Actinomycetes and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and discloses an actinomycete, which is streptomyces aureoflavus CYS4-5, is preserved in China General Microbiological Culture Collection Center (CGMCC), is preserved on October 17, 2023, and has the preservation number of CGMCC No.28506. The actinomycete has the advantages that the actinomycete can be used for preparing an actinomycete culture medium; the strain has the potential of preventing and treating salvia miltiorrhiza root rot, has obvious bacteriostatic activity on gaeumannomyces graminis, rhizoctonia cerealis, exserohilum turcicum, sclerotinia sclerotiorum, wheat root rot, fusarium graminearum and botryosphaeria dothidea, and plays an important role in biological prevention and treatment of diseases of crops such as wheat, corn, rape and grapes.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms and relates to actinomycetes and applications thereof. Background Art

[0002] Salvia miltiorrhiza, also known as red ginseng, is one of the earliest and most commonly used Chinese medicinal materials in traditional Chinese medicine. Salvia miltiorrhiza root rot is a major disease in Salvia miltiorrhiza production. In recent years, with the expansion of continuous cropping and planting area, the damage has become serious. Chemical control is the main means of prevention and control of Salvia miltiorrhiza root rot, but the prevention and control effect is limited, and the irrational application of chemical pesticides will have a serious impact on the environment and the quality of medicinal materials.

[0003] A potential way to reduce the negative impact of the continued use of fertilizers and pesticides on the environment is to use plant growth promoting rhizobacteria (PGPR). Soil actinomycetes, as the microbial resource that produces the most types of antibiotics and the richest secondary metabolites, have been used in the agricultural field as an important biocontrol factor for plant disease biological control. Biocontrol agents made from actinomycetes have the advantages of being non-toxic, residue-free, good compatibility with the environment, long effective period for disease prevention, and non-toxic to non-target microorganisms. Summary of the invention

[0004] The invention aims at the technical problem of biological control of Salvia miltiorrhiza root rot and provides an actinomycete. The strain CYS4-5 has the potential to control the Salvia miltiorrhiza root rot and also has obvious antibacterial activity against wheat take-all pathogen, wheat sheath blight pathogen, corn leaf blight pathogen, rapeseed sclerotinia pathogen, wheat root rot pathogen, wheat fusarium spore pathogen and grape spore pathogen, and plays an important role in the biological control of diseases of crops such as wheat, corn, rapeseed and grape.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] In the first aspect, the present invention provides an actinomycete, which is Streptomyces aureoverticillatus CYS4-5, deposited at the General Microbiology Center of China National Microbiological Culture Collection Administration, date of deposit: October 17, 2023, and deposit number: CGMCC No.28506.

[0007] In a second aspect, the present invention provides the use of the above-mentioned actinomycetes in biological control of crops.

[0008] In a third aspect, the present invention provides the use of the above-mentioned actinomycetes in preventing and treating Salvia miltiorrhiza root rot.

[0009] In a fourth aspect, the present invention provides the use of the above-mentioned actinomycetes in preventing and controlling wheat take-all disease, wheat sheath blight, corn leaf spot disease, rapeseed sclerotinia rot, wheat root rot, wheat fusarium rust, and grape stem cavity disease.

[0010] In a fifth aspect, the present invention provides a biocontrol agent comprising the fermentation liquid or metabolites of the above-mentioned actinomycetes.

[0011] Compared with the prior art, the present invention has the following beneficial effects:

[0012] The invention extracts an actinomycete with obvious antagonistic effect from soil, which is Streptomyces aureoverticillatus CYS4-5. The actinomycete has the potential to prevent and treat root rot of salvia miltiorrhiza, provides strain resources for biocontrol agents for root rot of salvia miltiorrhiza, and provides a new approach for biological control of root rot of salvia miltiorrhiza. The strain also has obvious antibacterial activity against wheat take-all pathogen, wheat sheath blight pathogen, corn leaf blight pathogen, rapeseed sclerotinia pathogen, wheat root rot pathogen, wheat fusarium spore pathogen, and grape cavitation pathogen, and plays an important role in biological control of diseases of crops such as wheat, corn, rapeseed, and grape. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The figure shows the colony morphology of the strain CYS4-5 of the present invention on GS (Gao's medium No. 1).

[0014] Figure 2 The hyphae and spore morphology of the strain CYS4-5 of the present invention.

[0015] Figure 3 This is the antibacterial effect of the strain CYS4-5 of the present invention on the pathogen TD-1.

[0016] Figure 4 The present invention shows the antibacterial effect of the fermentation liquid of the strain CYS4-5 on the pathogenic bacteria TD-1.

[0017] Figure 5 The figure is the result of the antibacterial spectrum determination of the strain CYS4-5 of the present invention.

[0018] Figure 6 The figures show the culture morphologies of the strain CYS4-5 of the present invention on different culture media.

[0019] Figure 7 The phylogenetic tree of the strain CYS4-5 of the present invention and related strains.

[0020] Figure 8 The following are the potted plant emulation test results of the strain CYS4-5 of the present invention. DETAILED DESCRIPTION

[0021] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means known to those skilled in the art. The test methods in the following examples are conventional methods unless otherwise specified.

[0022] Example 1

[0023] 1 Experimental materials and methods

[0024] 1.1 Test materials

[0025] Test plants: Salvia miltiorrhiza variety: “Zhengdan No. 1”, provided by Henan Academy of Agricultural Sciences.

[0026] The plant pathogens tested included: wheat take-all pathogen (Gaeumannomyces tritici), wheat gray mold pathogen (Botrytis cinerea), wheat sheath blight pathogen (Rhizoctonia cerealis), corn leaf blight pathogen (Exserohilum turcicum), rapeseed sclerotinia pathogen (Sclerotinia sclerotiorum), wheat root rot pathogen (Bipolaris sorokiniana), wheat head blight pathogen (F. pseudograminearum), and grape cavity pathogen (Botryosphaeria dothidea). The above target pathogens were provided by the Plant Disease Biological Control Laboratory of Henan Agricultural University.

[0027] 1.2 Screening of actinomycetes

[0028] 1.2.1 Isolation and purification of strains

[0029] Soil samples were collected from corn fields (wheat-corn rotation) in Mianchi County, Sanmenxia City, Henan Province. The gradient dilution method was used to separate and purify the actinomycetes distributed in the soil. After the soil samples were gradient diluted with sterile water, 200 μL of dilutions of 10 -3 , 10 -4 and 10 -5 The soil suspension was evenly spread on Gao's No. 1 plate, and then the plate was placed in a 28℃ biochemical incubator for inversion and cultured, and different gradients were repeated 3 times. After a single colony of actinomycetes grew, a single colony of different shape, color, and size was picked up with an inoculation loop for streaking and re-culture.

[0030] 1.2.2 Confrontation training

[0031] The screening of biocontrol bacteria was carried out by the plate confrontation method. A 5 mm diameter cake of TD-1 (Fusarium oxysporum), the root rot pathogen of Salvia miltiorrhiza, was inoculated in the center of a 9 cm diameter PDA plate. At the same time, an inoculation loop was used to dip actinomycetes and streaked symmetrically 2 cm from the edge of the plate. The plate was incubated upside down in a 28°C incubator for 7 days, and repeated 3 times. The antibacterial effect of actinomycetes on pathogens was observed.

[0032] 1.2.3 Determination of antibacterial effect of fermentation broth

[0033] Preparation of fermentation liquid: Pick 6 newly activated CYS4-5 bacterial blocks and inoculate them into liquid GS medium, shake and incubate at 28°C, 180r / min for 3 days to obtain the CYS4-5 seed liquid. Inoculate the seed liquid into new liquid GS medium at a ratio of 10%, shake and incubate for 5 days, and collect the bacterial liquid, which is the fermentation liquid for later use.

[0034] The growth rate method was used to determine the antibacterial effect of the biocontrol bacteria fermentation broth on the pathogen TD-1: the biocontrol bacteria fermentation broth after shaking culture for 5 days was filtered with a bacterial filter and set aside; treatment group: PDA culture medium and the filtrate of the biocontrol bacteria fermentation broth were mixed in a 1:1 ratio and poured onto the plate.

[0035] Control group: PDA medium and Gould's medium filtrate were mixed in a ratio of 1:1 and poured onto a plate. Fresh TD-1 bacterial block was inoculated in the center of the plate and cultured in a 28°C incubator for 7 days before observation.

[0036] 1.2.4 Determination of antibacterial spectrum

[0037] The antagonistic activity of strain CYS4-5 was determined using Gaeumannomyces tritici, Botrytis cinerea, Rhizoctonia cerealis, Exserohilum turcicum, Sclerotinia sclerotiorum, Bipolaris sorokiniana, F. pseudograminearum and Botryosphaeria dothidea as target bacteria, respectively. The specific method is shown in 1.1.

[0038] 1.3 Classification and identification of strains

[0039] 1.3.1 Observation of culture characteristics

[0040] The purified strain was inoculated on seven culture media, including GS (Gao's medium No. 1), PDA (potato dextrose agar medium), ISP1 (tryptone yeast extract medium), ISP2 (yeast extract maltose agar medium), ISP4 (starch inorganic salt medium), ISP5 (glycerol aspartate medium), and ISP7 (tyrosine glycerol medium), and cultured at 28°C for 7-30 days. The colony morphology of the strain in different culture media was observed.

[0041] 1.3.2 Physiological and biochemical characteristics determination

[0042] Refer to the methods described in the Manual of Identification of Common Bacterial Systems (2001) to determine the strain's ability to utilize different carbon sources (glucose, fructose, sucrose, galactose, maltose, mannitol, xylose), different nitrogen sources (leucine, serine, tryptophan, glycine, tyrosine, alanine, KNO3), produce H2S, produce melanin, degrade starch, degrade phosphate, degrade potassium, and liquefy gelatin.

[0043] 1.3.3 16srRNA gene sequence analysis

[0044] The genomic DNA of actinomycetes was extracted by CTAB method. The genomic DNA of actinomycetes was used as template, and PCR amplification was performed using actinomycete-specific primers based on the 16S rRNA gene of actinomycetes (upstream primer: 5′-AGAGTTTGATCCTGGCTCAG-3′; downstream primer: 5′-GGTTACCTTGTTACGACTT-3′). The system was: 2×PCR mix 25μL, primers (23F / 1492R) 1μL each, DNA template 2μL, sterile water 21μL; the program was: 95℃5min; 94℃1min, 59℃30s, 72℃1.5min, 35 cycles; and then 72℃ for 10min extension. The PCR amplification products were detected by 1% agarose gel electrophoresis and then submitted to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequence of the successfully sequenced 16S rRNA gene was compared with the GenBank database by BLAST to determine the species with the closest relationship. The 16S rRNA gene sequences of related species were downloaded from the NCBI database, and the phylogenetic tree was constructed using the neighbor-joining method in MEGA7 software, and cluster analysis was performed.

[0045] 2 Results and analysis

[0046] 2.1 Isolation of strains

[0047] An actinomycete was isolated and purified from soil samples by the dilution plate method and named CYS4-5. The colony morphology of strain CYS4-5 on GS (Gao's medium No. 1) is as follows Figure 1 As shown in Figure 2, the spores of strain CYS4-5 were oblong, arranged in straight chains, and spores were produced at the top of the spore chains. The aerial hyphae were alternate and nearly upright, with smooth branches and no constriction, as shown in Figure 2. Figure 2 shown.

[0048] 2.1.1 Results of confrontation training

[0049] The results of confrontation culture test are as follows Figure 3 As shown, strain CYS4-5 has obvious antagonistic activity against pathogen TD-1, with an inhibition rate of about 49.16%.

[0050] 2.1.2 Fermentation broth confrontation

[0051] The antibacterial effect of fermentation broth is as follows Figure 4 As shown, the fermentation broth of strain CYS4-5 had obvious antagonistic activity against pathogenic bacteria TD-1, with an inhibition rate of about 45.95%.

[0052] 2.1.3 Determination of antibacterial spectrum

[0053] The results of the antibacterial spectrum test were as follows Figure 5 As shown, strain CYS4-5 has obvious antibacterial activity against wheat sheath blight, wheat take-all pathogen, wheat head blight, corn leaf blight, gray mold, rapeseed sclerotinia, wheat root rot, and grape stem cavity pathogen, indicating that the strain has a broad-spectrum antibacterial activity.

[0054] 2.2 Classification and Identification

[0055] 2.2.1 Observation of culture characteristics

[0056] The different morphologies of strain CYS4-5 on GS, PDA, ISP1, ISP2, ISP4, ISP5, and ISP7 media are shown in Figure 2. Figure 6 shown.

[0057] from Figure 6It can be seen that strain CYS4-5 grows poorly on ISP4 medium and grows well on the other 6 mediums. On GS medium, aerial mycelia are orange-red, and the mycelia in the base are dark red. On PDA medium, aerial mycelia are orange-red, and the mycelia in the base are pink. On ISP1 medium, aerial mycelia are orange-yellow, and the mycelia in the base are orange-yellow. On ISP2 medium, aerial mycelia are pink, and the mycelia in the base are pink. On ISP4 medium, aerial mycelia are pink, and the mycelia in the base are pink. On ISP5 medium, aerial mycelia are orange-yellow, and the mycelia in the base are orange-yellow. On ISP7 medium, aerial mycelia are orange-yellow, and the mycelia in the base are orange-red.

[0058] 2.2.2 Physiological and biochemical test results

[0059] The strain's utilization of different carbon and nitrogen sources; production of H2S, melanin, starch dissolution, gelatin liquefaction and other physiological and biochemical abilities are shown in Table 1. It can be seen that strain CYS4-5 can utilize carbon sources such as glucose, sucrose, maltose, mannitol, but cannot utilize carbon sources such as fructose, xylose, and galactose. It can utilize nitrogen sources such as leucine, serine, glycine, alanine, and potassium nitrate, but cannot utilize nitrogen sources such as tyrosine and ammonium nitrate. It has the ability to dissolve starch, produce H2S, liquefy gelatin, and fix nitrogen. When the salt content is higher than 2.5%, the strain cannot grow.

[0060] Table 1 Physiological and biochemical test results of strain CYS4-5

[0061]

[0062]

[0063] 2.2.3 16S rRNA identification

[0064] The 16S rRNA gene sequence of strain CYS4-5 is 1450 bp in length (see SEQ ID NO. 1), and the phylogenetic tree is as follows Figure 7 As shown, it can be seen that strain CYS4-5 is most closely related to Streptomyces aureoverticillatus.

[0065] 2.3 Results of biological control test

[0066] The results of potted plant protection test are as follows Figure 8 The results showed that 30 days after inoculation, the plants inoculated with TD-1 spore solution alone showed typical symptoms of root rot of Salvia miltiorrhiza, such as short plants, black fibrous roots and black stem bases; the plants inoculated with CYS4-5 fermentation solution and then TD-1 spore solution grew well, with well-developed and bright red roots.

[0067] The disease index statistics are shown in Table 2. The results show that the disease index of the group inoculated with TD-1 spore solution alone was 12.5, and the disease index of the group inoculated with TD-1 spore solution after being treated with the fermentation liquid of strain CYS4-5 was 2.78. The control effect of strain CYS4-5 on root rot of Salvia miltiorrhiza reached 77.76%. This shows that the actinomycete strain CYS4-5 can significantly reduce the occurrence of root rot of Salvia miltiorrhiza and has good biocontrol potential.

[0068] Table 2 Statistical results of disease index of strain CYS4-5 for the control of Salvia miltiorrhiza root rot

[0069] deal with Disease index Control effect (%) TD-1 12.5 / TD-1+CYS4-5 2.78 77.76

[0070] The embodiments described above are only preferred embodiments of the present invention and are only used to explain the present invention, not to limit the scope of implementation of the present invention. For those skilled in the art, other implementation methods can certainly be easily made by replacement or modification based on the technical contents disclosed in this specification. Therefore, all changes and improvements made on the principles of the present invention should be included in the scope of the patent application of the present invention.

Claims

1. An actinomycete, characterized in that The actinomycetes are Streptomyces aureus ( Streptomyces aureoverticillatus )CYS4-5, depository unit: General Microbiology Center of China Microbiological Culture Collection Administration, deposit date: October 17, 2023, deposit number: CGMCC No.28506.

2. Use of the actinomycetes according to claim 1 in biological control of crops.

3. Use of the actinomycetes according to claim 1 in preventing and treating root rot of Salvia miltiorrhiza.

4. Use of the actinomycete according to claim 1 in preventing and controlling wheat take-all disease, wheat sheath blight, corn leaf spot disease, rapeseed sclerotinia rot, wheat root rot, wheat fusarium head blight, and grape cavity disease.

5. A biocontrol agent, characterized in that: A fermentation broth or metabolite containing the actinomycete according to claim 1.

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