACC as a unique nitrogen source, the complex bacteria have ACC deaminase activity. The results are shown in table 8 and fig. 10.

The results show that the complex bacteria can grow on a culture medium with ACC as the only nitrogen source, which indicates that the complex bacteria have ACC deaminase activity.

Production of NH ₃ Activity: sucking 1mL of stress-tolerant growth-promoting microbial agent (prepared by mixed fermentation) and inoculating into a test tube containing 10m L of peptone water (10 g of peptone, 5g of NaCl and 1000mL of distilled water, pH 7.6), culturing at 28 ℃ for 5d, adding 0.5m of S reagent into each tube, and indicating that the composite bacteria have NH production if yellow brown precipitate appears ₃ Activity, no formation of NH if no tan precipitate is present ₃ Activity, 3 replicates per experiment. The results are shown in table 8 and fig. 10.

The result shows that the chromogenic reaction of the cultured complex bacteria is positive, which indicates that the complex bacteria can produce NH ₃ Activity of (2).

Inorganic phosphorus dissolving capacity: absorbing 5 mu L of stress-resistant growth-promoting microbial agent (prepared by mixed fermentation) and inoculating the stress-resistant growth-promoting microbial agent to a Pikovasky's phosphorus-dissolving culture medium (NaCl 0.3g and MgSO) ₄ ·7H ₂ O 0.3g、MnSO ₄ 0.03g、KCl 0.3g、(NH ₄ ) ₂ SO ₄ 0.5g、 FeSO ₄ ·7H ₂ O 0.03g、Ca ₃ (PO ₄ ) ₂ 5g, 10g of glucose, 20g of agar and 1000mL of distilled water) on a sterilized filter paper sheet with the diameter of 0.6cm, and culturing for 5 days at 28 ℃, wherein if a transparent ring appears around a colony, the composite bacteria have the capability of dissolving inorganic phosphorus. The diameter D (cm) of the transparent ring and the diameter D (cm) of the colony are measured, and the D/D value is calculated, wherein the larger the ratio is, the stronger the inorganic phosphorus dissolving capacity is, and each experiment is repeated for 3 times. The results are shown in table 8 and fig. 10.

The result shows that the periphery of the bacterial colony of the compound bacteria has an obvious phosphorus-dissolving ring, which indicates that the compound bacteria has the capability of dissolving inorganic phosphorus.

Potassium-releasing ability: absorbing 5 mu L of stress-resistant growth-promoting microbial agent (prepared by mixed fermentation) and inoculating the stress-resistant growth-promoting microbial agent on a potassium-dissolving solid culture medium (5 g of sucrose and Na) ₂ HPO ₄ 0.2g、MgSO ₄ ·7H ₂ O 0.5g、FeCl ₃ 0.005g、CaCO ₃ 0.1g, 1g of potassium feldspar powder, 20g of agar and 1000mL of distilled water) on a sterilized filter paper sheet with the diameter of 0.6cm, and culturing at 28 ℃ for 5 days, wherein a transparent ring is positive if the periphery of the composite bacterial colony appears. The diameter D (cm) of the transparent circle and the diameter D (cm) of the colony were measured, and the D/D value was calculated, and the larger the ratio, the stronger the potassium-resolving power, and each experiment was repeated 3 times. The results are shown in table 8 and fig. 10:

the result shows that the periphery of the composite bacterial colony has an obvious transparent hydrolysis ring, which indicates that the composite bacteria has the potassium-dissolving capability.

Protease-producing ability: 5 mul of the complex microbial inoculum is absorbed and inoculated on a sterilized filter paper sheet which is flatly stuck on a screening culture medium (15 g of skim milk powder, 20g of agar and 1000mL of distilled water) of protease producing bacteria and has the diameter of 0.6cm, and the culture is carried out for 5 days at the temperature of 28 ℃, and a transparent ring appears around a bacterial colony and is positive. The results in FIG. 7 show that the complex bacteria have a clear hydrolytic ring around the colony, which indicates that the complex bacteria have the ability to produce protease. The diameter D (cm) of the clearing zone and the diameter D (cm) of the colonies were measured and the D/D value was calculated, the larger the ratio, the stronger the protease-producing ability, and each experiment was repeated 3 times. The results are shown in table 8 and fig. 10.

The result shows that the periphery of the bacterial colony of the compound bacteria has obvious transparent hydrolysis rings, and the compound bacteria have the capability of producing protease.

The capability of producing cellulase: absorbing 5 mu L of stress-resistant growth-promoting microbial agent (prepared by mixed fermentation) and inoculating the stress-resistant growth-promoting microbial agent on a CMC culture medium (CMC-Na 10g, KNO) ₃ 2g、MgSO ₄ ·7H ₂ O 0.3g、FeSO ₄ ·7H ₂ O 0.03g、NaCl 0.5g、K ₂ HPO ₄ 1g of agar, 20g of agar and 1000mL of distilled water) on a sterilized filter paper sheet with the diameter of 0.6cm, culturing at 28 ℃ for 5 days, dripping 0.5% Congo red solution to stain for 1 hour, covering the plate with 1moL/LNaCl solution to decolorize for 10min, and determining that a transparent circle appears around a bacterial colony as positive. The diameter D (cm) of the transparent ring and the diameter D (cm) of the colony are measured, and the D/D value is calculated, wherein the larger the ratio is, the stronger the cellulase production capacity is, and each experiment is repeated for 3 times. The results are shown in table 8 and fig. 10:

the result shows that the periphery of the composite bacterial colony has an obvious transparent hydrolysis ring, which indicates that the composite bacteria has the capability of producing the cellulase.

TABLE 8 results of the stress-resistant growth-promoting microbial inoculum growth-promoting characteristics test

Note: "+" indicates that the complex microbial inoculum has the growth promoting property.

Example 6

Test method for tolerance of different microbial agents to abiotic stress

In the following steps 1) to 3), the microbial inoculum G2 is obtained by shake culturing the G2 seed solution prepared in the example 2 at 28 ℃ and 180r/min for 2d until the viable bacteria concentration of G2 is (1-6) × 10 ⁸ CFU/mL; the microbial inoculum SF1 is obtained by shake culturing SF1 seed solution prepared in example 2 at 28 deg.C and 180r/min for 7d until SF1 viable bacteria concentration is (1-6) × 10 ⁸ CFU/mL; the microbial inoculum SF1+ G2 is obtained by mixing the G2 seed solution SF1 prepared in the embodiment 2 in equal volume, and then culturing for 7d in a shaking table at the temperature of 28 ℃ and at the speed of 180r/min until the total viable bacteria concentration of G2 and SF1 is (1-6) multiplied by 10 ⁸ CFU/mL. The OD600 value of more than 0.1 indicates that the microbial inoculum has stress toleranceOf the cell.

1) And (3) determination of acid and alkali resistance: the acid and alkali resistance of different microbial agents (SF 1, G2, SF1+ G2) is inspected under the conditions of different pH values (3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0), and the intensity of the acid and alkali resistance of the microbial agents is represented by the absorbance at 600 nm. The same concentration (OD) ₆₀₀ = 1) inoculants SF1, G2 and SF1+ G2 are respectively inoculated in co-culture media with different pH values (3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0) according to 1% inoculation amount, after shaking table culture is carried out at 28 ℃ and 180r/min, the absorbance of the inoculants at 600nm under different conditions is measured, and the higher the absorbance is, the stronger the acid and alkali resistance of the inoculants is. Each set of conditions was repeated with 3 sets. The results are shown in Table 9.

TABLE 9 acid and alkali resistance test results

Note: different capital letters in the same column indicate significant differences (P < 0.05).

As can be seen from table 9, the stress-tolerant growth-promoting complex microbial agents SF1+ G2 and the single microbial agents SF1 and G2 are not tolerant at pH =3.0, but the stress-tolerant growth-promoting complex microbial agents SF1+ G2 are significantly tolerant to pH =4.0, pH =5.0, pH =6.0, and pH =10.0 as compared to the single microbial agents SF1 and G2. The acid and alkali resistance of the stress-resistant growth-promoting compound microbial agent is obviously higher than that of a single microbial agent.

2) Determination of drought resistance: the drought resistance of different microbial agents (SF 1, G2, SF1+ G2) is examined under the conditions of different PEG-6000 (0, 2, 5, 10 and 15 percent), and the strength of the drought resistance of the microbial agents is represented by the absorbance at 600 nm. The same concentration (OD) ₆₀₀ = 1) inoculants SF1, G2 and SF1+ G2 are respectively inoculated in co-culture media of different PEG-6000 (0, 2, 5, 10 and 15%) according to the inoculation amount of 1%, after shaking bed culture is carried out at 28 ℃ and 180r/min, the absorbance of the inoculants at 600nm under different conditions is measured, and the higher the absorbance is, the stronger the drought resistance of the inoculants is. Each set of conditions was repeated with 3 sets.

TABLE 10 drought resistance test results

Degree of drought	0％PEG	2％PEG	5％PEG	10％PEG	15％PEG
						SF1	1.17±0.0014C	1.29±0.0021C	0.95±0.039C	0.63±0.010C	0.41±0.013C
G2	2.39±0.0059B	2.35±0.0061A	2.14±0.0028B	1.61±0.0040B	1.45±0.028B
						SF1+G2	2.44±0.0047A	2.24±0.0026B	2.25±0.0026A	2.17±0.014A	2.02±0.013A

As is clear from Table 10, the stress-tolerant growth-promoting complex microbial agents SF1+ G2 had a certain drought stress tolerance, and the 5% PEG, 10% PEG, 15% PEG drought tolerance was significantly improved as compared with the single agents SF1, G2. The stress-tolerant growth-promoting compound microbial agent is shown to have the drought-tolerant capability remarkably higher than that of a single microbial agent.

3) And (3) measuring the capability of resisting different salinity: the salt tolerance of different microbial agents (SF 1, G2, SF1+ G2) is examined under the condition of sodium chloride (0, 50, 100, 200 and 400 mM) with different concentrations, and the intensity of the salt tolerance of the microbial agents is represented by the absorbance at 600 nm. The same concentration (OD) ₆₀₀ = 1) inoculants SF1, G2 and SF1+ G2 are respectively inoculated in co-culture media of sodium chloride (0, 50, 100, 200 and 400 mM) with different concentrations according to the inoculation amount of 1%, after shaking table culture is carried out at 28 ℃ and 180r/min, the absorbance of the inoculants at 600nm under different conditions is measured, and the higher the absorbance is, the stronger the salt tolerance of the inoculants is. Each set of conditions was repeated with 3 sets.

TABLE 11 salinity tolerance test results

Salinity	0mMNaCl	50mMNaCl	100mMNaCl	200mMNaCl	400mMNaCl
						SF1	1.19±0.028B	1.14±0.0037C	0.88±0.011C	0.92±0.0036C	0.83±0.011C
G2	2.43±0.0087A	2.42±0.0063B	2.42±0.0042A	2.39±0.0021A	2.39±0.0019A
						SF1+G2	2.47±0.0091A	2.46±0.0028A	2.34±0.0042B	2.23±0.0053B	2.25±0.0058B

As can be seen from Table 11, the biomass of the stress-resistant growth-promoting compound microbial inoculum SF1+ G2 is not significantly changed compared with the biomass of a single microbial inoculum SF1 under different salinity conditions, which indicates that the compound microbial inoculum has good salt-resistant stability and higher salt stress-resistant property.

Example 7 examination of the stability of different types of microbial Agents

Respectively carrying out acid-base, heat and ultraviolet irradiation treatment on different types of microbial inoculum, adopting a flat plate confrontation experiment, taking target pathogenic bacterium sclerotinia sclerotiorum DS-8 as an indicator bacterium, and investigating the stability of the different microbial inoculum according to the bacteriostasis rate of the microbial inoculum.

1) Influence of pH value on microbial inoculum stability

5mL of the same concentration (OD) were taken ₆₀₀ = 1), adjusting the pH of different microbial agents (SF 1, G2, SF1+ G2) in a sterilized centrifuge tube with sodium hydroxide and hydrochloric acid solutions to 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, and 11.0, respectively, placing the sterilized centrifuge tube at room temperature, after 2 hours, determining the inhibition rates of the microbial agents subjected to different acid and alkali treatments by the method in the step 1) of example 3, and examining the acid and alkali resistance characteristics of different types of microbial agents according to the inhibition rates.

TABLE 12 results of the influence of pH on the stability of the microbial inoculum

Note: different lower case letters indicate significant difference under the same pH condition (P < 0.05).

As can be seen from table 12, the treatment with pH =3.0 has a certain inhibitory effect on the bacteriostatic effect of different types of microbial agents, indicating that the treatment under the conditions has a certain influence on the stability of the microbial agents. Under the conditions of pH =4.0 and pH =5.0, the stress-resistant growth-promoting compound microbial inoculum has a significantly higher bacteriostatic rate than that of a single microbial inoculum, which indicates that the stability of the compound microbial inoculum SF1+ G2 is significantly greater than that of the single microbial inoculum under the conditions of pH =4.0 and pH = 5.0; the stress-resistant growth-promoting compound microbial agent has no obvious difference in bacteriostasis rate compared with a single microbial agent under other pH conditions, but the whole phenomenon shows that the bacteriostasis rate of the compound microbial agent is slightly higher than that of the single microbial agent.

2) Influence of temperature on the stability of the inoculum

Respectively taking 5mL of the same concentration (OD) ₆₀₀ = 1) microbial Agents (SF 1, G2, SF1+ G2) in sterilized centrifuge tubes, after treatment in water bath (30 deg.C, 40 deg.C, 50 deg.C, 60 deg.C, 70 deg.C, 80 deg.C) for 2h, the procedure of example 3 was followedThe method in the step 1) is used for measuring the bacteriostasis rates of the bactericides treated at different temperatures, and the heat-resisting characteristics of different types of bactericides are investigated according to the bacteriostasis rates.

TABLE 13 results of temperature effect on microbial inoculum stability

Note: different lower case letters indicate significant difference (P < 0.05) under the same temperature condition.

As can be seen from Table 13, when the temperature of the water bath is higher than 60 ℃, the bacteriostatic rates of different types of microbial inoculum show a decreasing trend. The stress-resistant growth-promoting compound microbial agent has the bacteriostasis rate which is obviously higher than that of a single microbial agent at the temperature of 60 ℃ and is not obviously different from that of a G2 single microbial agent at the temperature of 70 ℃ and 80 ℃, but the whole bacteriostasis rate is higher than that of the G2 single microbial agent, so that the stability of the compound microbial agent SF1+ G2 under the heat treatment condition is improved compared with that of the single microbial agent.

3) Influence of ultraviolet irradiation on stability of microbial inoculum

5mL of the same concentration (OD) were taken ₆₀₀ = 1) different microbial agents (SF 1, G2, SF1+ G2) are shaken evenly and flatly in a sterilized empty culture dish, the culture dish is respectively irradiated under a 30w ultraviolet lamp for 5min, 10min, 15min, 20min, 25min and 30min, the method in 1) in the embodiment 3 is adopted to measure the bacteriostasis rate of the microbial agents after treatment of different irradiation time, and the ultraviolet irradiation resistance characteristics of different types of microbial agents are examined according to the size of the bacteriostasis rate.

TABLE 14 Effect of UV irradiation on microbial inoculum stability

Note: different lower case letters under the same uv irradiation indicate significant difference (P < 0.05).

As can be seen from Table 14, the bacteriostatic ratios of different microbial agents are in a descending trend along with the increase of the irradiation time, which indicates that ultraviolet irradiation has a certain influence on the stability of the microbial agents. Under the conditions of ultraviolet irradiation for 10min and 25min, the stress-resistant growth-promoting compound microbial inoculum has the bacteriostasis rate obviously higher than that of a single microbial inoculum, and the bacteriostasis rates of the compound microbial inoculum under other different irradiation time conditions are not obviously different from that of the single microbial inoculum but are slightly higher than that of the single microbial inoculum, so that the stability of the compound microbial inoculum under the ultraviolet irradiation is higher than that of the single microbial inoculum.

Example 8

Influence of stress-resistant growth-promoting compound microbial agent on germination of liquorice seeds and growth of seedlings

Pretreatment of liquorice seeds: selecting full and uniform Glycyrrhrizae radix seed, and adding 85% concentrated H ₂ SO ₄ Soaking for 45min, stirring at variable times, washing with distilled water for 3 times, and adding 0.1% H ₂ O ₂ Sterilizing for 10min, washing with distilled water for several times until no viscosity exists, cleaning, placing in a beaker, soaking in distilled water for 6-8 hr to make the seeds fully absorb water, and standing by.

Experiment design: the method adopts completely random design, and 4 treatments are respectively set, namely a control CK (sterilized distilled water), a microbial inoculum G2 (the preparation method is the same as that in example 6), a microbial inoculum SF1 (the preparation method is the same as that in example 6), and a stress-resistant growth-promoting microbial inoculum (SF 1+ G2) in example 3. Selecting full and uniform liquorice seeds which absorb water fully, sucking surface water, diluting the three groups of treated microbial inoculum with sterilized distilled water until OD value is 1 (indicating concentration is consistent), and then soaking the liquorice seeds respectively; the seeds are respectively soaked in different treatment solutions (sterilized distilled water, a microbial inoculum G2, a microbial inoculum SF1 and a stress-resistant growth-promoting microbial inoculum) for 3 hours, and then the seeds are uniformly placed in culture dishes (9 cm multiplied by 3cm, 3mL of sterilized distilled water is added into each dish for moisture preservation) on which double-layer sterile filter paper is padded for germination, and 40 seeds are placed in each dish. The experimental conditions were light/dark (12/12h, 28/20 ℃ C.). Distilled water was added to a constant mass by weighing every day to maintain constant matrix conditions. After germination, determining the germination rate, germination index, seedling activity index and dry weight of each treated seed; the lengths of the embryonic axis and the embryonic root of the licorice seedlings were measured with a ruler, and the thicknesses of the embryonic axis and the embryonic root of the seedlings were measured with a vernier caliper, and the results are shown in Table 15.

TABLE 15 Germination test results of Glycyrrhiza uralensis Fisch

Note: different lower case letters in the same row indicate significant difference (P < 0.05).

The results in table 15 show that the stress-resistant growth-promoting microbial agent has a remarkable promoting effect on the germination of liquorice seeds and the growth of seedlings, and compared with a control, the stress-resistant growth-promoting microbial agent can remarkably increase the germination rate and the germination index of the liquorice seeds, the root length of seedlings, the thickness of radicles and hypocotyls, the vitality index of the seedlings and the dry weight of the seedlings after being treated by the stress-resistant growth-promoting compound microbial agent; compared with the single G2 bacterial liquid treatment, the seedling activity index is obviously increased; compared with the SF1 single bacterial liquid treatment, the germination index, the dry weight, the embryonic root length and the embryonic axis thickness of the seedlings are obviously increased.

The stress-resistant growth-promoting compound microbial agent formed by compounding the streptomyces SF1 and the bacillus cereus G2 can obviously improve the stress-resistant capability of plants and promote the growth of the plants, and has more obvious and outstanding corresponding effect compared with the microbial agent formed by single streptomyces SF1 and single bacillus cereus G2.

Although the present invention has been described in detail with reference to the above embodiments, it is only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and all of the embodiments are included in the scope of the present invention.

Sequence listing

<110> Ningxia medical university

<120> stress-resistant growth-promoting compound microbial agent and application thereof

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<170> SIPOSequenceListing 1.0

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aaagcatcag agatggtgcc ccccttgtgg tcgggtgaca ggtggtgcat ggctgtcgtc 1020

agctcgtgtc gtgagatgtt gggttaagtc ccgcaacgag cgcaaccctt gttctgtgtt 1080

gccagcatgc ccttcggggt gatggggact cacaggagac tgccggggtc aactcggagg 1140

aaggtgggga cgacgtcaag tcatcatgcc ccttatgtct tgggctgcac acgtgctaca 1200

atggcaggta caatgagctg cgataccgca aggtggagcg aatctcaaaa agcctgtctc 1260

agttcggatt ggggtctgca actcgacccc atgaagtcgg agttgctagt aatcgcagat 1320

cagcattgct gcggtgaata cgttcccggg ccttgtacac accgcccgtc acgtcacgaa 1380

agtcggtaac acccgaagcc ggtggcccaa 1410

<210> 2

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

cagagtttga tcctggctag gaggtgatcc agccgca 37

<210> 3

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

agtttgatcm tggctcaggg ttaccttgtt acgactt 37

Claims

1. The stress-tolerant growth-promoting compound microbial agent is characterized in that the effective components of the stress-tolerant growth-promoting compound microbial agent comprise one or more of thallus, bacterial liquid and metabolite of Streptomyces (Streptomyces sp.) and Bacillus cereus.

2. The stress-resistant growth-promoting composite microbial inoculant according to claim 1, wherein the streptomyces comprises streptomyces SF1, and the preservation number is CGMCC No.23417; the bacillus cereus comprises bacillus cereus G2, and the preservation number is CGMCC No.16671.

3. The stress-tolerant growth-promoting composite microbial inoculant according to claim 2, wherein the concentrations of streptomyces SF1 and bacillus cereus G2 in the stress-tolerant growth-promoting microbial inoculant are (1-6) x 10 ⁸ CFU/mL。

4. A stress-tolerant growth-promoting complex microbial inoculant according to claim 2 or 3, wherein the effective concentration of the stress-tolerant growth-promoting complex microbial inoculant is (1-6) x 10 ⁸ CFU/mL。

5. Use of the stress-tolerant growth-promoting complex microbial inoculant according to any one of claims 1 to 4 for one or more of enhancing abiotic stress tolerance of a plant, enhancing biotic stress tolerance of a plant and promoting plant growth.

6. Use according to claim 5, wherein the abiotic stress includes one or more of high acid-base, drought, high salt, high temperature and UV stress.

7. Use according to claim 5, wherein the biotic stress comprises a plant disease.

8. The use according to claim 7, wherein the plant disease comprises one or more of plant root rot, plant gray mold, plant leaf spot, plant anthracnose and plant leaf spot.

9. Use according to claim 5, wherein the promotion of plant growth comprises promotion of seed germination and/or seedling growth.

10. The use according to any one of claims 6 to 9, wherein the plant comprises a medicinal plant.