A strain of Streptomyces with biological control effect and a disease-resistant growth-promoting bacterial agent and their application

Abstract

The object of the present invention is to provide a strain of Streptomyces with biocontrol effect and a disease-resistant and growth-promoting bacterial agent and their applications. The strain of the present invention can effectively control plant diseases and promote plant growth. The Streptomyces SF1 of the present invention has antagonistic effects on a variety of fungal pathogens, and can effectively control a variety of plant diseases including plant root rot, plant leaf spot, plant anthracnose and plant phyllosticta; at the same time, the Streptomyces SF1 can metabolize and produce a variety of growth-promoting substances such as indole acetic acid, NH3, ACC deaminase, nitrogen fixation, siderophore production, inorganic phosphorus solubilization, organic phosphorus solubilization, protease production, potassium solubilization and cellulase, and has the effect of promoting plant growth.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial agent preparation, and specifically relates to a strain of Streptomyces with biological control effect and an anti-disease growth-promoting agent and applications thereof. Background Art

[0002] Plant diseases refer to pathological phenomena in which plants cannot grow and develop normally due to environmental discomfort or infection by pathogens, resulting in a significant reduction in their yield and quality. A large number of studies have shown that plant diseases occur in a wide range, cause widespread damage, and are of many types. Most of them are caused by pathogenic fungi, which are highly contagious and difficult to eradicate. At the same time, due to the influence of abiotic factors such as drought and soil salinization, as well as unreasonable planting patterns such as long-term continuous cropping, plants grow slowly, resulting in a significant reduction in their yield and quality.

[0003] For a long time, chemical pesticides and fertilizers have played a historic role in preventing and controlling plant diseases, increasing plant yields and ensuring human food safety. However, while reducing plant diseases, chemical pesticides and fertilizers also have adverse effects on human health, the environment and biodiversity. Therefore, in order to comply with the development trend of "pure natural, pollution-free and pollution-free" green organic products and achieve the national goal of zero growth in chemical pesticides and fertilizers, it is particularly urgent to improve the prevention and control of plant diseases.

[0004] In order to overcome the pollution problem of chemical pesticides and fertilizers in plant disease prevention and control, microbial agents prepared from microorganisms and their metabolites play an increasingly important role in plant disease prevention and control and promoting plant growth, and there are more and more strains and application types of microbial agents in the prior art. Actinomycetes, as a disease-resistant and growth-promoting microorganism, have the advantages of good prevention effect, broad spectrum of action, and rich types of antibiotics compared with bacteria and fungi, and have obvious application potential in preventing and controlling plant diseases and promoting plant growth. Therefore, it is of great significance to screen strain resources with disease-resistant and growth-promoting properties and develop new microbial agents that can gradually replace chemical pesticides and fertilizers. Summary of the invention

[0005] The purpose of the present invention is to provide a strain of Streptomyces with biological control effect and an anti-disease growth-promoting bacterial agent and their application. The strain of the present invention can effectively prevent and control plant diseases and promote plant growth.

[0006] The invention provides a strain of Streptomyces sp. SF1 with biological control effect. The preservation number of the Streptomyces sp. SF1 is CGMCC No.23417.

[0007] The present invention also provides a disease-resistant and growth-promoting bacterial agent, the active ingredients of which include the Streptomyces SF1 described in the above technical solution.

[0008] Preferably, the concentration of Streptomyces SF1 in the disease-resistant growth-promoting bacterial agent is (1-6)×10 8 CFU / mL.

[0009] The present invention also provides the use of the Streptomyces SF1 described in the above technical scheme or the disease-resistant and growth-promoting bacterial agent described in the above technical scheme in preventing and controlling plant diseases and / or promoting plant growth.

[0010] Preferably, the plants include medicinal plants.

[0011] Preferably, the medicinal plants include angelica, codonopsis, astragalus, wolfberry, yam and liquorice.

[0012] Preferably, the pathogens of the plant diseases include fungal pathogens.

[0013] Preferably, the pathogenic bacteria include one or more of Fusarium oxysporum, Sclerotinia sclerotiorum, Rhizoctonia solani, Alternaria sordella, Colletotrichum oxysporum and Phyllosporium.

[0014] Preferably, the plant diseases include one or more of plant root rot, plant leaf spot, plant anthracnose and plant leaf spot.

[0015] Preferably, the promoting plant growth comprises promoting seed germination and / or seedling growth.

[0016] Beneficial effects:

[0017] The present invention provides a strain of Streptomyces sp. SF1 with biological control effect and an anti-disease and growth-promoting bacterial agent, and biological preservation has been completed. The Streptomyces sp. SF1 and its bacterial agent described in the present invention have antagonistic effects on a variety of fungal pathogens, and can effectively prevent and treat a variety of plant diseases including plant root rot, plant leaf spot, plant anthracnose and plant leaf spot; at the same time, the Streptomyces sp. SF1 can metabolize and produce a variety of growth-promoting substances such as indoleacetic acid, NH3, ACC deaminase, nitrogen fixation, siderophore production, inorganic phosphorus solubilization, organic phosphorus solubilization, protease production, potassium solubilization and cellulase, and has the effect of promoting plant growth.

[0018] Biological deposit information

[0019] Streptomyces sp. SF1 was deposited in the General Microbiology Center (CGMCC) of China Microorganism Culture Collection on September 15, 2021. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and the deposit number is CGMCC No. 23417. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0021] Figure 1 This is the colony morphology of Streptomyces SF1;

[0022] Figure 2 This is the antagonistic colony diagram of the disease-resistant growth-promoting bacteria agent of Example 2 against Rhizoctonia solani, Fusarium oxysporum and Sclerotinia sclerotiorum;

[0023] Figure 3 The antagonistic colony diagram of the antimicrobial spectrum of the disease-resistant growth-promoting bacterial agent in Example 2 (Alternaria suis, Colletotrichum oxysporum and Phyllosporium dioscorea);

[0024] Figure 4 This is a safety evaluation diagram for disease-resistant growth-promoting bacteria;

[0025] Figure 5 This is a graph showing the results of an in vitro disease prevention test of disease-resistant growth-promoting bacteria;

[0026] Figure 6 This is a comparison chart of colonies in the colonization experiment of disease-resistant and growth-promoting bacteria in the tested root tissues;

[0027] Figure 7 This is a study diagram of the growth-promoting properties of Streptomyces SF1;

[0028] Figure 8 This is the standard curve of IAA production by Streptomyces SF1 in Example 4;

[0029] Fig. 9 This is a diagram showing the effect of disease-resistant and growth-promoting bacteria on licorice seed germination.

[0030] exist Figures 4 to 6 Among them, A represents Angelica, B represents Astragalus, and C represents Codonopsis.

[0031] In the above-mentioned figures, SF1 is used to represent the disease-resistant growth-promoting bacteria or Streptomyces SF1, and the description of the embodiments of the corresponding figures shall prevail. DETAILED DESCRIPTION

[0032] The invention provides a strain of Streptomyces sp. SF1 with biological control effect. The preservation number of the Streptomyces sp. SF1 is CGMCC No.23417.

[0033]

[0034] The colony morphology of Streptomyces SF1 described in the present invention is: the colony is light yellow, nearly circular, with regular edges, the colony is small and does not extend widely, the surface is smooth and opaque, the colony is tightly bound to the culture medium and is not easy to pick up. Figure 1 shown.

[0035] The Streptomyces SF1 described in the present invention has the characteristics of producing citric acid, esterase, oxidase, catalase, urease and indoleacetic acid; the acid and alkali resistance range is: pH 5.0-9.0, and the optimal growth pH is 7.0; the temperature tolerance range is 15-37°C, and the optimal growth temperature is 28°C; the available carbon sources include D-mannitol, D-galactose, D-xylose, D-glucose, D-maltose, D-fructose, D-sorbitol and sucrose; and the available nitrogen sources include urea, glutamine, glycine, ammonium sulfate and histidine.

[0036] The Streptomyces SF1 described in the present invention has an antagonistic effect on a variety of fungal pathogens, and can effectively prevent and control a variety of plant diseases including plant root rot, plant leaf spot, plant anthracnose and plant leaf spot; at the same time, the Streptomyces SF1 can metabolize and produce a variety of growth-promoting substances such as indoleacetic acid, NH3, ACC deaminase, nitrogen fixation, siderophore production, inorganic phosphorus solubility, organic phosphorus solubility, protease production, potassium solubility and cellulase, and has the effect of promoting plant growth.

[0037] The present invention also provides a disease-resistant and growth-promoting bacterial agent, the active ingredients of which include the Streptomyces SF1 described in the above technical solution.

[0038] The concentration of Streptomyces SF1 in the disease-resistant growth-promoting bacterial agent of the present invention is preferably (1-6)×10 8 CFU / mL.

[0039] The present invention also provides a method for preparing the above-mentioned disease-resistant growth-promoting agent, comprising the following steps: inoculating the activated Streptomyces SF1 into a liquid culture medium, culturing for 5 to 7 days, and obtaining the disease-resistant growth-promoting agent. The liquid culture medium of the present invention is preferably Gao's No. 1 liquid culture medium. In the embodiment, Gao's No. 1 liquid culture medium is used as an example for illustration, and comprises the following components: soluble starch 20g, KNO31g, K2HPO40.5g, NaCl 0.5g, MgSO40.5g, FeSO40.01g, distilled water 1000mL, natural pH. The culture temperature of the present invention is preferably 15 to 37°C, more preferably 28°C; the culture time is preferably 5 to 7d, more preferably 7d. The present invention preferably further comprises dividing the activated Streptomyces SF1 into block cakes and then inoculating them into the liquid culture medium. The diameter of the block cake of the present invention is preferably 0.6cm. The present invention does not specifically limit the activation method, and the conventional activation method in the art can be used. The concentration of Streptomyces SF1 in the disease-resistant growth-promoting bacterial agent prepared by the present invention is preferably (1 to 6)×10 8 CFU / mL.

[0040] The present invention also provides the use of the Streptomyces SF1 described in the above technical solution or the disease-resistant growth-promoting bacterial agent described in the above technical solution in preventing and controlling plant diseases and / or promoting plant growth. The plants described in the present invention preferably include medicinal plants, more preferably include angelica, codonopsis, astragalus, wolfberry, yam and licorice, and further preferably angelica, astragalus, codonopsis and licorice.

[0041] The pathogens of the plant diseases described in the present invention preferably include fungal pathogens, more preferably include Fusarium oxysporum, Sclerotinia sclerotiorum, Rhizoctonia solani, Alternaria sordella, Colletotrichum oxysporum and Phyllostomium, and further preferably include Fusarium oxysporum, Sclerotinia sclerotiorum and Rhizoctonia solani.

[0042] The plant diseases described in the present invention preferably include plant root rot, plant leaf spot, plant anthracnose and plant leaf spot, and more preferably include angelica root rot, astragalus root rot, codonopsis root rot, astragalus leaf spot, wolfberry anthracnose and yam leaf spot.

[0043] The promoting of plant growth of the present invention preferably includes promoting seed germination and seedling growth; the promoting of seed germination preferably includes improving seed germination rate, germination potential and germination index; the promoting of seedling growth includes increasing the length of seedling radicle, promoting radicle thickening, improving seedling vigor and increasing seedling dry weight.

[0044] When the disease-resistant growth-promoting bacterial agent of the present invention is applied to actual production, the present invention has no special limitation on the use of the disease-resistant growth-promoting bacterial agent, and the conventional use of microbial agents in the art can be adopted.

[0045] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0046] In the following examples, unless otherwise specified, the reagents and materials used are all available from commercial sources.

[0047] The Rhizoctonia solani, Fusarium oxysporum, Sclerotinia sclerotiorum, Alternaria thunbergii, Colletotrichum oxysporum, and Leaf Spot Mildew of yam described in the embodiments of the present invention are all conventional strains, and the names of DG-13, HQI-5, DS-8, HQ-6, TJD, TJA, TJCE, and WHN7 are only used to distinguish the infecting pathogens on different plants.

[0048] Example 1: Isolation and identification of Streptomyces SF1

[0049] 1) Select healthy licorice seeds with full grains, soak the seeds in 85% concentrated sulfuric acid solution by mass for 45 minutes, stir them irregularly, rinse them with sterile water 3 times, then soak them in 0.1% hydrogen peroxide solution for 10 minutes, rinse them with sterile water 5 times, and then soak them in sterile water for 3 hours, and then transfer them to the clean bench to disinfect the seeds on the surface. Under sterile conditions, soak them in 75% ethanol solution by volume for 5 minutes, 1% hydrogen peroxide solution for 30 seconds, 5% sodium hypochlorite solution for 10 seconds, and rinse them with sterile water 10 times. The surface-sterilized licorice seeds are placed in a sterile culture dish pre-laid with sterile absorbent paper, and placed on a sterile clean bench to dry naturally. (To ensure thorough disinfection, take 200 μL of the washing water from the last washing of licorice seeds and spread it on NA solid medium (10.0 g of peptone, 3.0 g of beef extract, 5.0 g of sodium chloride, 17 g of agar, 1000 mL of distilled water). The plate was placed at 28°C for one week and no bacteria were found to grow.) Put the treated licorice seeds in a sterile mortar and add 10 mL of sterile water to grind. Take 200 μL of the suspension and spread it evenly on Gao's No. 1 solid medium. After culturing in an artificial climate box at 28°C for 5 days, pick a single colony of the strain and purify it by streaking to obtain a pure culture strain, named SF1.

[0050] The isolated strains were inoculated by streaking on different culture media and cultured at 28°C for 5 to 7 days. The color and growth status of aerial hyphae, intrabasal hyphae and soluble pigment of the colony were recorded. The results are shown in Table 1.

[0051] Table 1 Growth of SF1 in different culture media

[0052] Culture medium Aerial hyphae Mycelium in the base Soluble pigments Growth status Gao's medium No.1 Yellowish white tan Light yellow +++ Czapek medium Yellowish white Dark brown none +++ Agar nutrient medium Off-white Off-white none +++ PDA medium Off-white Dark brown none +++ ISP2 medium milky Light yellow none +++ ISP3 medium Off-white Off-white Light yellow +++ ISP4 medium White Taupe Light yellow +++

[0053] As can be seen from Table 1, the SF1 strain of the present invention can grow well in different culture media, and the colors of its aerial hyphae and intrabasal hyphae in different culture media are different, but mainly yellow-brown and yellow-white, and the soluble pigment conditions are also different, indicating that SF1 is a single microorganism with good stability.

[0054] 2) The physiological and biochemical characteristics of the isolated SF1 strain were determined with reference to the Bergey's Manual of Bacterial Identification and the Manual of Identification of Common Bacterial Systems. The results are shown in Table 2.

[0055] Table 2 Physiological and biochemical characteristics of SF1 strain

[0056]

[0057]

[0058] Note: “+” represents positive; “-” represents negative.

[0059] As shown in Table 2, SF1 has the characteristics of producing citrate, esterase, oxidase, catalase, urease, and indoleacetic acid; its acid and alkali resistance pH range is 5.0-9.0, and the optimal growth pH is 7.0; its temperature tolerance range is 15-37℃, and the optimal growth temperature is 28℃; the carbon sources that can be used are D-mannitol, D-galactose, D-xylose, D-glucose, D-maltose, D-fructose, D-sorbitol, and sucrose; the nitrogen sources that can be used are urea, glutamine, glycine, ammonium sulfate, and histidine. Based on the above basic experiments, it is shown that SF1 belongs to the genus Streptomyces.

[0060] 3) DNA was extracted using a DNA extraction kit (purchased from Shanghai Biotech Co., Ltd., catalog number SK8255), and PCR amplification was performed on the extracted DNA using a 16S rDNA bacterial universal primer pair (SEQ ID No. 2 forward primer 7F-1540R: CAGAGTTTGATCCTGGCTAGGAGGTGATCCAGCCGCA, SEQ ID No. 3 reverse primer 27F-1492R: AGTTTGATCMTGGCTCAGGGTTACCTTGTTACGACTT). The PCR reaction system was: template DNA 0.5 μL, 10× buffer (with Mg 2+)2.5μL, dNTP (10mM) 1μL, Taq enzyme (5U / μL) 0.2μL, each primer (10μM) 0.5μL; reaction program: 95℃5min; (94℃30s, 57℃30s, 72℃90s), 30 cycles; 72℃10min. After PCR amplification, 1% agarose gel electrophoresis was used to detect the PCR product, and the amplified product was sent to Sangon (Shanghai) Co., Ltd. for sequencing. After BLAST comparison analysis of the NCBI database, the similarity between this strain and Streptomyces dioscoristrain A217 reached 99.57%, so the strain was identified as Streptomyces sp. and named SF1.

[0061] Example 2 Preparation of disease-resistant growth-promoting bacterial agents and their inhibitory effects on plant pathogens

[0062] 1) Preparation of disease-resistant growth-promoting agent: After activating the SF1 strain, use a sterile puncher to punch out a bacterial cake with a diameter of 0.6 cm containing SF1 colonies. Use a sterile toothpick to pick up a piece of bacterial cake and inoculate it into a 250 mL Erlenmeyer flask containing 100 mL of Gao's No. 1 liquid culture medium. Cultivate at 28°C and 180 r / min for 5 to 7 days until the concentration of Streptomyces SF1 reaches 1×10 8 CFU / mL~6×10 8 CFU / mL to obtain disease-resistant growth-promoting agents.

[0063] 2) Determination of the antibacterial rate of disease-resistant and growth-promoting agents: Use a sterile inoculation loop to pick up pathogenic bacteria (slant culture preservation) and inoculate them into potato dextrose agar (PDA) medium (12.0 g potato extract, 20.0 g glucose, 14.0 g agar, 1000 mL distilled water, natural PH), seal the medium, and culture it in a 28°C constant temperature box. After 5 days of cultivation, the fungi basically cover the entire culture medium and are in a good active state. The plate confrontation method was used to take the pathogenic fungus Rhizoctonia solani DG-13 of Angelica sinensis root rot, the pathogenic fungus Fusarium oxysporum HQI-5 of Astragalus root rot and the pathogenic fungus Sclerotinia sclerotiorum DS-8 of Codonopsis pilosula root rot that had been cultured for 5 days. A 0.6 cm hole puncher was used to make a bacterial cake, which was inverted in the center of potato dextrose agar (PDA) solid culture medium. At the same time, a sterile filter paper with a diameter of 0.6 cm was flatly pasted on four points on the same cross line 2 cm away from the center of the bacterial cake, and 5 μL of the disease-resistant and growth-promoting bacteria agent was inoculated on it (test group), and an equal amount of sterile distilled water was added as the control group (see Figure 1 Each treatment was repeated 3 times, and the culture was carried out at 28°C until the colonies of the pathogens stopped growing. The diameter of the pathogen colonies was measured by the cross method and the inhibition rate was calculated. The results are shown in Table 3 and Figure 2 shown.

[0064] Antibacterial rate (%) = (D1-D2) / (D1-D3) × 100%;

[0065] Among them, D1 represents the diameter of the pathogen colony in the control group (inoculated with an equal amount of sterilized distilled water); D2 represents the diameter of the pathogen colony in the test group (inoculated with anti-disease and growth-promoting bacteria); and D3 represents the diameter of the pathogen cake (0.6 cm).

[0066] Table 3 Results of the experiment on antagonism of pathogenic bacteria by anti-disease growth-promoting bacteria

[0067]

[0068] From Table 3 and Figure 2 It can be seen that the disease-resistant and growth-promoting bacteria have a significant inhibitory effect on Rhizoctonia solani DG-13, Fusarium oxysporum HQI-5 and Sclerotinia sclerotiorum DS-8, and the inhibition rates are 63.21±1.53%, 64.84±1.35% and 74.19±2.88%, respectively.

[0069] 3) The antibacterial spectrum of the disease-resistant growth-promoting agent (the pathogen of Astragalus leaf spot disease, Alternaria suis HQ-6, the pathogen of wolfberry anthracnose, Colletotrichum oxysporum TJD, the pathogen of wolfberry anthracnose, Colletotrichum oxysporum TJA, the pathogen of wolfberry anthracnose, Colletotrichum oxysporum TJCE, the pathogen of wolfberry anthracnose, Colletotrichum oxysporum WHN7, the pathogen of yam leaf spot disease, Leaf spot of yam) was determined by the method in step 2), and the results are shown in Table 4 and Figure 3 shown.

[0070] Table 4 Antimicrobial spectrum test results of anti-disease growth-promoting bacteria

[0071]

[0072] From Table 4 and Figure 3 It can be seen that the disease-resistant growth-promoting bacterial agent of the present invention has a wide antibacterial spectrum, and has a strong inhibition rate against Alternaria suis HQ-6, Colletotrichum oxysporum TJD, Colletotrichum oxysporum TJA, Colletotrichum oxysporum TJCE, Colletotrichum oxysporum WHN7 and Leaf Spot of Dioscorea opposita. Figure 1 The results further indicate that the disease-resistant and growth-promoting bacterial agent with Streptomyces SF1 as the effective active ingredient of the present invention has a wide range of antibacterial activity and a significant antibacterial effect on each tested pathogen.

[0073] Example 3 Evaluation of the safety and disease prevention efficacy of anti-disease growth-promoting bacteria

[0074] Preparation of test materials: Take the roots of healthy test plants (Angelica, Astragalus, Codonopsis) and rinse them with running water for 30 minutes, then soak them in 75% alcohol for 20 seconds, 3% sodium hypochlorite solution for 3 minutes, and then rinse them with sterile distilled water for 5 times; use a sterile surgical blade to cut the surface-sterilized roots into 5 mm thick slices for later use.

[0075] Preparation of pathogen spore suspension: The test pathogens DG-13, HQI-5, and DS-8 were inoculated on fresh PDA solid medium and cultured at 28°C for 7 days. 100 mL of sterile distilled water was poured in and the spores were gently washed by shaking. The washing solution was filtered through sterile gauze into a sterile triangular flask. The concentration of the spore suspension was determined using a hemocytometer and made into 10 6 / mL suspension for later use.

[0076] 1) Safety evaluation of disease-resistant and growth-promoting microbial agents

[0077] The sterilized and sliced ​​roots of the test plants were placed in a sterile culture dish containing sterilized filter paper and 1 mL of sterile water was added to keep them moist. 20 μL of the disease-resistant and growth-promoting bacteria in Example 2 (referred to as SF1) was inoculated by bacterial liquid spot inoculation, and the same amount of distilled water was inoculated with CK. After sealing, the dish was cultured at 28°C in the dark. After 7 days, the disease index of each treatment was calculated according to the severity grading standard, and the incidence rate was calculated. Each treatment was repeated 9 times. The severity grading standard is shown in Table 5, and the results are shown in Table 6 and Figure 4 .

[0078] Disease index = Σ(number of diseased plants at each level × severity level) / (total number of tested plants × highest level) × 100%; the highest level is 5.

[0079] Incidence rate = (number of diseased plants / total number of tested plants) × 100%

[0080] Table 5 Severity grading standards

[0081]

[0082] Table 6 Safety evaluation results of Streptomyces SF1

[0083]

[0084] From Table 6 and Figure 4 It can be concluded that after inoculation with the disease-resistant growth-promoting bacteria of the present invention, the number of diseases and the incidence rate of each test plant were not significantly different from those in the CK group, indicating that the disease-resistant growth-promoting bacteria of the present invention are non-pathogenic and can continue to be developed and utilized.

[0085] 2) In vitro disease prevention test of disease-resistant and growth-promoting bacteria

[0086] The sterilized and sliced ​​roots of the test plants were placed in a sterile culture dish containing sterilized filter paper with 1 mL of sterile water added for moisture retention. The experiment set up three treatments: CK (only inoculated with an equal amount of sterile distilled water), inoculated with pathogenic bacteria spore suspension (respectively recorded as DG-13, HQI-5, DS-8), and the disease-resistant growth-promoting bacteria agent in Example 2 plus the pathogenic bacteria spore suspension (respectively recorded as SF1+DG-13, SF1+HQI-5, SF1+DS-8). The disease-resistant growth-promoting bacteria agent plus pathogenic bacteria spore suspension group in Example 2 was: 20 μL of disease-resistant growth-promoting bacteria agent was inoculated by bacterial liquid spot inoculation, and 24 hours after spot inoculation, 20 μL of pathogenic bacteria spore suspension was inoculated in the same way, sealed and placed in dark culture at 28°C, and after 7 days, the disease index of each treatment and the relative prevention effect of the disease-resistant growth-promoting bacteria agent were calculated according to the severity classification standard, and the incidence rate was statistically calculated. Each treatment was repeated 9 times, and the results are shown in Tables 7 and Figure 5 .

[0087] Relative protective effect (%) = (disease index of roots inoculated with pathogens only - disease index of roots inoculated with disease-resistant growth-promoting bacteria and pathogens in vitro) / disease index of roots inoculated with disease-resistant growth-promoting bacteria and pathogens in vitro × 100%.

[0088] Table 7 In vitro disease prevention test results of disease-resistant growth-promoting bacteria

[0089]

[0090] From Table 7 and Figure 5 It can be seen that inoculation of disease-resistant and growth-promoting bacteria can significantly reduce the incidence of the test plants, and the relative protective effects on Angelica sinensis, Astragalus membranaceus, and Codonopsis pilosula are 93.33%, 86.67%, and 73.33%, respectively.

[0091] 3) Colonization experiment of disease-resistant and growth-promoting bacteria in the test root tissue

[0092] The test tissue samples obtained in Example 3, 2) were preserved, the test root tissue samples CK (inoculated with an equal amount of sterile distilled water only) and the test root tissue samples treated with the disease-resistant growth-promoting bacteria agent plus the pathogen spore suspension in Example 2, the endophytes were isolated, and the colonization of Streptomyces SF1 in the test root tissues after different treatments was compared.

[0093] Isolation of endophytes in test root tissues: Take the test root tissues with different treatments after the experiment, and then grind them in a sterile mixing cup. Take 2g into a sterile mortar, add liquid nitrogen until the root powder is just covered, and then grind for 2-3min. Repeat twice, take 1ml of the supernatant into a sterile test tube, and then dilute it to 10 with sterile water. -3 and 10 -4Two gradients, 200 μL were taken and spread on Gao's solid medium No. 1, and placed in a constant temperature box at 28℃ for 5-7 days. The presence or number of SF1 colonies was used to measure whether SF1 was colonized in the test root tissue. The results are shown in Figure 6 .

[0094] Depend on Figure 6 It can be seen that there is no Streptomyces SF1 in the endophytes isolated from the test plants of Angelica sinensis and Codonopsis pilosula in the CK group, and there is a very small amount of Streptomyces SF1 in the endophytes of Astragalus membranaceus. However, after inoculation with the disease-resistant and growth-promoting bacteria in Example 2, Streptomyces SF1 can be isolated from the in vitro root tissues of the test plants (Angelica sinensis and Codonopsis pilosula) and the number of Streptomyces SF1 is significantly increased (Astragalus membranaceus).

[0095] Example 4 Study on the Growth-Promoting Properties of Streptomyces SF1

[0096] 1) Ability to produce IAA (indoleacetic acid): A single colony of the SF1 strain was selected and inoculated into Gao's liquid medium No. 1 containing 0.5 g / L tryptophan. The culture was shaken at 28°C and 180 r / min for 5 days, and then centrifuged at 9000 r / min for 10 min. 2 mL of the supernatant was added with 4 mL of Salkowski reagent (a mixture of 0.5 mM FeCl3 and 50 mL H2SO4). The culture was placed at 28°C in the dark for 30 min and then turned red. This indicated that Streptomyces SF1 could produce IAA. Figure 7 shown.

[0097] Quantitative determination of IAA (indoleacetic acid) production: The ability of Streptomyces SF1 to produce IAA (indoleacetic acid) was quantitatively determined using UV-visible spectrophotometry. The IAA standard curve data are shown in Table 8 and Figure 8 .

[0098] Table 8 Standard curve data of IAA production by Streptomyces SF1

[0099]

[0100] The OD of IAA (indoleacetic acid) produced by Streptomyces SF1 was measured. 530 =1.2967, and substituting it into the standard curve, it was calculated that the IAA content could reach 47.27 mg / L.

[0101] 2) Ability to produce iron carriers: Pick a single colony of Streptomyces SF1 and inoculate it on a universal CAS medium for detecting iron carriers (chrome azurol 60.5 mg, hexadecyl trimethylammonium bromide 72.9 mg, FeCl2·6H2O 2.645 mg, peptone 4.5 g, glucose 9 g, beef extract powder 2.7 g, NaCl 4.5 g, agar 20 g, distilled water 1000 mL) plate, and culture it at 28°C for 5 days. If a transparent circle appears around the colony, it is positive. Determine the diameter of the transparent circle D (cm) and the colony diameter d (cm), and calculate the D / d value. The larger the ratio, the greater the activity of producing iron carriers. Each experiment was repeated 3 times. The results are as follows Figure 7 shown.

[0102] The results showed that there was an obvious yellow transparent circle around the Streptomyces SF1 strain, indicating that the strain had the ability to produce siderophores.

[0103] 3) Nitrogen fixation: A single colony of Streptomyces SF1 was streaked and inoculated on an Ashby nitrogen-free solid medium (mannitol 10 g, NaCl 0.2 g, CaCO3 5 g, KH2PO4 0.2 g, FeSO4·7H2O 0.1 g, CaSO4·2H2O 0.1 g, agar 20 g, distilled water 1000 mL) plate, and cultured at 28°C for 5 days to observe the growth of the strain. Figure 7 The results in the study showed that Streptomyces SF1 grew well under nitrogen-free conditions and had nitrogen-fixing activity. Each experiment was repeated 3 times.

[0104] 4) Production of ACC deaminase activity: A single colony of Streptomyces SF1 was streaked and inoculated on ADF solid medium (KH2PO4 4 g, Na2HPO4 6 g, MgSO4·7H2O 0.2 g, FeSO4·7H2O 0.1 g, H3BO3 10 μg, MnSO4 10 μg, ZnSO4 70 μg, CuSO4 50 μg, MoO3 10 μg, glucose 2 g, gluconic acid 2 g, citric acid 2 g, agar 20 g, distilled water 1000 mL), and the growth of the strain was observed after culturing at 28°C for 5 days. Figure 7 The results showed that Streptomyces SF1 could still grow on a medium with ACC as the sole nitrogen source after three transfers and had the activity of producing ACC deaminase. Each experiment was repeated three times.

[0105] 5) NH3 production activity: A single colony of Streptomyces SF1 was selected and inoculated into a test tube containing 10 mL of peptone water (10 g of peptone, 5 g of NaCl, 1000 mL of distilled water, PH 7.6), and cultured at 28°C for 5 days. 0.5 mL of Nessler's reagent was added to each tube. If a yellow-brown precipitate appeared, it indicated that the strain had NH3 production activity. If no yellow-brown precipitate appeared, it indicated that the strain had no NH3 production activity. Each experiment was repeated 3 times. Figure 7The results showed that the color reaction of Streptomyces SF1 was positive after cultivation, indicating that Streptomyces SF1 had the activity of producing NH3.

[0106] 6) Ability to dissolve inorganic phosphorus: Pick a single colony of Streptomyces SF1 and inoculate it on Pikovasky's phosphate dissolving medium (NaCl 0.3g, MgSO4·7H2O 0.3g, MnSO4 0.03g, KCl 0.3g, (NH4)2SO4 0.5g, FeSO4·7H2O 0.03g, Ca3(PO4) 25g, glucose 10g, agar 20g, distilled water 1000mL), and culture it at 28℃ for 5 days. If a transparent circle appears around the colony, it means that Streptomyces SF1 has the ability to dissolve inorganic phosphorus. Determine the diameter of the transparent circle D (cm) and the colony diameter d (cm), and calculate the D / d value. The larger the ratio, the stronger the ability to dissolve inorganic phosphorus. Each experiment was repeated 3 times. Figure 7 The results showed that there was an obvious phosphate-dissolving zone around the colonies of Streptomyces SF1, indicating that the strain had the ability to dissolve inorganic phosphorus.

[0107] 7) Ability to dissolve organic phosphorus: Pick a single colony of Streptomyces SF1 and inoculate it on a Montana organic phosphorus medium ((NH4)2SO4 0.5g, NaCl 0.3g, KCl 0.3g, glucose 10g, FeSO4·7H2O 0.03g, MnSO4 0.03g, CaCO3 5g, MgSO4·7H2O 0.3g, lecithin 0.2g, agar 20g, distilled water 1000mL), culture at 28℃ for 5 days, and the appearance of a transparent circle around the colony is positive. Determine the diameter of the transparent circle D (cm) and the colony diameter d (cm), and calculate the D / d value. The larger the ratio, the stronger the ability to dissolve organic phosphorus. Each experiment was repeated 3 times. Figure 7 The results showed that a phosphate-dissolving zone appeared around Streptomyces SF1, indicating that the strain had the ability to dissolve organic phosphorus.

[0108] 8) Potassium-solubilizing ability: A single colony of Streptomyces SF1 was picked and inoculated on a potassium-solubilizing solid culture medium (5 g sucrose, 0.2 g Na2HPO4, 0.5 g MgSO4·7H2O, 0.005 g FeCl3, 0.1 g CaCO3, 1 g potassium feldspar powder, 20 g agar, 1000 mL distilled water), and cultured at 28°C for 5 days. A transparent circle around the colony was considered positive. The diameter D (cm) of the transparent circle and the colony diameter d (cm) were measured, and the D / d value was calculated. The larger the ratio, the stronger the potassium-solubilizing ability. Each experiment was repeated 3 times. Figure 7 The results showed that there was an obvious transparent hydrolysis zone around Streptomyces SF1, indicating that the strain had the ability to hydrolyze potassium.

[0109] 9) Protease production ability: Pick a single colony of Streptomyces SF1 and spot-inoculate it on the protease-producing bacteria screening medium (15 g skim milk powder, 20 g agar, 1000 mL distilled water), culture it at 28°C for 5 days, and the appearance of a transparent circle around the colony is positive. Figure 6 The results show that there is an obvious transparent hydrolysis zone around Streptomyces SF1, which indicates that the strain has the ability to produce protease. The diameter of the transparent zone D (cm) and the colony diameter d (cm) were measured, and the D / d value was calculated. The larger the ratio, the stronger the ability to produce protease. Each experiment was repeated 3 times. Figure 7 The results showed that there was an obvious transparent hydrolysis zone around Streptomyces SF1, indicating that the strain had the ability to produce protease.

[0110] 10) Cellulase production capacity: A single colony of Streptomyces SF1 was picked and inoculated on CMC medium (CMC-Na 10g, KNO 32g, MgSO4·7H2O 0.3g, FeSO4·7H2O 0.03g, NaCl 0.5g, K2HPO4 1g, agar 20g, distilled water 1000mL), cultured at 28℃ for 5 days, then 0.5% Congo red solution was added for staining for 1h, and then 1 mol / L NaCl solution was used to cover the plate for decolorization for 10min. If a transparent circle appeared around the colony, it was positive. The diameter D (cm) of the transparent circle and the colony diameter d (cm) were measured, and the D / d value was calculated. The larger the ratio, the stronger the cellulase production capacity. Each experiment was repeated 3 times. Figure 7 The results showed that there was an obvious transparent hydrolysis zone around Streptomyces SF1, indicating that the strain had the ability to produce cellulase.

[0111] The test results of the growth-promoting properties of Streptomyces SF1 are shown in Table 9, where the D / d value represents the ratio of the transparent halo diameter D (cm) to the colony diameter d (cm). Where D represents the transparent halo diameter, and d represents the colony diameter. The larger the D / d, the stronger the growth-promoting properties of the strain.

[0112] Table 9 Test results of growth-promoting properties of Streptomyces SF1

[0113]

[0114] Note: “*” indicates that Streptomyces SF1 has this growth-promoting property.

[0115] From Table 9 and Figure 7 It can be seen that the Streptomyces SF1 described in the present invention has the characteristics of producing IAA, ammonia, ACC deaminase, nitrogen fixation, siderophore production, inorganic phosphorus solubilization, organic phosphorus solubilization, protease production, potassium solubilization and cellulase production, indicating that it has the ability to promote plant growth.

[0116] Example 5 Application of disease-resistant growth-promoting bacteria to the growth of licorice

[0117] Licorice seed pretreatment: Select licorice seeds with full grains and uniform size, first soak them with 85% concentrated H2SO4 for 45 minutes, stir occasionally, then rinse them with distilled water for 3 times, and then disinfect them with 0.1% H2O2 for 10 minutes. Finally, rinse them with distilled water several times until they are no longer sticky. After washing, place them in a beaker and soak them in distilled water for 6-8 hours to allow the seeds to fully absorb water and set aside.

[0118] Experimental design: A completely randomized design was adopted, with a total of 2 treatments, namely the control CK (sterile distilled water) and the disease-resistant growth-promoting bacteria in Example 2. Select fully water-absorbed, plump and uniform licorice seeds, dry the surface moisture, place them in different treatment solutions (sterile distilled water, disease-resistant growth-promoting bacteria) and soak them for 3 hours respectively, and then evenly place them in culture dishes (9cm×9cm×3cm, 3mL sterile distilled water was added to each dish to keep it moist) padded with double-layer sterile filter paper for germination, 40 seeds per dish. The experimental conditions were light / dark (12 / 12h, 28 / 20℃). Distilled water was added to a constant mass by weighing every day to maintain constant substrate conditions. After germination, the germination potential, germination rate, germination index and seedling vitality index of each treatment seed were measured; the length of the licorice seedling embryo and radicle was measured with a ruler, and the thickness of the seedling embryo and radicle was measured with a vernier caliper. The results are shown in Tables 10 and Fig. 9 .

[0119] Table 10 Licorice seed germination experimental results

[0120] Measuring indicators CK Disease-resistant growth-promoting bacteria Germination rate (%) 70.28％±6.5％b 81.94％±5.6％a Germination potential 0.70±0.065b 0.82±0.056a Germination Index 55.08±4.57b 66.18±4.87a Radicle length (cm) 1.49±0.19b 2.35±0.22a Germ length (cm) 2.21±0.26a 2.58±0.13a Radicle diameter (mm) 0.69±0.077b 0.88±0.037a Germ diameter(mm) 1.50±0.028a 1.53±0.086a Seedling Vitality Index 211.69±22.21b 307.70±23.19a Dry weight (g / dish) 0.21±0.0195b 0.26±0.0095a

[0121] Note: Different lowercase letters in the same column indicate significant differences (P<0.05).

[0122] From Table 10 and Fig. 9 It can be seen that the disease-resistant and growth-promoting bacteria have a significant promoting effect on the germination of licorice seeds and the growth of seedlings. Compared with the control, the germination rate, germination potential, germination index of licorice seeds, seedling radicle length, radicle thickness, seedling vitality index and seedling dry weight can be significantly increased after treatment with the bacterial solution.

[0123] It can be seen from the above examples that the disease-resistant and growth-promoting bacterial agent with the Streptomyces SF1 described in the present invention as the effective active ingredient can effectively prevent and control plant diseases and has the effect of promoting plant growth.

[0124] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention. Sequence Listing <110> Ningxia Medical University <120> A strain of Streptomyces with biological control effect and a disease-resistant growth-promoting bacterial agent and their application <160> 3 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1410 <212> DNA <213> Artificial Sequence <400> 1 atggctcagg acgaacgctg gcggcgtgct taacacatgc aagtcgaacg atgaaccact 60 tcggtgggga ttagtggcga acgggtgagt aacacgtggg caatctgccc ttcactctgg 120 gacaagccct ggaaacgggg tctaataccg gataacactc tcgcaggcat ctgtgggggt 180 tgaaagctcc ggcggtgaag gatgagcccg cggcctatca gcttgttggt gaggtagtgg 240 ctcaccaagg cgacgacggg tagccggcct gagagggcga ccggccacac tgggactgag 300 acacggccca gactcctacg ggaggcagca gtggggaata ttgcacaatg ggcgaaagcc 360 tgatgcagcg acgccgcgtg agggatgacg gccttcgggt tgtaaacctc tttcagcagg 420 gaagaagcga aagtgacggt acctgcagaa gaagcgccgg ctaactacgt gccagcagcc 480 gcggtaatac gtagggcgca agcgttgtcc ggaattattg ggcgtaaaga gctcgtaggc 540 ggtctgtcgc gtcggatgtg aaagcccggg gcttaacccc gggtctgcat tcgatacggg 600 cagactagag tgtggtaggg gagatcggaa ttcctggtgt agcggtgaaa tgcgcagata 660 tcaggaggaa caccggtggc gaaggcggat ctctgggcca ttactgacgc tgaggagcga 720 aagcgtgggg agcgaacagg attagatacc ctggtagtcc acgccgtaaa cggtgggcac 780 taggtgttgg cgacattcca cgtcgtcggt gccgcagcta acgcattaag tgccccgcct 840 ggggagtacg gccgcaaggc taaaactcaa aggaattgac gggggcccgc acaagcagcg 900 gagcatgtgg cttaattcga cgcaacgcga agaaccttac caaggcttga catcgcccgg 960 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 <400> 2 cagagtttga tcctggctag gaggtgatcc agccgca 37 <210> 3 <211> 37 <212> DNA <213> Artificial Sequence <400> 3 agtttgatcm tggctcaggg ttaccttgtt acgactt 37

Claims

1. A strain of Streptomyces with biocontrol effect ( Streptomyces sp.) SF1, characterized in that The deposit number of the Streptomyces SF1 is CGMCC No.23417.

2. A disease-resistant growth-promoting bacterial agent, characterized in that: The active ingredients of the disease-resistant and growth-promoting bacterial agent include the Streptomyces SF1 described in claim 1.

3. The disease-resistant growth-promoting bacterial agent according to claim 2, characterized in that: The concentration of Streptomyces SF1 in the disease-resistant growth-promoting bacterial agent is (1-6)×10 8 CFU / mL.

4. Use of the Streptomyces SF1 according to claim 1 or the disease-resistant growth-promoting bacterial agent according to any one of claims 2 to 3 in preventing and controlling plant diseases and / or promoting plant growth; The pathogenic bacteria of the plant disease are one or more of Fusarium oxysporum, Sclerotinia sclerotiorum, Rhizoctonia solani, Alternaria sordella, Colletotrichum oxysporum and Phyllosporium dioscorea; The plant is a medicinal plant.

5. The use according to claim 4, characterized in that: The medicinal plants include angelica, codonopsis, astragalus, wolfberry, yam and liquorice.

6. The use according to claim 4 or 5, characterized in that: The plant diseases are one or more of plant root rot, plant leaf spot and plant anthracnose.

7. The use according to claim 4 or 5, characterized in that: The plant disease is plant leaf spot disease.

8. The use according to claim 4 or 5, characterized in that: The promoting plant growth includes promoting seed germination and / or seedling growth.

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